WO2014091255A1 - Transgenic plants - Google Patents

Abstract

The present invention relates to transgenic plants and in particular to transgenic plants having one or more modified genes resulting in phenotypic changes in leaf anatomy and/or vein spacing within the plant, from the C3 photosynthetic pathway to C4. The invention also relates to the genes which give rise to said phenotypic changes.

Description

TRANSGENIC PLANTS

FIELD OF THE INVENTION The present invention relates to transgenic plants and in particular to transgenic plants having one or more modified genes resulting in phenotypic changes in leaf anatomy and/or vein spacing within the plant. The invention also relates to the genes which give rise to said phenotypic changes. BACKGROUND TO THE INVENTION

Many commercially important plants such as the crops rice and wheat use the C3 photosynthetic pathway. In these C₃ plants, chloroplasts accumulate Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCo) and fix carbon dioxide (C0₂) in the Calvin-Benson cycle. Carbon fixation directly from the air by RuBisCo is limited in its efficiency by a number of factors, most notably by a competing reaction whereby RuBisCo fixes oxygen (0₂) rather than C0₂.

The C₄ photosynthetic pathway which is found in plants such as maize is an elaboration of the above-described C₃ pathway. In such C₄ plants, photosynthetic reactions are split between two morphologically distinct cell-types known as bundle sheath (BS) and mesophyll (M) cells. Specifically, C0₂ is initially fixed in the M cells into a 4-carbon compound (either malate or aspartate). The C₄ compound subsequently diffuses into the BS cells, where it is decarboxylated to release C0₂ that is refixed by RuBisCo to provide a substrate for carbohydrate synthesis. This two-step process maintains an elevated concentration of C0₂ within the BS cells that is able to out-compete 0₂ at the active site of RuBisCo. The efficiency of the C photosynthetic pathway depends on the correct organisation of BS and M cells, which are typically arranged into concentric wreaths around leaf veins to form what is known as Kranz anatomy, whereby veins are separated by two BS and two M cells in a repeating pattern:

V-BS-M-M-BS-V C₄ metabolism based on Kranz anatomy is far more productive than C₃ metabolism. It would therefore be highly advantageous if C traits could be introduced into C3 plants such as rice, oat, barley and/or wheat in order to increase productivity and thus crop yields.

Although Kranz anatomy apparently evolved multiple times, the underlying genetic mechanisms which pattern Kranz anatomy have not been disclosed. There is therefore a need for an understanding of the genetic process or processes which pattern Kranz anatomy in C₄ plants.

SUMMARY OF THE INVENTION

The present invention addresses the above need by providing one or more of the maize genes set out in Table 3. The accession numbers are provided, together with the accession numbers of the Arabidopsis orthologues of these sequences. A subset of these genes is provided in Table 4 (i.e. GRMZM2G016477, GRMZM2G027068, GRMZM2G045883, GRMZM2G081816, GRMZM2G052102, GRMZM2G1 14998, GRMZM2G171852, GRMZM2G140694, GRMZM2G028046, GRMZM2G136494, GRMZM2G074032, GRMZM2G129261 , GRMZM2G143723, GRMZM2G134998, GRMZM2G15001 1 , GRMZM2G132794, GRMZM2G172657, GRMZM2G123900, GRMZM2G471089, GRMZM2G132794, GRMZM2G163975, GRMZM2G151542, GRMZM2G039074, GRMZM2G178182, GRMZM2G045883, GRMZM2G140669, GRMZM2G472945, GRMZM2G178102, GRMZM2G131516, GRMZM2G469304, GRMZM2G172657, GRMZM2G374986, GRMZM2G002280, GRMZM2G1 19359, GRMZM5G8931 17, GRMZM2G15001 1 , GRMZM2G136494, GRMZM2G028046, GRMZM2G098988, GRMZM2G377217, GRMZM2G148467, GRMZM2G021573, GRMZM2G040924, GRMZM2G171365, GRMZM2G146688, GRMZM2G425236, GRMZM2G417229, GRMZM2G069365, GRMZM5G887276, GRMZM2G098813, GRMZM2G1 1 1045, AC215201 .3_FG008, GRMZM2G399072, GRMZM2G318592, GRMZM2G097275, GRMZM2G095899, GRMZM2G462623, GRMZM2G015666, GRMZM2G126018, GRMZM5G850129, GRMZM2G312419, GRMZM2G061734, GRMZM2G082586, GRMZM2G01 1463, GRMZM2G480386, GRMZM2G089819, GRMZM2G159953, GRMZM2G163724, GRMZM2G151955, GRMZM2G039934, GRMZM2G046316, GRMZM2G034155, GRMZM2G1 14276, GRMZM2G028643, GRMZM2G0591 17, GRMZM2G478876, GRMZM2G344857, GRMZM2G087243, GRMZM2G133716, GRMZM2G061314, GRMZM2G1 12210, GRMZM2G023051 , GRMZM2G139324, GRMZM2G077219, GRMZM2G1 14893, and GRMZM2G109

Over-expression of one or more of these genes or their orthologues may lead to the formation of ectopic veins and an increase in leaf vein density that is similar to Kranz anatomy. Here, a gene can be said to be "over-expressed" when the mRNA or protein corresponding to that gene is expressed in a temporally, spatially or quantitatively different manner relative to the wild-type allele or in the case where an ectopic copy of that gene (either host-derived or derived from a different plant species or synthesised with or without modification of codon usage) is expressed in any host tissue.

Thus, in a related embodiment, the invention provides a transgenic plant which over- expresses one or more of the maize genes set out in Table 3. In a further embodiment, the invention provides a transgenic plant which over-expresses one or more of the maize genes set out in Table 4. Orthologues of these sequences from other plant species may also be used. The Arabidopsis orthologue is provided. In particular, the transgenic plant may have a leaf which contains an increased number or density of veins with respect to a wild-type plant of the same species at the same stage of development as the transgenic plant. The transgenic plant can be any member of the kingdom Plantae comprising all C₃ plants including cereal crops (such as rice, wheat, oat and barley); or biofuel crops.

In particular, the vein spacing within one or more leaves of the transgenic plant may be altered with respect to a wild-type plant of the same species at the same stage of development as the transgenic plant. Although vein density is increased, the vein spacing within one or more leaves of the transgenic plant may not necessarily be uniform. Veins within one or more leaves of the transgenic plant may be closer together with respect to said wild-type plant. Veins within one or more leaves of the transgenic plant may be separated by fewer cells than the veins of said wild-type plant. Vein spacing may also be decreased by reducing the volume of cells between veins.

Such morphological changes with respect to said wild-type plant can be readily determined using light microscopy.

The transgenic plant may have a phenotype which is indicative of Kranz anatomy.

In one embodiment, the transgenic plant of the invention over-expresses a maize LRR repeat kinase (GRMZM2G016477). Orthologues of this sequence from other plant species may also be used.

The maize LRR repeat kinase (or its orthologue) may be present in combination with one or more of the other genes (or their orthologue) from Table 3 or Table 4.

The present invention also provides a method of making a transgenic plant, the method comprising introducing one or more of the genes herein identified into a plant such that the gene will be over-expressed. The plant may be any member of the kingdom Plantae comprising all C₃ plants including cereal crops such as rice, wheat, oat and barley. The gene to be introduced may be maize LRR repeat kinase (GRMZM2G016477). Orthologues of this sequence from other plant species may also be used. The maize LRR repeat kinase (or its orthologue) may be present in combination with one or more of the other genes (or their orthologue) from Table 3 or Table 4.

The one or more genes herein identified may be introduced via transformation with Agrobacterium, using suitable techniques known within the art (e.g. as set out in Nishimura et al. Nature Protocols (2006), 1 (6):2796-2802, which is incorporated by reference).

In a further embodiment, the present invention provides a transgenic plant seed. The transgenic plant seed will comprise one or more of the genes herein identified (or their orthologues). The transgenic plant seed will be capable of producing a transgenic plant which will over-express said one or more genes. The gene may be LRR repeat kinase (GRMZM2G016477). Orthologues of this sequence from other plant species may also be used. The maize LRR repeat kinase (or its orthologue) may be present in combination with one or more of the other genes (or their orthologue) from Table 3 or Table 4.

In yet a further embodiment, the invention provides a shoot, root, cutting or seedling from the transgenic plant of the invention. In a further embodiment, the invention provides the isolated maize genes (or their Arabidopsis orthologues) from Table 3 or Table 4. One or more of these genes (or their orthologues) may be present in a vector or other means suitable for causing over-expression of said gene or genes within a transgenic plant of the invention. The gene or genes (or their orthologues) may be present with a promoter. The promoter may be the endogenous promoter (i.e. maize for the maize gene(s)). Alternatively, the gene or genes (or their orthologues) may be present with another promoter (e.g. the maize ubiquitin promoter).

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:

Figure 1A shows a schematic and images of the tissues sampled for use in transcriptome sequencing;

Figure 1 B through to Figure 1 F shows transverse sections of various samples from the leaf tissue; Figure 1 G through to Figure 1 K shows transverse sections of various samples from the husk tissue; Figure 2 shows a comparative profile analysis of transcriptomes in developing leaf primordia: Figure 2 shows a Table displaying descending (D1 -D3), ascending (A1 - A3) and neutral transcriptome profiles. The total number of genes in each foliar (F) and husk (H) profile is shown along with the number of genes encoding transcription factors (TFs). The specificity of TFs with respect to each profile is shown, along with reference to the number of TFs that are shared with other profiles;

Figure 3 shows distinct transcription factor cohorts associated with early differentiation events in foliar leaf primordia. Transcription factors identified in the FA2 profile that are specific to foliar leaves, and have no previously identified role in early leaf development are shown. Transcripts reported to be BS or M localized are indicated;

Figure 4A shows proposed regulators of Kranz patterning. Accession numbers and gene family are indicated, along with the sub-family deduced from phylogenetic analysis. Where previously unnamed, genes have been given names on the basis of phylogenetic relationships. Presence in specific primordia profiles, and presence or absence in the list of putative positive regulators of Kranz is indicated; Figure 4B shows a proposed model of the genetic pathway regulating Kranz development: it is postulated that six genes are expressed in developing procambium and/or vasculature and that their role is to regulate leaf venation patterns. The SHR1 gene is additionally expressed in BS cells, as is the SHR target gene SCR1 . SCR1 is also expressed in M cells but at a lower level than in BS cells, establishing a gradient of expression emanating from the vein. We propose that the SHR/SCR gradient is fixed by the cell-specific accumulation of the SHR interacting proteins RAVEN 1 (BS) and JAY1 (M). BS cells are thus specified by the combined action of SHR1 , SCR1 , and RVN1 whereas M cells are specified by the interaction between SCR1 and JAY1 . Superimposed upon this SHR module is a gradient of DLK gene expression;

Figure 5 shows PCR results for a number of transgenic lines by comparison with a wild-type (negative control) and the plasmid carrying the transgene (positive control); Figure 6: 6A, 6B, 6C and 6D show hand sections from fully expanded WT (6A, 6C) and transgenic line 80.13 (6B, 6D) leaves. Figure 6E shows inter-veinal distance in microns (μιη) between two intermediate veins positioned between the closest and second closest lateral veins to the midrib, measured in three separate leaves of each of three transgenic lines (80.10, 80.12, 80.13). Figure 6F shows the number of leaf veins per mm of leaf width in the same three leaves.

EXAMPLES

Example 1: Identifying regulators of Kranz anatomy

The development of Kranz anatomy has been described from a histological perspective but the mechanisms underpinning the patterning of Kranz tissues are not known.

In maize, BS cells surrounding the veins, plus the middle layer of M cells in the leaf are derived from the same cell lineage as the leaf veins. As such, genes that regulate leaf venation patterns in maize will regulate whether or not Kranz anatomy is formed. If a vein is formed, it develops with associated BS cells and if veins are formed at appropriate intervals only two M cells will separate each vein/BS unit. The timing of vein formation is such that the leaf midvein is initiated first, developing from the shoot into the leaf primordium. The time interval is measured in plastochron (P); lateral veins begin to appear at the base of Plastochron 2 (P2) primordia and develop toward the leaf tip. Intermediate veins develop during P5 and lead to the vein patterning that is characteristic of Kranz anatomy; these veins are initiated at the tip of P5 primordia and by the end of P5 they have reached the base of the leaf sheath. Anastomoses or fusions at the developing ligule result in fewer intermediate veins in the sheath than in the blade. Kranz anatomy is therefore a feature of the leaf blade but not of the leaf sheath. This is apparent in the husk leaves that surround the ear, where most or all of the tissue is sheath tissue. Veins in husk leaf sheaths are surrounded by a wreath of BS cells but each vascular bundle is then separated by around 20 M cells rather than the two associated with Kranz anatomy.

A range of biological samples representing different developmental trajectories and anatomical traits were harvested as follows.

Plant material and growth conditions

Maize inbred line B73 was grown in soil in a greenhouse with a diurnal light regime of 16 hours of light (supplemented to 300 μιηοΙ m^"2 sec^"1) and 8 hours of darkness and an average daytime temperature of 28 degrees C and an average night time temperature of 20 degrees C.

Samples

Figure 1A depicts the position on the plant from which the samples were taken, together with the overall morphology of the tissue sampled.

For sample purposes, tissue was harvested as required from 2, 4 or 8 week old plants and placed directly into liquid nitrogen. In the case of husk samples, the prophyll was discarded prior to immersion in liquid nitrogen.

Foliar and husk leaf samples were both harvested at 5 stages of development. Foliar tissues were all harvested from 2 week old seedlings in which the 5th leaf was just emerging. The 5 foliar samples and the 5 husk samples covered the range from plastochron (P)1 to P9 and are set out in the table below.

old plants

HP5 P5 husk primordia from ear forming nodes of 4-week old plants

HI inner husk leaf from 8 week old plants

HE outermost husk leaf from 8 week old plants

FP5, Fl and FE leaves were all harvested above the ligule to ensure that only the blade (Kranz) tissue was represented. The husk samples were harvested from the same plants as the foliar samples, from the axils of P9 and P10 leaves. The husk leaves did not form blades and thus all such samples comprised only sheath (non- Kranz) tissue.

Leaf samples were fixed overnight in FAA (4% formaldehyde, 5% acetic acid, 50% ethanol) and embedded in Paraplast Plus as described in the method of Langdale JA (1994) In situ hybridisation, The Maize Handbook (eds. Freeling and Walbot), pp. 165-179, which is hereby incorporated by reference. Sections were stained with Safranin/Fast Green and viewed with a Leica DMRB microscope.

Transverse sections of all 10 samples were examined by light microscopy. The results are shown in Figure 1 B to Figure 1 K. For both foliar and husk samples, P1 and P2 primordia have cells with high cytoplasm to cell volume ratios and procambium associated with the midvein is minimally visible (Figure 1 B and 1 G). By P4 the midvein and developing lateral veins can be readily distinguished and the distance between them is smaller in foliar (Figure 1 C) than in the comparative-stage husk (Figure 1 H) primordia. At P5, foliar primordia have initiated intermediate veins and each vascular centre is surrounded by a wreath of small BS cells and is separated from the next centre by 1 to 2 intervening M cells (Figure 1 D). In husk samples, no intermediate veins can be seen to be formed and cell division and expansion in M cells increases the space and cell number between the veins: from 5 at P4, to around 10 at P5 (Figure 1 1) and ultimately to around 15 or more in mature husk leaves (Figure 1J and 1 K). Vein spacing does not change after P5 in foliar leaves and thus in Fl (Figure 1 E), which shows P7 foliar leaf, and FE (Figure 1 F), which shows P9 foliar leaf, the classical cellular Kranz arrangement is visible (i.e. V- BS-M-M-BS-V). In expanded husk leaves BS cells and chloroplasts within them are much smaller than in foliar leaves (Figure 1 K). Very few chloroplasts are visible in HI leaves (Figure 1 J). Table 1 shows a summary of key anatomical differences between the respective tissues. It was noted that HP samples were essentially arrested at the time of harvesting but that FP, FP3/4 and FP5 samples were undergoing rapid cell division and growth.

Accordingly the present inventors have postulated that positive regulators of Kranz anatomy in maize will act between P2 and P5 in foliar leaf primordia but have reduced activity or be inactive in husk leaf primordia.

Identifying putative genes

An overview of the changes in gene activity during the patterning of Kranz anatomy was carried out using lllumina sequencing to generate transcriptomes of developing foliar (Kranz) and husk sheath (non-Kranz) primordia. Transcriptome profiles were then compared with the later stages of development where initiation or maintenance of C metabolism is being superimposed on pre-established anatomy. A number of genes that are active during early leaf development were identified and from these positive regulators of Kranz patterning have been isolated and tested. The results reveal novel cohorts of genes that are active during early leaf development (as opposed to activity of a master regulator). From within these cohorts a regulatory network has been identified which is associated with the patterning of Kranz anatomy. RNA extraction and cDNA synthesis

Multiple individual samples were pooled prior to RNA extractions. For each of the two sequencing replicates, N=1200 (FP); 300 (FP3/4); 100 (FP5); 12 (Fl); 12 (FE); 2400 (HP); 300 (HP3/4); 100 (H5); 12 (HI) and 12(HE).

RNA was extracted using the mirVana miRNA Isolation Kit (Applied Biosystems). RNA integrity was analysed by formaldehyde gel electrophoresis. cDNA library preparation and sequencing were carried out at the Beijing Genome Centre (BGI). Each RNA sample was treated in the same manner prior to paired end lllumina sequencing. Total RNA was first treated with DNase I and then purified over an oligo-dT column. The enriched mRNA was then sheared and converted into cDNA using standard lllumina protocols. The cDNAs were subsequently ligated to lllumina adaptors and subjected to limited PCR as part of the lllumina library preparation. 72bp paired end reads were generated for all samples.

Transcript quantification and differential gene expression analysis Paired end reads were subject to quality-based trimming using the FASTX toolkit described in Goecks et al. Genome Biol (2010), 1 1 (8):R86, which is incorporated by reference; setting the PHRED quality threshold at 20 and discarding reads less than 21 nucleotides in length. Transcripts were quantified using RSEM as described in Li et al, BMC Bioinformatics (201 1 ), 12:323, which is incorporated by reference; and the predicted coding sequences from version 5b of the Maize genome. For differential gene expression analysis, expected transcript counts originating from the same gene locus were summed and all possible pairwise comparisons between replicated samples were performed using DESeq as described in Anders et al. Genome Biol (2010), 1 1 (10):R106, which is incorporated by reference. In all cases, differentially expressed genes were identified as those genes with a Benjamini- Hochberg corrected p-value of less than 0.05 as described in Benjamini et al. Journal of the Royal Statistical Society Series B (Methodological) 1995. 57(1 ):289- 300, which is incorporated by reference. Data analysis

To identify novel associations between gene cohorts and the earliest patterning processes that operate in maize leaf primordia, all genes that were detected in foliar and husk primordia samples were assigned to one of seven profiles (Figure 2). Each profile was designed to identify genes associated with specific aspects of leaf development. For example, descending profiles should contain genes required for meristem function and for very early events in leaf development such as midvein formation and adaxial/abaxial axis formation. In contrast, ascending profiles should contain genes required for the differentiation of lateral leaf veins, patterning of intermediate veins, specification of cell-types and early plastid biogenesis.

The A2 and A3 profiles were designed to identify genes required for patterning leaf venation and regulating cell-type differentiation in foliar leaves. The presence of genes in the FA2/3 profiles that are related to those that regulate xylem (e.g. XYLEM NAC DOMAIN"! ) and phloem (ALTERED PHLOEM 1 ) differentiation was confirmed. The presence of foliar-specific AUXIN RESPONSE FACTOR (ARF) and AUX/IAA homologs in the FA3 profile was confirmed. Very little is known about the patterning of leaf venation or the regulation of BS and M differentiation, and it is apparent that most of the genes in the FA2 profile have not been previously characterized and/or annotated. With this in mind, there are 16 foliar-specific genes of note that fall into four cohorts (Figure 3). First, there are three bHLH genes that may regulate cellular differentiation given the role of other bHLH proteins in patterning processes. Notably, transcripts encoded by two of these genes were detected in maize BS cells, but only when samples also included vascular tissue. One of the genes is related to a target of the auxin dependent transcription factor MONOPTEROS. Second, there is a novel bZIP gene, and three DoF Zn Finger genes that share clades with Arabidopsis DoF AFFECTING GERMINATION (DAG)-like and HIGH CAMBIAL ACTIVITY2 (HCA2) genes, all of which are expressed in vascular tissue. In this case, transcripts of the two DAG-like genes were detected both in isolated BS cells and in BS cells associated with vasculature. Third, two GRAS family SHORTROOT (SHR)-like genes are present, homologs of which play a role in radial patterning around vasculature in the Arabidopsis root. Fourth and most notable are seven C2H2 Zn Finger proteins. Two are related to the previously characterized Arabidopsis gene DEFECTIVELY ORGANIZED TRIBUTARI ES (DOT5) that regulates vascular patterning, and three are related to SHOOT GRAVITROPISM 5 (SGR5) and JACKDAW (JKD) proteins - known components of the SHR pathway in Arabidopsis. Transcripts of one of the JKD-related genes accumulate specifically in M cells and those of the second accumulate specifically in BS cells. The remaining two C2H2 ZnF proteins are related to MRP1 interacting (MRPI) proteins. MRPI proteins interact with MRP through a C-terminal domain that is shared with SHR target proteins. The phylogenetic relationships and expression profiles of all of these genes thus position them as likely regulators of vascular patterning and cellular differentiation in maize foliar leaves.

In addition to the profile analysis described above, transcriptome filters were designed (based on the anatomical and developmental analyses presented in Figure 1 ) that would identify positive regulators of Kranz in maize tissues. For positive regulators it was postulated that genes would be expressed at higher levels in foliar leaves than in husk leaves and that patterning genes would be expressed prior to P5. It was further postulated that if genes were expressed in the expanding leaf, transcript levels would be higher in the immature basal regions of the leaf than in more distal regions. The expanding leaf gradient dataset was generated by remapping the read data from Li et al. Nat Genet (2010). 42: 1060-1067. which is hereby incorporated by reference. The data were re-mapped to version 5b of the maize genome.

Table 2 shows the decision and filtration steps that were carried out to identify genes of interest involved in regulating Kranz anatomy (the p-value cut-off used was 0.05). It can be seen that following the final step 3, 283 putative positive regulators of Kranz anatomy were identified.

The putative positive regulators are presented in Table 3, together with a list of 16 transcription factors discussed further below.

In the list of 283, GO term enrichment identified a large proportion (cohort) of genes associated with e.g. the cytoskeleton and with ribosomes/protein synthesis. This was expected, since the filtration criteria selected genes that were most highly expressed in developing foliar primordia and FP3 to FP5 are actively dividing tissues. These 283 genes may play a role in regulating Kranz anatomy, but at present genes with a role in the regulation of transcription (e.g. nucleus, DNA binding) or of cell-signalling are of greater interest. 70 genes with putative regulatory roles were identified from within the list of 283. The presence of the ZmScarecrow 1 gene within this list is supportive of a role for this subset in vascular patterning, as ZmScrl is known to be expressed in early vascular development and in BS cells (as set out in Lim et a/, Plant Mol Biol (2005), 59:619-630, which is incorporated by reference). Similar support is demonstrated by the presence within this list of the C2H2 zinc finger encoding gene GRMZM2G15001 1 , because it is homologous to Defectively Organised Tributaries (DOTS), which is required for appropriate patterning of veins in the Arabidopsis leaf (as set out in Petricka et al. The Plant Journal (2008). 56:251 -263. which is incorporated by reference).

16 transcription factors were identified through profile classification of gene expression in leaf primordia (Figure 3) and 283 potential regulators were identified through comparative analysis within a broader developmental context (Table 3). Both approaches were used to ensure that genes involved in all stages of Kranz differentiation were identified. In this regard, the development of Kranz anatomy involves three distinct but undoubtedly overlapping stages. First, procambium is initiated at regular intervals across the mediolateral leaf axis. Second, concentric wreaths of BS and M cells are specified around each vein. Finally, components of the photosynthetic machinery accumulate in a BS and M cell-specific manner. None of the genes encoding C4 photosynthetic enzymes are represented in the leaf primordia samples. As such, the data provide insight into the patterning of leaf venation and the specification of distinct BS and M cell-types, prior to the induction of a functional C4 cycle. Of the 16 transcription factors revealed by the FA2 profile (Figure 3), six were also identified in the 283 list generated above. It is postulated that these six transcription factors play a role in patterning leaf venation. Three are homologs of Arabidopsis genes known to be involved in vascular development (SHR, DOT5), two encode proteins with a domain that is conserved in SHR target proteins, and the sixth is related to the bHLH protein SPEECHLESS which regulates stomatal spacing in the Arabidopsis epidermis. The maize bHLH gene may thus have been recruited to regulate vein spacing in sub-epidermal layers. Transcripts of all six genes accumulate to higher levels in foliar than husk primordia, as would be expected given the higher vein density in foliar leaves.

Also present within this list is LRR repeat kinase (GRMZM2G016477). It was decided that this gene would be tested within a C₃ plant.

Example 2: Over-expression of the LRR kinase

Creation of transgenic rice

Callus of the rice variety Kitaake was transformed with the construct MS80 using Agrobacterium tumefaciens. MS80 comprised a modified pVec8 binary vector that included the maize Ubiquitin promoter and intron placed upstream of the coding region of GRMZM2G016477 (a maize LRR kinase encoding gene). Putative transformants were selected and regenerated on hygromycin. A number of regenerated lines were assayed by genomic PCR for presence of the transgene.

Figure 5 shows that the same size fragment is amplified from genomic DNA extracted from lines 80-10, 80-12 and 80-13 as with MS80 plasmid DNA (positive control). No fragments are amplified from genomic DNA extracted from the wild-type (WT) negative control. The amplified fragments from lines 80-10, 80-12 and 80-13 were sequenced and shown to correspond to regions of GRMZM2G016477. It was accordingly concluded that the three lines are transgenic and are likely to be expressing the maize LRR kinase.

Observed phenotypic effects

The phenotype of each line was observed with respect to a wild-type. Samples were cut from the middle of expanded leaf blades of TO (primary transformed) plants that had been regenerating for 6-8 weeks, at least 4 weeks of which had been on soil in a glasshouse. Transverse sections were prepared by hand sectioning under water with a razor blade. Sections were place on a microscope slide and observed under UV light. The results are shown in Figure 6. 6A, 6B, 6C and 6D show hand sections of leaves from fully expanded WT (6A, 6C) and transgenic 80.13 (6B, 6D) lines. Figure 6E shows the inter-veinal distance in microns (μιη) between two intermediate veins positioned between the closest and second closest lateral veins to the midrib, measured in three separate leaves of three independent transgenic lines.

Figure 6F shows number of leaf veins per mm of leaf width in same three leaves as in Figure 6E.

Although none of the transgenic rice lines uniformly displays classical Kranz anatomy, it is clear that the lines 80.12 and 80.13 have veins that are more closely spaced together (i.e. with fewer cells between the veins) by comparison with the wild-type.

Summary

C₃ plant crops such as rice lack the Kranz anatomy and lack the C₄ photosynthetic pathway. We show here that certain genes are associated with regulation of Kranz anatomy and, when over-expressed in C₃ transgenic plants, lead to phenotypic changes that increase vascular density, modify leaf anatomy and may result in a more efficient photosynthetic pathway.

It is proposed in particular that six transcription factors play a role in patterning leaf venation (Figure 4). Three are homologs of Arabidopsis genes known to be involved in vascular development (SHR, DOT5), two encode proteins with a domain that is conserved in SHR target proteins, and the sixth is related to the bHLH protein SPEECHLESS which regulates stomatal spacing in the Arabidopsis epidermis. The maize bHLH gene may thus have been recruited to regulate vein spacing in sub- epidermal layers. Notably transcripts of all six genes accumulate to higher levels in foliar than husk primordia (Figure 4A), as would be expected given the higher vein density in foliar leaves. Once procambial centres have been initiated in developing leaf primordia, our data indicate that a SHR-related pathway specifies BS and M cell identity. In Arabidopsis roots the SHR gene is expressed in the vasculature and SHR protein is transported to the encircling endodermal cell layer. Movement of SHR to the next cell layer (the cortex) is prevented by an interaction between SHR and its downstream target SCR in the endodermis. Boundaries between the endodermis and cortex are maintained by cell-specific compartmentalization of JKD-related proteins. This scenario is reproduced in the context of developing Kranz tissues in that transcripts of ZmScr and of two JKDrelated genes (that we name ZmJayl and ZmRavenI ) accumulate differentially in BS and M cells. ZmScrl and ZmRvnl transcripts accumulate preferentially in BS cells whereas ZmJayl transcripts are enriched in M cells. Genes encoding four other direct targets of SHR (two F-box proteins, a receptor like kinase and an unknown protein) were also identified in the list of putative positive regulators of Kranz anatomy. We therefore propose that Kranz anatomy in maize is patterned by a SHR-related developmental module (Figure 4B). Superimposed upon the SHR module is an expression gradient of DoF AFFECTING GERMINATION (DAG)-like genes that we have named ZmDIkI and ZmDlk2. There is no predicted function for these genes but their classification profile and differential expression in BS and M cells warrants their inclusion in the proposed model. In summary the combinatorial action of just six transcription factors could explain the differentiation of distinct BS and M cell in C4 leaves. Importantly these six genes map directly on to the existing model of Kranz development in that there is a gradient emanating from the veins and BS and M cell-specific factors that interact with that gradient. The data herein thus suggest that a pre-existing developmental module that operates to specify cell-types in the radial axis of the root was co-opted during the evolution of Kranz anatomy to pattern BS and M cell-types around leaf veins. Table 1. Key anatomical traits and transcriptome profiles of foliar and husk leaf samples. Signature genes are those where transcripts were detected at significantly higher levels in the named sample as compared to the other nine samples (p=0.05).

Table 2. Filtration steps to identify putative regulators of Kranz anatomy. All tests were carried out at a p-value cut-off of 0.05.

Positive Regulators

All genes

N = 35,770

Step 1:

FP or FP3/4 or FP5 significantly > FE + HP + HI +HE

Genes must pass test in 2 out of 3 FP samples.

N = 918

Step 2:

FP3/4 or FP5 significantly > or not different to FP

FP3/4 not significantly < FP

HP3/4 not significantly > FP3/4

HP5 not significantly > FP5

HP not significantly > FP

HP5 significantly < FP3/4

HP significantly < FP3/4

FP5 or FP3/4 > 0

HI not significantly > FP, FP3/4, FP5, FI or FE

HE not significantly > FP, FP3/4, FP5, FI or FE

FE not significantly > FP, FP3/4, FP5 or FI

N = 334

Step 3:

If in leaf gradient dataset:

Base not significantly < 1cm, 4cm or tip

lcm not significantly < 4cm, tip

4cm not significantly < tip

N = 283

Table 3: utative ositive re ulators of Kranz anatomy

GRMZM5G893117 Growth-regulating factor Os02g53690

GRMZM2G150011 Zinc finger C2H2 type AT1G13290 Os09g 13680

GRMZM2G136494 ZnF C2H2 domain AT1G75710 Os04g59380

GRMZM2G028046 Zinc finger C2H2 type AT1G75710 Os11g06840

GRMZM2G098988 bHLH T AT1G29950 Os03g39432

GRMZM2G377217 WRKY TF AT2G44745 Os02g43560,Os04g46060

GRMZM2G148467 SBP protein TF AT1G27370 Os04g46580

GRMZM2G021573 AP2 TF AT4G37750 Os03g56050,Os07g03250

GRMZM2G040924 R2R3 Myb TF AT3G61250 Os02g42870

GRMZM2G171365 MADS box TF AT2G45660 Os10g39130

GRMZM2G146688 AP2 domain TF AT4G37750 Os03g56050

GRMZM2G425236 Zinc finger homeodomain protein AT4G24660 Os03g50920

GRMZM2G417229 Zinc finger homeodomain protein Os02g47770

GRMZM2G069365 Zn finger homeodomain AT4G24660 Os02g47770

GRMZM5G887276 Myb TF SHAQKYF class Os10g39550

GRMZM2G098813 floricaula/leafy-like 1 , TF AT5G61850 Os04g51000

GRMZM2G111045 P-type R2R3 Myb protein AT3G61250, AT1G34670 Os02g42870

AC215201 .3 FG008 HLH TF AT2G40435 Os05g27090

GRMZM2G131577 NAC domain TF AT1G17880 Os03g01910

GRMZM2G399072 AP2 TF Os03g 12950

GRMZM2G318592 ZnF C2H2 domain Os06g44200

GRMZM2G097275 SBP protein TF AT1G27370, AT5G43270 Os02g04680

GRMZM2G095899 HLH TF AT4G37850 Os03g46860

GRMZM2G462623 DP-1 TF AT5G03415 Os01g48700

GRMZM2G015666 bHLH TF AT1G72210 Os09g29360

GRMZM2G126018 SBP domain TF AT2G42200 Os09g31438

GRMZM5G850129 Growth regulating factor AT3G 13960 Os06g10310

GRMZM2G312419 R2R3 Myb TF AT1G69560 Os01g16810

GRMZM2G061734 SBP domain TF -TGA1 AT5G50570 Os09g32944

GRMZM2G082586 bHLH TF AT1G72210 Os09g29360

Auxin-related 6

GRMZM2G129413 aid auxin import carrier 1 AT2G38120 Os03g 14080

GRMZM2G098643 Auxin efflux, ZmPINIa protein AT1G77110 Os05g50140

GRMZM2G121309 auxin-responsive Aux/IAA family member AT3G 16500 Os02g 13520

AT1G23160.AT2G23170,

GRMZM2G053338 indole-3-acetic acid amido synthetase AT1G28130 Os01 g55940,Os07g47490

GRMZM2G011463 Auxin inducible - SAUR family Os09g26590 Flavin-containing monooxygenase

GRMZM2G480386 YUCCA-like - role in auxin biosynthesis AT4G28720 Os03g06654

Kinases 13

GRMZM2G089819 Brassinosteroid LRR receptor kinase AT3G56100, AT3G51740 Os11g01620,Os12g01700

GRMZM2G016477 LRR repeat kinase AT1G75640 Os04g48760

GRMZM2G159953 Lectin family receptor kinase AT5G03140 Os06g 17490

GRMZM2G163724 LRR RLK AT4G37250 Os09g02250

LRR-RLK (LRR-RLKII subfamily -

GRMZM2G151955 development and disease) AT5G16000, AT1G71830 Os08g34380,Os06g 16330

GRMZM2G039934 TDR/PXY, LRR-LRK AT3G24240 Os05g51740

GRMZM2G046316 LRR-RLK AT5G45800 Os02g 14480,Os06g35200

GRMZM2G034155 LRR-RLK AT4G20270 Os07g05740

GRMZM2G114276 LRR-RLK AT1G75640 Os04g48760

GRMZM2G028643 LRR-kinase AT3G 19230 Os03g08610

Kinases Arabidopsis accession

GRMZM2G059117 LRR RLK AT3G56370 Os03g21730

GRMZM2G478876 Serine/threonine kinase AT4G34500 Os03g 12680

Phosphatidylinositol-4-phosphate 5-kinase

GRMZM2G344857 4 AT2G26420 Os11g04840

Post-transcriptional

regulation 6

Small nuclear ribonucleoprotein-associated

GRMZM2G021149 protein B AT5G44500 Os07g07220

PolyA polymerase like (polynucleotide

GRMZM2G065097 adenylyltransferase activity) AT2G45620 Os02g02970

PolyA polymerase like (polynucleotide

GRMZM2G132340 adenylyltransferase activity) AT2G45620 Os02g02970

3-5 exonuclease/ nucleic acid binding

GRMZM2G152774 protein Os01g47180

GRMZM2G117642 Endoribonuclease AT3G20390 Os07g33240

GRMZM2G149520 DEAD/DEAH box helicase family protein AT5G62190 Os09g34910

DNA regulation 6

Nucleic acid binding protein (Alba family - Alba binds DNA without sequence

GRMZM2G075505 specificity) AT3G07030 Os03g06980

GRMZM2G369260 delta DNA polymerase Os08g06620

H/ACA ribonucleoprotein complex subunit

3-like protein (nucleor protein family

GRMZM2G133838 required for 18s rRNA production) AT2G20490 Os02g40514

AC196278.2 FG003 DNA polymerase subunit AT5G63960 Os11g08330

GRMZM2G026580 DNA pol AT4G24790 Os12g19180

AC196278.2 FG004 DNA polymerase subunit AT5G63960 Os11g08330 Heat Shock and

molecular

chaperone 5

GRMZM2G040890 Heat shock related AT5G57710 Os02g54720

GRMZM2G032547 Heat shock protein like AT4G29920 Os04g23220

AC216247.3 FG001 Heat shock TF (B-4d like) AT1G46264 Os03g25120

GRMZM2G129987 chaperone protein dnaJ 6 AT3G12170 Os02g 10220

GRMZM2G359779 Heat shock protein like AT4G29920 Os04g23220

Intra and inter

cellular

signalling/membrane

trafficking 10

DEM protein like (Vacuolar

GRMZM2G363474 import/degradation) AT3G 19240 Os02g54780

GRMZM2G143253 rac GTPase activating protein AT4G03100 Os07g22580

Dynamin like (GTPases involved in

endocytosis, organelle division,

GRMZM2G010306 cytokinesis) AT1G53140 Os01g54420

GRMZM5G819523 Glutaredoxin family AT5G03870 Os04g33680

Armadillo/beta catenin like (cell-cell

GRMZM2G087243 interactions and signalling) AT2G45720 Os10g37850

Patellin 5 (carrier protein may be involved

GRMZM2G086637 in membrane trafficking during cytokinesis) AT3G51670 Os05g27820

GRMZM2G323936 rac GTPase activating protein AT4G03100 Os12g34840

C2 domain protein (Ca2+ binding -

GRMZM2G064852 intracellular signalling/membrane traffiking) AT5G 12970 Os05g30750

Forkhead domain (putative nuclear

GRMZM2G133716 signalling domain) AT1G34355 Os11g07050

GRMZM2G329710 Fasciclini domain (possible cell adhesion) AT5G06920 Os02g28970

Ribosomal 14

GRMZM2G166963 60S ribosomal protein AT1G26880 Os09g24690

GRMZM2G360677 40S ribosomal protein AT5G59240 Os02g28810,Os04g28180

GRMZM2G081102 60S ribosomal protein AT3G49010 Os06g02510

GRMZM2G113720 60S ribosomal protein AT2G34480 Os05g49030

GRMZM2G088060 60S ribosomal protein AT2G 19730 Os01 g51020,Os02g57540

GRMZM2G336875 40S ribosomal protein AT5G59240 Os04g28180

AC230013.2 FG007 60S ribosomal protein L18a AT2G34480, AT1G29965 Os05g49030

GRMZM2G177720 40S ribosomal protein AT1G07770 Os02g27760

GRMZM2G086906 40S ribosomal protein AT3G10610 Os10g27190

GRMZM2G065868 60S ribosomal protein AT4G18100 Os08g41300,Os09g32500

AC225147.4 FG002 40S ribosomal protein S23 AT5G02960 Os03g60400 GRMZM2G007695 60S ribosomal protein L4/L1 AT3G09630 Os07g08330

GRMZM2G027232 50S ribosomal protein L11 AT4G35490 Os10g32870

GRMZM2G325749 50S ribosomal protein AT5G27820 Os01g 15290

F-box (not TF

specified) and

Ubiquitin related 5

GRMZM2G002830 ubiquitin-conjugating enzyme X AT3G20060 Os01g 16650

GRMZM2G007057 Ubiquitin carrier protein AT3G20060 Os01g 16650

GRMZM2G016323 ubiquitin-specific protease 23 AT5G57990 Os06g08530

GRMZM2G440543 F-box protein GID2 AT5G481 0 Os03g 10040

F-box cyclin like domain, similar to GID2

GRMZM2G040278 protein AT5G48170 Os03g 10040

Cytoskeletal 22

GRMZM2G136838 Kinesin heavy chain AT3G43210 Os02g43130

GRMZM2G030284 Microtubule associated protein AT5G51600 Os05g47970

GRMZM2G034828 Kinesin heavy chain AT3G20150 Os03g39020

GRMZM2G082384 Kinesin like AT5G02370 Os05g38480

GRMZM2G150984 phragmoplast-associated kinesin AT4G 14330 Os02g56540

Xlp2 targeting domain in some isoforms

GRMZM2G325783 (Xlp2 binds centromeres) AT1G03780 Os07g32390

GRMZM2G157616 Kinesin like AT1G18550 Os01g42070

Xlp2 targeting domain in some isoforms

GRMZM2G154904 (Xlp2 binds centromeres) AT1G03780 Os07g32390

GRMZM2G301194 Kinesin heavy chain AT2G47500 Os11g44880

GRMZM2G176047 Kinsesin motor protein AT1G72250, AT2G22610 Os03g02290,Os12g42160

GRMZM5G887077 Kinesin heavy chain AT5G27000 Os11g44880

GRMZM2G033785 Centromeric protein E (motor protein) AT1G59540 Os11g35090

Xklp2 like (MAP associated with

GRMZM2G091543 centrosome separation) AT5G15510 Os03g11400

Xlp2 targeting protein (Xlp2 binds

GRMZM2G100473 centromeres) AT5G15510 Os03g 11400

GRMZM5G832989 Microtubule associated protein AT1G68060 Os02g50320

GRMZM2G083475 Kinesin motor protein like AT3G45850 Os05g02670

Actin binding formin-homologyl like AT5G67470, AT3G25500

GRMZM2G040965 (cytoskeletal rearrangements) AT2G43800 Os09g34180

GRMZM2G141208 Microtubule associated protein AT4G26760 Os05g33890

GRMZM2G471186 Kinesin like AT3G23670 Os02g28850

GRMZM2G318849 Microtubule associated protein AT2G07170 Os09g38710

Actin binding formin-homologyl like

GRMZM2G132486 (cytoskeletal rearrangements) AT5G67470 Os09g34180

GRMZM2G136219 Kinesin like Os04g30720 Chromatin structure 6

GRMZM2G164020 Histone H1 AT2G30620

GRMZM5G866734 RCC AT1G19880 Os04g56720

High motility group (non-histone chromatin

GRMZM2G060253 component) AT4G23800 Os02g15810

GRMZM2G081474 Histone deacetylase AT4G38130 Os06g38470

NHP2-like protein 1 (Ribosomol family;

GRMZM2G140799 funcitonally similar to HMG proteins) AT5G20160 Os03g 13800

Regulator of Chromatin Condensationl

and Znf-FYVE domains (thought to have a

GRMZM2G170289 role in membrance trafficking) AT1G65920 Os02g45980

Cell cycle 15

GRMZM2G073003 Cyclin laZm AT5G06150 Os01g59120

GRMZM2G047143 mitotic spindle checkpoint component AT3G25980 Os04g40940

mitotic spindle checkpoint component

GRMZM2G009913 mad3 AT2G33560 Os02g 10020

GRMZM2G096184 chromosome condensation protein -like AT5G37630 Os06g 14460

GRMZM2G034647 Cyclinl AT5G06150 Os05g41390

Fizzy related WD-repeat cell cycle

GRMZM5G821639 regulatory protein AT5G 13840 Os03g03150,Os01 g 74146

syntaxin-related protein KNOLLE (role in

GRMZM2G131525 cell plate formation during cytokinesis) AT1G08560 Os03g52650

GRMZM2G073671 Cyclin B AT1G20610 Os06g511 10

Shugoshin-1 (centromeric cohesion in Pro

GRMZM2G020974 - Met transition) Os10g31930

GRMZM2G145879 CDK-activating kinase AT1G73690 Os05g32600

GRMZM2G363437 Cyclin A2 AT1G47230 Os03g41100

GRMZM2G140633 D-type cyclin AT5G65420, AT5G10440 Os09g29100

GRMZM2G079290 Phytosulfokines 2 (role in cell division) Os12g05260

replication factor C 40kDa subunit (AAA

AC235534.1 FG001 ATPase family) AT1G63160 Os04g48060

GRMZM2G178215 Cyclin A like AT1G47210, AT5G43080 Os03g 11040,Os12g39210,Os03g41 100

Cell wall related 4

Peptidoglycan binding lysin motif, erwinia

GRMZM2G168588 induced protein 1 AT2G17120 Os09g37600

Peptidoglycan binding lysin motif, erwinia

GRMZM2G154301 induced protein 1 AT1G21880 Os06g 10660

GRMZM2G004955 Expansin like family (cell wall extension) AT3G45970 Os06g50960

GRMZM2G040517 Extensin like Os05g45460

Heavy metal binding 3 GRMZM2G049693 multicopper oxidase AT4G 12420 Os08g05820

GRMZM2G155281 Metal ion binding AT5G50740 Os04g32030,Os02g30650

GRMZM2G113863 Copper chaperone (heavy metal transport) Os07g47480

Carbohydrate

metabolism 12

GRMZM2G015886 Cellulose synthase AT5G64740 Os08g06380

GRMZM2G009282 Glucosidase AT3G45940 Os06g46340

GRMZM2G127309 Glucanase AT1G09460 Os03g61780

GRMZM2G455642 endo-1 ,4-beta-glucanase AT1G23210 Os02g50040

GRMZM2G076049 endo-1 ,4-beta-glucanase AT1G70710, AT1G23210 Os06g 14540

GRMZM5G824920 Glucan endo-1 , 3-beta-glucosidase 3 AT1G66250 Os03g12140

GRMZM2G097207 glucan endo-1 , 3-beta-glucosidase 6 AT4G31140 Os08g 12800

GRMZM2G095725 beta-fructofuranosidase AT3G52600, AT2G36190 Os02g33110,Os04g33740,Os04g33720

GRMZM2G081151 beta-amyrin synthase AT2G07050, AT5G36150 Os11g35710,Os11 g18194

GRMZM2G123714 glucan endo-1 , 3-beta-glucosidase AT1G18650 Os03g54910

GRMZM2G162333 Pectinesterase AT5G47500 Os04g46740

GRMZM2G071339 Pectinesterase AT5G 19730 Os01g53990

Other metabolic

enzymes 18

GRMZM2G120085 Serine protease AT5G67360, AT2G04160 Os03g04950,Os09g26920

GRMZM2G052507 serine carboxypeptidase AT1G28110 Os11 g31980,Os04g32540

GRMZM5G879749 serine carboxypeptidase AT3G02110, AT4G30610 Os02g55130

Zf-DHHC protein (palmitoyltransferase -

GRMZM2G176270 anchors proteins in membranes) AT4G01730 Os12g16210

GRMZM5G870067 cytokinin-O-glucosyltransferase 2 AT1G22360 Os02g36830

GRMZM2G090441 Glycosyl hydrolase/chitinase AT4G19810 Os04g30770

GRMZM2G431504 Leucoanthocyanidin reductase AT1G61720 Os04g53920

GRMZM2G143625 stearoyl-ACP desaturase AT3G02610 Os04g31070

AC209819.3 FG005 Methyltransferase AT4G35160 Os12g25450

Ethylene synthesis (ACC oxidase,

GRMZM2G013448 catalyses ACC to ethylene) AT2G 19590 Os01g39860

GRMZM2G084547 peptidyl-prolyl cis-trans isomerase 1 AT2G 18040 Os04g56800

GRMZM2G064701 Omega-6 fatty acid desaturase AT3G12120 Os07g23410

GRMZM2G114140 Glycosyl hydrolase AT5G55180 Os02g53200,Os07g35480

GRMZM2G132154 Serine carboxypeptidase AT1G43780 Os11g10750 SET domain (methyltransferases, often of

G MZM2G091916 histones) AT1G26760 Os03g07260

GRMZM2G063909 Citrate synthase 4 AT2G44350 Os11g33240

GRMZM2G108032 Glycosyl hydrolase family 10 protein AT4G38650 Os03g14010

GRMZM2G122172 aldehyde dehydrogenase AT3G48000 Os01g40860

Nucleotide

metabolism 3

GRMZM2G144504 Nucleotide exchange factor AT5G05940 Os01g62990

Ribonucleotide reductase (formation of

GRMZM2G155546 deoxyribonucleotides from ribonucleotides) AT3G27060 Os06g03720

Guanine nucleotide-binding protein beta

GRMZM2G038032 subunit-like protein AT1G18080 Os05g47890

Transporters and ion

channels 4

Voltage dependent anion channel (possibly

GRMZM2G146670 mitochondrial) AT3G01280 Os09g 19734

Uracil permease (membrane transport

GRMZM2G115635 protein) AT2G34190 Os01g55500

MATE family efflux protein (transparent

GRMZM2G470075 testa like) AT3G21690, AT1G61890 Os03g37490,Os03g37640

GRMZM2G303244 Mechanosensitive ion channel AT1G53470 Os04g47320

Misc 8

GRMZM2G042604 Proteasome subunit AT5G66140 Os09g36710

GRMZM2G061314 LRR protein binding protein AT1G78230 Os03g09070

Nascent polypeptide-associated complex

GRMZM2G465333 subunit AT3G 12390 Os05g31000

Lipid transfer domain, Cortical cell

GRMZM2G094639 delineating protein AT4G 12520 Os04g46810

GRMZM2G112795 Wound inducible protein (very short) AT3G07230

GRMZM2G014499 Xylem serine proteinase 1 precursor AT5G51750 Os09g361 10

GRMZM2G168807 Major ampullate spidroin AT2G33510 Os02g31140

GRMZM2G164090 Gibberellin regulated protein 2 AT1G75750 Os05g35690

Misc domains 35

GRMZM2G153488 Defensin precursor Os02g41904

GRMZM2G112210 Dirigent-like protein Os01g24960

GRMZM2G077227 IQ domain protein AT2G26180 Os01g09790

GRMZM2G138840 WD repeat domain AT2G38630 Os01g60200

GRMZM2G000608 Armadillo repeat superfamily AT1G54385 Os11g37100

Tetratricopepeitde repeat protein (similar to

GRMZM2G050268 male sterility5 family protein) AT3G51280 Os09g36740

12.5% similarity to Medtr2g080180.1 :

Legume lectin, beta domain Replication

GRMZM2G416677 factor-A AT1G23790 Os01g01890 Tetratricopepeitde repeat protein (similar to

GRMZM2G000825 male sterility5 family protein) AT3G51280 Os09g36740

19.0% similarity to Medtr2g080180.1 :

Legume lectin, beta domain Replication

GRMZM2G115499 factor-A AT4G 13370 Os12g02710

Reticulon domain (unknown function,

GRMZM2G109159 associates with ER) AT2G20590 Os02g 19990

GRMZM2G477139 Thaumatin like protein AT4G36010 Os10g05660

GO. Biological process: cytokinesis by cell

plate formation(RCA), microtubule

cytoskeleton organization(RCA), pollen

tube growth(RCA); Molecular function:

GRMZM2G376282 structural constituent of cell wall(RCA) Os03g07810

Query vinculin family domain (PM actin

GRMZM2G000254 microfilament attachment) Os05g25620

DVL domain - suggested role in plant

GRMZM2G078164 developlment AT2G39705 Os03g 16600 nuclear localisation signal domains; weakly

similar to a homeodomain TF 51.8%

similarity to LOC_Os04g 44640.1 : ulpl

GRMZM2G465208 protease family, C-terminal catalytic dom AT5G37010 Os04g44640

Transposon protein (also annotated as an

GRMZM2G129540 asparaginase) AT5G08100 Os03g40070

GRMZM2G040359 anther-specific proline-rich protein APG AT5G41890 Os04g48800

Kelch domain protein (often associate with

GRMZM2G070553 actin) AT5G 18590 Os11g43590

Simiilar to putative bZIP (DUF630,

GRMZM2G074377 DUF632) AT1G52320 Os05g32760

GRMZM2G464011 signal-peptide; transmembrane_regions

GRMZM2G162020 IQ domain protein AT4G00820 Os08g02250

GRMZM2G002147 Armadillo repeat superfamily AT4G 15830 Os01g70560 calreticulin superfamily 25.5% simiarity to

Sb10g008510.1 : weakly similar to Putative

pollen-specific LIM dom; 15.9% similarity to

LOC_Os06g 13030.1 : OsLIM - LIM domain

GRMZM2G114113 protein, putative actin-bin AT5G17160 Os02g47130

FH2 superfamily 40.3% similarity to

POPTR_0009s05710.1 : similar to myosin- related; similar to low simi; 34.7% similarity

to Medtr4g 104600.1 : Gonadotropin, beta

chain; 34.5% similarity to AT1G51405.1 :

GRMZM2G324467 myosin-related AT1G51405 Os03g11770

GRMZM2G051738 Similar to DNA binding AT hook protein AT4G 12080 Os11g05160

62.6% similarity to Medtr1g013340.1 :

GRMZM2G012224 Galactose-binding like AT3G08030 Os03g59300

GRMZM2G476523 thaumatin-like protein AT1G73620 Os06g47600

Pfam:10497 Zinc-finger domain of

monoamine-oxidase A repressor R1 ;

39.2% similarity to Sb05g021980.1 : similar

to AT hook motif family protein, expressed;

23.3% similarity to 30205.m001596:

GRMZM2G127656 ubiquitin-protein ligase, putative Os03g50420

Proline rich extensin domain - no BLAST

GRMZM2G160466 function Os03g20350

GRMZM2G124744 Pyrophosphatase domain

GRMZM2G008764 IQ domain protein AT2G26180 Os01g51230 G1 (long sterile Iemma1)/Light sensitive

GRMZM2G459645 hypocotyls like, DUF640 AT1G07090 Os04q43580

GRMZM2G173280 Remorin, C terminal region (PM protein) AT1G30320 Os12g41940

GRMZM2G146809 Defensin precursor AT2G02120 Os02g41904

Protease inhibitor/seed storage/LTP family

GRMZM2G170969 protein Os01g68580

Other 41

Panther:21726 PHOSPHATIDYLINOSITOL

N-

ACETYLGLUCOSAMINYLTRANSFERASE SUBUNIT P (DOWN SYNDROME

CRITICAL REGION PROTEIN 5)-

GRMZM2G017536 R ELATED AT3G58650 Os03g 19080

Unknown 345aa predicted to be

GRMZM2G081521 chloroplast localization, middle reliability AT5G13100 Os01g01295

GO. Biological process: chromatin

silencing(RCA), histone H3K9

methylation(RCA), regulation of DNA

replication(RCA), regulation of G2/M

transition of mitotic cell cycle(RCA);

Cellular component: plasma

GRMZM2G107737 membrane(IDA) AT5G48310 Os02g 10490,Os02g52280

Panther:21726 PHOSPHATIDYLINOSITOL

N-

ACETYLGLUCOSAMINYLTRANSFERASE SUBUNIT P (DOWN SYNDROME

CRITICAL REGION PROTEIN 5)-

GRMZM2G115825 R ELATED AT3G58650 Os03g 19080

16.3% similarity to AT5G66230.1 :

Chalcone-flavanone isomerase family

protein; 28.6% similarity to

Medtr8g090020.1 : Sugar transporter

GRMZM2G067298 superfamily AT5G66230 Os10g42660

GO. Cellular component: chromatin(IEA),

nucleus(IEA); Molecular function:

GRMZM2G150408 nucleosomal DNA binding(IEA) Os03g04550

37.9% similarity to AT5G66230.1 :

Chalcone-flavanone isomerase family

protein; 54.4% similarity to

Medtr8g090020.1 : Sugar transporter

GRMZM2G057690 superfamily AT5G66230, AT3G51230 Os10g42660

56.9% similarity to Sb04g004570.1 : similar

GRMZM2G041310 to Dentin sialophosphoprotein-like Os02g07180

69.8% similarity to Bradi1g07270.1 : protein

GRMZM2G029314 kinase activity (Blast2GO) Os03g56070

17.5% similarity to AT1G58220.1 :

GRMZM2G023051 Homeodomain-like superfamily protein Os06g46990

GO. Biological process: histone

phosphorylation(RCA), cell

proliferation(RCA), DNA dependent DNA

replication initiation(RCA), regulation of cell

cycle(RCA), regulation of DNA

GRMZM2G120440 replication(RCA) Os02g47130

GO. Biological process: cytokinesis by cell

plate formation(RCA), histone

phosphorylation(RCA), microtubule

cytoskeleton organization(RCA), regulation

GRMZM2G177596 of cell proliferation(RCA) AT4G02800 Os04g 15800

GRMZM2G172758 GO. Biological process: Molecular Os03g04550

GRMZM2G061655 Alpha-crystallin B chain Os12g39100 GO. Biological process: cytokinesis by cell

plate formation(RCA), microtubule

GRMZM2G142609 cytoskeleton organization(RCA) AT3G52110 Os03g07810

67.0% similarity to Bradi1g07270.1 : protein

GRMZM2G050741 kinase activity (Blast2GO) AT4G 17000 Os03g56070

GRMZM5G896728 Unknown 183aa No localization prediction AT5G49170 Os05g29080

GO. Molecular function: calcium ion

GRMZM2G052412 binding(IEA)

GO. Biological process: cellular membrane

fusion(RCA), endoplasmic reticulum

unfolded protein(RCA), heat

acclimation(RCA), negative regulation of

programmed cell death(RCA), protein

targeting to membrane(RCA), regulation of

planttype hypersensitive response(RCA),

cellular response to retinoic acid(IEP),

negative regulation of transcription from

RNA polymerase II promoter(IMP),

negative regulation of transcription, DNA

dependent(IMP), pronephros

development(IMP); Cellular component:

intracellular(IEA); Molecular function:

nucleic acid binding(IEA), zinc ion

GRMZM2G397927 binding(IEA) AT5G57123 Os06g44980

GRMZM2G146143 mucin 13, cell surface associated AT1G16520 Os03g45760

GO. Biological process: response to

GRMZM2G139324 brassinosteroid stimulus(RCA) Os12g39100

Unknown (very short) 54aa predicted to be

GRMZM2G019280 mitochondrion localization, middle reliability

GRMZM2G155340 X-RAY INDUCED TRANSCRIPT 1 AT1G14630 Os08g36380

49.8% similarity to LOC_Os04g44640.1 :

ulpl protease family, C-terminal catalytic

GRMZM2G026558 domain containing protein, expressed AT5G37010 Os04g44640

GRMZM2G088543 signal-peptide; transmembrane_regions Os02g33790

28.7% similarity to Medtr3g 101900.1 : Zinc

finger, C2H2-type; 35.7% similarity to

GRMZM2G077219 AT2G30370.1 : allergen-related AT3G 13898 Os02g51950

Unknown 222aa predicted to be

mitochondrion localization, but with low

GRMZM2G062424 reliability AT1G14680 Os02g58470

GRMZM2G150859 signal-peptide; transmembrane_regions AT1G22030 Os01g65420

GRMZM2G024927 Unknown 153aa No localization prediction

52.4% similarity to LOC_Os10g40584.1 :

zinc finger family protein, putative,

expressed; 24.4% similarity to

Sb05g004260.1 : similar to Zinc finger,

GRMZM2G114893 C2H2 type, putative AT1G62520 Os10g40584

43.8% similarity to AT3G57860.1 : UV-B-

GRMZM2G089517 insensitive 4-like AT3G57860 Os02g37850

36.5% similarity to 29917.m001997:

Microtubule-associated protein futsch,

putative; 27.9% similarity to

Bradi3g02910.1 : protein serine/threonine

GRMZM2G070515 kinase activity (Blast2GO) Os09g32540

Pfam:02362 B3 DNA binding domain;

GO:0006355 regulation of transcription,

DNA-dependent; GO:0003677 DNA

GRMZM2G109480 binding AT5G42700, AT3G19184 Os03g08620 76.6% similarity to Bradi4g01260.1 : zinc

ion binding (Blast2GO); Panther: 12972

GRMZM2G300788 DOWNSTREAM NEIGHBOR OF SON AT3G54750 Os12g42700 vacuolar protein sorting 72 homolog;

putative glycoside hydrolase; Mucin related

GRMZM5G849149 29B AT1G16520 Os03g45760

GRMZM2G496319 transmembrane_regions

72.7% similarity to LOC_Os03g40780.1 :

transport protein-related, putative,

GRMZM2G466394 expressed AT1G49870 Os03g40780

Unknown 99aa predicted to be

GRMZM2G527028 mitochondrion localization, middle reliability

25.0% similarity to Bradi1g13530.1 :

microtubule plus-end binding (Blast2GO);

23.7% similarity to LOC_Os03g44760.5:

GRMZM2G300786 SWITCH1 , putative, expressed Os12g42830

Unknown 99aa predicted to be

mitochondrion localization, but with VERY

GRMZM5G862623 low reliability

Unknown 252aa predicted to be

mitochondrion localization, but with VERY

GRMZM2G011010 low reliability

Table 4: genes of particular interest

GRMZM2G123900 GRMZM2G399072

GRMZM2G471089 GRMZM2G318592

GRMZM2G132794 GRMZM2G097275

GRMZM2G163975 GRMZM2G095899

GRMZM2G151542 GRMZM2G462623

GRMZM2G039074 GRMZM2G015666

GRMZM2G178182 GRMZM2G126018

GRMZM2G045883 GRMZM5G850129

GRMZM2G140669 GRMZM2G312419

GRMZM2G472945 GRMZM2G061734

GRMZM2G178102 GRMZM2G082586

GRMZM2G131516 GRMZM2G011463

GRMZM2G469304 GRMZM2G480386

GRMZM2G172657 GRMZM2G089819

GRMZM2G374986 GRMZM2G016477

GRMZM2G002280 GRMZM2G159953

GRMZM2G119359 GRMZM2G163724

GRMZM5G893117 GRMZM2G151955

GRMZM2G150011 GRMZM2G039934

GRMZM2G136494 GRMZM2G046316

GRMZM2G028046 GRMZM2G034155

GRMZM2G098988 GRMZM2G1 14276

GRMZM2G377217 GRMZM2G028643

GRMZM2G148467 GRMZM2G059117

GRMZM2G021573 GRMZM2G478876

GRMZM2G040924 GRMZM2G344857

GRMZM2G171365 GRMZM2G087243

GRMZM2G146688 GRMZM2G133716

GRMZM2G425236 GRMZM2G061314

GRMZM2G417229 GRMZM2G1 12210

GRMZM2G069365 GRMZM2G023051

GRMZM5G887276 GRMZM2G139324

GRMZM2G098813 GRMZM2G077219

GRMZM2G111045 GRMZM2G1 14893

AC215201.3 FG008 GRMZM2G109480

GRMZM2G027068

GRMZM2G081816 GRMZM2G052102

GRMZM2G114998

GRMZM2G171852

GRMZM2G140694

GRMZM2G074032

GRMZM2G129261

GRMZM2G 143723

GRMZM2G134998

Claims

1 . A transgenic plant which over-expresses one or more maize genes (or their orthologues in any other plant species) selected from the list consisting of:

GRMZM2G016477 GRMZM2G027068 GRMZM2G045883, GRMZM2G081816, GRMZM2G052102 GRMZM2G1 14998 GRMZM2G171852, GRMZM2G140694, GRMZM2G028046 GRMZM2G136494 GRMZM2G074032, GRMZM2G129261 , GRMZM2G143723 GRMZM2G134998 GRMZM2G15001 1 , GRMZM2G132794, GRMZM2G172657 GRMZM2G123900 GRMZM2G471089, GRMZM2G132794, GRMZM2G163975 GRMZM2G151542 GRMZM2G039074, GRMZM2G178182, GRMZM2G045883 GRMZM2G140669 GRMZM2G472945, GRMZM2G178102, GRMZM2G131516 GRMZM2G469304 GRMZM2G172657, GRMZM2G374986, GRMZM2G002280 GRMZM2G1 19359 GRMZM5G8931 17, GRMZM2G15001 1 , GRMZM2G136494 GRMZM2G028046 GRMZM2G098988, GRMZM2G377217, GRMZM2G148467 GRMZM2G021573 GRMZM2G040924, GRMZM2G171365, GRMZM2G146688 GRMZM2G425236 GRMZM2G417229, GRMZM2G069365, GRMZM5G887276 GRMZM2G098813, GRMZM2G1 1 1045, AC215201 .3_FG008, GRMZM2G399072 GRMZM2G318592 GRMZM2G097275, GRMZM2G095899, GRMZM2G462623 GRMZM2G015666 GRMZM2G126018, GRMZM5G850129, GRMZM2G312419 GRMZM2G061734 GRMZM2G082586, GRMZM2G01 1463, GRMZM2G480386 GRMZM2G089819 GRMZM2G159953, GRMZM2G163724, GRMZM2G151955 GRMZM2G039934 GRMZM2G046316, GRMZM2G034155, GRMZM2G1 14276 GRMZM2G028643 GRMZM2G0591 17, GRMZM2G478876, GRMZM2G344857 GRMZM2G087243 GRMZM2G133716, GRMZM2G061314, GRMZM2G1 12210 GRMZM2G023051 GRMZM2G139324, GRMZM2G077219, GRMZM2G1 14893 and GRMZM2G109480.

The transgenic plant of claim 1 , which is a C₃ plant. 3. The transgenic plant of claim 2, which is a cereal crop such as rice, wheat, oat, or barley; or a biofuel crop.

4. The transgenic plant of any one of the preceding claims, wherein the transgenic plant comprises at least one leaf which comprises one or more ectopic veins or increased vascular density compared to a wild-type plant. 5. The transgenic plant of any one of the preceding claims, wherein the vein spacing or number within one or more leaves of the transgenic plant is altered with respect to a wild-type plant.

6. The transgenic plant of claim 5, wherein the altered vein spacing comprises any one or more of the following:

(i) uneven vein spacing with respect to said wild-type plant;

(ii) veins closer together with respect to said wild-type plant; and/or

(iii) veins separated by fewer or smaller cells than the veins of said wild- type plant.

7. The transgenic plant of any one of the preceding claims, wherein the transgenic plant has a phenotype which is indicative of Kranz anatomy.

8. The transgenic plant of any one of the preceding claims, wherein the transgenic plant of the invention over-expresses LRR repeat kinase (GRMZM2G016477) or its orthologue in any other plant species; optionally in combination with one or more of the other genes recited in claim 1 .

9. An isolated maize gene (or its orthologue in any other plant species) selected from the list consisting of: GRMZM2G016477, GRMZM2G027068,

GRMZM2G045883, GRMZM2G081816, GRMZM2G052102, GRMZM2G1 14998,

GRMZM2G171852, GRMZM2G140694, GRMZM2G028046, GRMZM2G136494,

GRMZM2G074032, GRMZM2G129261 , GRMZM2G143723, GRMZM2G134998,

GRMZM2G15001 1 , GRMZM2G132794, GRMZM2G172657, GRMZM2G123900, GRMZM2G471089, GRMZM2G132794, GRMZM2G163975, GRMZM2G151542,

GRMZM2G039074, GRMZM2G178182, GRMZM2G045883, GRMZM2G140669,

GRMZM2G472945, GRMZM2G178102, GRMZM2G131516, GRMZM2G469304,

GRMZM2G172657, GRMZM2G374986, GRMZM2G002280, GRMZM2G1 19359, GRMZM5G8931 17, GRMZM2G 15001 1 , GRMZM2G136494, GRMZM2G028046,

GRMZM2G098988, GRMZM2G377217, GRMZM2G148467, GRMZM2G021573,

GRMZM2G040924, GRMZM2G171365, GRMZM2G146688, GRMZM2G425236,

GRMZM2G417229, GRMZM2G069365, GRMZM5G887276, GRMZM2G098813, GRMZM2G1 1 1045, AC215201.3_FG008, GRMZM2G399072, GRMZM2G318592,

GRMZM2G097275, GRMZM2G095899, GRMZM2G462623, GRMZM2G015666,

GRMZM2G126018, GRMZM5G850129, GRMZM2G312419, GRMZM2G061734,

GRMZM2G082586, GRMZM2G01 1463, GRMZM2G480386, GRMZM2G089819,

GRMZM2G159953, GRMZM2G163724, GRMZM2G151955, GRMZM2G039934, GRMZM2G046316, GRMZM2G034155, GRMZM2G1 14276, GRMZM2G028643,

GRMZM2G0591 17, GRMZM2G478876, GRMZM2G344857, GRMZM2G087243,

GRMZM2G133716, GRMZM2G061314, GRMZM2G1 12210, GRMZM2G023051 ,

GRMZM2G139324, GRMZM2G077219, GRMZM2G1 14893, and GRMZM2G109480.

A vector comprising one or more of the isolated genes of claim 9.

1 1 . A method of making a transgenic plant, the method comprising introducing

(i) one or more of the genes of claim 9; or

(ii) the vector of claim 10;

into a plant such that the gene will be over-expressed.

12. The method of claim 1 1 , wherein the transgenic plant is a C₃ plant. 13. The method of claim 12, wherein the transgenic plant is a cereal crop such as rice, wheat, oat, or barley; or a biofuel crop.

14. The method of any one of claims 1 1 to 13, wherein the gene to be introduced is maize LRR repeat kinase (GRMZM2G016477) or its orthologue in any other plant species; optionally in combination with one or more of the other genes recited in claim 9.

15. A transgenic plant seed comprising one or more of the maize genes (or their orthologues in any other plant species) selected from the list consisting of:

GRMZM2G016477 GRMZM2G027068 GRMZM2G045883, GRMZM2G081816, GRMZM2G052102 GRMZM2G1 14998 GRMZM2G171852, GRMZM2G140694, GRMZM2G028046 GRMZM2G136494 GRMZM2G074032, GRMZM2G129261 , GRMZM2G143723 GRMZM2G134998 GRMZM2G15001 1 , GRMZM2G132794, GRMZM2G172657 GRMZM2G123900 GRMZM2G471089, GRMZM2G132794, GRMZM2G163975 GRMZM2G151542 GRMZM2G039074, GRMZM2G178182, GRMZM2G045883 GRMZM2G140669 GRMZM2G472945, GRMZM2G178102, GRMZM2G131516 GRMZM2G469304 GRMZM2G172657, GRMZM2G374986, GRMZM2G002280 GRMZM2G1 19359 GRMZM5G8931 17, GRMZM2G15001 1 , GRMZM2G136494 GRMZM2G028046 GRMZM2G098988, GRMZM2G377217, GRMZM2G148467 GRMZM2G021573 GRMZM2G040924, GRMZM2G171365, GRMZM2G146688 GRMZM2G425236 GRMZM2G417229, GRMZM2G069365, GRMZM5G887276 GRMZM2G098813, GRMZM2G1 1 1045, AC215201 .3_FG008, GRMZM2G399072 GRMZM2G318592 GRMZM2G097275, GRMZM2G095899, GRMZM2G462623 GRMZM2G015666 GRMZM2G126018, GRMZM5G850129, GRMZM2G312419 GRMZM2G061734 GRMZM2G082586, GRMZM2G01 1463, GRMZM2G480386 GRMZM2G089819 GRMZM2G159953, GRMZM2G163724, GRMZM2G151955 GRMZM2G039934 GRMZM2G046316, GRMZM2G034155, GRMZM2G1 14276 GRMZM2G028643 GRMZM2G0591 17, GRMZM2G478876, GRMZM2G344857 GRMZM2G087243 GRMZM2G133716, GRMZM2G061314, GRMZM2G1 12210 GRMZM2G023051 GRMZM2G139324, GRMZM2G077219, GRMZM2G1 14893 and GRMZM2G109480.

16. The transgenic seed of claim 15, wherein the transgenic seed is from a C₃ plant.

17. The transgenic seed of claim 16, wherein the transgenic seed is from a cereal crop such as rice, wheat, oat, or barley; or a biofuel crop.

18. The transgenic seed of any one of claims 15 to 17, wherein the gene is maize LRR repeat kinase (GRMZM2G016477) or its orthologue in any other plant species; optionally in combination with one or more of the other genes recited in claim 15. 19. The transgenic seed of any one of claims 15 to 18, wherein the transgenic plant seed will be capable of producing a transgenic plant which will over-express said one or more genes.

20. A shoot, root, cutting or seedling from the transgenic plant of any one of claims 1 to 8 or the seed of any one of claims 15 to 19.

21 . The transgenic plant of any one of claims 1 to 8, or the isolated gene of claim 9, or the vector of claim 10, or the seed of any one of claims 15 to 19, or the shoot, root cutting or seedling of claim 20, wherein the orthologue is Arabidopsis.