WO2016094362A1 - Polynucleotides, expression cassettes and methods of making plants with increased yield - Google Patents

Abstract

The invention provides polynucleotides and combinations of polynucleotides that can be used in operable association with a promoter to increase yield in a plant. Also provided are methods of improving plant yield by expression of polynucleotides or combination of polynucleotide.

Description

POLYNUCLEOTIDES, EXPRESSION CASSETTES AND METHODS OF MAKING

PLANTS WITH INCREASED YIELD

RELATED APPLICATION INFORMATION

This Application claims the benefit of U.S. Provisional Application No. 62/090420, filed 1 1 December 2014, the contents of which are incorporated herein by reference.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821 , entitled 73637-WO-REG-ORG-P-1_Sequence_Listing_ST25.txt, 44 kilobytes in size, generated on December 1 , 2015 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.

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] The increasing world population and the dwindling supply of arable land available for agriculture fuels the need for research in the area of increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilize selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are often labor intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant's genome. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.

Summary of the Invention

[0003] One embodiment of the present invention is a nonnaturally occurring expression cassette suitable for expressing polynucleotides comprising sedoheptulose- 1 ,7-bisphosphotase (SBPase), the large and small subunit of ADP-glucose

pyrophosphorylase (AGPase-) and fructose-1 ,6-bisphosphate aldolase (FBPase) in a plant. In some embodiments of the invention the expression cassette is introduced into a plant. A further embodiment of the invention is the simultaneous expression of

SBPase, the large and small subunit of AGPase, and FBPase. This simultaneous expression may be accomplished by transforming a plant or plant part with an

expression cassette or recombinant vector comprising SBPase, the large and small subunits of AGPase and FBPase. In other embodiments, the simultaneous expression or stacking of the polynucleotides is accomplished by introducing into a first plant at least one or more of the polynucleotides, introducing at least one or more of the polynucleotides into a second, third or fourth additional plant and then crossing the various plants produced to combine the transgenes into a seed or plant containing at least SBPase, the large and small subunit of AGPase and optionally FBPase. The small and large subunits of AGPase may be isoforms native to a chloroplast.

[0004] The nonnaturally occurring 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 a C3 plant. In other embodiments the plant is selected from the group consisting of wheat, tobacco, soybean, spinach, sugar beet, sunflower, rapeseed, rice and Arabidopsis. In some embodiments the plant is a soybean plant.

[0005] 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.

[0006] The polynucleotides of the nonnaturally occurring expression cassette may be selected from the group consisting of: a) polynucleotides comprising SEQ ID NOS: 1 , 3, 5, and 7; b) polynucleotides encoding the polypeptides comprising SEQ ID NOS: 2, 4, 6, and 8; c) polynucleotides having at least 80% identity to SEQ ID NOS: 1 , 3, 5, and 7; d) polynucleotides encoding polypeptides having at least 80% identity to ID NOS: 2, 4, 6, and 8; and e) polynucleotides capable of hybridizing under stringent conditions to polynucleotides comprising SEQ ID NOS: 1 , 3, 5, and 7.

[0007] The promoters used to drive expression of SBPase, the large subunit of AGPase, the small subunit of AGPase and optionally FBPase, may include constitutive promoters, tissue-preferred promoters and developmentally preferred promoters. Some embodiments of the invention may utilize promoters regulated by light or promoters with leaf specific expression to increase levels of FBPase, SBPase and AGPase in leaves. For some embodiments of the invention, the promoters are selected from the group consisting of an OsLHCH3 promoter, an OsLHCA4 promoter, an OsPSAK promoter and an OsPSID promoter. In addition, the promoters may be selected from promoters having the same or similar expression pattern as a promoter selected from the group consisting of an OsLHCH3 promoter, an OsLHCA4 promoter, an OsPSAK promoter and an OsPSID promoter.

[0008] The expression cassettes may be used to increase yield or increase pod retention in a plant or plant population or a C3 plant or C3 plant population. The increase in yield and/or pod retention may occur without a significant increase in the rate of photoassimilation.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0009] SEQ ID NO: 1 is fructose-bisphosphate aldolase, chloroplast precursor (ALDP) from rice, polynucleotide sequence.

[00010] SEQ ID NO: 2 is fructose-bisphosphate aldolase from rice, polypeptide. 90 [00011] SEQ ID NO: 3 is Sedoheptulose-1 ,7-bisphosphatase from rice, polynucleotide.

[00012] SEQ ID NO: 4 is Sedoheptulose-1 ,7-bisphosphatase from rice, polypeptide.

[00013] SEQ ID NO: 5 is ADP-glucose pyrophosphorylase from rice, 95 AGPS2a small subunit, polynucleotide.

[00014] SEQ ID NO: 6 is ADP-glucose pyrophosphorylase from rice, AGPS2a small subunit, polypeptide.

[00015] SEQ ID NO: 7 is ADP-glucose pyrophosphorylase from rice, AGPI3, large subunit, polynucleotide,

loo [00016] SEQ ID NO: 8 is ADP-glucose pyrophosphorylase from rice,

AGPI3, large subunit, polypeptide.

[00017] SEQ ID NO

[00018] SEQ ID NO

[00019] SEQ ID NO

105 [00020] SEQ ID NO

[00021] SEQ ID NO

rice, polynucleotide.

[00022] SEQ ID NO

rice, polynucleotide

110 [00023] SEQ ID NO

from rice, polynucleotide

[00024] SEQ ID NO

from rice, polynucleotide

[00025] SEQ ID NO

115 [00026] SEQ ID NO

fused to a soy-optimized Kozak sequence.

[00027] SEQ ID NO: 19 is OsLHC4 first exon from rice, polynucleotide.

[00028] SEQ ID NO: 20 is OsLHC4 first intron from rice, polynucleotide.

[00029] SEQ ID NO: 21 is OsLHC4 terminator from rice, polynucleotide. 120 [00030] SEQ ID NO: 22 is TMV-07 tobacco mosaic virus enhancer fused to a soy-optimized Kozak sequence.

[00031] SEQ ID NO: 23 is OsPsak first exon from rice, polynucleotide.

[00032] SEQ ID NO: 24 is OsPsak first intron from rice, polynucleotide.

[00033] SEQ ID NO: 25 is OsPsak terminator from rice, polynucleotide. 125 [00034] SEQ ID NO: 26 is NtADH translational enhancer based on the

tobacco alcohol dehydrogenase gene sequence with soy optimized Kozak sequence.

[00035] SEQ ID NO: 27 is OsPSID terminator from rice.

[00036] SEQ ID NO: 28 is TMV-omega translational enhancer complex, M14 version with a soy-optimized Kozak sequence

130

Detailed Description of the Invention

[00037] 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 135 and complete, and will fully convey the scope of the invention to those skilled in the art.

[00038] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[00039] Moreover, the present invention also contemplates that in some 140 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.

[00040] Based on the inherent limitations of photosynthesis, the following is 145 a list of target traits for potential genetic engineering in a plant (Murchie et al., (2009) New Phytol 181 : 532; von Caemmerer and Evans, (201 1 ) Plant Physiol 154: 589). For example, the list may include photochemical, biophysical, metabolic, physiological and developmental traits such as: the kinetics of photoprotection relaxation (Zhu et al., (2004) J Exp Bot 55: 1 167); mesophyll conductance to C0₂ (Heckwolf et al., (201 1 ) 150 Plant J 67(5): 795); rubisco carboxylation and oxygenation rates and specificities (Griffiths, (2006) Nature 441 : 940); electron transport rate and NADP/ATP production/homeostasis (Kramer and Evans, (201 1 ) Plant Physiol 155: 70; Kiirats et al., (2009) Func Plant Biol 36: 893); RuBP regeneration rate (Raines, (2006) Plant Cell Environ 29: 331 ); feedback control between carbon metabolism and light reactions;

155 sugar synthesis and carbon allocation; sink strength and source-sink signaling; photorespiration (Stitt et al., (2010) Plant J 61 : 1067); single-cell and whole-leaf C4 photosynthesis (Edwards et al., (2004) Annu Rev Plant Biol 55: 173; Hibberd et al., (2008) Curr Opin Plant Biol 1 1 : 228).

[00041] The four pathways of the primary photosynthetic carbon

160 metabolism - the Calvin cycle for RuBP regeneration, photorespiration, starch synthesis and sucrose synthesis - together represent a crucial step between carbon assimilation and growth. The output efficiency is limited upstream by photosystem II (PSIl) activity and the generation of chemical energy~ATP and reducing power NADH, and downstream by sink strength and metabolite transport efficiency. All three steps are

165 intricately connected by mutual feedback and feed forward regulation, and together represent a good example of a complex web of control at biochemical and physiological levels (Murchie et al., (2009) New Phytol 181 : 532; Stitt et al., (2010) Plant J 61 : 1067). Such a web of interaction confers great physiological adaptability by enabling plant to respond to the environment. Such remarkable inherent homeostasis and plasticity also

170 renders plants very resistant to genetic modification via gene insertion, and few single- gene insertions are expected to improve agricultural productivity. The steps to enhance photosynthetic output are: (i) to identify and engineer certain control points (e.g. Rubisco) and (ii) to pyramid a coordinated set of changes that are additive or synergistic to yield significant enhancement.

175 [00042] Computer or mathematical models help integrate knowledge of photosynthesis, enable prediction of system response to environmental perturbation (e.g. increased O₂ and CO₂), and identify the enzymes that exert significant control of metabolite fluxes (von Caemmerer and Farquhar, (1981 ) Planta 153: 376; Laisk and Edwards, (2000) Photosynth Res 66: 199; Poolman et al., (2000) J Exp Bot 51 Spec

180 No: 319; Farquhar et al., (2001 ) Plant Physiol 125: 42). Recently, Zhu et al., ((2007) Plant Physiol 145: 513) developed a model grouping these four pathways of the primary photosynthetic carbon metabolism, and applied an evolutionary algorithm to determine respective enzyme investments for maximum photosynthetic output without nitrogen addition. Their hypothesis was that plants have not had enough time to adapt to current

185 atmospheric CO₂ levels, and would likely not adapt to projected increases over the next several decades. The result is sub-optimal photoassimilation in most C3 species. Their results recommended a decrease in photorespiratory pathway enzymes, and significant increases in Rubisco, sedoheptulose-1 ,7-bisphosphatase and fructose-1 ,6- bisphosphate aldolase (RuBP regeneration) and in ADP-glucose pyrophosphorylase

190 (sink capacity). Gain- and loss-of-function studies in tobacco and potato support the hypothesis that manipulation of these enzymes can indeed modulate photoassimilation, at least in green house and growth chamber conditions, (Haake et al., (1999) Plant J 17: 479; Miyagawa et al., (2001 ) Nature Biotech 19: 965; Lefebvre et al., (2005) Plant Physiol 138: 451 ; Smidansky et al., (2007) Planta 225: 965).

195 [00043] There are examples of transgenic manipulation of FBPase, SBPase and AGPase, individually. See, for example, Lefebvre et al., (2005) Plant Physiol 138: 451 and Olcer et al., (2001 ) Plant Physiol 125; 982 for past work targeting SBPase. Other articles consider expression of a bifunctional cyanobacterial FBPase/SBPase (Miyagawa et al., (2001 ) Nature Biotech 19: 965; Wood, (2002) Plant Sci 7(1 ): 9). There

200 is a large body of work concerning genetic manipulation of AGPase to increase yield.

This work targets the isoform expressed during seed development, for example, please see Smidansky et al., (2007) Planta 225: 965. In the present application, transgenic expression of sedoheptulose-biphosphatase, the large and small subunits of ADP- glucose pyrophosphatase and optionally fructose-bispohosphate aldolase did not result

205 in a measurable increase in photoassimilation in C3 plants but did result in increased yield of seed pods and promoted early senescence of Inner canopy (lower) leaves. The embodiments of the invention disclosed herein may be used to increase yield in any monocot or dicot plant, for example, but not limited to, soybean, tobacco, sunflower, rapeseed, spinach, sugar beet, rice, wheat, or Arabidopsis. Embodiments of the

210 invention may include methods for increasing yield in a C3 plant or plant population.

[00044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not

215 intended to be limiting of the invention.

[00045] 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 herein 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

220 to limit the scope of the present invention, which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth. As used

225 herein, the word "or" means any one member of a particular list and also includes any combination of members of that list (i.e., includes also "and").

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

230 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, preferably 10 percent up or down (higher or lower). With regard to a temperature the term "about" means ± 1 °C, preferably ± 0.5 °C. Where the term "about" is used in the context of this invention (e.g., in combinations with temperature or molecular weight values) the exact value (i.e.,

235 without "about") is preferred.

The term "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or

components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

240 As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of" when used in a claim of this invention is not intended to be interpreted to be equivalent

245 to "comprising."

The term "modulate" (and grammatical variations) refers to an increase or decrease. As used herein, the terms "increase," "increases," "increased," "increasing" and similar terms indicate an elevation of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 75%, 80%,85%, 90%, 95%, 100%, 150%,

250 200%, 300%, 400%, 500% or more as compared to a control (e.g., a plant that does not comprise at least one isolated nucleic acid of the present invention).

As used herein, the terms "reduce," "reduces," "reduced," "reduction" and similar terms mean a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 75%, 80%,85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%,

255 500% or more as compared to a control (e.g., a plant that does not comprise at least one isolated nucleic acid of the present invention). In particular embodiments, the reduction results in no or essentially no {i.e., an insignificant amount, e.g., less than about 10%, less than about 5% or even less than about 1 %) detectable activity or amount.

260 [00046] "Expression cassette" as used herein means a nucleic acid

molecule capable of directing expression of a particular polynucleotide or

polynucleotides in an appropriate host cell, comprising a promoter operably linked to the polynucleotide or polynucleotides of interest which is/are operably linked to terminators. It also typically comprises polynucleotides required for proper translation of the

265 polynucleotide or polynucleotides of interest. The expression cassette may also

comprise polynucleotides not necessary in the direct expression of a polynucleotide of interest but which are present due to convenient restriction sites for removal of the cassette from an expression vector. The expression cassette comprising the

polynucleotide(s) of interest may be chimeric, meaning that at least one of its

270 components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e. the particular polynucleotide of the expression cassette does not occur naturally in the host cell and 275 must have been introduced into the host cell or an ancestor of the host cell by a transformation process known in the art. The expression of the polynucleotide(s) in the expression cassette is generally under the control of a promoter. In the case of a multicellular organism, such as a plant, the promoter can also be specific or preferential to a particular tissue, or organ, or stage of development. An expression cassette, or

280 fragment thereof, can also be referred to as "inserted polynucleotide" or "insertion

polynucleotide" when transformed into a plant.

[00047] 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

285 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.

[00048] Additional embodiments of the invention include methods of producing a transgenic plant or methods of increasing yield in a plant comprising

290 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.

[00049] The term "chimeric construct", "chimeric gene", "chimeric

295 polynucleotide" or chimeric nucleic acid" (and similar terms) as used herein refers to a construct or molecule comprising two or more polynucleotides of different origin assembled into a single nucleic acid molecule. The term "chimeric construct", "chimeric gene", "chimeric polynucleotide" or "chimeric nucleic acid" refers to any construct or molecule that contains (1 ) polynucleotides {e.g., DNA) , including regulatory and coding

300 polynucleotides that are not found together in nature (i.e., at least one of

polynucleotides is heterologous with respect to at least one of its other polynucleotides), or (2) polynucleotides encoding parts of proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Further, a chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid may comprise regulatory

305 polynucleotides and coding polynucleotides that are derived from different sources, or comprise regulatory polynucleotides and coding polynucleotides derived from the same source, but arranged in a manner different from that found in nature. In a preferred aspect of the present invention the chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid comprises an expression cassette comprising a

310 polynucleotides of the present invention under the control of regulatory polynucleotides, particularly under the control of regulatory polynucleotides functional in plants.

[00050] The term "heterologous" when used in reference to a gene or nucleic acid refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous gene or

315 heterologous coding region may include a gene or coding region from one species

introduced into another species. A heterologous coding region may also include a coding region native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.). Heterologous coding regions further may comprise plant polynucleotides that

320 comprise cDNA forms of a protein coding region; the cDNAs may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). In one aspect of the invention, heterologous coding regions are distinguished from endogenous plant coding regions in that the heterologous coding region polynucleotides are typically joined to

325 polynucleotides comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous coding region or with a plant coding region polynucleotide in the chromosome, or are

associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed). Further, in embodiments, a

330 "heterologous" polynucleotide is a polynucleotide not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.

[00051] "Chromosomally-integrated" refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes or coding

335 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.

340 [00052] A "coding region" or "coding region polynucleotide" is a

polynucleotide that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein. It may constitute an "uninterrupted coding polynucleotide", i.e., lacking an intron, such as in a cDNA, or it may include one or more introns bounded by

345 appropriate splice junctions. An "intron" is a polyribonucleotide which is contained in the primary transcript but which is removed through cleavage and religation of the RNA within the cell to create the mature mRNA that can be translated into a protein.

[00053] "Contiguous" is used herein to mean nucleic acid sequences that are immediately preceding or following one another.

350 [00054] The term "expression" when used with reference to a

polynucleotide, such as a gene, ORF or portion thereof, or a transgene in plants, refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein where applicable (e.g. if a

355 gene encodes a protein), through "translation" of mRNA. Gene expression can be

regulated at many stages in the process. For example, in the case of antisense or dsRNA constructs, respectively, expression may refer to the transcription of the antisense RNA only or the dsRNA only. In some embodiments, "expression" refers to the transcription and stable accumulation of sense (mRNA) or functional RNA.

360 "Expression" may also refer to the production of protein.

[00055] A "gene" is defined herein as a hereditary unit consisting of a polynucleotide that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristic or trait in an organism.

[00056] "Genetic engineering", "transformation" and "genetic modification"

365 are all used herein as synonyms for the transfer of isolated, nonnaturally occurring or synthetic genes or expression cassettes into the DNA, usually the chromosomal DNA or genome, of another organism. [00057] The term "genotype" refers to the genetic constitution of a cell or organism. An individual's "genotype for a set of genetic markers" includes the specific

370 alleles, for one or more genetic marker loci, present in the individual. As is known in the art, a genotype can relate to a single locus or to multiple loci, whether the loci are related or unrelated and/or are linked or unlinked. In some embodiments, an individual's genotype relates to one or more genes that are related in that the one or more of the genes are involved in the expression of a phenotype of interest (e.g., a quantitative trait

375 as defined herein). Thus, in some embodiments a genotype comprises a sum of one or more alleles present within an individual at one or more genetic loci of a quantitative trait. In some embodiments, a genotype is expressed in terms of a haplotype (defined herein below).

[00058] The term "heterologous" when used in reference to a gene or

380 nucleic acid refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous gene or

heterologous coding region may include a gene or coding region from one species introduced into another species. A heterologous coding region may also include a coding region native to an organism that has been altered in some way (e.g., mutated,

385 added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.). Heterologous coding regions further may comprise plant polynucleotides that comprise cDNA forms of a plant coding region; the cDNAs may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). In one aspect of the invention,

390 heterologous coding regions are distinguished from endogenous plant coding regions in that the heterologous coding region polynucleotides are typically joined to

polynucleotides comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous coding region or with plant coding region polynucleotide in the chromosome, or are associated

395 with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed). Further, in embodiments, a "heterologous" polynucleotide is a polynucleotide not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.

400 [00059] The terms "homology", "sequence similarity" or "sequence identity" of nucleotide or amino acid sequences mean a degree of identity or similarity of two or more sequences and may be determined conventionally by using known software or computer programs such as the Best-Fit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.

405 5371 1 ). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981 ), to find the best segment of identity or similarity between two sequences. Sequence comparison between two or more polynucleotides or polypeptides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity.

410 The comparison window is generally from about 20 to 200 contiguous nucleotides. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit to determine the degree of DNA sequence homology, similarity or identity, the default setting may be used, or an

415 appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Similarly, when using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.

420 [00060] The term "isolated" or "nonnaturally occurring", when used in the context of the nucleic acid molecules, polynucleotides or expression cassettes of the present invention, refers to a polynucleotide that is identified within and nonnaturally occurring/separated from its chromosomal polynucleotide context within the respective source organism. A nonnaturally occurring nucleic acid or polynucleotide is not a nucleic

425 acid as it occurs in its natural context, if it indeed has a naturally occurring counterpart.

In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA, which are found in the state they exist in nature. For example, a given polynucleotide (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes. The nonnaturally occurring nucleic acid molecule may be present in single-stranded or

430 double-stranded form. Alternatively, it may contain both the sense and antisense

strands (i.e., the nucleic acid molecule may be double-stranded). In a preferred embodiment, the nucleic acid molecules of the present invention are understood to be nonnaturally occurring. "5' non-coding sequence" refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA 435 upstream of the translation 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).

[00061] "3' non-coding sequence" refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and 440 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).

445 [00062] The phrase "nucleic acid" or "polynucleotide" refers to any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA polymer or polydeoxyribonucleotide or RNA polymer or polyribonucleotide), modified oligonucleotides (e.g., oligonucleotides comprising bases that are not typical to biological RNA or DNA, such as 2'-O-

450 methylated oligonucleotides), and the like. In some embodiments, a nucleic acid or polynucleotide can be single-stranded, double-stranded, multi-stranded, or

combinations thereof. Unless otherwise indicated, a particular nucleic acid or

polynucleotide of the present invention optionally comprises or encodes complementary polynucleotides, in addition to any polynucleotide explicitly indicated.

455 [00063] 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). 460 [00064] "Operably linked" refers to the association of polynucleotides on a single nucleic acid fragment so that the function of one affects the function of the other. For example, a promoter is operably linked with a coding polynucleotide or functional RNA when it is capable of affecting the expression of that coding polynucleotide or functional RNA (i.e., that the coding polynucleotide or functional RNA is under the

465 transcriptional control of the promoter). Coding polynucleotide in sense or antisense orientation can be operably linked to regulatory polynucleotides.

[00065] "Overexpression" refers to the level of expression in transgenic organisms that exceeds levels of expression in normal or untransformed organisms.

"Primary transformant" and "TO generation" refer to transgenic plants that are of

470 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 "T1 , 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

475 secondary transformants with other transformed or untransformed plants.

[00066] The terms "protein," "peptide" and "polypeptide" are used

interchangeably herein.

[00067] "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

480 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

485 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

490 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". "Tissue-specific promoter" or "tissue-preferred promoter" refers to regulated promoters that are not expressed in all plant cells but only or preferentially in one or

495 more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These terms 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. Those skilled in the art will understand

500 that tissue-specific promoters need not exhibit an absolute tissue-specificity, but

mediate transcriptional activation in most plant parts at a level of about 1 % or less of the level reached in the part of the plant in which transcription is most active. "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

505 pathogen.

[00068] "Regulatory sequences" or "regulatory nucleic acids" refer to nucleotide sequences that contribute to the activity of a given gene as it relates to imRNA production, stability and translatability. Regulatory sequences include enhancers, promoters, translational enhancer sequences, introns, terminators and

510 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

515 sequence from the OsLHCA3 gene is isolated from the same coding sequence or the OsLHCA3 gene. Alternatively, the promoter could be from the OsLHCA3 gene, the intron from the OsLHCA4 gene and the terminator from the OsPSID gene. Light regulatory nucleic acids are regulatory elements that respond to light and are therefore light inducible.

520 [00069] "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 imRNA through a process called splicing, which joins the exons to form an imRNA. For purposes of the presently disclosed subject matter, the definition of the term "intron" 525 includes modifications to the nucleotide sequence of an intron derived from a target gene.

[00070] "Exon" refers to a section of transcribed DNA that is maintained in imRNA. 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 530 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.

[00071] 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

535 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.

[00072] As used herein, gene or trait "stacking" is combining desired genes or traits into one transgenic plant line. As one approach, plant breeders stack transgenic traits by making crosses between parents that each have a desired trait and then

540 identifying offspring that have both of these desired traits (so-called "breeding stacks").

Another way to stack genes is by transferring two or more genes into the cell nucleus of a plant at the same time during transformation. Another way to stack genes is by re- transforming a transgenic plant with another gene of interest. For example, gene stacking can be used to combine two different insect resistance traits, an insect

545 resistance trait and a disease resistance trait, or a herbicide resistance trait (such as, for example, Bt1 1 ). The use of a selectable marker in addition to a gene of interest would also be considered gene stacking.

[00073] Substantially identical: the phrase "substantially identical," in the context of two nucleic acid or protein sequences, refers to two or more sequences or

550 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 a 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,

555 950, 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.

[00074] For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences

560 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

565 the preferred program.

[00075] 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

570 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.

575 [00076] "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

580 (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^QC lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH.

585 Typically, under high stringency conditions a probe will hybridize to its target subsequence, but to no other sequences.

[00077] 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

590 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^QC, with the hybridization being carried out overnight. An example of very high stringency wash conditions is 0.1 M NaCI at 72^QC for about 15 minutes. An example of high stringency

595 wash conditions is a 0.2x SSC wash at 65^QC 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 1 x SSC at 45^QC for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is

600 4-6x SSC at 40^QC 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^QC. High stringency conditions can also be achieved with the addition of destabilizing agents such as formamide. In

605 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

610 codon degeneracy permitted by the genetic code.

[00078] Low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1 % SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1 X to 2X SSC (20X SSC = 3.0 M NaCI/0.3 M trisodium. citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 615 45% formamide, 1 .0 M NaCI, 1 % SDS at 37°C, and a wash in 0.5X to 1 X SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1 % SDS at 42°C, and a wash in 0. 1 X SSC at 60 to 65°C.

[00079] The following are examples of sets of hybridization/wash conditions that may be used to identify homologous nucleotide sequences that are substantially

620 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 NaPO₄, 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 NaPO₄, 1 mM EDTA at 50 °C with washing in 1 X SSC, 0.1 % SDS at 50 °C; 7% sodium dodecyl sulfate (SDS),

625 0.5 M NaPO₄, 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 NaPO₄, 1 mM EDTA at 50 °C with washing in 0.1 X SSC, 0.1 % SDS at 50 °C, or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 0.1 X SSC, 0.1 % SDS at 65 °C.

[00080] Specificity is typically the function of post-hybridization washes, the

630 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

635 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

640 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);

645 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 1 1 , 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

650 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 ,

655 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).

[00081] The "terminus" includes the 3'-untranslated sequence and the 3'

660 non-transcribed sequence, which extends 0.5 to 1 .5 kb downstream of the transcription termination site. The terminus may include 3' regulatory sequence.

[00082] A "transcriptional or synthetic gene 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

665 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.

[00083] 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 .

670 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.

[00084] The term "transformation" refers to the transfer of a nucleic acid

675 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

680 selection media following transformation.

[00085] "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

685 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.

690 [00086] 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 imRNA upstream (5') of the translation start codon. The translational enhancer sequence may affect processing of the primary transcript to imRNA, imRNA stability or translation efficiency.

695 [00087] As used herein, the term "recombinant" refers to a form of nucleic acid (e.g. DNA or RNA) and/or protein and/or an organism that would not normally be found in nature and as such was created by human intervention. Such human intervention may produce a recombinant nucleic acid molecule and/or a recombinant plant. As used herein, a "recombinant DNA molecule" is a DNA molecule comprising a

700 combination of DNA molecules that would not naturally occur together and is the result of human intervention, e.g., a DNA molecule that is comprised of a combination of at least two DNA molecules heterologous to each other, and/or a DNA molecule that is artificially synthesized and comprises a polynucleotide that deviates from the polynucleotide that would normally exist in nature, and/or a DNA molecule that

705 comprises a transgene artificially incorporated into a host cell's genomic DNA and the associated flanking DNA of the host cell's genome. An example of a recombinant DNA molecule is a DNA molecule resulting from the insertion of the transgene into a plant's genomic DNA, which may ultimately result in the expression of a recombinant RNA and/or protein molecule in that organism. As used herein, a "recombinant plant" is a

710 plant that would not normally exist in nature, is the result of human intervention, and contains a transgene and/or heterologous DNA molecule incorporated into its genome. As a result of such genomic alteration, the recombinant plant is distinctly different from the related wildtype plant.

[00088] "Vector" is defined to include, inter alia, any plasmid, cosmid, phage

715 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,

720 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. plant, mammalian, yeast or fungal cells).

[00089] 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

725 the plant, which comprises a cell wall but may also refer to a protoplast. The plant cell may be in the 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

730 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,

735 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 740 can be used.

[00090] The term "C3 plant" refers to plants which fix CO₂ using a C3 pathway of photosynthesis for converting carbon dioxide derived from the air and ribulose bisphosphate into 3-phosphoglycerate. Examples of C3 plants include, but are not limited to, monocotyledonous plants such as rice, wheat, and barley, as well as,

745 dicotyledonous plants such as soybeans, tobacco, potatoes, and sweet potatoes. The term "C4 plant" refers to plants that obtain carbon dioxide from malate rather than directly from the air. Examples of C4 plants include, but are not limited to, maize, sugar cane, millet and sorghum.

[00091] As used herein, "plant tissue", "plant cell", "plant material," "plant

750 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.

755 [00092] 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

760 sequences, intron, exon, and/or even comprise intron and exons of the associated genomic gene.

[00093] As used herein the phrase "plant biomass" refers to the amount (measured in grams of air-dried or Heat-dried tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the plant yield or the yield per 765 growing area.

[00094] As used herein, "yield" may include reference to bushels per acre of a grain crop at harvest, as adjusted for grain moisture (15.5% typically for maize, for example), and the volume of biomass generated (for forage crops such as alfalfa and plant root size for multiple crops). Grain moisture is measured in the grain at harvest. 770 The adjusted test weight of grain is determined to be the weight in pounds per bushel, adjusted for grain moisture level at harvest. Biomass is measured as the weight of harvestable plant material generated. Yield can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen

775 fixation, efficiency of nutrient assimilation, carbon assimilation, plant architecture, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Yield of a

780 plant of the can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tons per acre, or kilo per hectare. For example, corn yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture

785 adjusted basis, for example at 15.5 percent moisture. Moreover a bushel of corn is defined by law in the State of Iowa as 56 pounds by weight, a useful conversion factor for corn yield is: 100 bushels per acre is equivalent to 6.272 metric tons per hectare. Other measurements for yield are common practice in the art. In certain embodiments of the invention yield may be increased in stressed and/or non-stressed conditions.

790 [00095] The term "modulate" (and grammatical variations) refers to an increase or decrease.

[00096] As used herein, the terms "increase," "increases," "increased," "increasing" and similar terms indicate an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.

795 [00097] Where appropriate, the heterologous nucleotide sequence may be optimized for increased expression in a transformed plant, e.g., by using plant preferred codons. Methods for synthetic optimization of nucleic acid sequences are available in the art. The nucleotide sequence can be optimized for expression in a particular host plant or alternatively can be modified for optimal expression in monocots. See, e.g., EP

800 0 359 472, EP 0 385 962, WO 91 /16432; Perlak et al., Proc. Natl. Acad. Sci. USA (1991 ) 88, 3324, and Murray et al., Nuc. Acids Res. (1989) 17, 477, and the like. Plant preferred codons can be determined from the codons of highest frequency in the proteins expressed in that plant. Genes synthesized with such plant preferred codons are often referred to as "codon optimized" for that plant species. In some embodiments

805 of the invention, rice or monocot gene sequences coding for FBPase, SBPase, AGPase small subunit and AGPase large subunit are codon optimized for expression in a dicot, such as, soybean. It is understood in the art that any FBPase, SBPase or AGPase sequence could be modified for expression in any plant species.

[00098] The Kozak consensus sequence, is a sequence which occurs on

810 eukaryotic imRNA and has the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G' (Kozac (1978) Nucleic Acids Res. 15: 8125; Kozak M (2002) Gene 299:1 - 34). The Kozak consensus sequence plays a major role in the initiation of the translation process. A preferred Kozak consensus sequence can be determined for a species from

815 the highest frequency nucleic acid sequence around the translation start site in the genes expressed by that species. In some embodiments of the invention, rice gene sequences coding for FBPase, SBPase, AGPase small subunit and AGPase large subunit are modified to provide soy-optimized Kozak sequences. . It is understood in the art that any FBPase, SBPase or AGPase sequence could be modified for improved

820 translation efficiency by providing a species preferred Kozak sequence.

[00099] A "recombinant vector" is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

825 [000100] As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally

830 occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or

835 RNAs comprising unusual bases, such as inosine or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or

840 metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.

[000101] A protein "isoform" is any of several different forms of the same protein. Different forms of a protein may be produced from related genes, or may arise

845 from the same gene by alternative splicing. A large number of isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic differences between alleles of the same gene. These occur at specific individual nucleotide positions within a gene.

[000102] Proteins "native to chloroplast" are either encoded in the nuclear genome and transported the chloroplast or encoded in the chloroplast genome. They

850 perform their primary function in the chloroplast.

[000103] By "host cell" is meant a cell, which comprises a heterologous nucleic acid sequence of the invention, which contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian or

855 mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells, including but not limited to sunflower, soybean, tobacco, wheat, alfalfa, rice, cotton, rapeseed, spinach, sugar beet, Arabidopsis and tomato. A particularly preferred monocotyledonous host cell is a soybean host cell.

[000104] Leaves and roots may "undergo senescence", as part of the

860 biological aging of the plant. As part of senescence, leaves and roots may die off when they are no longer efficient enough for nutrient acquisition. The nutrients from these senescent tissues are transferred to another part of the plant. "Early or premature senescence", refers herein, to senescence that happens earlier in a modified or transgenic plant as compared to a control plant.

865 [000105] "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

870 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

875 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

880 acids".

[000106] A "plant promoter" is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. "Constitutive plant promoter" refers to a

885 promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant (referred to as "constitutive expression"). "Regulated plant promoter" refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially- regulated manner, and includes tissue-specific, tissue-preferred and inducible

890 promoters. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Some promoters preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids or sclerenchyma. Such promoters are referred to as "tissue preferred". A "cell type" promoter primarily

895 drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" plant promoter is a promoter, which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, drought stress, abiotic stress, biotic stress or the presence of light. Promoters "regulated by light"

900 include promoters that have increased transcription in the presence of light. Promoters regulated by light may include, but are not limited to, promoters regulating transcription of genes coding for proteins involved in photosynthesis such as the genes involved in of photosystem I, photosystem II and the Calvin cycle. In general, promoters regulated by light drive high levels of transcription in green tissue such as leaf, stem, or seedling and

905 low levels of transcription in other tissues such as, root, seed or embryo. Another type of promoter is a developmental^ regulated promoter, for example, a promoter that drives expression during pollen development.

[000107] Suitable tissue-specific promoters include, but not limited to, leaf- specific promoters [such as described, for example, by Yamamoto et al. (1997) Plant J.

910 12:255-265; Kwon et al. (1994) Plant Physiol. 105:357-67,; Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23:1 129-1 138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90:9586-9590,], seed-preferred promoters [e.g., from seed specific genes (Simon, et al. (1985) Plant Mol. Biol. 5. 191 ; Scofield, et al. (1987) J. Biol. Chem. 262: 12202;

915 Baszczynski, et al. (1990) Plant Mol. Biol. 14: 633), Brazil Nut albumin (Pearson' et al.

(1992) Plant Mol. Biol. 18: 235-245), legumin (Ellis, et al. (1988) Plant Mol. Biol. 10: 203-214), Glutelin (rice) (Takaiwa, et al. (1986) Mol. Gen. Genet. 208: 15-22,; Takaiwa, et al. (1987) FEBS Letts. 221 : 43-47), Zein (Matzke et al., Plant Mol Biol (1990) 143: 323-32), napA (Stalberg, et al. (1996) Planta 199: 515-519), Wheat SPA (Albanietal,

920 Plant Cell (1997) 9: 171 -184), sunflower oleosin (Cummins, et al. (1992) Plant Mol. Biol.

19: 873-876)], endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet (1989) 216:81 -90; NAR 17:461 -2), wheat a, b and g gliadins (EMBO3:1409-15, 1984), Barley Itrl promoter, barley B1 , C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), Barley 925 DOF (Mena et al. (1998) The Plant Journal, 1 16(1 ): 53-62), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al. ( 1998) Plant J. 13: 629-640), rice prolamin NRP33, rice -globulin Glb-1 (Wu et al. (1998) Plant Cell Physiology 39(8) 885-889), rice alpha-globulin REB/OHP-1 (Nakase et al.( 1997) Plant Mol. Biol. 33: 513-S22), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-

930 46, 1997), sorgum gamma-kafirin (PMB 32:1029-35, 1996)], embryo specific promoters [e.g., rice OSH1 (Sato et al., Proc. Nati. Acad. Sci. USA, 93: 81 17-8122), KNOX (Postma-Haarsma of al. (1999) Plant Mol. Biol. 39:257-71 ), rice oleosin (Wu et al (1998) J. Biochem., 123:386)], and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al. (1990) Plant Mol. Biol. 15, 95-109), LAT52 (Twell et al.

935 (1989) Mol. Gen Genet. 217:240-245), apetala-3].

[000108] Promoters showing a high level of activity in photosynthetic tissue with may be useful in some embodiments of the invention. Examples of such promoters include the ribulose-1 ,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch {Larix laricina), the pine cab6 promoter (Yamamoto et al.

940 (1994) Plant Cell Physiol. 35:773-778), the Cab-1 gene promoter from wheat (Fejes et al. (1990) Plant Mol. Biol. 15:921 -932), the CAB-1 promoter from spinach (Lubberstedt et al. (1994) Plant Physiol. 104:997-1006), the cabl R promoter from rice (Luan et al. (1992) Plant Cell 4:971 -981 ), the pyruvate orthophosphate dikinase (PPDK) promoter from maize (Matsuoka et al. (1993) Proc Natl Acad Sci USA 90:9586-9590), the tobacco

945 Lhcb1 ^*2 promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), the Arabidopsis thaliana SUC2 sucrose-H⁺ symporter promoter (Truernit et al. (1995) Planta 196:564- 570), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Other promoters that drive transcription in stems, leafs and green tissue are described in U.S. Patent Publication No. 2007/0006346, herein

950 incorporated by reference in its entirety.

[000109] A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and correct imRNA polyadenylation. The termination region may be derived from the same native sequence as the transcriptional initiation region, may be

955 native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign to the promoter, the DNA sequence of interest, the plant host, or any combination thereof). Appropriate transcriptional terminators are those that are known to function in plants and include the CAMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea

960 rbcs E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator may be used.

[000110] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.

965 [000111] Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.

[000112] Various intron sequences have been shown to enhance

970 expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild- type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al. (1987) Genes Develop. 1 : 1 183-

975 1200). In the same experimental system, the intron from the maize bronze 1 gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

[000113] A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in

980 dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "omega sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. (1987) Nucl. Acids Res. 15: 8693-871 1 ; Gallie DR, Walbot, V. (1992) Nucleic Acids Res 20:4631 -4638; Skuzeski et al. (1990) Plant Molec. Biol. 15: 65-79). Other leader

985 sequences known in the art include but are not limited to: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chain binding 990 protein (BiP) leader, (Macejak, D. G., and Samow, P. (1991 ) Nature 353: 90-94; untranslated leader from the coat protein imRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L. (1987) Nature 325:622-625) and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al. (1991 ) Virology 81 :382-385). See also, Della-Cioppa et al. (1987) Plant Physiology 84:965-968.

995 [000114] Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein looo (e.g. Comai et al. (1988) J. Biol. Chem. 263: 15104-15109). These signal sequences can be fused to heterologous gene products to affect the import of heterologous products into the chloroplast (van den Broeck, et al. (1985) Nature 313: 358-363). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2

1005 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled "Expression With Chloroplast Targeting" in Example 37 of U.S. Pat. No: 5,639,949.

[000115] The above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with loio heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.

[000116] In order to ensure the localization in the plastids it is conceivable to use one of the following transit peptides: of the plastidic Ferredoxin: NADP⁺

1015 oxidoreductase (FNR) of spinach which is enclosed in Jansen et al. (Current Genetics 13 (1988), 517-522). In particular, the sequence ranging from the nucleotides -171 to 165 of the cDNA sequence can be used, which comprises the 5' non-translated region as well as the sequence encoding the transit peptide. Another example is the transit peptide of the waxy protein of maize including the first 34 amino acid residues of the

1020 mature waxy protein (Klosgen et al., (1989) Mol. Gen. Genet. 217, 155-161 ). It is also possible to use this transit peptide without the first 34 amino acids of the mature protein. Furthermore, the signal peptides of the ribulose bisphosphate carboxylase small subunit (Wolter et al. (1988) Proc. Natl. Acad. Sci. USA 85, 846-850; Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91 , 12760-12764), of the NADP malate dehydrogenase

1025 (Galiardo et al. (1995) Planta 197, 324-332), of the glutathione reductase (Creissen et al. (1995) Plant J. 8, 167-175) or of the R1 protein Lorberth et al. (Nature Biotechnology 16, (1998), 473-477) can be used.

[000117] As used herein, the phrase "percent identical", or "percent similarity" in the context of two nucleic acid sequences or two polypeptide sequences,

1030 refers to two or more sequences or subsequences that have in some embodiments at least 60% {e.g., 60, 63, 65, 67, or 69%), in some embodiments at least 70% {e.g., 70, 73, 75, 77, or 79%), in some embodiments at least 80% {e.g., 80, 83, 85, 86, 87, 88, or 89%), in some embodiments at least 90% {e.g., 90, 91 , 92, 93, 94, 95, 96, 97, or 98%), and in some embodiments at least 99% nucleotide or amino acid identity or similarity,

1035 respectively, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The percent identity exists in some embodiments over a region of the sequences that is at least about 50 nucleotides in length, in some embodiments over a region of at least about 100 nucleotides in length, in some embodiments over a region of at least about

1040 250 nucleotides in length, in some embodiments over a region of at least about 500 nucleotides in length, in some embodiments over a region of at least about 1000 nucleotides in length, and in some embodiments, the percent identity exists over at least about 1500 residues. In some embodiments, the percent identity exists over the entire length of one or both of the sequences.

1045 [000118] 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 1050 calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[000119] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm disclosed in Smith & Waterman, 1981 , by the homology alignment algorithm disclosed in Needleman & Wunsch, 1970, by the 1055 search for similarity method disclosed in Pearson & Lipman, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and T FAST A in the GCG® WISCONSIN PACKAGE®, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Ausubel et al., 2002; Ausubel et al., 2003.

1060 [000120] 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. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI) of the United States National Institutes of Health (NIH). This algorithm involves

1065 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-valued 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. See generally, Altschul et al., 1990. These initial neighborhood word hits act as seeds for initiating searches to find

1070 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

1075 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 1080 speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1 , 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 wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff & Henikoff, 1992.

1085 [000121] 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, 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

1090 occur by chance. For example, a test nucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is in some embodiments less than about 0.1 , in some embodiments less than about 0.01 , and in some embodiments less than about 0.001 . In some embodiments, the similarity of two

1095 sequences refers to the similarity between the sequences over the entire length of one or both of the sequences.

[000122] Two nucleotide sequences can also be considered to be substantially identical when the two sequences hybridize to each other under stringent conditions. A non-limiting example of "stringent" hybridization conditions include lioo conditions represented by a wash stringency of 50% formamide with 5x Denhardt's solution, 0.5% SDS and 1 x SSPE at 42 °C. "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. An extensive guide to the

1105 hybridization of nucleic acids is found in Tijssen 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 (1993). In some representative embodiments, two nucleotide sequences considered to be substantially identical hybridize to each other under highly mo stringent conditions. Generally, highly stringent 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.

[000123] As used herein, the term "polypeptide" encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise.

ins The following enzymes and associated referenced accession number and

sequences may find use in various embodiments as described herein:

Fructose-bisphosphate aldolase (FBPase, EC 4.1 .2.13) catalyzes the reaction D- fructose 1 ,6-bisphosphate to glycerone phosphate and D-glyceraldehyde 3-phosphate. A representative nucleic acid encoding fructose-bisphosphate aldolase in Arabidopsis is

1120 fructose-bisphosphate aldolase (GenBank Accession No. NM_001 124981 .1 ). Many FBPases are known in the art and have been isolated from plant, microbial and animal sources. Plant FBPases have been isolated from, for example, rice, pea, spinach, wheat, and maize. See for example, Konishi, et al., (2004) Plant Mole Bio 56(6): 839- 848; and Schnarrengerger, C. and Kruger, I. (1986) Plant Phys 80(2): 301 -304.

1125 [000124] Sedoheptulose-bisphosphatase (SBPase, EC 3.1 .3.37) catalyses the reaction sedoheptulose 1 ,7-bisphosphate and H₂O to sedoheptulose 7-phosphate and phosphate. A representative sedoheptulose-bisphosphatase in Arabidopsis is sedoheptulose-1 ,7-bisphosphatase (GenBank Accession No. NM_1 15438). SBPases have been isolated from a variety of organisms including, but not limited to, Arabidopsis,

1130 Chlamydomonas, tobacco, rice, pea, and mulberry.

[000125] ADP-glucose pyrophosphatase (AGPase, glucose-1 -phosphate adenylytransferase EC 2.7.7.27) catalyses the reaction ATP and a-D-glucose 1 - phosphate to diphosphate and ADP-glucose. In plants this enzyme is comprised of a large and a small subunit. A representative ADP-glucose pyrophosphatase small

1135 subunit in Arabidopsis is glucose-1 -phosphate adenylyltransferase small subunit

(ADG1 , GenBank Accession No. NM_124205). A representative of ADP-glucose pyrophosphatase large subunit in Arabidopsis is glucose-1 -phosphate

adenylyltransferase large subunit (APL3, GenBank Accession No. AY059862). For a review of plant AG Pases, please see Ballicora, et. al. (2004) Photosynthesis Research

1140 79:1 -24. 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

1145 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

1150 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,

[000126] Transient transformation methods, stable transformation methods,

1155 and virus-mediated methods.

[000127] "Transient transformation" in the context of a polynucleotide is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant.

[000128] By "stably introducing" or "stably introduced" in the context of a

1160 polynucleotide introduced into a plant is intended the introduced polynucleotide is stably incorporated into the plant genome or organelle, and thus the plant is stably

transformed with the polynucleotide.

[000129] "Stable transformation" or "stably transformed" is intended to mean that a polynucleotide, for example, a nucleotide construct described herein, introduced

1165 into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive

generations.

[000130] Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes

1170 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

1175 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

1180 dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7):

1099-1 104 (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).

[000131] Methods for regeneration of plants are also well known in the art.

1185 For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and

microprojectiles. 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

1190 representative plastid transformation technique.

[000132] 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

1195 Publication No. 2006/026001 1 , herein incorporated by reference.

[000133] 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

1200 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. 2006026001 1 , herein incorporated by reference.

1205 [000134] 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

1210 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.

1215 [000135] 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 are preferably 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

1220 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.,

1225 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).

[000136] Alternatively one or more of the polynucleotides of the invention could be used to transform a plant plastid, such as, for example, a chloroplast. Plastid

1230 transformation technology is extensively described in U.S. Patent Nos. 5,451 ,513;

5,545,817 and 5,545,818, all of which are hereby incorporated by reference in their entireties; in PCT application no. WO 95/16783, which is hereby incorporated by reference in its entirety; and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 , 7301 -7305, which is also hereby incorporated by reference in its entirety. 1235

Examples

[000137] The following examples have been included to illustrate modes of the invention. Certain aspects of the following examples are described in terms of techniques and procedures found or contemplated by the present co-inventors to work

1240 well in the practice of the invention. In light of the present disclosure 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 may be employed without departing from the scope of the invention.

[000138] Example 1 : Identification of trait protein coding sequences

1245 [000139] Rice was selected as the donor organism for FBPase, SBPase and

AGPase. The FBPase is GenBank Accession Q40677.2 (Tsutsumi et al., 1994). The SBPase selected is GenBank Accession Q84JG8. Two papers (Odhan et al., 2005; Lee et al., 2007) describe the rice AGPase gene family. According to these papers the leaf isoform, which localizes to plastids, consists of the large subunit gene AGPL3

1250 (GenBank Accession BAG91362.1 ) and the small subunit gene AGPS2a (GenBank Accession AK071826.1 ). The coding sequence of these genes was optimized for efficient codon utilization in a dicot, such as, soybean. The optimized polynucleotide sequences are described in SEQ ID NOS: 1 , 3, 5 and 7. The resulting polypeptide sequences are described in SEQ ID NOS: 2, 4, 6 and 8.

1255 [000140] Example 2: Identification of trait regulatory sequences

[000141] It was determined by using the Zhu evolutionary algorithm, (please see, Zhu et al., ((2007) Plant Physiol 145: 513) that AGPase will need to be boosted approximately 4 fold higher than endogenous levels, SBPase will need to be boosted the approximately 4 fold higher than endogenous levels, and FBPase will need to be

1260 boosted approximately 2 fold higher than endogenous levels.

[000142] Promoters were selected based on linkage to photoassimilation and whether they provide the appropriate spatial and temporal regulation. Probe sequences listed in Table 1 were based well characterized proteins involved in plant

photosynthesis. The amino acid sequences for Hordeum vulgare (barley) Photosystem I

1265 reaction center subunit psaD (PSID) with Swiss-Prot ID P36213.1 , the Hordeum vulgare Photosystem I reaction center subunit psaK (PSAK) with Swiss-Prot ID P36886.1

(formerly Swiss-Prot ID A48527), the Pisum sativum (pea) light harvesting protein of photosystem I LHCA3(LHCA3) with Genbank ID AAA84545.1 , and the Hordeum vulgare chlorophyll a/b-binding protein precursor LHCA4 (LHCA4) with Genbank ID

1270 AAF90200.1 were used in a tBLASTn search of rice gene databases to find the

corresponding rice genes. cDNAs representing the nearest rice homologs are indicated in Table 1 . gDNAs for each gene were identified, annotated and used to define their corresponding regulatory sequence. EST and gene expression profiling data in Table 2 confirm each gene is primarily active in green tissue.

1275 [000143] The following promoters/terminators were identified and then

isolated from rice. The OsLHCA3 promoter from a rice light harvesting protein of photosystem I gene (SEQ ID NO: 9), the OsLHCA4 promoter from a rice chlorophyll a/b-binding protein precursor (SEQ ID NO: 1 0); the OsPSAK promoter from a rice Photosystem I reaction center subunit gene (SEQ ID NO: 1 1 ) and the OsPSID promoter

1280 from a rice Photosystem I reaction center subunit gene (SEQ ID NO: 1 2).

Table 1 . Identification of rice genes linked to photoassimilation

1285 Table 2. Characterization of rice genes linked to photoassimilation EST ANALYSIS EXPRESSION PROFILING b

green EB/I 26K 50K

tissue M Chi Chi

Rice a seed

Gene P P

Total

EST ro lea E

s (%) ot (%) leaf root EB f root B

PSI D

ferredoxin

docking 53. 1 .

protein 31 87.1 3.2 0.0 52.9 1 .9 1 .7 4 1 .3 0 psaK PSI 61 . 1 .

protein 100 81 .0 0.0 4.0 37.7 1 .4 1 .2 9 1 .3 4

LHCA3 48. 1 .

protein 100 100.0 0.0 0.0 57.5 4.4 3.4 5 2.1 5

LHCA4 42. 1 .

protein 100 100.0 0.0 0.0 56.6 2.6 1 .7 9 1 .3 2 includes leaf, stem, seedling, green shoot

Semiquantitative score

EB = embryo

IM = immature seed

1290

[000144] Based on the above information the AGPase AGPS2a subunit was operably linked to the OsLHCA3 promoter (SEQ ID NO: 9), OsLHCA3 first exon (SEQ ID NO: 13), first intron (SEQ ID NO: 14), OsLHCA3 second exon (SEQ ID NO: 15), OSLHCA3 second intron (SEQ ID NO: 16) and OsLHCA3 terminator (SEQ ID NO: 17),

1295 after the cassette was modified to include a TMV-Ω translational enhancer (Gallie DR, Walbot, V. (1992) Nucleic Acids Res 20:4631 -4638) and terminate in a soy-optimized Kozak sequence (SEQ ID NO: 18). The AGPase AGPL3 subunit was operably linked to the OsLHCA4 promoter (SEQ ID NO: 10), OsLHCA4 first exon (SEQ ID NO: 19), OsLHCA4 first intron (SEQ ID NO: 20) and OsLHCA4 terminator (SEQ ID NO: 21 ) after

1300 the vector is modified to include the TMV-Ω M15 sequence and terminate in a soy- optimized Kozak sequence (SEQ ID NO: 22). The FBPase was operably linked to the OsPSAK promoter (SEQ ID NO: 1 1 ), the OsPSAK first exon (SEQ ID NO: 23), the OsPSAK first intron (SEQ ID NO: 24) and OsPSAK terminator (SEQ ID NO: 25) after the vector was modified to include the NtADH translational enhancer and terminate in a

1305 soy optimized Kozak sequence (SEQ ID NO: 26). The SBPase was operably linked to the OsPSID promoter (SEQ ID NO: 13) and OsPSID terminator (SEQ ID NO: 27) after it was modified to include the TMV-Ω M14 sequence and a soy-optimized Kozak

sequence (SEQ ID NO: 28).

[000145] The expression cassettes were sequentially ligated to a binary

1310 vector for agrobacterium-mediated transformation. This vector also includes a

polyphenol oxidase expression cassette for plant selection (Li X, et. al. (2003) Plant Physiol 133:736-747). The trait gene ligation order from right border to left border was FBPase, SBPase and AGPase. An eFMV/e35S transcriptional enhancer complex is near the right border. This strategy enables coordinate expression of the four genes

1315 required for the trait. Each cassette is optimized for high protein expression.

[000146] Example 3: Production of transgenic tobacco

[000147] The DNA of Example 2 was inserted into tobacco following standard agrobacterium-mediated plant transformation procedure (Li X, et. al. (2003) Plant Physiol 133:736-747). Based on primary and secondary TaqMan analysis, 42

1320 single-copy, backbone free events were produced. Twenty-one of these events set seed. Transgene activity was assessed by qRT-PCR on TO leaf samples. For each plant, the tip region of the youngest fully expanded leaf was sampled and transcript abundance relative to endogenous tobacco Alcohol Dehydrogenase 1 (ADH1 ) was determined for all four trait genes (Jian, B., et. al. (2008) BMC Mol Biol 9: 59). The

1325 average transcript abundance for each trait gene ranged from 1316 to 2978 in the

fertile, single-copy, backbone free events. TO events produced transcripts from all four trait genes between 0.5-fold and 5-fold of ADH1 levels, and the relative abundance was PSID:SBP > LHC3:AGPS >LHC4:AGPL > PSAK:FBA. It is notable that respective transcript levels among the 4 trait genes were highly consistent between events, and

1330 that position effect likely accounted for expression variation between events. The

observed average increase in trait gene expression was close to the engineering objective. Based on TO qRT-PCR data, one low (A123A), one medium (A148A), and three high expressers (A1 17A; A126A and A156A) were selected for further analysis.

[000148] Example 4: T1 segregation and T+ expression analysis

1335 [000149] T1 seed for events A1 17A, A123A, A126A, A148A and A156A were surface sterilized and spread on plates containing Gamborg's B5 media plus 2% sucrose and either none, 100 nM or 200 nM butafenacil (Li X, et. al. (2003) Plant Physiol 133:736-747). The T1 seed germination ratios on Gamborg's B5 Gelzan plates containing the PPO herbicide (please see Table 4) were consistent with single insertions, and germination rates were between 82 and 97%.

Table 3.

^*Days after imbibing.

[000150] Trait gene transcription was robust in T1 plants but did not correlate well with TO generation data. Of the five events chosen for T1 analysis, three events

(A1 17A, A126A and A156A) were selected for in-depth physiological analyses at the T2 generation. Trait gene activity was detected across three generations.

[000151] Example 5: Trait temporal expression and transcript

processing 1350 [000152] These events were further analyzed by looking at the (i) the

circadian transcript abundance of each trait gene and (ii) the pre-mRNA splicing efficiency of intron-containing trait transcripts.

[000153] The regulatory sequence used to express the three genes was active in green tissue and was light regulated. Transcript abundance should peak early

1355 to mid-afternoon. Transcript levels were measured for each trait gene by qRT-PCR in leaf samples collected every 4 hours, over a 24 hour period. The transcript levels were low during the night and increased from dawn to a peak at around 1500. Afterwards transcript levels declined. This suggests that the rice regulatory sequences functioned as expected in transgenic tobacco. Three of the four trait gene expression cassettes

1360 (PsAK:FBPase; LHC3:AGPSS; and LHC4:AGPLS), produced disrupted transcripts. The introns used in the expression cassettes were derived from rice sequences. To determine if the rice (monocot) intron donor-acceptor (GT-AG) sites are recognized and the efficiently spliced in tobacco cells (dicot) (Hanley, B.A., Schuler, M.A. (1988) Nucleic Acids Res 16: 7159-7176; Goodall, G.J., Filipowicz, W. (1991 ) EMBO J 10: 2635-2644),

1365 the first strand cDNA was synthesized by reverse transcription of imRNA, and primers from the flanking introns were used to detect their presence by PCR. The PCR products were also sequenced to confirm the agarose gel electrophoresis results. PCR products corresponding to the expected spliced transcript were recovered for all three trait genes in two different events. The PsaK:FBPase and LHC4:AGPLS trait genes produce a

1370 significant proportion (between 30% and 50%) of unspliced transcript. An unspliced version of the LHC3:AGPSS transcript was not detected, but approximately 30 to 40% was a mis-spliced form that contained 48 additional nucleotides upstream of the expected AG donor site was identified. Such mis-splicing of monocot introns in dicot systems is consistent with previous reports (Hanley, B.A., Schuler, M.A. (1988) Nucleic

1375 Acids Res 16: 7159-7176; Goodall, G.J., Filipowicz, W. (1991 ) EMBO J 10: 2635-2644).

Together the data indicate that the rice introns were recognized, but may not be efficiently processed in tobacco. Despite this observation a significant portion (between 50 and 70%) of trait transcripts were present in the correct, mature forms indicating that the trait is functional at the molecular genetic level. 1380 [000154] Example 6: Fructose 1 ,6-bisphosphate aldolase activity in

transgenic tobacco leaf tissue

[000155] Tips from the youngest fully expanded leaf of homozygous trait positive and null plants were sampled. Protein extracts were prepared from 130-180 mg fresh weight tissue using a common extraction buffer (Iwaki, T., et. al. (1991 ) Plant Cell

1385 Physiol 32: 1083-1091 ; Muller-Rober, B., et. al. (1992) EMBO J 1 1 : 1229-1238;

Harrison, E.P., et. al. (1998) Planta 204: 27-36). To quantify aldolase activity, the rate of FBP cleavage was followed by oxidation of NADH via absorbance at 340 nm in a coupled-enzyme assay. To reduce biological variation when comparing homozygous trait positive and null plants, consistent plant growth conditions (illumination, irrigation,

1390 fertilization and similar plant orientation) were maintained. To reduce technical variation during sample preparation and the activity assay, null and trait positive plants samples were alternatively processed.

[000156] FBP aldolase activity in homozygous trait positive and null T2 plant leaf extracts could not be distinguished between the trait positive and null plants.

1395 Despite high coefficients of variation (CV) in this assay, we found very similar total

aldolase activity between homozygous and null plants. A possible explanation is the assay's inability to distinguish cytosolic and plastidial FBP isoforms, although some reports indicate the plastidal isoform accounts for up to 90% of cellular activity (Haake et al., (1999) Plant J 17: 479; Miyagawa et al., (2001 ) Nature Biotech 19: 965; Lefebvre

1400 et al., (2005) Plant Physiol 138: 451 ; Smidansky et al., (2007) Planta 225: 965)

[000157] Example 7: Analysis of photosynthetic apparatus

[000158] Several experiments to physiologically assess the engineered trait were conducted. The purpose was to determine if homozygous trait positive plants had distinct photosynthetic properties compared to null plants. To ensure detection of trait

1405 effects in homozygous trait positive plants, when compared to null plants, leaf samples (for protein quantification and biochemical assays) and physiological experiments were conducted between 1400 and 1600 hours.

[000159] Chlorophyll fluorescence was measured in young plants (leaves 1 to 4) then CO₂ assimilation rates were measured in the youngest fully developed leaf

1410 (leaves 5 to 7) of more mature tobacco plants. These experiments included 12 homozygous trait positive and 12 null plants from the T2 generation of events A1 17A, A126A and A156A.

[000160] Chlorophyll fluorescence was measured as a diagnostic for in vivo photosynthetic activity (Baker, N.R. (2008) Annu Rev Plant Biol 59: 89-1 13). Leaves on

1415 3-4 weeks old tobacco plants at growth stages 12 to 17 (Lancashier, P.D., et. al. (1991 ) Ann. appl. Biol. 1 19: 561 -601 are flat and horizontally oriented to the light source.

[000161] Using the theory of chlorophyll fluorescence measurements (CFM), Fq'/Fm' was calculated, which provides a diagnostic of PSIl operating efficiency. This estimates the linear electron transport rate, thus the NADH and ATP consumption rate,

1420 thus the RuBP regeneration rate. This is a good indicator of changes in the quantum yield of CO₂ assimilation (Baker, 2008) and was previously shown to correlate with increased SBPase activity and the CO₂ assimilation rate in young tobacco leaves (Lefebvre, S., et. al. (2005) Plant Physiol. 138: 451 -460). The Fv/Fm was also calculated, which represents the PSIl maximum quantum efficiency. Table 4 shows that

1425 the homozygous trait positive plants were not significantly different from null plants.

[000162] Table 4a and b. In vivo photosynthetic activity of young plants leaves assayed by chlorophyll fluorescence. T2 progeny of A117A-10 (null), A117A-11 (horn), A126-1 (null) and A126-5 (horn) T1 plants were analyzed. No significant differences (Students t-test p<0.05) were found. Data are the mean ± SD (n=12). Note

1430 that 'horn' is 'homozygous trait positive'.

Table 4a.

Table 4b.

1435

[000137] The CO₂ photoassimilation rate was assayed on 2.5 cm² source leaf patches in older plants by infra-red gas analysis (IRGA). The CIRAS-2 IRGA device was fixed to a tripod to gently clamp the gas exchange cuvette to leaves and minimize data noise generated by plant handling. The environment applied to the leaf patch was

1440 programmed to mimic the growth chamber environment (400 μιηοΙ mol^"1 CO₂; 26 °C; ambient humidity) to assess steady-state photosynthesis under standard growth conditions. The initial analysis examined the youngest fully expanded leaf of

homozygous trait positive and null T1 plants (4<n<6). There was no significant difference in photoassimilation between homozygous trait positive and null plants.

1445 Measurements were then taken from a larger population of T2 plants. In addition, plants subjected to sub-optimal growth temperatures for 18 hours (12 °C and 37 °C) prior to each measurement. Although temperature affects the observed photoassimilation rate, there was no significant difference between homozygous trait positive and null plants.

[000138] The CIRAS-2 IRGA system can vary CO₂ levels applied to the leaf

1450 patch from 10 to 1500 μιηοΙ-ιτιο ¹. The photoassimilation (A) response to intracellular CO₂ (Ci) reports the in vivo regulation and limitation of photosynthetic activity.

Specifically, at low C, (10-300 μιηοΙ mol^"1) rubisco catalytic activity is the limiting factor, and at intermediate C, (300-700 μηποΙ mol^"1) and high (700-1300 μηποΙ mol^"1) the RuBP regeneration rate and triose-phosphate utilization become rate-limiting, respectively.

1455 [000139] SBP is a critical control point in RuBP regeneration, and several reports show that SBPase over-expression has a positive effect on photoassimilation (A) and plant growth (Miyagawa, Y., et. al. (2001 ) Nature Biotech 19: 965-969;

Lefebvre, S., et. al. (2005) Plant Physiol. 138: 451 -460). In addition, SBPase over- expression had the highest transcription among the four trait genes comprising this 1460 photosynthesis enhancement construct. Therefore, A/C, curves were constructed to determine how the trait effects RuBP regeneration.

[000140] A total of 15 A/C, curves, 7 null and 8 homozygous trait positive, were built using T1 plants representing 5 selected events. The data clearly showed no significant difference between homozygous trait positive plants and null plants. There 1465 was a slight but insignificant decrease in trait positive photoassimilation (A) at

intermediate C, levels corresponding to the RuBP regeneration limiting phase.

[000141] Example 8: Plant growth assessment

[000142] Various plant growth parameters were measured to assess the effect of trait gene expression on plant growth and development. These included leaf 1470 chlorophyll content (SPAD meter value), leaf size (length and width) and plant shoot height.

[000143] Harrison and co-workers found reduced chlorophyll content in SBPase-antisense tobacco plants, indicating that SBPase influences chlorophyll content (Harrison, E.P., et. al. (1998) Planta 204: 27-36). Chlorophyll content was measured at

1475 the tip of the youngest fully expanded leaf using a SPAD meter. T1 plants representing 5 events were assayed, and there is no significant difference in chlorophyll content between trait positive and null plants. Although SPAD meter data are not as robust as direct chlorophyll extraction/quantification (Harrison, E.P., et. al. (1998) Planta 204: 27- 36), the assay is non-destructive and provides a good first approximation. In addition,

1480 the data show that plant growth conditions (soil, irrigation, nutrition, light, temperature etc.) were highly homogenous.

[000144] The size of the youngest fully expanded leaf and plant height were measured with a ruler. Some significant differences in leaf properties between trait positive and null plants for events A123A, A126A and A156A were observed, but the

1485 pattern was not consistent and the sample size was small. No significant differences in plant shoot height were observed between trait positive and null plants. The low data variance indicates a very high level of plant homogeneity in this growth environment (e.g. the fully developed leaf #6 at 35 days).

[000145] Although the differences between homozygous trait positive and

1490 null plants were very small and mainly non-significant, small differences in photoassimilation rates may produce a significant effect on overall plant biomass

accumulation over 45 days of growth. To test this, shoot biomass was measured (grams DW) in T1 and T2 plants. T1 A126A trait positive plants have a small, but significant increase in shoot biomass, but this was not observed for other events. Furthermore, no 1495 significant difference between A126A trait positive and null T2 plants were observed, and plant biomass was surprisingly consistent among the T2 plants.

[000146] Example 9: Closed chamber monitoring of whole-plant gas exchange

[000147] It may not be possible to detect small, but significant changes in

1500 photoassimilation using an IRGA device An additional test was devised using large

hypobaric chambers (Wheeler, R.M., et. al. (201 1 ) Adv Space Res 47:1600-1607) to monitor with high precision plant CO₂ demand, night time respiration and transpiration of a 29 plant population throughout development. Event A126A was chosen for these experiments because, among all the measurements performed, this event presented

1505 some significant differences between homozygous trait positive and null plants in leaf size and shoot biomass. The data in this study were collected on a chamber basis, one chamber contained A126A-5 (homozygous trait positive) T2 plants and a second chamber contained the of A126A-1 (null) T2 plants. The experiment was repeated twice.

[000148] The study phases were germination, thinning, growth, response to

1510 environment and maturation. Excess seed were germinated for each chamber to ensure establishment of a uniform population. The plants were thinned to 30 per chamber, after which the chambers were sealed for the duration of the study. At the end of the study 29 plants developed in each chamber. Several plant growth-related chamber parameters were monitored during the study including atmospheric CO₂ and O₂, CO₂ demand to

1515 maintain a 400 ppm set point and condensate. The CO₂ data were used to calculate two photosynthetic rates. The first is CO₂ draw down that occurs at the beginning of the light period, in which the CO₂ released during the dark period is reacquired. The second is steady state photosynthesis, in which the CO₂ required to maintain an [CO₂]_at_m of 400 ppm. The CO₂ data were also used to calculate the night time respiration, by monitoring

1520 CO₂ released during the dark period. The condensate data were used to calculate daily transpiration rates. The mean condensate data collected during the germination period was used to establish instrument background, for the daily transpiration rate

calculations. Both a dark period and light period transpiration rate was calculated. The daily steady state photosynthetic and transpiration rates were used to calculate daily

1525 water use efficiency.

[000149] The general trend in both replicates is that the null plants outperformed the homozygous trait positive plants in terms of daily net CO₂ assimilated, daily CO₂ assimilation rates and night time respiration rates. Plant response to change in both CO₂ level and temperature were examined. Homozygous trait positive and null

1530 plants responded similarly to the environmental perturbations. This contributed to the increased biomass produced by the null plants relative to the homozygous trait positive plants. Table 5 shows that across both replications the nulls produced approximately 30% more aerial biomass, or about 4 kg. Taken together with data presented in previous examples, a general conclusion is that the trait does not work. Plant

1535 photoassimilation by all measures was lower in trait positive plants, relative to null

plants. Although there was no observed difference in photoassimilation observed, Table 5 shows the number of developing reproductive structures was significantly increased by the end of the study. In both replications the homozygous trait positive plants produced significantly more healthy seed pods than the null plants. In replication

1540 1 (chambers 2 & 3), the difference was more than 3:1 and in replication 2 (chambers 4 & 5) the trait positive plants produced -72% more pods. In replication 1 , an unexplained ethylene spike in the null chamber at the transition to reproductive development likely caused significant pod abortion. This was not observed in replication 2, and ethylene eventually returned to comparable levels in replication 1 . Nevertheless, the difference in

1545 pod set between the transgenics and nulls was highly significant. Although not to be limited by theory, this observation suggests that the simultaneous overexpression of an FBPase, and SBPase and the large and small subunit of an AGPase resulted in greater partitioning of photoassimilate to reproductive structures, rather than increased photoassimilation itself.

1550

Table 5. Summary of biomass production in Precision Chambers. Data are the mean ± SD (n=29) Fresh Dry weight DW:FW Seed pods Seed pods Seed pods

Chamber weight (g) (fl) ratio per plant per gram FW per gram DW

588.5 ± 0.096 ± 0.027 ± 0.285 ±

HC-2 286.4 55.0 ± 27.9 0.020 19.9 ± 18.7 0.021 0.224

442.3 ± 0.113 ± 0.184 ± 1 .583 ±

HC-3 244.6 50.2 ± 28.5 0.011 70.8 ± 48.3 0.154 1 .244 p-value

(t-test) 0.04118063 0.51865947 0.00020546 0.00000204 0.00000131 0.00000088

619.3 ± 0.094 ± 0.052 ± 0.548 ±

HC-4 214.2 58.1 ± 22.8 0.014 34.9 ± 26.7 0.033 0.323

479.5 ± 0.111 ± 0.112 ±

HC-5 243.9 51 .9 ± 26.6 0.019 60.2 ± 42.4 0.062 1 .01 ± 0.487 p-value

(t-test) 0.0239980 0.3488579 0.0002618 0.0086275 0.0000264 0.0000792

[000150] Seed pod production on an aerial biomass basis was examined.

1555 Comparing aerial biomass on a plant basis did not distinguish homozygous trait positive from null plants. However trait positive plants had a significantly higher dry weight:fresh weight ratio. The senesced leaves were collected and weighed in each chamber. The homozygous trait positive plants shed significantly more leaf biomass than the null plants, indicating that the homozygous trait positive plants had an early leaf senescence

1560 phenotype. The difference in number of seed pods per plant, alone or as a function of aerial biomass, was statistically significant.

[000151] Early leaf senescence between the homozygous trait positive and null plants was not observed in initial growth chamber and greenhouse work. Possible explanations are that the growth chamber and greenhouse grown plants were managed

1565 in individual pots, which constrained growth and made it difficult to distinguish homozygous trait positive from null plants. In the closed chamber environment access to water and nutrients, including CO₂ was not limiting. However the plants produced a canopy and had to compete for access to available light. The plants also had to cope with increasing levels of oxygen.

1570 [000152] Example 10: Expression cassette performance in monocots and dicots

[000153] Several maize transformation vectors were constructed and used to test the expression cassettes found in the tobacco transformation vectors. Table 6 outlines the components in each vector. There are subtle differences in sequence but 1575 the global expression control elements are derived from the same source gene. For example some promoters don't include a translational enhancer, since these tend to inhibit expression cassette activity in maize.

[000154] Transgenic maize were generated using each binary vector in

Table 6, and leaf tissue from primary transgenic plants, or the initial regenerants, was 1580 sampled for qRT-PCR analysis. Only single-copy, backbone-free events were analyzed.

The results in Table 7 show that all four expression cassettes are transcriptionally active in tobacco and maize. The activity level varies between constructs, and the maize variation is likely due to the coding sequence. The data show that these expression tools are effective in both maize and tobacco.

1585

Table 6. Binary vectors used to evaluate light regulated expression cassettes from rice. Each expression cassette consists of a promoter and a terminator. The suffix indicates version number.

1590 Table 7. Performance of light regulated expression cassettes in transgenic plants.

Various trait genes were used to generate qRT-PCR data. The data are reported as the ratio of the signal from the trait gene and the signal from an endogenous control gene multiplied by 1000. In tobacco the endogenous control gene is alcohol dehydrogenase 1595 and in maize the endogenous control gene is EF1 -alpha.

All references cited herein, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries {e.g.,

GEN BANK® database entries and all annotations available therein) are incorporated 1600 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

WHAT IS CLAIMED IS:

1 . A nonnaturally occurring expression cassette comprising:

a) a sedoheptulose-1 ,7-bisphosphotase recombinant polynucleotide

operably linked to a light regulated promoter;

b) an ADP-glucose pyrophosphorylase large subunit polynucleotide

operably linked to a light regulated promoter;

c) an ADP-glucose pyrophosphorylase small subunit polynucleotide

operably linked to a light regulated promoter; and

d) a fructose-1 ,6-bisphosphate aldolase polynucleotide operably linked to a light regulated promoter.

2. The expression cassette of claim 1 , wherein the promoter of a) is a Photosystem I reaction center promoter; the promoter of b) is a chlorophyll a/b-binding protein precursor protein; the promoter of c) is a light harvesting protein of photosystem I and the promoter of d) is a promoter isolated from a Photosystem I reaction center subunit promoter.

3. The expression cassette of claim 2, wherein the promoter of a) is an OsPSID promoter; the promoter of b) is an OsLHCA4 promoter; the promoter of c) is an OsLHCA3 promoter and the promoter of d) is a OsPSAK promoter.

4. The expression cassette of claim 3, wherein the OsPSID promoter is SEQ ID NO: 13; the OsLHCA4 promoter is SEQ ID NO: 10; the OsLHCA3 promoter is SEQ ID NO: 9 and the OsPSAK promoter is SEQ ID NO: 1 1 .

5. The expression cassette of claim 1 , wherein the promoters of b) and c) increase the expression of ADP-glucose pyrophosphorylase to approximately 4 fold higher than in a control plant.

6. The expression cassette of claim 1 , wherein the promoter of d) increases the expression of sedoheptulose-1 ,7-bisphosphotase to approximately 4 fold higher than in a control plant.

7. The expression cassette of claim 2, wherein the promoter of d) increases the expression of fructose

8. The expression cassette of claim 4, wherein all of the promoters are light

regulated promoters.

9. The expression cassette of claim 4, wherein all of the promoters are leaf

preferred promoters.

10. The expression cassette of claim 4, wherein one or more of the promoters is a light regulated promoter and at least one of the promoters is a leaf preferred

1640 promoter.

1 1 . The expression cassette of any one of claims 1 -7, wherein the ADP-glucose

pyrophosphorylase subunits are selected from isoforms native to a chloroplast.

12. The expression cassette of any one of claims 1 -9, wherein the polynucleotides are selected from the group consisting of:

1645 a. polynucleotides comprising SEQ ID NOS: 1 , 3, 5, and 7;

b. polynucleotides encoding the polypeptides comprising SEQ ID NOS: 2, 4, 6, and 8;

c. polynucleotides having at least 80% identity to SEQ ID NOS: 1 , 3, 5, and 7;

1650 d. polynucleotides encoding polypeptides having at least 80% identity to

SEQ ID NOS: 2, 4, 6, and 8; and

e. polynucleotides capable of hybridizing under stringent conditions to

polynucleotides comprising SEQ ID NOS: 1 , 3, 5, and 7.

13. A host cell comprising the expression cassette of any one of claims 1 -9. 1655

14. A host cell of claim 1 1 , wherein the cell is a plant cell.

15. A transgenic C3 plant or seed comprising the expression cassette of any one of claims 1 -10.

16. The transgenic plant of claim 13, wherein the plant is a monocot.

17. The transgenic plant of claim 13, wherein the plant is a dicot.

1660 18. The transgenic plant of claim 13, wherein the transgenic plant is selected from the group consisting of wheat, tobacco, soybean, spinach, sugar beet, sunflower, rapeseed, rice, and Arabidopsis.

19. The transgenic plant of claim 16, wherein the transgenic plant is soybean.

20. A method for producing a transgenic plant comprising regenerating a transgenic 1665 plant from the plant cell according to claim 12.

21 . A method for producing the transgenic plant comprising crossing a transgenic plant comprising the expression cassette of any one of claims 1 -10 with a non- transgenic plant and selecting for a progeny plant comprising the expression cassette of any one of claims 1 -10.

22. A method of increasing yield in a plant comprising the steps of:

A) introducing the expression cassette of any one of claims 1 -10 into a plant cell;

B) regenerating a plant from the plant cell; and

C) growing a plant having increased yield.

23. The method of any one of claims 18-20, wherein the plants have an increased number of seed pods.

24. The method of any one of claims 18-20, wherein the plant's lower leaves

undergo early senescence.

25. The method of any one of claims 18-20 wherein the promoters are selected from the group consisting of constitutive promoters, leaf preferred promoters and light regulated promoters.

26. The method of claim 23, wherein as least one of the promoters is a leaf preferred promoter.

27. The method of claim 23, wherein at least one of the promoters is a light regulated promoter.

28. The method of claim 23, wherein all of the promoters are light regulated

promoters.

29. The method of claim 23, wherein all the promoters are leaf preferred promoters.

30. The method of claim 23, wherein the promoters are a combination of one or more light regulated promoters and one or more leaf preferred promoters.

31 . The method of claim 23, wherein the promoters are selected from the group

consisting of an OsLHCH3 promoter, an OsLHCA4 promoter, a OsPSAK promoter and an OsPSID promoter.

32. The method of any one of claims 18-20, wherein the polynucleotides have been optimized for expression in a dicot.

33. The method of any one of claims 18-20, wherein the ADP-glucose

pyrophosphorylase subunits are selected from isoforms native to a chloroplast.

34. The method of any one of claims 18-20, wherein the polynucleotides are selected from group consisting of:

a. polynucleotides comprising SEQ ID NOS: 1 , 3, 5, and 7;

1700 b. polynucleotides encoding the polypeptides comprising SEQ ID NOS: 2, 4,

6, and 8;

c. polynucleotides having at least 80% identity to SEQ ID NOS: 1 , 3, 5, and 7;

d. polynucleotides encoding polypeptides having at least 80% identity to ID 1705 NOS: 2, 4, 6, and 8; and

e. polynucleotides capable of hybridizing under stringent conditions to

polynucleotides comprising SEQ ID NOS: 1 , 3, 5, and 7.

35. A method for making a transgenic seed comprising a sedoheptulose-1 ,7- bisphosphotase, an ADP-glucose pyrophosphorylase large subunit, an ADP-

1710 glucose pyrophosphorylase small subunit and a fructose-1 ,6-bisphosphate

aldolase:

a. introducing an expression cassette into a first plant, wherein the

expression cassette comprises at least one or more polynucleotides selected from the group consisting of fructose-1 ,6-bisphosphate aldolase,

1715 sedoheptulose-1 ,7-bisphosphotase, ADP-glucose pyrophosphorylase large subunit and ADP-glucose pyrophosphorylase small subunit;

b. introducing an expression cassette into a second plant, wherein the

expression cassette comprises at least one or more polynucleotides selected from the group consisting of fructose-1 ,6-bisphosphate aldolase,

1720 sedoheptulose-1 ,7-bisphosphotase, ADP-glucose pyrophosphorylase large subunit and ADP-glucose pyrophosphorylase small subunit;

c. crossing the first plant with the second plant; and

d. obtaining seed from the plant resulting from the cross in c), wherein the seed comprises fructose-1 ,6-bisphosphate aldolase, sedoheptulose-1 ,7-

1725 bisphosphotase, ADP-glucose pyrophosphorylase large subunit and

ADP-glucose pyrophosphorylase small subunit.

36. The method of claim 33 wherein the promoters are selected from the group

consisting of constitutive promoters, leaf preferred promoters and light regulated promoters.

1730 37. The method of claim 34, wherein as least one of the promoters is a leaf preferred promoter.

38. The method of claim 34, wherein at least one of the promoters is a light regulated promoter.

39. The method of claim 34, wherein all of the promoters are light regulated 1735 promoters.

40. The method of claim 34, wherein all the promoters are leaf preferred promoters.

41 . The method of claim 34, wherein the promoters are a combination of one or more light regulated promoters and one or more leaf preferred promoters.

42. The method of claim 34, wherein the promoters are selected from the group

1740 consisting of an OsLHCH3 promoter, an OsLHCA4 promoter, an OsPSAK

promoter and an OsPSID promoter.

43. The method of any one of claims 33-40, wherein the polynucleotides have been optimized for expression in a dicot.

44. The method of any one of claims 33-41 , wherein the ADP-glucose

1745 pyrophosphorylase subunits are selected from isoforms native to chloroplast.

45. The method of claim 33, wherein the polynucleotides code for polypeptides

described in SEQ ID NOS: 2, 4, 6, and 8.

46. A method for increasing yield in a plant without a significant increase in the rate of photoassimilation, the method comprising expressing in a plant a

1750 sedoheptulose-1 ,7-bisphosphotase, an ADP-glucose pyrophosphorylase large subunit, an ADP-glucose pyrophosphorylase small subunit and a fructose-1 ,6- bisphosphate aldolase.

47. The method of claim 20 wherein the yield in a plant is increased without a

significant difference in photoassimilation between transgenic and null plants.

1755