WO2015084969A1

WO2015084969A1 - Plants with improved drought tolerance

Info

Publication number: WO2015084969A1
Application number: PCT/US2014/068392
Authority: WO
Inventors: Patrick S. Schnable; Sanzhen LIU; Frank HOCKHOLDINGER; Josefine NESTLER
Original assignee: Iowa State University Research Foundation, Inc.; University Of Bonn
Priority date: 2013-12-03
Filing date: 2014-12-03
Publication date: 2015-06-11

Abstract

Disclosed herein is a method for increasing drought tolerance in a plant. The method includes introducing into the plant a recombinant DNA construct that includes a heterologous promoter operably linked to a polynucleotide that encodes a RTH5 family member. The method can be performed in monocots and dicots. Also, disclosed herein are plants and expression cassettes that include recombinant DNA that contains a heterologous promoter operably linked to polynucleotides that encodes an RTH5 family member.

Description

PLANTS WITH IMPROVED DROUGHT TOLERANCE

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application No. 61/91 1,275, filed on December 3, 2013 and the benefit of U.S. provisional Application No. 61/91 1,826, filed on December 4, 2013. Each application is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on December 3, 2014 as a text file named

"36446_0084Pl_Sequence_Listing.txt," created on November 25, 2014, and having a size of 92,427 bytes is hereby incorporated by reference.

FIELD

The field of disclosure relates to plant breeding and genetics and, in particular, relates to recombinant DNA constructs useful in plants for conferring improved agronomic traits.

BACKGROUND

Improving agronomic traits in crop plants is beneficial to farmers. Several factors influence crop yield. Abiotic stress is the primary cause of crop loss worldwide, causing average yield losses of more than 50% for major crops. Among the various abiotic stresses, drought is a major factor that limits crop productivity worldwide. Exposure of plants to a water-limiting environment during various developmental stages appears to activate various physiological and developmental changes. Molecular mechanisms of abiotic stress responses and the genetic regulatory networks of drought stress tolerance have been studied.

Natural responses to abiotic stress vary among plant species and among varieties and cultivars within a plant species. Certain species, varieties or cultivars are more tolerant to abiotic stress such as drought than others. Transgenic approaches are needed for improving drought tolerance in crop plants.

SUMMARY

Compositions and methods to increase drought tolerance and to increase nutrient uptake in plants are disclosed. Compositions comprise recombinant RTH5 family polynucleotides, RTH5 polypeptides, expression cassettes, plants and seeds. The methods of the invention comprise increasing the expression of an RTH5 polypeptide in a plant of interest. Any method for increasing the expression of the RTH5 polypeptide is encompassed. That is plants can be transformed with a DNA construct comprising an RTH5 polynucleotide operably linked with a heterolgous promoter that drives expression in plant roots.

Alternatively, expression levels of the endogenous RTH5 polypeptide in the plant can be increased by methods available in the art to enhance the expression of endogenous genes. Expression constructs comprising an RTH5 polynucleotide as well as plants and seed having increased levels of an RTH5 polypeptide are provided.

Expression of RTH5 polypeptides increases root hair formation and growth.

Increased expression of RTH5 polypeptides in the root of a plant thus results in increased drought tolerance and increased nutrient uptake.

The following embodiments are encompassed by the present invention:

1. A method for increasing root hair formation and growth in a plant, said method comprising increasing the expression of an RTH5 polypeptide in roots of said plant.

2. The method of embodiment 1, wherein said RTH5 polypeptide is an endogenous RTH5 polypeptide.

3. The method of embodiment 2, wherein said expression is increased by alterations to the genome of the plant said alterations comprising additions, deletions, and/or substitutions of nucleotides into the genome.

4. The method of embodiment 1, wherein said expression is increased by transforming said plant with a recombinant DNA construct comprising a polynucleotide encoding an RTH5 polypeptide operably linked to a heterologous promoter that drives expression in a plant root.

5. A method for increasing drought tolerance in a plant, said method comprising introducing into said plant a recombinant DNA construct, said construct comprising a heterologous promoter that drives expression in a plant root operably linked to a

polynucleotide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

(b) a nucleotide sequence encoding the amino acid sequence set forth in

SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13;

(c) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein said polynucleotide encodes a polypeptide having NADPH oxidase activity; and (d) a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13, wherein said polynucleotide encodes a polypeptide having NADPH oxidase activity.

6. A method for enhancing nutrient uptake in a plant, said method comprising introducing into said plant a recombinant DNA construct, said construct comprising a heterologous promoter that drives expression in a plant root operably linked to a

(a) a nucleotide sequence comprising SEQ ID NO: 2;

(b) a nucleotide sequence encoding the amino acid sequence set forth in

SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13;

(c) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein said polynucleotide encodes a polypeptide having NADPH oxidase activity; and

(d) a nucleotide sequence encoding an amino acid sequence having at least

90% sequence identity to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13, wherein said polynucleotide encodes a polypeptide having NADPH oxidase activity.

7. The method of embodiment 5 or 6, wherein said polynucleotide is stably integrated into the genome of the plant.

8. The method of any one of embodiments 5-7, wherein said plant is a plant cell.

9. The method of any one of embodiments 5-8, wherein said plant is a dicot.

10. The method of embodiment 9, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

11. The method of any one of embodiments 5-8, wherein said plant is a monocot. 12. The method of embodiment 11, wherein said monocot is maize, sugarcane, wheat, rice, barley, sorghum, millet, switchgrass, or rye.

13. A seed of the plant produced by the method of any one of embodiments 1-12.

14. A plant comprising a recombinant DNA construct comprising a heterologous promoter that drives expression in a plant root operably linked to a polynucleotide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13; (c) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein said polynucleotide encodes a polypeptide having NADPH oxidase activity; and

(d) a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13, wherein said polynucleotide encodes a polypeptide having NADPH oxidase activity.

15. The plant of embodiment 14, wherein said plant is a cell.

16. The plant of embodiment 14 or 15, wherein said plant is a monocot.

17. The plant of embodiment 16, wherein said monocot is maize, sugarcane, wheat, rice, barley, sorghum, millet, switchgrass, or rye.

18. The plant of embodiment 14 or 15, wherein said plant is a dicot.

19. The plant of embodiment 18, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

20. The plant of any one of embodiments 14-19, wherein said polynucleotide is stably incorporated into the genome of the plant.

21. A seed of the plant of embodiment 20.

22. An expression cassette comprising a polynucleotide operably linked to a heterologous promoter that drives expression in a plant root, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13;

23. The expression cassette of embodiment 22, wherein said plant is a monocot. 24. The expression cassette of embodiment 23, wherein said monocot is maize, sugarcane, wheat, rice, barley, sorghum, millet, switchgrass, or rye.

25. The expression cassette of embodiment 22, wherein said plant is a dicot.

26. The expression cassette of embodiment 25, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa. 27. A method of selecting or identifying an allelic variant of rth5 in a maize plant, the method comprising the steps of:

a. obtaining a population of maize plants, wherein said maize plants exhibit an alteration of at least one agronomic characteristic; wherein the at least one agronomic characteristic is selected from the group consisting of increased root hair formation and growth, increased drought tolerance, and enhanced nutrient uptake;

b. evaluating allelic variations with respect to the polynucleotide sequence encoding a protein comprising SEQ ID NO:3, or in the genomic region that regulates the expression of the polynucleotide encoding the protein;

c. associating allelic variations with said alteration of at least one agronomic characteristic; and

d. selecting or identifying an allelic variant that is associated with said alteration of at least one agronomic characteristic.

28. A method of selecting or identifying a first maize plant or a first maize germplasm that has one or more beneficial alleles of rth5, the method comprising:

(a) screening a plurality of maize plants or a plurality of maize germplasm for

at least one polymorphism within a marker locus, wherein the marker locus is:

(a) a first polynucleotide having at least 90% and less than 100% nucleotide sequence identity with SEQ ID NO: 1 or 2; or

(b) a second polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% and less than 100% sequence identity to SEQ ID NO:3, wherein expression of the first or second polynucleotide in a maize plant results in a phenotype comprising an alteration of at least one agronomic characteristic when compared to a control maize plant; wherein the at least one agronomic characteristic is selected from the group consisting of increased root hair formation and growth, increased drought tolerance, and enhanced nutrient uptake; and wherein the control maize plant comprises:

(c) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 2; or

(d) a polynucleotide encoding the amino acid sequence of SEQ ID NO:3;

(b) identifying a first maize plant or a first maize germplasm comprising the at least one polymorphism of the marker locus; and

(c) selecting the first maize plant or first maize germplasm of step (b).

29. A method of reducing soil erosion in a crop field, said method comprising introducing into a plant a recombinant DNA construct, said construct comprising a heterologous promoter that drives expression in a plant root operably linked to a polynucleotide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

30. A method of increasing infection of a plant with a desirable microorganism, said method comprising introducing into said plant a recombinant DNA construct, said construct comprising a heterologous promoter that drives expression in a plant root operably linked to a polynucleotide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

(c) a nucleotide sequence having at least 90% sequence identity to SEQ ID

NO: 2, wherein said polynucleotide encodes a polypeptide having NADPH oxidase activity; and

31. The method of embodiment 30, wherein the beneficial microorganism is rhizobium or Pse dotnonas putida KT2440.

32. A method of increasing the resistance to root pathogens in a plant, said method comprising reducing the expression of an RTH5 family member, or inhibiting the function of the RTH5 family member.

33. The method of embodiment 32, wherein the maize root pathogen is Fusarium species, like F. verticilUoides or F. graminearu. BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.

Figure 1 shows the mapping of rth5. (A) Map-based cloning experiments positioned the rth5 gene within a 280 kb interval of chromosome 3. (B) Sequencing revealed a G to A (G>A) substitution in the last exon of candidate gene GRMZM2G426953 resulting in a cysteine by tyrosine (C>Y) substitution in the protein sequence for the rth.5-1 allele at position 821. The positions of MM transposon insertions in three additional alleles, rth.5-2, rth5-3, and rth5-4, identified in transposon tagged populations, are shown. (C) Alignment of the C-terminal 50 amino acids of human, yeast and Arabidopsis NADPH oxidases and wild- type and mutant RTH5. TM: trans-membrane, NADPHox: NADPH oxidase domain, EF hand: Ca²⁺ binding site, Fe red: Ferric reduction domain, FAD / NAD: FAD / NAD binding site, Hs: Homo sapiens, Sc: Saccharomyces cerevisiae, At: Arabidopsis thaliana, Zm: Zea mays.

Figure 2 shows a phylogenetic reconstruction of NADPH oxidases of maize, rice, Arabidopsis, and soybean. The phylogenetic relations of RTH5 were reconstructed by blasting the RTH5 sequence against the maize (Zea mays, maizesequence.org), rice (Oryza sativa, rice.plantbiology.msu.edu), soybean (Glycine max, soybean.org) and Arabidopsis thaliana (arabidopsis.org) databases. MEGA4 was used for alignment and tree generation via the Neighbor Joining algorithm. Two subfamilies were discriminated by presence or absence of several conserved domains in group I and II . Monocot specific clades / sub clades are highlighted with gray boxes.

Figure 3 shows an alignment of RTH5 and its homologs from maize, soybean, rice and Arabidopsis. Proteins are listed according to their order in the phylogenetic tree (Figure 2). Sequences were aggregated in units of ten amino acids (aa). Five and more aa per unit are depicted as boxes, >5 as lines. Color code: red - conserved in both groups, blue - conserved in group I, green - conserved in group II, dark gray - annotated functional domain, light gray - no functional annotation.

Figure 4 shows tissue-specific expression of rth5 transcripts and accumulation of

RTH5 proteins. (A) Expression of the rth5 gene family members in root hairs. (B) rth5 expression in different maize tissues. All expression levels are relative to the Myosin gene (Genbank AC: 486090G09.xl). Error bars indicate standard deviation; fold changes are indicated relative to root hairs. -value obtained via Student's t-test, -value * < 0.05, n = 3. R A in situ hybridization of rt/z5 antisense (C) and sense (D) probes on paraffin- embedded cross-sections. (E-J) Immunohistochemistry was performed on paraffin-embedded cross (E, F) and longitudinal sections of the differentiation (G, H) or elongation zone (I, J). (E, C, F) Sections incubated with anti-RTH5 antibody. (B, D, F) Control sections incubated with buffer. Arrows indicate root hairs. Scale bars in C, D: 50 μιη, E-J: 100 μιη. For all experiments three-days-old B73 seedling were used.

Figure 5 shows the expression of the rth5 gene in various tissues in the normalized expression value of FPKM (fragments per kilobase of exon per million of fragments mapped). The data was downloaded from qteller.com as of 4/1/2013.

Figure 6 shows an expression analysis of maize rboh genes. Quantitative RT-PCR analysis of different root tissues of all maize rboh gene family members in three-day-old B73 seedlings is shown. Coleoptilar nodes and leaves were harvested from seven-day-old seedlings. Expression was calculated (n = 3) relative to reference gene myosin. Error bars represent standard deviation. Please note the different relative expression levels on the Y- axes of the different genes have been adjusted to highlight tissue-specific expression differences for individual genes.

Figure 7 is a schematic model for the predicted function of RTH5 in root hair initiation and growth. (A) Shows the suggested function of RTH5 in root hair initiation. A trichoblast-specific signal can activate the RTH5 protein through a small GTPase or calcium- dependent protein kinase (CDPK) to facilitate trichoblast differentiation. (B) Illustration of the role of RTH5 in tip growth by producing apoplastic superoxide, which is rapidly converted into hydrogen peroxide. Apoplastic peroxidases (PER) generate hydroxyl radicals, which are cleaving celluloses and hemicelluloses leading to cell wall loosening. The acidic cell wall (CW) allows oriented growth at the softened site. PM: plasma membrane.

Summary of SEP ID NOS

At3g45810.1 7

At5g60010.1 8

LOC_Os01g61880.1 9

Glyma07g 15690.1 10

Glymal8g39500.1 11

LOC_Os05g38980.1 12

Glymal 8g39500. l_variant 13

DETAILED DESCRIPTION

Methods for increasing the expression of an RTH5 polypeptide in a plant are provided. The methods comprise transforming the plant with a DNA construct comprising an RTH5 polynucleotide operably linked to a heterologous promoter that drives expression in a plant root. Alternatively the plant can be altered to increase the levels of expression of the endogenous RTH5 polypeptide. Such methods for altering the expression of the endogenous gene are known in the art and are encompassed by the present invention.

Using the methods and compositions of the present invention, drought tolerance and/or the uptake of nutrients in a plant can be increased. In particular, polynucleotides that encode polypeptides from the Roothairless5 (RTH5) family are provided. Polypeptides from the RTH5 family promote root hair formation and growth.

Root hairs are important for the uptake of water and nutrients in plants. As shown herein, the ROOTHAIRLESS5 (RTH5) family of polynucleotides and polypeptides are important for the formation of root hairs. Expression of RTH5 polypeptides in plant roots promotes the formation and growth of root hairs, thus improving water and nutrient uptake by the plant.

As referred to herein, the "RTH5 family" refers to a family of polynucleotides and polypeptides that have high sequence identity with and include the Zea mays L. (Maize) roothairless5 (rthS) gene, cDNA, or polypeptide described herein as SEQ ID NOs: 1, 2, and 3, respectively, and promote the formation and growth of root hairs in a plant. An "RTH5 family member" refers to a specific polynucleotide or polypeptide included in the RTH5 family (an RTH5 polynucleotide or RTH5 polypeptide, respectively). As an example, maize roothairless5 is an RTH5 family member. The maize roothairless5^''^'' and "rt/z5" genomic sequence, cDNA, and polypeptide are provided in SEQ ID NOs: 1, 2, and 3, respectively.

Polynucleotides of the invention are polynucleotides encoding RTH5 family members and include without limitation, the polynucleotides corresponding to SEQ ID NOs: 1 and 2, as well as active fragments and variants thereof. Polypeptides of the invention include members of the RTH5 family. Such RTH5 polypeptides include those set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, and active fragments and variants thereof. RTH5 family members include polynucleotides having sequences with about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5% or more sequence identity to SEQ ID NO: 2 and which encode RTH 5 polypeptides that have the ability to promote root hair formation and growth in a plant.

RTH5 family members also include polypeptides having sequences with about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5% or more sequence identity to SEQ ID NO: 3 and which promote root hair formation and growth.

The RTH5 family demonstrates nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase or NOX) activity. As referred to herein, "NADPH oxidase activity" or "NOX activity" refers to the enzymatic activity of the polypeptide that produces the reactive oxygen species (ROS) superoxide (0₂ ^") using an electron donor. Without wishing to be limited to a particular mechanism of action, ROS and ROS-related proteins are thought to play an important role in root hair tip growth. The methods of the invention utilize RTH5 family polynucleotides to increase the expression of RTH5 family polypeptides, which advantageously increase root hair formation and growth.

In general, concentration and/or activity of the RTH5 family member is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell which did not have the sequence of the invention introduced. The increased expression in the present invention can occur during and/or subsequent to growth of the plant to the desired stage of development. In specific embodiments, the levels and/or activity of RTH5 polypeptides of the present invention are increased in monocots, particularly maize.

The expression level of the RTH5 family polypeptide may be measured directly, for example, by assaying for the level of the RTH5 family polypeptide in the plant, or indirectly, for example, by measuring the NAPDH oxidase activity of the RTH5 family polypeptide in the plant. Methods of assaying for NOX activity are known in the art. For example, NADPH oxidase activity can be measured by such methods as detecting superoxide and/or hydrogen peroxide in the roots, as disclosed in the experimental section and as described in the art. See, for example, Foreman et al. (2003) Nature 422:442-446, herein incorporated by reference. Root hair cells include long tubular projections referred to herein as "root hairs." Root hairs are thought to aid plants in nutrient uptake, anchorage, and microbial interactions. Root hair growth is divided into three phases: first, defined swelling to form a bulge; second, transition to tip growth; and finally, tip growth by oriented exocytosis. Root hairs increase the surface area on a plant root, thereby increasing the ability of the root to take up water and nutrients.

Root hair formation and growth can be measured by phenotypic analysis. For example, root hair formation and growth can be measured using microscopy techniques described in the experimental section and as described by Foreman et al. (2003) Nature 422:442-446, herein incorporated by reference.

The formation and growth of root hairs leads to increased drought tolerance. As referred to herein, "drought" refers to a decrease in water availability to a plant that, especially when prolonged, can cause damage to the plant or prevent its successful growth (e.g., limiting plant growth or seed yield). Accordingly, "drought tolerance" is a trait of a plant to survive under drought conditions over prolonged periods of time without exhibiting substantial physiological or physical deterioration.

Embodiments of the present methods and compositions promote "increased drought tolerance" of a plant. Increased drought tolerance is measured relative to a reference or control plant, and is a trait of the plant to survive under drought conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar drought conditions. Typically, when a transgenic plant comprising a recombinant DNA construct in its genome exhibits increased drought tolerance relative to a reference or control plant due to the presence of the construct, the reference or control plant does not comprise in its genome the recombinant DNA construct.

One of ordinary skill in the art is familiar with protocols for simulating drought conditions and for evaluating drought tolerance of plants that have been subjected to simulated or naturally occurring drought conditions. For example, one can simulate drought conditions by giving plants less water than normally required or no water over a period of time, and one can evaluate drought tolerance by looking for differences in physiological and/or physical condition, including (but not limited to) vigor, growth, size, or root length, or in particular, leaf color or leaf area size. Other techniques for evaluating drought tolerance include measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates. See, for example, WO 2013/006345 herein incorporated by reference in its entirety. One can also evaluate drought tolerance by the ability of a plant to maintain sufficient yield (at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% yield) in field testing under simulated or naturally occurring drought conditions (e.g., by measuring for substantially equivalent yield under drought conditions compared to non-drought conditions, or by measuring for less yield loss under drought conditions compared to a control or reference plant).

In addition, increased formation and growth of root hairs leads to increased nutrient uptake. As referred to herein, "nutrient uptake" refers to a plant's ability it to remove nutrients from a soil or growth medium. "Increased nutrient uptake" refers to a plant's ability to remove nutrients from a soil or growth medium relative to a reference or control plant, and is a trait of the plant wherein the plant demonstrates an improved agronomic characteristic compared to the reference or control plant. Typically, when a transgenic plant comprising a recombinant DNA construct in its genome exhibits increased nutrient uptake relative to a reference or control plant due to the presence of the construct, the reference or control plant does not comprise in its genome the recombinant DNA construct. Nonlimiting examples of nutrients taken up by plant roots include nitrogen, phosphorus, potassium, and carbon.

"Agronomic characteristic" or "agronomic parameter" is a measurable trait including but not limited to, abiotic stress tolerance, greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, early seedling vigor and seedling emergence under low temperature stress.

It is understood and herein contemplated that the plants and germplasms disclosed herein can be identified as having an alternation in at least one agronomic characteristic through the identification of a polymorphism in a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, or 99.5% identity to SEQ ID NO: 1 or 2 or the identification of a polymorphism in a polynucleotide that encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, or 99.5% identity to SEQ ID NOs: 3, 4, 5, 6, 7, 8, 910, 11, 12, or 13. Thus, in one aspect, disclosed herein are methods of selecting or identifying a first maize plant or a first maize germplasm that has one or more beneficial alleles of rth5, the method comprising:

(a) screening a plurality of maize plants or a plurality of maize germplasm for

at least one polymorphism within a marker locus, wherein the marker locus is:

(b) a second polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% and less than 100% sequence identity to SEQ ID NO:3, wherein expression of the first or second polynucleotide in a maize plant results in a phenotype comprising an alteration of at least one agronomic characteristic when compared to a control maize plant; wherein the at least one agronomic characteristic is selected from the group consisting of increased root hair formation and growth, increased drought tolerance, and enhanced nutrient uptake; and wherein the control plant comprises:

(c) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 2; or (d) a polynucleotide encoding the amino acid sequence of SEQ ID NO:3

(c) selecting the first maize plant or first maize germplasm of step (b).

Abiotic stress can be at least one condition selected from the group consisting of: drought, water deprivation, flood, high light intensity, high temperature, low temperature, salinity, etiolation, defoliation, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, UV irradiation, atmospheric pollution (e.g., ozone) and exposure to chemicals (e.g., paraquat) that induce production of reactive oxygen species (ROS).

"Increased stress tolerance" of a plant is measured relative to a reference or control plant, and is a trait of the plant to survive under stress conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar stress conditions.

A plant with "increased stress tolerance" can exhibit increased tolerance to one or more different stress conditions.

"Stress tolerance activity" of a polypeptide indicates that over-expression of the polypeptide in a transgenic plant confers increased stress tolerance to the transgenic plant relative to a reference or control plant.

Methods of assaying increased nutrient uptake are known in the art. For example, one can grow a transgenic plant comprising a recombinant DNA construct and evaluate nutrient uptake by looking for differences in physiological and/or physical condition, including (but not limited to) vigor, growth, size, or root length, leaf color, leaf area size, or crop yield. Other methods include measurement of the kinetics of plant root nutrient uptake. See, for example, the methods reviewed by Bassirirad (2000) New Phytol. 147: 155-169, incorporated by reference herein.

In another embodiment, increased root hair formation and growth can reduce soil erosion in crop fields. It is recognized that root hairs help cling to soil particles and are therefore important to preventing soil erosion.

In addition, increased root hair formation and growth is expected to promote the infection of the plant with beneficial microorganisms, such as rhizobium and Pseudomonas putida KT2440. Rhizobia enter the plant via root hairs, which results in root nodule formation and increased nitrogen fixation.

In addition, plants are known to interact with a wide range of rhizosphere-colonizing bacteria. These are attracted to root surfaces by chemical components in root exudates, which are rapidly assimilated into microbial biomass (Rangel-Castro JI, et al. (2005) Environ

Microbiol 7: 828-838). This so-called rhizosphere effect supports bacterial cell densities in the root vicinity up to 100-fold greater than in surrounding soil (Whipps JM et al. (2001) J Exp Bot 52: 487-51 1).

In another embodiment, it is recognized that the reduction of RTH5 family expression or mutation in RTH5 family members can result in reduced susceptibility of the plant to plant pathogens which infect the plant roots. For example, Barssica are susceptible to

Plasmodiophora brassicae, which is the pathogen responsible for clubroot. Clubroot is caused when P. brassicae enter the plant via the root hairs. Therefore, it is thought that reducing the number of root hairs can limit the mode of entry of plant pathogens.

The sequences disclosed herein as SEQ ID NOs: 2 and 3 have been previously identified as respiratory burst oxidase homolog A (rbohA). See, Lin et al. (2009) J. Exp. Bot. 60:3221-3238. While regulation of the rbohA encoded NADPH oxidase (NOX) by a mitogen-activated protein kinase cascade signaling was demonstrated in maize leaves, rth5/rbohA has not been previously identified to be involved in root hair formation and growth.

Thus, in one aspect, disclosed herein are methods of increasing the resistance to root pathogens in a plant, said method comprising reducing the expression of an RTH5 family member, or inhibiting the function of the RTH5 family member. Maize root pathogens can be any organzims that cause a dileterious effecto on a plant and can infect the plant throught the root or root hairs. On non-limiting list of maize root pathogens include, but are not limited to, Fusarium species, like F. verticillioides and F. graminearu.

The RTH5 polynucleotides disclosed herein can be provided in expression cassettes for expression in a plant of interest. An "expression cassette" comprises a recombinant nucleic acid that is operably linked to a heterologous promoter that drives expression of the nucleic acid. A "recombinant" nucleic acid is made by a combination of two otherwise separated segments of nucleic acid sequence, for example, by chemical synthesis or by the manipulation of isolated segments of polynucleic acids by genetic engineering techniques. The term "recombinant DNA construct" refers to any agent such as a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or double- stranded DNA or RNA nucleotide sequence, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule where one or more DNA sequences have been linked in a functionally operative manner. Such recombinant DNA constructs are capable of introducing a 5' regulatory sequence or promoter region and a DNA sequence for a selected gene product into a cell in such a manner that the DNA sequence is transcribed into a functional mRNA that is translated and therefore expressed. A

recombinant DNA construct (and an expression cassette) may comprise a polynucleotide of interest linked to a heterologous polynucleotide such as, for example, a heterologous promoter.

The cassette can include 5' and 3' regulatory sequences operably linked to an RTH5 polynucleotide. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the RTH5 polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

In preferred embodiments, the expression cassette includes a polynucleotide operably linked to a root-preferred heterologous promoter. The polynucleotide can include a nucleotide sequence encompassed by SEQ ID NO: 2. In some embodiments the nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, or 99.5% sequence identity to SEQ ID NO: 2, and the polynucleotide encodes a polypeptide having NADPH oxidase activity. In some embodiments the nucleotide sequence is the sequence of SEQ ID NO: 2.

In other embodiments, the expression cassette includes a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In some embodiments, the nucleotide sequence encodes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, or 99.5% sequence identity to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13, wherein said polynucleotide encodes a polypeptide that has NADPH oxidase activity. In some embodiments the polynucleotide encodes a polypeptide having the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

The expression cassette can include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), an RTH5 family polynucleotide as disclosed herein, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the RTH5 family polynucleotide of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the RTH5 family polynucleotide of the invention can be heterologous to the host cell or to each other. As used herein,

"heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a "chimeric gene" comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

While it may be optimal to express the sequences using heterologous promoters, the native promoter sequences can be used. Such constructs can change expression levels of RTH5 family proteins in the plant or plant cell. Thus, the phenotype of the plant or plant cell can be altered. The termination region can be native with the transcriptional initiation region, can be native with the operably linked RTH5 family polynucleotide of interest, can be native with the plant host, or can be derived from another source (i.e., foreign or heterologous) to the promoter, the RTH5 polynucleotide of interest, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefacien , such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mo/. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91 : 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. ( i) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy -chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology

81 :382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments can be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers can be employed to join the DNA fragments or other manipulations can be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, can be involved.

Promoters useful in the methods of the invention include those promoters that drive expression of a polypeptide in roots of a plant. Constitutive promoters can be used such as the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313 :810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81 :581-588); MAS (Velten et al. (1984) EMBO J. 3 :2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608, 149; 5,608, 144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608, 142; and

6, 177,611.

A "root-preferred" promoter, as referred to herein, is a promoter which favors spatial expression of a polynucleotide of interest in the root of a plant compared to expression in other plant tissue within the same plant. Root-preferred promoters can be heterologous or native to the plant receiving the polynucleotide of interest. Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol.

20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root- inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(l):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1' gene, fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the Vf£NOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Patent Nos. 5,837,876; 5,837,848; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5, 1 10,732; 5,023, 179; and 7,554,005.

Other root preferred promoters include the following: the maize NAS2 promoter, the maize Cyclo promoter (US 2006/0156439, published July 13, 2006), the maize ROOTMET2 promoter (WO05063998, published July 14, 2005), the CR1BIO promoter (WO06055487, published May 26, 2006), the CRWAQ81 promoter (WO05035770, published April 21, 2005) and the maize ZRP2.47 promoter (NCBI accession number: U38790; GI No.

1063664),

The above list of promoters is not meant to be limiting. Any root-preferred promoter or promoter that drives expression in the root of a plant can be used with the polynucleotides disclosed herein.

The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Many selectable markers are known in the art and any can be used in the practice of the invention.

Embodiments found herein encompass isolated, substantially purified, or recombinant polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a polynucleotide can encode protein fragments that retain the biological activity of the native protein and hence promote root hair formation and growth. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide encoding the proteins disclosed herein.

A fragment of an RTH5 family polynucleotide that encodes a biologically active portion of an RTH5 family protein disclosed herein can encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 contiguous amino acids, or up to the total number of amino acids present in a full-length RTH5 family protein, for example, 852, 789, 850, 774, 822, 842, 912, 886, 843, 799, 860, 819, and 820 amino acids for SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, respectively. Fragments of an RTH5 family polynucleotide that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of an RTH5 family protein.

Thus, a fragment of an RTH5 family polynucleotide can encode a biologically active portion of an RTH5 family protein, or it can be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of an RTH5 family protein can be prepared by isolating a portion of one of the RTH5 family polynucleotide disclosed herein, expressing the encoded portion of the RTH5 family protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the RTH5 family protein. Polynucleotides that are fragments of an RTH5 family nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, or 3,600 contiguous nucleotides, or up to the number of nucleotides present in a full-length RTH5 family polynucleotide disclosed herein, for example, 3,592, 3,691, 3,009, 3,674, and 2,974 nucleotides for SEQ ID NO: 2.

"Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the RTH5 family polypeptides as disclosed herein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotide, such as those generated, for example, by using site-directed mutagenesis but which still encode a RTH5 family protein. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, isolated polynucleotides that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, and 13, respectively, are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5% or more sequence identity. "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, root hair formation and growth and/or NADPH oxidase activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation.

Biologically active variants of a native RTH5 family protein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.5% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention can differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, as few as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

In one aspect, disclosed herein are methods of selecting or identifying an allelic variant of rth5 in a maize plant, the method comprising the steps of a) obtaining a population of maize plants, wherein said maize plants exhibit an alteration of at least one agronomic characteristic; wherein the at least one agronomic characteristic is selected from the group consisting of increased root hair formation and growth, increased drought tolerance, and enhanced nutrient uptake; b) evaluating allelic variations with respect to the polynucleotide sequence (for example by performing the sequence identity comparisons described above) encoding a protein comprising SEQ ID NO:3, or in the genomic region that regulates the expression of the polynucleotide encoding the protein; c) associating allelic variations with said alteration of at least one agronomic characteristic; and d) selecting or identifying an allelic variant that is associated with said alteration of at least one agronomic characteristic.

The proteins disclosed herein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the RTH5 family proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367- 382; U.S. Patent No. 4,873, 192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.

Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.

Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants continue to possess the desired root hair formation and growth activity and/or NADPH oxidase activity. The mutations that are made in the DNA encoding the variant must not place the sequence out of reading frame and optimally do not create complementary regions that produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein.

However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays. That is, the activity can be evaluated by, for example, phenotypic analysis of root hair formation and growth and/or by assaying for NADPH oxidase activity.

Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different RTH5 family members coding sequences can be

manipulated to create a new RTH5 family member possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest can be shuffled between the rth5 gene disclosed herein and other rboh and rth genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K_m in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751 ; Stemmer (1994) Nature 370:389-391 ; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore ei al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391 :288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458. The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", and, (d) "percentage of sequence identity."

(a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 1 1-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA).

Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73 :237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet ei a/. (1988) Nucleic Acids Res. 16: 10881- 90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol.

24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff ( 1989) Proc. Natl. Acad. Sci. USA 89: 10915).

(c) As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative

substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

(d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

Sequence alignments and percent identity calculations may be determined using the MEGALIGN® program of the LASERGENE® bioinformatics computing suite

(DNASTAR® Inc., Madison, WI). Multiple alignment of the sequences provided herein may be performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5: 151 153) with the default parameters (GAP PENALTY=10, GAP LENGTH

PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP

PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS

SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain "percent identity" and "divergence" values by viewing the "sequence distances" table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

Alternatively, the Clustal W method of alignment may be used. The Clustal W method of alignment (described by Higgins and Sharp, CABIOS. 5: 151-153 (1989); Higgins, D. G. et al, Comput. Appl. Biosci. 8: 189-191 (1992)) can be found in the MEGALIGN® v6.1 program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Default parameters for multiple alignment correspond to GAP

PEN ALT Y= 10, GAP LENGTH PENALTY=0.2, Delay Divergent Sequences=30%, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB. For pairwise alignments the default parameters are Alignment=Slow-Accurate, Gap

Penalty=10.0, Gap Length=0.10, Protein Weight Matrix=Gonnet 250 and DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain "percent identity" and "divergence" values by viewing the "sequence distances" table in the same program.

The use of the term "polynucleotide" is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. "Introducing" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus -mediated methods.

"Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Set USA 83 :5602-5606, Agrobacterium-mediated transformation (U.S. Patent No. 5,563,055 and U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.

3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Patent Nos.

4,945,050; U.S. Patent No. 5,879,918; U.S. Patent No. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)

Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Patent Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311 :763-764; U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The

Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250- 255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, W099/25821, W099/25854, WO99/25840, W099/25855, and W099/25853, all of which are herein incorporated by reference. Briefly, the expression cassette disclosed herein can be contained in transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.

It is therefore recognized that methods of the present invention do not depend on the incorporation of the entire polynucleotide into the genome, only that the plant or cell thereof is altered as a result of the introduction of the polynucleotide into a cell. In one embodiment of the invention, the genome may be altered following the introduction of the polynucleotide into a cell. For example, the polynucleotide, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides into the genome. While the methods of the present invention do not depend on additions, deletions, and substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprises at least one nucleotide.

As indicated, the invention also encompasses plants where the endogenous RTH5 expression is enhanced. Thus, the level and/or activity of an RTH5 polypeptide can be increased by altering the gene encoding the RTH5 family polypeptide or its promoter. See, e.g., Kmiec, U.S. Patent 5,565,350; and Zarling et al, PCT/US93/03868. Therefore mutagenized plants that carry mutations in RTH5 family genes, where the mutations increase expression of the RTH5 family gene or increase the root hair formation and growth activity of the encoded RTH5 family polypeptide are provided.

Levels of endogenous RTH5 can be increased by methods known in the art. For example, endogenous RTH5 can be increased by the introduction of sequences of interest that upregulate endogenous expression. Such sequences of interest can include heterologous promoters, siRNA targeted to genomic regulatory elements, enhancer elements, and/or booster sequences. See, e.g., U. S. Patent 5,939,541 ; U.S. Patent 6,576,442; US

2012/0036594; and WO 1991/009955. Other methods of inserting sequences of interest to upregulate endogenous expression can include the use of various meganucleases to target polynucleotides. Such meganucleases are set forth in WO 2009/114321 (herein incorporated by reference), which describes "custom" meganucleases. See, also, Gao et al. (2010) Plant Journal 1 : 176-187. Additional methods of inserting sequences of interest to upregulate endogenous expression of a sequence of interest that can be employed, include but are not limited to the use of ZnFingers, meganucleases, and, TAL nucleases. See, for example, WO2010079430, WO201 1072246, and US20110201 118, each of which is herein

incorporated by reference in their entirety.

Other embodiments include methods of detection and use of polymorphisms in the maize Rth5 promoter that are associated with increased expression of the RTH5 polypeptide.

The cells that have been transformed can be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

As used herein, the term plant also includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.

In some embodiments, the composition is a seed of a plant comprising a recombinant DNA construct that includes a promoter that drives expression in roots of a plant operably linked to a polynucleotide that encodes an RTH5 family member as described herein.

The recombinant DNA constructs disclosed herein can be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum {Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria itatica), finger millet (Eleusine coracanaj), sunflower (Hetianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solatium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.

Conifers that can be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea giauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska

yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean and sugarcane plants are optimal, and in yet other embodiments corn plants are optimal.

Another embodiment is a method for transforming a cell (or microorganism) comprising transforming a cell (or microorganism) with any of the isolated polynucleotides or recombinant DNA constructs of the present invention. The cell (or microorganism) transformed by this method is also included. In particular embodiments, the cell is eukaryotic cell, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell. The microorganism can be Agrobacterium, e.g. Agrobacterium tumefaciens or Agrobacterium rhizogenes.

Another embodiment is a recombinant DNA construct (and corresponding cells, plants, seeds and methods) comprising a promoter (heterologous or native) that drives expression in a plant root operably linked to a polynucleotide, wherein said polynucleotide encodes a polypeptide that comprises the following regions: four trans-membrane (TM) domains, two EF hand motifs, FAD and NAD cofactor binding sites, a ferric reductase domain, and the NADPH oxidase domain, wherein each of these regions have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the corresponding regions from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13, wherein said polypeptide has NADPH oxidase activity. The polypeptide can be further characterized by having nine additional amino acids in the FAD cofactor domain, and by lacking 15 amino acids in the NADPH binding region, as compared to the dicot group I proteins (AT3G45810; AT5G60010;

Glyma07gl5690; Glymal8g39500) of the subclade that includes RTH5. EXAMPLES

Example 1 ; Cloning and characterization of the maize roothairless5 gene, which controls root hair initiation and outgrowth and encodes a monocot-specific NADPH oxidase involved in superoxide production

The roothairless5 gene controls both root hair length and density

An EMS mutagenesis screen yielded a recessive mutant (Schnable Lab Accession # 1350) that affects root hair development but has no other obvious effects on plant growth and development. Genetic crosses demonstrated that this mutant is not allelic to the previously described rthl, rth2 and rth3 mutants (Wen et al. (1994) Am. J. Bot. 81 :833-842). The affected gene and reference allele were designated rth5 and rth5-l, respectively. Compared to wild-type, the rth5 mutant displays significantly shorter root hairs. Root hair length was determined by WinRhizo software (n=12) and density of wild-type and rth5 primary roots was counted and calculated for per mm² (n=3), and then analyzed via eSEM (environmental scanning electron microscopy). P- values were obtained via Student's t-test. The length of root hairs on primary roots of 4-day-old (4 d) rth5 mutants was significantly decreased to 4% of wild-type length (p-value <0.001). Moreover, in these mutant seedlings root hair density was significantly reduced to 64% of wild-type density (p-value <0.001). In 10 d seedlings it is possible to also observe root hairs on seminal and shoot-borne roots. The lengths and numbers of the root hairs on these roots including primary, seminal, crown and lateral were also significantly reduced in the mutant (p-value <0.001, n=12).

Map-based cloning of rth5 identified an NADPH oxidase

Using Sequenom-based bulked segregant analysis (BSA) (Liu et al. (2010) Genetics

184: 19-26) the rth5 locus was mapped to the long arm of chromosome 3. By analyzing F₂ and FiBC populations (Methods) the rth5 gene was mapped to the interval 180.1-180.4 Mb of chromosome 3 (Refgenl), flanked by IDP (insertion deletion polymorphism) markers IDP4064 (SEQ ID NO: 16) and C3.184743 (SEQ ID NO: 18) (Table 1). This interval harbors only five gene models (4a53), including GRMZM2G426953, an NADPH oxidase (NOX), which contains a G to A transition at position 2462, resulting in a Cysteine to Tyrosine conversion at amino acid position 821 close to the C-terminus of the protein (Figure IB). This amino acid exchange is the result of a G-to-A transition relative to the B73 allele, which is characteristic of EMS-induced mutations. This cysteine is conserved among all 26 inbred parents of the maize NAM population.

The affected cysteine residue is also conserved among divergent NADPH oxidases ranging from plants to yeast and human (Figure 1C). Hence, an alteration of this conserved residue in the predicted NAD substrate binding region can lead to a functional deficiency in the rth5 mutant.

Confirmation of rth5 identity via the generation of independent alleles To confirm that the mutation in candidate gene GRMZM2G426953 indeed confers the phenotype of the rth5 mutant, several novel putative Mutator alleles were isolated from Pioneer's trait utility system of corn (TUSC, Bensen et al. (1995) The Plant Cell 7:75-84). Among these putative Mu insertion alleles, three displayed the roothairless phenotype and each of these alleles contained a Mutator insertion in the candidate gene as demonstrated by sequencing. Allele rth5-3 contains a Mul insertion in exon 5 (Figure IB) while rth5-2 and rth5-4 contain MuDR insertions in exons 3 and 5, respectively (Figure IB).

The rth5 gene structure

The B73 allele of the rth5 gene consists of 14 exons and 13 introns (Figure IB) encoding a 3,792 bp mRNA (including 5' and 3' UTR). This mRNA encodes an 852 amino acid protein with a predicted molecular weight of ~96 kDa.

The RTH5 protein is predicted to contain four trans -membrane (TM) domains, two

EF hand motifs, FAD and NAD cofactor binding sites, a ferric reductase domain, and the NADPH oxidase domain characteristic for NADPH oxidases (Figure IB).

The NADPH oxidase family in maize

Based on sequence similarity with the RTH5 protein 17 respiratory burst oxidases (Rbohs) were identified in the maize filtered gene set (FGS, 4a53), a set of high confidence genes. Phylogenetic reconstructions based on the full-length protein sequences of all known members of the maize, Arabidopsis, soybean and rice NADPH oxidase gene families (Figure 2) were performed. Two major groups of NADPH oxidases were observed. All the NADPH oxidases exhibit diverse sequences in their N-terminus. The smaller group II is characterized by less conservation or the absence of the NADPH oxidase domain and a different conserved sequence in the NAD cofactor binding region (Figure 3). Interestingly, no soybean and two rice and only one maize proteins were included in group II, whereas eight Arabidopsis RBOH proteins belong to that subgroup. Along with two other maize and two rice RBOHs RTH5 is a member of a monocot-specific sub-clade of group I (Figure 2). No homeolog of RTH5 resulting from an ancient genome duplication was found in the maize genome. In silico searches for syntenic homologs (genomeevolution.org/CoGe) identified the rice gene

Os01g61880, which was also the closest homolog of RTH5 in the phylogenetic tree (Figure 2). RTH5 and its two closest maize homologs RBOH 11 and RBOH 13 are located in a subgroup of the phylogenetic tree characterized by the presence of a few additional amino acids at the end of the FAD cofactor binding site (Figure 3). The rth5 gene is preferentially expressed in root hairs

Tissue-specific expression oirth5 and all 17 Rboh family members was examined via qRT-PCR in six different seedling tissues including the cap of the primary root, elongation zone, differentiation zone without root hairs, isolated root hairs, and the coleoptilar node and the first leaf. Rth5 transcripts accumulated in all tested tissues, but the significantly highest signal was detected in root hairs (Figure 4A). Moreover, the qTeller tool was used to compare the accumulation oirth5 transcripts in RNA-Seq data from a wide range of tissues and organs. The highest expression values were observed in seedling roots and shoots (Figure 5). Expression data of the remaining Rboh family members in these tissues are summarized in Figure 6. The relative expression levels of all 17 Rboh genes in root hairs were determined via quantitative real time PCR (qRT-PCR). The rth5 gene displayed the highest expression, accounting for 52% of the total Rboh expression in maize root hairs (Figure 4A). Rbohll and Rbohl3, the closest homologs of rth5 were only very weakly expressed in root hairs.

RNA in situ hybridization was used to study root tissue-specific expression patterns. Cross sections hybridized with an in vitro transcribed rth5 RNA antisense probe resulted in a signal in root hairs (Figure 4C), whereas the sense probe yielded no signal (Figure 4D).

The RTH5 protein is localized in epidermal cells of the primary root

Immunohistochemical experiments were performed to localize the RTH5 protein in situ. Cross-sections of wild-type primary roots with emerging root hairs were incubated with the RTH5 antibody (Figure 4E) or buffer solution (Figure 4F) followed by the secondary antibody. A signal of the RTH5 antibody was detected in all epidermis cells and in root hairs; whereas the control sections displayed no signal.

In longitudinal sections an RTH5 signal was detected in root hairs and all epidermis cells of the root hair zone (Figure 4G). Interestingly, an RTH5 signal was also detected in longitudinal sections of the elongation zone, which does not form root hairs (Figure 41). The control sections showed no detectable color reaction (Figure 4H & 4J).

Both, cross- and longitudinal sections were performed for wild-type and rth5 mutant primary roots, but displayed no difference in signal intensity or tissue organization apart from the root hair defect. The rth5 mutant accumulates less ROS during root hair tip growth

Superoxide is a very short-lived radical that is rapidly converted into hydrogen peroxide (i.e., NADPH oxidase generates superoxide which in turn permutates into hydrogen peroxide). Staining for both molecules was performed in wild-type and rth5 mutant seedlings and presence or absence of high ROS signals in root hair tips were quantified. While 74% of wild-type root hairs displayed the superoxide signal at the tip, the frequency was significantly reduced to 24% in rth5 root hairs. Similarly, 77% and 71% of wild-type root hairs exhibited a high hydrogen peroxide signal in the tips of root hairs as detected using DAB or H₂DCF-DA, respectively, while only 27% and 21% of rth5 root hairs displayed this signal using the same dyes. These differences were significant in Fisher^'s exact test at p <0.001.

RNA-Seq of rth5 vs. wild-type

To understand the influence of rth5 on global patterns of transcription, RNA-Seq was performed on 6-day-old roots from the rth5 mutant and wild-type seedlings (Methods). Two biological replicates were analyzed for each genotype. Approximately 98% of raw reads from each sample passed the quality check and trimming procedure. 3.7-4.7 million reads per replicate (-88% of the post-trimmed reads) were uniquely and confidently mapped to the B73 reference genome (Table 2) (Methods). Consistent with the results from the qRT-PCR experiment, the rth5 gene was preferentially expressed in wild-type vs rth5 root hairs with a relatively small fold change (1.4). Among 21,007 genes that have >20 RNA-Seq reads across all four biological replicates, 1,257 genes differentially expressed genes (DEGs) were identified at a 10% false discovery rate (FDR). 634 and 623 DEGs were significantly up- and down-regulated, respectively. A gene ontology (GO) enrichment analysis of the 1,257 DEGs revealed statistically significant over-representation of many functional categories, including monooxygenase activity, peroxidase activity, oxidoreductase activity, response to oxidative stress, heme binding, phospholipid-translocating ATPase activity, cellulose synthase activity, and lipid biosynthetic process (Table 3).

Separate gene ontology (GO) enrichment analyses of the 634 and 623 up and down- regulated DEGs indicate that DEGs are significantly over-represented in some functional categories in both DEG groups, such as monooxygenase activity and heme binding (Table 3). Genes associated with response to oxidative stress, response to biotic stimulus, as well as sucrose metabolic process are significantly enriched in the up-regulated DEG group; whereas genes involved in cellulose biosynthetic process, phospholipid-translocating ATPase activity, and myosin complex are significantly enriched in the down-regulated DEG group. Overall, these RNA-Seq results are consistent with the view that rth5/rbohA functions in producing ROS required for elongation of root hairs.

Of 17 maize rboh homologous genes, 14 are at the detectable level in the RNA-Seq data. Six rboh genes are DEGs (Table 4). Statistically, the rboh genes are 7.2x over- represented in the DEGs (χ² test, p-value=1.5xl0^~7). Four and two rboh genes were up- and down-regulated in the mutant relative to the wild-type, respectively. One DEG, rbohl (GRMZM2G065144) was up-regulated >8 fold in the mutant.

Table 2. Summary of Reads from the RNA-Seq Experiment

Sample Number of raw Number of remaining trimmed Number of mapped reads Number of uniquely & confidently reads reads (% raw reads) (% trimmed reads) mapped reads (% trimmed reads) mut repl 5,301,418 5,197,927 (98.0%) 5,086,480 (97.9%) 4,569,445 (87.9%) mut repl 4,436,707 4,352,149 (98.1%) 4,260,690 (97.9%) 3,833,694 (88.1%) wt_repl 4,232,619 4,152,065 (98.1%) 4,065,642 (97.9%) 3,666,583 (88.3%) wt_rep2 5,437,462 5,329,121 (98.0%) 5,217,366 (97.9%) 4,694,561 (88.1%)

Table 3. Significantly Over-Represented GOs in Differentially Expressed Genes (DEGs)

;o

Table 4. Expression of Maize Rboh Homologous Genes

GenelD Symbol wt_repl wt_rep2 mut_repl mut_rep2 mut-wt_log2FC mut-wt_pvalue mut-wt_qvalue mut-wt_sig :

GRMZM2G065144 Rboh7 10 24 217 192 3.61 0.000 0.000 yes

GRMZM2G426953 RTH5 409 540 365 286 -0.53 0.000 0.000 yes

GRMZM2G034896 Rboh6 243 328 398 322 0.35 0.001 0.029 yes

GRMZM2G037993 RbohAlike 45 96 107 107 0.63 0.002 0.046 yes

GRMZM2G441541 RbohD 72 84 60 40 -0.63 0.004 0.070 yes

GRMZM2G358619 Rbohl2 3 6 13 16 1.71 0.004 0.072 yes

GRMZM2G043435 RbohC 525 723 537 542 -0.19 0.038 0.294 no

GRMZM2G300965 RbohlO 14 17 18 28 0.59 0.122 0.504 no

GRMZM2G089291 Rboh8 31 35 29 20 -0.42 0.162 0.566 no

GRMZM2G138152 RbohB 294 378 345 274 -0.1 0.293 0.702 no

GRMZM2G448185 Rbohl4 146 152 120 151 -0.12 0.446 0.808 no

GRMZM2G022547 Rboh5 414 632 610 466 0.06 0.494 0.833 no

GRMZM2G147966 Rboh9 42 52 47 40 -0.1 0.679 0.913 no

GRMZM2G169201 Rbohl5 131 153 141 139 0 0.973 0.996 no

GRMZM2G068557 RbohFR 0 0 0 0 NA NA NA NA

GRMZM2G323731 Rbohll 0 0 0 0 NA NA NA NA

GRMZM2G401179 Rbohl3 0 0 0 0 NA NA NA NA

a: melting temperature

The maize rth5 mutant displays significantly shortened root hairs and reduced root hair density while aboveground development remains unaffected. Hence, rth5 specifically controls both the elongation of root hairs and the specification of epidermis cells or the initiation of root hairs. More specifically, root hair elongation in the mutant rth5 is affected during the transition from bulge to tip growth. In contrast to rth5, root hairs of the rth3 mutant are characterized by disrupted bulges while, rthl and rth2 root hairs fail to elongate after transition to tip growth (Wen and Schnable (1994) Am. J. Bot. 81 :833-842). The rth5 mutant is like rth2 and rth3 (Wen and Schnable (1994) Am. J. Bot. 81 :833-842), which are specifically affected in root hair formation. In contrast, the maize mutants rthl (Wen and Schnable (1994) Am. J. Bot. 81 :833-842), dill and diU (Lid et al, (2004) Planta 218:370- 378) which are defective in root hair formation also display pleiotropic effects during development. The rthl mutation displays general growth abnormalities during development (Wen and Schnable (1994) Am. J. Bot. 81 :833-842) while dill and diU control cell division orientation in the aleurone, leaf and root epidermis. The rth3 mutant shows no difference in root hair density as compared to wild-type, while the rthl and rth2 mutants form twice as many hairs as wild-type plants. In contrast, the maize mutant rth5 forms fewer root hairs as compared to wild-type primary roots indicating that the rth5 gene is involved in not only root hair elongation but also in epidermis specification and/or root hair initiation.

In rth5 all root types including primary, seminal, lateral, and shoot-borne roots display defects in root hair formation, indicating that the RTH5 protein is critical for root hair formation in all root types. A general defect in root hair formation affecting all root types has also been observed for the rthl, rth2 and rth3 mutants (Hochholdinger et al. (2008) Plant J. 54:888-898, (Wen and Schnable (1994) Am. J. Bot. 81 :833-842). In contrast to this general mechanism of root hair formation in all root types, the control of lateral root formation is root-type specific. For instance, ruml (Woll et al. (2005) Plant Physiol. 139: 1255-1267) and Irtl (Hochholdinger and Feix (1998) Plant J. 16:247-255) display root-type specific defects, that are confined to embryonic roots. In these mutants postembryonic shoot-borne roots do not display any lateral root defects.

The RTH5 protein is characterized by several functional domains (Figure IB) including four trans-membrane domains, responsible for the membrane embedding, and two EF hand motifs, which are required for Ca²⁺ regulation. Moreover, the FAD and NAD domains confer binding to the cofactor FAD and the substrate NADPH, respectively.

Regulation of the RBOHA/RTH5 NADPH oxidase by a mitogen-activated protein kinase cascade controlled by abscisic acid signaling was demonstrated in maize leaves (Lin et al. (2009) J. Exp. Bot. 60:3221-3238).

Consistent with this co-factor binding site and functional assignment, the rth5 gene encodes a predicted NADPH oxidase (NOX). The NOX protein family is present in most eukaryotic species (Bedard et al. (2007) Biochimie 89: 1 107-11 12). The rth5 mutant was induced by EMS, which resulted in a Guanine to Adenine substitution in the genomic sequence changing the cysteine (C) residue in amino acid position 821 to tyrosine (Y) (Figure IB). Cysteine residues often form intra- or inter-protein disulfide bridges to enable secondary structures. As this cysteine residue is highly conserved among a very diverse set of species (Figure 1C), it is likely that the C821Y mutation is disabling a functionally important protein domain. A point mutation of this conserved C in the human NADPH oxidase 2 (NOX2) gene results in the genetic disorder chronic granulomatous disease (Kawahara et al. (2007) BMC Evol. Biol. 7: 109). This disease affects cells of the immune system, which have difficulties in forming reactive oxygen compounds, including superoxide used to kill certain ingested pathogens.

Phylogenetic reconstruction of RBOH (RESPIRATORY BURST OXIDASE

HOMO LOG) proteins of rice, maize, soybean, and Arabidopsis revealed the existence of two subgroups. These subgroups are distinguished by inter-group differences in the sequences of the NAD binding region, which are conserved within each group (Figure 3). Ten Arabidopsis RBOH proteins are classified to group I, which is consistent with previous annotations of superoxide-producing NOX proteins (Kawahara et al. (2007) BMC Evol. Biol. 7: 109).

Interestingly, all soybean proteins are assigned to group I, while eight of 18 members of the Arabidopsis RBOH protein family are classified in group II. According to their annotation, group II proteins are often connected to iron deficiency and can function as ferric reductases which are NOX proteins that do not produce superoxide (Ivanov et al. (2012) Mol. Plant 5:27-42). The nine members of the group I subclade that includes RTH5 are characterized by nine additional amino acids in the FAD cofactor domain as compared to the remaining group I proteins. Within this subclade, all five proteins in the monocot-specific group of this subclade lack 15 amino acids in their NADPH binding region. Both, the additional amino acids in the FAD, and the missing amino acids in the NAD region can confer altered binding to cofactors or substrates leading to specialized functions.

While several types of NOX proteins are found in mammals ranging from ancestral types to peroxidase-containing DUOXs, in plants only homologs of EF -hand-containing NOX5-types have been identified (Bedard et al. (2007) Biochimie 89: 1107-11 12). Human NOX proteins consist of two transmembrane heterodimers (gp9 l^phox and p22^phox) and four regulatory subunits (p40^phox, p47^phox, p67^phox, and Rac2) in the cytoplasm (Lam et al. 2010). Thus far, no homologs for the regulatory subunits p47^phox, p67^phox, or p22^phox have been found in plants (Bedard et al. (2007) Biochimie 89: 1107-1 112).

The human NOX2 proteins have been shown to play a role in the production of superoxide as a first defense response to microorganisms invading neutrophils and macrophages (Lam et al. (2010) Seminars in Immunopathology 32:415-430, Nauseef (2008) J. Biol. Chem. 283: 16961-16965). Plant NOX proteins have been shown to function in plant immunity and several developmental processes. Pepper and tobacco NOX proteins were demonstrated to function in plant immunity (Wi et al. (2012) Plant Physiol. 159:251-265, Yi et al. (2010) New Phytol. 185:701-715). Aerchenyma formation after waterlogging was shown to be conferred by the maize NOX protein GRMZM2G300965 (Yamauchi et al. (2011) Plant Signal Behav. 6:759-761), which showed specific expression in the root cap and the elongation zone (Figure 5, RBOH10). Finally, the Arabidopsis RBOHD and RBOHF are involved in ABA-dependent stomatal closure (Kwak et al. (2003) EMBO J22:2623-2633) and lead to an oxidative burst following infection (Galletti et al. (2008) Plant Physiol 148: 1695-1706).

The Arabidopsis RBOH protein RBOHC (RHD2), which is distantly related to RTH5 also controls root hair growth. Arabidopsis plants defective in rhd2 gene function form very short root hairs that do initiate bulges, but that do not elongate (Schiefelbein and Somerville (1990) Plant Cell 2:235-243). Moreover, such plants display stunned root growth (Foreman et al. (2003) Nature 422:422-446), an effect that was not observed for rth5 mutant roots. Rhd2 is expressed in root epidermal cells, but also in the cortical cells of the elongation- and differentiation zone (Foreman et al. (2003) Nature 422:422-446). Consistent with its mutation in the C-terminus of the protein, the expression of the rth.5-1 mutant reference allele was only slightly reduced as compared to wild-type primary roots relative to the Myosin gene (Genbank AC: 48090G09.xl). Similarly, protein levels detected by immunohistochemistry and Western blot analyses using three-days-old seedlings did not show any obvious change between wild-type and rth5-l mutants (for example, see Figure 4). Nevertheless, although expression and protein abundance are not dramatically affected, this mutation significantly affects the function of the protein as illustrated by the mutant phenotype.

Expression of rth5 was detected in several shoot and root tissues at low levels with expression maxima in root hairs (Figure 5 and Figure 5). Similarly, the rice rboh genes are also expressed in multiple plant organs and tissues (Wong et al. (2007) Plant Cell 19:4022- 4034). In maize root hairs the rth5 gene accounts for 52% of the expression of all 17 rboh genes and hence the majority of NADPH oxidase transcripts in root hairs. The RTH5 protein was detected preferentially in root hairs and epidermis cells, but weaker signals were also detected in root cortex cells (Figure 4). In two studies mainly focusing on the influence of hormones on NADPH oxidase activity in maize leaves it was demonstrated that expression of rth5 (rbohA) is induced after abscisic acid and brassinosteroid treatment as well as hydrogen peroxide application (Lin et a/.(2009) J. Exp. Bot. 60:3221-3238, Zhang et al. (2010) J. Exp. Bot. 61 :4399-441 1). These experiments suggest that RTH5 may play a role in hormone- mediated oxidative defense in leaves.

Arabidopsis root hairs display high Ca²⁺ and superoxide concentrations in their growing tips (Foreman et al. (2003) Nature 422:422-446). The tips of root hairs of the mutant rth5 displayed significantly less superoxide staining and accumulate less hydrogen peroxide (p-value <0.001). These results indicate that as a consequence of a functional defect of the RTH5 protein, the rth5 mutant is unable to establish high levels of ROS in the tips of root hairs. Not all growing tips of wild-type root hairs displayed staining for superoxide and hydrogen peroxide. Some root hairs may have died during the staining procedure; some may not have taken up the indicator chemicals. Consistent with expression data the presence of RTH5 homologous RBOH proteins may account for the residual ROS content detected in the mutant (Figure 6).

A model for the function of RTH5 in root hair elongation is summarized in Figure 7B. Superoxide molecules, localized in the root hair tips produced by RTH5 are transformed rapidly into hydrogen peroxide, which in turn is converted to hydroxyl radicals by apoplastic peroxidases (Liszkay et al. (2003) Planta 217:658-667). These hydroxyl radicals are cleaving polysaccharides (Fry (1998) Biochem. J. 332:507-515) and therefore cause cell wall loosening, thereby enabling polarized cell growth (Bibikova et al. (1998) Development 125:2925-2934, iszkay et al. (2004) Plant Physiol. 136:3114-3123).

In Arabidopsis the molecular interactions resulting in position-dependent root hair initiation are well characterized (reviewed in: Hochholdinger and Nestler (2012)

Encyclopedia of Genetics, Elsevier: New York, vol. 5, p. 349-352). In contrast, the molecular network underlying random root hair patterning in maize remains enigmatic (Clowes (2000) New Phytologist 146: 83-94). The reduced root hair density observed in mutant seedlings indicates that RTH5 is involved not only in root hair tip growth, but also in root hair initiation or epidermis specification (Figure 7). The presence of RTH5 in all epidermal cells is consistent with this hypothesis that superoxide is only produced upon activation of RTH5, which thus acts as a signal converter, similar to the human NOX2 (Lam et al. (2010) Seminars in Immunopathology 32:415-430), which subsequently promotes root hair initiation. This is supported by the finding of selective root hair cell superoxide staining in the maize differentiation zone. In contrast, all epidermis cells display superoxide staining in the elongation zone (Liszkay et al. (2004) Plant Physiol. 136:3114-3123). Residual root hair initiation in the rth5 mutant might be controlled by RTH5-independent pathways. Due to the lack of functional RTH5 proteins these root hairs fail the transition to tip growth.

Experimental Procedures

Plant material and growth conditions

The mutation was initially induced by EMS (Ethyl methanesulfonate) mutagenesis of the Pioneer inbred line 1 14748/AD. Mutants used in the present study were progeny of plants backcrossed 10 times to the inbred line B73 (Schnable laboratory stock OOg- 1542-1).

Kernels were surface sterilized as described previously (Nestler et al. (2011) J.

Proteome Res. 10:2525-2537) and germinated in moist paper rolls in a 16 h light, 8 h dark cycle at 26 °C in distilled water. Binocular imaging and environmental scanning electron microscopy

A Zeiss Stemi SV8 Binocular coupled with a Powershot G2 camera was used to document root hairs at a 20x magnification. Root hair length was determined via WinRhizo Software. A Philips XL30 FEI environmental scanning electron microscope (eSEM) was used for surface illustration of fresh four-day-old primary roots without fixation or tissue drying.

Bulked segregant analysis (BSA)

Sequenom-based BSA (bulked segregant analysis) (Liu et al. (2010) Genetics 184: 19- 26) was used to map the rth5 gene. 89 individuals from an F₂-family were phenotypically divided into two bulks: mutant and non-mutant. Equal quantities of root tissue were pools from each individual in each bulk. DNA was extracted from the two tissue bulks and then subjected to Sequenom-based SNPtyping using -1,000 SNP markers.

Fine-scale mapping of rth5

Multiple markers located on the long arm of chromosome 3 were used to genotype 80 rth5 mutant individuals from an F2-population. An rth5 mutant individual in a B73 genetic background was crossed with the Mol7 inbred line and the resulting Fi-seeds were backcrossed to Mol7 to create an FiBC population. Two SNP (single nucleotide

polymorphism) markers, SNP2673 (SEQ ID NO: 15) and SNP6262 (SEQ ID NO: 19), were used to genotype 512 FiBC individuals. In total, 65 recombinants were identified between SNP2673 and SNP6262, each of which was self-pollinated. The root hair phenotypes of self- pollinated progeny were scored to infer whether or not specific FiBCi recombinants carried the rth5 mutant allele. In parallel, the recombinants were genotyped with a series of molecular markers (Figure 1A). Based on an analysis of the resulting genotyping and phenotyping data, the rth5 mutant was mapped to the 179.9-182.3 Mb interval of

chromosome 3 in AGPvl. To further narrow down the rth5 interval, a larger FiBC population ( =~5,000) was genotyped with SNP markers SNP90373 (SEQ ID NO: 14) and MJ09352 (SEQ ID NO: 17). In total, 440 recombinants between these two marker loci were identified and genotyped for rth5 as described above. After genotyping all available recombinants (65 + 440) with 35 SNP markers (15 of which are co-dominant in this population) the rth5 gene was mapped to the interval 180.1-180.4 Mb.

Identification of novel rth5 mutant alleles

Three novel alleles, rth5-2, rth5-3, and rth5-4, were identified by a reverse genetic screen of Mutator stocks at Pioneer Hi-Bred. Confirmation of the candidate seedlings displaying a roothairless phenotype was performed by PCR mapping of the Mutator insertion with a general Mutator (Mu) oligonucleotide primer (Dietrich et al. 2002) and an rth5- specific oligonucleotide primer (5 ' -GCACATCTCCCGGATAAATTG-3 ' ) (SEQ ID NO: 54). The amplification product contained 39 bp beyond the Mu TIR sequence sufficient to identify the Mutator element (Dietrich et al. (2002) Genetics 160:697-716). Mu insertions were found in positions 1300, 1931, and 1968 bp of the genomic rth5 sequence counting from the ATG start codon in the alleles, rth5-2, rth5-3, and rth5-4, respectively.

RTH5 protein domain prediction

Several bioinformatics tools were used to predict protein domains: InterProScan, MyHits (EMBL- myhits.isb-sib.ch), SMART (simple modular architecture research tool- smart, embl-heidelberg.de) and TMHMM. Domains predicted by at least two search tools were summarized in the protein domain structure (Figure IB).

Phylogenetic analyses

The protein sequences of RTH5 homologs were obtained by blasting the RTH5 sequence against species-specific databases at maizesequence.org, rice.plantbiology.msu.edu, soybean.org, and TAIR.org. The DNA Baser software was used to combine all sequences into a Fasta file which was used for alignment and calculation of a phylogenetic tree by MEGA4.0 (Tamura et al. (2007) Mol. Biol. Evol. 24: 1596-1599) using the Neighbor joining algorithm (Bootstrap, 1000 replications). RNA in situ hybridization

Primary roots of three-day-old seedlings were fixed and embedded in paraffin as described previously (Hochholdinger et al. (2008) Plant J. 54:888-898). The RNA probe for hybridization was generated by amplification of 282 bp from the 3 '-end of the rth5 gene with the oligonucleotide primers forward 5 '-GTGTACCCGAAGATCCGATG-3 '(SEQ ID NO: 55), and reverse 5 ' -GACAGCTCGGGCAGAAAGAC-3 ' (SEQ ID NO: 56). The amplicon was cloned into the pGEM T-easy vector (Promega,) in sense and antisense directions. In vitro transcription of the probes and Digoxigenin-labeling was performed using the SP6 polymerase (NEB) according to the manufacturer's protocol. RNA in situ hybridization was performed according to (Jackson (1992) Mol. Plant Pathol. Oxford 163-174). A Zeiss Axioplan 2 microscope in combination with a Photometries Cool Snapcamera (Roper Scientific GmbH) was used for image acquisition.

Quantitative RT-PCR

For qRT-PCR, RNA was extracted from approximately 100 mg of different maize tissues with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's protocol, including on-column DNase I digestion (Fermentas). Isolation and harvesting of root hairs was performed as described previously (Nestler, et al. (201 1) J. Proteome Res 10:2525- 2537). cDNA was prepared from 500 ng total RNA by using the qScript cDNA Super Mix (Quanta Biosciences). qRT-PCR experiments were performed using MESA Green or Blue qPCR Mastermix Plus for SYBR Assay no ROX kit (Eurogentec). All experiments were conducted in three biological replicates and three technical replicates per biological replicate in a CFX384 Real-Time PCR Detection System (Bio-Rad). The efficiency for every oligonucleotide primer pair (summarized in Table 5) was determined by a dilution series. Expression was calculated relative to the reference gene myosin (Genbank

AC486090G09.xl), which has previously been used as expression standard in maize root assays (Dembinsky et al. (2007) Plant Physiol. 145: 575-588).

Antibody preparation and immune histochemistry

A polyclonal antibody was produced by incubating rabbits with a specific RTH5 peptide with the amino acid sequence VAGMRPGRMTRMQSSAQM (SEQ ID NO: 57). Sample preparation was slightly modified from the RNA in situ hybridization protocol. Fixation was performed using 4% formamide solution. After sectioning, the samples were deparaffined using RotiClear (Roth) and rehydrated using decreasing ethanol concentrations (90%, 50%, 25%) in Microtubules stabilizing buffer (MtSB) (Albertini et al. (1984) Eur. J. Cell. Biol. 33 : 134-143). Unspecific binding was blocked by 3% BSA in lx MtSB, followed by incubation with the RTH5 antibody (1 : 100 in lx MtSB) overnight at 4 °C. After six wash steps in lx MtSB the samples were incubated for 2 h with the secondary antibody ZytoChem Plus AP Polymer anti-Rabbit (ZytoMed) according to the manufacturer's protocol. After washing with a buffer containing 100 mM Tris, 100 mM NaCl, and 50 mM MgCi₂, the signal was detected using the Roche NBT/BCIP stock solution by incubation in the dark for 5-10 min. The reaction was stopped by three water incubations. Images were obtained as described for RNA in situ hybridization experiments.

Detection of superoxide and hydrogen peroxide

Superoxide was detected by incubating root samples in 0.5 mM Nitro blue tetrazolium chloride (NBT) in 0.1 M KC1/ 0.1 M NaCl solution which forms an insoluble blue formazan precipitate upon reaction with superoxide (Bielski et al. (1980) J. Phys. Chem-US 84:830- 833). To detect hydrogen peroxide two dyes were used: DAB (3,3-diaminobenzidine) and 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA). DAB forms a brown precipitate when oxidized by peroxidase activity (Thordal-Christensen et al. (1997) Plant J. 1 1 : 1 187-1 194). Seedlings were incubated overnight in lmg/ml DAB dissolved in water. H₂DCF-DA emits a green fluorescence that corresponds to cytoplasmic ¾(¾ levels (Keston and Brandt (1965) Anal. Biochem. 11 : 1-5). The stock solution was prepared by dissolving 1 mg DCHF-DA in 1 ml DMSO, and mixing with 1 ml H₂O. To obtain the working solution a dilution in 200 ml H₂0 was prepared.

Three-day-old seedlings were incubated for 45 min in the dark in petri dishes, each of which contained a detection solution. The signal was detected by mounting primary roots in water on a microscopic slide covered by a 60 mm cover slip. Excitation of DCHF-DA was performed at 488 nm. Emission was detected at 525 nm using a Zeiss Axioplan 2 microscope.

RNA-Seq experiment

Seeds from an F₂-family segregating for rth5 after nine generations of backcrossing to the inbred B73 were grown in water-soaked germination paper in an incubator at 25 °C. 3 cm root tips were collected from 6-day-old rth5 mutants and wild-type siblings. Two mutant and two wild-type pools were collected. Each pool consisted of root tissues from six individuals. RNA was extracted using R easy mini kits (Qiagen) with DNasel treatment per the manufacturer's protocol. RNA quality was analyzed using a Bioanalyer 2100 RNA Nano chip. RNA-Seq libraries were constructed using an Illumina RNA-Seq sample preparation kit following the manufacturer's protocol. In total four libraries were prepared (two mutant and two wild-type) and sequenced on an Illumina HiSeq2000 at the Iowa State University DNA facility, generating 99 bp single-end reads (GenBank accession no. SRP020528).

Trimming and mapping of RNA-Seq reads

Raw reads were subjected to quality checking and trimming to remove low quality bases using a custom trimming pipeline (Liu et al. (2012) PLoS ONE 7:e36406). Trimmed reads were aligned to the B73 reference genome (B73Ref2) using GSNAP (Wu and Nacu (2010) Bioinformatics 26:873-881) , allowing at most 2 mismatches every 50 bp and 2 bp tails per 50 bp. Only uniquely mapped reads were used for subsequent analyses. The read depth of each gene in the filtered gene set (ZmB73_5b_FGS) was computed based on the coordinates of mapped reads and the annotated locations of genes in the B73 reference genome.

Differential expression analysis of RNA-Seq

Genes with an average of at least five uniquely mapped reads across the four samples were tested for differential expression between the rth5 mutant and wild-type using the R package QuasiSeq (http://cran.r-project.org/web/packages/QuasiSeq). The negative binomial QLSpline method implemented in the QuasiSeq package was used to compute a p-value for each gene. The 0.75 quantile of reads from each sample was used as the normalization factor (Bullard et al. (2010) BMC Bioinformatics 1 1 :94). An approach for controlling for multiple testing (Benjamini and Hochberg (1995) J. Roy. Statistical Society, Series B 57:289-300) was used to convert ^-values to -values. To approximately control the false discovery rate (FDR) at 10%, only genes with -values < 0.1 were declared to be differentially expressed.

Gene ontology (GO) enrichment analysis of significantly differentially expressed genes

The software GOseq (Young et al. (2010) Genome Biol. 1 1 :R14) was used to perform the GO analysis for differentially expressed genes (DEGs), up-regulated DEGs, and down- regulated DEGs in the mutants, ^-values for over-representation of DEGs for each GO category were generated by randomly sampling the same number of differentially expressed genes 10,000 times. Genes were weighted with their read counts from all samples to account for the potential bias in power to detect differential expression in genes with different levels of read counts. Example 2; Preparation of cDNA Libraries and Isolation and Sequencing of cDNA Clones

mRNAs can be isolated using the Qiagen® RNA isolation kit for total R A isolation, followed by mR A isolation via attachment to oligo(dT) Dynabeads from Invitrogen (Life Technologies, Carlsbad, CA), and sequencing libraries can be prepared using the standard mR A-Seq kit and protocol from Illumina, Inc. (San Diego, CA). In this method, mRNAs are fragmented using a ZnC12 solution, reverse transcribed into cDNA using random primers, end repaired to create blunt end fragments, 3 ' A-tailed, and ligated with Illumina paired-end library adaptors. Ligated cDNA fragments can then be PCR amplified using Illumina paired- end library primers, and purified PCR products can be checked for quality and quantity on the Agilent Bioanalyzer DNA 1000 chip prior to sequencing on the Genome Analyzer II equipped with a paired end module.

Reads from the sequencing runs can be soft-trimmed prior to assembly such that the first base pair of each read with an observed FASTQ quality score lower than 15 and all subsequent bases are clipped using a Python script. The Velvet assembler (Zerbino et a\. (2008) Genome Research 18:821-9) can be run under varying kmer and coverage cutoff parameters to produce several putative assemblies along a range of stringency. The contiguous sequences (contigs) within those assemblies can be combined into clusters using Vmatch software (available on the Vmatch website) such that contigs which are identified as substrings of longer contigs are grouped and eliminated, leaving a non-redundant set of longest "sentinel" contigs. These non-redundant sets can be used in alignments to

homologous sequences from known model plant species.

In cases where the sequence assemblies are in fragments, the percent identity to other homologous genes can be used to infer which fragments represent a single gene. The fragments that appear to belong together can be computationally assembled such that a translation of the resulting nucleotide sequence returns the amino acid sequence of the homologous protein in a single open-reading frame. These computer-generated assemblies can then be aligned with other polypeptides of the invention. The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

SEQUENCES

SEQ ID NO: 1 : Maize (rth5 genomic sequence) (Maize Genetics and Genomics Database Gene ID: RMZM2G426953)

gcttcctctt gcccccccac cccaccccac cgccaccacc cacccctcgc ccgcaggtgg 60 gcccagcctc catcctcctc ctctgagagc gagccggcgg cggcgagcgg cattgcgggt 120 tatctggtgg agcgggacat ggcggggtac ggggaccggc gatcgccgcc gctggagggc 180 atcaccgtcg acggggccag cagggcccaa tcgcccgggg cgggtgcggg ggcgggggcg 240 gggaggctgc cgccgccgcc aggaggcttc gcgcgcgggc tcatgaagca gccgtcgcgg 300 ctggcgtccg gggtgcggca gttcgcttcg cgtgtgtcga tgaaggtgcc cgagggcgtg 360 gccgggatgc ggccgggacg gatgacgcgg atgcagtcta gcgcgcagat ggggctccgt 420 ggcttgcgct tcctcgacaa gacctccggc ggcaaggagg gatggaaggc cgtcgagcgc 480 cgcttcgacg acatggccaa gggcagcgga cgactccaga aggagagctt cggcaagtgc 540 atcggtaagc aaagtgctag tgtgctgctt gctcttgctc catcgagatt gcctcttctg 600 tcggttggcc acatgctgcc taagtgcttg gtgtgatcca gactggaaat aaaccccaaa 660 taatcgatag ttgagacgct gataagaacc gctaggcgta ggcactcata tgacagcaat 720 tgttaatatg tgcatcgaat ctctttcctt gcttttagga attcttgttt gctttacttt 780 tttttttgag tgtttcaaag agctctcatg gagtcatggt actgcaatga actgtgcctg 840 tttccaccaa gcctacctaa ctgagatgag atccaccgtc attcgctccc caagggtctt 900 cccaaatcat gctgaaagaa aagactcttc ccaaatttta atgaagtttc tccccaaagt 960 ggtcctttag cactaatcgg tcagctcgga caaacaagat tctagctagt aacgccatgg 1020 gccaccacca tgtaattcta tcccatgctt tcatctgcca gcaagcgtaa aatcatttgc 1080 tggcgagctg ttcgtgttgc tggtgcccgt ggcgtgatca atggatgatc cgtggctgac 1140 tgactgatct cattcccaag gcatggggga ctccaaggag tttgccggcg agctgttcgt 1200 ggcgctggcg cggaggcgga gcctggagcc ggaggatggc atcaccaaag agcagctcaa 1260 ggagttctgg gaggagatga ccgaccagaa cttcgactcg cggctgcgca ttttctttga 1320 catgtaaagc tccaccgcgc cctcgttccc ggcccaacgg acatgttcat cgttaggcta 1380 ctgataaaac gaattcccct ctgcgtctga ctcaggtgcg acaagaacgg cgatgggatg 1440 ctcacggaag atgaggtcaa agaggtgagc gacggcaact tttcgcgatt tggatgacca 1500 aatatttagg agtggacgaa cttattaaat tatcaacaaa ttaatttgtt ttttaatttg 1560 ttccccccgg caggttatta tactgagcgc gtcagcgaac aagctagcca agctgaaggg 1620 tcacgccgcg acctacgcgt ccctgatcat ggaagagctc gacccggacg accgcggcta 1680 cattgaggtc gtgcttgcag aaacaacatt ttgctttctg ttcgtatcca acagcgataa 1740 gtggtacgcg ttgcttatac aaccaaacgg tgtgatgcag atttggcaac tggagacgtt 1800 gctccggggc atggtgagcg cgcaggctcc cgagaagctg aagcggacga cgtcgagcct 1860 ggcgcggacg atgatcccgt cgcggtaccg gaacccgctg aagcggcacc tgtccaagac 1920 ggtggacttc atccacgaga actggaagcg gatatggctc gtgacgctgt ggctggtcgt 1980 caacgtcgcc ctgttcgtgt acaagttcga gcagtacaag cgccgaaccg cgttccaggt 2040 gatgggctac tgcgtctgcg tcgccaaggg tgccgctgag atcctcaagc tcaacatggc 2100 tctcatcctg ctgcccgtct gccggaatac gctgacgacg ctcaggtcca ccgcgctcag 2160 ccatgtcata cccttcgatg acaacatcaa cttccacaag gtcatcgcgc tgtccatcgc 2220 gatcgccaca gcgatccaca cgctcgcaca cgtgacctgc gacttcccaa ggctgatcag 2280 ctgcccgacg gacaagttca tggccacctt ggggtccaac ttccactaca agcagccgac 2340 ttacctgggc ttgctggaga gcacacccgg ggttaccgga atcctcatga tcatcataat 2400 gtccttctcc ttcacgctgg caacacattc cttcaggcgg agtgtggtga agctgccatc 2460 gccgctacac caccttgccg gtttcaatgc cttctggtac gctcaccacc tgctggtcct 2520 tgcgtatgtc ctgctggtgg tgcactccta cttcatattc ctcaccaggg agtggtacaa 2580 gaagacggta aacctcggac accttgcttt gagatgaaac tcattctccc ctctgcatgt 2640 tcagtcagtg ttgggagcag caaaataaca tatggtctca attgtttatt cagacatgga 2700 tgtacctgat tgtccctgtc ctcttctatg cctgtgaaag agtcatcagg aaatttcgtg 2760 agaacaacta ccatgcggga attgtgaggg taagctattg aactaaattg atttggttca 2820 ttctgagttt ctggctggta attgttaatc aaattctgtt atgtatatgc aggcagcaat 2880 ttatccggga gatgtgctct ctattcacat gaagaagcca cagggtttca agtacaagag 2940 tgggatgtat ctgtttgtta aatgcccaga agtctcgccc ttcgagtggt accatttcat 3000 taattgttgc atacatctcc aagaaataat ttatctgtgg ttcttgctta ccgttctctc 3060 atatttgcag gcaccccttc tctataactt cggcaccagg cgatgactac ttgagtgtgc 3120 atatccgtac gctgggtgac tggacatccg aactgcggat gctttttggg aaggttagtt 3180 tgaggcagca aaagtgttaa gagttggaag agtaacaata ctgttttttg aatggcagaa 3240 taatgtggac tatcagaaat gtataatgtg gattattcgc acaggcttgc caggcacaag 3300 taacttccaa gaaggctacc cttacaagac ttgaaactac agttgtggca gacgcccaga 3360 cagaggacac taggtgetta cctttctttc attttttcac accctatagt ttgattggtt 3420 ttagagaaac tactgeaact ttaagatttt attaacctgg tcagctttgg tgtgtttgct 3480 ttaggtttcc caaggtctac atagaeggge catacggtgc accagcacaa aattacagga 3540 aatatgacat tettctgett attggccttg gaataggagc aactcctttc atcagcatac 3600 tgaaggatat gttgaacaac ctaaaatcca acgaagtaat ttcccttttt aatgeattat 3660 ttcgatactg ttaagaaaat caaggatagc tgataaccgt actgagttga ttaaacagga 3720 ggtggaaagc atccacggct ctgagatagg cagcttcaag aacaatggtc caggaagggc 3780 ttacttctac tgggtcacca gagagcaagg atctttcgaa tggttcaaag gagtcatgaa 3840 tgaggttgca gggagegate acagegtact gtctcactct catttctctt aaatcttaat 3900 tacagcgtat gttcataaac taagttgaaa tetggctget gcagaatgtt atagagatgc 3960 acaattacct gaecagegtg tatgaagaag gtgacgcaag gtcagctctg attgecatgg 4020 tacagtcact teagegtget aaaaacggcg tggatatcgt ctccggcagc aaggtatacg 4080 ataatttccc ttttgctcaa ctgtaaagtg ttttgeatat gctatctgtt ttggttaacg 4140 gagcactctg ttgcagattc gaacacattt tgcaagacca aactggagga aggtattctg 4200 tgatttggcc agcgcacaca agaactctcg cataggtaag ccacttgtca gtgaacatcg 4260 agtgacacga ttttcagatt cagtacaaca gttaatgttt tctctaaaaa aaaattcagg 4320 agttttctat tgtggatctc cgacgctcac aaaacaactg aaggatcttt cgaaagaatt 4380 cagccagaca accacaaccc ggttccattt ccacaaagag aacttctgag acgtgtaccc 4440 gaagatccga tggactggaa acataattgt atagggaaaa aatacgatag cattggcata 4500 gcagatttag ttttacaagt tttgatgtat gcggggttgt acaaaatatg tgtagaaagc 4560 tagatgtcac catcatacat agattctgaa atgcttgcag ctatatatat tgeattgeat 4620 aagtgaaacc acttgcttcc taggataacc gagttctaga tctcctgctt tgaacacgea 4680 aattttctct tttcagtctt tctgcccgag ctgtcattgc attgegaata agggtgetat 4740 tctgtttcct agaagattct tgtgtatcat ettgeactgg cacaagtgcc acagaacctg 4800 gtgtgtcttc agcgccctga gaatttccag gtgeattect gttctttgga ggcctgcctc 4860 tttttttagg ctgettatea gcagataaag atgtatcttt tegaggectg cccctcttcc 4920 taggttgatt actagcctgc aagccgtcca ctctaactat cgccatctta ttgtttgtat 4980 catttccctg ggatagtgta actatatgtg atgttttctc catactgtca tccactgtag 5040 catatatttt ccttggtcta cccacctttg gtttggagtc agaaactgat cccttaagtt 5100 tgcacttgtc gataacaagt gttttaagtg gaacagttat gtccaatggc agtagaactt 5160 tctcaagttc acgccattca agatcttcct ggtctccatc atcataaatg acggtgtacc 5220 agttgctttc aatgtcatac ttggccactt tcccgacgaa atatgtgtct ccaaaaagct 5280 tacgcacttt cctcccttct aaccattcac caaaacgagg atcaacatga gattctgegt 5340 cattttttac tgcagattga gaaccagttg tgcaatcaaa aggaaaatct ggaacgecat 5400 tgataatgat ggcaggctct ttaggaactt tgattataga tgtagtttct gaetcttget 5460 tgccatcagc cccctccatg gttttctata tggaaaatat ggaagaatca ggaaacagat 5520 teat 5524

SEQ ID NO: 2: Maize (rth5 cDNA sequence) (GenBank Accession No: DQ855284) gtgggcccag cctccatcct cctcctctga gagegagecg geggeggega gcggcattgc 60 gggttatctg gtggagcggg acatggcggg gtaeggggae cggcgatcgc cgccgctgga 120 gggcatcacc gtcgacgggg ccagcagggc ccaatcgccc ggggcgggtg egggggeggg 180 ggeggggagg ctgccgccgc cgecaggagg cttcgcgcgc gggctcatga agcagccgtc 240 gcggctggcg tccggggtgc ggcagttcgc ttcgcgtgtg tcgatgaagg tgcccgaggg 300 cgtggccggg atgeggcegg gaeggatgae gcggatgcag tctagcgcgc agatggggct 360 ccgtggcttg cgcttcctcg acaagacctc eggeggcaag gagggatgga aggcegtega 420 gcgccgcttc gacgacatgg ccaagggcag cggacgactc cagaaggaga getteggcaa 480 gtgeategge atgggggact ccaaggagtt tgeeggegag ctgttcgtgg cgctggcgcg 540 gaggeggage ctggagccgg aggatggcat caccaaagag cagctcaagg agttctggga 600 ggagatgacc gaccagaact tcgactcgcg getgegcatt ttctttgaca tgtgcgacaa 660 gaaeggegat gggatgetea eggaagatga ggtcaaagag gttattatac tgagcgcgtc 720 agegaacaag ctagccaagc tgaagggtca cgccgcgacc tacgcgtccc tgatcatgga 780 agagctcgac ccggacgacc geggctacat tgagatttgg caactggaga cgttgctccg 840 gggcatggtg agegegcagg ctcccgagaa getgaagegg aegaegtega gcctggcgcg 900 gacgatgatc ccgtcgcggt accggaaccc getgaagegg cacctgtcca agacggtgga 960 cttcatccac gagaactgga ageggatatg gctcgtgacg ctgtggctgg tegtcaaegt 1020 cgccctgttc gtgtacaagt tcgagcagta caagcgccga accgcgttcc aggtgatggg 1080 ctactgcgtc tgcgtcgcca agggtgeege tgagatcctc aagctcaaca tggctctcat 1140 cctgctgccc gtctgccgga ataegctgae gaegctcagg tccaccgcgc teagecatgt 1200 catacccttc gatgacaaca tcaacttcca caaggtcatc gcgctgtcca tcgcgatcgc 1260 cacagcgatc cacacgctcg cacacgtgac ctgcgacttc ccaaggctga tcagctgccc 1320 gacggacaag ttcatggcca ccttggggtc caacttccac tacaagcagc cgacttacct 1380 gggcttgctg gagagcacac ccggggttaa cggaaacctc atgatcatca taatgtcctt 1440 ctccttcaag ctggcaacac attccttcag gcggagtgtg gtgaagctgc catcgccgct 1500 acaccacctt gccggtttca atgccttctg gtacgctcac cacctgctgg tccttgcgta 1560 tgtcctgctg gtggtgcact cctacttcat attcctcacc agggagtggt acaagaagac 1620 gacatggatg tacctgattg tccctgtcct cttctatgcc tgtgaaagag tcatcaggaa 1680 atttcgtgag aacaactacc atgcgggaat tgtgagggca gcaatttatc cgggagatgt 1740 gctctctatt cacatgaaga agccacaggg tttcaagtac aagagtggga tgtatctgtt 1800 tgttaaatgc ccagaagtct cgcccttcga gtggcacccc ttctctataa cttcggcacc 1860 aggcgatgac tacttgagtg tgcatatccg tacgctgggt gactggacat ccgaactgcg 1920 gatgcttttt gggaaggctt gccaggcaca agtaacttcc aagaaggcta cccttacaag 1980 acttgaaact acagttgtgg cagacgccca gacagaggac actaggtttc ccaaggtcta 2040 catagacggg ccatacggtg caccagcaca aaattacagg aaatatgaca ttcttctgct 2100 tattggcctt ggaataggag caactccttt catcagcata ctgaaggata tgttgaacaa 2160 cctaaaatcc aacgaagagg tgggaagcat ccacggctct gagataggca gcttcaagaa 2220 caatggtcca ggaagggctt acttctactg ggtcaccaga gagcaaggat ctttcgaatg 2280 gttcaaagga gtcatgaatg aggttgcagg gagcgatcac agcaatgtta tagagatgca 2340 caattacctg accagcgtgt atgaagaagg tgacgcaagg tcagctctga ttgccatggt 2400 acagtcactt cagcgtgcta aaaacggcgt ggatatcgtc tccggcagca agattcgaac 2460 acattttgca agaccaaact ggaggaaggt attctgtgat ttggccagcg cacacaagaa 2520 ctctcgcata ggagttttct attgtggatc tccgacgctc acaaaacaac tgaaggatct 2580 ttcgaaagaa ttcagccaga caaccacaac ccggttccat ttccacaagg aaaacttctg 2640 agacgacgtg tacccgaaga tccgatggac tggaaacata attgtatagg gaaaaaatac 2700 gatagcattg gcatagcaga tttagtttta caagttttga tgtatgcggg gttgtacaaa 2760 atatgtgtag aaagctagat gtcaccatca tacatagatt ctgaaatgct tgcagatata 2820 tatattgcat tgcataagtg aaaccacttg cttcctagga taaccgagtt ctagatctcc 2880 tgctttgaac acgcaaattt tctcttttca gtctttctgc ccgagctgtc attgcattgc 2940 gaataagggt gctattctgt ttcctagaag attcttgtgt atcatcttgc actggcacaa 3000 gtgccacaga acctggtgtg tcttcagcgc cctgagaatt tccaggtgca ttcctgttct 3060 ttggaggcct gcctcttttt ttaggctgct tatcagcaga taaagatgta tcttttcgag 3120 gcctgcccct cttcctaggt tgattactag cctgcaagcc gtccactcta actatcgcca 3180 tcttattgtt tgtatcattt ccctgggata gtgtaactat atgtgatgtt ttctccatac 3240 tgtcatccac tgtagcatat attttccttg gtctacccac ctttggtttg gagtcagaaa 3300 ctgatccctt aagtttgcac ttgtcgataa caagtgtttt aagtggaaca gctatgtcca 3360 atggcagtag aactttctca agttcacgcc attcaagatc ttcctggtct ccatcatcat 3420 aaatgacggt gtaccagttg ctttcaatgt catacttggc cactttcccg acgaaatatg 3480 tgtctccaaa aagcttacgc actttcctcc cttctaacca ttcaccaaaa cgaggatcaa 3540 catgagattc tgcgtcattt tttactgcag attgagaacc agttgtgcaa tc 3592

SEQ ID NO: 3 : Maize (RTH5 amino acid sequence) (GenBank Accession No: DQ855284)

Met Ala Gly Tyr Gly Asp Arg Arg Ser Pro Pro Leu Glu Gly lie Thr

1 5 10 15

Val Asp Gly Ala Ser Arg Ala Gin Ser Pro Gly Ala Gly Ala Gly Ala

20 25 30

Gly Ala Gly Arg Leu Pro Pro Pro Pro Gly Gly Phe Ala Arg Gly Leu

35 40 45

Met Lys Gin Pro Ser Arg Leu Ala Ser Gly Val Arg Gin Phe Ala Ser

50 55 60

Arg Val Ser Met Lys Val Pro Glu Gly Val Ala Gly Met Arg Pro Gly 65 70 75 80

Arg Met Thr Arg Met Gin Ser Ser Ala Gin Met Gly Leu Arg Gly Leu

85 90 95

Arg Phe Leu Asp Lys Thr Ser Gly Gly Lys Glu Gly Trp Lys Ala Val

100 105 110

Glu Arg Arg Phe Asp Asp Met Ala Lys Gly Ser Gly Arg Leu Gin Lys

115 120 125

Glu Ser Phe Gly Lys Cys lie Gly Met Gly Asp Ser Lys Glu Phe Ala 130 135 140 Gly Glu Leu Phe Val Ala Leu Ala Arg Arg Arg Ser Leu Glu Pro Glu 145 150 155 160

Asp Gly lie Thr Lys Glu Gin Leu Lys Glu Phe Trp Glu Glu Met Thr

165 170 175

Asp Gin Asn Phe Asp Ser Arg Leu Arg He Phe Phe Asp Met Cys Asp

180 185 190

Lys Asn Gly Asp Gly Met Leu Thr Glu Asp Glu Val Lys Glu Val He

195 200 205

lie Leu Ser Ala Ser Ala Asn Lys Leu Ala Lys Leu Lys Gly His Ala 210 215 220

Ala Thr Tyr Ala Ser Leu He Met Glu Glu Leu Asp Pro Asp Asp Arg 225 230 235 240

Gly Tyr He Glu He Trp Gin Leu Glu Thr Leu Leu Arg Gly Met Val

245 250 255

Ser Ala Gin Ala Pro Glu Lys Leu Lys Arg Thr Thr Ser Ser Leu Ala

260 265 270

Arg Thr Met He Pro Ser Arg Tyr Arg Asn Pro Leu Lys Arg His Leu

275 280 285

Ser Lys Thr Val Asp Phe He His Glu Asn Trp Lys Arg He Trp Leu 290 295 300

Val Thr Leu Trp Leu Val Val Asn Val Ala Leu Phe Val Tyr Lys Phe 305 310 315 320

Glu Gin Tyr Lys Arg Arg Thr Ala Phe Gin Val Met Gly Tyr Cys Val

325 330 335

Cys Val Ala Lys Gly Ala Ala Glu He Leu Lys Leu Asn Met Ala Leu

340 345 350 lie Leu Leu Pro Val Cys Arg Asn Thr Leu Thr Thr Leu Arg Ser Thr

355 360 365

Ala Leu Ser His Val He Pro Phe Asp Asp Asn He Asn Phe His Lys 370 375 380

Val lie Ala Leu Ser He Ala He Ala Thr Ala He His Thr Leu Ala 385 390 395 400

His Val Thr Cys Asp Phe Pro Arg Leu He Ser Cys Pro Thr Asp Lys

405 410 415

Phe Met Ala Thr Leu Gly Ser Asn Phe His Tyr Lys Gin Pro Thr Tyr

420 425 430

Leu Gly Leu Leu Glu Ser Thr Pro Gly Val Thr Gly He Leu Met He

435 440 445

lie lie Met Ser Phe Ser Phe Thr Leu Ala Thr His Ser Phe Arg Arg 450 455 460

Ser Val Val Lys Leu Pro Ser Pro Leu His His Leu Ala Gly Phe Asn 465 470 475 480

Ala Phe Trp Tyr Ala His His Leu Leu Val Leu Ala Tyr Val Leu Leu

485 490 495

Val Val His Ser Tyr Phe He Phe Leu Thr Arg Glu Trp Tyr Lys Lys

500 505 510

Thr Thr Trp Met Tyr Leu He Val Pro Val Leu Phe Tyr Ala Cys Glu

515 520 525

Arg Val He Arg Lys Phe Arg Glu Asn Asn Tyr His Ala Gly He Val 530 535 540

Arg Ala Ala He Tyr Pro Gly Asp Val Leu Ser He His Met Lys Lys 545 550 555 560

Pro Gin Gly Phe Lys Tyr Lys Ser Gly Met Tyr Leu Phe Val Lys Cys

565 570 575 Pro Glu Val Ser Pro Phe Glu Trp His Pro Phe Ser He Thr Ser Ala

580 585 590

Pro Gly Asp Asp Tyr Leu Ser Val His He Arg Thr Leu Gly Asp Trp

595 600 605

Thr Ser Glu Leu Arg Met Leu Phe Gly Lys Ala Cys Gin Ala Gin Val 610 615 620

Thr Ser Lys Lys Ala Thr Leu Thr Arg Leu Glu Thr Thr Val Val Ala

625 630 635 640

Asp Ala Gin Thr Glu Asp Thr Arg Phe Pro Lys Val Tyr He Asp Gly

645 650 655

Pro Tyr Gly Ala Pro Ala Gin Asn Tyr Arg Lys Tyr Asp He Leu Leu

660 665 670

Leu lie Gly Leu Gly He Gly Ala Thr Pro Phe He Ser He Leu Lys

675 680 685

Asp Met Leu Asn Asn Leu Lys Ser Asn Glu Glu Val Glu Ser He His

690 695 700

Gly Ser Glu He Gly Ser Phe Lys Asn Asn Gly Pro Gly Arg Ala Tyr

705 710 715 720

Phe Tyr Trp Val Thr Arg Glu Gin Gly Ser Phe Glu Trp Phe Lys Gly

725 730 735

Val Met Asn Glu Val Ala Gly Ser Asp His Ser Asn Val He Glu Met

740 745 750

His Asn Tyr Leu Thr Ser Val Tyr Glu Glu Gly Asp Ala Arg Ser Ala

755 760 765

Leu He Ala Met Val Gin Ser Leu Gin Arg Ala Lys Asn Gly Val Asp

770 775 780

He Val Ser Gly Ser Lys He Arg Thr His Phe Ala Arg Pro Asn Trp

785 790 795 800

Arg Lys Val Phe Cys Asp Leu Ala Ser Ala His Lys Asn Ser Arg He

805 810 815

Gly Val Phe Tyr Cys Gly Ser Pro Thr Leu Thr Lys Gin Leu Lys Asp

820 825 830

Leu Ser Lys Glu Phe Ser Gin Thr Thr Thr Thr Arg Phe His Phe His

835 840 845

Lys Glu Asn Phe

850

SEQ ID NO: 4: GRMZM2G401179_P01_variant; corn; 21-aa at position 215 removed to get better alignment

MTSSAGYADGGTGGLGDGDPPVPPLRKQPSRIASGMRRLASKVSAAVPEMRGLKRTHSGTQSGLRGLRFLDKTSA GKDGWKSVEKRFDEMNTEGRLQRENFAKCIGMADSNEFASEVFVALARRTHINPDDGVTKEQLKQFWEEMTDQNF DSRLRIFFDMCDKNGDGKLTEDEVKEVIVLSASANKLAKLKKHAATYASLIMEELDPDHRGYIEIWQLETLLRGM VTASAPPEKMNMASASLARTMVPSSHRSPLQRRINKAVDFVHENWKRIWVLSLWGVLCIALFVFKFIQYRRRAVF EVMGYCVCIAKGAAETLKLNMALILLPVCRNTLTRLRSTALSKVVPFDDNINFHKVIALAIAIGSATHTLAHVLC DFPRLVACPKDKFMEKLGPFFNYVQPTWPTLLASI PGWTGILLILIMSFSFTLATHSFRRSVVKLPSPLHHLAGF NAFWYAHHLLVIAYVLLVMHSYFIFLTKQWYKRTTWMYLAVPVVFYASERSIRKIREQSYRVSI IKAAIYPGNVL SLYMTKPPGFKYKSGMYMFVKCPDVSPFEWHPFSITSAPGDDYLSVHIRTLGDWTSELRNLFGKACEAEVTSKKA TLARLETTWAHGLAEDTRFPKVFIDGPYGAPAQNYRKYDILLLIGLGIGATPFIS ILKDLLNNIKSNEEMHDAE LGCSLKTNGPGRAYFYWVTREQGSFEWFKGVMNDVAESDRDDVIEMHNYLTSVYEEGDARSALIAMVQSLQHAKN GVDIVSGSKIRTHFARPNWRKVFSDLANAHKNSRIGVFYCGSPTLTKTLRDLSIEFSSTTTTRFHFHKENF

SEQ ID NO: 5: GRMZM2G323731_P01_ Maize (amino acid)

Met Ala Ser Ser Ser Gly Tyr Ala Asp Gly Gly Thr Gly Gly Leu Gly

1 5 10 15

Asp Gly Asp Pro Pro Val Pro Ser Met Arg Lys Gin Pro Ser Arg He

20 25 30

Ala Ser Gly Met Arg Arg Leu Ala Ser Lys Val Ser Ala Ala Val Pro

35 40 45

Glu Met Arg Gly Leu Lys Arg Thr Gin Ser Gly Ala Gin Ser Gly Leu

50 55 60 Arg Gly Leu Arg Phe Leu Asp Lys Thr Ser Ala Gly Lys Asp Gly Trp 65 70 75 80

Lys Thr Val Glu Lys Arg Phe Asp Glu Met Ser Thr Asp Gly Arg Leu

85 90 95

Gin Arg Glu Asn Phe Ala Lys Cys He Gly Met Ala Asp Ser Lys Glu

100 105 110

Phe Ala Ser Glu Val Phe Val Ala Leu Ala Arg Arg Arg His He Asn

115 120 125

Pro Asp Asp Gly Val Thr Lys Glu Gin Leu Lys Glu Phe Trp Glu Glu 130 135 140

Met Thr Asp Gin Asn Phe Asp Ser Arg Leu Arg He Phe Phe Asp Met 145 150 155 160

Cys Asp Lys Asn Gly Asp Gly Lys Leu Thr Glu Asp Glu Val Lys Glu

165 170 175

Val lie Val Leu Ser Ala Ser Ala Asn Lys Leu Ala Lys Leu Lys Lys

180 185 190

His Ala Ala Thr Tyr Ala Ser Leu He Met Glu Glu Leu Asp Pro Asp

195 200 205

His Arg Gly Tyr He Glu He Trp Gin Leu Glu Thr Leu Leu Arg Gly 210 215 220

Met Val Thr Ala Ser Gly Pro Thr Asn Met Gly Ala Ser Ala Ser Leu 225 230 235 240

Ala Arg Thr Met Val Pro Ser Ser His Arg Thr Pro Leu Gin Arg Arg

245 250 255

Met Asn Lys Ala Val Asp Leu Val His Glu Asn Trp Lys Arg He Trp

260 265 270

Val Leu Ser Leu Trp Gly Val Leu Asn Met Ala Leu Phe Val Phe Lys

275 280 285

Phe Thr Gin Tyr Arg Arg Arg Ala Val Phe Glu Val Met Gly Tyr Cys 290 295 300

Val Cys lie Ala Lys Gly Ala Ala Glu Thr Leu Lys Leu Asn Met Ala 305 310 315 320

Leu lie Leu Leu Pro Val Cys Arg Asn Thr Leu Thr Arg Leu Arg Ser

325 330 335

Thr Ala Leu Ser Lys Val Val Pro Phe Asp Asp Asn He Asn Phe His

340 345 350

Lys Val lie Ala Leu Ala He Ala He Gly Ser Ala Thr His Thr Leu

355 360 365

Ala His Val Leu Cys Asp Phe Pro Arg Leu Val Ala Cys Pro Lys Asp 370 375 380

Lys Phe Met Glu Lys Leu Gly Pro Phe Phe Asn Tyr Ala Gin Pro Thr 385 390 395 400

Trp Ala Thr Leu Leu Ser Ser He Pro Gly Trp Thr Gly He Leu Leu

405 410 415 lie Leu lie Met Ser Phe Ser Phe Thr Leu Ala Thr His Ser Phe Arg

420 425 430

Arg Ser Val Val Lys Leu Pro Ser Pro Leu His His Leu Ala Gly Phe

435 440 445

Asn Ala Phe Trp Tyr Ala His His Leu Leu Val He Ala Tyr Val Leu 450 455 460

Leu Val Met His Ser Tyr Phe He Phe Leu Thr Lys Gin Trp Tyr Lys 465 470 475 480

Arg Thr Thr Trp Met Tyr Leu Ala Val Pro Val Val Phe Tyr Ala Ser

485 490 495

Glu Arg Ser He Arg Arg He Arg Glu Lys Ser Tyr Arg Val Ser He

500 505 510 lie Lys Ala Ala He Tyr Pro Gly Asn Val Leu Ser Leu Tyr Met Lys

515 520 525

Lys Pro Thr Ser Phe Lys Tyr Lys Ser Gly Met Tyr Met Phe Val Lys 530 535 540 Cys Pro Asp Val Ser Pro Phe Glu Trp His Pro Phe Ser lie Thr Ser 545 550 555 560

Ala Pro Gly Asp Asp Tyr Leu Ser Val His lie Arg Thr Leu Gly Asp

565 570 575

Trp Thr Ser Glu Leu Arg Asn Leu Phe Gly Lys Ala Cys Glu Ala Glu

580 585 590

Val Thr Ser Lys Lys Ala Thr Leu Ala Arg Leu Glu Thr Thr Val Val

595 600 605

Ala His Gly Leu Ala Glu Asp Thr Arg Phe Pro Lys Val Phe lie Asp

610 615 620

Gly Pro Tyr Gly Ala Pro Ala Gin Asn Tyr Arg Lys Tyr Asp lie Leu 625 630 635 640

Leu Leu lie Gly Leu Gly lie Gly Ala Thr Pro Phe lie Ser lie Leu

645 650 655

Lys Asp Leu Leu Asn Asn lie Lys Ser Asn Glu Glu Met Gin Ser Met

660 665 670

His Asp Thr Glu Leu Gly Cys Ser Phe Lys Thr Asn Gly Pro Gly Arg

675 680 685

Ala Tyr Phe Tyr Trp Val Thr Arg Glu Gin Gly Ser Phe Glu Trp Phe

690 695 700

Lys Gly Val Met Asn Asp Val Ala Glu Ser Asp His Asp Asp Val lie 705 710 715 720

Glu Met His Asn Tyr Leu Thr Ser Val Tyr Glu Glu Gly Asp Ala Arg

725 730 735

Ser Ala Leu lie Ala Met Val Gin Ser Leu Gin His Ala Lys Asp Gly

740 745 750

Val Asp lie Val Ser Gly Ser Lys lie Arg Thr His Phe Ala Arg Pro

755 760 765

Asn Trp Arg Lys Val Phe Ser Asp Leu Ala Asn Ala His Lys Asn Ser

770 775 780

Arg lie Gly Val Phe Tyr Cys Gly Ser Pro Thr Leu Thr Lys Thr Leu 785 790 795 800

Arg Asp Leu Ser lie Glu Phe Ser Ser Thr Thr Thr Thr Arg Phe His

805 810 815

Phe His Lys Glu Asn Phe

820

SEQ ID NO: 6: GRMZM2G401179_P01_Maize

Met Thr Ser Ser Ala Gly Tyr Ala Asp Gly Gly Thr Gly Gly Leu Gly

1 5 10 15

Asp Gly Asp Pro Pro Val Pro Pro Leu Arg Lys Gin Pro Ser Arg lie

20 25 30

Ala Ser Gly Met Arg Arg Leu Ala Ser Lys Val Ser Ala Ala Val Pro

35 40 45

Glu Met Arg Gly Leu Lys Arg Thr His Ser Gly Thr Gin Ser Gly Leu

50 55 60

Arg Gly Leu Arg Phe Leu Asp Lys Thr Ser Ala Gly Lys Asp Gly Trp 65 70 75 80

Lys Ser Val Glu Lys Arg Phe Asp Glu Met Asn Thr Glu Gly Arg Leu

85 90 95

Gin Arg Glu Asn Phe Ala Lys Cys lie Gly Met Ala Asp Ser Asn Glu

100 105 110

Phe Ala Ser Glu Val Phe Val Ala Leu Ala Arg Arg Thr His lie Asn

115 120 125

Pro Asp Asp Gly Val Thr Lys Glu Gin Leu Lys Gin Phe Trp Glu Glu

130 135 140

Met Thr Asp Gin Asn Phe Asp Ser Arg Leu Arg lie Phe Phe Asp Met 145 150 155 160

Cys Asp Lys Asn Gly Asp Gly Lys Leu Thr Glu Asp Glu Val Lys Glu

165 170 175 Val lie Val Leu Ser Ala Ser Ala Asn Lys Leu Ala Lys Leu Lys Lys 180 185 190

His Ala Ala Thr Tyr Ala Ser Leu He Met Glu Glu Leu Asp Pro Asp

195 200 205

His Arg Gly Tyr He Glu Val His Leu Ala Leu Phe Ala Cys Leu Ser 210 215 220

Arg Ser Ser He Leu Thr Glu Asp Arg Lys Gin He Trp Gin Leu Glu 225 230 235 240

Thr Leu Leu Arg Gly Met Val Thr Ala Ser Ala Pro Pro Glu Lys Met

245 250 255

Asn Met Ala Ser Ala Ser Leu Ala Arg Thr Met Val Pro Ser Ser His

260 265 270

Arg Ser Pro Leu Gin Arg Arg He Asn Lys Ala Val Asp Phe Val His

275 280 285

Glu Asn Trp Lys Arg He Trp Val Leu Ser Leu Trp Gly Val Leu Cys 290 295 300

lie Ala Leu Phe Val Phe Lys Phe He Gin Tyr Arg Arg Arg Ala Val 305 310 315 320

Phe Glu Val Met Gly Tyr Cys Val Cys He Ala Lys Gly Ala Ala Glu

325 330 335

Thr Leu Lys Leu Asn Met Ala Leu He Leu Leu Pro Val Cys Arg Asn

340 345 350

Thr Leu Thr Arg Leu Arg Ser Thr Ala Leu Ser Lys Val Val Pro Phe

355 360 365

Asp Asp Asn He Asn Phe His Lys Val He Ala Leu Ala He Ala He 370 375 380

Gly Ser Ala Thr His Thr Leu Ala His Val Leu Cys Asp Phe Pro Arg 385 390 395 400

Leu Val Ala Cys Pro Lys Asp Lys Phe Met Glu Lys Leu Gly Pro Phe

405 410 415

Phe Asn Tyr Val Gin Pro Thr Trp Pro Thr Leu Leu Ala Ser He Pro

420 425 430

Gly Trp Thr Gly He Leu Leu He Leu He Met Ser Phe Ser Phe Thr

435 440 445

Leu Ala Thr His Ser Phe Arg Arg Ser Val Val Lys Leu Pro Ser Pro 450 455 460

Leu His His Leu Ala Gly Phe Asn Ala Phe Trp Tyr Ala His His Leu 465 470 475 480

Leu Val He Ala Tyr Val Leu Leu Val Met His Ser Tyr Phe He Phe

485 490 495

Leu Thr Lys Gin Trp Tyr Lys Arg Thr Thr Trp Met Tyr Leu Ala Val

500 505 510

Pro Val Val Phe Tyr Ala Ser Glu Arg Ser He Arg Lys He Arg Glu

515 520 525

Gin Ser Tyr Arg Val Ser He He Lys Ala Ala He Tyr Pro Gly Asn 530 535 540

Val Leu Ser Leu Tyr Met Thr Lys Pro Pro Gly Phe Lys Tyr Lys Ser 545 550 555 560

Gly Met Tyr Met Phe Val Lys Cys Pro Asp Val Ser Pro Phe Glu Trp

565 570 575

His Pro Phe Ser He Thr Ser Ala Pro Gly Asp Asp Tyr Leu Ser Val

580 585 590

His lie Arg Thr Leu Gly Asp Trp Thr Ser Glu Leu Arg Asn Leu Phe

595 600 605

Gly Lys Ala Cys Glu Ala Glu Val Thr Ser Lys Lys Ala Thr Leu Ala 610 615 620

Arg Leu Glu Thr Thr Val Val Ala His Gly Leu Ala Glu Asp Thr Arg 625 630 635 640

Phe Pro Lys Val Phe He Asp Gly Pro Tyr Gly Ala Pro Ala Gin Asn

645 650 655 Tyr Arg Lys Tyr Asp lie Leu Leu Leu lie Gly Leu Gly lie Gly Ala

660 665 670

Thr Pro Phe lie Ser lie Leu Lys Asp Leu Leu Asn Asn lie Lys Ser

675 680 685

Asn Glu Glu Met His Asp Ala Glu Leu Gly Cys Ser Leu Lys Thr Asn

690 695 700

Gly Pro Gly Arg Ala Tyr Phe Tyr Trp Val Thr Arg Glu Gin Gly Ser 705 710 715 720

Phe Glu Trp Phe Lys Gly Val Met Asn Asp Val Ala Glu Ser Asp Arg

725 730 735

Asp Asp Val lie Glu Met His Asn Tyr Leu Thr Ser Val Tyr Glu Glu

740 745 750

Gly Asp Ala Arg Ser Ala Leu lie Ala Met Val Gin Ser Leu Gin His

755 760 765

Ala Lys Asn Gly Val Asp lie Val Ser Gly Ser Lys lie Arg Thr His

770 775 780

Phe Ala Arg Pro Asn Trp Arg Lys Val Phe Ser Asp Leu Ala Asn Ala 785 790 795 800

His Lys Asn Ser Arg lie Gly Val Phe Tyr Cys Gly Ser Pro Thr Leu

805 810 815

Thr Lys Thr Leu Arg Asp Leu Ser lie Glu Phe Ser Ser Thr Thr Thr

820 825 830

Thr Arg Phe His Phe His Lys Glu Asn Phe

835 840

SEQ ID NO: 7: At3g45810.1_ Arabidopsis thaliana

Met Lys Asn Asn Lys Lys Val Gly Thr Glu Asp Ser Thr Lys Trp Met

1 5 10 15

Leu Glu Ser Val Glu lie Asp Pro Lys Gly Asp Ser Ser Val Lys Gin

20 25 30

Pro Glu Ser Thr lie Asn Ser Asn Asn Pro Glu Ser Ser Gly Ala Gly

35 40 45

Gly Gly lie Leu Lys Asn Val Ser Lys Asn Leu Ala Val Gly Ser lie

50 55 60

lie Arg Ser Met Ser Val Asn Lys Trp Arg Lys Ser Gly Asn Leu Gly 65 70 75 80

Ser Pro Ser Thr Arg Lys Ser Gly Asn Leu Gly Pro Pro Leu Pro Val

85 90 95

Ser Gin Val Lys Arg Pro Gly Pro Gin Arg Val Glu Arg Thr Thr Ser

100 105 110

Ser Ala Ala Arg Gly Leu Gin Ser Leu Arg Phe Leu Asp Arg Thr Val

115 120 125

Thr Gly Arg Glu Arg Asp Ser Trp Arg Ser lie Glu Asn Arg Phe Asn

130 135 140

Gin Phe Ala Val Asp Gly Arg Leu Pro Lys Asp Lys Phe Gly Val Cys 145 150 155 160 lie Gly Met Gly Asp Thr Leu Glu Phe Ala Ala Lys Val Tyr Glu Ala

165 170 175

Leu Gly Arg Arg Arg Gin lie Lys Thr Glu Asn Gly lie Asp Lys Glu

180 185 190

Gin Leu Lys Leu Phe Trp Glu Asp Met lie Lys Lys Asp Leu Asp Cys

195 200 205

Arg Leu Gin lie Phe Phe Asp Met Cys Asp Lys Asp Gly Asp Gly Lys

210 215 220

Leu Thr Glu Glu Glu Val Lys Glu Val lie Val Leu Ser Ala Ser Ala 225 230 235 240

Asn Arg Leu Val Asn Leu Lys Lys Asn Ala Ala Ser Tyr Ala Ser Leu

245 250 255 lie Met Glu Glu Leu Asp Pro Asn Glu Gin Gly Tyr lie Glu Met Trp 260 265 270

Gin Leu Glu Val Leu Leu Thr Gly He Val Ser Asn Ala Asp Ser His

275 280 285

Lys Val Val Arg Lys Ser Gin Gin Leu Thr Arg Ala Met He Pro Lys 290 295 300

Arg Tyr Arg Thr Pro Thr Ser Lys Tyr Val Val Val Thr Ala Glu Leu 305 310 315 320

Met Tyr Glu His Trp Lys Lys He Trp Val Val Thr Leu Trp Leu Ala

325 330 335

Val Asn Val Val Leu Phe Met Trp Lys Tyr Glu Glu Phe Thr Thr Ser

340 345 350

Pro Leu Tyr Asn He Thr Gly Arg Cys Leu Cys Ala Ala Lys Gly Thr

355 360 365

Ala Glu He Leu Lys Leu Asn Met Ala Leu He Leu Val Pro Val Leu 370 375 380

Arg Arg Thr Leu Thr Phe Leu Arg Ser Thr Phe Leu Asn His Leu He 385 390 395 400

Pro Phe Asp Asp Asn He Asn Phe His Lys Leu He Ala Val Ala He

405 410 415

Ala Val He Ser Leu Leu His Thr Ala Leu His Met Leu Cys Asn Tyr

420 425 430

Pro Arg Leu Ser Ser Cys Pro Tyr Asn Phe Tyr Ser Asp Tyr Ala Gly

435 440 445

Asn Leu Leu Gly Ala Lys Gin Pro Thr Tyr Leu Gly Leu Met Leu Thr 450 455 460

Pro Val Ser Val Thr Gly Val Leu Met He He Phe Met Gly He Ser 465 470 475 480

Phe Thr Leu Ala Met His Tyr Phe Arg Arg Asn He Val Lys Leu Pro

485 490 495

He Pro Phe Asn Arg Leu Ala Gly Phe Asn Ser Phe Trp Tyr Ala His

500 505 510

His Leu Leu Val He Ala Tyr Ala Leu Leu He He His Gly Tyr He

515 520 525

Leu He He Glu Lys Pro Trp Tyr Gin Lys Thr Thr Trp Met Tyr Val 530 535 540

Ala He Pro Met Val Leu Tyr Ala Ser Glu Arg Leu Phe Ser Arg Val 545 550 555 560

Gin Glu His Asn His Arg Val His He He Lys Ala He Val Tyr Ser

565 570 575

Gly Asn Val Leu Ala Leu Tyr Met Thr Lys Pro Gin Gly Phe Lys Tyr

580 585 590

Lys Ser Gly Met Tyr Met Phe Val Lys Cys Pro Asp He Ser Lys Phe

595 600 605

Glu Trp His Pro Phe Ser He Thr Ser Ala Pro Gly Asp Glu Tyr Leu 610 615 620

Ser Val His He Arg Ala Leu Gly Asp Trp Thr Ser Glu Leu Arg Asn 625 630 635 640

Arg Phe Ala Glu Thr Cys Glu Pro His Gin Lys Ser Lys Pro Ser Pro

645 650 655

Asn Asp Leu He Arg Met Glu Thr Arg Ala Arg Gly Ala Asn Pro His

660 665 670

Val Glu Glu Ser Gin Ala Leu Phe Pro Arg He Phe He Lys Gly Pro

675 680 685

Tyr Gly Ala Pro Ala Gin Ser Tyr Gin Lys Phe Asp He Leu Leu Leu 690 695 700

He Gly Leu Gly He Gly Ala Thr Pro Phe He Ser He Leu Lys Asp 705 710 715 720

Met Leu Asn Asn Leu Lys Pro Gly He Pro Lys Thr Gly Gin Lys Tyr

725 730 735 Glu Gly Ser Val Gly Gly Glu Ser Leu Gly Gly Ser Ser Val Tyr Gly 740 745 750

Gly Ser Ser Val Asn Gly Gly Gly Ser Val Asn Gly Gly Gly Ser Val

755 760 765

Ser Gly Gly Gly Arg Lys Phe Pro Gin Arg Ala Tyr Phe Tyr Trp Val 770 775 780

Thr Arg Glu Gin Ala Ser Phe Glu Trp Phe Lys Gly Val Met Asp Asp 785 790 795 800 lie Ala Val Tyr Asp Lys Thr Asn Val He Glu Met His Asn Tyr Leu

805 810 815

Thr Ser Met Tyr Glu Ala Gly Asp Ala Arg Ser Ala Leu He Ala Met

820 825 830

Val Gin Lys Leu Gin His Ala Lys Asn Gly Val Asp He Val Ser Glu

835 840 845

Ser Arg He Arg Thr His Phe Ala Arg Pro Asn Trp Arg Lys Val Phe 850 855 860

Ser Glu Leu Ser Asn Lys His Glu Thr Ser Arg He Gly Val Phe Tyr 865 870 875

Cys Gly Ser Pro Thr Leu Val Arg Pro Leu Lys Ser Leu Cys Gin Glu

885 890 895 Phe Ser Leu Glu Ser Ser Thr Arg Phe Thr Phe His Lys Glu Asn Phe

900 905 910

SEQ ID NO: 8: At5g60010.1_ Arabidopsis thaliana

Met Lys Ser Asn Thr Pro Thr Glu Asp Ser Thr Lys Trp Met Leu Glu

1 5 10 15

Ser Val Glu He Asp Ser Met Gly Glu Ser Ser Ser Lys Glu Pro Glu

20 25 30

He Asn Leu Asn Lys Asn Glu Gly Gly Leu Lys Lys Asn Ala Ser Arg

35 40 45

Asn Leu Gly Val Gly Ser He He Arg Thr Leu Ser Val Ser Asn Trp

50 55 60

Arg Lys Ser Gly Asn Leu Gly Ser Pro Ser Thr Arg Lys Ser Gly Asn 65 70 75 80

Leu Gly Pro Pro Thr Asn Ala Val Pro Lys Lys Thr Gly Pro Gin Arg

85 90 95

Val Glu Arg Thr Thr Ser Ser Ala Ala Arg Gly Leu Gin Ser Leu Arg

100 105 110

Phe Leu Asp Arg Thr Val Thr Gly Arg Glu Arg Asp Ala Trp Arg Ser

115 120 125

He Glu Asn Arg Phe Asn Gin Phe Ser Val Asp Gly Lys Leu Pro Lys

130 135 140

Glu Lys Phe Gly Val Cys He Gly Met Gly Asp Thr Met Glu Phe Ala 145 150 155 160

Ala Glu Val Tyr Glu Ala Leu Gly Arg Arg Arg Gin He Glu Thr Glu

165 170 175

Asn Gly He Asp Lys Glu Gin Leu Lys Leu Phe Trp Glu Asp Met He

180 185 190

Lys Lys Asp Leu Asp Cys Arg Leu Gin He Phe Phe Asp Met Cys Asp

195 200 205

Lys Asn Gly Asp Gly Lys Leu Thr Glu Glu Glu Val Lys Glu Val He

210 215 220

Val Leu Ser Ala Ser Ala Asn Arg Leu Gly Asn Leu Lys Lys Asn Ala 225 230 235 240

Ala Ala Tyr Ala Ser Leu He Met Glu Glu Leu Asp Pro Asp His Lys

245 250 255

Gly Tyr He Glu Met Trp Gin Leu Glu He Leu Leu Thr Gly Met Val

260 265 270

Thr Asn Ala Asp Thr Glu Lys Met Lys Lys Ser Gin Thr Leu Thr Arg 275 280 285

Ala Met lie Pro Glu Arg Tyr Arg Thr Pro Met Ser Lys Tyr Val Ser 290 295 300

Val Thr Ala Glu Leu Met His Glu Asn Trp Lys Lys Leu Trp Val Leu 305 310 315 320

Ala Leu Trp Ala lie lie Asn Val Tyr Leu Phe Met Trp Lys Tyr Glu

325 330 335

Glu Phe Met Arg Asn Pro Leu Tyr Asn lie Thr Gly Arg Cys Val Cys

340 345 350

Ala Ala Lys Gly Ala Ala Glu Thr Leu Lys Leu Asn Met Ala Leu He

355 360 365

Leu Val Pro Val Cys Arg Lys Thr Leu Thr lie Leu Arg Ser Thr Phe 370 375 380

Leu Asn Arg Val Val Pro Phe Asp Asp Asn lie Asn Phe His Lys Val 385 390 395 400 lie Ala Tyr Met lie Ala Phe Gin Ala Leu Leu His Thr Ala Leu His

405 410 415 lie Phe Cys Asn Tyr Pro Arg Leu Ser Ser Cys Ser Tyr Asp Val Phe

420 425 430

Leu Thr Tyr Ala Gly Ala Ala Leu Gly Asn Thr Gin Pro Ser Tyr Leu

435 440 445

Gly Leu Met Leu Thr Ser Val Ser lie Thr Gly Val Leu Met He Phe 450 455 460

Phe Met Gly Phe Ser Phe Thr Leu Ala Met His Tyr Phe Arg Arg Asn 465 470 475 480 lie Val Lys Leu Pro Lys Pro Phe Asn Val Leu Ala Gly Phe Asn Ala

485 490 495

Phe Trp Tyr Ala His His Leu Leu Val Leu Ala Tyr He Leu Leu He

500 505 510 lie His Gly Tyr Tyr Leu lie lie Glu Lys Pro Trp Tyr Gin Lys Thr

515 520 525

Thr Trp Met Tyr Leu Ala Val Pro Met Leu Phe Tyr Ala Ser Glu Arg 530 535 540

Leu Phe Ser Arg Leu Leu Gin Glu His Ser His Arg Val Asn Val He 545 550 555 560

Lys Ala lie Val Tyr Ser Gly Asn Val Leu Ala Leu Tyr Val Thr Lys

565 570 575

Pro Pro Gly Phe Lys Tyr Lys Ser Gly Met Tyr Met Phe Val Lys Cys

580 585 590

Pro Asp Leu Ser Lys Phe Glu Trp His Pro Phe Ser He Thr Ser Ala

595 600 605

Pro Gly Asp Asp Tyr Leu Ser Val His lie Arg Ala Leu Gly Asp Trp 610 615 620

Thr Thr Glu Leu Arg Ser Arg Phe Ala Lys Thr Cys Glu Pro Thr Gin 625 630 635 640

Ala Ala Ala Lys Pro Lys Pro Asn Ser Leu Met Arg Met Glu Thr Arg

645 650 655

Ala Ala Gly Val Asn Pro His lie Glu Glu Ser Gin Val Leu Phe Pro

660 665 670

Lys lie Phe lie Lys Gly Pro Tyr Gly Ala Pro Ala Gin Asn Tyr Gin

675 680 685

Lys Phe Asp lie Leu Leu Leu Val Gly Leu Gly He Gly Ala Thr Pro 690 695 700

Phe He Ser He Leu Lys Asp Met Leu Asn His Leu Lys Pro Gly He 705 710 715 720

Pro Arg Ser Gly Gin Lys Tyr Glu Gly Ser Val Gly Gly Glu Ser He

725 730 735 Gly Gly Asp Ser Val Ser Gly Gly Gly Gly Lys Lys Phe Pro Gin Arg

740 745 750 Ala Tyr Phe Phe Trp Val Thr Arg Glu Gin Ala Ser Phe Asp Trp Phe 755 760 765

Lys Gly Val Met Asp Asp He Ala Glu Tyr Asp Lys Thr His Val He 770 775 780

Glu Met His Asn Tyr Leu Thr Ser Met Tyr Glu Ala Gly Asp Ala Arg 785 790 795 800

Ser Ala Leu He Ala Met Val Gin Lys Leu Gin His Ala Lys Asn Gly

805 810 815

Val Asp He Val Ser Glu Ser Arg He Arg Thr His Phe Ala Arg Pro

820 825 830

Asn Trp Arg Lys Val Phe Ser Glu Leu Ser Ser Lys His Glu Ala Cys

835 840 845

Arg He Gly Val Phe Tyr Cys Gly Ser Pro Thr Leu Val Arg Pro Leu 850 855 860

Lys Glu Leu Cys Gin Glu Phe Ser Leu Glu Ser Ser Thr Arg Phe Thr 865 870 875

Phe His Lys Glu Asn Phe

885

SEQ ID NO: 9: LOC_Os01g61880.1_ Oryza sativa

Met Ala Ser Pro Tyr Asp His Gin Ser Pro His Ala Gin His Pro Ser

1 5 10 15

Gly Leu Pro Arg Pro Pro Gly Ala Gly Ala Gly Ala Ala Ala Gly Gly

20 25 30

Phe Ala Arg Gly Leu Met Lys Gin Pro Ser Arg Leu Ala Ser Gly Val

35 40 45

Arg Gin Phe Ala Ser Arg Val Ser Met Lys Val Pro Glu Gly Val Gly

50 55 60

Gly Met Arg Pro Gly Gly Gly Arg Met Thr Arg Met Gin Ser Ser Ala 65 70 75 80

Gin Val Gly Leu Arg Gly Leu Arg Phe Leu Asp Lys Thr Ser Gly Gly

85 90 95

Lys Glu Gly Trp Lys Ser Val Glu Arg Arg Phe Asp Glu Met Asn Arg

100 105 110

Asn Gly Arg Leu Pro Lys Glu Ser Phe Gly Lys Cys He Gly Met Gly

115 120 125

Asp Ser Lys Glu Phe Ala Gly Glu Leu Phe Val Ala Leu Ala Arg Arg

130 135 140

Arg Asn Leu Glu Pro Glu Asp Gly He Thr Lys Glu Gin Leu Lys Glu 145 150 155 160

Phe Trp Glu Glu Met Thr Asp Gin Asn Phe Asp Ser Arg Leu Arg He

165 170 175

Phe Phe Asp Met Cys Asp Lys Asn Gly Asp Gly Met Leu Thr Glu Asp

180 185 190

Glu Val Lys Glu Val He He Leu Ser Ala Ser Ala Asn Lys Leu Ala

195 200 205

Lys Leu Lys Gly His Ala Ala Thr Tyr Ala Ser Leu He Met Glu Glu

210 215 220

Leu Asp Pro Asp Asp Arg Gly Tyr He Glu He Trp Gin Leu Glu Thr 225 230 235 240

Leu Leu Arg Gly Met Val Ser Ala Gin Ala Ala Pro Glu Lys Met Lys

245 250 255

Arg Thr Thr Ser Ser Leu Ala Arg Thr Met He Pro Ser Arg Tyr Arg

260 265 270

Ser Pro Leu Lys Arg His Val Ser Arg Thr Val Asp Phe Val His Glu

275 280 285

Asn Trp Lys Arg He Trp Leu Val Ala Leu Trp Leu Ala Val Asn Val

290 295 300

Gly Leu Phe Ala Tyr Lys Phe Glu Gin Tyr Glu Arg Arg Ala Ala Phe 305 310 315 320

Gin Val Met Gly His Cys Val Cys Val Ala Lys Gly Ala Ala Glu Val

325 330 335 Leu Lys Leu Asn Met Ala Leu He Leu Leu Pro Val Cys Arg Asn Thr

340 345 350

Leu Thr Thr Leu Arg Ser Thr Ala Leu Ser His Val He Pro Phe Asp

355 360 365

Asp Asn lie Asn Phe His Lys Val He Ala Ala Thr He Ala Ala Ala 370 375 380

Thr Ala Val His Thr Leu Ala His Val Thr Cys Asp Phe Pro Arg Leu 385 390 395 400 lie Asn Cys Pro Ser Asp Lys Phe Met Ala Thr Leu Gly Pro Asn Phe

405 410 415

Gly Tyr Arg Gin Pro Thr Tyr Ala Asp Leu Leu Glu Ser Ala Pro Gly

420 425 430

Val Thr Gly He Leu Met He He He Met Ser Phe Ser Phe Thr Leu

435 440 445

Ala Thr His Ser Phe Arg Arg Ser Val Val Lys Leu Pro Ser Pro Leu 450 455 460

His His Leu Ala Gly Phe Asn Ala Phe Trp Tyr Ala His His Leu Leu 465 470 475 480

Val Leu Ala Tyr Val Leu Leu Val Val His Ser Tyr Phe He Phe Leu

485 490 495

Thr Arg Glu Trp Tyr Lys Lys Thr Thr Trp Met Tyr Leu He Val Pro

500 505 510

Val Leu Phe Tyr Ala Cys Glu Arg Thr He Arg Lys Val Arg Glu Asn

515 520 525

Asn Tyr Arg Val Ser He Val Lys Ala Ala He Tyr Pro Gly Asn Val 530 535 540

Leu Ser Leu His Met Lys Lys Pro Pro Gly Phe Lys Tyr Lys Ser Gly 545 550 555 560

Met Tyr Leu Phe Val Lys Cys Pro Asp Val Ser Pro Phe Glu Trp His

565 570 575

Pro Phe Ser He Thr Ser Ala Pro Gly Asp Asp Tyr Leu Ser Val His

580 585 590 lie Arg Thr Leu Gly Asp Trp Thr Thr Glu Leu Arg Asn Leu Phe Gly

595 600 605

Lys Ala Cys Glu Ala Gin Val Thr Ser Lys Lys Ala Thr Leu Ser Arg 610 615 620

Leu Glu Thr Thr Val Val Ala Asp Ala Gin Thr Glu Asp Thr Arg Phe 625 630 635 640

Pro Lys Val Leu He Asp Gly Pro Tyr Gly Ala Pro Ala Gin Asn Tyr

645 650 655

Lys Lys Tyr Asp He Leu Leu Leu He Gly Leu Gly He Gly Ala Thr

660 665 670

Pro Phe He Ser He Leu Lys Asp Leu Leu Asn Asn He Lys Ser Asn

675 680 685

Glu Glu Val Glu Ser He His Gly Ser Glu He Gly Ser Phe Lys Asn

690 695 700

Asn Gly Pro Gly Arg Ala Tyr Phe Tyr Trp Val Thr Arg Glu Gin Gly 705 710 715 720

Ser Phe Glu Trp Phe Lys Gly Val Met Asn Asp Val Ala Glu Ser Asp

725 730 735

His Asn Asn He He Glu Met His Asn Tyr Leu Thr Ser Val Tyr Glu

740 745 750

Glu Gly Asp Ala Arg Ser Ala Leu He Ala Met Val Gin Ser Leu Gin

755 760 765

His Ala Lys Asn Gly Val Asp He Val Ser Gly Ser Arg He Arg Thr 770 775 780 His Phe Ala Arg Pro Asn Trp Arg Lys Val Phe Ser Asp Leu Ala Asn 785 790 795 800 Ala His Lys Asn Ser Arg lie Gly Val Phe Tyr Cys Gly Ser Pro Thr

805 810 815

Leu Thr Lys Gin Leu Lys Asp Leu Ser Lys Glu Phe Ser Gin Thr Thr

820 825 830

Thr Thr Arg Phe His Phe His Lys Glu Asn Phe

835 840

SEQ ID NO: 10: Glyma07gl 5690.1_ Glycine max

Arg Lys Met Met Arg Thr Glu Ser Gly Ala Ala Arg Gly lie Lys Gly

1 5 10 15

Leu Arg Phe Leu Asp Arg Thr Val Thr Gly Arg Glu Thr Asp Ala Trp

20 25 30

Lys Ser lie Glu Lys Arg Phe Thr Gin Asn Ala Val Asp Gly Lys Leu

35 40 45

Thr Lys Asp Lys Phe Gly Thr Cys Met Gly Met Gly Ala Glu Ser Lys

50 55 60

Asp Phe Ala Gly Glu Leu Tyr Glu Ala Leu Ala Arg Arg Arg Lys Val 65 70 75 80

Tyr Ala Glu Asn Gly lie Ser Leu Asp Glu Ala Lys Val Phe Trp Glu

85 90 95

Asp Met Thr Asn Lys Asp Phe Glu Ser Arg Leu Gin Val Phe Phe Asp

100 105 110

Met Cys Asp Lys Asn Gly Asp Gly Lys Leu Ser Glu Asp Glu Val Lys

115 120 125

Glu Val lie Val Leu Ser Ala Ser Ala Asn Lys Leu Gly Asn Leu Lys

130 135 140

Met His Ala Asp Gly Tyr Ala Ser Leu lie Met Glu Glu Leu Asp Pro 145 150 155 160

Asp His Asn Gly Tyr lie Glu lie Trp Gin Leu Glu Thr Leu Leu Lys

165 170 175

Glu Met Val Ser Ser Glu Glu Gly Thr Lys Lys Leu Asp Gin Cys Arg

180 185 190

Ala Met Thr Leu Ser Lys Ala Met lie Pro Ser Lys Tyr Arg Thr Pro

195 200 205

Val Ser Lys Phe Leu Ser Lys Thr Thr Glu Phe Ala Leu Asp Lys Trp

210 215 220

Lys Lys lie Trp Val Phe Ala Leu Trp Leu Ala lie Asn Leu Val Leu 225 230 235 240

Phe lie Trp Lys Phe Lys Gin Tyr Arg Glu Lys Lys Ala Phe Gin Val

245 250 255

Met Gly Tyr Cys Leu Cys Phe Ala Lys Gly Ala Ala Glu Thr Leu Lys

260 265 270

Phe Asn Met Ala Leu lie Val Leu Thr Met Cys Arg Arg Thr Leu Thr

275 280 285

Lys Leu Arg Gly Ser Phe Leu Ser Arg lie lie Pro Phe Asp Asp Asn

290 295 300

lie Asn Phe His Lys Thr lie Ala Val Ala Val Val lie Gly Thr Phe 305 310 315 320 lie His Val Met Met His lie Thr Cys Asp Phe Pro Arg Leu lie Ser

325 330 335

Cys Pro Glu Asn Lys Phe Phe Ser lie Phe Gly Asp Gly Phe Asn Tyr

340 345 350

Glu Gin Pro Thr Tyr Tyr Thr Leu Val Lys Ser lie Pro Gly Leu Thr

355 360 365

Gly lie Leu Met Val Leu lie Met Ala Phe Thr Phe Thr Leu Ala Thr

370 375 380

His Tyr Phe Arg Lys Ser Val Val Lys Leu Pro Ser Pro Leu His Arg 385 390 395 400

Leu Ala Gly Phe Asn Ala Phe Trp Tyr Ala His His Leu Leu He Val

405 410 415 Val Tyr He Leu Leu He He His Gly Tyr Phe Leu Phe Leu Thr Lys

420 425 430

Glu Trp Asn Lys Lys Thr Thr Trp Met Tyr Leu Val Val Pro Leu Ala

435 440 445

Leu Tyr Ala Phe Glu Arg He His Pro Phe Phe Arg Ser Lys Asp His 450 455 460

Arg Val Ser He He Lys Ala He He Tyr Thr Gly Asn Val Leu Ala 465 470 475 480

Leu Tyr Met Thr Lys Pro Gin Gly Phe Lys Tyr Glu Ser Gly Met Tyr

485 490 495

Leu Phe Val Lys Cys Pro Asp He Ser Thr Phe Glu Trp His Pro Phe

500 505 510

Ser He Thr Ser Ala Pro Gly Asp Asp Tyr Leu Ser Val His He Arg

515 520 525

Thr Leu Gly Asp Trp Thr Thr Glu Leu Lys Asn Thr Phe Ala Gin Val 530 535 540

Cys Glu Pro His Asn Ala Gin Pro Arg Lys Gly Asn Leu Met Arg Met 545 550 555 560

Glu Thr Arg Ala Pro Asn Ser Thr Tyr Asn His Pro Ser Lys Ser Arg

565 570 575

He Arg Tyr Pro Lys He Leu He Lys Gly Pro Tyr Gly Ala Pro Ala

580 585 590

Gin Ser Tyr Lys Asn Tyr Asp Val Leu Phe Leu He Gly Leu Gly He

595 600 605

Gly Ala Thr Pro Met He Ser He Leu Lys Asp Met Leu Asn Asn Met 610 615 620

Lys Ser Glu Ser Pro Lys Glu Val Ser Gly Arg Leu Tyr He Leu Phe 625 630 635 640

Leu Gin Gly Thr Tyr Met Gin Asp Ser Val Pro Ser Ser Asn Ser Asp

645 650 655

Asp Gin He Lys Lys Gly Pro Glu Arg Ala Tyr Phe Tyr Trp Val Thr

660 665 670

Arg Glu Gin Ser Ser Phe Glu Trp Phe Lys Gly Val Met Asp Asp He

675 680 685

Ala Asp Tyr Asp Cys Asp Asn He He Glu Met His Asn Tyr Leu Thr

690 695 700

Ser Val Tyr Glu Glu Gly Asp Ala Arg Ser Ala Leu He Ala Met He 705 710 715 720

Gin Arg Leu Gin His Ala Lys Asn Gly Val Asp Val Val Ser Glu Ser

725 730 735

Arg He Arg Thr His Phe Ala Arg Pro Asn Trp Lys Lys Val Phe Thr

740 745 750

Glu Leu Ala Asn Ala His Gin Ser Ser Arg He Gly Val Phe Tyr Cys

755 760 765

Gly Ser Pro Thr Leu Thr Lys Thr Leu Lys Glu Leu Cys His Glu Phe

770 775 780

Ser Leu Lys Ser Ser Thr Arg Phe Gin Phe His Lys Glu Asn Phe 785 790 795

SEQ ID NO: 1 1 : Glymal8g39500.1_ Glycine max

Met Ser Thr Glu Lys Gly Gly Lys Asp Glu Ser Ser Thr Trp He Leu

1 5 10 15

Glu Gly He Asp He Asp Pro He Val Glu Arg Pro Lys Arg Asp Asp

20 25 30

Glu Ala Pro Pro Asn Gly He Met Gly Asn Asn Gly Gly Arg Lys Met 35 40 45

Met Arg Ala Glu Ser Gly Ala Ala Arg Gly He Lys Ser Leu Arg Phe 50 55 60

Leu Asp Arg Thr Val Thr Gly Lys Glu Ala Asp Ala Trp Lys Ser He 65 70 75 80

Glu Lys Arg Phe Thr Gin Asn Ala Val Asp Gly Lys Leu Thr Lys Asp

85 90 95

Lys Phe Gly Thr Cys Met Gly Met Gly Ala Glu Ser Lys Asp Phe Ala

100 105 110

Gly Glu Leu Tyr Glu Ala Leu Ala Arg Arg Arg Asn Val Cys Ala Glu

115 120 125

Asn Gly He Thr Leu Asp Glu Val Lys Val Phe Trp Glu Asp Met Thr 130 135 140

Asn Arg Asp Leu Glu Ser Arg Leu Gin Val Phe Phe Asp Met Cys Asp 145 150 155 160

Lys Asn Gly Asp Gly Arg Leu Ser Glu Glu Glu Val Lys Glu Val He

165 170 175

Val Leu Ser Ala Ser Ala Asn Lys Leu Gly Asn Leu Lys Val His Ala

180 185 190

Asp Ala Tyr Ala Ser Leu He Met Glu Glu Leu Asp Pro Asp His Asn

195 200 205

Gly Tyr He Glu Val Arg Ser Glu Lys Phe Leu Leu Leu Ser Asn Phe 210 215 220

He Glu Phe Tyr He Asn Leu His Leu Leu Ala Met Thr Leu Ser Arg 225 230 235 240

Ala Met He Pro Ser Lys Tyr Arg Thr Pro Val Ser Lys Phe Leu Ser

245 250 255

Thr Thr Ala Glu Phe Ala Leu Asp Lys Trp Lys Lys He Trp Val Val

260 265 270

Ala Leu Trp Leu Ala He Asn Leu Val Leu Phe He Trp Lys Phe Lys

275 280 285

Gin Tyr Arg Glu Arg Glu Ala Phe Lys Val Met Gly Tyr Cys Leu Cys 290 295 300

Phe Ala Lys Gly Ala Ala Glu Thr Leu Lys Phe Asn Met Ala Leu He 305 310 315 320

Val Leu Thr Met Cys Arg Arg Thr Leu Thr Lys Leu Arg Gly Ser Phe

325 330 335

Leu Asn Arg He He Pro Phe Asp Asp Asn He Asn Phe His Lys Thr

340 345 350

He Ala Val Ala Val Val He Gly Thr Phe He His Val Met Met His

355 360 365

He Thr Cys Asp Phe Pro Arg Leu He Ser Cys Pro Glu Asn Lys Phe 370 375 380

Met Ser He Leu Gly Gin Asp Phe Asn Tyr Glu Gin Pro Thr Phe Tyr 385 390 395 400

Thr Leu Leu Lys Ser He Leu Gly Val Thr Gly He Leu Met Val Leu

405 410 415

Leu Met Ala Phe He Phe Thr Leu Ala Thr His Tyr Phe Arg Lys Ser

420 425 430

Val Val Lys Leu Pro Leu Ser Leu His Arg Leu Ala Gly Phe Asn Ala

435 440 445

Phe Trp Tyr Ala His His Leu Leu He Val Val Tyr He Leu Leu He 450 455 460

He His Gly Tyr Phe Leu Phe Leu Thr Lys Glu Trp Asp Lys Lys Thr 465 470 475 480

Thr Trp Met Tyr Leu Val Val Pro Leu Val Leu Tyr Ala Phe Glu Arg

485 490 495 He His Pro Phe Phe Arg Gly Lys Asp His Arg Val Ser He He Lys

500 505 510

Ala He He Tyr Gly Asn Val Leu Ala Leu Tyr Met Thr Lys Pro 515 520 525

Gin Gly Phe Lys Tyr Lys Ser Gly Met Tyr He Phe Val Lys Cys Pro 530 535 540

Asp lie Ser Ser Phe Glu Trp His Pro Phe Ser He Thr Ser Ala Pro 545 550 555 560

Gly Asp Asp Tyr Leu Ser Val His He Arg Thr Leu Gly Asp Trp Thr

565 570 575

Thr Glu Leu Lys Asn Lys Phe Thr Gin Val Cys Glu Pro His Ser Ala

580 585 590

Gin Pro Arg Lys Gly Asn Leu Met Arg Met Glu Thr Arg Ala Pro Ser

595 600 605

Ser Asn Tyr Asn His Ser Ser Asn Ser Ser He Arg Tyr Pro Lys He 610 615 620

Leu lie Lys Gly Pro Tyr Gly Ala Pro Ala Gin Ser Tyr Lys Asn Tyr 625 630 635 640

Asp Val Leu Met Leu He Gly Leu Gly He Gly Ala Thr Pro Met He

645 650 655

Ser He Leu Lys Asp Met Leu Asn Asn Met Lys Ser Glu Ser Pro Lys

660 665 670

Glu Val Ser His Arg Leu Tyr He Leu Phe Trp Leu Ala Ala Tyr Val

675 680 685

Tyr Leu Ser Leu Leu Val Glu He He Phe Ser Lys Thr Phe Lys Gly 690 695 700

Thr Tyr Met Gin Asp Ser Asp His Ser Tyr His Leu Asp Asp Gin He 705 710 715 720

Lys Lys Gly Pro Glu Arg Ala Tyr Phe Tyr Trp Val Thr Arg Glu Gin

725 730 735

Ser Ser Phe Glu Trp Phe Lys Gly Val Met Asp Asp He Ala Asp Tyr

740 745 750

Asp His Asp Asn He He Glu Met His Asn Tyr Leu Thr Ser Val Tyr

755 760 765

Glu Glu Gly Asp Ala Arg Ser Ala Leu He Ala Met He Gin Lys Leu 770 775 780

Gin His Ala Lys Asn Gly Val Asp Val Val Ser Glu Ser Arg He Arg 785 790 795 800

Thr His Phe Ala Arg Pro Asn Trp Lys Lys Val Phe Thr Gin Leu Ala

805 810 815

Asn Ala His Gin Ser Ser Arg He Gly Val Phe Tyr Cys Gly Ser Pro

820 825 830

Thr Leu Thr Lys Thr Leu Lys Glu Leu Cys Leu Glu Phe Ser Leu Asn

835 840 845

Ser Ser Thr Arg Phe Gin Phe His Lys Glu Asn Phe

850 855 860

SEQ ID NO: 12: LOC_Os05g38980.1_ Oryza sativa

Met Ala Gly Asp Tyr Val Asp Val Pro Leu Gly Gly Gly Gly Gin Ser

1 5 10 15

Thr Leu Pro Pro Val Ala Pro Leu Lys Lys Gin Pro Ser Arg Leu Ala

20 25 30

Ser Gly Met Lys Arg Leu Ala Ser Met Val Pro Asp Thr Met Lys Leu

35 40 45

Lys Arg Thr His Ser Ser Ala Gin Pro Ala Leu Arg Gly Leu Arg Phe

50 55 60

Leu Asp Lys Thr Ser Ala Gly Lys Asp Gly Trp Lys Asn Val Glu Lys 65 70 75 80

Arg Phe Asp Glu Met Ser Ala Asp Gly Arg Leu Pro Gin Glu Ser Phe

85 90 95

Ala Lys Cys He Gly Met Ala Asp Ser Lys Glu Phe Ala Ser Glu Val 100 105 110

Phe Val Ala Leu Ala Arg Arg Arg Ser He Lys Pro Glu Asp Gly He

115 120 125

Thr Lys Glu Gin Leu Lys Glu Phe Trp Glu Glu Leu Thr Asp Gin Asn 130 135 140

Phe Asp Ser Arg Leu Arg He Phe Phe Asp Met Cys Asp Lys Asn Gly 145 150 155 160

Asp Gly Gin Leu Thr Glu Asp Glu Val Lys Glu Val He Val Leu Ser

165 170 175

Ala Ala Ala Asn Lys Leu Ala Lys Leu Lys Ser His Ala Ala Thr Tyr

180 185 190

Ala Ser Leu He Met Glu Glu Leu Asp Pro Asp His Arg Gly Tyr He

195 200 205

Glu He Trp Gin Leu Glu Thr Leu Leu Arg Gly Met Val Thr Ala Gin 210 215 220

Gly Pro Pro Glu Lys Val Lys Leu Ala Ser Ala Ser Leu Ala Arg Thr 225 230 235 240

Met Val Pro Ser Ser His Arg Ser Pro Met Gin Arg Arg Phe Asn Lys

245 250 255

Thr Val Asp Phe He His Glu Asn Trp Lys Arg He Trp Val Leu Ser

260 265 270

Leu Trp Ala He Leu Asn He Ala Leu Phe Met Tyr Lys Phe Val Gin

275 280 285

Tyr Ser Arg Arg Asp Ala Phe Gin Val Met Gly Tyr Cys Val Cys He 290 295 300

Ala Lys Gly Ala Ala Glu Thr Leu Lys Leu Asn Met Ala Val He Leu 305 310 315 320

Leu Pro Val Cys Arg Asn Thr Leu Thr Arg Leu Arg Ser Thr Ala Leu

325 330 335

Ser Lys Val Val Pro Phe Asp Asp Asn He Asn Phe His Lys Val He

340 345 350

Ala Leu Thr He Ala He Gly Ala Ala Thr His Thr Leu Ala His Val

355 360 365

Thr Cys Asp Phe Pro Arg Leu Val Ser Cys Pro Arg Asp Lys Phe Glu 370 375 380

Ala Thr Leu Gly Pro Tyr Phe Asn Tyr Val Gin Pro Thr Tyr Ser Ser 385 390 395 400

Leu Val Ala Ser Thr Pro Gly Trp Thr Gly He Leu Met He Leu He

405 410 415

Met Ser Phe Ser Phe Thr Leu Ala Thr His Ser Phe Arg Arg Ser Val

420 425 430

Val Lys Leu Pro Ser Pro Leu His His Leu Ala Gly Phe Asn Ala Phe

435 440 445

Trp Tyr Ala His His Leu Leu Val He Ala Tyr He Leu Leu Val Leu 450 455 460

His Ser Tyr Phe He Phe Leu Thr Lys Gin Trp Tyr Asn Arg Thr Thr 465 470 475 480

Trp Met Phe Leu Ala Val Pro Val Leu Phe Tyr Ser Cys Glu Arg Thr

485 490 495

He Arg Arg Val Arg Glu Ser Ser Tyr Gly Val Thr Val He Lys Ala

500 505 510

Ala He Tyr Pro Gly Asn Val Leu Ser He His Met Asn Lys Pro Ser

515 520 525

Ser Phe Lys Tyr Lys Ser Gly Met Tyr Met Phe Val Lys Cys Pro Asp 530 535 540

Val Ser Pro Phe Glu Trp His Pro Phe Ser He Thr Ser Ala Pro Gly 545 550 555 560

Asp Asp Tyr Leu Ser Val His He Arg Thr Leu Gly Asp Trp Thr Thr

565 570 575 Glu Leu Arg Asn Leu Phe Gly Lys Ala Cys Ala Gin Val Ser Ser 580 585 590

Lys Lys Ala Thr Leu Ala Arg Leu Glu Thr Thr He He Ala Asp Gly

595 600 605

Leu Lys Glu Glu Thr Cys Phe Pro Lys Val Phe He Asp Gly Pro Phe

610 615 620

Gly Ala Pro Ala Gin Asn Tyr Lys Lys Tyr Asp He Leu Leu Leu He

625 630 635 640

Gly Leu Gly He Gly Ala Thr Pro Phe He Ser He Leu Lys Asp Leu

645 650 655

Leu Asn Asn He Lys Ser Asn Gly Asp Val Gin Ser Thr His Asp Ala

660 665 670

Glu Leu Gly Cys Thr Phe Lys Ser Asn Gly Pro Gly Arg Ala Tyr Phe

675 680 685

Tyr Trp Val Thr Arg Glu Gin Gly Ser Phe Glu Trp Phe Lys Gly Val

690 695 700

Met Asn Asp Val Ala Glu Ser Asp His Asp Asn Val He Glu Met His

705 710 715 720

Asn Tyr Leu Thr Ser Val Tyr Glu Glu Gly Asp Ala Arg Ser Ala Leu

725 730 735

He Ala Met Val Gin Ser Leu Gin His Ala Lys Asn Gly Val Asp He

740 745 750

Val Ser Gly Ser Lys He Arg Thr His Phe Ala Arg Pro Asn Trp Arg

755 760 765

Lys Val Phe Ser Asp Leu Ala Asn Ala His Gin Asn Ser Arg He Gly

770 775 780

Val Phe Tyr Cys Gly Ser Pro Thr Leu Thr Lys Met Leu Arg Asp Leu

785 790 795 800

Ser Leu Glu Phe Ser Gin Thr Thr Thr Thr Arg Phe His Phe His Lys

805 810 815

Glu Asn Phe

SEQ ID NO: 13: Glymal8g39500.1_variant; soybean; 19-aa at position 683 removed to get better alignment.

MSTEKGGKDESSTWILEGIDIDPIVERPKRDDEAPPNGIMGNNGGRKMMRAESGAARGIKSLRFLDRTVTGKEAD AWKSIEKRFTQNAVDGKLTKDKFGTCMGMGAESKDFAGELYEALARRRNVCAENGITLDEVKVFWEDMTNRDLES RLQVFFDMCDKNGDGRLSEEEVKEVIVLSASANKLGNLKVHADAYASLIMEELDPDHNGYIEVRSEKFLLLSNFI EFYINLHLLAMTLSRAMI PSKYRTPVSKFLSTTAEFALDKWKKIWVVALWLAINLVLFIWKFKQYREREAFKVMG YCLCFAKGAAETLKFNMALIVLTMCRRTLTKLRGSFLNRI I PFDDNINFHKTIAVAWIGTFIHVMMHITCDFPR LISCPENKFMSILGQDFNYEQPTFYTLLKSILGVTGILMVLLMAFIFTLATHYFRKSVVKLPLSLHRLAGFNAFW YAHHLLIVVYILLI IHGYFLFLTKEWDKKTTWMYLWPLVLYAFERIHPFFRGKDHRVSI IKAI IYTGNVLALYM TKPQGFKYKSGMYIFVKCPDISSFEWHPFSITSAPGDDYLSVHIRTLGDWTTELKNKFTQVCEPHSAQPRKGNLM RMETRAPSSNYNHSSNSS IRYPKILIKGPYGAPAQSYKNYDVLMLIGLGIGATPMISILKDMLNNMKSESPKEVS HRLYILFFKGTYMQDSDHSYHLDDQIKKGPERAYFYWVTREQSSFEWFKGVMDDIADYDHDNI IEMHNYLTSVYE EGDARSALIAMIQKLQHAKNGVDWSESRIRTHFARPNWKKVFTQLANAHQSSRIGVFYCGSPTLTKTLKELCLE FSLNSSTRFQFHKENF

Claims

What is claimed is:

1. A method for increasing drought tolerance in a plant, said method comprising introducing into said plant a recombinant DNA construct, said construct comprising a root- preferred promoter operably linked to a polynucleotide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

2. A method for enhancing nutrient uptake in a plant, said method comprising introducing into said plant a recombinant DNA construct, said construct comprising a root- preferred promoter operably linked to a polynucleotide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

3. The method of claim 1 or 2, wherein said polynucleotide is stably integrated into the genome of the plant.

4. The method of claim 1, 2, or 3, wherein said plant is a plant cell.

5. The method of claim 1, 2, or 3, wherein said plant is a dicot.

6. The method of claim 5, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

7. The method of claim 1, 2 or 3, wherein said plant is a monocot.

8. The method of claim 7, wherein said monocot is maize, sugarcane, wheat, rice, barley, sorghum, or rye.

9. A seed of the plant produced by the method of any one of claims 1-8.

10. A plant comprising a recombinant DNA construct comprising a root-preferred promoter operably linked to a polynucleotide, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2;

11. The plant of claim 10, wherein said plant is a cell.

12. The plant of claim 10, wherein said plant is a monocot.

13. The plant of claim 12, wherein said monocot is maize, sugarcane, wheat, rice, barley, sorghum, or rye.

14. The plant of claim 10, wherein said plant is a dicot.

15. The plant of claim 14, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

16. The plant of any one of claims 10 to 15, wherein said polynucleotide is stably incorporated into the genome of the plant.

17. A seed of the plant of claim 16.

18. An expression cassette comprising a polynucleotide operably linked to a heterologous promoter that drives expression of the polynucleotide in a root of a plant, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence comprising SEQ ID NO: 2; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or 13;

19. The expression cassette of claim 18, wherein said plant is a monocot.

20. The expression cassette of claim 19, wherein said monocot is maize, sugarcane, wheat, rice, barley, sorghum, or rye.

21. The expression cassette of claim 18, wherein said plant is a dicot.

22. The expression cassette of claim 21, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

23. A method of selecting an allelic variant of rth5 in a maize plant, the method comprising the steps of:

d. selecting an allelic variant that is associated with said alteration of at least one agronomic characteristic.

24. A method of selecting a first maize plant or a first maize germplasm that has one or more beneficial alleles of rth5, the method comprising:

(a) screening a plurality of maize plants or a plurality of maize germplasm for

at least one polymorphism within a marker locus, wherein the marker locus is:

(a) a first polynucleotide having at least 90% and less than 100% nucleotide sequence identity with SEQ ID NO: 1 or 2; or (b) a second polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% and less than 100% sequence identity to SEQ ID NO:3, wherein expression of the first or second polynucleotide in a maize plant results in a phenotype comprising an alteration of at least one agronomic characteristic when compared to a control maize plant; wherein the at least one agronomic characteristic is selected from the group consisting of increased root hair formation and growth, increased drought tolerance, and enhanced nutrient uptake; and wherein the control maize plant comprises:

(c) a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 2; or

(d) a polynucleotide encoding the amino acid sequence of SEQ ID NO:3;

(c) selecting the first maize plant or first maize germplasm of step (b).