WO2008110848A2

WO2008110848A2 - Plant responses

Info

Publication number: WO2008110848A2
Application number: PCT/GB2008/050186
Authority: WO
Inventors: Phil Mullineaux; Ulrike Bechthold
Original assignee: Plant Bioscience Limited
Priority date: 2007-03-15
Filing date: 2008-03-17
Publication date: 2008-09-18
Also published as: GB0704984D0; US8445747B2; US20140007294A1; WO2008110848A3; BRPI0808735A2; US20100138958A1

Abstract

The present invention relates to methods and uses for improving traits in plants which are important in the field of agriculture. In particular, the metho ds and uses of the invention use a plant Hsf to increase plant productivity, water use efficiency, dro ught or pathogen resistance.

Description

Plant responses

Field of the invention

The present invention relates to methods and uses for improving traits in plants which are important in the field of agriculture. In particular, the methods and uses of the invention can be employed to increase plant productivity, for example by improving the way in which plants make use of the water resources available to them or by conferring pathogen resistance.

Background of the invention

External conditions that adversely affect growth, development or productivity trigger a wide range of plant responses, such as altered gene expression, cellular metabolism and changes in growth rates and crop yields. There are two types of stress: biotic stress is imposed by other organisms, such as a pathogen, whereas abiotic stress arises from an excess or deficit in the physical or chemical environment, such as drought, salinity, high or low temperature or high light. Biotic and abiotic stresses can reduce average plant productivity by 65% to 87%, depending on the crop.

An example of biotic stress is pathogen infection. Plants have evolved defensive mechanisms, such as the induction of the expression of specific resistance genes upon infection. It is known that resistance is heritable and plant breeders have been breeding varieties of crop plants with disease resistance ever since. However, pests and pathogens have also developed ways to compromise plant resistance. Pathogens are adaptive by their ability to evolve strains that defeat the resistance genes deployed in crop plants by plant breeders. This has led to the need of continually updating and replacing varieties with different genes or combinations of genes for resistance in response to the ever-changing pathogen populations. Therefore, new ways of improving pathogen resistance are needed (Crute et al 1998, Cook et al 1996).

In addition to pathogen infection, plants are exposed to varying environmental conditions. One important factor in the development of plants and thus in agriculture is the availability of water. Water is essential for crop production because plants require water for growth and tissue expansion. Thus, the supply of fresh water is essential for all forms of agriculture, although the amount of water required varies greatly between different agricultural types and climatic regions.

There has been limited success with conventional breeding to improve the way in which plants use the water resources available. Genetic engineering is therefore considered an alternative. Several genes that regulate drought response have been identified in the model plant Arabidopsis, These are categorised as responsive to dehydration and early response to dehydration genes (Valliyodan et al 2006). One of the factors identified in regulating cold and drought stress responsive gene expression in Arabidopsis is a family of transcription factors termed DREB, which interact with a dehydration responsive element. Overexpression of DREB results in significant drought tolerance under water limited conditions. However, resistance to drought often compromises development of these transgenic plants under normal conditions. It has been shown that overexpression of DREB1/CBF and DREB2A driven by the CaMV 35S promoter causes growth retardation under normal conditions (Vailiyodan et a! 2006, Sakuma et al 2006, Qiang et al 2000). Thus, there has so far been no success in genetically modifying plants so that they show improved and more efficient use of water under normal non-drought conditions as well as under water deficit conditions.

Although increasing drought tolerance is desirable in the face of global warming, from an agricultural point of view, drought resistance is usually linked to low productivity, and is thus of limited use in agricultural production. Also, severe water deficits are generally rare in viable agriculture. Therefore, reducing the amount of water used per unit yield is now seen as the most promising way forward.

This is increasingly important due to the rising amounts of water which are used in agriculture and the changing climate. Globally, some 2.7 x 10³ km³ of water were used in agriculture in 2000. It is estimated that the production of 1 kg of wheat requires 1 m³ of water, and 1 kg of rice requires at least 1.2 m³ of water. In the 15 countries of the EU in 2003, an area equivalent to 15.5% of the arable and permanent crop area was irrigated, and irrigation comprised over half of the total water consumption (EEA 2003). Even within the humid, temperate climate of England, 147 kha of outdoor crops were irrigated in 2001 (about 3% of the cropped area), using 131 x 10⁶ m³ of water (Morison et al 2008; Rijsberman. 2004 ; Richards 2004 ; Food and Agriculture Organisation (FAO) 2003; Parry et al 2005.)

Thus, how to reduce agricultural water use and make water resources more sustainable is an increasingly urgent question. There is a need to develop crops that require less water to produce sufficient yield under normal conditions in addition to showing improved drought resistance. The amount of yield produced per unit water used is referred to as 'water productivity', a well known term in agriculture (Morison et al., 2008).

All eukaryotic organisms respond to an increase in the ambient temperature with the expression of a group of proteins known as heat shock proteins (HSPs). Key factors in the regulation of the expression of Hsp genes are the heat shock transcription factors (Hsfs) that act by binding to a highly conserved palindromic heat shock response sequence in the promoters of the target genes. In addition to mediating the response to heat stress, Hsfs are thought to be involved in cellular responses to oxidative stress, heavy metals and other stress responses (Panchuk et al 2002, Panikulangara et al 2004). It is known that the basic structure of Hsfs and of their promoter recognition site is conserved throughout the eukaryotic kingdom (Kotak et ai 2004, Miller and Mittler 2006). Hsfs have a modular structure with a highly conserved N- terminal DNA binding and a C- terminal activation domain. Other conserved domains include an oligomerisation domain, a nuclear localisation sequence and a nuclear export sequence. Thus, Hsfs are easily recognised by their conserved motifs essential for their function as transcription factors (Kotak et al 2004, Miller and Mittler 2006, Nover et al 2001).

Yeast and Drosophila contain only one Hsf gene, while vertebrates are thought to have three Hsf genes. In plants, Hsf genes have been identified in many species, for example maize, the model plant Arabidopsis thaliana (21 Hsfs), soybean (34 Hsfs), rice (23 Hsfs), barley, potato, tomato (18Hsfs) and others. Hsfs within the plant kingdom are highly conserved and divided into three classes (A, B and C). For example, it has been found that a class of Hsfs in Arabidopsis is closely related to Hsf from rice and to Hsfs identified from ESTs in barley, potato, tomato and soy bean (Nover et al 2001 and Kotak et al 2004).

The invention is aimed at solving or at least mitigating the problems discussed above by introducing and expressing a gene sequence encoding a plant heat shock transcription factor.

Summary of the invention

The invention relates to methods and uses for improving a plant's tolerance to abiotic or biotic stress, not including heat stress. The method comprises introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant. In particular, the invention provides methods and uses for improving traits in plants which are important in the field of agriculture selected from the group comprising improved productivity, preferably growth or yield, water use efficiency, water productivity, drought tolerance or pathogen resistance. In one aspect, the invention provides a method for improving plant productivity comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.

The invention also provides a method for improving water use efficiency in plants comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.

Furthermore, there is provided a method for conferring pathogen resistance in plants comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.

The invention also relates to uses of a plant Hsf in improving plant productivity, plant water use efficiency, water productivity, drought tolerance or pathogen resistance. In one embodiment, plant water use efficiency and water productivity are improved under normal, non drought conditions.

Detailed description

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. In a first aspect, the invention relates to a method for improving plant productivity comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf in said plant.

Plant productivity can be assessed by measuring plant growth or plant yield. Preferably, the term is used to describe an improvement in yield. This can be assessed by measuring seed yield, such as increased seed biomass or increased number of seeds. It can be improved by increasing water productivity.

According to the different aspects and embodiments of the invention, the plant into which a plant Hsf of plant origin is introduced may be any monocot or dicot plant.

A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species. In one embodiment, the plant is oilseed rape.

Also included are biofuel and bioenergy crops such as rape/canola, linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, unseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).

A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat sorghum, rye, onion, leek, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species.

Preferably, the plant into which a plant Hsf is introduced is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.

Preferred plants are maize, wheat, rice, oilseed rape, sorghum, soybean, potato, tomato, barley, pea, bean, field bean, lettuce, broccoli or other vegetable brassicas or poplar.

The polynucleotide according to the different aspects and embodiments of the invention comprises or consists of a plant heat shock transcription factor gene, i.e. a plant Hsf. The term plant heat shock transcription factor gene or plant Hsf refers to a nucleic acid sequence which encodes a plant heat shock transcription factor. The Hsf gene can be from genomic DNA and therefore contain introns, a cDNA copy synthesised from the Hsf3 mRNA or could be a completely synthetic copy of the coding sequence made by assembly of chemically synthesised oligonucleotides. The plant heat shock transcription factor gene sequence can be isolated from a plant and inserted into a vector/expression cassette for transformation, for example by using an artificial plant chromosome.

Within the scope of the invention is also a derivative of a Hsf gene, such as a mutant/mutated gene, chimeric gene or gene shuffled variant. For example, the mutant gene may be modified so that the resulting protein is constitutively active and cannot be inhibited by other components of the Hsf signalling pathway. The derivative gene expressed a protein which is biologically active. It may have 80% or more sequence homology with the wild type gene. Thus, the methods and uses of the invention also relates to methods and uses employing a Hsf derivative.

The Hsf polynucleotide is a transgene that is introduced in the plant. This can be carried out by various methods as known in the field of plant genetic engineering, for example using transformation with Agrobacterium or particle bombardment.

The plant heat shock transcription factor gene may be an exogenous gene, such as one or more Arabidopsis Hsf, overexpressed in a different plant species. Alternatively, the plant Hsf may be an endogenous plant Hsf, i.e. a plant Hsf that is endogenous to the plant in which it is introduced and overexpressed.

In one embodiment of the different aspects of the invention, the exogenous plant Hsf may originate from any plant, for example a family or species listed above and expressed in a different plant species according to the invention. There is a structural high similarity between Hsfs in the plant kingdom. Plant Hsfs are conserved throughout the plant kingdom and can be identified due to their conserved domains. Plant Hsfs are divided into three groups A, B and C. Thus, according to the invention, the plant Hsf may be selected from group A, B or C. For example, the plant Hsf may be an Arabidopsis Hsf, a tomato Hsf, such as LpHsfAI , LpHsfA2, LpHsfA3 or LpHsfBL Alternatively, the plant Hsf may be derived from rice, wheat, pea, maize, tobacco or any crop cereal. Non limiting examples of known Hsfs which can be used according to the invention are given in tables 1 and 2.

OsHsfAia AK100430 Os03g0854500

OsHsfA2c AK072391 Os10g0419300 OsHsfA2b AK101824 Os07g0178600

OsHsfA2a AK069579 ABF98829

OsHsfA1a2d AK066844 Os03g0161900

OsHsfA2e AK068660 Os03g0795900

OsHsfA3 AK101934 XM_466050

OsHsfA4b AK109856 Os01g0749300

OsHsfA4d AK100412 Os05g0530400

OsHsfAδ AK065643 Os02g0496100

OsHsfA7 AK064271 Os01g0571300

OsHsfAΘ AK064271 Os03g0224700

OsHsfBI AK101182 Os09g0456800

OsHsfB2a Os04g0568700

OsHsfB2b AK101700 Os08g0546800

OsHsfB2c AK106525 Os09g0526600

OsHsfB4a P0461 F06.21

OsHsfB4b AK063952 Os07g0640900

OsHsfB4c Os09g0455200

OsHsfB4d AK069479 ABF96133

OsHsfCia AK066316 Os01g0625300

OsHsfCib AK106488 Os01g0733200

OsHsfC2a Os02g0232000

OsHsfC2b Os06g0553100

Arabidopsis thaliana

AtHsfAla AT4G 17750

AtHsfAlb AT5G16820

AtHsfAid AT1 G32330

AtHsfAiθ AT3G02990

AtHsfA2 AT2G26150

AtHsfA3 AT5G03720

AtHsfA4a AT4G18880

AtHsfA4c AT5G45710 AtHsfAS AT4G 13980

AtHsfAβa AT5G43840

AtHsfA6b AT3G22830

AtHsfA7a AT3G51910

AtHsfA7b AT3G63350

AtHsfAδ AT1G67970

AtHsfA9 AT5G54070

AtHsfBI AT4G36990

AtHsfB2a AT5G62020

AtHsfB2b AT4G11660

AtHsfB3 AT2G41690

AtHsfB4 AT1G46264

AtHsfCI AT3G24520

Tomato

Lycopersicon esculentum

LpHsfAI X67600 LpHsfA2 X67601 LpHsfA3 AF208544 LpHsfBI X55347

Homology searches of plant sequence databases such as the expressed sequence tag (EST) cDNA databases

(http://www.ncbi.nlm.nih.gov/genomes/PLANTS/PlantESTBLAST.shtml73847) using the HSF3 derived amino acid sequence readily detects many highly significant homologies corresponding to HSFs in the query species. In the examples below the amino acid sequence of HSF3 was used to query the following databases with a significance value (E) of less than 1e^"10 . The values and hits, including the identification number are all shown in table 2 below. Soybean gb|AW569256 11 sι64gO9 y1 Gm-M 030 Glycine max cDNA clone 290 4e-78 gb|AW569138 11 sι63gO9 y1 Gm-r1030 Glycine max cDNA clone 287 2e-77 gb|BM086093.11 sah35dO7 y1 Gm-c1036 Glycine max cDNA clone 238,1e-62 gb|CA938396 1 ] sav31h12 y1 Gm-c1048 Glycine max cDNA clone 213 5e-55 gb|BM521654 1 | sak60e12 y1 Gm-c1036 Glycine max cDNA clone 211 2e-54 gb|BM527729 1 | sal65bO5 y1 Gm-c1061 Glycine max cDNA clone 209 5e-54 gb|AW395668 1 | sg73g10 y1 Gm-c1007 Glycine max cDNA clone 201 2e-51 gb|CA801396 1 | sauO5bO3 y2 Gm-c1062 Glycine max cDNA clone 195 1e-49 gb|AW508573 1 j sι33fO1 y1 Gm-M 030 Glycine max cDNA clone 190 4e-48 gb|BI786160 1 | saι33fO4 y1 Gm-c1065 Glycine max cDNA clone 189 6e-48 gb|AW164509 1 | se74f12 y1 Gm-c1023 Glycine max cDNA clone 166 7e-41 gb|BI471764 1 | sae83dO2 y3 Gm-c1065 Glycine max cDNA clone 162 1e-39 gb|BE611683 1 | sq86gO5 y1 Gm-c1049 Glycine max cDNA clone 160 4e-39 gb|AW132703 1 | seO9aO8 y1 Gm-c1013 Glycine max cDNA clone 159 6e-39 gblBE347442 11 sp38dO2 y 1 Gm-c1043 Glycine max cDNA done 151 2e-36 gb|AW203851 1 | sf38h11 y1 Gm-c1028 Glycine max cDNA clone 149 9e-36 gb|BG839442 1 |Gm01_17a10_A_lGm01_AAFC_ECORC_G!ycιne_max_cold_„ 147 2e-35 gbSBG352891 1 | sab92fO8 y1 Gm-c1040 Glycine max cDNA clone 146 6e-35 gb[BG789771 1 [ sae55cO3 y1 Gm-c1051 Glycine max cDNA clone 142 1e-33 gb[AW596493 11 sj13aO9 y1 Gm-c1032 Glycine max cDNA clone 141 2e-33 gb[BM523618 11 sam86dO1 y2 Gm-c1087 Glycine max cDNA clone 139 7e-33 gb|BM188104 11 saj84gO5 y1 Gm-c1074 Glycine max cDNA clone 139 7e-33 gb|BM094717 11 saj19hO6 y1 Gm-c1066 Glycine max cDNA clone 139 7e-33 gb[AI900223 1 | scO2fO5 y1 Gm-c1012 Glycine max cDNA clone 139 7e-33 gb[BM732569 11 sal78hO7 y1 Gm-c1061 Glycine max cDNA clone 136 6e-32 gb|BI498205 1 | sag17cO1 y1 Gm-c1080 Glycine max cDNA clone 136 6e-32 gb|BU764266 11 sas54gO3 y1 Gm-c1023 Glycine max cDNA clone 134 3e-31 gb|BQ094759 1 | san51d12 y1 Gm-c1052 Glycine max cDNA clone 134 3e-31 gb|BI894096 1 | saι60a12 y1 Gm-c1068 Glycine max cDNA clone 134 3e-31 gb|AW703969 1 | sk14gO8 y1 Gm-c1023 Glycine max cDNA clone 134 4e-31 gb|BM527450 1 ] sal62aO4 y1 Gm-c1061 Glycine max cDNA clone 133 6e-31 gb|BG405291 1 | sac50e11 y1 Gm-c1062 Glycine max cDNA clone 133 6e-31 gb|BG840046 1 | Gm01_08b12_F,Gm01_AAFC_ECORC_Glycιne_max_cokL . 132 1e-30 gb|CA936104 1 | savO5g11 y1 Gm-c1048 Glycine max cDNA clone 130 3e-30 gb|BE346810 11 sp31eO1 y1 Gm-c1042 Glycine max cDNA clone 130 3e-30 gb|BQ094171 1 | san43bO7 y1 Gm-c1052 Glycine max cDNA clone 130 4e-30 gb|BE020791 1 | sm52hO9 y1 Gm-c1028 Glycine max cDNA clone 127 3e-29 gb|CA850642 11 D04F08 seq cDNA Peking library 2, 4 day SCN3 126 6e-29 ~ gb|BM886719 1 | sam29cO6 y1 Gm-c1068 Glycine max cDNA clone 126 6e-29 gb|BU577235 11 sar67dO3 y1 Gm-c1074 Glycine max cDNA clone 126 8e-29 gb|BF071322 1 [ st45aO8 y1 Gm-c1067 Glycine max cDNA clone 125 1e-28 gb|CX711571 1 | gmrtDrNS01_35-D_M13R_F05_037 s2 Water stressed 116 6e-26 gbjBQ474006 1 | sap25bO6 y1 Gm-c1082 Glycine max cDNA clone 115 1 e-25 gb|CO984075 1 [ GM89021A1G02 r1 Gm-r1089 Glycine max cDNA 109 7e-24 gb[BE019974.11 sm38b12 y1 Gm-c1028 Glycine max cDNA clone 108 1e-23 gb|CX711887 1 | gmrtDrNS01_39-D_M13R_C04_028 s3 84 7 5e-22 gb|BF067962.11 st79cO6 y1 Gm-c1054 Glycine max cDNA clone 103 5e-22 gb|BU548776 11 GM880016B20F09 Gm-r1088 Glycine max cDNA 103 7e-22 gb|CA953210 1 | sav53hO2 y1 Gm-c1069 Glycine max cDNA clone 102 9e-22 gb|CA801977 11 sau28a12 y1 Gm-c1062 Glycine max cDNA clone 100 3e-21 gb|AW756148 11 sl16eO7.y1 Gm-c1036 Glycine max cDNA clone 99 4 1e-20 gb|CD403874 1 | Gm_ck26662 Soybean induced by Salicylic Acid G 90 9 4e-18 gb|BE330669 11 so82h05 y1 Gm-c1040 Glycine max cDNA clone 89.4 1e-17 gb|BI469342 1 | sail 0f07 y1 Gm-d 053 Glycine max cDNA clone 89 0 1e-17 gb|BM271159 1 [ sakO5hO6 y 1 Gm-d 074 Glycine max cDNA clone 87 0 5e-17 gb|DY577402 1 | sgs2c pkOO1 j19 DupontLib Glycine max cDNA 5", m 86 7 7e-17 gb|CX705290 1 | gmrtDrNS01_40-B_M13R_H02_002 s2 85 9 1e-16 gb|AW508846 1 | sι41a12 y1 Gm-r1030 Glycine max cDNA clone GEN . 85 1 2e-16 gb|BU578607 1 | sar59bO5 y1 Gm-c1074 Glycine max cDNA clone 82 8 1 e-15 gb|BQ473641 1 | sap15g12 y1 Gm-c1082 Glycine max cDNA clone 82 0 2e-15 gb|BE348040 1 | sp10e12 y1 Gm-c1042 Glycine max cDNA clone 79 7 8e-15 gb|BF425514 1 j su56fO5 y1 Gm-c1069 Glycine max cDNA clone 79 3 1e-14 gb|BQ628408 1 | sap46fO3 y1 Gm-c1087 Glycine max cDNA clone 79 0 1e-14 gbjBM269600 1 ] sakO1 hO5 y1 Gm-c1074 Glycine max cDNA clone 76 6 7e-14 gb|AW620962 1 | sj98bO3 y1 Gm-c1023 Glycine max cDNA clone 76 6 7e-14 gb[AW704152 11 sk28bO2 y1 Gm-c1028 Glycine max cDNA clone 75 1 2e-13 gb|B!316569 1 | safO5a1O y1 Gm-c1065 Glycine max cDNA clone 74 3 3e-13 gb|BU760760 1 | sas58bO7 y1 Gm-c1023 Glycine max cDNA clone 72 8 1e-12 gb|BE475593.1 | sp78eO5 y1 Gm-c1044 Glycine max cDNA clone 68 2 3e-11

Barley gb|BI951809 1 ] HVSMEm0003A03f Hordeum vulgare green seedling 241 2e-63 gb|DN182018 1 | HO22J02S HO Hordeum vulgare cDNA clone HO22J02 239 1e-62 dbj|AV833112 1 | AV833112 K Sato unpublished cDNA library 235 2e-61 dbj|AV941967 1 | AV941967 K Sato unpublished cDNA fibrary201 2e-51 gb[BU967095 1 | HB03E12r BC Hordeum vulgare subsp vulgare 193 6e-49 gb|BU967280 1 j HB03N01 r BC Hordeum vulgare subsp. vulgare 190 7e-48 gb|CA002527 1[ HS07M12r HS Hordeum vulgare subsp. vulgare 177 4e-44 gb|BQ466839 1 | HS01 L22T HS Hordeum vulgare subsp. vulgare 177 4e-44 gb|BM373839 2| EBma03_SQ002_N22_R maternal, 8 DPA176 1e-43 gb|BQ466452 1 | HT02J04r HT Hordeum vulgare subsp vulgare 176 1e-43 gb|BQ762325 1 | EBro01_SQ005_B18 _R root, 3 week,175 2e-43 gb|BF628773.2[ HVSMEb0008B1 Of Hordeum vulgare seedling shoot 164 5e-40 gb|BF264338 2| HV_CEa0009C13f Hordeum vulgare seedling green 159 1e-38 gb|DN183502 11 HO17K20S HO Hordeum vulgare cDNA clone HO17K20 . 156 8e-38 gb|BQ466741.1 ] HS01 H02T HS Hordeum vulgare subsp vulgare 154 3e-37 gb|DN156902.1 | GCN003J14u GCN Hordeum vulgare cDNA clone GCNO. . 154 5e-37 gb|BQ660530.1 | HI04A10u HI Hordeum vulgare subsp. vulgare 152 1e-36 gb|CB870232.11 HC13L22w CH Hordeum vulgare cDN A clone HC13L22 . 152 2e-36 gb|BE216310 2| HV_CEb0010C13f Hordeum vulgare seedling green 151 3e-36 gb|BQ740081 11 HC04G06 HC Hordeum vulgare subsp. vulgare cDNA150 6e-36 gb|BI959876 1 | HVSMEn0022C22f Hordeum vulgare rachis EST 146 8e-35 gb|BQ660415 1 | HI02G20u HI Hordeum vulgare subsp vulgare 143 7e-34 gb|CA019131 1 | HV10O05r HV Hordeum vulgare subsp vulgare 138 3e-32 gb|CA028721 1 | HZ63A24r HZ Hordeum vulgare subsp vulgare 137 4e-32 gb|BU969351 1 | HB11 E12r BC Hordeum vulgare subsp vulgare 136 9e-32 gb|BF616419 2[ HVSM Ec0007lO4f Hordeum vulgare seedling shoot 134 6e-31 gb|BI948455 1 | HVSMEI0009K13f Hordeum vulgare spike EST 133 1e-30 dbj|BY838837 1 | BY838837 Etiolated seedling shoot Hordeum 132 2e-30 dbj|BY847793.1 | BY847793 Seminal root Hordeum vulgare subsp 132 2e-30 gb|CA003800 1 | HS15J20r HS Hordeum vulgare subsp vulgare 132 2e-30 gb|CA001818 1 | HS05K18r HS Hordeum vulgare subsp. vulgare 132 2e-30 gb[BM369160 2| EBem07_SQ002_L22_R embryo, 28 DPA 132 2e-30 dbjjBY853068 1 | BY853068 Germination shoots Hordeum vulgare 129 2e-29 dbj[BY847188 1 | BY847188 Seminal root Hordeum vulgare subsp.129 2e-29 gb|BF264951 3| HV_CEa001 ON 1Of Hordeum vulgare seedling green . . 128 3e-29 gb|BQ739839.1 | HB04B12 HB Hordeum vulgare subsp vulgare cDNA. 125 3e-28 gb|BQ758567.1 | EBma07_SQ002_K03_R maternal, 21 DPA124 6e-28 dbj|BY848814.1 | BY848814 Seminal root Hordeum vulgare subsp 121 4e-27 gb|DN183559.1 | HO17C03S HO Hordeum vulgare cDNA clone HO17C03 95.1 1e-26 gb|CV063030.1 | BNEL85h11 Barley EST endosperm hbrary Hordeumi 19 1e-26 gb|CV059324.1 | BNEL46h 12 Barley EST endosperm library Hordeum 119 1e-26 gb|CV055928 1 | BNEL12A4 Barley EST endosperm library Hordeum 119 1e-26 gb|BM098554 2| EBem08_SQ003_G12_R embryo, 40 DPA 117 7e-26 gb|BM098700 2| EBem08_SQ003_N07_R embryo, 40 DPA116 1e-25 gb|CB873818 11 HC13L22y CH Hordeum vulgare cDNA clone HC13L22 115 2e-25 gb|BE602936 2[ HVSMEhO"! 01 B12f Hordeum vulgare 5-45 DAP spike 115 3e-25 dbj|BY847001 1 | BY847001 Seminal root Hordeum vulgare subsp 93 2 6e-25 gb|CA001991.1 | HS06D04r HS Hordeum vulgare subsp. vulgare 113 8e-25 gb|CA008447.1 | HU10P22r HU Hordeum vulgare subsp vulgare 112 1e-24 gb|CV057176.1 | BNEL24h11 Barley EST endosperm library Hordeum 111 3e-24 gb|BE216016 3[ HV_CEb0009C04f Hordeum vulgare seedling green 77 8 9e-24 dbj|BY849120 1 j BY849120 Germination shoots Hordeum vulgare 109 1e-23 gb|CV057937.1 | BNEL32e5 Barley EST endosperm library Hordeum 107 6e-23 gb|BQ759041 1 | EBma07_SQ003_H17_R maternal, 21 DPA102 1e-21 dbj|BY849968 1 j BY849968 Germination shoots Hordeum vulgare 102 2e-21 gb|BQ758691 1 | EBmaO7_SQOO2_D17_R maternal, 21 DPA 100 9e-21 dbj|BY850372 1 j BY850372 Germination shoots Hordeum vulgare 90 9 6e-18 gb|BQ463252 1 | HI04i11 r HI Hordeum vulgare subsp vulgare 90 5 7e-18 dbj|AV834862 1 j AV834862 K Sato unpublished cDNA library Ho 89 7 1e-17 gb|BU990279 1 | HF24J17r HF Hordeum vulgare subsp vulgare cDNA 88 6 3e-17 dbj|BY854029 1 j BY854029 Germination shoots Hordeum vulgare 84 0 7e-16 gb|CA001926 1 | HS06A02r HS Hordeum vulgare subsp vulgare cDNA 84 0 7e-16 gb|BM815949 1 | HB108G05_SK ab1 HB Hordeum vuigare subsp 84 0 7e-16 gb|BF619429 2| HVSM Ec0003F06f Hordeum vulgare seedling shoot 84 0 7e-16 gb|BF630021 2| HVSMEb0007L02f Hordeum vulgare seedling shoot 84 0 7e-16 gb|BF265538 11 HV_CEa0012M6f Hordeum vulgare seedling green 82 0 3e-15 dbj|BJ477649 1 | BJ477649 K Sato unpublished cDNA Iιbrary77 0 8e-14 dbj|BJ469794 1 | BJ469794 K Sato unpublished cDNA Iιbrary68 9 2e-13 gb|BU997524 1 | HI08D24r HI Hordeum vulgare subsp vulgare 69 7 1e-11

Maize

gb|EE046500 1 j ZM_BFc0116O04 r ZM_BFc Zea mays cDN A clone 354 3e-97 gb|DR827048 11 ZM_BFb0070B11 f ZM_BFb Zea mays cDNA 3¹, mRNA seq 320 7e-87 gb|EE153197 1 ! ZM_BFc0057D18 f ZM_BFc Zea mays cDNA clone 301 4e-81 gb|EE176144 1 ) ZM_BFcO155L14 f ZM_BFc Zea mays cDNA clone 300 1e-80 gb|EE016215 1 j ZM_BFc0066O22 r ZM_BFc Zea mays cDNA clone 283 1e-75 gb|DR814130 1 | ZM_BFb0043K05 r ZM_BFb Zea mays cDNA 5', mRNA seq 251 3e-66 gb|EE044700 1 | ZM_BFc0113119 r ZM_BFc Zea mays cDNA clone 237 8e-62 gb|CO466103 11 MZCCL20041 E02 g Maize Endosperm cDNA Library 228 3e-59 gb|CX129539.11 QCD4fO5 yg QCD Zea mays cDNA clone QCD4TO5, mRNA 223 2e-57 gb|EE0207581| ZM_BFc0074B16 rZM_BFc Zea mays cDNA clone 213 1e-54 gb[DR8300421| ZM_BFb0077D11 rZM_BFb Zea mays cDNAδ¹, mRNA seq 213 1e-54 gb[EE0369521| ZM_BFc0100H21 rZM_BFc Zea mays cDNA clone 205 3e-52 gb|EE1746271| ZM_BFc0153H07 rZM_BFc Zea mays cD NA clone 2001e-50 gb|EE1686411| ZM_BFcO143L05 rZM_BFc Zea mays cDNA clone 2001e-50 gb|EC8780821| ZM_BFc0012B11 r ZM_BFc Zea mays cDNA clone 2001e-50 gb|DV5317081| ZM_BFb0221P10 rZM_BFb Zea mays cDNA 5\ mRNA seq 200 1e-50 gb|DR8193131] ZM_BFbO055J22 r ZM_BFb Zea mays cDNA 5', mRNA seq 200 1e-50 gb|DR8143131| ZM_BFb0043O15 rZM_BFb Zea mayscDNA 5', mRNAseq 200 1e-50 gb|DV5211451| ZM_BFb0206F11 rZM_BFb Zea mayscDNA 5', mRNAseq 199 2e-50 gb|EE023205.1| ZM_BFc0078F16 rZM_BFc Zea mayscDNA clone 197 5e-50 gb|DT6447961| ZM-BFbOI 04H15 r ZM-BFb Zea mays cDNA 5', mRNAseq 197 5e-50 gb|EE045614.1| ZM-BFcO 115D04 r ZM-BFc Zea mays cDNA clone 1296e-50 gb|EE184383.1| ZM-BFc0168M02 r ZM-BFc Zea mays cDNA clone 197 7e-50 gb|EB6763771| ZM_BFb0340P21.rZM_BFb Zea mayscDNA 5¹, mRNAseq 197 9e-50 gb|EB6411341| ZM_BFbO33OIO4 r ZM-BFb Zea mayscDNA 5¹, mRNA seq 197 9e-50 gb|DV514179.1| ZM_BFb0196A08 r ZM-BFb Zea mays cDNA 5', mRNAseq 197 9e-50 gb|DT93881811 ZM_BFbO120H03 r ZM-BFb Zea mays cDNA 5', mRNA seq 197 9e-50 gb|EC8782821| ZM_BFc0012G03 rZM_BFc Zea mays cDNA clone 194 6e-49 gb|DR8033121| ZM_BFb0027O21.r ZM-BFb Zea mays cDNA 5', mRNAseq 192 2e-48 gb|DV5361831| ZM_BFbO228J07 r ZM-BFb Zea mayscDNA 5\ mRNAseq 192 3e-48 gb|EE01658711 ZM_BFc0067H20.r ZM_BFc Zea mays cDNA clone 110 1e-47 gb|EE0375961| ZM_BFcO101H15 rZM_BFc Zea mays cDNA clone 188 3e-47 gb|EE1810561| ZM_BFc0163K15 rZM_BFc Zea mays cDNA clone 1862e-46 gb|EB82174911 ZM_BFbO383A18 r ZM_BFb Zea mays cDNA 5', mRNA seq 184 5e-46 gb|DR972034.1| ZM_BFb0095A05 r ZM_BFb Zea mays cDNA 5', mRNA seq 182 3e-45 gb|EE0467471| ZM_BFcO119D19 rZM_BFc Zea mayscDNA clone 1792e-44 gb|EC8986311| ZM_BFcO043J13 rZM_BFc Zea mayscDNA clone 1002e-41 gb|EE0162071| ZM_BFc0066O16 rZM_BFcZea mays cDNA clone 1662e-40 gb|EE1727611| ZM_BFc0150H22 rZM_BFc Zea mays cDNA clone 971 3e-40 gb|EC89347911 ZM_BFcO035LO5 r ZM_BFc Zea mays cDNA clone 1654e-40 gb|CA8287161| 1114032E05 y21114 - Unigene IV from Maize 164 7e-40 gb3DY68942511 ZM_BFbO285C17 r ZM_BFb Zea mays cDNA 5', mRNA seq 162 2e-39 gb[DV538370.1| ZM_BFb0231M05 r ZM-BFb Zea mays cDNAS¹, mRNAseq 162 2e-39 gb[DR96572311 ZM_BFb0085M05 r ZM-BFb Zea mays cDNA 5', mRNA seq 162 2e-39 gb|DV167013.1| ZM_BFbO164N15 r ZM-BFb Zea mayscDNA 5¹, mRNAseq 161 4e-39 gb|DR82023011 ZM_BFb0058D10 r ZM_BFb Zea mays cDNA 5¹, mRNA seq 161 4e-39 gb|BU0795651| 946145D05 y1946 -tassel pπmordium prepared 161 4e-39 gb|EE1650471j ZM_BFcO137E21 rZM_BFcZea mays cDNA clone 1616e-39 gb|EE0374881[ ZM_BFc0101 E17 rZM_BFc Zea mays cDNA clone 1607e-39 gb|DV5198771| ZM_BFb0204H22 r ZM_BFb Zea mayscDNAS', mRNAseq 160 7e-39 gb|DV508968.1| ZM_BFbO187M19 rZM_BFb Zea mays cDNA5^r, mRNAseq 160 7e-39 gb|DV168574.11 ZM_BFb0167E09 rZM_BFb Zea mays cDNA 5', mRNAseq 160 7e-39 gb|DV033268.1| ZM_BFbO157G21 rZM_BFb Zea mays cDNA 5¹, m RNA seq 160 7e-39 gb|DV019914.1| ZM_BFbO138A11 rZM_BFb Zea mays cDNA 5', mRNAseq 160 7e-39 gb|DT6414381| ZM_BFbOO99IO1.rZM_BFb Zea mays cDNA5', mRNAseq 160 7e-39 gb|DR970291.11 ZM_BFb0092H08 r ZM_BFb Zea mays cDNA 5', mRNA seq 160 7e-39 gb|DR969885.11 ZM_BFb0091 N01 r ZM_BFb Zea mays cDNA 5', mRNA seq 160 7e-39 gb|DR82826811 ZM_BFb0072L05 r ZM_^BFb Zea mays cDNA 5', mRNA seq 160 7e-39 gb|EE0269881| ZM_BFc0084J01 rZM_BFc Zea mays cDNA clone ZM_ . 160 9e-39 gb|DN5605881| ME24-A03-T3-96-R1 E7PCR Zea mays cDNA clone E7 . 160 1e-38 gb|BM5002101| PAC000000000320 Pioneer AF-1 array Zea mays cDNA, 160 1e-38 gb|DV032779.1| ZM_BFb0156L04 rZM_BFb Zea mays cDNAS¹, mRNAseq 159 2e-38 gb|DR82847611 ZM_BFb0073E20 r ZM_BFb Zea mays cDNA 5", mRNA seq 159 2e-38 gb|CA8302061 [ 1117003H06.y 11117 - Unigene V from Maize 159 2e-38 gb|EC891458.1| ZM_BFc0032K08 rZM_BFc Zea mays cDNA clone 158 4e-38 gb|EC8794861| ZM_BFc0014C20.rZM_BFc Zea mays cDNA clone 1584e-38 gbjCO451095.1l MZCCL10160C11.g Maize Endosperm cDNA Library 901 7e-38 gb|BQ295716.1| 1091041H03 y11091157 8e-38 gb|CO525958.1| 3530JM72J _F12 y_13530 - FuIi length cDNA 154 7e-37 gb|CO46602211 MZCCL20042D10 g Maize Endosperm cDN A Library 154 9e-37 gb[DY2403931| ZM_BFb0259N07 rZM_BFbZea mays cDNA5', mRNA seq 153 1e-36 gb[EE0442341| ZM_BFcO112N15 rZM_BFc Zea mays cDNA clone 1523e-36 gb[EE020950.1| ZM_BFc0074H05.r ZM_BFc Zea mays cDNA clone 1501e-35 gb[DR80788711 ZM_BFbO034F24.r ZM_BFb Zea mays cDNA 5', mRNA seq 150 1 e-35 gb|CF002965.1| QBH17eO7 xg QBH Zea mays cDNA clone QBH17eO7, mRN 147 6e-35 gb|EC8865191| ZM_BFc0025E04.r ZM_BFc Zea mays cDNA clone 147 1e-34 gb|CO51874011 3530_1_122_1_E05 y_13530 - Full length cDNA 146 1e-34 gb|CF2448201| 3530_1_5_1_A07 y_23530 - Fui! length cDNA 146 1e-34 gb|EE015293.11 ZM_BFc0064K18 rZM_BFc Zea mays cDNA clone 144 7e-34 gb|CF62955511 zmrws48 J)A20-002-g05 s0 zmrws48 Zea mays cDNA 3', 140 8e-33 gb|CD4435211| EL01N0427E10 b Endosperm_4 Zea mays cDNA, mRNA se 140 8e-33 gb|EE0139681| ZM_BFcOO62H12 rZM_BFc Zea mays cDNA clone 140 1e-32 gb|EE0139671| ZM_BFc0062H12.f ZM_BFc Zea mays cDNA clone 140 1e-32 gb|DR9671391| ZM_BFb0087N19 rZM_BFb Zea mays cDNA 5', mRNAseq 140 1e-32 gb[AW065936 11 687003D06 y1 687 140 1e-32 gb|BQ295608 1 | 1091038B11 y1 1091 140 1e-32 gb|EC880371 1 | ZM_BFcOO15IO1 r ZM_BFc Zea mays cDNA clone 139 3e-32 gb|DY688753 1 | ZM_BFbO284B21 r ZM_BFb Zea mays cDNA 5\ mRNA seq 139 3e-32 gb|DY624757 11 ZM_BFb0347H01 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32 gb|DV539151 11 ZM_BFbO232N24 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32 gb|DV537032 11 ZM_BFbO229M13 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32 gb|DV535973 1 ] ZM_BFbO228E13 r ZM_BFb Zea mays cDNA S¹, mRNA seq 139 3e-32 gb|DV535514 1 j ZM__BFbO227J2O r ZM_BFb Zea mays cDNA 5¹, mRNA seq 139 3e-32 gb|DV530458 1 [ ZM_BFb0220E01 r ZM_BFb Zea mays cDNA S', mRNA seq 139 3e-32 gb|DV515662 1 | ZM_BFbO198F14 r ZM_BFb Zea mays cDNA δ¹, mRNA seq 139 3e-32 gb|DV507107 11 ZM_BFb0185C03 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32 gb|DV025493.11 ZM_BFbO146B22 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32 gb|DV025290.1 | ZM_BFb0145N07 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32 gb|DT946133 11 ZM_BFbO133K13 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32 gb|DT645771 1 | ZM_BFb0105P04 r ZM_BFb Zea mays cDNA 5', mRNA seq 139 3e-32

Wheat

gb|DR735777 1 | FGAS081411 Triticum aestivum 319 2e-86 dbj|CJ649060 11 CJ649060 Y Ogihara unpublished cDNA library 238 4e-62 gb|DR740109 11 FGAS085037 Triticum aestivum FGAS Library 197 9e-50 gb[CD881399 1 | F1 103B17F010329 F1 Triticum aestivum cDNA 194 6e-49 dbj|CJ655373 1 | CJ655373 Y Ogihara unpublished cDNA library 189 2e-47 gb|CD884036 1 | F1 115E10F010507 F1 Tπticum aestivum cDNA 186 2e-46 gb|CV762533 11 FGAS056922 Triticum aestivum FGAS Library 2 185 5e-46 gb|DR740033 1 | FGAS084961 Triticum aestivum FGAS Library 2 184 6e-46 gb|BI479783 1 | WHE3452_A08_A16ZS Wheat pre-anthesis spike cDNA 175 5e-43 gb|CD919573 1 j G608 113N06F010911 G608 Triticum aestivum CDNA164 7e-40 gb|CD939626 1 | OV 114E09F010312 OV Tπticum aestivum cDNA 162 4e-39 emb|AL812570 1 | AL812570 e 310 Triticum aestivum cDNA cione 157 1e-37 gb|CD871372 1 | AZO2 118B14F010207 AZO2 Triticum aestivum cDNA 157 1e-37 gb|CD909725 11 G468 113G04F010820 G468 Triticum aestivum cDNA 154 7e-37 dbj|BJ304701 1 j BJ304701 Y Ogihara unpublished cDNA library 152 3e-36 gb[CV766170 1 | FGAS060557 Triticum aestivum FGAS Library 2 152 3e-36 gb|BE488911 1 [ WHE1077_G04_N07ZS Wheat unstressed seedling 152 4e-36 gb|BE418746 1 | SCL074 E11 R990812 ITEC SCL Wheat Leaf Library 151 8e-36 gb|CD868305 1 | AZO2 108I04F001113 AZO2 Triticum aestivum cDNA 148 5e-35 gb|CD918044 1 | G608 107M05F010905 G608 Triticum aestivum cDNA 147 1e-34 gb|CA733194 1 | wlpic pk007 b24 wlpic Triticum aestivum cDNA 147 1e-34 gb|CK208279 1 | FGAS019979 Triticum aestivum FGAS Library 5 146 2e-34 dbj|CJ627951 1 j CJ627951 Y Ogthara unpublished cDNA library 145 4e-34 gb|CK213161 1 | FGAS025066 Triticum aestivum FGAS Library 6 143 2e-33 gb|CV781279 1 | FGAS075690 Triticum aestivum FGAS Library 2 137 9e-32 gbjCK196713 1 | FGAS005173 Triticum aestivum FGAS Library 3 137 1e-31 gbjCD894087 1 [ G118 125F14F010828 G118 Triticum aestivum cDNA 137 1e-31 gbjCA741191 1 | wialc pkOOI g8 wialc Triticum aestivum cDNA 137 1e-31 gb[BQ240791 1 | TaE05012H01 R TaE05 Triticum aestivum cDNA 137 1e-31 gb[BU100082 1 | WHE3314_H10_O20ZS Chinese Spring wheat drought 137 1e-31 emb[AJ601747 11 AJ601747 T05 Triticum aestivum cDNA clone 135 3e-31 gb[BU100475 1 | WHE3353J305JM09ZS Chinese Spring aluminum-stre 135 3e-31 gb|BE426590 1 | WHE0336J=08J-16ZS Wheat unstressed seedling 135 4e-31 gb[CD924507 1 | G750 113E16F010706 G750 Triticum aestivum cDNA 135 6e-31 gb|BF200512 1 | WHE0825-0828_B15_B15ZS 134 1e-30 gb|CV762050 1 | FGAS056439 Triticum aestivum FGAS Library 132 4e-30 gb|CD868306 11 AZ02 108I04R010328 AZO2 Triticum aestivum cDNA 132 4e-30 gb|CD924855 1 | G750 114O18F010706 G750 Triticum aestivum CDNA131 6e-30 gb|BE471094 1 | WHE0284_H12_O24ZS 130 1e-29 gb|CV780245 1| FGAS074654 Triticum aestivum FGAS Library 2 128 5e-29 emb|AJ602329 1 | AJ602329 T05 Tπticum aestivum cDNA clone 127 9e-29 gb|CV768109 1 | FGAS062500 Triticum aestivum FGAS Library 2 127 2e-28 gb|CD881419 1 | F1 103C19F010328 F1 Triticum aestivum cDNA 125 3e-28 gb|CD920551 1 | G608 117J13F010912 G608 Triticum aestivum CDNA125 4e-28 dbj|CJ660695 11 CJ660695 Y Ogihara unpublished cDNA library 123 2e-27 dbj|CJ696176 1 | CJ696176 Y Ogihara unpublished cDNA library 122 3e-27 gb|DR736601 1 | FGAS081971 Triticum aestivum FGAS Library 5 120 2e-26 dbj|CJ627959 11 CJ627959 Y Ogihara unpublished cDNA library 119 2e-26 dbj|CJ501729 1 | CJ501729 Y Ogihara unpublished cDNA library 118 5e-26 gb|CV780793 1 | FGAS075204 Triticum aestivum FGAS Library 2 118 7e-26 gb|CA701457 1 | wkm2c pkOO6 e15 wkm2c Triticum aestivum cDNA 105 8e-26 gb|CV775774 1 | FGAS070178 Triticum aestivum FGAS Library 2 102 3e-25 gb|CD919574 11 G608 113N06R011027 G608 Triticum aestivum cDNA 115 4e-25 gb|BE499216 1 j WHE0972_H11_O22ZS Wheat pre-anthesis spike cDNA 112 4e-24 gb|CK216192 1 | FGAS028177 Triticum aestivum FGAS Library 6 110 1e-23 gb|CA632168 1 | w!e1 n pk0062 f9 wle1 n Triticum aestivum cDNA 110 1e-23 emb|AL816629 1 j AL816629 I 226 Triticum aestivum cDNA clone 67 4 4e-23 gb|CV758782 1 | FGAS053164 Tπtιcum aestivum FGAS Library 2 107 1e-22 gb|BE406522 1 | WHE0417_f11_k21zB 106 3e-22 gb|CA732800 1 | wlpic pkOO4 j3 wlpic Triticum aestivum cDNA 103 2e-21 gb|CA697753 11 wik4 pkOOIO g3 wlk4 Tπticum aestivum cDNA 61 6 5e-21 gb|CD882214 1 | F1 105L19F010330 F1 Tπticum aestivum cDNA 101 7e-21 gb|CA657309 1 | wlmO pkOO34 e2 wlmO Triticum aestivum cDNA 97 8 1e-19 dbj|CJ712793 1 | CJ712793 Y Ogihara unpublished cDNA library 93 6 2e-18 dbj|CJ519157 1 | CJ519157 Y Ogihara unpublished cDNA library 92 O 5e-18 dbj|CJ712794 1 | CJ712794 Y Ogihara unpublished cDNA library 91 7 7e-18 gb|CD918480 1 | G608 109J07F010907 G608 Triticum aestivum cDNA 91 7 7e-18 gb|BQ839307 1 | WHE4164_F06_K12ZS Wheat CS whole plant cDNA 90 1 2e-17 emb|AL818825 1 | AL818825 I 125 Triticum aestivum cDNA clone 87 0 2e-16 gb|BF202987 1 j WHE1768_A07_A14ZS Wheat pre-anthesis spike cDNA 84 7 9e-16 gb|CD925960 1 | G750 119H10F010711 G750 Tπtιcum aestivum cDNA 84 0 1e-15 dbj[CJ608117 1 | CJ608117 Y Ogihara unpublished cDNA library 82 4 4e-15 emb|AJ602330 11 AJ602330 T05 Triticum aestivum cDNA clone E08 82 4 4e-15 gb|CK211694 1 | FGAS023548 Triticum aestivum FGAS Library 6 81 6 7e-15 gb|BE470775.1 | WHE0281_A11_B22ZS 81 6 7e-15 gb|CA610399 1 | wr1 pkO119 f3 wr1 Triticum aestivum cDNA 65 9 9e-15 gb|DY741996 1 | EST0565 Cold treated wheat cDNA library 81 3 1 e-14 gb|CA596291 1 | wpaic pkO12.m23 wpa1c Tπticum aestivum cDNA 81 3 1e-14 gb|BE427506 1 | PSR7104 ITEC PSR Wheat Pericarp/Testa 76 6 1e-14 dbj|CJ640745 1 j CJ640745 Y Ogihara unpublished cDNA library 77 8 1e-13 gb|CA620714 11 wl1 n pkO067 b9 wl1 n Triticum aestivum cDNA 75 9 4e-13 emb|AJ603615 11 AJ603615 T07 Triticum aestivum cDNA clone 75 5 5e-13 dbj|BJ232988 11 BJ232988 Y Ogihara unpublished cDNA hbrary72 4 4e-12 gb|CA645001 11 wre1 n pk0086 b5 wre1 n Triticum aestivum cDNA 71 2 1 e-11 gb|CA484478 11 WHE4307_A11_B21ZS Wheat meiotic anther cDNA 67 0 2e-10

Potato

gb|CK253280 1 [ EST736917 potato callus cDNA library, normaliz 324 2e-88 gb[CK267715 11 EST713793 potato abiotic stress cDNA library S 323 3e-88 gb|CK246340 1 | EST729977 potato callus cDNA library, normaliz 308 1e-83 gb|CK244536.1 | EST728173 potato callus cDNA library, normaliz 306 6e-83 gb|BG890899 11 EST516750 cSTD Solanum tuberosum cDNA done cS 304 2e-82 gb|CK246138 1 | EST729775 potato callus cDNA library, normaliz 303 3e-82 gb|CK245930 11 EST729567 potato callus cDNA library, normaliz 295 2e-80 gb|CK277468 11 EST723546 potato abiotic stress cDNA library S 292 7e-79 gb|CK253321 11 EST736958 potato callus cDNA library, normaiiz . 246 2e-76 gb|CK251625 11 EST735262 potato callus cDNA library, normaiiz . 267 3e-71 gb|CK251428 11 EST735065 potato callus cDNA library, normahz . 263 5e-70 gbjBQ510589 2| EST618004 Generation of a set of potato cDNA c 243 5e-64 gb|DN922927 1 | 44403 2 Common Scab-Challenged Tubers Solanum 219 8e-57 gbjBG599468 1 | EST504363 cSTS Solanum tuberosum cDNA clone cS 214 3e-55 gb|CK269104 1 ] EST715182 potato abiotic stress cDNA library S 212 1e-54 gb|CK262462 1 | EST708540 potato abiotic stress cDNA library S 212 1e-54 gb|CK252508 1 | EST736145 potato callus cDNA library, normaiiz 212 1e-54 gb|CK245866 1 | EST729503 potato callus cDNA library, normaiiz 212 1e-54 gb|CV429240 1 [ 51723 1 After-Cooking Darkening C Solanum tube 211 2e-54 gb| BG890138 11 EST515989 cSTD Solanum tuberosum cDNA clone cS 211 2e-54 gb|CV431358 1 [ 55572 1 After-Cooking Darkening C Soianum tube 209 1e-53 gb|BF459947 11 068G10 Mature tuber lambda ZAP Solanum tuberos 205 1e-52 gb|CK264318 11 EST710396 potato abiotic stress cDNA library S 204 3e-52 gb| BG594178 11 EST492856 cSTS Solanum tuberosum cDNA clone cS 203 4e-52 gb|BI406849 11 182A06 Mature tuber lambda ZAP Solanum tuberos 201 2e-51 gb|BG595575 11 EST494253 cSTS Solanum tuberosum cDNA clone cS 200 5e-51 gb|CK719979 11 20306 Swollen Stolon Solanum tuberosum cDNA, mRNA 198 1e-50 gb|BG886998 11 EST512849 cSTD Solanum tuberosum cDNA clone cS 198 1e-50 gb|BG890352 11 EST516203 cSTD Solanum tuberosum cDNA clone cS 195 2e-49 gb|CN213018 1 | 26561 Suspension culture Solanum tuberosum cDNA, 194 3e-49 gb|EG012052 11 STDB004A01 u STDB Solanum tuberosum cDNA clone 194 4e-49 gb|BG596222 11 EST494900 cSTS Solanum tuberosum cDNA clone cS 168 5e-49 gb|CV474156 1 | 22487 1 Developing Tubers Solanum tuberosum cD . 192 1e-48 gb|Bl 176643 11 EST517588 cSTE Solanum tuberosum cDNA clone cS 190 4e-48 gb|BG890868 11 EST516719 cSTD Solanum tuberosum cDNA clone cS . 190 5e-48 gb|BE922055.11 EST425824 potato leaves and petioles Solanum t 189 9e-48 gb|DN923069 11 44928 2 Common Scab-Challenged Tubers Solanum 188 1e-47 gb|BG887370 1 ] EST513221 cSTD Solanum tuberosum cDNA clone cS 187 4e-47 gb|BG351853 11 135A04 Mature tuber lambda ZAP Solanum tuberos 186 7e-47 gb|BG591987 1 j EST499829 P infestans-challenged leaf Solanum 180 4e-45 gb|BG889138 1 [ EST514989 cSTD Solanum tuberosum cDNA clone cS 180 5e-45 gb|CK277133 11 EST723211 potato abiotic stress cDNA library S 179 9e-45 gb|CK269720 11 EST715798 potato abiotic stress cDNA library S 179 9e-45 gb|CK851489 11 11654 Stolon Solanum tuberosum cDNA, mRNA sequenc 119 1e-44 gb|CN216247 1 | 30125 Suspension culture Solanum tuberosum cDNA, 178 2e-44 gb|CK256967 1 | EST740604 potato callus cDNA library, normaliz 178 2e-44 gb|CK255235 1 | EST738872 potato callus cDNA library, normaliz 178 2e-44 gb|CK249408 11 EST733045 potato callus cDNA library, normaliz 178 2e-44 gb|CK261460 11 EST707538 potato abiotic stress cDNA library S 177 4e-44 gb|BQ121678 2| EST607254 mixed potato tissues Solarium tuberos 176 6e-44 gb|BG886969 11 EST512820 cSTD Solanum tuberosum cDNA clone cS 175 2e-43 gb|BG888694 11 EST514545 cSTD Solanum tuberosum cDNA clone cS. 172 1e-42 gb[BF459641.1 | 062F02 Mature tuber lambda ZAP Solanum tuberos 172 1e-42 gb|BF153546 11 028C10 Mature tuber lambda ZAP Solanum tuberos 172 1e-42 gb|BG597894 1 | EST496572 cSTS Solanum tuberosum cDNA clone cS 171 3e-42 gb|BG595042 11 EST493720 cSTS Solanum tuberosum cDNA clone cS 169 7e-42 gb|CV430232 11 53385 1 After-Cooking Darkening C Solanum tube 169 9e-42 gb|BG890527 11 EST516378 cSTD Solanum tuberosum cDNA clone cS 168 2e-41 gb|CV471720 1 | 44928 1 Common Scab-Challenged Tubers Solanum . 167 4e-41 gb|BQ116126 2| EST601702 mixed potato tissues Solanum tuberos . 97 4 1e-40 gb|BQ511608 2j EST619023 Generation of a set of potato cDNA c 163 5e-40 gb[CV496124 1 | 73841.1 Cold Sweetening B Solanum tuberosum cD 163 7e-40 gb|CK720145 11 20511 Swollen Stolon Solanum tuberosum cDNA, mRNA 162 1e-39 gb|BI176663 1 \ EST51760S cSTE Solanum tuberosum cDNA clone cS 161 2e-39 gb|BG888216 1 | EST514067 cSTD Solanum tuberosum cDNA clone cS 160 3e-39 gb|CK250720 1 ] EST734357 potato callus cDNA library, normaliz . 160 4e-39 gb|BG886541 11 EST512392 cSTD Solanum tuberosum cDNA clone cS 160 6e-39 gb|CV471787 1 j 45004 1 Common Scab-Challenged Tubers Solanum 159 7e-39 gb|BI405991.1 | 150C03 Mature tuber lambda ZAP Solanum tuberos . 159 1e-38 gb|DV623092.1 | 92505 1 Cold Sweetening C Solanum tuberosum cD . 155 1e-37 gb[BE342382 1 [ EST395226 potato stolon, Cornell University So. 152 2e-36 gb|BI178192 11 EST519137 CSTE Solanum tuberosum cDNA clone cS 151 2e-36 gb|BQ509197 2| EST616612 Generation of a set of potato cDNA c 150 3e-36 gb|AW906822.11 EST342945 potato stolon, Cornell University So 150 4e-36 gb|DN941337 11 55572 2 After-Cooking Darkening C Solanum tube 150 6e-36 gb|CN216727.1 | 30658 Suspension culture Solanum tuberosum cDNA, 149 1e-35 gb|CK261481.1 | EST707559 potato abiotic stress cDNA library S 146 6e-35 gb|BQ510562 2\ EST617977 Generation of a set of potato cDNA c . 146 8e-35 gb|CK851608 1 | 11803 Stolon Solanum tuberosum cDNA, mRNA sequenc 145 1e~34 gb|BQ505416 2| EST612831 Generation of a set of potato cDNA c 145 1e-34 gb|BG593665 11 EST492343 cSTS Solanum tuberosum cDNA done cS 145 1e-34 gb|DN909107 11 57843 2 Developing Tubers Solanum tuberosum cD 145 2e-34 gb|DN907365 11 22487 2 Developing Tubers Solanum tuberosum cD 145 2e-34 gb|CV477893 1 | 57843.1 Developing Tubers Solanum tuberosum cD. . 145 2e-34 gb|CK274767 1 j EST720845 potato abiotic stress cDNA library S .. 143 5e-34 gb[CK258841 1 j EST742478 potato callus cDNA library, normaliz... 143 5e-34 gb|CK258748 1 j EST742385 potato callus cDNA library, normaliz... 143 7e-34 gb|BE920995.1 | EST424764 potato leaves and petioles Solanum t 142 9e-34 gb|DN923089.1 | 45004 2 Common Scab-Challenged Tubers Solanum . 142 1e-33 gb|BF187134 1 | EST443421 potato stolon, Cornell University So 141 3e-33 gb|BF187133.1 | EST443420 potato stolon, Cornell University So , 141 3e-33 gb|CK263459.1 | EST709537 potato abiotic stress cDNA library S . 140 4e-33 gb|AW906840.11 EST342963 potato stolon, Cornell University So... 139 1e-32 gbjCK256905.11 EST740542 potato callus cDNA library, normaliz 139 1 e-32 gb|CV434757.1 | 58247.1 Suspension culture Solanum tuberosum c. 137 3e-32 gbjCK27S836.1 l EST724914 potato abiotic stress cDNA library S . 137 3e-32 gb[BF052865.1| EST438095 potato leaves and petioles Solanum t . 137 3e-32 gb|BE471540.11 EST416393 potato stolon, Cornell University So.. 134 3e-31 gb|DV625610 1 | 95977 1 Coid Sweetening C Solanum tuberosum cD 132 1e-30 gb|DN849475 11 13215 2 Stolon Solanum tuberosum cDNA clone 13 . 132 1e-30

Table 2

In one embodiment of the different aspects of the invention, an Arabidopsis Hsf selected from the Arabidopsis Hsf listed above, is overexpressed in another plant. The Arabidopsis Hsf is selected from the group comprising AtHsfAla, AtHsfAlb, AtHsfAid, AtHsfAle, AtHsfA2, AtHsfA3, AtHsfA4a, AtHsfA4c, AtHsfAS, AtHsfAθa, AtHsfA6b, AtHsfA7a, AtHsfA/b, AtHsfA8, AtHsfA9, AtHsfBI , AtHsfB2a, AtHsfB2b, AtHsfB3, AtHsfB4 or AtHsfCl In one embodiment, the Arabidopsis Hsf is AtHSFAI b. The full sequence of AtHSFAI b is shown in figure 1 (SEQ No 1).

The plant in which the Hsf is overexpressed may be any plant as listed herein. Preferably, the Arabidopsis Hsf, for example AtHSFAI b is overexpressed in a crop, for example a cereal, such as wheat, rice, barley, maize, oat sorghum, rye, onion, leek, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species. However, the applicability of the invention is not limited to the sequence shown in Seq ID No 1 as a skilled person would understand that other Hsfs isolated or derived from Arabidopsis or from other plants can also be used. Any combination of a plant Hsf for example as listed herein, in another plant, for example as listed herein, is within the scope of the invention.

In another embodiment of the different aspects of the invention, an endogenous plant Hsf may be overexpressed according to the methods and uses of the invention. For example, a tomato Hsf may be overexpressed in tomato, a wheat Hsf may be expressed in wheat, a rice Hsf may be overexpressed in rice. Plants and their one or more Hsf may be selected from any plant, such as from one of the families or species listed above.

Overexpression according to the invention means that the transgene is expressed at a level that is higher than expression driven by its endogenous promoter. For example, overexpression may be carried out using a strong promoter, such as the cauliflower mosaic virus promoter (CaMV35S), the rice actin promoter or the maize ubiquitin promoter or any promoter that gives enhanced expression. Alternatively, enhanced or increased expression can be achieved by using transcription or translation enhancers or activators and may incorporate enhancers into the gene to further increase expression. Furthermore, an inducible expression system may be used, such as a steroid or ethanol inducible expression system. The coding sequence may be on a monocistronic or polycistronic messenger RNA. Also envisaged is ectopic expression, i.e. gene expression in a tissue in which it is normally not expressed

According to the different aspects of the invention, plant characteristics are increased or improved. This is understood to mean an increase or improvement in plant productivity, water use efficiency, water productivity, drought tolerance or pathogen resistance compared to the level as found in a wild type plant.

According to one embodiment of the first aspect of the invention, the method increases water productivity. Thus, the method can be used to increase water productivity. As used herein, water productivity describes the amount of yield produced per unit of water (for example ml or I) used. The transgenic plants as described herein require a lower amount of water than a wild type plant to produce the same amount of yield under normal non drought conditions where water is not at a shortage. Thus, according to the invention, water productivity can be improved under non drought conditions. For example, water productivity can be improved under non drought conditions by expression of the Arabidopsis Hsf is AtHSFAIb in another plant as defined herein.

In a different embodiment of first aspect of the invention, the method improves plant productivity under water deficit conditions. Thus, the method of the first aspect confers plant drought tolerance.

Water deficit or water limited conditions as used herein refer to conditions where water is at a shortage. This includes conditions where water is at a shortage compared to the normal average of water available to a plant grown in the particular environment, for example due to a change in climate or unseasonable weather. It also refers to conditions where water is generally known to be scarce, for example in arid climatic zones. Water shortage for a prolonged period of time is known as drought.

In another embodiment of the method of the first aspect of the invention, the method of the invention confers pathogen resistance. Plants with ability to resist infection by a particular pathogen are referred to as having increased resistance to that pathogen. Pathogens according to the different aspects of the invention include any viral, bacterial, fungi or animal pathogens, such as nematodes or insects, which infect plants. In one embodiment, the pathogen may be Pseudomonas syringae pv. Tomato, turnip crinkle virus or Hyaloperonospora parasitica. Fungal pathogens according to the invention include, but are not limited to the rust fungi (order Uridenales) e.g. Puccinia graminis, Pυccinia striiformis (yellow rust) P. recondite and other Puccinia species, flax rust (Melampsora tini); Rhizoctonia sp. or Phakospora pachyrhizi (Soybean rust), the powdery mildew fungi (order Erysiphales, e.g barley powdery mildew (Blumeria graminis); Erisyphe sp. (infects legumes, trees and shrubs), Leveillula sp. (infects Solanaceae), Golovinomyces sp. (infect Cucurbits and Compositae), Podosphaera sp. (infects Rosaceae); Fusarium sp. Verticillium sp., Rice blast fungus Magnoporthe grisea or Potato blight (Phytophtora infestans).

Bacterial pathogens according to the invention include, but are not limited to Pseudomonas syringae (various pathovars), Xanthomonas sp. (e.g. X. campestris infects Brassicas, X. axonopodis causes citrus canker).

Viral pathogens according to the invention include, but are not limited to Tobacco mosaic virus (Solanaceae), tomato spotted wilt virus, rice tungrovirus, maize rough dwarf virus. Maize streak virus, cucumber mosaic virus, potato viruses X and Y, brome mosaic virus, pepper mild mottle virus, pea seed borne mosaic virus or pea ennation virus.

In a second aspect, the invention relates to a method for improving water use efficiency in plants comprising introducing and over-expressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant. In particular, water use efficiency can be improved under non drought conditions. For example, water use efficiency can be improved under non drought conditions by expression of the Arabidopsis Hsf is AtHSFAI b in another plant as defined herein.

The term water use efficiency as used herein relates to the plants ability of using a water supply efficiently under normal or water deficit conditions. Because the plants according to the invention use water more efficiently than a wild type plant, they show drought resistance and thus prolonged lifespan under water limiting conditions. However, the inventors have also surprisingly found that the plants according to the invention use water more efficiently under normal non-drought conditions compared to wild type plants. As shown in the examples, plants according to the invention require less amount of water than wild type plants to survive and produce yield, thus they use the water supply more efficiently. It will be appreciated that the term normal conditions refers to conditions which are not exceptional, i.e. conditions in which water is not limited. Drought conditions are not normal conditions as water is at a deficit. It will also be appreciated that what in detail is to be understood by normal conditions depends on the plant concerned and on the climatic zone in which the plant is grown.

Thus, in one embodiment of the second aspect of the invention, the method increases water productivity. The method as described in the second aspect of the invention can thus be used to increase water productivity.

In another embodiment, the method improves water use efficiency under water deficit conditions. Therefore, the method increases plant drought tolerance, As shown in examples 2 to 7, plants transformed with a gene sequence encoding a plant HSF polypeptide whose expression is regulated by a strong promoter have improved resistance to prolonged periods of water shortage, i.e. drought conditions. Wild type plant survival rates are very low under these conditions whereas the transgenic plants survive and produce yield.

In another aspect, the invention relates to a method for increasing water productivity. Water productivity can be increased under normal conditions, i.e. conditions where water is not iimited. In a further aspect, the invention provides a method for conferring drought resistance

In a further aspect of the invention, the invention relates to the use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving plant productivity.

Another aspect of the invention relates to the use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving water use efficiency. In particular, this use relates to improving water use efficiency under normal non drought conditions or under drought conditions. Therefore, according to the invention, a plant Hsf can be used to improve water productivity, thereby enabling the plant to use less water than a wild type plant. Thus, the amount of water used in irrigation of crop plants can be reduced. In addition, the use according to the invention also provides that a plant Hsf can be used to improve water use efficiency under water deficit conditions, such as drought conditions.

The invention also provides the use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving water productivity and the use in conferring drought tolerance.

In a final aspect, the invention provides the use of a polynucleotide sequence comprising or consisting of a plant Hsf in conferring pathogen resistance. The pathogen may be selected from those described herein.

The invention is further described by reference to the non-limiting figures and examples.

Figures

Figure 1 shows SEQ ID No 1 , the full length genomic sequence of AtHSFAI b.

Figure 2 shows the conserved domains of Arabidopsis Hsfs.

Figure 3 shows wild type and 35S-Hsf3 (left) plants which were not watered for 2 weeks. Transgenic plants are marked with an arrow.

Figure 4 shows plants which were not watered for 2.5 weeks and then rewatered. The picture was taken 48 hours after rewatering. Transgenic plants are marked with an arrow. Figures 5a and 12 show a decline in pot water content for 35S-Hsf3 and wild type (WT) plants under drought conditions in glasshouse. Withdrawal of water began at day 0. Re-watering commenced when the pot water content had attained 33 gm (indicated by the arrows). Note that this took 24 hours longer for 35S-Hsf3 plants. Figure 5b shows the quantum efficiency of photosynthetic electron transport (Fq//FnrT) in 35S-Hsf3 versus wild type plants just prior to recommencing watering as indicated.

Figure 6 shows the total seed yield after 10-11 days of drought and then subsequent re-watering until seed set. Under well-watered conditions, in both sets of conditions, 35S-Hsf3 plants yield better than WT. This differential is maintained after a moderate drought stress.

Figure 7 shows the vegetative and reproductive biomass as well as seed yield of plants kept at different soil water contents (40% and 80%). In both soil water contents Hsf3 plants show higher seed yield and reproductive biomass, but reduced vegetative biomass.

Figures 8a, 8b, 13a, 13b and 15 Wild type and transgenic plants overexpressing Hsf3 infected with virulent Pseudomonas syringae pv. tomato DC3000 8a) Diseased leaves were scored as those showing a yellowing 7 days after inoculation. 8b) number of bacteria (colony forming units) recovered after 5 days of infection. * = significant difference (p < 0.05).

Figures 9a to 9d and 14 to 14c Wild type and transgenic plants overexpressing Hsf3 infected with Hyaloperonospora parasitica a) diseases symptoms as scored by different disease classes: I healthy leaves, II- chlorotic lesions, ill leaves with sporulation and IV leaves with chlorotic lesions and sporulation The HSF3 plants have mainly healthy leaves and at most leaves that show chlorotic lesions. The fungus is prevented from sporulating on HSF3 leaves b) callose deposition at pathogen entry point scored using epifluoresnce microscopy by counting coincidence of spore presence and cailose deposition. The more callose with spores the higher the resistance, as seen in the 35S-Hsf3 plants. Figure 10 Northern blot analysis. Wild type and transgenic plants overexpressing Hsf3 infected with turnip crinkle virus. Viral RNA accumulation in leaves of HSf3 plants is delayed compared with wild type.

Figures 11 and 16 show foliar H₂O₂ levels during rosette development in 35S- Hsf3 and wild type plants.

Figure 17 is the expression construct used to transform Brassica napus.

Figure 18 shows a measurement of H₂O₂ of primary Brassica napus transgenics transformed with the construct as shown in figure 17

Figure 19 shows that PR1 gene expression is significantly higher in the 35S: HSF3 12.2 line compared with controls. Gene expression data was analysed in the primary Brassica napus transgenics transformed with the construct as shown in figure 17 using qRT-PCR.

Figure 20 shows APX2 gene expression in Brassica napus transgenics transformed with the construct as shown in figure 17. Examples

35S-Hsf3 (35SAthsfA1 b) plants display a number of characteristics which can be classified into two major traits, both elicited by overexpression of AtHsf3 and which interact to improve water productivity of these plants.

The plants show altered growth and development under well-watered conditions that mimic plants growing under mild water deficit conditions. Under a range of water deficit conditions these characteristics ensure that Hsf3 plants outperform wild type plants in terms of seed yield and survival. Plants show increased biomass of reproductive structures and seed production at the expense of leaf biomass, under both well-watered and drought conditions. The early flowering of Hsf3 plants under both well-watered and drought stress, altered senescence of source leaves and the consequent maintenance of photosynthetic efficiency are key factors in the increased seed yield of 35S- AtHSF3 plants under both well-watered conditions and a range of soil water deficit conditions.

35S-AtHsf3 (35SAthsfA1 b) plants also show reduced transpiration. An elevated apoplastic H₂O₂ level in plants over-expressing Hsf3 may, in part, be responsible for the trait by altering stomatai guard ceil function such that water loss through stomatai pores is reduced. H₂O₂ produced in guard cells has been implicated in the ABA signal transduction pathway leading to stomatai closure. Without wishing to be bound by theory, the inventors suggest that apoplastic H₂O₂ from elsewhere in the leaf can also influence guard cell function, importantly, in these plants reduced rates of transpiration did not lead to a sufficiently reduced CO₂ assimilation rate as to reduce growth and yield.

Example 1

Creating a construct and transforming plants

The creation of the plant described here has been published (Prandl et al 1998). Briefly, a full length 1.7 kb cDNA fragment containing the entire Hsf3 coding sequence was inserted as a BamH1 fragment into the binary Ti vector pBI121 , base on the well-known vector pBIN19. This procedure fused the cDNA to the CaMV 35S promoter. The mRNA would also contain a GUS coding sequence followed by a nos polyadenylation sequence. Moncistronic constructs fused to 35S promoter have also been created for transformation into crop plant species.

Example 2

Plant growth in response to drought and well watered conditions

Plants were grown in controlled environment rooms under short day (8h light/16hdark) at 22⁰C and 60% relative humidity. The Wild type (right) and 35S:AtHSF3 (left) plants are shown in figure 3. The plants were not watered for 2 weeks in a controlled environment room. These plants were typical in their response.

As shown in figure 4, the 35S AtHSF3 plants recover sufficiently from prolonged drought stress so that upon re-watering they flower and set seed. Wild type plants do not recover.

It was also shown that 35S-AtHSF3 plants have higher leaf temperatures. Under well-watered conditions, 35S-AtHSF3 plants have a 1.5 - 2.5⁰C higher leaf temperature, implying a reduced transpiration rate and a lower stomatal conductance.

Plants were grown in individual pots for 4 weeks in the controlled environment room as described above. After 4 weeks plants were transferred to the glass house under long day and water was withdrawn for half of the pots. The plants were left to dry out to a similar soil water content of ~35% and were then re- watered.

When control and 35S-AtHsf3 plants were re-watered at the times indicated in figure 5, seed yield was substantially higher in 35S-AtHsf3 plants than wild type (WT) plants (Table 3), a differential that was also observed in watered controls (Table 3). Since the amount of water provided was the same, these data represent a real increase in water productivity of the 35S-AtHsf3 plants compared with WT control. Thus, 35S-AtHsf3 plants have a higher seed yield in weil-watered conditions and after a mild water deficit stress.

Genotype Treatment Seed Yield (mg n P plant¹) 5S-AtHSF3 Watered 361 ± 84 4 0.022

WT Watered 156 ± 86 4 5S-AtHSF3 mild water deficit 368 + 31 4 0.0005

WT mild water deficit 156± 38 4 Table 3: Total seed yield from plants subjected to drought in a containment glasshouse. The water stress conditions were those applied in Fig. 5a up to the times indicated, when re-watering started. Controls were kept well-watered throughout the experiment.

Furthermore, rosette and reproductive structure biomass was measured. This was done by at the end of the life cycle. Plants were bagged and these bags contained all stem, pods, seeds etc above the rosette. The rosettes were harvested and bagged separately. The seed was threshed from the stems. All chaff was collected. The chaff, rosettes and soil pots were placed in a drying oven at 70 degrees Celsius for 72hrs. These were weighed. Seed was weighed and counted. Dry weight measurements of the above ground vegetative (rosette) and reproductive (stalks and pods) parts and seed yield were determined under two different soil water contents, (see Figure 7)

The experiments shows that 35S-AtHsf3 rosette biomass was reduced but the biomass of all aerial parts was increased in the same plants.

Example 3

Measurement of photosynthetic electron transport

This was done by measuring the maximum efficiency (Fv/Fm) of photosystem II. Whole rosette or leaf Fv/Fm values were taken to indicate their response to the drought treatment using a chlorophyll a fluorescence imaging system (Fluorimager; Technologica Ltd, Colchester, UK) as described by Barbagallo et al 2003.

CO2 gas exchange measurements were carried out on leaves of 5 week old plants using a CIRAS-2 (PP systems, Hitchin, U. K) CO2/H2O Infra Red Gas Analyser. Photosynthetic electron transport rates higher in drought-stressed 35S-AtHSF3 plants. The quantum efficiency of photosynthetic electron transport showed no difference between the genotypes in watered conditions, but was 15% better after 11 days of drought in 35S-HSF3 versus wild type (see Fig. 5b). There were no differences between 35S-AtHSF3 and wild type in the response of photosynthetic CO₂ exchange to intercellular CO₂ concentration when well watered. These parameters were used to evaluate transgenic plants.

Example 4

Measurement of flowering

Growth conditions were as described earlier. The first 4 weeks plants were grown in controlled environment rooms after which plants were transferred to the glass house and water level was maintained at either 40% or 80% soil water content. Measurement of flowering time was done by noting the time of the visible (by naked eye) of first appearance of the floral apical meristem as the number of days post germination until the terminal flower opened. 35S-Hsf3 plants fully flowered (terminal flower open) on average 7 days earlier than the CoI-O plants.

it was shown that 35S-AtHsf3 plants showed on average 7 days early flowering. During flowering and seed set, leaves of 35S-AtHS F3 plants show delayed senescence until siliques yellowed under glasshouse conditions. This may mean that in 35S-Hsf3 flowering plants, source leaves could maintain a supply of photosynthate to ensure an increased reproductive biomass and the supply to developing seed for longer, and this was a contributing factor for the observed increase in seed yield in 35S-AtHsf3 plants, and reproductive biomass.

Example 5

Measurement of H₂O₂ content 100mg of leaf materia! was extracted in 0.1 M HCI and the supernatant of this extraction was purified using activated charcoal. Analysis of H₂O₂ levels was by spectrophotometry using the Amplex Red kit from Invitrogen. It was shown that the 35S-AtHsf3 plants have up to 3 times the foliar H₂O₂ content of wild type plants. This increased H₂O₂ accumulates only in the apoplast and is generated by enhanced activity of apoplastic reticuline (carbohydrate) oxidase. The enhanced H₂O₂ levels stimulate an increase in both ascorbate peroxidase and cell wall peroxidase activities.

Example 6

Measurement of ABA content

1 g of leaf material was harvested and extracted in methanol. The supernatant of this extraction is dried down and samples were dissolved in diethylether/methanoi. The diethylether phase is put through a NH₂ SPE column and washed subsequently with chloroform/isopropanol before resuspending in diethylether and acetic acid. Analysis of ABA content was carried out using Gas chromatography combined with mass spectrometry (GC/MS) adapted from a method described by (Muller et al 2002).

The experiments showed that ABA content is no different from wild type plants under well-watered conditions and significantly our microarray data show no alteration of ABA-responsive genes (other than APX2).

Example 7

Microarray experiment

Rosettes from 5 week old CoI-O and HSF3 plants were harvested and RNA was extracted from 2 different biological samples. Gene expression analysis was carried out using Arabidopsis 3 whole genome oligonucleotide-based microarrays from Agilent cRNA labelling and hybridisation of the arrays was according to the manufacturers' instructions. Hsf3/WT

Name Locus Classification fold change

At5g03720 At-HSFA3 3.3

HSF4 At4g36990 At-HSFBI 2.5

HSF5 At1g67970 At-HSFA8 2.1

HSF6 At5g62020 At-HSFB2a 2

Table 4

Example 3

Pathogen infections

Pseudomonas syringae pv tomato (DC3000-) infection was carried out on 5 week old plants. Whole rosettes were dipped in a solution containing 5*10⁷ colony forming units (cfu)/ ml. Leaf material was harvested at the beginning (day 0) and 5 days post inoculation to determine the bacterial proliferation by grinding in MgSO4 and plating a dilution series on KB plates containing Rifampicin (50mg/L) and Cycloheximtde (100 mg/L). Bacterial proliferation is calculated as the difference in cfu between day 5 and day 0. Symptoms were scored 6 days post inoculation (yellowing leaves).

Hyaloperonospora parasitica infection was carried out at a concentration of 5^*10⁴ spore/ml. The fungal spores are obtained from leaves of infected plants and are extracted in water and diluted to the right concentration. Leaves of 3 week old plants are inoculated with the fungus by spraying a fine layer of liquid onto each leaf. Disease symptoms and callose formation are scored 7 days post inoculation.

Turnip crinkle virus infection was carried out on 3 week old plants. 2.5 ul of 0.1 ug/ul viral RNA in bentonite buffer were gently rubbed into three leaves of each plants. Systemic leaves are harvested at different times post inoculation and RNA is extracted and checked for viral RNA replication via Northern blotting. As a loading control, the blot is probed with 18S rRNA.

Example 9

Rapeseed (Brassica napus), also known as rape or oilseed rape, is a bright yeflow flowering member of the family Brassicaceae (mustard or cabbage family). Rapeseed is grown for the production of animal feed, vegetable oil for human consumption, and biodiesel; leading producers include the European Union, Canada, the United States, Australia, China and India. In one set of experiments, a construct overperessing HSF3 from Arabidopsis was used to transform rapeseed.

Brassica napus ecotype Q6 was transformed with a 35S:Af.HSF3 construct and an empty vector control (35S:Kan) (see figure 17). Eight months post transformation, 7 empty vector plants and 2 HSF3 transformed lines were recovered onto soil, Transformants were screened for the presence of the transgene using a 35S forward and HSF3 reverse primer, using 35S: Kan, empty vector controls and Arabidopsis HSF3 plant DNA as a positive control. Oniy one of the HSF3 transgenics (35S:HSF3 12.2) amplifies the 35S-HSF3 junction PCR product. The empty vector controls are also negative.

H₂O₂ of primary transgenics was measured (see figure 18). Foliar H₂O₂ levels are increased in line 35S:HSF3 12.2 compared to the empty vector controls.

Stomatal conductance and photosynthetic rate of the primary transgenics were also measured. Stomatal conductance is reduced in the 35S:HSF3 12.2 plant. Despite their reduction in stomatal conductance, the linear phase of photosynthesis is not affected in the 35S:HSF3 12.2 plants, however, photosynthesis saturates at lower levels compared to the empty vector controls and the second HSF3 line. We also analysed the transgenic plants by thermal imaging of primary transgenic lines of oils seed rape. It was found that the HSF3 transgenic (35S: HSF3 12.2) shows a 1⁰C warmer leaf temperature across the plant compared with flanking empty vector controls. This indicates less evaporative water loss by transpiration in the HSf3 transgenic compared with controls. This agrees with the lowered stomatal conductance values for the same line and is consistent with the observations made in 35S:HSF3 Arabidopsis plants.

Furthermore, gene expression data was analysed in the primary transgenics using qRT-PCR. PR1 gene expression is significantly higher in the 35S: HSF3 12.2 line compared with controls. This indicates activated pathogen defences.

We also analaysed APX2 gene expression. Increased expression in the 35S: HSF3 12.2 line shows functioning HSF3 and, from a physiological point of view, again indicates a change in leaf water status.

The data demonstrates that transformation of Brassica napus with a 35S:AMHSF3 construct overexpressing HSF3 from Arabidopsis produces similar results to those observed when overexpressing HSF3 from Arabidopsis in Arabidopsis.

List of references

Barbagallo et al 2003, Plant Physiol. 132, 485-493

Cook R J, 1998. PNAS. 95, 9711-9712.

Crute I R et al., 1996. The Plant Cell. 18, 1747-1755.

EEA 2000 Europe's environment: the third assessment. Chapter 8: Water.

Copenhagen. Available at: http://reports.eea.europa.eu/environmentai assessment report 2003 10/en

FAO 2003 Review of World Water Resources by Country. Water Reports 23,

123 pp. Rome:FAO.

Hu et at, 2006, PNAS 103, 35, 1287-12992 Kotak S et al., 2004. The Plant Journal 39, 98-112. Miller G et al., 2006. Animals of Botany. 98, 279-288. Morison JIL. et al. (2008) Proc Royal Society series B 363: 639-658. Muller et al 2002, Planta 216, 44-56 Nover L et al., 2001 Cell Stress & Chaperones. 6, 177-189. Panchuck I et al., 2002 Plant Physiology. 129, 838-853.

Panikulangara T et al., 2004. Plant Physiology. 136, 3148-3158 Parry M.A.J, et ai 2O05.Annals of Applied Biology 147, 211-226. Prandl R et al 1998 Molecular and General Genetics 259: 269-278. Qiang L et al., 2000. Chinese Science Bulletin. 45, 970-975. Rijsberman, F. 2004 Water scarcity - fact or fiction? R. A. Fischer, N. Turner, J. Angus, L. Mclntyre, M. Robertson, A. Borrell & D. Lloyd (eds) New Directions for a Diverse Planet. Proceedings of the 4th Internationa! Crop Science Congress, Brisbane, Australia, 26 September - 1 October, 2004. Available at: www.cropscience.org.au/icsc2004

Richards, R.A. 2004 Physiological traits used in the breeding of new cultivars for water-scarce environments. R. A. Fischer, N. Turner, J. Angus, L. Mclntyre, M. Robertson, A. Borrell & D. Lloyd (Eds) New Directions for a Diverse Planet. Proceedings of the 4th International Crop Science Congress, Brisbane, Australia, 26 September - 1 October, 2004. Available at: www.cropscience.orq.au/icsc2004 Sakuma Y et al., 2006. The Plant Cell. 18, 1292-1309. Vailiyodan B et al., 2006. Current Opinion in Plant Biology. 9, 1-7.

Claims

CLAIMS:

1. A method for improving water use efficiency and/or water productivity in plants comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.

2. A method for improving plant productivity comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant,

3. A method according to claim 1 or claim 2 wherein the method increases water use efficiency and/or water productivity under normal non drought conditions.

4. A method according to claim 1 or claim 2 wherein the method is used to improve water productivity under normal non drought conditions.

5. A method according to claim 1 or 2 wherein the method improves plant productivity under water deficit conditions.

6. A method according to claim 1 or 2 wherein the method is used to improve plant productivity under water deficit conditions.

7. A method according to claim 1 or claim 2 or wherein the method is used to confer plant drought tolerance.

8. A method according to claim 2 wherein the method confers pathogen resistance.

9. A method for conferring pathogen resistance in plants comprising introducing and overexpressing a polynucleotide sequence comprising or consisting of a plant Hsf into said plant.

10. A method according to a preceding claim wherein said plant is a cereal.

1 1. A method according to any of claims 1 to 10 wherein said plant is selected from maize, wheat, rice, oilseed rape, sorghum, soybean, cotton, potato, tomato or poplar.

12. A method according to a preceding claim wherein the plant Hsf is an Arabidopsis plant Hsf.

13. A method according to claim 12 wherein the Arabidopsis Hsf is selected from the group comprising AtHsfAla, AtHsfAl b, AtHsfAid, AtHsfAl e, AtHsfA2, AtHsfA3, AtHsfA4a, AtHsfA4c, AtHsfA5, AtHsfAθa, AtHsfA6b, AtHsfA7a, AtHsfA7b, AtHsfAδ, AtHsfA9, AtHsfBI , AtHsfB2a, AtHsfB2b, AtHsfB3, AtHsfB4 or AtHsfd .

14. A method according to claim 13 wherein the Arabidopsis Hsf is AtHsfAl b.

15. Use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving plant productivity.

16. Use of a polynucleotide sequence comprising or consisting of a plant Hsf in improving water use efficiency.

17. A use according to claim 15 or 16 for improving water productivity.

18. A use according to claim 17 where conditions are non drought conditions.

19. A use according to claim 15 or 16 for drought tolerance.

20. Use of a polynucleotide sequence comprising or consisting of a plant Hsf in conferring pathogen resistance.

21. A use according to any of claims 15 to 20 wherein said plant is a cereal.

22. A use according to any of claims 15 to 21 wherein the plant Hsf is an Arabidopsis plant Hsf.

23. A use according to claim 22 wherein the Arabidopsis Hsf is selected from the group comprising AtHsfAl a, AtHsfAl b, AtHsfAid, AtHsfAle, AtHsfA2, AtHsfA3, AtHsfA4a, AtHsfA4c, AtHsfAδ, AtHsfA6a, AtHsfA6b, AtHsfA7a, AtHsfA7b, AtHsfA8, AtHsfA9, AtHsfBI , AtHsfB2a, AtHsfB2b, AtHsfB3, AtHsfB4 or AtHsfd .

24. A use according to claim 23 wherein the Arabidopsis Hsf is AtHsfAl b.

25. A use according to any of claims 15 to 24 wherein said plant is selected from maize, wheat, rice, oilseed rape, sorghum, cotton, soybean, potato, tomato or poplar.

26. A method or use according to a preceding claim wherein the plant Hsf is a plant Hsf derivative. \