WO2013067259A2

WO2013067259A2 - Regulatory nucleic acids and methods of use

Info

Publication number: WO2013067259A2
Application number: PCT/US2012/063169
Authority: WO
Inventors: Michael Nuccio
Original assignee: Syngenta Participations Ag
Priority date: 2011-11-03
Filing date: 2012-11-02
Publication date: 2013-05-10
Also published as: CN103998611A; CA2853490A1; WO2013067259A3; WO2013067252A1; HUP1400505A2; MX2014005375A; AU2012332343A1

Abstract

The present invention relates generally to the field of molecular biology and describes nucleic acids encoding regulatory elements capable of affecting expression of a coding sequence. The regulatory elements described herein may be used to direct the expression of a heterologous coding region in the green tissues and upon exposure to light in plants. The invention may also be used to create transgenic plants having increased yield.

Description

REGULATORY NUCLEIC ACIDS AND METHODS OF USE

FIELD OF THE INVENTION

[0001] The present invention relates to the fields of agriculture, plant breeding or genetic engineering for plants with increased yield.

BACKGROUND

[0002] A critical component of plant biotechnology is the use of promoters with unique spatial and temporal activity profiles to express agronomically important genes in crop plants so that genes of interest are expressed at optimal levels in appropriate tissues. In agricultural biotechnology, plants can be modified according to one's needs. One way to accomplish this is by using modern genetic engineering techniques. For example, by introducing a gene of interest into a plant, the plant can be specifically modified to express a desirable phenotypic trait. For this, plants are transformed most commonly with a heterologous gene comprising a promoter region, a coding region and a termination region. When genetically engineering a heterologous gene for expression in plants, the selection of a promoter is often a factor. While it can be desirable to express certain genes constitutively, i.e. throughout the plant at all times and in most tissues and organs, other genes are more desirably expressed only in response to particular stimuli or confined to specific cells or tissues.

SUMMARY OF THE INVENTION

[0003] One embodiment of the invention is an isolated, regulatory nucleic acid comprising a regulatory nucleic acid having at least 90 percent, 95 percent, 98 percent or greater sequence identity to the nucleotide sequences set forth in SEQ ID NO: 1, 4, 7, 10 or 13; a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13 or a functionally equivalent fragment thereof ; or a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, or 13; wherein the regulatory nucleic acid directs transcription of an operably linked polynucleotide.

[0004] In another embodiment of the invention, the nucleic acid contains one or more motifs selected from the group consisting of a BOXIIPCCHS motif, a CIACADIANLELHC motif, a GT1 CONSENSUS motif, an IBOX motif, an IBOXCORE motif, an IBOXCORENT motif, an INRNTPSADB motif, a SORLIP1AT motif and a SORLIP2AT motif.

[0005] The nucleic acid may be a functionally equivalent fragment comprising at least 200, 300 or 400 base pairs of SEQ ID NO: 1, 4, 7, 10 or 13. In some embodiments the nucleic acid may be operably linked to an intron. The intron may be selected from the group consisting of SEQ ID NO: 2, 5, 8, 11 and 14. In addition, the nucleic acid may be operably linked to a terminator. Alternatively, the terminator may be selected from nucleic acids described by SEQ ID NOS: 3, 6, 9, 12 and 15. In one embodiment, the promoter, intron and terminator are isolated from the same coding region. Alternatively, the promoter, intron and terminator may be isolated from more than one coding region.

[0006] Another embodiment is an expression cassette comprising a first nucleic acid comprising a regulatory nucleic acid having at least 90 percent, 95 percent, 98 percent or greater sequence identity to the nucleotide sequences set forth in SEQ ID NO: 1, 4, 7, 10 or 13; a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13 or a functionally equivalent fragment thereof ; or a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, or 13; wherein the regulatory nucleic acid directs transcription of an operably linked polynucleotide; a second nucleic acid to be transcribed, wherein said first and second nucleic acids are heterologous to each other and are operably linked; and a terminator operably linked 3' to the nucleic acid to be transcribed. The second nucleic acid may be selected from the group comprising a pest resistance nucleic acid, a disease resistance nucleic acid, an herbicide resistance acid, a value-added trait nucleic acid, a photoassimilation regulated nucleic acid, a yield nucleic acid and a stress tolerant nucleic acid. In addition, the heterologous coding region may be expressed green tissue or light regulated, such that, transcription of the coding region is induced in the presence of light.

[0007] In another embodiment, a plant, plant tissue, or plant cell comprising any of the above described expression cassettes. The plant, plant tissue, or plant cell can be a monocot or from monocot, such as, maize. [0008] Another embodiment is a method of expressing a heterologous coding region comprising providing a regulatory nucleic acid having at least 90 percent, 95 percent, 98 percent or greater sequence identity to the nucleotide sequences set forth in SEQ ID NO: 1, 4, 7, 10 or 13; a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13 or a functionally equivalent fragment thereof ; or a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, or 13 operably linked to a heterologous coding region; and creating a plant, plant tissue, or plant cell comprising the nucleic acid, wherein the heterologous coding region is expressed. The heterologous coding region may be expressed in green tissue or light regulated such that, transcription of the coding region is induced in the presence of light. The plant, plant tissue, plant cell or a portion thereof may be a monocot, from a monocot, maize or from maize.

[0009] Another embodiment includes a plant, plant tissue, plant cell, or portion thereof made by the method of expressing a heterologous coding region comprising providing a regulatory nucleic acid having at least 90 percent, 95 percent, 98 percent or greater sequence identity to the nucleotide sequences set forth in SEQ ID NO: 1, 4, 7, 10 or 13; a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13 or a functionally equivalent fragment thereof ; or a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, or 13 operably linked to a heterologous coding region; and creating a plant, plant tissue, or plant cell comprising the nucleic acid, wherein the heterologous coding region is expressed. Included is the progeny, seed, or grain produced by the plant, plant tissue, plant cell, or portion thereof.

[0010] Another embodiment is an isolated nucleic acid comprising SEQ ID NO: 2, 5, 8, 11 or 14 or a terminator comprising SEQ ID NO: 3, 6, 9, 12, 15 or a functional fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a plasmid map of 19862 showing SoFBP, SoPRK, and ZmPEPC expression cassettes in a binary vector, "pr-" prefix denotes a promoter; "i-" prefix denotes an intron; "e-" prefix denotes an enhancer; "c-" prefix denotes a coding sequence; "t-" prefix denotes a terminator. [0012] FIG. 2 is a plasmid map of 19863 showing SoFBP, SbPPDK, and SbNADP-MD expression cassettes in a binary vector, "pr-" prefix denotes a promoter; "i-" prefix denotes an intron; "e-" prefix denotes an enhancer; "c-" prefix denotes a coding sequence; "t-" prefix denotes a terminator.

DETAILED DESCRIPTION OF THE INVENTION

[0013] It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0014] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0015] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0016] A promoter is a region which facilitates the transcription of a specific gene or coding region. Transcription factors bind to promoter regions at specific sequences. Binding motifs for transcription factors can be predicted in promoter sequence. Some motifs are annotated as light inducible, i.e. transcription of the gene or coding region occurs upon exposure to light. In some embodiments of the invention, the promoters described contain one or more motifs selected from the group consisting of a BOXIIPCCHS motif, CIACADIANLELHC motif, GT1CONSENSUS motif, IBOX motif, IBOXCORE motif, IBOXCORENT motif, INRNTPSADB motif,

LRENPCABE motif, SORLIP1AT motif, SORLIP2AT and SORLIP5AT motif.

[0017] The promoter, intron and terminator sequences and methods of use disclosed herein may be used in combination with any one of the following elements such as enhancers, upstream elements, and/or activating sequences from the 5' flanking regions of plant expressible structural genes. In some embodiments of this invention, the regulatory nucleic acids comprise a promoter, a first ex on, an intron, and optionally a second ex on or fragment thereof. Alternatively, the regulatory nucleic acids may combine a promoter, intron and terminator. These regulatory nucleic acids may or may not be derived from the same locus of a non-trans genie plant. The regulatory nucleic acids may comprise the first or 5' most exon of the locus, the 5' most intron and the second exon immediately downstream of the 5' most intron in the genome of the non- transgenic plant.

[0018] In some embodiments, the invention provides an expression cassette that may be used to drive expressions of heterologous genes or coding regions for increasing yield, or improving resistance to herbicides, pests, disease or drought. Some embodiments provide expression cassettes to express heterologous genes or coding regions in response to light. This expression may occur in green tissues such as leaves.

[0019] The expression cassettes may be introduced in to host cells, including plant cells. The plant cell may be regenerated into a plant comprising the expression cassettes. The plant may be a monocot or dicot plant. In some embodiments, the plant is selected from the group consisting of maize, sugarcane, sorghum, amaranth, other grasses and sedges. In some embodiments the plant is a maize plant.

[0020] Additional embodiments of the invention include methods of producing a transgenic plant or methods of increasing yield in a plant comprising introducing one of the expression cassettes of the invention into a plant and producing or regenerating a transgenic plant. The transgenic plant may be crossed with a non-transgenic plant and then selected for a progeny plant comprising one of the expression cassettes of the invention.

[0021] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, plant species or genera, constructs, and reagents described as such. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. As used herein the singular forms "a", "and", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art.

[0022] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent.

[0023] As used herein, the word "or" means any one member of a particular list and also includes any combination of members on that list.

[0024] "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of protein from an endogenous gene or a transgene.

[0025] "Cis-element" refers to a cis-acting transcriptional regulatory element that confers an aspect of the overall control of gene expression. A cis-element may function to bind transcription factors, trans-acting protein factors that regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one cis-element. Cis-elements can be identified by a number of techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5' end or internal to a promoter; DNA binding protein analysis using DNase I footprinting, methylation interference,

electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis with known cis-element motifs by conventional DNA sequence comparison methods. The fine structure of a cis-element can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Cis-elements can be obtained by chemical synthesis or by isolation from promoters that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation.

[0026] "Chimeric" is used to indicate that a DNA sequence, such as a vector or a gene, is comprised of two or more DNA sequences of distinct origin that are fused together by recombinant DNA techniques resulting in a DNA sequence, which does not occur naturally.

[0027] "Chromosomally-integrated" refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes or coding regions are not

"chromosomally integrated", they may be "transiently expressed." Transient expression of a gene or coding region refers to the expression of a gene or coding region that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus. [0028] "Coding sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An "intron" is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation, or splicing, of the RNA within the cell to create the mature mRNA that can be translated into a protein.

[0029] "Constitutive promoter" refers to a promoter that is able to express the gene or coding region that it controls in all or nearly all of the plant tissues during all or nearly all

developmental- stages of the plant, thereby generating "constitutive expression" of the gene or coding region.

[0030] "Co-suppression" and "sense suppression" refer to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially identical transgene, endogenous genes or endogenous coding sequences.

[0031] "Contiguous" is used herein to mean nucleic acid sequences that are immediately preceding or following one another.

[0032] "Expression" refers to the transcription and stable accumulation of mRNA.

Expression may also refer to the production of protein.

[0033] "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.

[0034] A "coding region" usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA, a nontranslated RNA or a microRNA.

[0035] "Gene" refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein. The term "chimeric gene" refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences that are not found together in nature or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory nucleic acids and coding sequences that are derived from different sources, or comprise regulatory nucleic acids and coding sequences derived from the same source, but arranged in a manner different from that found in nature. A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed.

Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism or a gene not in its natural location that has been introduced into the organism by gene transfer or transformation.

[0036] "Gene silencing" refers to homology-dependent suppression of viral genes, transgenes, or endogenous nuclear genes. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover (degradation) of RNA species homologous to the affected genes. (English, et al., 1996, Plant Cell 8: 179-1881). Gene silencing includes virus- induced gene silencing (Ruiz et al, 1998, Plant Cell 10:937-946).

[0037] "Heterologous DNA Sequence" is a DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.

[0038] "Inducible promoter" refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen.

[0039] "5' non-coding sequence" refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. (Turner et al, 1995, Molecular Biotechnology, 3:225).

[0040] "3' non-coding sequence" refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The

polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. (1989, Plant Cell, 1 :671-680).

[0041] The term "nucleic acid" refers to a polynucleotide of high molecular weight which can be single-stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. A "genome" is the entire body of genetic material contained in each cell of an organism. The term "nucleotide sequence" refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.

[0042] The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

[0043] "Operably linked" refer to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Alternatively, sequences can be operably linked without being physically connected. For example, in an expression cassette a promoter may be operably linked to a terminator. Coding sequences in sense or antisense orientation can be operably linked to regulatory nucleic acids.

[0044] "Overexpression" refers to the level of expression in transgenic organisms that exceeds levels of expression in normal or untransformed organisms.

[0045] "Preferred expression", "Preferential transcription" or "preferred transcription" interchangeably refers to the expression of gene products that are preferably expressed at a higher level in one or a few plant tissues (spatial limitation) and/or to one or a few plant developmental stages (temporal limitation) while in other tissues/developmental stages there is a relatively low level of expression. [0046] "Primary transformant" and "TO generation" refer to transgenic plants that are of the same genetic generation as the tissue that was initially transformed (i.e., not having gone through meiosis and fertilization since transformation). "Secondary transformants" and the "Tl, T2, T3, etc. generations" refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.

[0047] The terms "protein, " "peptide" and "polypeptide" are used interchangeably herein.

[0048] "Promoter" refers to a nucleic acid, which controls the expression of a coding sequence or gene by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter regulatory sequences" or "promoter regulatory nucleic acids" can comprise proximal and more distal upstream elements. Promoter regulatory nucleic acids influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory nucleic acids include enhancers, untranslated leader sequences, introns, exons, polyadenylation signal sequences and terminators. They include natural and synthetic sequences as well as sequences that can be a combination of synthetic and natural sequences. An "enhancer" is a nucleotide sequence that can stimulate promoter activity and can be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. The primary sequence can be present on either strand of a double- stranded DNA molecule, and is capable of functioning even when placed either upstream or downstream from the promoter. The meaning of the term "promoter" includes "promoter regulatory sequences" or "promoter regulatory nucleic acids".

[0049] "Regulatory sequences" or "regulatory nucleic acids" refer to nucleotide sequences that contribute to the activity of a given gene as it relates to mRNA production, stability and translatability. Regulatory sequences include enhancers, promoters, translational enhancer sequences, introns, terminators and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. When a regulatory sequence is a combination of regulatory sequence elements, such as, a promoter, intron and terminator, the regulatory sequence elements are isolated from the same gene or different genes. For example, a promoter, intron and terminator sequence from the ZmPRKl gene is isolated from the same coding sequence or the ZmPRKl gene. Alternatively, the promoter could be from the ZmPRKl gene, the intron from the ZmSBP gene and the terminator from the ZmPGK gene. Light regulatory nucleic acids are regulatory elements that respond to light and are therefore light inducible.

[0050] "Intron" refers to an intervening section of transcribed DNA that occurs almost exclusively within a eukaryotic gene, but which is not translated to amino acid sequences in the gene product. The introns are removed from the pre-mature mRNA through a process called splicing, which joins the exons to form an mRNA. For purposes of the presently disclosed subject matter, the definition of the term "intron" includes modifications to the nucleotide sequence of an intron derived from a target gene.

[0051] "Exon" refers to a section of transcribed DNA that is maintained in mRNA. Exons generally carry the coding sequence for a protein or part of the coding sequence. Exons are separated by intervening, non- coding sequences (introns). For purposes of the presently disclosed subject matter, the definition of the term "exon" includes modifications to the nucleotide sequence of an exon derived from a target gene.

[0052] A "terminator" refers to a nucleic acid capable of stopping gene transcription by RNA polymerase. Terminators typically consist of the 3'-UTR of a gene or coding sequence and about 1 kb of downstream sequence. For a review on terminators, please see, Richard and Manley (2009) Genes & Dev. 23: 1247-1269.

[0053] Substantially identical: the phrase "substantially identical," in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, 80%, 90%, 95%, and 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The substantial identity may exist over a region of the sequence that is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 residues in length. The sequences may be substantially identical over the entire length of the coding regions. Furthermore, substantially identical nucleic acid or protein sequences perform substantially the same function.

[0054] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity lor the test sequence(s) relative to the reference sequence, based on the designated program parameters. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

[0055] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by visual inspection (see generally, Ausubel et al., infra); by improved FASTA "Improved tools for biological sequence comparison" Pearson, et. al. (1988) PNAS 85(8):2444-8; MUSCLE:

multiple sequence alignment with high accuracy and high throughput" Edgar, (2004) Nucleic Acids Res. 32(5): 1792-1797.

[0056] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive value threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. 89: 10915).

[0057] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1 , 0.01, and 0.001.

[0058] For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein can be made using the BLASTN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.

[0059] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent hybridization conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0060] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York. Generally, high stringency hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Typically, under high stringency conditions a probe will hybridize to its target subsequence, but to no other sequences.

[0061] The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very high stringency conditions are selected to be equal to the T_m for a particular probe. An example of high stringency hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of very high stringency wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes. An example of high stringency wash conditions is a 0.2x SSC wash at 65°C for 15 minutes {see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes {e.g., about 10 to 50 nucleotides), high stringency conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion

concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. High stringency conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific

hybridization. Nucleic acids that do not hybridize to each other under high stringency conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. [0062] Low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in IX to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium. citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 42°C, and a wash in 0. 1 X SSC at 60 to 65°C.

[0063] The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C; 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C; 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C; 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.

[0064] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T_m can be approximated from the equation of Meinkoth and Wahl Anal. Biochem. 138:267-284 (1984); TM 81.5°C + 16.6 (log M) +0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T is reduced by about 1°C for each 1% of mismatching; thus, T_m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T_m can be decreased 10°C. Generally, high stringency conditions are selected to be about 19°C lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, very high stringency conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (T_m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (T_m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (T_m). Using the equation, hybridization and wash compositions, and desired T, variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part 1, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley - Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2^nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0065] The "terminus" includes the 3 '-untranslated sequence and the 3' non-transcribed sequence, which extends 0.5 to 1.5 kb downstream of the transcription termination site. The terminus may include 3' regulatory sequence.

[0066] "Tissue specific promoter" refers to regulated promoters that do not transcribe DNA in all plant cells but only in one or more cell types in specific organs (such as leaves, roots or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.

[0067] A "transcriptional cassette" will comprise in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a

transcriptional and translational termination region functional in plants. The termination region may be native or physically or genetically linked with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.

[0068] The "transcription initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e. further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0069] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. "Transiently transformed" refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance. "Stably transformed" refers to cells that have been selected and regenerated on a selection media following transformation.

[0070] "Transformed / transgenic / recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non- transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

[0071] The term "translational enhancer sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon. The translational enhancer sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.

[0072] "Vector" is defined to include, inter alia, any plasmid, cosmid, phage or

Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic species (e.g. higher plant, mammalian, yeast or fungal cells). [0073] The term "plant" refers to any plant, particularly to agronomically useful plants (e.g. seed plants), and "plant cell" is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized units such as for example, a plant tissue, or a plant organ differentiated into a structure that is present at any stage of a plant's development. The promoters and compositions described herein may be utilized in any plant. Examples of plants that may be utilized in contained embodiments herein include, but are not limited to, maize (corn), wheat, rice, barley, soybean, cotton, sorghum, beans in general, rape/canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, tropical sugar beet, Brassica spp., cotton, coffee, sweet potato, flax, peanut, clover; vegetables such as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, Brussel sprouts, peppers, and pineapple; tree fruits such as citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, and coconut; and flowers such as orchids, carnations and roses. Other plants useful in the practice of the invention include perennial grasses, such as switchgrass, prairie grasses, Indiangrass, Big bluestem grass, miscanthus and the like. It is recognized that mixtures of plants can be used.

[0074] As used herein, "plant tissue", "plant cell", "plant material," "plant part" or "plant portion thereof means plant cells, plant protoplasts, plant cell tissue cultures, differentiated and undifferentiated tissues from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, tubers, rhizomes and the like.

[0075] A transcription regulating nucleic acid may comprise at least one promoter sequence localized upstream of the transcription start of the respective gene and is capable of inducing transcription of downstream sequences. The transcription regulating nucleic acid may comprise the promoter sequence of said genes but may further comprise other elements such as the 5'- untranslated sequence, enhancer sequences, intron, ex on, and/or even comprise intron and exons of the associated genomic gene.

[0076] Promoters can comprise several regions that play a role in function of the promoter. Some of these regions are modular, in other words they can be used in isolation to confer promoter activity or they can be assembled with other elements to construct new promoters. The first of these promoter regions lies immediately upstream of the coding sequence and forms the "core promoter region" containing consensus sequences, normally 20-70 base pairs immediately upstream of the coding sequence. The core promoter region typically contains a TATA box and often an initiator element as well as the initiation site. The precise length of the core promoter region is not fixed. Such a region is normally present, with some variation, in most promoters. The core promoter region is often referred to as a minimal promoter region because it is functional on its own to promote a basal level of transcription.

[0077] The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. The core region acts to attract the general transcription machinery to the promoter for transcription initiation. However, the core promoter region is typically not sufficient to provide promoter activity at a desired level. A series of regulatory sequences, often upstream of the core, constitute the remainder of the promoter. The regulatory sequences can determine expression level, the spatial and temporal pattern of expression and, for a subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemicals and hormones). Regulatory sequences can be short regions of DNA sequence 6-100 base pairs that define the binding sites for trans-acting factors, such as transcription factors. Regulatory sequences can also be enhancers, longer regions of DNA sequence that can act from a distance from the core promoter region, sometimes over several kilobases from the core region. Regulatory sequence activity can be influenced by trans-acting factors including but not limited to general transcription machinery, transcription factors and chromatin assembly factors. Transcription factor binding "motifs" represent the differences in the sequence that a transcription factor binds in different promoters by using IUPAC codes to represent the degenerate positions such as "R" represents "A" or "G".

[0078] As used herein, a "control plant" may be a non-transgenic plant of the parental line used to generate a transgenic plant herein. A control plant may in some cases be a transgenic plant line that includes an empty vector or marker gene, but does not contain the recombinant

polynucleotide of the present invention that is expressed in the transgenic plant being evaluated. A control plant in other cases is a transgenic plant expressing the gene with a constitutive promoter. In general, a control plant is a plant of the same line or variety as the transgenic plant being tested, lacking the specific trait-conferring, recombinant DNA that characterizes the transgenic plant. Such a progenitor plant that lacks that specific trait-conferring recombinant DNA can be a natural, wild-type plant, an elite, non-trans genie plant, or a transgenic plant without the specific trait-conferring, recombinant DNA that characterizes the transgenic plant. The progenitor plant lacking the specific, trait-conferring recombinant DNA can be a sibling of a transgenic plant having the specific, trait-conferring recombinant DNA. Such a progenitor sibling plant may include other recombinant DNA.

[0079] Highly active light regulated, green tissue preferred expression cassettes are desirable tools for bioengineering plants for a number of traits, for example, improved yield or drought tolerance. Genes expressed in these cassettes could contribute to photosynthesis or cause the plant to make better use of the energy produced by photosynthesis. Light regulated promoters might be found driving the expression of native genes for photosystem I, photosystem II, or Calvin Cycle proteins. The amino acid sequences for Hordeum vulgare Photosystem I reaction center subunit psaD with Swiss-Prot ID P36213.1, the Hordeum vulgare Photosystem I reaction center subunit psaK with Swiss-Prot ID P36886.1 (formerly Swiss-Prot ID A48527), the Pisum sativum light harvesting protein of photosystem I LHCA3 with Genbank ID AAA84545.1, and the Hordeum vulgare chlorophyll a/b-binding protein precursor LHCA4 with Genbank ID AAF90200.1 were used in a tBLASTn search of a proprietary rice genome database to find rice genes corresponding to the barley and pea genes. Public rice genome sequences are available including on the World Wide Web at rice.plantbiology.msu.edu.

[0080] One approach to designing plant expression cassettes is to try to get all of the regulatory sequence guiding the expression of a gene by including 5' flanking sequence, 3' flanking sequence and intron sequence. Genome analysis shows that genes are dispersed at 4-6 kb intervals in rice (Delseny, Current Opinion Plant Biology 6 (2): 101-105 (2003)). A plant gene can be broken into three basic components: the promoter, the coding sequence and the terminator. The promoter may consist of 5'-upstream regulatory (non-transcribed) sequence, generally 1.0-2.5 kb, and the 5'-UTR. The coding sequence consists of the exons and introns between the translation start and stop codons. The terminator consists of the 3'-UTR and about 1 kb of downstream sequence. These components contain virtually all of the necessary gene regulatory information and can be used to design transgene expression cassettes that replicate or recapitulate the expression profile of a gene from which the transgene regulatory sequence was derived. This model has been applied in both dicots (U.S. Pat. No. 6100450) and monocots (U.S. Pat. No. 8129588). [0081] Each cassette is based on a unique plant gene derived from rice, maize, or sugar cane. Construct design is modeled on plant gene structure, described above. Where possible, attention was paid to transcribed sequence to reduce the occurrence of sequence repeats of more than 15 nucleotides. Modifications were achieved by substituting adenosine for thymidine or cytidine for guanidine (and vice versa) at 15 base intervals, except in introns, to minimize gene silencing (Carrington et al., Science 301 (5631): 336-338 (2003)). Also sequence surrounding the intended translation start codon can be optimized following the guidelines of Kozak (Kozak, Gene 299( 1-2): 1-34(2002)). This design strategy eliminates repetitive sequence that could trigger gene silencing and produces a construct that looks more like plant genomic DNA and less like plant pathogen DNA. The constructs are assembled in a binary vector and transformed into maize using standard agrobacterium procedures.

[0082] Expression cassettes can be introduced into the plant cell in a number of art-recognized ways. The term "introducing" in the context of a polynucleotide, for example, a nucleotide construct of interest, is intended to mean presenting to the plant the polynucleotide in such a manner that the polynucleotide gains access to the interior of a cell of the plant. Where more than one polynucleotide is to be introduced, these polynucleotides can be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into the host cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, only that the polynucleotide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotides into plants are known in the art including, but not limited to, transient transformation methods, stable transformation methods, and virus-mediated methods.

[0083] Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642), and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

[0084] Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojec tiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.

[0085] Many vectors are available for transformation using Agrobacterium tumefaciens . These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). For the construction of vectors useful in Agrobacterium

transformation, see, for example, US Patent Application Publication No. 2006/0260011, herein incorporated by reference.

[0086] Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and

electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. For the construction of such vectors, see, for example, US Application No. 20060260011, herein incorporated by reference.

[0087] Transformation techniques for plants are well known in the art and include

Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.

[0088] The plants obtained via transformation with a nucleic acid sequence of the present invention can be any of a wide variety of plant species; however, the plants used in the method of the invention can be selected from the list of agronomically important target crops set forth supra. The expression of a gene of the present invention in combination with other

characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis. (1983); Mayo O., The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseases and Insect Pests, Springer- Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).

[0089] It is specifically contemplated that one could mutagenize a promoter to potentially improve the utility of the elements for the expression of transgenes in plants. The mutagenesis of these elements can be carried out at random and the mutagenized promoter sequences screened for activity in a trial-by-error procedure. Alternatively, particular sequences which provide the promoter with desirable expression characteristics, or the promoter with expression enhancement activity, could be identified and these or similar sequences introduced into the sequences via mutation. It is further contemplated that one could mutagenize these sequences in order to enhance their expression of transgenes in a particular species. The means for mutagenizing a DNA segment encoding a promoter sequence of the current invention are well-known to those of skill in the art. As indicated, modifications to promoter or other regulatory element may be made by random, or site-specific mutagenesis procedures. The promoter and other regulatory element may be modified by altering their structure through the addition or deletion of one or more nucleotides from the sequence which encodes the corresponding unmodified sequences. [0090] Mutagenesis may be performed in accordance with any of the techniques known in the art, such as, and not limited to, synthesizing an oligonucleotide having one or more mutations within the sequence of a particular regulatory sequence. In particular, site-specific mutagenesis is a technique useful in the preparation of promoter mutants, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered.

[0091] Where a clone comprising a promoter has been isolated in accordance with the instant invention, one may wish to delimit the essential promoter regions within the clone. One efficient, targeted means for preparing mutagenizing promoters relies upon the identification of putative regulatory elements within the promoter sequence. This can be initiated by comparison with promoter sequences known to be expressed in similar tissue specific or developmentally unique manner. Sequences which are shared among promoters with similar expression patterns are likely candidates for the binding of transcription factors and are thus likely elements which confer expression patterns. Confirmation of these putative regulatory elements can be achieved by deletion analysis of each putative regulatory sequence followed by functional analysis of each deletion construct by assay of a reporter gene which is functionally attached to each construct. As such, once a starting promoter sequence is provided, any of a number of different deletion mutants of the starting promoter could be readily prepared.

[0092] Functional equivalent fragments may be 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more base pairs. Narrowing the

transcription regulating nucleic acid to its essential, transcription mediating elements can be realized in vitro by trial-and-error deletion mutations, or in silico using promoter element search routines. Regions essential for promoter activity often demonstrate clusters of certain, known promoter elements. Such analysis can be performed using available computer algorithms such as PLACE ("Plant Cis-acting Regulatory UNA Elements"; Higo Nucl. Acids Res. 27 (1): 297-300 (1999), the BIOBASE database "Transfac" Wingender Nucl. Acids Res. 29 (1): 281-283 (2001) or the database PlantCARE Lescot Nucl. Acids Res. 30 (1): 325-327 (2002).

[0093] For example, functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter Proc Natl Acad Sci USA 87:5387-5391(1990); Terzaghi WB, Cashmore AR Light-regulated transcription Annu Rev Plant Physiol Plant Mol Biol 46:445-474 (1995); Nakashima K, Fujita Y, Katsura K, Maruyama K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis. Plant Mol Biol. 60: 51-68 (2006); Piechulla B, Merforth N, Rudolph B Identification of tomato Lhc promoter regions necessary for circadian expression Plant Mol Biol 38:655-662 (1998); Villain P, Mache R, Zhou DX The mechanism of GT element-mediated cell type-specific transcriptional control J Biol Chem 271 :32593-32598 (1996); Le Gourrierec J, Li YF, Zhou DX Transcriptional activation by Arabidopsis GT-1 may be through interaction with TFIIA-TBP-TATA complex Plant J 18:663-668 (1999); Buchel AS, Brederode FT, Bol JF, Linthorst HJM Mutation of GT-1 binding sites in the Pr-IA promoter influences the level of inducible gene expression in vivo Plant Mol Biol 40:387-396 (1999); Zhou DX Regulatory mechanism of plant gene transcription by GT-elements and GT-factors Trends in Plant Science 4:210-214 (1999); Giuliano G, Pichersky E, Malik VS, Timko MP, Scolnik PA, Cashmore AR An evolutionarily conserved protein binding sequence upstream of a plant light regulated gene. Proc Natl Acad Sci USA 85:7089-7093 (1988); Donald RGK, Cashmore AR Mutation of either G box or 1 box sequences profoundly affects expression from the Arabidopsis rbcS-ΙΑ promoter. EMBO J 9:1717-1726 (1990); Rose A, Meier I, Wienand U The tomato I-box binding factor LeMYBI is a member of a novel class of Myb-like proteins Plant J 20: 641-652 (1999); Martinez-Hernandez A, Lopez-Ochoa L, Arguello-Astorga, G,Herrera-Estrella L. Functional properties and regulatory complexity of a minimal RBCS light- responsive unit activated by phytochrome, cryptochrome, and plastid signals. Plant Physiol. 128:1223-1233 (2002); Nakamura M, Tsunoda T, Obokata J Photosynthesis nuclear genes generally lack TATA-boxes: a tobacco photosystem I gene responds to light through an initiator Plant J 29: 1-10 (2002); Castresana C, Garcia-Luque I, Alonso E, Malik VS, Cashmore AR Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia EMBO J 7: 1929-1936 (1988); Hudson ME, Quail PH.

Identification of promoter motifs involved in the network of phytochrome A-regulated gene expression by combined analysis of genomic sequence and microarray data. Plant Physiol. 133: 1605-1616 (2003); Jiao Y, Ma L, Strickland E, Deng XW. Conservation and Divergence of Light-Regulated Genome Expression Patterns during Seedling Development in Rice and Arabidopsis. Plant Cell. 17: 3239-3256 (2005)).

[0094] Promoter activity can be routinely confirmed by expression assays, for example, as described in the Examples section herewith. In addition, modification of promoter sequences without loss of activity is routine in the art. For example, the well-known CaMV 35S promoter has been shown to retain promoter activity when fragmented into two domains, with Domain A (-90 to +8) able to confer expression primarily in root tissues (Benfrey et. ah, (1989) EMBO J 8(8):2195-2202 and Domain B (-343 to -90) conferring expression in most cell types of leaf, stem and root vascular tissues. A CaMV promoter has been truncated to a -46 promoter and still retains, although reduced, correct promoter activity (Odell et. ah, (1985) Nature 313:810-812).

[0095] Welsch et. ah describe the creation of multiple deletion fragments of an Arabidopsis thaliana phytoene synthase gene promoter (Welsch et. ah (2003) Planta 216:523-534). Using truncation studies, Welsch et. ah showed that as little as 11% of the promoter needed to be retained in order to observe some promoter activity. The deletion analysis of promoters from the cab 1A, cab IB, cab8 and cab 11 genes from the tomato light harvesting complex of genes determined which deletion would affect circadian expression (Piechulla, et. ah (1998) Plant Molecular Biology 38:655-662). A deletion of approximately 775 bp could be made from a 1058 bp plant promoter designated AtEXP18 without significantly reducing promoter activity (Cho and Cosgrove (2002) Plant Cell 14:3237-3253). In addition, the authors showed that numerous substitution mutations could be made in a fragment of AtEXP18, while retaining full promoter activity and in some cases increasing activity.

[0096] The invention disclosed herein provides polynucleotide molecules comprising regulatory element fragments that may be used in constructing novel chimeric regulatory elements. Novel combinations comprising fragments of these polynucleotide molecules and at least one other regulatory element or fragment can be constructed and tested in plants and are considered to be within the scope of this invention. Thus the design, construction, and use of chimeric regulatory elements is one embodiment of this invention. Promoters of the present invention include homologues of cis elements known to effect gene regulation that show homology with the promoter sequences of the present invention. These cis elements include but are not limited to light regulatory elements.

[0097] Functional equivalent fragments of one of the transcription regulating nucleic acids described herein comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 base pairs of a transcription regulating nucleic acid as described by SEQ ID NOS. 1 to 15. Equivalent fragments of transcription regulating nucleic acids, which are obtained by deleting the region encoding the 5 '-untranslated region of the mRNA, would then only provide the (untranscribed) promoter region. The 5 '-untranslated region can be easily determined by methods known in the art (such as 5 '-RACE analysis). Accordingly, some of the transcriptions regulating nucleic acids, described herein, are equivalent fragments of other sequences.

[0098] As indicated above, deletion mutants of the promoter of the invention also could be randomly prepared and then assayed. Following this strategy, a series of constructs are prepared, each containing a different portion of the promoter (a subclone), and these constructs are then screened for activity. A suitable means for screening for activity is to attach a deleted promoter or intron construct which contains a deleted segment to a selectable or screenable marker, and to isolate only those cells expressing the marker gene. In this way, a number of different, deleted promoter constructs are identified which still retain the desired, or even enhanced, activity. The smallest segment which is required for activity is thereby identified through comparison of the selected constructs. This segment may then be used for the construction of vectors for the expression of exogenous genes.

[0099] An expression cassette as described herein may comprise further regulatory elements. The term in this context is to be understood in the broad meaning comprising all sequences which may influence construction or function of the expression cassette. Regulatory elements may, for example, modify transcription and/or translation in prokaryotic or eukaryotic organisms. The expression cassette described herein may be downstream (in 3 '-direction) of the nucleic acid sequence to be expressed and optionally contain additional regulatory elements. Each additional regulatory element may be operably liked to the nucleic acid sequence to be expressed (or the transcription regulating nucleotide sequence). Additional regulatory elements may comprise additional promoters, minimal promoters, promoter elements, or transposon elements which may modify or enhance the expression regulating properties. The expression cassette may also contain one or more introns, one or more exons and one or more terminators.

[00100] Furthermore, it is contemplated that promoters combining elements from more than one promoter may be useful. For example, U.S. Pat. No. 5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with a histone promoter. Thus, the elements from the promoters disclosed herein may be combined with elements from other promoters. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive (Odell Nature 313: 810 - 812 (1985)), temporally regulated, spatially regulated, tissue specific, and spatial temporally regulated. Using the regulatory elements described herein, numerous agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below.

1. Pests or Disease Resistance Nucleic Acids, For Example:

[00101] (A) Plant disease resistance nucleic acids. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae). A

developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo . alpha.- 1 ,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-. alpha.- 1,4-D-galacturonase. See Lamb et al., Bio/Technology 10: 1436 (1992). The cloning and characterization of a gene which encodes a bean

endopolygalacturonase-inhibiting protein is described by Toubart et al., Plant J. 2: 367 (1992). A molecule that stimulates signal transduction. For example, see the disclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess et al., Plant Physiol.104: 1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone. A hydrophobic moment peptide. See PCT application W095/16776 (disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT application W095/18855 (teaches synthetic antimicrobial peptides that confer disease resistance), the respective contents of which are hereby incorporated by reference. A membrane permease, a channel former or a channel blocker. For example, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993), of heterologous expression of a cecropin-.beta. lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum. A viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id. An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland, 1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments). A virus- specific antibody. See, for example, Tavladoraki et al., Nature 366: 469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack. A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10: 305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.

[00102] (B) Pest Resistance Nucleic Acids. A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence of a Bt .delta. -endotoxin gene.

Moreover, DNA molecules encoding .delta.-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998. A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes. A vitamin-binding protein, such as avidin. See PCT application US93/06487 the contents of which are hereby incorporated by. The application teaches the use of avidin and avidin homologues as larvicides against insect pests. An enzyme inhibitor, lor example, a protease inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci. Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus .alpha.-amylase inhibitor). An insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344: 458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone. An insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Res. Comm.163: 1243 (1989) (an allostatin is identified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encoding insect-specific, paralytic neurotoxins. Insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 116: 165 (1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insect toxic peptide. An enzyme responsible for a hyperaccumulation of a monterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity. An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO 93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol.23: 691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al., Plant Mole. Biol. 21: 673 (1993), who provide the nucleotide sequence of the parsley ubi4- 2 polyubiquitin gene. 2. Herbicide Resistance Nucleic Acids, lor Example:

[00103] An herbicide that inhibits the growing point or meristem, such as an

imidazalinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee et al., EMBO J. 7: 1241 (1988), and Miki et al., Theor Appl. Genet. 80: 449 (1990), respectively. Glyphosate (resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cycloshexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah et al., which discloses the nucleotide sequence of a form of EPSP which can confer glyphosate resistance. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession No. 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. European patent application No. 0 333 033 and U.S. Pat. No. 4,975,374 describe nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in European application No. 0 242 246; De Greef et al., Bio/Technology 7: 61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. Exemplary of genes conferring resistance to phenoxy proprionic acids and cycloshexones, such as sethoxydim and haloxyfop, are the Accl-Sl, Accl-S2 and Accl-S3 genes described by Marshall et al., Theor. Appl. Genet. 83: 435 (1992). An herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al., Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al., Biochem. J. 285: 173 (1992).

3. Value-added Trait Nucleic Acids, For Example:

[00104] Modified fatty acid metabolism, for example, by transforming a plant with an antisense gene of stearoyl-ACP desaturase to increase stearic acid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci. USA 89: 2624 (1992). Introduction of a phytase-encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For example, see Van Hartingsveldt et al., Gene 127: 87 (1993), for a disclosure of the nucleotide sequence of an Aspergillus niger phytase gene. Modified carbohydrate composition effected, for example, by transforming plants with a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810 (1988) (nucleotide sequence of Streptococcus mutans fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Pen et al., Bio/Technology 10: 292 (1992) (production of transgenic plants that express Bacillus licheniformis .alpha.- amylase), Elliot et al., Plant Molec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes) and Fisher et al., Plant Physiol. 102: 1045 (1993) (maize endosperm starch branching enzyme II).

4. Photoassimilation Regulation Nucleic Acids, For Example:

[00105] Any of the enzymes or genes involved in the C₃, C₄ or CAM

photosynthesis/photorespiration pathway may be operably linked to any of the regulatory nucleic acids described herein. Enzymes may include rubisco (ribulose bisphosphate

carboxylase/oxygenase, EC 4.1.1.39), phosphoglycollate phosphatase (EC 3.1.3.18), (S)-2- hydroxy-acid oxidase (EC 1.1.3.15), glycine transaminase (EC 2.6.1.4), serine-glyoxylate aminotransferase (EC 2.6.1.45), glycerate dehydrogenase (EC 1.1.1.29), glycerate kinase (2.7.1.31); phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), NADP-dependent malic enzyme (NADPMD) or malate dehydrogenase (EC 1.1.1.40, EC 1.1.1.82), phosphoglycerate kinase (PGK, EC 2.7.2.3), sedoheptulose-l,7-bisphosphatase (SBP, EC 3.1.3.37), fructose-1, 6- bisphosphate phosphatase (FBPase, EC 3.1.3.11), phosphoribulokinase (PRK, EC 2.7.1.19), pyruvate, orthophosphate dikinase (PPDK, EC 2.7.9.1), and the like. Numerous examples of the photoassimilation regulation genes can be found in the literature. The BRENDA database (brenda.enzymes.org) can be queried for sequence information on many of the genes involved in the photosynthesis/photorespiration pathways. In particular, examples of PRK, SBP, PGK and NADPME from maize can be found in WO2012061585, which is hereby incorporated by reference. Typical C₃ plants include wheat, rice, soybean and potato. Typical C₄ plants are primarily monocotyledonous plants include maize, sugarcane, sorghum, amaranth, other grasses and sedges. Typical CAM plants are pineapple, epiphytes, succulent xerophytes, hemiepiphytes, lithophytes, terrestrial bromeliads, wetland plants, Mesembryanthemum crystallinum, Dodoneaea viscosa, and Sesuvium portulacastrum. It is possible to express photoassimilation regulation genes from one type of plant in another. For example, C₄-cycle enzymes have been introduced into C₃ plants. For a review, please see Hausler, et.al. (2002) J of Experimental Botany, Vol. 53, No. 369, pp. 591-607).

5. Yield Increasing or Stress Tolerant Nucleic Acids

[00106] There are a number of nucleic acids that may provide improved yield, such as, improved grain yield or biomass. In addition, there are a number of nucleic acids that improve a plants ability to yield under a number of abiotic stresses, such as, drought, salinity, heat, reduced nitrogen, shade tolerance and the like. For example, US Patent Nos. 7,030,294; 6,686,516;

6,566,511, 5,925,804; 6,833,490; 7,247,770 and US Patent Publication No. 2010/0205692, describe the use of genes of the trehalose pathway for increasing yield and improving stress tolerance. US Patent Nos. 7,109,033; 7,692,065; 7,732,667 and US Patent Publication Nos. 2003/303589; 2003/299859 describe a number of plant genes for improving a plant's response to stress. Additional genes capable of conferring stress tolerance include, LNT1 gene for improving NUE (WO 2010/031312); GMWRKY54 gene (WO 2009/057061); genes for inhibiting ammonia (US Patent Publication No. 2011/0030099); OsGATA for nitrogen use efficiency (US Patent No. 7,554,018) and the like.

[00107] The foregoing examples described herein are for illustrative purposes only and are not intended to be limiting.

[00108] The following Examples provide illustrative embodiments. In light of the invention and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently claimed subject matter.

[00109] Unless indicated otherwise, The recombinant DNA steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, linking DNA fragments, transformation of E. coli cells, growing bacteria, and sequence analysis of recombinant DNA, are carried out as described by Sambrook (1989).

EXAMPLE 1 : REGULATORY SEQUENCES TO TARGET CANDIDATE GENE EXPRESSION

[00110] A series of plant expression cassettes were designed to deliver robust trait gene expression in either mesophyll or bundle sheath cells. A combination of proteomic data (Majeran, et. al. (2005) Plant Cell 17: 3111-3140) and expression profiling data was used to identify candidate regulatory nucleic acids based on the expression patterns of genes of interest, and six novel expression cassettes were identified (Coneva V, et. al. (2007) J of Exp Botany 58:3679-3693). Each cassette is composed of regulatory nucleic acids including the promoter, first intron, a 5 '-untranslated sequence and terminator sequences. In addition the promoter terminates with a translational enhancer derived from the tobacco mosaic virus omega sequence (Gallie and Walbut (1990) Nucleic Acids Res 20(17): 4631-4638) and a maize-optimized Kozak sequence (Kozak (2002) Gene 299: 1-34). The terminator consists of 3 '-untranslated sequence starting just after the translation stop codon and 3 '-non-transcribed sequence. The following regulatory nucleic acid candidates were identified from maize:

Candidate Gene Name Maize Gene Chip Expression in probe Cell Type phosphribulokinase-2 ZmPRK-2 Zm000129 at Bundle sheath phosphribulokinase ZmPRK-1 Zm003395 at Bundle sheath sedoheptulose-l,7-bisphosphatase ZmSBP Zm009018 at Bundle sheath phosphoglycerate kinase ZmPGK Zm008627 at Mesophyll

NADP-dependent malic enzyme ZmNADPME MZENDMEX_at Mesophyll [00111] The following nucleic acids were isolated:

[00112] Specific base substitutions were made to eliminate internal Xhol, SanDI, Ncol,

Sacl, RsrII and Xmal restriction endonuclease sites. In addition base substitutions were used to eliminate ATGs and insert stop codons in the 5 '-untranslated sequence. The promoters were flanked with XhoI/SanDI at the 5 '-end and Ncol on the 3 '-end. The terminators were flanked with Sacl at the 5 '-end and RsrII/Xmal on the 3 '-end. Cassettes were ligated sequentially as RsrII/SanDI fragments into binary vector cut with RsrII.

EXAMPLE 2: EXPRESSION CASSETTES AND COMBINATIONS

[00113] To test the expression pattern of the identified regulatory nucleic acids, a three- gene and a four-gene expression cassette binary vector containing genes selected to be used to increase the C4 photosynthesis output were designed. The three gene C4 photosynthesis enhancement construct and the four gene C4 photosynthesis enhancement construct are shown below. The gene number indicates order, starting at the right border of the T-DNA and extending to the left border. The PRK-1, PRK-2, SBP, PGK and NADPME sequences from maize can be found in WO2012061585, which is hereby incorporated by reference. The regulatory nucleic acids include the promoter, intron and terminator from the same gene source. For example, the expression vector of ZmPRK-1 includes the prZmPRK-1 , the iZmPRKl and the tZmPRKl. The three gene binary vector is 19862 and is shown in Figure 1. The four gene binary vector is 19863 and is shown in Figure 2.

Vector 19862

Number Trait Gene Regulatory Translational SEQ ID nucleic acid enhancer NO

1 Fructose- 1,6-bisphosphatase (SoFBP) ZmPRK-1 eTMV-06 19

2 phosphoribulokinase (SoPRK) ZmSBP eTMV-06 20

3 phosphoenolpyruvate carboxylase ZmPGK eTMV-07 21

(ZmPEPC)

Vector 19863

Number Trait Gene Regulatory Translational SEQ ID nucleic acid enhancer NO

1 Fructose- 1,6-bisphosphatase (SoFBP) ZmPRK-2 eTMV-08 22

2 phosphoribulokinase (SoPRK) ZmNADPME eNtADH-02 23

3 pyruvate, orthophosphate dikinase ZmPEPC 24

(SbPPDK)

4 NADP-malate dehydrogenase ZmPGK eTMV-07 25

(SbNADP-MD)

EXAMPLE 3: PLANT TRANSFORMATION

[00114] Constructs 19862 and 19863 were used for Agrobacterium-mediated maize transformation. Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. For this example, all media constituents were essentially as described in Negrotto et al., supra. However, various media constituents known in the art may be substituted.

[00115] The genes used for transformation were ligated into a vector suitable for maize transformation. Vectors used in this example contain the phosphomannose isomerase (PMI) gene for selection of transgenic lines (Negrotto et al., supra), as well as the selectable marker phosphinothricin acetyl transferase (PAT) (U.S. Patent No. 5,637,489). Briefly, Agrobacterium strain LBA4404 (pSBl) containing a plant transformation plasmid was grown on YEP (yeast extract (5 g/L), peptone (lOg/L), NaCl (5g/L), 15g/l agar, pH 6.8) solid medium for 2 - 4 days at 28°C. Approximately 0.8X 10⁹ Agrobacterium were suspended in LS-inf media supplemented with 100 μΜ As (Negrotto et al., supra). Bacteria were pre-induced in this medium for 30-60 minutes.

[00116] Immature embryos from A188 or other suitable genotype were excised from 8 -

12 day old ears into liquid LS-inf + 100 μΜ As. Embryos were rinsed once with fresh infection medium. Agrobacterium solution is then added and embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred, scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate were transferred to LSDc medium supplemented with cefotaxime (250 mg/1) and silver nitrate (1.6 mg/1) and cultured in the dark for 28°C for 10 days.

[00117] Immature embryos, producing embryogenic callus were transferred to

LSD1M0.5S medium. The cultures were selected on this medium for about 6 weeks with a subculture step at about 3 weeks. Surviving calli were transferred to Regl medium supplemented with mannose. Following culturing in the light (16 hour light/ 8 hour dark regiment), green tissues were then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago 111.) containing Reg3 medium and grown in the light.

[00118] Plants were assayed for PMI, PAT, one candidate gene coding sequence and vector backbone by TaqMan. Plants that were positive for PMI, PAT and the candidate gene markers, and negative for vector backbone were transferred to the greenhouse. Expression for all trait expression cassettes was assayed by qRT-PCR. Fertile, single copy events were identified and maintained. EXAMPLE 4: IDENTIFYING ELEMENTS WITHIN THE PROMOTER SEQUENCES

[00119] The known transcription factor binding motifs on the promoter sequences were predicted using the PLACE (PLAnt Cis-acting regulatory DNA Elements) Web Signal Scan program on the World Wide Web at dna.affrc.go.jp/PLACE/signalscan.html. See Higo et al., Nucleic Acids Research, 27(1): 297-300(1990); and Prestridge, CABIOS, 7:203-206 (1991). The identification of such motifs provided important information about the candidate promoter. For example, some motifs are associated with informative annotation such as (but not limited to) "light inducible binding site" or "stress inducible binding motif and can be used to select with confidence a promoter that is able to confer light inducibility or stress inducibility to an operably linked transgene, respectively. Motifs annotated as essential for light regulation e.g. BOXIIPCCHS, necessary for circadian expression e.g. CIACADIANLELHC, conserved sequence upstream of light-regulated genes e.g. GBOXLERBCS, binding site in many light- regulated genes e.g. GT1CONSENSUS, light regulation e.g. IBOX, light regulation e.g. IBOXCORE, associated with light-responsive promoter regions e.g. IBOXCORENT, light- responsive transcription e.g. INRNTPSADB, light responsive element e.g. LREBOXIIPCCHS 1 , light regulatory element e.g. LRENPCABE, involved in light induction e.g. MNF1ZMPPC1, light responsiveness e.g. PALBOXAPC, sequences over-represented in light-induced promoters e.g. SORLIP1AT, SORLIP2AT, and SORLIP5AT support the light regulated function of promoters as well as identify the location of functional elements responsible for light regulated function allowing promoter fragments and variants to be designed that will retain light regulated function by retaining functional motifs.

TABLE OF IDENTIFIED PROMOTER ELEMENTS

MOTIF

SEQUENCE BEGIN END STRAND MOTIF Name Sequence

prPepC-11 26 31 + IBOX GATAAG prPepC-11 26 30 + IBOXCOPvE GATAA prPepC-11 243 248 + GT 1 CONS ENS US GPvWAAW prPepC-11 272 276 - IBOXCOPvE GATAA prPepC-11 387 396 + CIACADIANLELHC CAANNNNATC prPepC-11 486 493 + INPvNTPSADB YTCANTYY prPepC-11 525 530 + IBOX GATAAG prPepC-11 525 529 + IBOXCOPvE GATAA prPepC-11 525 531 + IBOXCOPvENT GATAAGPv prPepC-11 610 617 + INPvNTPSADB YTCANTYY prPepC-11 624 631 - INPvNTPSADB YTCANTYY prPepC-11 663 670 + INPvNTPSADB YTCANTYY prPepC-11 672 676 - SOPvLIPlAT GCCAC prPepC-11 704 709 + GTICONSENSUS GPvWAAW prPepC-11 747 752 + GTICONSENSUS GPvWAAW prPepC-11 793 798 + GTICONSENSUS GPvWAAW prPepC-11 843 848 + GTICONSENSUS GPvWAAW prPepC-11 1069 1074 + GTICONSENSUS GPvWAAW prPepC-11 1072 1079 - INPvNTPSADB YTCANTYY prPepC-11 1256 1260 - IBOXCOPvE GATAA prPepC-11 1260 1267 + INPvNTPSADB YTCANTYY prPepC-11 1277 1281 + IBOXCOPvE GATAA prPepC-11 1389 1396 + INPvNTPSADB YTCANTYY prPepC-11 1496 1503 + MNF1ZMPPC1 GTGCCCTT prPepC-11 1517 1523 - IBOXCOPvENT GATAAGPv prPepC-11 1518 1523 - IBOX GATAAG prPepC-11 1519 1523 - 1BOXCORE GATAA prPepC-11 1558 1564 - IBOXCOPvENT GATAAGR prPepC-11 1559 1564 - IBOX GATAAG prPepC-11 1560 1564 - IBOXCOPvE GATAA prPepC-11 1671 1680 + CIACADIANLELHC CAANNNNATC prPepC-11 1700 1707 + INPvNTPSADB YTCANTYY prPepC-11 1712 1718 - IBOXCOPvENT GATAAGR prPepC-11 1713 1718 - IBOX GATAAG prPepC-11 1714 1718 - IBOXCOPvE GATAA prPepC-11 1781 1785 - IBOXCOPvE GATAA prPepC-11 1803 1807 + SOPvLIPlAT GCCAC prPepC-11 1827 1836 + CIACADIANLELHC CAANNNNATC prPepC-11 1855 1859 + SOPvLIPlAT GCCAC prPepC-11 1873 1878 - GT1CONSENSUS GRWAAW prPepC-11 2219 2223 + SOPvLIPlAT GCCAC prPepC-11 2286 2290 + SORLIP2AT GGGCC prPepC-11 2295 2300 - GT1CONSENSUS GRWAAW prPepC-11 2308 2312 + SOPvLIPlAT GCCAC prPepC-11 2379 2388 + CIACADIANLELHC CAANNNNATC prPepC-11 2431 2436 - GT1CONSENSUS GRWAAW prPepC-11 2444 2449 - GT1CONSENSUS GRWAAW prPepC-11 2479 2483 - SORLIP2AT GGGCC prPepC-11 2553 2562 + CIACADIANLELHC CAANNNNATC prPepC-11 2617 2621 + SORLIP2AT GGGCC prPepC-11 2618 2622 - SORLIP2AT GGGCC prZmNADPME 31 35 - 1BOXCORE GATAA prZmNADPME 54 58 + SORLIP1AT GCCAC prZmNADPME 147 152 + GTICONSENSUS GRWAAW prZmNADPME 408 412 + SORLIPIAT GCCAC prZmNADPME 472 476 - SORLIPIAT GCCAC prZmNADPME 476 481 + PALBOXAPC CCGTCC prZmNADPME 523 528 + IBOX GATAAG prZmNADPME 523 527 + IBOXCORE GATAA prZmNADPME 523 529 + IBOXCORENT GATAAGR prZmNADPM 567 571 - SORLIP2AT GGGCC prZmNADPME 646 650 + SORLIPIAT GCCAC prZmNADPME 862 871 + CIACADIANLELHC CAANNNNATC prZmNADPME 876 880 - SORLIP2AT GGGCC prZmNADPME 906 911 + GTICONSENSUS GRWAAW prZmNADPME 956 961 + GTICONSENSUS GRWAAW prZmNADPME 1055 1059 + SORLIPIAT GCCAC prZmNADPME 1094 1099 + GTICONSENSUS GRWAAW prZmNADPME 1134 1139 + GTICONSENSUS GRWAAW prZmNADPME 1151 1155 - SORLIPIAT GCCAC prZmNADPME 1 153 1157 - SORLIP2AT GGGCC prZmNADPME 1179 1184 + GTICONSENSUS GRWAAW prZmNADPME 1180 1185 + GTICONSENSUS GRWAAW prZmNADPME 1261 1266 + GTICONSENSUS GRWAAW prZmNADPME 1303 1308 + GTICONSENSUS GRWAAW prZmNADPME 1554 1559 + PALBOXAPC CCGTCC prZmNADPME 1558 1563 + PALBOXAPC CCGTCC prZmNADPME 1637 1641 - SORLIP2AT GGGCC prZmNADPME 1667 1671 + SORLIP1AT GCCAC prZmNADPME 1747 1751 + SORLIP1AT GCCAC prZmNADPME 1806 1810 + SORLIP1AT GCCAC prZmPGK 149 158 - CIACADIANLELHC CAANNNNATC prZmPGK 335 339 + SORLIP1AT GCCAC prZmPGK 679 684 + GTl CONSENSUS GRWAAW prZmPGK 855 859 - SORLIP1AT GCCAC prZmPGK 909 913 + SORLIP1AT GCCAC prZmPGK 987 994 - INRNTPSADB YTCANTYY prZmPGK 992 997 + GT1CONSENSUS GRWAAW prZmPGK 1189 1194 + GTl CONSENSUS GRWAAW prZmPGK 1198 1205 - INRNTPSADB YTCANTYY prZmPGK 1209 1214 - PALBOXAPC CCGTCC prZmPGK 1241 1246 + GT1CONSENSUS GRWAAW prZmPGK 1251 1255 + SORLIP1AT GCCAC prZmPGK 1352 1357 + IBOX GATAAG prZmPGK 1352 1356 + IBOXCORE GATAA prZmPGK 1375 1379 - IBOXCORE GATAA prZmPGK 1394 1399 - GT1CONSENSUS GRWAAW prZmPGK 1512 1516 - SORLIP2AT GGGCC prZmPGK 1522 1526 + SORLIP1AT GCCAC prZmPGK 1573 1577 + SORLIP1AT GCCAC prZmPGK 1662 1667 - GT1CONSENSUS GRWAAW prZmPGK 1673 1677 - SORLIPIAT GCCAC prZmPGK 1682 1686 + SORLIP2AT GGGCC prZmPGK 1690 1694 + SORLIPIAT GCCAC prZmPGK 1820 1824 + SORLIP2AT GGGCC prZmPGK 1863 1872 + CIACADIANLELHC CAANNNNATC prZmPGK 1880 1884 + SORLIPIAT GCCAC prZmPrkl 1 8 - INRNTPSADB YTCANTYY prZmPrkl 57 62 - GT1CONSENSUS GRWAAW prZmPrkl 138 142 - IBOXCORE GATAA prZmPrkl 207 216 - CIACADIANLELHC CAANNNNATC prZmPrkl 262 267 - GT1CONSENSUS GRWAAW prZmPrkl 316 321 - GT1CONSENSUS GRWAAW prZmPrkl 416 429 - PALBOXPPC CCGTCC prZmPrkl 469 473 + IBOXCORE GATAA prZmPrkl 590 595 + GT1CONSENSUS GRWAAW prZmPrkl 597 602 + GT1CONSENSUS GRWAAW prZmPrkl 603 608 + GT1CONSENSUS GRWAAW prZmPrkl 604 609 + GT1CONSENSUS GRWAAW prZmPrkl 618 623 + GT1CONSENSUS GRWAAW prZmPrkl 808 812 + SORLIPIAT GCCAC prZmPrkl 851 857 - BOXIIPCCHS ACGTGGC prZmPrkl 851 859 - GBOXLERBCS MCACGTGGC prZmPrkl 851 860 - LREBOXIIPCCHS1 TCCACGTGGC prZmPrkl 851 855 + SORLIPIAT GCCAC prZmPrkl 915 919 + SORLIP2AT GGGCC prZmPrkl 937 941 + SORLIPIAT GCCAC prZmPrkl 1071 1076 - GTICONSENSUS GRWAAW prZmPrkl 1102 1107 + GTICONSENSUS GRWAAW prZmPrkl 1103 1108 + GTICONSENSUS GRWAAW prZmPrkl 1116 1121 + GTICONSENSUS GRWAAW prZmPrkl 1144 1149 + GTICONSENSUS GRWAAW prZmPrkl 1204 1209 + GTICONSENSUS GRWAAW prZmPrkl 1260 1267 - INPvNTPSADB YTCANTYY prZmPrkl 1328 1335 - INPvNTPSADB YTCANTYY prZmPrkl 1392 1397 + GTICONSENSUS GRWAAW prZmPrkl 1461 1468 + INPvNTPSADB YTCANTYY prZmPrkl 1524 1529 + GTICONSENSUS GRWAAW prZmPrkl 1539 1543 + SORLIPIAT GCCAC prZmPrkl 1556 1563 + INPvNTPSADB YTCANTYY prZmPrkl 1570 1574 - IBOXCOPvE GATAA prZmPrkl 1606 1611 - GTICONSENSUS GRWAAW prZmPrkl 1617 1622 - GTICONSENSUS GRWAAW prZmPrkl 1638 1642 + SORLIPIAT GCCAC prZmPrkl 1664 1668 + SORLIP2AT GGGCC prZmPrkl 2133 2137 + SORLIP2AT GGGCC prZmPrkl 2243 2248 - IBOX GATAAG prZmPrkl 2244 2248 - IBOXCORE GATAA prZmPRK2 46 51 + GTICONSENSUS GRWAAW prZmPRK2 325 329 + SORLIPIAT GCCAC prZmPRK2 352 357 + GTICONSENSUS GRWAAW prZmPRK2 352 356 + IBOXCORE GATAA prZmPRK2 608 612 - IBOXCORE GATAA prZmPRK2 737 742 + GTICONSENSUS GRWAAW prZmPRK2 800 804 + SORLIP2AT GGGCC prZmPRK2 847 851 + SORLIP1AT GCCAC prZmPRK2 951 957 - BOXIIPCCHS ACGTGGC prZmPRK2 951 959 - GBOXLERBCS MCACGTGGC prZmPRK2 951 955 + SORLIP1AT GCCAC prZmPRK2 1052 1057 + GTICONSENSUS GRWAAW prZmPRK2 1052 1056 + IBOXCORE GATAA prZmPRK2 1102 1107 + GTICONSENSUS GRWAAW prZmPRK2 1201 1206 + GTICONSENSUS GRWAAW prZmPRK2 1225 1230 + GTICONSENSUS GRWAAW prZmPRK2 1494 1501 + INRNTPSADB YTCANTYY prZmPRK2 1528 1532 - IBOXCORE GATAA prZmPRK2 1537 1542 + GTICONSENSUS GRWAAW prZmPRK2 1552 1557 + GTICONSENSUS GRWAAW prZmPRK2 1605 1609 - IBOXCORE GATAA prZmPRK2 1649 1654 - GTICONSENSUS GRWAAW prZmPRK2 1662 1666 + SORLIP1AT GCCAC prZmPRK2 1983 1987 - SORLIP1AT GCCAC prZmPRK2 2127 2131 + SORLIP2AT GGGCC prZmPRK2 2237 2242 - IBOX GATAAG prZmPRK2 2238 2242 - IBOXCORE GATAA prZmPRK2 2245 2249 + SORLIP1AT GCCAC prZmSBP 127 133 1BOXCORENT GATAAGR prZmSBP 128 133 IBOX GATAAG prZmSBP 129 133 IBOXCOPvE GATAA prZmSBP 163 167 SORLIPIAT GCCAC prZmSBP 357 361 SORLIPIAT GCCAC prZmSBP 359 363 SORLIP2AT GGGCC prZmSBP 527 533 BOXIIPCCHS ACGTGGC prZmSBP 527 535 GBOXLEPvBCS MCACGTGGC prZmSBP 527 531 SORLIPIAT GCCAC prZmSBP 528 536 GBOXLERBCS MCACGTGGC prZmSBP 530 536 BOXIIPCCHS ACGTGGC prZmSBP 532 536 SORLIPIAT GCCAC prZmSBP 780 784 SORLIPIAT GCCAC prZmSBP 844 849 GTICONSENSUS GRWAAW prZmSBP 986 991 GTICONSENSUS GRWAAW prZmSBP 1007 1011 SORLIPIAT GCCAC prZmSBP 1044 1049 GTICONSENSUS GRWAAW prZmSBP 1052 1056 IBOXCORE GATAA prZmSBP 1143 1150 INRNTPSADB YTCANTYY prZmSBP 1148 1153 GTICONSENSUS GRWAAW prZmSBP 1192 1197 GTICONSENSUS GRWAAW prZmSBP 1195 1199 SORLIPIAT GCCAC prZmSBP 1417 1422 GTICONSENSUS GRWAAW prZmSBP 1427 1431 SORLIP2AT GGGCC prZmSBP 1476 1480 SORLIP2AT GGGCC prZmSBP 1479 1484 + PALBOXAPC CCGTCC prZmSBP 1490 1494 - SORLIPIAT GCCAC prZmSBP 1508 1512 + SORLIPIAT GCCAC prZmSBP 1547 1551 - SORLIP2AT GGGCC prZmSBP 1563 1568 - GTlCONSENSUvS GRWAAW prZmSBP 1601 1605 - IBOXCORE GATAA prZmSBP 1605 1610 - GT1CONSENSUS GRWAAW prZmSBP 1734 1738 - SORLIP2AT GGGCC prZmSBP 1760 1764 + SORLIP2AT GGGCC prZmSBP 1762 1766 + SORLIPIAT GCCAC prZmSBP 1795 1799 + SORLIPIAT GCCAC prZmSBP 1830 1835 - GT1CONSENSUS GRWAAW prZmSBP 1847 1851 - SORLIP2AT GGGCC

Nucleotide ambiguity code

(IUPAC)

Nomenclature Committee (1985) Eur. J. Biochem. 150: 1-5 EXAMPLE 5: EVALUATION OF IRAN GEN 1C PLANTS EXPRESSING

PHOTOASSIMILATION REGULATING NUCLEIC ACIDS

[00120] Plant photoassimilation can be assessed in several ways. The following prophetic example describes how the transgenic plants described above will be measured for changes in plant photoassimilation. First plant growth between hemizygous trait positive and null seedlings can be compared in V3 seedlings. In this assay, approximately 60 Bl plants are germinated in 4.5 inch pots and genotyped. About 17 days after germination the pot soil is saturated with water and the soil surface is sealed to prevent evaporation. Some seedlings are sacrificed to determine shoot mass (in both fresh and dry weight) at time zero. Pot mass is recorded daily to assess plant water demand. After 7 days shoots are harvested and weighed (both fresh and dry weight). Plant water utilization is corrected using a pot with no plant to report natural water loss. This protocol enables plant growth and water utilization to be compared between trait positive and null groups. Improved photoassimilation may enable the trait positive plants to accumulate more aerial biomass relative to null plants.

[00121] A second method is to measure photoassimilation using an infrared gas analysis

(IRGA) instrument. For example a CIRAS-2 IRGA device can be fixed to a tripod to gently clamp the gas exchange cuvette to leaves and minimize data noise generated by plant handling. Stomatal aperture is very sensitive to touch and plant movement. The environment applied to the leaf patch can be programmed to mimic a growth chamber environment (400 μπιοΐ mol^"1 C0₂; 26°C; ambient humidity) to assess steady-state photosynthesis under standard growth conditions. In this way photoassimilation between trait positive and null plants can be directly compared.

[00122] Although IRGA is a powerful and common tool to assess photosynthetic activity

(e.g. A/Ci curves), it has some caveats. First, it only assays a small leaf patch and does not provide information on whole-plant and canopy-level photosynthesis, which are ultimately required to determine trait function in an agronomic context. Second, many measurements are needed to determine A throughout plant development. Third, the general state of the photosynthetic apparatus depends on which leaf is assayed and when it is assayed, there is variability throughout the plant. Finally, it is an invasive technique requiring direct contact with the leaf. A component of the data generated is leaf response to the instrument. Taken together this creates high (10-15%) coefficients of variation. Hence, it may not be possible to detect small, but significant changes in photoassimilation using this device. [00123] To bypass these limitations, large hypobaric chambers such as the chambers at the

Controlled Environment Systems Research Facility at the University of Guelph, Ontario (Wheeler et al., 2011) can be used to monitor with high precision plant C0₂ demand, night time respiration and transpiration of a 30 plant population for periods lasting up to several weeks.

EXAMPLE 6: PRODUCTION OF TRANSGENIC MAIZE WITH CONSTRUCTS 19862 AND 19863

[00124] Transgenic maize events were produced according to Example 4, using binary vectors 19862 and 19863. A total of 32 single-copy, backbone free 19862 events were identified. A total of 22 single-copy, backbone free 19863 events were identified. Messenger RNA produced from each transgene was measured in seedling leaf tissue by qRT-PCR. The qRT-PCR data are reported as the ratio of the gene-specific (coding sequence) signal to that of the endogenous control signal times 1000. The regulatory nucleic acids used to generate the transgenic plants are active in green tissue and light regulated. Transcript abundance should peak early to mid-afternoon. Data for the constitutive expression cassettes are included as a benchmark for signal strength. It should be noted that the constitutive cassettes are active in far more leaf cells than the trait cassettes which are restricted to either mesophyll or bundle sheath cells.

TABLE DESCRIBING EXPRESSION LEVELS OF TRANSGENES OPERABLE LINKED TO TRANSCRIPTION REGULATORY NUCLEIC ACIDS.

ZmPGK ZmPEPC mesophyll 1240 720

ZmUbil PMI All 6990 6120

19863 22 35S/NOS PAT All 13100 12900 bundle

ZMPRK2 SoFBP sheath 484 276 bundle

ZmNADPME SoPPvK sheath 10200 5980

ZmPEPC SbPPDK mesophyll 3860 2820

SbNADP-

ZmPGK MD mesophyll 2270 1920

ZmUbil PMI All 4850 3200

[00125] TO seedling leaf tissue was sampled for qRT-PCR analysis roughly two weeks after transfer to soil (V3). Gene-specific TaqMan probes were used to determine transcript abundance. Data are reported relative to EFIA transcript, the internal control. Each event was assayed in quadruplicate. Data are the mean + standard deviation for each construct.

EXAMPLE 7: SEEDLING BIOMASS ACCUMULATION IN A GROWTH CHAMBER

[00126] Seedling growth can be used to determine if a trait has the potential to cause yield drag. We used this assay to determine if either the 19862 or 19863 traits reduced plant growth. Back-crossed seed were germinated and seedlings were evaluated in a growth chamber according to Example 5. Seedlings for each event were genotyped to establish trait segregation and organize transgenic and null groups. Trait segregation was confirmed as 1 null:l hemizygote, as expected, for each event. Data in the Table below summarize the results of several assays. For each event, growth of the transgenic seedlings could not be distinguished from the null seedlings. This indicates the trait is not impeding growth. The wild type plants are included as a benchmark. It should be noted that plants one generation removed from a parent regenerated through tissue culture tend to grow slower than non-transformed or wild type plants. The mean data suggest that the 19862 plants may be growing slower than the wild type plants but the difference is not statistically significant.

[00127] Transgenic Bl seed were germinated in 4.5 inch pots and genotyped. Plants for each event were organized into transgenic and null groups which were grown in a growth chamber. Shoots were harvested 24 days after planting. Shoots were dried in an oven at 89°C for 5 days then weighed. Data report the mean + standard deviation for each construct.

EXAMPLE 8: EVALUATION OF 19862 EVENTS IN CLOSED CHAMBERS

[00128] Closed growth chambers can be used to accurately assess whole plant photoassimilation and respiration. Hybrid seed that segregate for the 19862 trait were made for two events, and evaluated in large hypobaric chambers at the Controlled Environment Systems Research Facility at the University of Guelph as described in Example 5. Seed were germinated, genotyped and organized into trait positive and trait negative groups of 40 plants. Ten seedlings per group were weighed at the beginning of the experiment. Each group was placed in a hypobaric chamber and grown for 4 weeks. Identical growth conditions were programmed into each chamber. The Table below reports plant biomass accumulation. The A184A null plants did not differ from A184A transgenic plants. However the B027A transgenic plants significantly outperformed the corresponding null plants. Mean biomass production was 28% higher in the transgenic plants. Photoassiniilation and respiration data collected during the second week of the study illustrate the physiological basis for the difference in biomass. Figure 1 shows the B027A transgenic plants have a higher daily photoassiniilation rate and respire less at night. Both metrics indicate that transgenics are putting more carbon into biomass. The difference in respiration was not expected.

[00129] Fl hybrid seed were germinated and genotyped. Plants were organized into transgenic and null groups. Each group was cultivated in a large hypobaric chamber at the Controlled Environment Systems Research Facility at the University of Guelph. Shoots were harvested, dried and weighed. Initial biomass was determined for seedlings shortly after genotyping and represent shoot mass at the time beginning of the study. Data are the mean + standard deviation for each group.

[00130] Taken together the data illustrate that mathematical modeling is a useful tool for developing strategies to improve plant performance.

[00131] All references cited herein, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (e.g. , GENBANK® database entries and all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

Claims

CLAIMS What is claimed is:

1. An isolated, regulatory nucleic acid comprising

a. a regulatory nucleic acid having at least 90 percent or greater sequence identity to a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10 or 13; or b. a regulatory nucleic comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13 or a functionally equivalent fragment thereof ; or

c. a regulatory nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, or 13;

wherein said regulatory nucleic acid directs transcription of an operably linked polynucleotide.

2. The nucleic acid of claim 1 , wherein said sequence identity is at least 95 percent or

greater.

3. The nucleic acid of claim 1, wherein said sequence identity is at least 98 percent or

greater.

4. The nucleic acid of claim 1 , wherein said nucleic acid contains one or more motifs

selected from the group consisting of a BOXIIPCCHS motif, a CIACADIANLELHC motif, a GTl CONSENSUS motif, a IBOX motif, a IBOXCORE motif, a IBOXCORENT motif, a INRNTPSADB motif, a SORLIP1 AT motif and a SORLIP2AT motif.

5. The nucleic acid of claim 1, wherein the functionally equivalent fragment comprises at least 200 base pairs of SEQ ID NO: 1, 4, 7, 10 or 13.

6. The nucleic acid of claim 1 , wherein the functionally equivalent fragment comprises at least 300 base pairs of SEQ ID NO: 1, 4, 7, 10 or 13.

7. The nucleic acid of claim 1, wherein the functionally equivalent fragment comprises at least 400 base pairs of SEQ ID NO: 1, 4, 7, 10 or 13.

8. The nucleic acid of claim 1, wherein the nucleic acid is operably linked to an intron.

9. The nucleic acid of claim 9, wherein the intron is selected from the group consisting of SEQ ID NO: 2, 5, 8, 11 and 14.

10. The nucleic acid of claim 10, wherein the nucleic acid molecule is operably linked to a terminator.

11. The nucleic acid of claim 11 , wherein the terminator is selected from the group consisting of SEQ ID NO: 3, 6, 9, 12 and 15.

12. The nucleic acid of claim 11, wherein the promoter, intron and terminator are isolated from the same coding region.

13. The nucleic acid of claim 11, wherein the promoter, intron and terminator are isolated from more than one coding region.

14. An expression cassette comprising:

a. a first nucleic acid according to claim 1 ;

b. a second nucleic acid to be transcribed, wherein said first and second nucleic acids are heterologous to each other and are operably linked; and c. a terminator operably linked 3' to the nucleic acid to be transcribed.

15. The expression cassette of claim 14, wherein the second nucleic acid is selected from the group comprising a pest resistance nucleic acid, a disease resistance nucleic acid, an herbicide resistance acid, a value-added trait nucleic acid, a photoassimilation regulated nucleic acid, a yield nucleic acid and a stress tolerant nucleic acid.

16. The expression cassette of claim 14, wherein the heterologous coding region is expressed in green tissue and light regulated.

17. The expression cassette of claim 14, wherein the terminator is selected from the group consisting of SEQ ID NO: 3, 6, 9, 12 and 15.

18. The expression cassette of claim 14, wherein the first nucleic acid is operably linked to an intron.

19. The expression cassette of claim 18, wherein the intron is selected from the group

consisting of SEQ ID NO: 2, 5, 8, 11 and 14.

20. A plant, plant tissue, or plant cell comprising the expression cassette of claim 15.

21. The plant, plant tissue, or plant cell of claim 20, wherein the plant, plant tissue, or plant cell is a monocot or from a monocot.

22. The plant, plant tissue, or plant cell of claim 21, wherein the plant, plant tissue, or plant cell is maize or from maize.

23. A method of expressing a heterologous coding region in a plant, plant tissue or plant cell comprising: a. providing the nucleic acid of claim 1 operably linked to a heterologous coding region; and

b. creating a plant, plant tissue, or plant cell comprising the nucleic acid, wherein the heterologous coding region is expressed.

24. The method of claim 23, wherein the heterologous coding region is expressed when exposed to light.

25. The method of claim 23, wherein the plant, plant tissue, plant cell or a portion thereof is a monocot.

26. The method of claim 25, wherein the plant, plant tissue, plant cell or a portion thereof is maize.

27. A plant, plant tissue, plant cell, or portion thereof made by the method of claim 23.

28. Progeny, seed, or grain produced by the plant, plant tissue, plant cell, or portion thereof of claim 25.

29. An isolated nucleic acid comprising SEQ ID NO: 2, 5, 8, 11 or 14.

30. An isolated nucleic acid comprising a terminator comprising SEQ ID NO: 3, 6, 9, 12, 15 or a functional fragment thereof.