WO2007055826A1 - Modulation of fertility in monocots - Google Patents

Abstract

Materials and methods useful for controlling the unwanted spread of transgenic traits are disclosed. The methods can involve a female containing a transgene for a desired trait and a transgene that decreases expression of a cytosine methylation polypeptide, and a male-fertile plant carrying one or more transcription activators that activate expression of transgenes carried by the female. Pollination of the female by a male-fertile plant activates expression of both transgenes in the female. The resulting infertile seeds do not germinate, or germinate to form plants that are infertile.

Description

Modulation of Fertility in Monocots

TECHNICAL FIELD

The invention relates to methods and materials for maintaining the integrity of the germplasm of transgenic and conventionally bred plants. In particular, the invention pertains to methods and materials that can be used to minimize the unwanted transmission of transgenic traits.

INCORPORATION-BY-REFERENCE & TEXT

The material on the accompanying compact disc is hereby incorporated by reference into this application. The accompanying compact discs all contain one identical file, 18207-012WO1 - Sequence.txt, which was created on September 28, 2006. The file named 18207-012WO1 - Sequence.txt is 883 KB. The file can be accessed using Microsoft Word on a computer that uses Windows OS.

BACKGROUND Transgenic plants are now common in the agricultural industry. Such plants express novel transgenic traits such as insect resistance, stress tolerance, improved oil quality, improved meal quality and heterologous protein production. As more and more transgenic plants are developed and introduced into the environment, it is important to control the undesired spread of transgenic traits from transgenic plants to other traditional and transgenic cultivars, plant species and breeding lines.

While physical isolation and pollen trapping border rows have been employed to control transgenic plants under study conditions, these methods are cumbersome and are not practical for many cultivated transgenic plants. Effective ways to control the transmission and expression of transgenic traits without intervention would be useful for managing transgenic plants. SUMMARY

The present invention features methods and materials useful for controlling the transmission and expression of transgenic traits. The methods and materials of the invention facilitate the cultivation of transgenic plants without the undesired transmission of transgenic traits to other plants.

In one aspect, the invention features a method for making infertile seed. The method comprises permitting seed development to occur on a plurality of first monocotyledonous plants that have been pollinated by a plurality of second monocotyledonous plants. The first plants comprise a first nucleic acid, which comprises a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide. The second plants are male-fertile and comprise at least one activator nucleic acid comprising at least one coding sequence for a transcription activator that binds to the first recognition site. The transcription activator coding sequence has a regulatory region operably linked thereto. The seeds that develop on the first plants are infertile. The regulatory region for the transcription activator can be a constitutive promoter, or a broadly expressing promoter. The method can further comprise harvesting the seeds.

The first plants can further comprise a second nucleic acid. The second nucleic acid comprises a second transcription activator recognition site operably linked to a sequence to be transcribed. If so, the second plants can comprise an activator nucleic acid that encodes a first transcription activator that binds the first recognition site and a second transcription activator that binds the second recognition site. Alternatively, the second plants can comprise an activator nucleic acid encoding a first transcription activator that binds the first recognition site and a different activator nucleic acid encoding a second transcription activator that binds the second recognition site. The regulatory region of the first transcription activator can be a broadly expressing promoter and the regulatory region of the second transcription activator can be a maturing endosperm promoter. The promoter of the first transcription activator can be a p326 promoter. The promoter of the second transcription activator can be a 15 kD zein promoter, 16 IcD zein promoter, 19 kD zein promoter, 22 kD zein promoter, 27 kD zein promoter, Osgt-1 promoter, glutelin-1 gene promoter, /3-amylase gene promoter, or hordein gene promoter. The sequence to be transcribed can encode a preselected polypeptide. The seeds can have a statistically significant increase in the amount of the preselected polypeptide relative to seeds that lack the first nucleic acid. The preselected polypeptide can be an antibody or an enzyme. The plurality of first plants can be cytoplasmically male- sterile, or can be male-sterile due to nuclear male sterility.

The nucleic acid that decreases expression of a methylation status polypeptide can be an antisense nucleic acid to a sequence encoding a cytosine DNA methyltransferase. The nucleic acid that decreases expression of a methylation status polypeptide can be an antisense nucleic acid to a sequence encoding a decrease of DNA methylation (DDMl) polypeptide. The nucleic acid that decreases expression of a methylation status polypeptide can be transcribed into an interfering RNA to a sequence encoding a cytosine DNA methyltransferase. The nucleic acid that decreases expression of a methylation status polypeptide can be transcribed into an interfering RNA to a sequence encoding a DDMl polypeptide. The nucleic acid that decreases expression of a methylation status polypeptide can comprise all or part of the coding sequence for a polypeptide shown in Figures 1, 2 or 3.

In one aspect, the invention features a method for making a polypeptide. The method comprises providing seed produced by pollination of monocotyledonous plants and extracting a preselected polypeptide from the seed. Such seed comprises a first nucleic acid comprising a first recognition site for a transcription activator operably linked to a nucleic acid that decreases expression of a methylation status polypeptide, a second nucleic acid comprising a second recognition site for a transcription activator operably linked to a coding sequence for a preselected polypeptide, and an activator nucleic acid comprising at least one coding sequence for a transcription activator that binds to the recognition sites. Each of the at least one transcription activator has a regulatory region operably linked thereto. The seeds are infertile and have a statistically significant increase in the amount of the preselected polypeptide relative to seeds that do not contain the first nucleic acid. In another aspect, the invention features a method for making a polypeptide. The method comprises providing seed produced by pollination of monocotyledonous plants and extracting the preselected polypeptide from the seed. The seed comprises a first nucleic acid comprising a first recognition site for a transcription activator operably linked to a nucleic acid that decreases expression of a methylation status polypeptide, a second nucleic acid comprising a second recognition site for a transcription activator operably linked to a coding sequence for a preselected polypeptide, and at least one activator nucleic acid comprising a coding sequence for a transcription activator that binds to the first recognition site and a coding sequence for a transcription activator that binds to the second recognition site. Each of the transcription activators have a regulatory region operably linked thereto. The seeds are infertile and have a statistically significant increase in the amount of the preselected polypeptide relative to seeds that do not contain the first nucleic acid. The nucleic acid that decreases expression of a methylation status polypeptide can be an antisense nucleic acid to a sequence encoding a cytosine DNA methyltransferase or a decrease of DNA methylation polypeptide, an interfering RNA to a sequence encoding a cytosine DNA methyltransferase or a decrease in DNA methylation polypeptide, e.g., a full or partial sequence encoding a polypeptide shown in Figures 1, 2 or 3. In one aspect, the invention features a method for making infertile plants.

The method comprises permitting seed development to occur on a plurality of first monocotyledonous plants that have been pollinated by a plurality of second monocotyledonous plants, harvesting seeds from the plurality of first plants, and germinating the seeds. The first plants comprise a first nucleic acid, the first nucleic acid comprising a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide. The second plants are male- fertile, of the same species as the first plant, and comprise an activator nucleic acid comprising a regulatory region operably linked to a coding sequence for a first transcription activator that binds to the first recognition site. Plants that grow from the seeds are infertile. The regulatory region for the first transcription activator coding sequence can be a constitutive or broadly expressing promoter. The first plants can further comprise a second nucleic acid comprising a second transcription activator recognition site operably linked to a sequence to be transcribed. The first transcription activator can bind to the second recognition site. The second plants can further comprise a different activator nucleic acid encoding a second transcription activator that binds the second recognition site. The sequence to be transcribed can be a coding sequence for a preselected polypeptide.

The plants in the above methods can be a species from the genera Zea, Sorghum, Festuca, Festulolium, Panicum, Pannesetum, or Poa.

In one aspect, the invention features an article of manufacture comprising packaging material, a first type of monocotyledonous seeds within the packaging material and a second type of monocotyledonous seeds within the packaging material. The first type of seeds comprise at least one first nucleic acid comprising a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide. The second type of seeds comprise at least one activator nucleic acid comprising a regulatory region operably linked to a coding sequence for a transcription activator that binds to the first recognition site. Plants grown from the second type of seeds are male-fertile. The first type of seeds can further comprise a second nucleic acid comprising a second transcription activator recognition site operably linked to a sequence to be transcribed. The sequence to be transcribed can be a preselected polypeptide. Plants grown from the first type of seeds can be male-sterile. The ratio of the first type of seeds to the second type of seeds can be about 70:30 or greater. The at least one activator nucleic acid can encode a transcription activator that binds to the first recognition site, and a different transcription activator that binds to the second recognition site. The regulatory region for the transcription activator that binds the first recognition site can be a broadly expressing promoter and the regulatory region for the transcription activator that binds to the second recognition site can be a maturing endosperm promoter. In one aspect, the invention features a plant comprising a first nucleic acid comprising a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide, and at least one activator nucleic acid encoding a regulatory region operably linked to a transcription activator that binds to the first recognition site. The plant is infertile. The seeds or plants can be a species from the genera Zea, Sorghum, Festuca, Festulolium, Panicum, Pannesetum, or Poa.

The plant can further comprise a second nucleic acid comprising a second transcription activator recognition site operably linked to a sequence to be transcribed. The first and second nucleic acids can be a single nucleic acid molecule. The sequence to be transcribed can encode a preselected polypeptide. The at least one activator nucleic acid can comprise a transcription activator that binds to the first and second transcription activator recognition sites. Alternatively, the at least one activator nucleic acid can encode a transcription activator that binds to the first recognition site, and a different transcription activator that binds to the second recognition site. The regulatory region for the transcription activator that binds the first recognition site can be a broadly expressing promoter and the regulatory region for the transcription activator that binds to the second recognition site can be a maturing endosperm promoter. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows an alignment of cDNA ID 23631543, At METl, (SEQ ID NO: 33) with orthologous amino acid sequences gi37039880 (SEQ ID NO: 35); gi56130955 (SEQ ID NO: 37); gi 2895089 (SEQ ID NO: 39); gi2887280 (SEQ ID NO: 41); gi2895087 (SEQ ID NO: 43); gi2654108 (SEQ ID NO: 45); gi37201980 (SEQ ID NO: 47); gi 75219927 (SEQ ID NO: 49); gi50509186 (SEQ ID NO: 51); gi20977598 (SEQ ID NO: 53); gil 8653391 (SEQ ID NO: 55); and gi31126751 (SEQ ID NO: 57), and a consensus sequence.

Fig. 2 shows an alignment of cDNA ID 23965502 OsMETl (SEQ ID NO: 83) with orthologous amino acid sequences gi37201980 (SEQ ID NO: 85); gi50509186 (SEQ ID NO: 87); gi 75219927 (SEQ ID NO: 89); gi20977598 (SEQ ID NO: 91); gi2895089 (SEQ ID NO: 93); gi56130955 (SEQ ID NO: 95); gi2887280 (SEQ ID NO: 97); gi2895087 (SEQ ID NO: 99); gi2654108 (SEQ ID NO: 101); gi37039880 (SEQ ID NO: 103); gilO177145 (SEQ ID NO: 105); CeresClone 1368619 (SEQ ID NO: 107); gi7268119 (SEQ ID NO: 109); gi6523846 (SEQ ID NO: 111); gi7267541 (SEQ ID NO: 113); and gi4678387 (SEQ ID NO: 115), and a consensus sequence. Fig. 3 shows an alignment of cDNA ID 23505366 DDMl (SEQ ID NO: 2) with orthologous amino acid sequences gi 33086941 (SEQ ID NO: 4); gi 51536001 (SEQ ID NO: 6); Clone 1155680 (SEQ ID NO: 9); gi 18463957 (SEQ ID NO: 11); gi 34914698 (SEQ ID NO: 13); gi 37542688 (SEQ ID NO: 19); gi 45357049 (SEQ ID NO: 23); gi 23193481 (SEQ ID NO: 25); and gi 45357056 (SEQ ID NO: 27), and a consensus sequence.

DETAILED DESCRIPTION

The present invention is based on the discovery that an alteration in chromosomal 5' cytosine methylation status in monocotyledonous plants and plant cells can result in infertile seed and even total ablation of inflorescences. This discovery has led to novel means for effectively controlling the transmission of recombinant DNA-based traits from transgenic plants to other cultivars. A biocontainment system as described herein is based in part on the following components: 1) a transcription activator under the control of a selected plant promoter and 2) an upstream activation sequence (UAS) recognized by the transcription activator, that controls the expression of a methylation status polypeptide. These components, when combined genetically, result in transcription and expression of the methylation status polypeptide. Timely expression of such polypeptide results in the production of infertile seeds, i.e., seeds that are incapable of producing offspring. In some embodiments, infertile seeds do not germinate. In other embodiments, infertile seeds germinate and form seedlings that do not mature, e.g., seedlings that die before reaching maturity. In yet other embodiments, infertile seeds germinate and form mature plants that are incapable of forming seeds, e.g., that produce no floral structures or abnormal floral structures, or that cannot form gametes. The system is useful, inter alia, in species that can be readily cross- pollinated on a large scale.

I. Methods for Making Infertile Seeds and Plants

Infertile Seeds In one aspect, the invention features a method for making infertile seed. The method comprises permitting seed development to occur on a plurality of first monocotyledonous plants that have been pollinated by a plurality of second monocotyledonous plants. The first plants can be male-sterile and comprise a first nucleic acid. The first nucleic acid comprises a first transcription activator recognition site and a first promoter, that are operably linked to a nucleic acid that decreases expression of a methylation status polypeptide. An optional second nucleic acid may also be present. The second nucleic acid comprises a second transcription activator recognition site (UAS) and a second promoter, that are operably linked to a sequence of interest. The second plants are male-fertile and comprise at least one activator nucleic acid encoding at least one transcription activator and a promoter operably linked thereto. The transcription activator is effective for binding to one or both of the first and second recognition sites.

Upon pollination of the first, male-sterile plants by pollen from the second, male-fertile plants, seed development ensues. The activator nucleic acid carried by the pollen can be expressed prior to or during seed development, and the resulting transcription activator polypeptide activates transcription of the first nucleic acid in developing seeds on the male-sterile female plants. Transcription of the first nucleic acid causes seed infertility. Thus, unwanted spread of seeds produced by such pollination is effectively contained because all, or substantially all, of the seeds are infertile.

The activator nucleic acid carried by the pollen can also be expressed during or after maturation- of the resulting seeds, e.g., after germination and during vegetative development. The transcription activator polypeptide activates transcription of the first nucleic acid in vegetative tissue during plant growth. Transcription of the first nucleic acid results in seed infertility, i.e., either no, or substantially no, seeds are produced by such plants, or all, or substantially all, of the seeds that are produced are infertile.

In embodiments in which a second nucleic acid is present, all, or substantially all, of the resulting seeds have a statistically significant increase in the amount of the gene product of the sequence of interest, relative to seeds that do not contain or express the first nucleic acid. Seeds made in such a manner contain the first and second nucleic acids and the transcription activator nucleic acid. Unwanted spread of the gene product present in the seeds is contained because all, or substantially all, of the seeds are infertile. In some embodiments, the method comprises growing a plurality of plants of an apomictic species. The apomictic plants contain first and optional second nucleic acids as described above. Typically, the first nucleic acid comprises a first transcription activator recognition site and a first promoter, that are operably linked to a nucleic acid that decreases expression of a methylation status polypeptide. The second nucleic acid comprises a second transcription activator recognition site and a second promoter, each of which is operably linked to a sequence to be transcribed into a desired gene product. When such apomictic plants contain a second nucleic acid, they further comprise at least two activator nucleic acids that have different expression patterns. The second activator nucleic acid typically is expressed preferentially in tissues other than seed tissues and floral tissues, whereas the first activator nucleic acid typically is expressed preferentially in seed tissues, e.g., a second transcription activator for the second recognition site is operably linked to a vegetative tissue-specific promoter and a first transcription activator for the first recognition site is operably linked to a maturing endosperm promoter. In this embodiment, the second transcription activator polypeptide activates transcription of the second nucleic acid, and results in the production of a desired gene product in vegetative tissues, while transcription of the first nucleic acid confers seed infertility. Unwanted spread of the transgene responsible for the desired trait is effectively contained in such apomictic plants.

II. Nucleic Acids and Polypeptides

As used herein, nucleic acid refers to RNA or DNA, and can be single- or double-stranded. If single-stranded, a nucleic acid having a polypeptide coding sequence can be either the coding or the non-coding strand.

A nucleic acid can be made by, for example, chemical synthesis or the polymerase chain reaction (PCR). PCR refers to a procedure or technique in which target nucleic acids are amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach, C. & Dveksler, G., Eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid. Nucleic acids can be detected by methods such as ethidium bromide staining of agarose gels, Southern or Northern blot hybridization, PCR or in situ hybridizations. Hybridization typically involves Southern or Northern blotting (see, for example, sections 9.37-9.52 of Sambrook et ah, 1989, "Molecular Cloning, A Laboratory Manual", 2^nd Edition, Cold Spring Harbor Press, Plainview; NY). Probes should hybridize under high stringency conditions to a nucleic acid or the complement thereof. High stringency conditions can include the use of low ionic strength and high temperature washes, for example 0.015 M NaCl/0.0015 M sodium citrate (0.1X SSC), 0.1% sodium dodecyl sulfate (SDS) at 65⁰C. In addition, denaturing agents, such as formamide, can be employed during high stringency hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42⁰C.

As used herein, the term "percent sequence identity" refers to the degree of identity between any given query sequence and a subject sequence. A subject amino acid or nucleotide sequence typically has a length that is more than 80%, e.g., more than 82%, 85%, 87%, 89%, 90%, 93%, 95%, 97%, 99%, 100%, 105%, 110%, 115%, or 120%, of the length of the query sequence. A query nucleic acid or amino acid sequence is aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment).

ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: GIy, Pro, Ser, Asn, Asp, GIn, GIu, Arg, and Lys; residue- specific gap penalties: on. The output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi- align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw). To determine a "percent identity" between a query sequence and a subject sequence, the number of matching bases or amino . acids in the alignment is divided by the total number of matched and mismatched bases or amino acids, followed by multiplying the result by 100.

It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.

The term "exogenous" with respect to a nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non- natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration. For example, a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant. Such progeny are considered to contain the exogenous nucleic acid.

It will be appreciated that methods and compositions described herein may . be able to utilize non-transgenic plant cells or plants that carry a mutation in a methylation status polypeptide. For example, a plant carrying a T-DNA insertion, a deletion, a transversion mutation, or a transition mutation in the coding sequence for one of the aforementioned polypeptides can affect (e.g., decrease) genomic cytosine methylation levels.

III. Methylation Status Polypeptides

Methods and compositions described herein utilize a nucleic acid that decreases expression of a methylation status polypeptide, i.e., a polypeptide that affects the pattern and/or relative level of cytosine methylation within genomic DNA, either generally or in a segment thereof. Such polypeptides are capable of affecting the methylation status of DNA in vivo and in vitro, and changes in their expression can be used to bring about changes in the methylation status of DNA. These polypeptides can affect the methylation status of genomic DNA, a segment or portion of genomic DNA. Without being bound by theory, it is believed that decreasing expression of a methylation status polypeptide affects cytosine methylation status in chromatin and, via transcriptional gene activation and/or silencing, expression of one or more endogenous genes involved in gamete development, fertilization, and/or embryo development is altered, thereby resulting in infertile seeds. Polypeptides that affect methylation status are known to be present in a variety of organisms and are suitable for use in the methods described herein.

In some embodiments, such a polypeptide is a cytosine DNA methyltransferase. A number of methyl transferases (e.g., cytosine DNA methyltransferase) are known to catalyze the transfer of a methyl group to the C5 position of cytosine in DNA and play a role in the control of gene expression during development, including the polypeptide encoded by the Arabidopsis METl locus, the polypeptide encoded by the Arabidopsis MET2 locus, and orthologs thereof. See, e.g., SEQ ID NOS: 32-153, which describe orthologs and homologs of Arabidopsis and Oryza METl, and nucleic acids encoding them. .

In other embodiments, such a polypeptide is a decrease in DNA methylation 1 polypeptide (DDMl; SNF2 domain-containing proteins / helicase domain- containing proteins; e.g., At5g66750). See, e.g., SEQ ID NOS: 1-31, which describe orthologs and homologs of DDMl and nucleic acids encoding them. The DDMl polypeptide is found in the nucleosome, possesses an ATPase activity, and plays a role in methylation-dependent chromatin silencing. In some embodiments, a methylation status polypeptide is an ortholog, homolog or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, e.g., SEQ ID NOS: 6, 9, 11, 13 and 15, or the consensus sequence shown in Figure 3. In some embodiments, a methylation status polypeptide is an ortholog, homolog or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 33, e.g., SEQ ID NOS: 47, 49, 51 and 53, or the consensus sequence shown in Figure 1. In some embodiments, a methylation status polypeptide is an ortholog, homolog or variant of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 83, e.g., SEQ ID NOS: 85, 89, 91, 143 and 145, or the consensus sequence shown in Figure 2. In certain cases, a methylation status polypeptide comprises an amino acid sequence having about 80% or greater sequence identity to SEQ ID NO: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, or 115. Eighty percent sequence identity or greater can be about 82, 85, 87, 90, 92, 95, 96, 97, 98, 99, or 100% sequence identity to such a sequence. In some embodiments, a methylation status polypeptide comprises an amino acid sequence having about 80% or greater sequence identity to SEQ ID NO: 2, 4, 6, 9, 11, 13, 19, 23, 25, or 27. Eighty percent sequence identity or greater can be about 82, 85, 87, 90, 92, 95, 96, 97, 98, 99, or 100% sequence identity to such a sequence. Methylation status polypeptides that are suitable candidates for modulation can be identified in a variety of ways. For example, candidate methyltransferases can be screened to identify polypeptides that affect cytosine methylation by preparing nuclear extracts from axenic seedlings and incubating solubilized proteins from the extract with a hemi-methylated (CpI)_n substrate and radioactively labeled S-adenosyl-methionine. See, e.g., Kakutani et ah, Nucleic Acids Res. 93:12406- 12411 (1995).

Suitable methylation status polypeptides also can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify orthologs of the polypeptides having the amino acid sequence set forth in SEQ ID NOS: 2, 33 and 83. Sequence analysis can involve BLAST or PSI-BLAST analysis of nonredundant databases using amino acid sequences of known methylation status polypeptides. Those proteins in the database that have greater than 40% sequence identity can be candidates for further evaluation for suitability as methylation status polypeptides. If desired, manual inspection of such candidates can be. carried out. in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains suspected of being present in methylation status polypeptides. A consensus amino acid sequence for a methylation status polypeptide can be determined by aligning amino acid sequences {e.g., amino acid sequences related to SEQ ID NO: 2 and 83) from a variety of plant species and determining the most common amino acid or type of amino acid at each position. Consensus sequences are shown in Figures 1-3.

Typically, conserved regions of methylation status polypeptides exhibit at least 40% amino acid sequence identity {e.g., at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region of target and template polypeptides exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity. Amino acid sequence identity can be deduced from amino acid or nucleotide sequences. In certain cases, highly conserved domains have been identified within methylation status polypeptides. These conserved regions can be useful in identifying functionally similar methylation status polypeptides. Domains are groups of contiguous amino acids in a polypeptide that can be used to characterize protein families and/or parts of proteins. Such domains have a "fingerprint" or "signature" that can comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three-dimensional conformation. Generally, each domain has been associated with either a conserved primary sequence or a sequence motif. Generally these conserved primary sequence motifs have been correlated with specific in vitro and/or in vivo activities. Examples of domains that can be used to identify orthologous cytosine DNA methyltransferases include, without limitation, a methyltransferase catalytic activity domain, a "eukaryotic" domain, a PWWP domain, an Ado-Met binding site, a TS domain, a bromo-adjacent homology (BAH) domain, a Cys-rich domain, a GK repeat domain, a UBA domain, and a PC repeat domain.

The identification of conserved regions in a template, or subject, polypeptide can facilitate production of variants of wild type methylation status polypeptides. Conserved regions can be identified by locating a region within the primary amino acid sequence of a template polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing a variety of protein motifs and domains at sanger.ac.uk/Pfam and genome.wustl.edu/Pfam. A description of the information included at the Pfam database is described in Sonnhammer et al., 1998, Nucl. Acids Res. 26: 320-322; Sonnhammer et al, 1997, Proteins 28:405-420; and Bateman et al, 1999, Nucl Acids Res. 27:260-262.

Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate. For example, sequences from Arabidopsis and Zea mays can be used to identify one or more conserved regions.

In some embodiments, the amino acid sequence of a suitable subject polypeptide has greater than 40% sequence identity {e.g., > 40%, > 50%, > 60%, > 70% or > 80%) to the amino acid sequence of the query polypeptide. In some embodiments, the nucleotide sequence of a suitable subject nucleic acid has greater than 70% sequence identity (e.g., > 75%, > 80%, , >85%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, or > 99%) to the nucleotide. , sequence of the query nucleic acid. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It is also noted that the length value will always be an integer.

If desired, the classification of a polypeptide as a methylation status polypeptide can be determined by techniques known to those having ordinary skill in the art. These techniques can be divided into two general categories: global methylation analysis, and gene-specific methylation analysis. Global methylation analysis techniques, such as chromatographic methods and a methyl accepting capacity assay, allow the measurement of the overall level of methyl cytosines in genomic DNA. One global methylation analysis technique includes digesting total genomic DNA with Taql and labeling 5' terminal cytosines in the digest with radioactivity. The labeled DNA is then digested to mononucleotides and the amount of methylated and unmethylated cytosine is estimated using thin layer chromatography. See, e.g., Kakutani, et al., Nucl. Acids Res. 93: 12406-12411 (1995). In addition, techniques such as Restriction Landmark Genomic Scanning for Methylation (RLGS-M), and CpG island microarray can be used to identify unknown methylation hot-spots or methylated CpG islands in genomic DNA. Gene- specific methylation analysis techniques include the use of methylation sensitive restriction enzymes to digest DNA, followed by Southern detection or PCR amplification. For example, the methylation of single copy and repetitive sequences can be estimated from the digestion pattern observed in Southern blots of genomic DNA digested with Hpaϊl or Mspl. See, Jeddeloh et al, Ylant J. 9:5.79-586 (1996) and Finnegan et al., Proc. Natl. Acad. ScL USA 93:8449-8454 (1996). In addition, techniques based on bisulfite reaction are known, and include methylation specific PCR (MSP) and bisulfite genomic sequencing PCR. Other techniques include the use of hydrazine or potassium permanganate and ligation-mediated PCR. A recombinant construct utilized in the methods and compositions described herein contains a nucleic acid that decreases the amount of transcription or translation product of a gene encoding a methylation status polypeptide, e.g., decreases the stability of, reduces the accumulation of, or decreases the translation of, an mRNA for such a polypeptide. Examples of nucleic acids that can affect expression of a methylation status polypeptide include antisense nucleic acids, ^' ribozyme nucleic acids, or interfering RNA nucleic acids. Such nucleic acids are typically targeted in a plant or plant cell to a cytosine DNA methyltransferase or a DDMl polypeptide. As discussed below, such nucleic acids preferably are targeted to a cytosine DNA methyltransferase that is endogenous to the plant in which the nucleic acid will be introduced.

In some embodiments, a nucleic acid that decreases the amount of transcription or translation product of a gene encoding a methylation status polypeptide is transcribed into an antisense nucleic acid or an interfering RNA similar or identical to the sense coding sequence of SEQ ID NOS: 5, 7, 8, 14, 16,

18, 20, 22, 24, 26, 28 or 30. In some embodiments, a nucleic acid that decreases the amount of transcription or translation product of a gene encoding a methylation status polypeptide is transcribed into an antisense nucleic acid or an interfering RNA similar or identical to the sense coding sequence of SEQ ID NOS: 46, 48, 50, 52, 56, 58, 60, 66, 72, 76 or 78. In some embodiments, a nucleic acid that decreases the amount of transcription or translation product of a gene encoding a methylation status polypeptide is transcribed into an antisense nucleic acid or an interfering RNA identical to all or part of the sense coding sequence of SEQ ID NOS: 84, 86, 88, 90, 116, 118, 124, 126, 128, 130, 134, 138, 140, 142, 144, 148, 150 or 152. In such embodiments, the antisense nucleic acid or interfering RNA is from about 15 nucleotides to about 2,500 nucleotides in length, or any integer therebetween as described herein. For example, the length of the antisense nucleic acid or interfering RNA nucleic acid can be 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, or 1500 nucleotides. The antisense nucleic acid or interfering RNA can have a length in a range from 20 nucleotides to 100 nucleotides, or 20 nucleotides to 40 nucleotides, or 50 nucleotides to 250 nucleotides, or 100 nucleotides to 350 nucleotides, or 150 nucleotides to 500 nucleotides, or 250 nucleotides to 600 nucleotides, or 400 nucleotides to 800 nucleotides, or 500 nucleotides to 900 nucleotides, or 700 nucleotides to 1200 nucleotides or 900 nucleotides to 1300 nucleotides, or 1100 nucleotides to 1500 nucleotides, or 1300 nucleotides to 1600 nucleotides, or 1500 nucleotides to 1800 nucleotides, or 1800 nucleotides to 2100 nucleotides, or 2000 nucleotides to 2300 nucleotides, or 2100 nucleotides to 2400 nucleotides, or 2100 nucleotides to 2500 nucleotides.

Thus, for example, a suitable nucleic acid can be an antisense nucleic acid to one of the aforementioned genes encoding a methylation status polypeptide. Alternatively, the transcription product of a nucleic acid can be similar or identical to the sense coding sequence of a methylation status polypeptide, but is an RNA that is unpolyadenylated, lacks a 5' cap structure, or contains an unsplicable intron. In some embodiments, the nucleic acid is a partial or full-length coding sequence that, in sense orientation results in inhibition of the expression of an endogenous polypeptide by co-suppression. Methods of co-suppression using a full-length cDNA sequence as well as a paitial cDNA sequence are known in the art. See, e.g., U.S. Patent No. 5,231,020.

A suitable nucleic acid can be transcribed into an interfering RNA. Such an RNA can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure. One strand of the stem portion of a double stranded RNA can comprise a sequence that is similar or identical to the sense coding sequence of an endogenous polypeptide, and that is from about 15 nucleotides to about 2,500 nucleotides in length. The length of the nucleic acid sequence that is similar or identical to the sense coding sequence can be from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides. The other strand of the stem portion of a double stranded RNA can comprise an antisense sequence of an endogenous polypeptide, and can have a length that is shorter, the same as, or longer than the length of the corresponding sense sequence. The loop portion of a double stranded RNA can be from 10 nucleotides to 500 nucleotides in length, e.g., from 15 nucleotides to 100 nucleotides, from 20 nucleotides to 300 nucleotides, or from 25 nucleotides to 400 nucleotides in length. The loop portion of the RNA can include an intron. See, e.g., WO 98/53083; WO 99/32619; WO 98/36083; WO 99/53050; and US patent publications 20040214330 and 20030180945. See also, U.S. Patents 5,034,323; ^■ 6,452,067; 6,777,588; 6,573,099; and 6,326,527.

A suitable interfering RNA can be constructed as described in Brummell, et. al, Plant J. 33:793-800 (2003). Examples of RNAi nucleic acids are shown in SEQ ID NOS: 169 and 171. SEQ ID NO: 169 comprises about 0.6 kb of a rice cytosine DNA methyltransferase sense strand (N-terminal region) and an inverted repeat of a nos terminator sequence. SEQ ID NO: 171 comprises about 0.7 kb of a rice cytosine DNA methyltransferase sense strand (C-terminal region) and an inverted repeat of a nos terminator sequence. Nucleic acid sequences for the N and C- terminal domains of the rice cytosine DNA methyltransferase are shown in SEQ ID NOS: 170 and 172.

IV. Transcription activators

A transcription activator is a polypeptide that binds to a recognition site in DNA, resulting in an increase in the level of transcription from a promoter operably linked in cis with the recognition site. Suitable transcription activators include, without limitation, plant transcription activators, chimeric transcription activators and yeast transcription activators. Plant transcription activators typically are from a species that is in a different taxonomic genus from plants used in a method, are from a species that is geographically widely separated from plants used in a method, and/or are from a species where the timing or tissue specificity of naturally occurring expression differs from that occurring in plants used in a method. If desired, a transcription activator can be tested for its allergenic properties and those that are non-allergenic selected for use. Suitable transcription activators include YAP 1 , YAP2, SKO 1 , zinc finger protein MIG 1 , ABFl and UME6, all of which are from yeast. Other suitable transcription activators include AtERFl, AtERF2, AtERF5, CBFl and Athb-1, all of which are from plants. See, e.g., Fujimoto, S. Y. et al. (2000) Plant Cell 12:393-404; Stockinger, E. J. et al. (1997) Proc. Natl. Acad. Sci. USA 94:1035-40; and Aoyama, T. et al. ^1995^ Plant Cell 7: 1773-85.

Many transcription activators have discrete DNA binding and transcription activation domains. Thus, DNA binding domain(s) and transcription activation domain(s) of a suitable transcription activator can be derived from different sources, i.e., can be a chimeric transcription activator. For example, a transcription activator can have a DNA binding domain derived from the yeast gal4 gene and a transcription activation domain derived from the VP 16 gene of herpes simplex virus. In other embodiments, a transcription activator can have a DNA binding domain derived from a yeast HAPl gene and the transcription activation domain derived from VP 16. In yet other embodiments, a transcription activator can have a DNA binding domain derived from a yeast gal4 or HAPl gene and a transcription activation domain derived from a maize Cl gene. See, e.g., Guyer et al., Genetics 149:633-639 (1998). Transcription activation domains from the maize DOFl- and rice RISBZl transcription activators can also be used in a chimeric transcription activator. The Table below sets forth other plant transcription activation domains that can be used in a chimeric transcription activator.

Transcription Activation Domains

In some embodiments, a chimeric transcription activator contains a non- naturally occurring DNA -binding domain. Non-naturally occurring domains that selectively bind to a specific DNA sequence can be generated using methods known in the art. See, e.g., U.S. Pat. No. 5,198,346.

Populations of transgenic organisms or cells having a first nucleic acid construct and an activator nucleic acid can be produced by transformation, transfection, or genetic crossing. See, e.g., WO 97/31064.

In some embodiments, a single activator nucleic acid encodes two different transcription activators, one of which binds to the first recognition site and the other of which binds to the second recognition site. Alternatively, two different transcription activators can be encoded by separate nucleic acids. In either. case, each of the transcription activators can have a different expression pattern, e.g., the transcription activator for the first recognition site can be operably linked to a constitutive promoter and the transcription activator for the second recognition site can be operably linked to a maturing endosperm promoter. In other embodiments, both transcription activators are operably linked to different, maturing endosperm promoters.

V. Sequence of Interest

Typically, the desired gene product of a sequence of interest is a preselected polypeptide. A preselected polypeptide can be any polypeptide (i.e., 5 or more amino acids joined by a peptide bond). Plants have been used to produce a variety of preselected industrial and pharmaceutical polypeptides, that lead to the production of high value chemicals, modified and specialty oils, enzymes, and renewable non-foods such as fuels anaplastics, vaccines and antibodies. See e.g., Owen, M. and Pen, J. (eds.), 1996. Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins. John Wiley & Son Ltd.; Austin, S. et al., 1994. Annals NY Acad. ScL 721:234-242; Austin, S. et al., 1995. Euphytica 85: 381- 393; Ziegelhoffer, T. et al., 1998. Molecular Breeding. US Pat. No. 5,824,779 discloses phytase-protein-pigmenting concentrate derived from green plant juice. US Pat. No. 5,900,525 discloses animal feed compositions containing phytase derived from transgenic alfalfa. US Pat. No. 6,136,320 discloses vaccines produced in transgenic plants. U.S. 6,255,562 discloses insulin. U.S. Patent 5,958,745 discloses the formation of copolymers of 3-hydroxy butyrate and 3-hydroxyvalerate. U.S. Pat. No. 5,824,798 discloses starch synthases. U.S. Patent 6,303,341 discloses immunoglobulin receptors. U.S. Patent 6,417,429 discloses immunoglobulin heavy- and light-chain polypeptides. U.S. Patent 6,087,558 discloses the production of proteases in plants. U.S. Patent 6,271,016 discloses an anthranilate synthase gene for tryptophan overproduction in plants. Thus, a preselected polypeptide can be an industrial enzyme such as alpha-amylase, gluoamylase or glucose isomerase, or a pharmaceutical polypeptide such as an antibody or a polypeptide in Table 1. A preselected polypeptide can be an antibody or antibody fragment. An antibody or antibody fragment includes a humanized or chimeric antibody, a single chain Fv antibody fragment, an Fab fragment, and an F(ab)₂ fragment. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region. Antibody fragments that have a specific binding affinity can be generated by known techniques. Such antibody fragments include, but are not limited to, F(ab')₂ fragments that can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by deducing the disulfide bridges of F(ab')₂ fragments. Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a single chain polypeptide. Single chain Fv antibody fragments can be produced through standard techniques, such as those disclosed in U.S. Patent No. 4,946,778. Plant glycans are often non-immunogenic in animals or humans. However, if desired, glycosylation sites can be identified in a preselected polypeptide, and relevant glycosyl transferases can be expressed in parallel with expression of the preselected polypeptide. Alternatively, it may be desirable to prevent glycosylation of a preselected polypeptide, by engineering N-acetylglucosaminyltransferase knock-out plants. If a preselected polypeptide is an antibody or antibody fragment, Asn-X-Ser/Thr sites in the antibody can be deleted. In some embodiments, the gene product of a sequence to be transcribed is one of the preselected polypeptides in the Table below.

Table 1.

In some embodiments, a sequence to be transcribed results in a desired gene product that is an RNA. Such an RNA, made from a sequence to be transcribed, can be useful for inhibiting expression of an endogenous gene. Suitable DNAs from which such an RNA can be made include an antisense construct and a co- suppression construct. Thus, for example, a sequence to be transcribed can be similar or identical to the sense coding sequence of an endogenous polypeptide, but is transcribed into a mRNA that is unpolyadenylated, lacks a 5' cap structure, or contains an unsplicable intron. Alternatively, a sequence to be transcribed can incorporate a sequence encoding a ribozyme. In some embodiments, a sequence to be transcribed can include a sequence that is transcribed into an interfering RNA. Such an RNA can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure. One strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of an endogenous polypeptide, and that is from about 10 nucleotides to about 2,500 nucleotides in length. The length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, e.g., from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides. The other strand of the stem portion of a double stranded RNA comprises an antisense sequence of an endogenous polypeptide, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence. The loop portion of a double stranded RNA can be from 10 nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides. The loop portion of the RNA can include an intron. See, e.g., WO 99/53050. See, e.g., WO 98/53083; WO 99/32619; WO 98/36083; and WO 99/53050. See also, U.S. Patent 5,034,323. Useful RNA gene products are described in, e.g., U.S. 6,326,527. In some embodiments, a preselected polypeptide is a polypeptide that confers herbicide resistance on plants expressing the polypeptide. Herbicide resistance is also sometimes referred to as herbicide tolerance. Polypeptides conferring resistance to a herbicide that inhibits the growing point or meristem, sixch as an imidazolinone or a. sulfonylurea can be suitable. Exemplary polypeptides in this category code for mutant ALS and AHAS enzymes as described, for example, in U.S. 5,767,366 and 5,928,937. U.S. 4,761,373 and 5,013,659 are directed to plants resistant to various imidazolinone or sulfonamide herbicides. U.S. Pat. No. 4,975,374 relates to plant cells and plants containing a gene encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that are known to inhibit GS, e.g. phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,162,602 discloses plants resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The resistance is conferred by an altered acetyl coenzyme A carboxylase(ACCase).

Polypeptides for resistance to glyphosate (sold under the trade name Roundup®) are also suitable. See, for example, U.S. Pat. No. 4,940,835 and U.S. Pat. No. 4,769,061. U.S. Pat. No. 5,554,798 discloses transgenic glyphosate resistant maize plants, in which resistance is conferred by an altered 5-enolpyruvyl- 3-phosphoshikimate (EPSP) synthase. Such polypeptides can confer resistance to glyphosate herbicidal compositions, including without limitation glyphosate salts such as the trimethylsulphonium salt, the isopropylamine salt, the sodium salt, the potassium salt and the ammonium salt. See, e.g., U.S. Patents 6,451,735 and •

6,451,732.

Polypeptides for resistance to phosphono compounds such as glufosinate ammonium or phosphinothricin, and pyridinoxy or phenoxy propionic acids and cyclohexones are also suitable. See European application No. 0 242 246. See also,

U.S. Patents 5,879,903, 5,276,268 and 5,561,236.

Other herbicides include those that inhibit photosynthesis, such as a triazine and a benzonitrile (nitrilase). See U.S. Pat. No. 4,810,648. Other herbicides include

2,2-dichloropropionic acid, sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil. Also suitable are herbicides that confer resistance to a protox enzyme.

See, e.g., U.S. Patent Application No. 20010016956, and U.S. Patent 6,084,155. It will be recognized that more than one sequence to be transcribed can be present in some embodiments. For example, coding sequences for two preselected polypeptides may be present on the same or different nucleic acids, and encode polypeptides useful for manipulating a biosynthetic pathway. Alternatively, two coding sequences may be present and encode polypeptides found in a single protein, e.g., a heavy-chain immunoglobulin polypeptide and a light-chain immunoglobulin polypeptide, respectively.

VI. Markers

Optionally, marker genes can also be included in plants to help distinguish the infertile seeds and plants described herein from other types of plants. Such markers include genes that result in the production of colored products that are not normally found in other commercial crops. Such markers can also be used to confirm that pollination has occurred between first and second plants and/or to indicate that expression of a desired gene product is likely occurring. Marker genes comprise a coding sequence for a marker polypeptide operably linked to a UAS recognized by a transcription activator.

VI. Regulatory regions A recombinant nucleic acid construct disclosed herein typically includes one or more regulatory regions. The term "regulatory region" refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of the transcript or polypeptide product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and other regulatory regions that can reside within coding sequences, such as secretory signals and protease cleavage sites.

As used herein, the term "operably linked" refers to positioning of a regulatory region and a transcribable sequence in a nucleic acid so as to allow_. or facilitate transcription of the transcribable sequence. For example, to bring a coding sequence under the control of a promoter, it typically is necessary to position the translation initiation site of the translational reading frame of the coding sequence . _; . between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation start site, or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element such as an upstream element. Such elements include upstream activation regions (UARs) and, optionally, other DNA sequences that affect transcription of a polynucleotide such as a synthetic upstream element. The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell or tissue specificity. It is a routine matter for one of skill in the art to modulate expression by appropriately selecting and positioning promoters and other regulatory regions relative to an operably linked sequence. Some suitable promoters initiate transcription only, or predominantly, in certain cell types. For example, a promoter specific to a reproductive tissue (e.g., fruit, ovule, seed, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo, zygote, endosperm, integument, seed coat or pollen) can be used. A cell type or tissue-specific promoter, however, may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a cell type or tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos, et al., Plant Cell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044 (1988); Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., Plant Physiology 110: 1069-1079 (1996).

Examples of various classes of promoters are described below. Some of the promoters indicated below are described in more detail in U.S. Patent Application . .. Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,609; 60/583,691; 60/612,891; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 110/950,321; 0/957,569; 11/058,689; 11/097,589; 11/172,703; 11/208,308; 11/233,726; , 11/274,890; 11/360,017; 11/408,791; 11/414,142; PCT/US05/011105; PCT/US05/034308; and PCT/US05/23639. Nucleotide sequences of promoters are set forth in SEQ ID NOS: 154-168. It will be appreciated that a promoter may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a different classification based on its activity in another plant species. Constitutive Promoters

Constitutive promoters can promote transcription of an operably linked nucleic acid under most, but not necessarily all, environmental conditions and states of development or cell differentiation. Non-limiting examples of constitutive promoters that can be included in the nucleic acid constructs provided herein include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the mannopine synthase (MAS) promoter, the 1' or 2' promoters derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 35S promoter, and actin promoters such as the rice actin promoter, ubiquitin promoters such as the maize ubiquitin- 1 promoter.

Broadly Expressing Promoters A promoter can be said to be "broadly expressing" when it promotes transcription in many, but not all, plant tissues. For example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds. In certain cases, a broadly expressing promoter operably linked to a sequence can promote transcription of the linked sequence in a plant shoot at a level that is at least two times, e.g., at least 3, 5, 10, or 20 times, greater than the level of transcription in a developing seed. In other cases, a broadly expressing promoter can promote transcription in a plant shoot at a level that is at least two times, e.g., at least 3, 5, 10, or 20 times, greater than the level of transcription in a reproductive tissue of a flower. In view of the above, the CaMV 35S promoter is not considered a^' broadly expressing promoter. Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326, YP0158, YP0214, YP0380, PT0848, PTO633, YP0050, YP0144 and YP0190 promoters. Root-Specific Promoters

Root-specific promoters confer transcription only or predominantly in root tissue, e.g., root endodermis, root epidermis or root vascular tissues. Root-specific promoters include the YP0128, YP0275, PT0625, PT0660, PT0683 and PT0758 promoters. Promoter p32449 has preferential activity in roots, and somewhat less activity in other vegetative tissues. Other examples of root-specific promoters include the root specific subdomains of the CaMV 35 S promoter (Lam et al., Proc Natl Acad Sd USA 86:7890-7894 (1989)), root cell specific promoters reported by Conkling et al. Plant Physiol. 93: 1203-1211 (1990), and the tobacco RD2 gene promoter.

Maturing Endosperm-Specific Promoters

In some embodiments, promoters that preferentially drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm- specific promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Non-limiting examples of maturing endosperm-specific promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin gene promoter (Bustos et al, Plant Cell l(9):839-853 (1989)), the soybean trypsin inhibitor_.promoter (Riggs et al, Plant Cell l(6):609-621 (1989)), the ACP promoter (Baerson et al, Plant MoI Biol, 22(2):255-267 (1993)), the stearoyl-ACP desaturase gene (Slocombe et al, Plant Physiol 104(4): 167-176 (1994)), the soybean d subunit of /3-conglycinin promoter (Chen et al, Proc Natl Acad Sci USA 83:8560-8564 (1986)), the oleosin promoter (Hong et al, Plant MoI Biol 34(3):549-555 (1997)), zein promoters such as the 15 IcD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 IcD zein promoter and 27 kD zein promoter. Also suitable are the Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al, MoI Cell Biol. 13:5829-5842 (1993)), the β- amylase gene promoter, and the barley hordein gene promoter. Other maturing endosperm-specific promoters include the YP0092, PT0676 and PT0708 promoters. Ovary Tissue Promoters Promoters that are active in ovary tissues such as the ovule wall, e.g., mesocarp and carpel wall, can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, and the melon actin promoter. Embryo Sac/Early Endosperm Promoters To achieve embryo sac and/or early endosperm specific expression, regulatory regions can be used that preferentially drive transcription in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. A pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac or early endosperm specific promoters, although transcription typically decreases significantly in later endosperm development during the cellularization phase.

Expression in the zygote or developing embryo typically is not present with embryo sac or early endosperm promoters.

Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous- 1 (see, GenbankNo. U93215); Arabidopsis armycl (see, Urao (1996) Plant MoI Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBankNo. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Patent 6,906,244). Other promoters that may be suitable include those derived from the following genes: maize MACl (see, Sheridan (1996) Genetics, 142:1009-1020); maize Cat3 (see, GenBankNo. L05934; Abler (1993) Plant MoI. Biol, 22:10131-1038); Arabidopsis viviparous-1 (see, Genbank No. U93215); Arabidopsis atmycl (see, Urao ( 1996) Plant MoI. Biol. , 32:571-57; Conceicao (1994) Plant, 5:493-505). Other promoters include the following Arabidopsis promoters: YP0039, YPOlOl, YP0102, YPOI lO, YP0117, YPOl 19, YP0137, DME, YP0285 and YP0212. Other promoters that may be useful include the following rice promoters: p530cl0, pOsFIE2-2, pOsMEA, pOsYpl02, pOsYp285, having SEQ ID NOS: 162, 155, 156, 157, 158, respectively. Embryo-Specific Promoters

Regulatory regions that preferentially drive transcription in zygotic cells following fertilization can provide embryo-specific expression. Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart stage, but expression in late stage and maturing embryos is also suitable.

Embryo-specific promoters include the barley lipid transfer protein (Ltpl) promoter. Plant Cell Rep (2001) 20:647-654.

Photosvnthetically-Active Tissue Promoters Photosynthetically-active tissue promoters confer transcription only or predominantly in photosynthetically active tissue. Examples of such promoters include the ribulose-l,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricina), the pine cab6 promoter (Yamamoto et al, Plant Cell Physiol. 35:173-11% (1994)), the Cab-1 gene promoter from wheat (Fejes et al, Plant MoI. Biol. 15:921-932 (1990)), the CAB-I promoter from spinach (Lubberstedt et al, Plant Physiol. 104:997-1006 (1994)), the cablR promoter from rice (Luan et al, Plant Cell 4:971-981 (1992)), the pyruvate, orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al, Proc Natl Acad. Sd USA 90:9586-9590 (1993)), the tobacco Lhcbl*2 promoter (Cerdan et al, Plant MoI. Biol 33:245-255 (1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al, Planta. 196:564-570 (1995)), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS).

Basal Promoters

A basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation. Basal promoters frequently include a "TATA box" element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation. Basal promoters also may include a "CCAAT box" element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site. Other Promoters

Other classes of promoters include, but are not limited to, inducible promoters, such as promoters that confer transcription in response to external stimuli such as chemical agents, developmental stimuli, or environmental stimuli. Promoters designated YP0086, YP0188, YP0263, PT0758; PT0743; PT0829;

YPOl 19; and YP0096, as described in the above-referenced patent applications, may be useful.

Other Regulatory Regions

A 5' untranslated region (UTR) can be included in nucleic acid constructs described herein. A 5' UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide. A 3' UTR can be positioned between the translation termination codon and the end of the transcript. UTRs can have particular functions such as increasing mRNA message stability or translation attenuation. Examples of 3 ' UTRs include, but are not limited to polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.

A suitable enhancer is a cis-regulatory element (-212 to -154) from the upstream region of the octopine synthase (ocs) gene. Fromm et al., Plant Cell 1:977- 984 (1989). Recombinant nucleic acid constructs provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers. A marker gene can confer a selectable phenotype on a plant cell. For example, a marker can confer, biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or a herbicide (e.g., glyphosate, chlorosulfuron, glufosinate, or phosphinothricin). In addition, a recombinant nucleic acid construct can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of a polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, CT) sequences typically are expressed as a. fusion with the encoded polypeptide. Such tags can be inserted within the polypeptide, or at either the carboxyl or amino terminus.

It will be understood that more than one regulatory region may be present, in a recombinant nucleic acid, e.g., introns, enhancers, upstream activation regions, and inducible elements. Thus, more than one regulatory region can be operably linked to the sequence for a methylation status polypeptide.

VII. Regulatory Regions for Transcription Activators

For expression of a transcription activator that is to activate expression of a nucleic acid that decreases expression of a methylation status polypeptide, a transcription activator coding sequence is operably linked to a promoter from one of the classes described above. In some embodiments, a promoter is used that has greater expression in endosperm or embryo, and lower expression in other plant tissues. Such promoters include maturing endosperm and embryo sac/early endosperm promoters. However, in some embodiments, a constitutive promoter, a broadly expressing promoter, or a photosynthetically active tissue promoter is used.

VIII. Recognition Sites and Nucleic Acids that Decrease Expression of a Methylation Status Polypeptide

A nucleic acid that decreases expression of a methylation status polypeptide is operably linked to a recognition site for the transcription activator that is used to activate transcription of the nucleic acid. For example, a gal4 UAS recognition site would be operably linked to such a nucleic acid when a chimeric gal4-VP16 chimeric transcription activator is to be used to activate transcription. As another example, a Hapl recognition site is operably linked to such a nucleic acid when a chimeric Hapl -VP 16 chimeric transcription activator is to be used to activate transcription. It will be appreciated that more than one copy of a UAS can be operably linked to a nucleic acid that decreases expression of a methylation status polypeptide, i.e., 2, 3, 4, 5, or more than 5 copies of a UAS can be used in order to achieve the desired level of activation of the nucleic acid.

A nucleic acid that decreases expression of a methylation status polypeptide is also operably linked to a promoter. Typically, a basal promoter is used.

IX. Recognition Sites and Desired Gene Products

For expression of a sequence of interest, the sequence is operably linked to a recognition site for a transcription activator and, optionally, a promoter. The recognition site can be the same as that used with a nucleic acid that decreases expression of a methylation status polypeptide. Alternatively, a different recognition site (in conjunction with a different transcription activator) can be used. Different recognition sites for a sequence of interest and a methylation status nucleic acid can be used, for example, when it is desirable to delay expression of the sequence of interest relative to expression of the methylation status nucleic acid.

Other recognition sites and their cognate transcription activators that can be used include those listed in the Table below, all of which are from Saccharomyces cerevisiae. See, e.g., Fernandes et al., MoI. Cell Biol. 17:6982-93 (1997); Nehlin et al.. Nucleic Acids Res. 20:5271-8 (1992); Lundin et al, MoI. Cell Biol. 14:1979-85 (1994); Buchman et al., (1988) MoI. Cell. Biol. 8: 210-225 (1988); and Williams et al. Proc Natl Acad Sci U S A. 99:13431-62002 (2002).

Table 2.

A promoter suitable for being operably linked to a sequence of interest can, if desired, have greater expression in one or more tissues of a developing embryo or developing endosperm once activation by a transcription activator has occurred. For example, such a promoter can have greater expression in the aleurone layer, or parts of the endosperm such as chalazal endosperm.

If the gene product of a sequence of interest is targeted to endosperm and encodes a polypeptide, accumulation of the product can be facilitated by fusing certain amino acid sequences to the amino- or carboxy-terminus of the polypeptide. Such amino acid sequences include KDEL and HDEL, which facilitate targeting of the polypeptide to the endoplasmic reticulum. A histone can be fused to the polypeptide, which facilitates targeting of the polypeptide to the nucleus. Extensin can be fused to the polypeptide, which facilitates targeting to the cell wall. A seed storage protein can be fused to the polypeptide,. which facilitates targeting to protein bodies in the endosperm or cotyledons.

X. Transgenic Plants and Cells

A plant or plant cell used in the methods above contains one or more recombinant nucleic acid constructs as described herein. The plant or plant cell can be transformed and have the construct integrated into its genome, i.e., be stably transformed. Stably transformed cells typically retain the introduced nucleic acid sequence with each cell division. The plant or plant cells can also be transformed and have the construct not integrated into its genome. Such transformed cells are called transiently transformed cells. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after sufficient number of cell divisions. Either transiently transformed or stably transformed transgenic plants and plant cells can be useful in the methods described herein. Stably transformed plants are made by steps that include but are not limited to: introducing the recombinant nucleic acid construct into recipient plant cells, selecting or screening for plants derived from the recipient cells that have stably incorporated the nucleic acid, and identifying those stably transformed plants that express the gene product of the nucleic acid at a desired expression level. For example, the desired expression level for a recombinant nucleic acid that decreases the amount of transcription or translation product of a gene encoding a methylation status polypeptide, operably linked to a recognition site for a transcription activator, can be determined by standard techniques such as RT-PCR. As an alternative, the desired expression level for such a recombinant nucleic acid can be determined by allowing pollination of a stably transformed plant having such a nucleic acid with pollen from a plant expressing the cognate transcription activator. The formation of infertile seeds indicates that the desired expression level has been achieved.

Typically, transgenic plant cells used in methods described herein constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to , other species or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. Progeny includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F₁, F₂, F₃, F₄, F₅, F₆ and subsequent generation plants, or seeds formed on BC₁, BC₂, BC₃, and subsequent generation plants, or seeds formed on F₁B-C₁, F₁BC₂, FjBC₃, and subsequent generation plants. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct. In other embodiments, transgenic plant cells are grown in suspension culture, or tissue or organ culture, for production of a desired gene product. For the purposes of this invention, solid and/or liquid tissue culture techniques can be used. When using solid medium, transgenic plant cells can be placed directly onto the medium or can be placed onto a filter film that is then placed in contact with the medium. When using liquid medium, transgenic plant cells can be placed onto a floatation device, e.g., a porous membrane that contacts the liquid medium. Solid medium typically is made from liquid medium by adding agar. For example, a solid medium can be Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g., kinetin. Techniques for introducing exogenous nucleic acids into monocotyledonous plants are known in the art, and include, without limitation, Agrobacterium- mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Patents 5,538,880, 5,204,253, 6,329,571 and 6,013,863. If a cell or tissue culture is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.

The polynucleotides and vectors described herein can be used to transform a number of monocotyledonous plants and plant cell systems, including monocots such as banana, barley, bluegrass, date palm, fescue, field corn, garlic, millet, oat, . oil palm, onion, pineapple, popcorn, rice, rye, ryegrass, sorghum, sudangrass, sugarcane, sweet corn, switchgrass, turf grasses, and wheat. Thus, the methods and compositions described herein can be used with monocotyledonous plants such as those belonging to the orders Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanihales, Cyperάles, Eriocaulales, Hydrocharitales, Juncales, Liliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. The methods and compositions can be used over a broad range of plant species, including species from the monocot genera Agrostis, Allium, Ananas, Andropogon, Asparagus, Avena, Cynodon, Elaeis, Eragrostis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pennisetum, Phleum, Phoenix, Poa, Saccharum, Secale, Sorghum, Triticum, Zoysia and Zea.

If a plant is transformed with a first nucleic acid, the first nucleic acid preferably contains a full-length sequence or partial sequence of an endogenous cytosine DNA methyl transferase, so that expression of the endogenous gene is decreased. For example, when a first nucleic acid is stably transformed into a rice plant, the first nucleic acid preferably comprises a rice cytosine DNA methyltransferase sequence effective for decreasing expressing of the endogenous rice gene product. Similarly, when a first nucleic acid is stably transformed into a corn plant, the first nucleic acid preferably comprises a corn cytosine DNA methyltransferase sequence effective for decreasing expressing of the endogenous corn gene product.

A first plant containing a first nucleic acid as described herein, suitable for use in the invention, can be identified by crossing with one or more second plants containing a transcription activator as described herein, followed by selecting or screening for seed infertility in progeny. After a suitable first plant has been identified, the first nucleic acid can be introduced into other plants using, for example, standard breeding techniques.

Transgenic plants can have an altered phenotype as compared to a corresponding control plant that either lacks the transgene or does not express the transgene. Phenotypic effects can be evaluated relative to a control plant that does not express the exogenous polynucleotide of interest, such as a corresponding plant that is not transgenic for the exogenous polynucleotide of interest but otherwise is of the same genetic background as the transgenic plant of interest, or a corresponding plant of the same genetic background in which expression of the polypeptide is suppressed, inhibited, or not induced (e.g., where expression is under the control of an inducible promoter). A plant can be said "not to express" a polynucleotide when the plant exhibits less than 10% (e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) of the amount of mRNA exhibited by the plant of interest. Expression can be evaluated using methods including, for example, RT- PCR, Northern blots, Sl RNAse protection, primer extensions and chip assays. It should be noted that if a polynucleotide is expressed under the control of a tissue- specific or broadly expressing promoter, expression can be evaluated in the entire plant or in a selected tissue. Similarly, if a polynucleotide is expressed at a particular time, e.g., at a particular time in development or upon induction, expression can be evaluated selectively at a desired time period. Articles of Manufacture

A plant seed composition can contain seeds of a first type of plant and of a second type of plant. Seeds of the first type of plant are of a single hybrid, inbred, line or variety, as are seeds of the second type of plant. The proportion of seeds of each type of plant in a composition is measured as the number of seeds of a particular type divided by the total number of seeds in the composition, and can be formulated as desired to meet requirements based on geographic location, pollen quantity, pollen dispersal range, plant maturity, choice of herbicide, and the like. The proportion of the first type can be from about 70 percent to about 99.9 percent, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. The proportion of the second type can be from about 0.1 percent to about 30 percent, e.g., 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30%. When large quantities of a seed composition are formulated, or when the same composition is formulated repeatedly, there may be some variation in the proportion of each type observed in a sample of the composition, due to sampling error. In the present invention, such sampling error typically is about ± 5 % of the expected proportion, e.g., 90% ± 4.5%, or 5% ± 0.25%.

For example, a seed composition can be made from two corn hybrids. A first corn hybrid can constitute 92% of the seeds in the composition, is male-sterile, and carries a first nucleic acid construct comprising a nucleic acid that decreases the amount of transcription or translation product of a gene encoding a methylation status polypeptide, operably linked to a recognition site for a transcription activator. The second corn hybrid can constitute 8% of the seed in the composition, is male- fertile and carries an activator nucleic acid encoding a transcription activator that recognizes the transcription recognition site on the first nucleic acid construct. The transcription activator coding sequence is operably linked to a promoter as described herein. Such a seed composition can be used to grow plants that are suitable for practicing a method of the invention. See, e.g., PCT publications WO 2004/027038 A3 and WO 03/025172. In some embodiments, plants of the first type are male-sterile, e.g., pollen is either not formed or is nonviable, and plants of the second type are male-fertile. Suitable male-sterility systems are known, including cytoplasmic male sterility (CMS), nuclear male sterility, and genetic male sterility. Female parent plants containing CMS are particularly useful. In the case of rice, see, e.g., U.S. Patent 6,294,717. In the case of corn, a number of different methods of conferring male sterility are available, such as multiple mutant genes at separate locations within the genome that confer male sterility. Some nuclear male-sterility systems are reversible, i.e., plants are male sterile unless a plant-derived compound is applied to developing flowers. For example, certain plants exhibit nuclear male-sterility due to mutations affecting j asm onate biosynthesis. Fertility can be temporarily reversed for such plants by applying 12-oxophytodienoic acid or methyl jasmonate. See, Stintzi and Browse, Proc. Natl. Acad. Sci. USA 19: 10625-10630 (2000); and U.S. Patent Application 2003/0217388. Reversible nuclear male-sterility systems are also useful.

Alternatively, plants of both the first type and the second type can be male- fertile. In this case, plants of the first type can be pollinated by hand, using pollen from plants of the second type. In some embodiments, pollen-forming structures on plants of the first type are removed in order to prevent self-pollination of first plants, thereby permitting manual or natural pollination by pollen from second plants. One can also use gametocides to inhibit or prevent pollen formation on plants of the first type. Gametocides are chemicals that affect cells critical to male fertility, and that do not involve expression of a transgene to inhibit or prevent pollen formation. Typically, a gametocide affects fertility only in the plants to which the gametocide is applied. Application of the gametocide, timing of the application and genotype can affect the usefulness of the approach. See, U.S. Pat. No. 4,936,904. In some embodiments, plants are of a species that exhibits partial or complete self-incompatibility. When complete or nearly complete self- incompatibility is present, measures such as male sterility systems or removal of pollen-forming structures on plants of the first type may not be necessary. For example, both tetraploid and octaploid cultivars of switchgrass have pre- fertilization incompatibility systems. Martinez-Reyna and Vogel, Crop Sci. 42 : 1800- 1805

(2002). Thus, the first type of plant in a switchgrass seed composition need not be male-sterile. Rather, both the first and second types of plants can be male-fertile synthetic varieties, each carrying a nucleic acid construct(s) as described above.

Typically, seeds of each of the types is conditioned and bagged in packaging material by means known in the art to form an article of manufacture. Packaging material such as paper and cloth are well known in the art. Such a bag of seed preferably has a package label accompanying the bag, e.g., a tag or label secured to the packaging material, a label printed on the packaging material or a label inserted within the bag. The package label indicates that the seeds therein are a mixture of different plant types, e.g., two different varieties. The package label also may indicate the seed mixture contained therein incorporate transgenes that provide increased amounts of a desired gene product in one or more tissues of such plants,

In some embodiments, microparticles are used to mark one or more varieties in a seed composition. Microparticles adhered to individual seeds of a particular variety permit ready identification of that variety. A specific series of microparticle types can be used, each series adhered to seeds of a particular variety.

Alternatively, a single type of microparticles can be used, such a type adhered to seeds of only one of the varieties in a seed composition. As another alternative, microparticles can be used to mark an individual article of manufacture, e.g., by adhering microparticles to packaging material or to a package label accompanying the article. In the case of a plurality of articles, microparticles can be combined with or adhered to a packaging or shipping container that contains the plurality of articles.

Microparticles can be combined with a binder, for instance an adhesive or coating formulation. Suitable binder materials are known. The resulting particle/adhesive mixture can, for example, then be applied to the surface of seeds for identification purposes.

A marked seed(s) can be observed to determine the presence or absence of microparticles. If the microparticles are visible to the naked eye, the examination may be performed without additional equipment. For microparticles that are not easily visualized by the naked eye, equipment such as a light microscope or a magnifying glass may be used. Typically, microparticles can be examined using a common 4OX or I OOX microscope. The presence or absence of specific microparticles can be detected and recorded. An individual can perform the detection and recordation manually. Alternately, an automated system, e.g., a computerized system, can perform detection and recordation. Microparticles having a single colored layer can be used, recognizing that certain colors may not be suitable for particular seed coat colors. For example, a tan microparticle would render identification difficult if the marked variety had a tan seed coat color. Microparticles having two colored layers can be used. Dual layer microparticles can often provide a sufficient diversity of color combinations. Alternatively, a 5-layered particle can be used. If desired, microparticles can include visual enhancers. Suitable visual enhancers include, without limitation, pearlescent colorant, glitter, metal flake pigments and glass microspheres. Visual enhancers can provide microparticles with a higher localized reflectance and a more characteristic appearance, making the colored layer(s) of a microparticle are more easily distinguishable. Visual enhancers can also further differentiate color layers of one type of microparticle from another type of microparticle. For example, a visual enhancer can be added to distinguish one secondary color (i.e., orange, green, and purple) from another secondary color.

As an alternative to visually distinguishable characteristics, the layer(s) of different types of microparticles may be distinguished by machine-readable characteristics. Machine-readable characteristics can include magnetic characteristics, infrared or ultraviolet absorption characteristics, infrared or ultraviolet reflection characteristics, or fluorescence or visible light transmission characteristics. Plants grown from seeds in the composition typically have the same or very similar maturity, i.e., the same or very similar number of days from germination to crop seed maturation. In some embodiments, however, one or more of the seed types can have a different relative maturity compared to other varieties in the composition. The presence of plants of different relative maturities in a seed composition can be useful as desired to properly coordinate optimum pollen. receptivity of the first type of plants with optimum pollen shed from the second type of plants. Relative maturity of a hybrid, inbred, line or variety of a given crop species is classified by techniques known in the art.

In some embodiments, a plant seed composition of the invention comprises seeds of an apomictic plant species. Seeds of an apomictic species in such a composition constitute at least about 90% of the seeds in the composition, e.g., at least 91%, 93%, 95%, 97% or 99%. Typically the apomictic seeds are of a single variety. Apomictic plant species include facultative apomicts such as weeping lovegrass, Kentucky bluegrass, or bluestems, as well as obligate apomicts. Apomictic mechanisms in seeds of the composition can be classified as aposporous or diplosporous, found primarily in grasses, or adventitious embryony, found in primarily in citrus. Seeds of an apomictic plant species in such a composition contain nucleic acid constructs as discussed herein, and can be germinated and grown to form plants whose seeds are infertile. The infertility of the seed prevents unwanted spread of a desired transgenic trait present in such plants to other plants of the same species.

In some embodiments, a seed composition contains seeds of essentially a single plant type, e.g., a corn hybrid. The hybrid can be made by crossing two corn inbreds. The first corn inbred carries a first nucleic acid construct comprising a first nucleic acid that decreases the amount of transcription or translation product of a gene encoding a methylation status polypeptide, operably linked to an upstream activation region for a transcription activator. The second corn inbred carries an activator nucleic acid encoding a transcription activator that recognizes the upstream activation region on the first nucleic acid construct. The transcription activator coding sequence is operably linked to a broadly expressing promoter, e.g., p326. The F₁ progeny of the cross can be placed in packaging material and used to grow plants that are infertile. See, e.g., PCT publications WO 2004/027038 A3 and WO 03/025172.

In some embodiments in which a sequence of interest is present, plant cells are subjected to environmental conditions that facilitate the synthesis of increased amounts of the gene product of the sequence of interest. Environmental conditions under which a plant, or a plant or cell culture, is grown can be altered, e.g., by increasing the temperature, increasing the watering rate, or decreasing the watering rate, relative to a control temperature or watering rate. Other environmental conditions that can be altered in order to increase the amount or synthesis rate of a polypeptide include the concentration of salt, minerals, hormones, nitrogen, carbon, osmoticum, or known elicitors such as yeast extract, salicylic acid, and methyl jasmonate.

XI. Methods for Making a Polypeptide

In another aspect, the invention features a method for making a polypeptide. The method involves producing seed by allowing pollination of first plants with pollen from second plant as described herein. Such seed are infertile and can be identified by, e.g., the presence of a nucleic acid that decreases expression of a methylation status polypeptide, an activator nucleic acid and a sequence of interest that encodes a preselected polypeptide as described above. In some embodiments, there are two transcription activators present in male- fertile plants. A practitioner can produce seed by harvesting both male-sterile and male-fertile plants, or by harvesting seeds solely from the male-sterile plants. The choice depends upon, inter alia, whether the two types of parent plants are planted in rows or are randomly interplanted. However, either type of harvesting is encompassed by the invention. In some embodiments, seeds are provided by purchasing them from a grower following pollination.

In some embodiments, a method of making a polypeptide involves extracting the preselected polypeptide, or an endogenous polypeptide, from seed produced as described herein. Typically, such seeds have a statistically significant increase in the amount of the preselected polypeptide relative to seeds that do not contain or express the first nucleic acid. The choice of techniques to be used for carrying out extraction of a preselected polypeptide will depend on the nature of the polypeptide. For example, if the preselected polypeptide is an antibody, non- denaturing purification techniques may be used. On the other hand, if the preselected polypeptide is a high methionine zein, denaturing techniques may be used. The degree of purification can be adjusted as desired, depending on the nature of the preselected or endogenous polypeptide. For example, an animal feed having an increased amount of an endogenous polypeptide may have no purification, whereas a preselected antibody polypeptide may have extensive purification.

Typically, an increase in the amount of a polypeptide in cells of a transgenic plant or cell relative to a control plant or cell is considered statistically significant at p ≤0.05 with an appropriate parametric or non-parametric statistic, e.g., Chi-square test, Student's t-test, Mann- Whitney test, or F-test. In some embodiments, a difference in the amount of a polypeptide is statistically significant at p<0.01, p<0.005, orpO.001. An important advantage of certain methods and compositions described herein is that production of a preselected polypeptide can be limited to the time period after pollination. This is achieved when the polypeptide is expressed after plants that contain the sequence of interest are crossed with plants that possess an activation nucleic acid encoding a non-allergenic transcription activator that is commonly found in the food chain. . If plants that contain the sequence of interest . are male sterile, no pollen is produced by them. Rather, any pollen that leaves a production field comes from plants expressing the activator nucleic acid. Such pollen will not activate transcription if it pollinates plants outside the production field and any expression of the transcription activator in progeny will not lead to concern about allergic reactions.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1 - Rice Methyltransferase RNAi Constructs

An RNAi construct was made by operably linking a CaMV35S promoter to a sequence effective for being transcribed into an interfering RNA. The RNAi sequence comprised about 600 nucleotides of a rice cytosine DNA methyltransferase sense strand (N-terminal region) and an inverted repeat of a nos terminator sequence. The construct was made using standard molecular biology techniques and designated 35S::OsMETlNt-RNAi. The sequence of the 35S::rice MET: inverted nos construct is shown in SEQ ID NO: 169. The rice MET portion 5 of the construct is shown in SEQ ID NO: 170. The construct was inserted into a vector that contained a selectable marker gene conferring resistance to the herbicide Basta®.

A second RNAi construct was made by operably linking a CaMV35S promoter to a sequence effective for being transcribed into an interfering RNA. The

10 RNAi sequence comprised about 660 nucleotides of a rice cytosine DNA methyltransferase sense strand (C-terminal region) and an inverted repeat of a nos terminator sequence. The construct was made using standard molecular biology techniques and designated 35S::OsMETlCt-RNAi. The sequence of the 35S::rice - MET: inverted nos construct is shown in SEQ ID NO: 171. The rice MET portion

1.5 of the construct is shown in SEQ ID NO: 172. The construct was inserted into a vector that contained a selectable marker gene conferring resistance to the herbicide Basta®.

0

Example 2 - Analysis of ^"Seed Phenotypes in Transgenic Rice

The following symbols are used in this Example: Tl: plant regenerated from transformed tissue culture; T2: first generation progeny of self-pollinated Tl plants; T3: second generation progeny of self-pollinated T2 plants; T4: third generation 5 progeny of self-pollinated T3 plants.

Each RNAi construct vector of Example 1 was introduced into a tissue culture of the rice cultivar Kitaake by an Agrobacterium-mβdiaXed transformation protocol. Approximately 20 independent Tl transgenic plants were generated from each transformation, as well as for the control plasmid (empty vector NB42-35S- 0 RNAi). Preliminary phenotypic analysis indicated that Ti transformants did not show any significant phenotypic anomalies in vegetative organs, with a few exceptions where some plants appeared to be shorter than the rest of the Tl plants. However, any variation in plant height may be due to tissue culture stress.

Tl plants were allowed to self-pollinate, T2 seeds were germinated and plants grown in a greenhouse. The presence of the RNAi construct was confirmed by PCR. Most of the Tl plants displayed severe fertility defects. In some case, inflorescences developed to maturity, but pistil development arrested soon after pollination. In other cases, plants had no inflorescence. These defects were observed in the majority of Tl plants. Occasionally, it was possible to obtain a few fully developed seeds from each inflorescence. A summary of seed phenotypes observed in individual Tl plants is presented in Table 3.

Table 3.

1 = Plants designated as "CT" were transformed with 35S-OsMETlCt-RNAi; plants designated as "NT" were transformed with 35S-OsMETlNt-RNAi. 2 = small leaves

3 = small plant

4 = few leaves

5 = "no seeds" indicates that an inflorescence was present, but no seeds formed

Example 3 - OsMETl-I and OsMETl -2 Transcription in Inflorescence Tissues

Al Previously, it has been reported that expression of OsMETl-I and OsMETl- 2 is active in callus, root and inflorescence, and that the steady-state level of OsMETl-2 is 7- to 12-fold higher than that for OsMETl-I in these tissues. Teerawanichpan P, et al. Planta 218:337-49 (2004). In addition, it was reported that no transcript of OsMETl-2 was detectable in differentiated tissue (10-day-old leaf), and no expression for either gene was found in mature leaves.

Total RNA was isolated from whole-inflorescence tissues from eighteen of the Tl plants described in Example 2, and qRT-PCR analysis was performed using gene-specific primers for OsMETl-I and OsMETl-2. Inflorescence tissue collected from plants transformed with an empty vector was used as the control. The results are shown in Table 4. The majority of Tl CT and NT transgenic plants had reduced transcription of OsMETl genes. For example, endogenous inflorescence OsMETl-I transcript levels in plant CTl were diminished by up to 95%. Endogenous inflorescence OsMETl -2 transcript levels in plant CTl were reduced by up to 99%.

Table 4.

*: Amount of transcript in inflorescences relative expressed as a percentage of amount in controls — : Not done

Example 4 - Preparation of Transgenic Rice Containing Different Rice Methyltransferase RNAi Constructs

Rice cells were transformed as described in Example 2 with three different MET 1 KNAi constructs. Each construct was the same as the second construct described in Example 1, except that the 35S promoter was replaced with one of the following rice regulatory regions: pOsFIE2-2, pOsMEA and p530cl0. See, SEQ ID NOS: 155, 156 and 162, respectively.

Transgenic Ti plants from independent transformation events containing the p53OclO, pOsFIE2-2 and pOsMEA constructs are regenerated from tissue selected for Basta® resistance and are allowed to self-pollinate. Inflorescences from transgenic T₁ plants are analyzed for seed phenotypes as described in Example 2. Any Tl plants exhibiting a fertility defect are selected for further study.

Example 4 - Determination of ^' Ortholoz/Functional Homolog Sequences

A subject sequence was considered a functional homolog and/or ortholog of a query sequence if the subject and query sequences encode proteins having a similar function and/or activity. A process known as Reciprocal BLAST (Rivera et al, Proc.Natl. Acad. Sd. USA ,1998, 95:6239-6244) was used to identify potential functional homolog and/or ortholog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.

Before starting a Reciprocal BLAST process, a specific query polypeptide was searched against all peptides from its source species using BLAST in order to identify polypeptides having sequence identity of 80% or greater to the query polypeptide and an alignment length of 85% or greater along the shorter sequence in the alignment. The query polypeptide and any of the aforementioned identified polypeptides were designated as a cluster.

The main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search. In the forward search step, a query polypeptide sequence, "polypeptide A," from source species S^A was BLASTed against all protein sequences from a species of interest. Top hits were determined using an E-value cutoff of 10^~5 and an identity cutoff of 35%. Among the top hits, the sequence having the lowest E-value was designated as the best hit, and considered a potential functional homolog and/or ortholog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original query polypeptide was considered a potential functional homolog and/or ortholog as well.

This process was repeated for all species of interest.

In the reverse search round, the top hits identified in the forward search from all species were BLASTed against all protein sequences from the source species S^A. A top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit was also considered as a potential functional homolog and/or ortholog.

Functional homologs and/or orthologs were identified by manual inspection of potential functional homolog and/or ortholog sequences. Representative functional homologs and/or orthologs for SEQ ID NOS: 33, 83 and 2 are shown in

Figures 1 through 3, respectively. Percent identities to SEQ ID NOS: 33, 83 and 2 are shown below in Tables 5 through 7, respectively.

Table 5 AtMETl

Table 6 (OsMETl)

Table 7 (DPMI)

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the. appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for making infertile seed, said method comprising: a) permitting seed development to occur on a plurality of first monocotyledonous plants that have been pollinated by a plurality of second monocotyledonous plants, wherein said first plants comprise a first nucleic acid, said first nucleic acid comprising a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide, wherein said second plants are male- fertile and comprise at least one activator nucleic acid comprising at least one coding sequence for a transcription activator that binds to said first recognition site, said transcription activator coding sequence having a regulatory region operably linked thereto, and wherein said seeds that develop on said first plants are infertile.

2. The method of claim 1 , wherein said regulatory region for said transcription activator is a constitutive promoter.

3. The method of claim 2, wherein said regulatory region for said transcription activator is a broadly expressing promoter.

4. The method of claim 1 , wherein said first plants further comprise a second nucleic acid, said second nucleic acid comprising a second transcription activator recognition site operably linked to a sequence to be transcribed.

5. The method of claim 4, wherein said second plants comprise an activator nucleic acid that encodes a first transcription activator that binds said first recognition site and a second transcription activator that binds said second recognition site.

6. The method of claim 4, wherein said second plants comprise an activator nucleic acid encoding a first transcription activator that binds said first recognition site and a different activator nucleic acid encoding a second transcription activator that binds said second recognition site.

7. The method of claim 5 or 6, wherein said regulatory region of said first transcription activator is a broadly expressing promoter and said regulatory region of said second transcription activator is a maturing endosperm promoter.

8. The method of claim 7, wherein said promoter of said first transcription activator is a p326 promoter.

9. The method of claim 7, wherein said promoter of said second transcription activator is a 15 IcD zein promoter, 16 kD zein promoter, 19 kD zein promoter, 22 IcD zein promoter, 27 kD zein promoter, Osgt-1 promoter, glutelin-1 gene promoter, /3-amylase gene promoter, or hordein gene promoter.

10. The method of any of claims 4-9, wherein said sequence to be transcribed encodes a preselected polypeptide.

11. The method of claim 10, wherein said seeds have a statistically significant increase in the amount of said preselected polypeptide relative to seeds that lack said first nucleic acid.

12. The method of claim 10, wherein said preselected polypeptide is an antibody.

13. The method of claim 10, wherein said preselected polypeptide is an enzyme.

14. The method of any of claims 1-13, further comprising the step of harvesting said seeds.

15. The method of any of claims 1-14, wherein said plurality of first plants is cytoplasmically male-sterile.

16. The method of any of claims 1-14, wherein said plurality of first plants is male-sterile due to nuclear male sterility.

17. The method of any of claims 1-16, wherein said nucleic acid that decreases expression of a methylation status polypeptide is an antisense nucleic acid to an endogenous sequence encoding a cytosine DNA methyltransferase.

18. The method of any of claims 1-16, wherein said nucleic acid that decreases expression of a methylation status polypeptide is an interfering RNA to an endogenous sequence encoding a cytosine DNA methyltransferase.

19. The method of any of claims 1-16, wherein said nucleic acid that decreases expression of a methylation status polypeptide comprises all or part of the coding sequence for a polypeptide shown in Figures 1, 2 or 3.

20. The method of any of claims 1-19, wherein said first and second plants are a species from the genera Zea or Sorghum.

21. A method for making a polypeptide, said method comprising: a) providing seed produced by pollination of monocotyledonous plants, said seed comprising: i) a first nucleic acid comprising a first recognition site for a transcription activator operably linked to a nucleic acid that decreases expression of a methylation status polypeptide; ii) a second nucleic acid comprising a second recognition site for a transcription activator operably linked to a coding sequence for a preselected polypeptide; and iii) an activator nucleic acid comprising at least one coding sequence for a transcription activator that binds to said recognition sites, each said at least one transcription activator having a regulatory region operably linked thereto, wherein said seeds are infertile and have a statistically significant increase in the amount of said preselected polypeptide relative to seeds that do not contain said first nucleic acid; and b) extracting said preselected polypeptide from said seed.

22. A method for making a polypeptide, said method comprising: a) providing seed produced by pollination of monocotyledonous plants, said seed comprising: i) a first nucleic acid comprising a first recognition site for a transcription activator operably linked to a nucleic acid that decreases expression of a methylation status polypeptide; ii) a second nucleic acid comprising a second recognition site for a transcription activator operably linked to a coding sequence for a preselected polypeptide; and iii) at least one activator nucleic acid comprising a coding sequence for a transcription activator that binds to said first recognition site and a coding sequence for a transcription activator that binds to said second recognition site, each said transcription activator having a regulatory region operably linked thereto, wherein said seeds are infertile and have a statistically significant increase in the amount of said preselected polypeptide relative to seeds that do not contain said first nucleic acid; and b) extracting said preselected polypeptide from said seed.

23. The method of claim 22, wherein each said regulatory region for each said transcription activator coding sequence is a broadly expressing promoter.

24. The method of any of claims 21-23, wherein said nucleic acid that decreases expression of a methylation status polypeptide is an antisense nucleic acid to an endogenous sequence encoding a cytosine DNA methyl transferase.

25. The method of any of claims 21 -23, wherein said nucleic acid that decreases expression of a methylation status polypeptide is an interfering RNA to an endogenous sequence encoding a cytosine DNA methyltransferase.

26. The method of any of claims 21-25, wherein said plurality of first plants is cytoplasmically male-sterile.

27. The method of any of claims 21 -25, wherein said plurality of first plants is male-sterile due to nuclear male sterility.

28. The method of any of claims 21-27, wherein said plants are a species from the genera Zea or Sorghum.

29. A method for making infertile plants, said method comprising: a) permitting seed development to occur on a plurality of first monocotyledonous plants that have been pollinated by a plurality of second monocotyledonous plants, wherein said first plants comprise a first nucleic acid, said first nucleic acid comprising a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide, wherein said second plants are male-fertile, of the same species as said first plant, and comprise an activator nucleic acid comprising a regulatory region operably linked to a coding sequence for a first transcription activator that binds to said first recognition site; b) harvesting seeds from said plurality of first plants; and c) germinating said seeds, wherein plants that grow from said seeds are infertile.

30. The method of claim 29, wherein said regulatory region for said first transcription activator coding sequence is a constitutive or broadly expressing promoter.

31. The method of claim 29, wherein said first plants further comprise a second nucleic acid comprising a second transcription activator recognition site operably linked to a sequence to be transcribed.

32. The method of claim 31 , wherein said first transcription activator binds to said second recognition site.

33. The method of claim 31 , wherein said second plants further comprise a different activator nucleic acid encoding a second transcription activator that binds said second recognition site.

34. The method of any of claims 29-33, wherein said sequence to be transcribed is a coding sequence for a preselected polypeptide.

35. The method of any of claims 29-34, wherein said plurality of first plants is cytoplasmically male-sterile.

36. The method of any of claims 29-34, wherein said plurality of first plants is male-sterile due to nuclear male sterility.

37. The method of any of claims 29-36, wherein said first and second plants are a species from the genera Panicum.

38. The method of any of claims 29-36, wherein said first and second plants are a species from the genera Festuca, FestuloHum, Pannesetum, or Poa.

39. An article of manufacture comprising: a) packaging material; b) a first type of monocotyledonous seeds within said packaging material, said first type of seeds comprising at least one first nucleic acid comprising a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide; and c) a second type of monocotyledonous seeds within said container, said second type of seeds comprising at least one activator nucleic acid comprising a regulatory region operably linked to a coding sequence for a transcription activator that binds to said first recognition site, wherein plants grown from said second type of seeds are male-fertile.

40. The article of claim 39, wherein said first type of seeds further comprise a second nucleic acid comprising a second transcription activator recognition site operably linked to a sequence to be transcribed.

41. The article of claim 40, wherein said sequence to be transcribed is a preselected polypeptide.

42. The article of any of claims 39-41, wherein plants grown from said first type of seeds are male-sterile.

43. The article of any of claims 39-42, wherein the ratio of said first type of seeds to said second type of seeds is from about 70:30 to about 99.5:0.5.

44. The article of any of claims 40-43, wherein said at least one activator nucleic acid encodes a transcription activator that binds to said first recognition site, and a different transcription activator that binds to said second recognition site.

45. The article of claim 44, wherein said regulatory region for said transcription activator that binds said first recognition site is a broadly expressing promoter and said regulatory region for said transcription activator that binds to said second recognition site is a maturing endosperm promoter.

46. The article of any of claims 39-45, wherein said seeds are a species from the genera Panicum, Zea or Sorghum.

47. A plant comprising: a) a first nucleic acid comprising a first transcription activator recognition site operably linked to a nucleic acid that decreases expression of a methylation status polypeptide; and b) at least one activator nucleic acid encoding a regulatory region operably linked to a transcription activator that binds to said first recognition site, wherein said plant is infertile.

48. The plant of claim 47, further comprising a second nucleic acid comprising a second transcription activator recognition site operably linked to a sequence to be transcribed.

49. The plant of claim 48, wherein said first and second nucleic acids are a single nucleic acid molecule.

50. The plant of claim 48 or 49, wherein said sequence to be transcribed encodes a preselected polypeptide.

51. The plant of any of claims 48-50, wherein said at least one activator nucleic acid encodes a transcription activator that binds to said first and said second transcription activator recognition sites.

52. The plant of any of claims 48-50, wherein said at least one activator nucleic acid encodes a transcription activator that binds to said first recognition site, and a different transcription activator that binds to said second recognition site.

53. The plant of claim 52, wherein said regulatory region for said transcription activator that binds said first recognition site is a broadly expressing promoter and said regulatory region for said transcription activator that binds to said second recognition site is a maturing endosperm promoter.

54. The plant of any of claims 47-53, wherein said plant is a species from the genera Festuca, Festulolium, Panicum, Pannesetum, Poa, Sorghum or Zea.