# Patent

## Title
GRF3 mutants, methods and plants

## Abstract

The present disclosure provides a novel modified gene, rGRF3, or an ortholog thereof, which is shown to be decoupled from control by miR396, particularly in the presence of over-expression of at least one GIF gene, such as GIF1, AtGIF 2, AtGIF 3, Os11g40100, Os12g31350, Os03g52320 or combinations thereof. When present in a plant, the rGRF3 results in a phenotype of increased productivity (e.g. increased yield, increased biomass, increased stress resistance, increased seed production, increased seed yield, increased root growth, increased root elongation speed, delayed leaf senescence or increased drought tolerance and combinations thereof).

## Background
The instant application contains a Sequence Listing which has been submitted via EFS-web and is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
Plants exhibiting improved productivity and/or yield phenotypes and/or increased drought tolerance by introducing into such plants mutations in the GRF3 growth factor, or in a GRF3 orthologue, which mutants deregulate the GRF3 or GRF3 orthologue from miR396 control (optionally in combination with overexpression of at least one GIF gene).
BACKGROUND OF THE INVENTION
In contrast to animals, plants continue to produce new organs throughout their life cycle. The above-ground organs are derived from the shoot apical meristem (SAM), which includes a pool of stem cells residing at the growing tip of the plant. Proliferating SAM cells produce an excess of daughter cells that are either incorporated into the developing leaf primordia at the SAM periphery or become part of the shoot. The core machinery controlling the progression of the cell cycle in plants, as well as in other eukaryotes, relies on the activity of cyclin-dependent kinases (Inze and De Veylder, 2006). Many aspects of cell cycle regulation are highly conserved among eukaryotes. It is, however, the integration of the basic cell cycle mechanisms with the developmental program that generates the enormous phenotypic variation among multicellular organisms, a process that is much less understood (Inze and De Veylder, 2006).
In contrast to the indeterminate SAM in Arabidopsis thaliana, leaves are determinate organs that have a defined morphology. Leaf development involves the concerted action of various hormone signalling pathways and transcription factor networks. Some of the major transcriptional regulators involved in the control of cell proliferation in leaves include AINTEGUMENTA (Mizukami and Fischer, 2000), PEAPOD (White, 2006), JAGGED (Dinneny et al., 2004; Ohno et al., 2004), BLADE ON PETIOLE (Ha et al., 2003), TCPs (Nath et al., 2003) and GROWTH-REGULATING FACTORs (GRFs) (Kim et al., 2003).
To obtain their characteristic final size and shape, growth of the developing leaf needs to be tightly coordinated first through cell proliferation and then by cell expansion (Piazza et al., 2005; Tsukaya, 2006). Initially, cell proliferation is observed throughout the developing leaf (Donnelly et al., 1999). Then, the cell cycle stops at the tip of the leaf and a mitotic arrest front moves towards the base of the organ (Donnelly et al., 1999). Once cells cease to divide, they begin to enlarge and cell growth becomes the driving force regulating organ size (Piazza et al., 2005; Tsukaya, 2006).
Currently, little is known about the molecular mechanisms that coordinate cell proliferation throughout a developing leaf. A known regulator is the TCP gene CINCINNATA (CIN), which controls the progression of the mitotic arrest front in snapdragon (Nath et al., 2003). Mutations such as cin (Nath et al., 2003) and triple knock-outs of its Arabidopsis homologues tcp2/4/10 (Schommer et al., 2008) cause changes in leaf morphogenesis and uneven organ curvature due to excess cell proliferation at the leaf margins. Interestingly, five Arabidopsis TCPs (TCP2, 3, 4, 10 and 24), as well as CIN, have a target site for microRNA (miRNA) miR319 (Palatnik at al., 2003). Overexpression of miR319 causes the degradation of these TCPs and the generation of crinkled leaves similar to those observed in tcp loss-of-function mutants (Palatnik et al., 2003). Mutations in the target site of the TCPs that diminish the interaction with the miRNA affect leaf morphology in Arabidopsis (Palatnik et al., 2003; Palatnik at al., 2007) and leaf complexity in tomato (Ori at al., 2007), and are lethal in extreme cases (Palatnik at al., 2003).
The GRF family of transcription factors comprises nine members in Arabidopsis (Kim et al., 2003). Seven of them have a target site for miR396 (Jones-Rhoades and Bartel, 2004). Loss-of-function mutations in different GRFs or overexpression of miR396, which decreases GRF levels, have been shown to reduce cell number in Arabidopsis leaves (Horiguchi et al., 2005; Kim et al., 2003; Kim and Kende, 2004; Liu at al., 2009). The GRFs work together with GRF-INTERACTING FACTORs (G/Fs), a small gene family encoding proteins with homology to the human SYT transcriptional co-activator (Horiguchi at al., 2005; Kim and Kende, 2004). Inactivation of GIF1 (Kim and Kende, 2004), also known as ANGUSTIFOLIA 3 (AN3) (Horiguchi at al., 2005), produces narrower leaves as a result of a reduction in cell proliferation.
It has been disclosed by Rodriguez at al., Development 137, 103-112 (2010), that a microRNA, miR396, plays a role in the coordination of cell proliferation in Arabidopsis leaves. They showed that in leaf primordia, miR396 is expressed at low levels, but its expression steadily increases during organ development. They showed that miR396 antagonizes the expression pattern of its targets, the GROWTH-REGULATING FACTOR (GRF) transcription factors. miR396 was shown to accumulate preferentially in the distal part of young developing leaves, restricting the expression of GRF2 to the proximal part of the organ. This, in turn, was shown to coincide with the activity of the cell proliferation marker CYCLINB1;1. miR396 was shown to attenuate cell proliferation in developing leaves through the repression of GRF activity and a decrease in the expression of cell cycle genes. Furthermore, they reported that over-expression of miR396 in a mutant lacking GRF-INTERACTING FACTOR 1 (GIF1) severely compromised the shoot meristem. miR396 was found to be expressed at low levels throughout the meristem, overlapping with the expression of its target, GRF2. In addition, it was shown that overexpression of miR396 can reduce cell proliferation and the size of the meristem. Arabidopsis plants with an increased activity of the transcription factor TCP4, which reduces cell proliferation in leaves, were shown to have higher miR396 and lower GRF levels. Modified GRF2, which was mutated to interfere with the interaction with miR396, was shown to be independent of miR396 regulation to which the wild-type GRF2 was subject. These plants were reported to have slightly bigger leaves than those of wild-type, however these leaves were curved downwards which could be detrimental for light capture and photosynthesis. Those results indicated that miR396 levels can significantly restrict cell proliferation in plants.
In the present disclosure, it is shown that a mutant GRF3 (sometimes referred to herein as rGRF3) and mutant GRF3 orthologues (sometimes referred to herein as rGRF3 orthologues) are relieved of miR396 regulation, and that plants comprising the mutant GRF3 or mutant GRF3 orthologues have improved productivity and/or yield (including greater leaf area, greater cell numbers, increased biomass, increased stress resistance, delayed leaf senescence, increased seed production, increased seed yield, increased root growth, increased root elongation speed and greater tolerance to drought), whether compared to wild-type plants or to plants comprising a mutant GRF2 relieved of miR396 regulation. Furthermore, the leaves from mutant GRF3 plants or mutant GRF3 orthologue plants were not curved downwards as those of mutant GRF2. The slight increase in leaf area observed in mutant GRF2 plants were caused by increasing its level at least twenty-fold compared with the level of GRF2 in wild-type plants; however, just three to five times more mutant GRF3 compared with the level of GRF3 in wild-type plants has been observed to cause a much larger impact on leaf size and plant biomass.
When the GRF3 modification or GRF3 orthologue modification is combined in a plant overexpressing GIF1, these effects are greatly enhanced.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows nucleic acid constructs and sequences of relevance to this invention; top panel shows the sequence of GRF3 wild-type sequence in the region that is substantially complementary to miR396b, showing the binding affinity (L1G=−33.9 kcal/mole)(Upper nucleotide sequence SEQ ID NO: 118, lower nucleotide sequence SEQ ID NO: 125, and amino acid sequence SEQ ID NO: 120); middle panel shows the modified GRF3 sequence (rGRF3), which includes five base changes from the wild-type sequence {an A→U, a G→A, a U→A, a G→A and a A→G modification), all of which retain the native amino acid sequence, but which substantially destabilizes the interaction with the miR396b microRNA, (reducing the L1G to −14.4 kcal/mole)(Upper nucleotide sequence SEQ ID NO: 121, lower nucleotide sequence SEQ ID NO: 126 and amino acid sequence SEQ ID NO: 120); and the bottom panel shows a graphic of a 35S:G/F1 expression construct.
FIG. 2 shows the relative expression levels of GRF3 and GIF1 in transgenic Arabidopsis plants as estimated by RT-qPCR as well as in crosses between such transgenic plants, representing GRF3 levels in wild-type plants as having a relative value of 1, it can be seen that over-expression of miR396 (under the control of the 35S promoter), reduces the GRF3 expression, while the level of expression of GRF3 in rGRF3 transgenics is approximately five-fold the level of expression of GRF3 in wild type plants. This increase of GRF3 in transgenic plants expressing the mutant version is caused by the relief of the miRNA repression.
In the cross between rGRF3 and 35S:GIF1 plants, the rGRF3 expression is slightly (but not significantly) lower than the five-fold expression level seen in the rGRF3 plants. By comparison, the expression levels of GIF1, again representing levels in wild-type plants as 1, it is not significantly altered in the 35S:miR396 expressing plants, but is almost forty times the wild-type level in both rGRF3 plants and crosses between rGRF3×GIF1 plants. The measurements are triplicates±SEM
FIG. 3 shows the modification in leaf development observed in rGRF3 plants, 35S:GIF1 plants and rGRF3×35S:GIF1 plants. In the left panel, leaf area, fresh weight and dry weight were determined for fully expanded first leaves, which show the most easily observed changes; the right panel shows leaf phenotypes of developing plants in short days, while the bottom right panel shows plants grown in large pots in short day conditions.
FIG. 4 shows, in the top panel, delayed leaf senescence of rGRF3×35S:GIF1 crossed plants; in the bottom panel, delayed leaf senescence of an individual leaf is shown for fully expanded leaf 5, which was detached and incubated in the dark (dark induced senescence). The progression of senescence was followed by measuring chlorophyll fluorescence (Fv/Fm).
FIG. 5 shows leaf area of plants transformed with the wild-type version of GRF3 (GRF3) and/or with the miR396 resistant version of GRF3 (rGRF3).
FIG. 6 shows the fresh weight (FIG. 6A) and dry weight (FIG. 6B) of rGRF3, GRF2 and 35S:miR396 plants, all in long day conditions, with the vertical axis being in units of grams.
FIG. 7 shows a neighbour joining analysis of GRFs from Arabidopsis thaliana (AtGRF#), Oryza sativa (OsGRF#), Zea mays (ZmORF#), Glycine max (GmGRF), Populus trichocarpa (PtGRF), Prunus persica (PpGRF), Medicago truncatula (MtGRF) and Carica papaya (CpCRF) shown as an unrooted cladogram. Underlined: GRFs with a miR396 binding site: Labelled with an asterisk: GRFs with a FFD conserved motif.
FIG. 8 shows the distribution of QLQ, WRC and FDD protein motifs in GRFs from Arabidopsis thaliana (AtGRF#), Oryza sativa (OsGRF#), Zea mays (ZmORF#), Glycine max (GmGRF), Populus trichocarpa (PtGRF), Prunus persica (PpGRF), Medicago truncatula (MtGRF), and Carica papaya (CpGRF)
FIG. 9 shows a neighbour joining analysis of GIF from Arabidopsis thaliana and Oryza sativa shown as an unrooted cladogram: Sequences were retrieved from PlantTFDB 2.0 (http://planttfdb.cbi.pku.edu.cn).
FIG. 10 shows the detrimental leaf-shape changes (downward “rolling”) which are found with rGRF2, but not in rGRF3.
FIG. 11 shows that a mild increase in GRF3 (3×) causes a higher increase in productivity, e.g. biomass compared with a large accumulation of GRF2 (25×).
FIG. 12 shows that rGRF3 plants display higher rates of stem growth and stem biomass accumulation. Left: elongation of a 4.5 cm long stem segment in 10 days of wild type (wt) and rGRF3 plants.
FIG. 13 shows—rosette phenotypes of short-day grown plants. Note increased leaf size and biomass accumulation with plants according to the present invention.
FIG. 14 shows drought effects in the different transgenic plants.
FIG. 15 shows Arabidopsis GRFs are expressed in proliferative tissues. Left panel: GRF3 expression pattern during leaf development (DAS=days after sowing). Right panel: GRF3 is coexpressed with mitosis-specific genes during Arabidopsis development.
FIG. 16 shows that maize GRFs are co-expressed with mitosis-specific genes.
FIG. 17 shows an increase in plant size caused by Arabidopsis miR396-resistant GRF3.
A) 30 days old plants corresponding to independent transgenic plant lines: empty vector (WT, left), miR396-resistant GRF3 (rGRF3 centre) and wild-type GRF3 (GRF3, right). Note the bigger size of the rosettes transformed with the rGRF3.
B) Fully expanded first leaf area of the different transgenic plants depicted in (A). At least 50 independent plants were scored for each vector. Bars marked with different letters are significantly different as determined by ANOVA and Duncan's multiple range test (P<0.05).
FIG. 18 shows that tissue-specific expression improves rGRF3 performance in plant productivity. Area of fully expanded first leaf of transgenic plants expressing rGRF3 from different promoters: GRF3, ASYMMETRIC LEAVES 1 (AS1) or AINTEGUMENTA (ANT). At least 50 plants were scored for each vector. For AS1:rGRF3 and ANT:rGRF3 the data represent independent primary transgenics, whereas for GRF3:rGRF3 a representative line was used. Bars marked with different letters are significantly different as determined by Kruskal-Wallis and Dunn's multiple range test (P<0.05).
FIG. 19 shows an increase in stem diameter due to rGRF3. Stem diameter of transgenic plants expressing rGRF3 from different promoters: GRF3, ASYMMETRIC LEAVES 1 (AS1) and AINTEGUMENTA (ANT). Bars marked with different letters are significantly different as determined by Kruskal-Wallis and Dunn's multiple range test (P<0.05).
FIG. 20 shows uncoupling of effects on leaf size from those on timing of leaf-senescence using tissue specific promoters. As shown herein GRF3:rGRF3 increases leaf size and delays leaf senescence. The latter effect can be decoupled from the increase in leaf size if desired. Expression of rGRF3 from ANT and AS1 promoters significantly increased leaf size with a minor effect on leaf senescence. Dark-induced senescence of fully expanded leaf #5. Pictures were taken immediately after the full expanded leaves were cut from the rosette (Day 1) and after they were incubated 6 days in darkness (Day 6). For GRF3:rGRF3 a representative line was used, and for AS1:rGRF3 and ANT:rGRF3 vector the 4 primary transgenic plants with the biggest leaf area were selected.
FIGS. 21A to 212I show the nucleotide sequences of the Arabidopsis thaliana GRFs of which there are 9, namely: FIG. 21A, AtGRF1 (SEQ ID No. 40); FIG. 21B, AtGRF2 (SEQ ID No. 87); FIG. 21C, AtGRF3 (SEQ ID No. 2); FIG. 21D, AtGRF4 (SEQ ID No. 19); FIG. 21E, AtGRF5 (SEQ ID No. 41); FIG. 21F, AtGRF6 (SEQ ID No. 42); FIG. 21G, AtGRF7 (SEQ ID No. 43); FIG. 21H, AtGRF8 (SEQ ID No. 44), and; FIG. 21I, AtGRF9 (SEQ ID No. 45). The underlined section of the sequences represent the portion of the nucleotide sequence encoding the WRC (Trp, Arg, Cys) domain; and the underlined and bolded section of the sequences represent the miR396 target site, if one is present.
FIGS. 22A to 22I shows the amino acid sequences of the A. thaliana GRFs of which there are 9: FIG. 22A, AtGRF1 (SEQ ID No. 46); FIG. 22B, AtGRF2 (SEQ ID No. 47); FIG. 22C, AtGRF3 (SEQ ID No. 20); FIG. 22D, AtGRF4 (SEQ ID No. 21); FIG. 22E, AtGRF5 (SEQ ID No. 48); FIG. 22F, AtGRF6 (SEQ ID No. 49); FIG. 22G, AtGRF7 (SEQ ID No. 50); FIG. 22H, AtGRF8 (SEQ ID No. 51), and; FIG. 22I, AtGRF9 (SEQ ID No. 52), the underlined section of the sequences represent the portion of the amino acid sequence known as the WRC (Trp, Arg, Cys) domain; and the underlined and bolded section of the sequences represent the FFD motif.
FIGS. 23A to 23L show the nucleotide sequences of the Oryza sativa (rice) GRFs of which there are 12: FIG. 23A, OsGRF1 (SEQ ID No. 3); FIG. 23B, OsGRF2 (SEQ ID No. 4); FIG. 23C, OsGRF3 (SEQ ID No. 5); FIG. 23D, OsGRF4 (SEQ ID No. 6); FIG. 23E, OsGRF5 (SEQ ID No. 53); FIG. 23F, OsGRF6 (SEQ ID No. 54); FIG. 23G, OsGRF7 (SEQ ID No. 55); FIG. 23H, OsGRF8 (SEQ ID No. 56); FIG. 23I, OsGRF9 (SEQ ID No. 57); FIG. 23J, OsGRF10 (SEQ ID No. 58); FIG. 23K, OsGRF11 (SEQ ID No. 59), and; FIG. 23L, OsGRF12 (SEQ ID No. 60). The underlined and bolded section of the sequences represent the miR396 target site, if one is present.
FIGS. 24A-24L show the amino acid sequences of the Oryza sativa (rice) GRFs of which there are 12: FIG. 24A, (OsGRF1 (SEQ ID No. 22); FIG. 24B, OsGRF2 (SEQ ID No. 23); FIG. 24C, OsGRF3 (SEQ ID No. 24); FIG. 24D, OsGRF4 (SEQ ID No. 25); FIG. 24E, OsGRF5 (SEQ ID No. 61); FIG. 24F, OsGRF6 (SEQ ID No. 62); FIG. 24G, OsGRF7 (SEQ ID No. 63); FIG. 24H, OsGRF8 (SEQ ID No. 64); FIG. 24I, OsGRF9 (SEQ ID No. 65); FIG. 24J, OsGRF10 (SEQ ID No. 66); FIG. 24K, OsGRF11 (SEQ ID No. 67), and; FIG. 24L, OsGRF12 (SEQ ID No. 68).
FIGS. 25A to 25N show the nucleotide sequences of the Zea mays (maize) GRFs of which there are 14; FIG. 25A, ZmGRF1 (SEQ ID No. 7); FIG. 25B, ZmGRF2 (SEQ ID No. 69); FIG. 25C, ZmGRF3 (SEQ ID No. 8); FIG. 25D, ZmGRF4 (SEQ ID No. 70); FIG. 25E, ZmGRF5 (SEQ ID No. 9); FIG. 25F, ZmGRF6 (SEQ ID No. 10); FIG. 25G, ZmGRF7 (SEQ ID No. 11); FIG. 25H, ZmGRF8 (SEQ ID No. 71); FIG. 25I, ZmGRF9 (SEQ ID No. 12); FIG. 25J, ZmGRF10 (SEQ ID No. 72); FIG. 25K, ZmGRF11 (SEQ ID No. 13); FIG. 25L, ZmGRF12 (SEQ ID No. 73); FIG. 25M, ZmGRF13 (SEQ ID No. 74), and; FIG. 25N, ZmGRF14 (SEQ ID No. 14). The underlined and bolded section of the sequences represent the miR396 target site, if one is present.
FIGS. 26A to 26N show the amino acid sequences of the Zea mays (maize) GRFs of which there are 12; FIG. 26A, ZmGRF1 (SEQ ID No. 26); FIG. 26B, ZmGRF2 (SEQ ID No. 75); FIG. 26C, ZmGRF3 (SEQ ID No. 27); FIG. 26D, ZmGRF4 (SEQ ID No. 76); FIG. 26E, ZmGRF5 (SEQ ID No. 28); FIG. 26F, ZmGRF6 (SEQ ID No. 29); FIG. 26G, ZmGRF7 (SEQ ID No. 30); FIG. 26H, ZmGRF8 (SEQ ID No. 77); FIG. 26I, ZmGRF9 (SEQ ID No. 31); FIG. 26J, ZmGRF10 (SEQ ID No. 78); FIG. 26K, ZmGRF11 (SEQ ID No. 32); FIG. 26L, ZmGRF12 (SEQ ID No. 79); FIG. 26M, ZmGRF13 (SEQ ID No. 80), and; FIG. 26N, ZmGRF14 (SEQ ID No. 33).
FIG. 27 shows the nucleotide sequence for a GRF with high similarity to AtGRF3, namely Glycine max (soybean) GRF (GmGRF) (SEQ ID No. 16). The underlined and bolded section of the sequences represent the miR396 target site, if one is present.
FIG. 28 shows the nucleotide sequence for a GRF with high similarity to AtGRF3, namely Medicago truncatula GRF (MtGRF) (SEQ ID No. 17).
FIG. 29 shows the nucleotide sequence for a GRF with high similarity to AtGRF3, namely Populus trichocarpa GRF (PtGRF) (SEQ ID No. 18).
FIG. 30 shows the nucleotide sequence for a GRF with high similarity to AtGRF3, namely Prunus persica GRF (PpGRF) (SEQ ID No. 15).
FIG. 31 shows the amino acid sequence for a Medicago truncatula GRF (MtGRF) (SEQ ID No. 36); the underlined section of the sequences represent the portion of the amino acid sequence known as the WRC (Trp, Arg, Cys) domain; and the underlined and bolded section of the sequences represent the FFD motif.
FIG. 32 shows the amino acid sequence for a Glycine max (soybean) GRF (GmGRF) (SEQ ID No. 35); the underlined section of the sequences represent the portion of the amino acid sequence known as the WRC (Trp, Arg, Cys) domain; and the underlined and bolded section of the sequences represent the FFD motif.
FIG. 33 shows the amino acid sequence for a Populus trichocarpa GRF (PtGRF) (SEQ ID No. 37); the underlined section of the sequences represent the portion of the amino acid sequence known as the WRC (Trp, Arg, Cys) domain; and the underlined and bolded section of the sequences represent the FFD motif.
FIG. 34 shows the amino acid sequence for a Prunus persica GRF (PpGRF) (SEQ ID No. 34); the underlined section of the sequences represent the portion of the amino acid sequence known as the WRC (Trp, Arg, Cys) domain; and the underlined and bolded section of the sequences represent the FFD motif.
FIG. 35 shows the nucleotide sequence for the Arabidopsis GRF3 with a mutated miR396-target site (At-rGRF3) (SEQ ID No. 81); the shaded and underlined portion of the sequence is the mutated miR396-target site. The lower case refers to base substitutions to make the GRF resistant to miR396. For the avoidance of doubt when the mutant AtGRF3 is referred to herein unless stated otherwise it is this sequence that is being referred to. This sequence is also referred to herein as At-rGRF3 and rGRF3. This mutated At-rGRF3 was used herein to generate inter alia transgenic Arabidopsis plants.
FIG. 36 shows the nucleotide sequence for a Glycine max GRF with a mutated miR396-target site (Gm-rGRF) (SEQ ID No. 82); the shaded and underlined portion of the sequence is the mutated miR396-target site. The lower case refers to base substitutions to make the GRF resistant to miR396. This mutated Gm-rGRF was used herein to generate transgenic Arabidopsis plants.
FIG. 37 shows the nucleotide sequence for an Oryza sativa GRF4 with a mutated miR396-target site (Os-rGRF4.1) (SEQ ID No. 83); the shaded and underlined portion of the sequence is the mutated miR396-target site. The lower case refers to base substitutions to make the GRF resistant to miR396. This mutated Os-rGRF4 was used herein to generate transgenic Arabidopsis plants. This sequence is also referred to herein as Os-rGRF4.1 and rOsGRF4.1.
FIG. 38 shows similarity tables between At-GRF3 and GRFs from other plant species based on primary amino acid sequence. The global similarity between GRF3 and every GRF from At, Os and Zm (plus other highly similar GRFs from selected species) was scored using Needle (EMBOSS: http://www.ebi.ac.uk/Tools/psa/). Identity relates to when an identical amino acid is in the corresponding position; whereas similarity relates to when a conservative substitution of an amino acid is found in a corresponding position.
FIGS. 39A to 39G show the nucleotide sequence encoding JD16_GIF1 (including 35S promoter (nt 427-1295)—underlined section; GIF1 coding Sequence (nt 1310-1942)—section in italics and bold; and Terminator (nt 2106-2755)—section in bold and underline.
FIGS. 40A to 40H show the nucleotide sequence encoding RER32 GRF3 (SEQ ID No. 85) (including GRF3 Promoter (427-1707)—underlined section; 5′UTR (1708-1913)—lower case and italics; GRF3 Coding Sequence+Introns [in lower case] (1914-4231)—italics & bold; 3′UTR (4232-4454)—lower case and italics; and Terminator (4455-5105)—section in bold and underlined.
FIG. 41 shows a map of the vector 35S:GIF1 (JD16) (SEQ ID No. 84)—Vector size: 11332 pb Digest with bamHI and SalI. Products: 10682 and 650 pb.
FIG. 42 shows a map of the vector GRF3:GRF3r (RER32)—Vector size: 13642 pb Digest with XbaI and SalI. Products: 11962 and 1680 pb.
FIG. 43 shows that overexpression of GIF1, GIF2, and GIF3 promotes cell proliferation and leaf size and that GIF2 and GIF3 proteins are functional equivalents of GIF1 (see FIG. 43 in combination with FIG. 9).
FIG. 44 shows the maps of the two plasmids comprising rGRF3:GIF1 in pBRACT114. pBRACT114 is available from www.bract.org. The pBRACTs are based on the pGreen/pSoup vector system and the original reference for pGreen is: Hellens et al 2000.
FIG. 45 shows delayed leaf senescence in primary transgenic Arabidopsis plants by a mutated Arabidopsis GRF (At-rGRF3) and by a mutated soybean GRF (Gm-rGRF).
FIG. 46 shows that expression in Arabidopsis of GRF3 orthologues from soybean and from rice, when decoupled from miR396 regulation also increase plant biomass. The area of fully expanded first leaf of transgenic plants expressing GRF from Arabidopsis, soybean or rice was measured.
FIG. 47 shows the nucleotide sequence for a GRF with high similarity to AtGRF3, namely Carica papaya GRF (CpGRF) (SEQ ID No. 88).
FIG. 48 shows the amino acid sequence for a Carica papaya GRF (CpGRF) (SEQ ID No. 89); the underlined section of the sequences represent the portion of the amino acid sequence known as the WRC (Trp, Arg, Cys) domain; and the underlined and bolded section of the sequences represent the FFD motif.
FIG. 49 shows data comparing stem width 10 cm above soil level at flowering and maximum stem width at flowering in Brassica oleracea plants transformed with Arabidopsis rGRF3 and control plants (without the At rGRF3).
FIG. 50 shows expression of rGRF3 from tissue-specific promoters.
A) Top: Schematic representation of a construct expressing GRF3 as a translational fusion to GFP. Bottom: Expression pattern of GRF3-GFP fusion protein in leaves of different ages collected from GRF3:GRF3-GFP and GRF3:rGRF3-GFP plants. B) Expression level of GRF3 mRNA in apex and leaves of different ages. C) GUS staining of plants transformed with ANT:GUS and AS1:GUS reporters. Upper part, schematic representation of the reporters.
FIG. 51 shows the expression levels of rGRF3 under tissue-specific promoters and leaf area of transformants.
A) Expression levels of GRF3 in transgenic seedlings expressing GRF3 from different promoters. Determinations were carried out by RT-qPCR and normalized to wild-type plants. B) Area of fully expanded first and second leaves. C) Fully expanded first (left) and third (right) leaves.
FIG. 52 shows pictures of 40 day old plants expressing rGRF3 from their endogenous promoters and from the ANT and AS1 promoters.
FIG. 53 shows delayed senescence when rGRF3 is expressed under the control of its own promoter. Senescence is evident in wild-type and when rGRF3 is expressed under the control of AS1 and/or ANT.
A) Pictures of 50 day old rosettes. Note the delayed senescence of GRF3: rGRF3 plants and the normal development of AS1:GRF3 and ANT:GRF3. B) Senescence of an individual leaf is shown for fully expanded leaf 5, which was detached and incubated in the dark (dark induced senescence). The progression of senescence was quantitated by determining Fv/Fm
FIG. 54 shows leaf area plotted for independent primary transgenic plants. CHF3 is an empty vector control, rGRF3 with the FFD motif is the rGRF3 cDNA expressed from its own promoter. rGRF3 AAD is the cDNA of rGRF3 with three mutations in the FFD motif (FFDDW) that replace the two phenylalanine amino acids and the tryptophan with three alanine amino acids (AADDA).
FIG. 55 shows a comparison between plants expressing rGRF2 and rGRF3. As shown herein rGRF3 expression leads to the production of bigger plants than wild-type or rGRF2 expression. rGRF2 also generates distorted rosettes.
FIG. 56 is a table showing widest stem width at flowering and 10 cm stem weight for Brassica oleracea transformants expressing rGRF3 and control plants (TC).
FIG. 57 left, a graph showing root length at various days after sowing for wild-type Brassica oleracea, and two transgenic Brassica oleracea plants expressing rGRF3. Right, a graph showing root elongation speed for wild-type or two transgenic plants expressing rGRF3.



SUMMARY OF THE INVENTION
The present invention is predicated upon the surprising finding that a novel modified GRF3 gene, rGRF3, which is shown to be decoupled from control by miR396, particularly in the presence of over-expression of GIF1, can be used to significantly improve the biomass, improve stress resistance, improve drought tolerance, delay leaf senescence in plants. The improvement in biomass accumulation is surprisingly high and unexpectedly better than the only other reported miRNA decoupled GRF, namely rGRF2, while the tolerance to drought is unexpected from previously reported data.
The present inventors have also surprisingly found that orthologues of GRF3 which are also modified to be decoupled from control by miR396 also provide these surprising and unexpected effects.
In a first aspect there is provided an isolated nucleic acid encoding a modified growth regulatory factor (GRF)-3 or an orthologue thereof which nucleic acid is decoupled from control by miR396.
In another aspect there is provided a construct comprising the nucleic acid according to the present invention operably linked with a promoter and a terminator.
The present invention further provides a vector comprising the nucleic acid of the present invention or the construct according to the present invention.
In a further aspect the present invention provides a plant, plant cell or plant tissue comprising the nucleic acid according to the present invention, the construct according to the present invention or the vector according to the present invention.
In yet another aspect there is provided a method for using the nucleic acid according to the present invention which comprises introducing said nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention into a plant.
In another aspect of the present invention there is provided nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention for use in the manufacture of a plant with increased productivity and/or yield (including for example increased biomass, increased stress resistance, increased drought tolerance, increased seed production, increased seed yield, increased root growth, increased root elongation speed, delayed leaf senescence and combinations thereof).
In another aspect of the present invention there is provided a method of producing a plant with increased productivity and/or yield (including for example one or more of increased biomass, increased stress resistance, increased drought tolerance, delayed leaf senescence, increased seed production, increased seed yield, increased root growth, increased root elongation speed and combinations thereof) comprising transforming the plant with nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention.
A further aspect provides the use of a nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention in the manufacture of a plant for increasing productivity and/or yield (for example one or more of increasing biomass, increasing stress resistance, increasing drought tolerance, delaying leaf senescence, increasing seed production, increasing seed yield, increasing root growth, increasing root elongation speed or combinations thereof).
In another aspect the present disclosure provides a novel modified gene, rGRF3, which is shown to be decoupled from control by miR396, particularly in the presence of over-expression of GIF1.
Accordingly, it is an object of this invention to provide a novel modified GRF3 gene or a novel modified GRF3 orthologue gene.
It is a further object of this invention to provide novel plants comprising a modified GRF3 gene or a modified GRF3 orthologue gene.
It is a further object of this invention to provide novel plants comprising a modified GRF3 or a modified GRF3 orthologue in the presence of over-expression of GIF1.
It is yet a further object of this invention to provide a method for using the modified GRF3 or modified GRF3 orthologue disclosed herein.
It is a further object of this invention to provide a method for producing plants with a phenotype of increased productivity and/or yield (for example a phenotype of delayed leaf senescence, increased biomass, increased stress response, increased drought tolerance, increased seed production, increased seed yield increased root growth, increased root elongation speed or combinations thereof), as compared with either wild-type plants or plants comprising a modified GRF2 (rGRF2).
A further object of the present invention is to provide plants with a phenotype of increased productivity and/or yield (for example a phenotype of delayed leaf senescence, increased biomass, increased stress response, increased drought tolerance, increased seed production, increased seed yield increased root growth, increased root elongation speed or combinations thereof) without adverse side effects observed in plants expressing modified GRF2 (rGRF2), such as detrimental leave shape changes, e.g. curved leaves or downwardly rolling leaves.
Further objects and advantages of this invention will be appreciated by referring to the entire disclosure provided herein, and the appended claims.
DETAILED DISCLOSURE OF THE PREFERRED EMBODIMENTS OF THE INVENTION
Rodriguez et al. (2010) followed the expression pattern of miR396 directly using small RNA blots and in situ hybridization in apices, and indirectly through the differential expression of wild-type and miRNA-resistant GRF2-GUS reporters. miR396 was expressed at low levels in the meristem and leaf primordia, and then it steadily accumulated with the development of the leaf. In contrast, the GRFs, which are highly expressed in the SAM and young leaves, decreased during leaf development, in concert with the retreat of cell proliferation.
Temporal antagonistic patterns of expression have been observed for miR156 and miR172 and their targets, the SPL and AP2-like transcription factors, respectively (Chuck et al., 2007; Wu and Poethig, 2006). The heterochronic miR156 and miR172 networks correspondingly regulate juvenile to adult, and vegetative to reproductive phase transitions, which require decisions implicating the whole plant (Aukerman and Sakai, 2003; Chen, 2004; Chuck et al., 2007; Schmid et al., 2005; Wu and Poethig, 2006). The observations on miR396 indicated that this miRNA is also involved in the coordination of developmental events in plants; however, its role would be restricted to individual organs.
The Arabidopsis developmental program directs a basiplastic pattern, whereby leaf maturation begins at the tip and then proceeds towards the base of the organ (Donnelly et al., 1999). Cell division occurs first throughout the primordia and then a mitotic arrest front moves from the tip to the base of the leaf, so that cells in the distal part of the leaf stop cycling and begin to expand, while cells at the base continue to proliferate (Donnelly et al., 1999). Rodriguez et al.'s results showed that the distal part of the leaf accumulates more miR396 and a gradient of miRNA activity proceeds towards the base of the organ. That result was supported by small RNA blots and the observed retreat of the wild-type GRF2-GUS reporter, which then matched the pattern of a CYCB1;1 reporter. Those observations prompted those authors to implicate the repression of GRF expression by miR396 as a component of the mitotic arrest front.
Similar spatial patterns of expression for GRF2 mRNA and miR396 in the meristem and leaf primordial have been observed, indicating that there is co-expression of the miRNA and its target at this early stage. The situation was different, however, at later stages of leaf development. The wild-type GRF2-GUS reporter was active only in the proximal part of young developing leaves, whereas the rGRF2-GUS reporter was expressed throughout the leaf. This qualitative change in the expression of wild-type GRF2-GUS was paralleled by a large increase in miR396, whose levels change by up to 10-30-fold in leaves with different developmental ages. Interestingly, the decrease in GRF expression occurred before miR396 reached its maximum level, indicating that a partial increase in the miRNA is sufficient to repress the GRFs in vivo; however, it cannot be ruled out that additional factors that act in concert with miR396 may participate in this process.
It has been proposed that miRNAs could have both qualitative effects, leading to complete elimination of their targets, and more subtle quantitative effects (Bartel and Chen, 2004). In plants, these quantitative interactions have been proposed for miR169 (Cartolano et al., 2007) and miR156 (Wang et al., 2008), miR319 (Ori et al., 2007) and miR164 (Baker et al., 2005; Nikovics et al., 2006), and their targets. From a mechanistic point of view, it is tempting to speculate that miR396 has dual functions during leaf development: it might quantitatively regulate GRF expression in the SAM and leaf primordia, while causing a large qualitative effect contributing to the clearance of GRF activity from older organs. This latter functional role in clearing GRF transcripts might explain the continued rise in miR396 levels, even after cell proliferation has ceased. On the other hand, the potential quantitative regulation of GRF activity during early leaf development might play a relevant role in the fine-tuning of cell proliferation, it has been shown that modifications of the balance between miR396 and GRF2 levels have important consequences for the final number of cells in the organ.
miR396 was first identified because of its conservation between A. thaliana and rice (Jones-Rhoades and Bartel, 2004). miR396 and GRFs with an miR396 target site are present in many plant species (Axtell and Bartel, 2005; Jones-Rhoades and Bartel, 2004), indicating an ancient origin for the miR396-GRF regulatory network. The function of the GRFs as regulators of cell number in leaves is well established based on the phenotypes of grf (Horiguchi et al., 2005; Kim et al., 2003; Kim and Lee, 2006) and gif (Horiguchi et al., 2005; Kim and Kende, 2004) mutants, and plants with high miR396 levels (Liu et al., 2009).
Rodriguez et al. (2010) extended these observations and found that the GRFs regulate cell proliferation in the SAM, which at least partially explains the lack of a functional meristem in an3-1 mutants overexpressing miR396 (this study) and in grf multiple knock-outs (Kim et al., 2003; Kim and Lee, 2006). Analysis of the transcriptome of moderate miR396 overexpressers has shown that the downregulation of mitosis-specific genes is one of the main molecular effects of high miR396 levels. However, the GRFs themselves do not change their expression during the cell cycle (Menges et al., 2005) and future work will be required to identify the mechanisms underlying the activity of the GRFs.
Measurements of the GRFs by RT-qPCR indicated that miR396 targets and non-targets are turned off at similar stages of leaf development, and that they act redundantly. Previous studies in which promoters have been fused directly to a GUS reporter have shown that the transcription of the GRF genes can occur in different regions of the leaf (Horiguchi et al., 2005). Rodriguez et al. observed that the post-transcriptional control of GRF2 by miR396 contributes significantly to its final expression pattern, and concluded that it is possible that the miRNA also plays a key role in adjusting the expression of other GRFs.
The snapdragon TCP gene CIN has been shown to be expressed in a dynamic pattern during leaf development and to regulate cyclin expression (Nath et al., 2003). CIN-like genes from Arabidopsis, which are regulated by miR319, have also been implicated in the coordination of cell proliferation and differentiation in leaves (Efroni et al., 2008; Koyama et al., 2007; Masuda et al., 2008; Palatnik et al., 2003; Schommer et al., 2008). An increase of TCP4 levels due to mutations that impair the interaction with miR319 produces smaller leaves (Efroni et al., 2008; Palatnik et al., 2003; Schommer et al., 2008).
Rodriguez et al. observed that plants expressing miR319-resistant forms of TCP4 induced miR396. As the quantitative balance between miR396 and the GRFs regulates cell number in leaves, the increase in miR396 caused by TCP4 might be responsible for at least part of the reduction in cell number in soj8 mutants. They observed, however, that the increase in TCP4 levels also caused a reduction in the GRFs that were not regulated by miR396 and GIF1, indicating an effect at the transcriptional level. Regulatory circuits in which a transcription factor causes both the transcriptional repression of target genes and the induction of an miRNA that in turn post-transcriptionally inhibits the same group of genes are well described in animals, where they are referred to as coherent feed-forward loops (Hornstein and Shomron, 2006).
miR319 overexpressers (Efroni et al., 2008; On et al., 2007; Palatnik et al., 2003) and tcp knock-outs (Nath et al., 2003; Schommer et al., 2008) have large changes in leaf morphology, as well as other phenotypic defects, such as a delay in flowering time (Palatnik et al., 2003). This indicates that the TCPs have functions that go beyond leaf development. However, it may be possible that the miR319-regulated TCPs recruit the miR396 network as part of their biological function. Rodriguez et al. proposed that the miR396 network could be a link between different developmental inputs or environmental stimuli and the components of the cell cycle machinery.
In this disclosure, the effects in plants of mutating GRF3 (and orthologues thereof) to produce a novel molecule, rGRF3 (or orthologues thereof), in a manner analogous to that for GRF2 reported by Rodriguez et al. are shown. Surprisingly, however, it is reported here that the result is a plant with a pronounced increase in productivity and/or yield (for example with a pronounced increase in biomass, increased stress response, delayed leaf senescence, increased seed production, increased seed yield, increased root growth, increased root elongation speed and/or increased tolerance to drought), whether compared to plants with wild-type (e.g. non-mutated) GRF3, wild type GRF2 or the mutated GRF2 (rGRF2) described in Rodriguez et al.
In addition, it is shown that where at least one GIF (e.g. GIF1) is overexpressed in the presence of the mutated GRF3 (rGRF3) or an orthologue thereof, these effects are enhanced.
Furthermore, the leaves from mutant GRF3 plants and/or mutant GRF3 orthologue plants were not curved downwards as those of mutant GRF2 (rGRF2) reported in Rodriguez et al.
A slight increase in leaf area can be observed in rGRF2 plants if its level is increased to at least twenty times the level of GRF2; however, a much larger impact on productivity (for example leaf size and plant biomass) can be seen in rGRF3 plants and rGRF3-orthologue plants with only three to five times more GRF3 or GRF3-orthologue.
Thus, per this disclosure, as shown in detail in the examples and experimental methods provided below, rGRF3 or orthologues thereof is/are produced comprising several synonymous mutations in the nucleic acid sequence—i.e. there is no change in the amino acid sequence of GRF3.
The result is a plant in which the repression otherwise achieved by miR396 is uncoupled from the rGRF3, and plants with increased productivity and/or yield (including with increased biomass, increased stress resistance, delayed leaf senescence and increased drought tolerance or combinations thereof) are thereby producible.
In a first aspect there is provided an isolated nucleic acid encoding a modified growth regulatory factor (GRF)-3 or an orthologue thereof which nucleic acid is decoupled from control by miR396.
The nucleic acid may be decoupled from control by miR396 by mutating the miR396 target site.
Preferably the mutated or modified nucleic acid is only modified in the miR396 target site, e.g. with the remainder of the gene being unmodified or not being mutated.
In a preferred embodiment, the modified nucleic acid is modified in such a way as to comprise conserved nucleic acid changes. In other words, the nucleic acid is modified such that there is no change in the amino acid sequence of the GRF3 or the GRF3 orthologue expressed by the nucleic acid.
The modification to the nucleic acid essentially decouples the nucleic acid (e.g. gene) from control by miR396.
Preferably the nucleic acid is decoupled from control by miR396 by mutating the nucleic acid in the miR396 target site.
Preferably the nucleic acid according to the present invention encodes a protein having the FFD motif.
In some embodiments preferably the nucleic acid according to the present invention encodes a protein having the FFD(D/E)WP motif.
For the avoidance of doubt “(D/E)” means that at that position there is either a D or an E residue. In other workds, FFD(D/E)WP (SEQ ID NO: 117) means FFDDWP (SEQ ID NO: 127) or FFDEWP (SEQ ID NO: 128).
In order to determine whether a GRF is a GRF3-orthologue in accordance with the persnt invention one may look for GRFs which encode a protein having the FFD, (e.g. FFD(D/E)WP) (SEQ ID NO: 117) motif.
GRF3-orthologues in accordance with the present invention will be GRFs which at least comprise a miR396 target site.
Suitably the miR396 target site (e.g. in the nucleic acid according the present invention, such as in the GRF3 gene or in the GRF3-orthologue gene) may have, comprise or consist of the following nucleotide sequence CGTTCAAGAAAGCCTGTGGAA (SEQ ID No. 1). In some embodiments this nucleotide sequence may be considered the wild-type miR396 target site sequence.
The GRF3-orthologue according to the present invention is preferably one or more of the following GRFs selected from the group consisting of: Arabidopsis thaliana GRF4; Oryza sativa GRF 1, 2, 3, 4, or 5; Zea mays GRF 1, 3, 5, 6, 7, 9, 11 or 14; Glycine max GRF; Medicago truncatula GRF; Populus trichocarpa GRF, Carica papaya GRF and Prunus persica GRF which have been decoupled from control by miR396.
In one embodiment, the GRF3-orthologues are ones which cluster with AtGRF3 in the cladogram depicted in FIG. 7. It has been found that these GRF3-orthologues function similarly to AtGRF3.
For the avoidance of doubt GRFs which cluster with either AtGRF2 or AtGRF9 are not of interest in the present application as it has been found that these GRFs do not function like AtGRF3.
A GRF3-orthologue in accordance with the present invention is one which has the same functionality as AtGRF3.
The term “orthologue” as used herein means genes of similar or same function but occurring in different species.
As shown in FIG. 7 the GRF3-orthologue may be preferably one that comprises a miR396 target site and which encodes for a protein having the FFD (e.g. FFD(D/E)WP) (SEQ ID NO: 117) motif.
The GRF3-orthologues in accordance with the present invention will be GRFs which at least comprise a miR396 target site.
The present invention relates to isolated nucleic acid according to any one of the preceding claims comprising i) a nucleotide sequence shown as SEQ ID No. 2 (AtGRF3); ii) or a nucleotide sequence which is at least 45%, preferably at least 50%, preferably at least 60%, preferably at least 65%, identical to SEQ ID No. 2; or iii) a nucleotide sequence which hybridises under stringent conditions with a nucleotide sequence of either i) or ii) wherein the nucleotide sequence comprises a modification in the miR396 target site to decouple the nucleic acid from control by miR396.
The isolated nucleic acid according to the present invention may comprise i) a nucleotide sequence shown as SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, or SEQ ID No. 19; ii) or a nucleotide sequence which is at least 45%, preferably at least 50%, preferably at least 60%, preferably at least 65%, identical to SEQ ID No. SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, or SEQ ID No. 19; or iii) a nucleotide sequence which hybridises under stringent conditions with a nucleotide sequence of either i) or ii) wherein the nucleotide sequence of i), ii) or iii) comprises a modification in the miR396 target site to decouple the nucleic acid from control by miR396.
The isolated nucleic acid according the present invention may comprise i) a nucleotide sequence encoding a polypeptide shown herein as SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37; ii) or a nucleotide sequence having at least 45%, preferably at least 50%, preferably at least 60%, preferably at least 65%, identity with the nucleotide sequence of i); or iii) a nucleotide sequence which hybridises under stringent conditions with a nucleotide sequence of either i) or ii) wherein the nucleotide sequence of i), ii) or iii) comprises a modification in the miR396 target site to decouple the nucleic acid from control by miR396.
Preferably the nucleic acid decoupled of miR396 control according to the present invention exhibits further enhancement in the presence of over-expression of at least one GIF gene (e.g. GIF1).
Over-expression of at least one GIF (e.g. GIF1) may be accomplished by transforming a plant, or a plant cell, or a plant tissue, with a construct comprising at least one GIF (e.g. GIF1) encoding sequence operably linked to a promoter.
In one embodiment the plant, plant cell or plant tissue comprises at least two, e.g. 2 or 3, over-expressed GIF genes.
The GIF gene in accordance with the present invention may be any suitable GIF gene, including AtGIF1 (sometimes referred to herein as GIF1), AtGIF 2, AtGIF 3, Os11g40100, Os12g31350, Os03g52320 or combinations thereof.
The GIF (e.g. GIF1) coding sequence may be under the control of a constitutive promoter, such as CaMV 35S promoter, or may be a tissue specific promoter.
As shown in detail in the examples and experimental methods provided below, rGRF3 or orthologues thereof may be produced comprising several synonymous nucleic acid changes—i.e. there is no change in the amino acid sequence of GRF3.
In one embodiment the modified GRF3 or orthologue thereof may comprise comprising at least one or all of the following base changes in the miR396 target site an A→U, a G→A, a U→G, a U→A, a G→C, a A→T, a G→A, a T→A, a G→A, a A→G modification. These changes may retain the native amino acid sequence, but substantially destabilize the interaction of miR396 with said rGRF3.
In one embodiment the modified GRF3 or orthologue thereof may comprise comprising at least one or all of the following base changes in the miR396 target site an A→U, a G→A, a U→A, a G→A, a A→G modification. These changes may retain the native amino acid sequence, but substantially destabilize the interaction of miR396 with said rGRF3.
In a preferred embodiment the modified GRF 3 or orthologue thereof comprises a modified miR396 target site having the following sequence:
CGTTCnAGAAAnCCnGTnGAn (SEQ ID No. 86), wherein n designates bases that have been modified (e.g. mutated) (e.g. compared with the wild-type sequence).
The modified GRF 3 or orthologue thereof comprises a modified miR396 target site having the following sequence: CGTTCtAGAAAaCCaGTaGAg (SEQ ID No. 38), wherein the lower case letters designates modified bases (e.g. compared with the wild-type sequence).
Mutant sequences can be produced by any known method and various methods are readily available to one of ordinary skill in the art. As one skilled in the art will appreciate, it is possible to produce numerous site directed or random mutations into a nucleotide sequence and to subsequently screen for improved functionality of the encoded polypeptide by various means.
Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
A suitable method is disclosed in Morinaga et al., (Biotechnology (1984) 2, p 646-649). Another method of introducing mutations in nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151).
One method for introducing mutations into a nucleotide sequence would be to use QuikChange® Site Directed Mutagenesis Kit from Stratagene.
In some embodiments Targeted Induced Local Lesions IN Genomes (TILLING) technology described in Colbert et al 2001 (Plant Physiology June 2001, Vol. 126, pp 480-484) may be used to screen for induced mutations, e.g. induced point mutations.
In another aspect there is provided a construct comprising the nucleic acid according to the present invention operably linked with a promoter and/or a terminator.
The promoter may be a constitutive promoter, such as CaMV 35S promoter, the native AtGRF3 promoter, or the native GRF3 orthologue promoter, or may be a tissue specific promoter.
In one embodiment the promoter may be a tissue specific promoter.
When it is desired to decouple the different functions of GRF3 (such as to decouple the increased biomass from delayed leaf senescence), preferably the nucleic acid according to the present invention is operably linked with a tissue specific promoter.
In addition the use of a tissue specific promoter can improve the performance of plant production and further improve productivity.
In some embodiments the tissues specific promoter may comprise a (or may be a) promoter which is transiently expressed during early leaf development.
In one embodiment the tissue specific promoter may comprise a (or may be a) ASYMMETRIC LEAVES 1 (AS-1) promoter or a AINTEGUMENTA (ANT) promoter.
A person skilled in the art would be aware of other suitable tissue specific promoters to target expression of the nucleic acid according to the present invention in the appropriate location of the plant. Without wishing to be bound by theory, a mutant GRF3 or mutant GRF3 orthologue uncoupled of miR396 control with or without the co-overexpression of GIF may modify cell number in leaves or other organs when the nucleic acids are expressed specifically in those tissues. Therefore, rGRF3 will only affect that part of the plant where expression occurs.
A person skilled in the art would also be aware that the temporal pattern and level of expression might also be modified. For example, the AS-1 promoter is active for a longer period of time than the ANT promoter, and thus generates bigger leaves when expressing the mutated GRF3 (rGRF3) or mutated GRF3 orthologue sequences.
Therefore the tissue specific promoter may be one which is spatially and/or temporally regulating expression.
The present invention further provides a vector comprising the nucleic acid of the present invention or the construct according to the present invention.
In a further aspect the present invention provides a plant, plant cell or plant tissue comprising the nucleic acid according to the present invention, the construct according to the present invention or the vector according to the present invention.
In one embodiment the plant, plant cell or plant tissue according the present invention may further comprise a modified GRF2 (rGRF2) which modified GRF2 is also decoupled from control by miR396. In other words, the GRF2 may also be mutated in miR396 target site in accordance with the present invention. For the avoidance of doubt this embodiment only relates the combination of the rGRF3 or rGRF3-orthologue in accordance with the present invention in combination with rGRF2.
AtGRF2 and AtGRF9 are not GRF3-orthologues in accordance with the present invention.
Hence there term “GRF3-orthologue” as used herein does not include AtGRF2 or AtGRF9.
Hence the nucleotide sequence according to the present invention does not comprise a nucleotide sequence comprising the nucleotide sequence shown herein as SEQ ID No. 87 or SEQ ID No. 45.
Likewise, the term “modified GRF3-orthologue” or “rGRF3-orthologue” as used herein does not include modified AtGRF2 or modified AtGRF9.
In one embodiment of the present invention the plant, plant cell or plant tissue may in addition over-express at least one GIF (e.g. GIF1).
Over-expression of at least one GIF (e.g. GIF1) may be accomplished by transforming said plant, or a plant cell, or a plant tissue, with a construct comprising the at least one GIF (e.g. GIF1) encoding sequence operably linked to a promoter.
In some embodiments the plant, plant cell or plant tissue according to the present invention may comprise more than one (e.g. two, for example three) nucleic acids according to the present invention.
By way of example only, the plant, plant cell or plant tissue according to the present invention may comprise more than one (e.g. two, for example three) rGRF3 genes and/or rGRF3-orthologues. For example the plant, plant cell or plant tissue according to the present invention may comprise rGRF3 in combination with one or more rGRF3-orthologues.
The term “GRF3” as used herein means the GROWTH-REGULATING FACTOR 3 obtainable (preferably obtained) from Arabidopsis thaliana. 
The term “rGRF3” as used herein means a mutated or modified GROWTH-REGULATING FACTOR 3 obtainable (preferably obtained) from Arabidopsis thaliana. Preferably the mutated or modified GROWTH-REGULATING FACTOR 3 has been mutated or modified to decouple it from control by miR396.
The term “GRF3-orthologue” as used herein may encompass one or more of the following GRFs selected from the group consisting of: Arabidopsis thaliana GRF4; Oryza sativa GRF 1, 2, 3, 4, or 5 Zea mays GRF 1, 3, 5, 6, 7, 9, 11 or 14; Glycine max GRF; Medicago truncatula GRF; Populus trichocarpa GRF; Carica papaya GRF and Prunus persica GRF.
The term “rGRF3-orthologue” as used herein may encompass one or more of the following GRFs selected from the group consisting of Arabidopsis thaliana GRF4; Oryza sativa GRF 1, 2, 3, 4 or 5; Zea mays GRF 1, 3, 5, 6, 7, 9, 11 or 14; Glycine max GRF; Medicago truncatula GRF; Populus trichocarpa GRF; Carica papaya GRF and Prunus persica GRF which have been decoupled from control by miR396.
The nucleic acid encoding a modified GRF-3 or an orthologue thereof may comprise introns or may exclude introns.
In one embodiment the nucleic acid encoding a modified GRF-3 or an orthologue thereof comprises introns. Without wishing to be bound by theory introns may enhance the expression of the transgenes.
In yet another aspect there is provided a method for using the nucleic acid according to the present invention which comprises introducing said nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention into a plant.
In another aspect of the present invention there is provided nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention for use in the manufacture of a plant with increased biomass, increased stress resistance, increased drought tolerance, delayed leaf senescence and combinations thereof.
In another aspect of the present invention there is provided a method of producing a plant with increased biomass, increased stress resistance, increased drought tolerance, delayed leaf senescence and combinations thereof comprising transforming the plant with nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention.
A further aspect provides the use of a nucleic acid according to the present invention or a construct according to the present invention or a vector according to the present invention in the manufacture of a plant for increasing biomass, increasing stress resistance, increasing drought tolerance, delaying leaf senescence or combinations thereof.
Preferably plants in accordance with the present invention have increased biomass, increased stress resistance, increased drought tolerance, delayed leaf senescence or combinations thereof.
The term “increased biomass” may comprise one or more of the following selected from the group consisting of: increased overall plant biomass, increased fresh weight, increased leaf area or size, increased root length, increased dry weight, increased stem growth, increased stem biomass, increased stem diameter, and increased stem width at flowering.
A surprising technical advantage of the use of rGRF3 or rGRF3 orthologues (which differs from use of rAtGRF2) is that the increased biomass, increased drought tolerance, delayed leaf senescence or combinations thereof occurs without detrimental leaf shape changes, e.g. downward rolling.
In some embodiments it may be preferable to uncouple increased biomass from delayed leaf senescence. The inventors have surprisingly found that this can be achieved by using tissue specific promoters.
The term “increased stress resistance” as used herein means the ability of a plant to remain productive (e.g. maintain or increase biomass, etc.) even in conditions which place the plant under stress, e.g. drought etc.
The terms “increased biomass”, “increased stress resistance”, “increased drought tolerance”, “delayed leaf senescence” “increased root growth”, “increased root elongation speed” mean increased or delayed compared with either wild-type plants (e.g. plants comprising a non-modified GRF3 or GRF3-orthologue) or plants comprising a modified GRF2 (rGRF2).
The terms “increased overall plant biomass”, “increased fresh weight”, “increased leaf area or size”, “increased dry weight”, “increased stem growth”, “increased stem biomass”, “increased stem diameter”, and “increased stem width at flowering” mean increased or delayed compared with either wild-type plants (e.g. plants comprising a non-modified GRF3 or GRF3-orthologue) or plants comprising a modified GRF2 (rGRF2).
The term “modified” as used herein may mean mutated. The term “modified” as used herein mean different from the wild-type.
The term “wild type” as used herein means a naturally-occurring nucleic acid. That is to say a nucleic acid found in an endogenous genetic code and isolated from its endogenous host organism which has not been mutated (i.e. does not contain base deletions, additions or substitutions) when compared with the genetic code of the host organism.
The vector according to the present invention may be an expression vector. The term “expression vector” means a construct capable of in vivo or in vitro expression.
Preferably, the expression vector is incorporated into the genome of a suitable host organism, e.g. plant. The term “incorporated” preferably covers stable incorporation into the genome.
The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism, e.g. plant.
The vectors for use in the present invention may be transformed into a suitable host cell, e.g. plant cell, as described below.
The vectors for use in the present invention may contain one or more selectable marker genes such as a gene which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell.
Thus, in a further embodiment, the invention provides a method of making nucleotide sequences of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host (e.g. plant) cell, and growing the host (e.g. plant) under conditions which bring about replication of the vector.
The term “operably linked” as used herein refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
The term “regulatory sequences” includes promoters and enhancers and other expression regulation signals.
The term “promoter” is used in the normal sense of the art, e.g. an RNA polymerase binding site.
The term “construct”—which is synonymous with terms such as “conjugate”, “cassette” and “hybrid”—includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter.
An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Sh1-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term “fused” in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
The construct may even contain or express a marker, which allows for the selection of the genetic construct.
For some applications, preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.
A host organism suitable for transformation with the nucleic acid of the present invention may be a plant. In this respect, the basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).
Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D. N. et al., (1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208.
Other techniques for transforming plants include ballistic transformation, the silicon whisker carbide technique (see Frame B R, Drayton P R, Bagnaall S V, Lewnau C J, Bullock W P, Wilson H M, Dunwell J M, Thompson J A & Wang K (1994) Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation, The Plant Journal 6: 941-948) and viral transformation techniques (e.g. see Meyer P, Heidmann I & Niedenhof I (1992) The use of cassava mosaic virus as a vector system for plants, Gene 110: 213-217).
Further teachings on plant transformation may be found in EP-A-0449375.
Plant cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc.
In a further aspect, the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant. The vector system may comprise one vector, but it may comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheung An et al., (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
One extensively employed system for transformation of plant cells uses the Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al., (1986), Plant Physiol. 81, 301-305 and Butcher D. N. et al., (1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208. After each introduction method of the desired promoter or construct or nucleotide sequence according to the present invention in the plants, the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985) 4:277-284.
The term GIF as used herein means GRF-INTERACTING FACTORs (GIFs), a small gene family encoding proteins with homology to the human SYT transcriptional co-activator (Horiguchi et al., 2005; Kim and Kende, 2004).
GIF1 (Kim and Kende, 2004) is also known as ANGUSTIFOLIA 3 (AN3).
In one embodiment preferably the GIF used in accordance with the present invention is GIF1. GIF1 may also be referred to herein as AtGIF1.
In one embodiment the GIF used in accordance with the present invention may be GIF1, wherein GIF1 i) comprises the amino acid shown herein as MQQHLMQMQPMMAGYYPSNVTSDHIQQYLDENKSLILKIVESQNSGKLSECAENQ ARLQRNLMYLAAIADSQPQPPSVHSQYGSAGGGMIQGEGGSHYLQQQQATQQQQ MTQQSLMAARSSMLYAQQQQQQQPYATLQHQQLHHSQLGMSSSSGGGGSSGLH ILQGEAGGFHDFGRGKPEMGSGGGGEGRGGSSGDGGETLYLKSSDDGN (SEQ ID No. 95) or an amino acid sequence having at least 80% identity therewith; or ii) is encoded by the nucleotide sequence:
ATGCAACAGCACCTGATGCAGATGCAGCCCATGATGGCTGGTTACTACCCCAG CAATGTTACCTCTGATCATATCCAACAGTACTTGGACGAAAACAAATCGTTGATT CTGAAGATTGTTGAGTCTCAAAACTCTGGAAAGCTTAGCGAATGCGCCGAGAAT CAAGCAAGGCTTCAACGCAACCTAATGTACCTAGCTGCAATAGCAGATTCTCAG CCTCAGCCACCAAGTGTGCATAGCCAGTATGGATCTGCTGGTGGTGGGATGAT TCAGGGAGAAGGAGGGTCACACTATTTGCAGCAGCAACAAGCGACTCAACAGC AACAGATGACTCAGCAGTCTCTAATGGCGGCTCGATCTTCAATGTTGTATGCTC AGCAACAGCAGCAGCAGCAGCCTTACGCGACGCTTCAGCATCAGCAATTGCAC CATAGCCAGCTTGGAATGAGCTCGAGCAGCGGAGGAGGAGGAAGCAGTGGTC TCCATATCCTTCAGGGAGAGGCTGGTGGGTTTCATGATTTTGGCCGTGGGAAG CCGGAAATGGGAAGTGGTGGTGGCGGTGAAGGCAGAGGAGGAAGTTCAGGGG ATGGTGGAGAAACCCTTTACTTGAAATCATCAGATGATGGGAATTGA (SEQ ID No. 39); or

iii) is encoded by a nucleotide sequence which is at least 70%, preferably 80%, more preferably 90%, even more preferably 95% identical with SEQ ID No. 39; or

iv) is encoded by a nucleotide sequence which hybridizes under stringent conditions with SEQ ID No. 39.

As can be seen from FIG. 9 a number of GIF sequences from Arabidopsis thaliana and Oryza sativa cluster together. It is envisaged that any one of these GIFs may be used in accordance with the present invention. Therefore the GIF for use in accordance with the present invention may be one or more of the GIFs designated Os11g40100, Os12g31350, Os03g52320 obtainable (preferably obtained) from Oryza sativa or may be one or more of the GIFs designated AtGIF1, AtGIF2 or AtGIF3 obtainable (preferably obtained) from Arabidopsis thaliana. 
In one embodiment the GIF used in accordance with the present invention may be AtGIF2, wherein AtGIF2 i) comprises the amino acid shown herein as MQQQQSPQMFPMVPSIPPANNITTEQIQKYLDENKKLIMAIMENQNLGKLAECAQY QALLQKNLMYLAAIADAQPPPPTPGPSPSTAVAAQMATPHSGMQPPSYFMQHPQA SPAGIFAPRGPLQFGSPLQFQDPQQQQQIHQQAMQGHMGIRPMGMTNNGMQHA MQQPETGLGGNVGLRGGKQDGADGQGKDDGK (SEQ ID No. 96) or an amino acid sequence having at least 80% identity therewith; or
ii) is encoded by the nucleotide sequence:
ATGCAGCAGCAGCAGTCTCCGCAAATGTTTCCGATGGTTCCGTCGATTCCCCCT GCTAACAACATCACTACCGAACAGATCCAAAAGTACCTTGATGAGAACAAGAAG CTGATTATGGCCATCATGGAAAACCAGAATCTCGGTAAACTTGCTGAGTGCGCC CAGTACCAAGCTCTTCTCCAGAAGAACTTGATGTATCTTGCTGCAATTGCTGATG CTCAACCCCCACCACCTACGCCAGGACCTTCACCATCTACAGCTGTCGCTGCC CAGATGGCAACACCGCATTCTGGGATGCAACCACCTAGCTACTTCATGCAACAC CCACAAGCATCCCCTGCAGGGATTTTCGCTCCAAGGGGTCCTTTACAGTTTGGT AGCCCACTCCAGTTTCAGGATCCGCAACAGCAGCAGCAGATACATCAGCAAGC TATGCAAGGACACATGGGGATTAGACCAATGGGTATGACCAACAACGGGATGC AGCATGCGATGCAACAACCAGAAACCGGTCTTGGAGGAAACGTGGGGCTTAGA GGAGGAAAGCAAGATGGAGCAGATGGACAAGGAAAAGATGATGGCAAGTGA (SEQ ID No. 90), or

iii) is encoded by a nucleotide sequence which is at least 70%, preferably 80%, more preferably 90%, even more preferably 95% identical with SEQ ID No. 90; or

iv) is encoded by a nucleotide sequence which hybridizes under stringent conditions with SEQ ID No. 90.

In one embodiment the GIF used in accordance with the present invention may be AtGIF3 wherein AtGIF3 i) comprises the amino acid shown herein as MQQSPQMIPMVLPSFPPTNNITTEQIQKYLDENKKLIMAILENQNLGKLAECAQYQA LLQKNLMYLAAIADAQPQPPAATLTSGAMTPQAMAPNPSSMQPPPSYFMQQHQAV GMAQQIPPGIFPPRGPLQFGSPHQFLDPQQQLHQQAMQGHMGIRPMGLNNNNGL QHQMHHHETALAANNAGPNDASGGGKPDGTNMSQSGADGQGGSAARHGGGDA KTEGK (SEQ ID No. 97) or an amino acid sequence having at least 80% identity therewith; or
ii) is encoded by the nucleotide sequence:
ATGCAGCAATCTCCACAGATGATTCCGATGGTTCTTCCTTCATTTCCGCCCACCA ATAATATCACCACCGAACAGATCCAAAAGTATCTTGATGAGAACAAGAAGCTGAT AATGGCGATCTTGGAAAATCAGAACCTCGGTAAACTTGCAGAATGTGCTCAGTA TCAAGCTCTTCTCCAGAAGAATTTGATGTATCTCGCTGCAATTGCGGATGCTCAA CCTCAGCCACCAGCAGCTACACTAACATCAGGAGCCATGACTCCCCAAGCAAT GGCTCCTAATCCGTCATCAATGCAGCCACCACCAAGCTACTTCATGCAGCAACA TCAAGCTGTGGGAATGGCTCAACAAATACCTCCTGGGATTTTCCCTCCTAGAGG TCCATTGCAATTTGGTAGCCCGCATCAGTTTCTGGATCCGCAGCAACAGTTACA TCAACAAGCTATGCAAGGGCACATGGGGATTAGACCAATGGGTTTGAATAATAA CAACGGACTGCAACATCAAATGCACCACCATGAAACTGCTCTTGCCGCAAACAA TGCGGGTCCTAACGATGCTAGTGGAGGAGGTAAACCGGATGGGACCAATATGA GCCAGAGTGGAGCTGATGGGCAAGGTGGCTCAGCCGCTAGACATGGCGGTGG TGATGCAAAAACTGAAGGAAAATGA (SEQ ID No. 91), or

iii) is encoded by a nucleotide sequence which is at least 70%, preferably 80%, more preferably 90%, even more preferably 95% identical with SEQ ID No. 91; or

iv) is encoded by a nucleotide sequence which hybridizes under stringent conditions with SEQ ID No. 91.

In one embodiment the GIF used in accordance with the present invention may be the GIF designated Os11g40100 wherein Os11g401001) comprises the amino acid shown herein as:
MQQQMAMPAGAAAAAVPPAAGITTEQIQKYLDENKQLILAILENQNLGKLAECAQY QAQLQKNLLYLAAIADAQPPQNPGSRPQMMQPGATPGAGHYMSQVPMFPPRTPL TPQQMQEQQQQQLQQQQAQALAFPGQMLMRPGTVNGMQSIPVADPARAADLQT AAPGSVDGRGNKQDATSEPSGTESHKSAGADNDAGGDIAEKS (SEQ ID No. 98) or an amino acid sequence having at least 80% identity therewith; or

ii) is encoded by the nucleotide sequence:

ATGCAGCAGCAGATGGCCATGCCGGCGGGGGCCGCCGCCGCCGCGGTGCCG CCGGCGGCCGGCATCACCACCGAGCAGATCCAAAAGTATTTGGATGAAAATAA ACAGCTAATTTTGGCCATCCTGGAAAATCAAAACCTAGGGAAGTTGGCTGAATG TGCTCAGTACCAAGCTCAGCTTCAAAAGAATCTCTTGTATCTGGCTGCCATTGCA GATGCCCAACCACCTCAGAATCCAGGAAGTCGCCCTCAGATGATGCAGCCTGG TGCTACCCCAGGTGCTGGGCATTACATGTCCCAAGTACCGATGTTCCCTCCAAG AACTCCCTTAACCCCACAACAGATGCAAGAGCAGCAGCAGCAGCAACTCCAGC AACAGCAAGCTCAGGCTCTAGCCTTCCCCGGCCAGATGCTAATGAGACCAGGT ACTGTCAATGGCATGCAATCTATCCCAGTTGCTGACCCTGCTCGCGCAGCCGAT CTTCAGACGGCAGCACCGGGCTCGGTAGATGGCCGAGGAAACAAGCAGGATG CAACCTCGGAGCCTTCCGGGACCGAGAGCCACAAGAGTGCGGGAGCAGATAA CGACGCAGGCGGTGACATAGCGGAGAAGTCCTGA (SEQ ID No. 92)), or

iii) is encoded by a nucleotide sequence which is at least 70%, preferably 80%, more preferably 90%, even more preferably 95% identical with SEQ ID No. 92; or

iv) is encoded by a nucleotide sequence which hybridizes under stringent conditions with SEQ ID No. 92.

In one embodiment the GIF used in accordance with the present invention may be the GIF designated Os12g31350 wherein Os12g313501) comprises the amino acid shown herein as:
MQQQPMPMPAQAPPTAGITTEQIQKYLDENKQLILAILENQNLGKLAECAQYQAQL QKNLLYLAAIADTQPQTTISRPQMVPHGASPGLGGQYMSQVPMFPPRTPLTPQQM QEQQLQQQQAQLLSFGGQMVMRPGWNGIPQLLQGEMHRGADHQNAGGATSEP SESHRSTGTENDGGSDFGDQS (SEQ ID No. 99) or an amino acid sequence having at least 80% identity therewith; or

ii) is encoded by the nucleotide sequence:

ATGCAGCAGCAGCCGATGCCGATGCCCGCGCAGGCGCCGCCGACGGCCGGAA TCACCACCGAGCAGATCCAAAAGTATCTGGATGAAAACAAGCAGCTTATTTTGG CTATTTTGGAAAATCAGAATCTGGGAAAGTTGGCAGAATGTGCTCAGTATCAAG CGCAGCTTCAGAAGAATCTCTTGTACTTGGCTGCAATTGCTGATACTCAACCGC AGACCACTATAAGCCGTCCCCAGATGGTGCCGCATGGTGCATCGCCGGGGTTA GGGGGGCAATACATGTCGCAGGTGCCAATGTTCCCCCCCAGGACCCCTCTAAC GCCCCAGCAGATGCAGGAGCAGCAGCTGCAGCAACAGCAAGCCCAGCTGCTC TCGTTCGGCGGTCAGATGGTTATGAGGCCTGGCGTTGTGAATGGCATTCCTCA GCTTCTGCAAGGCGAAATGCACCGCGGAGCAGATCACCAGAACGCTGGCGGG GCCACCTCGGAGCCTTCCGAGAGCCACAGGAGCACCGGCACCGAAAATGACG GTGGAAGCGACTTCGGCGATCAATCCTAA (SEQ ID No. 93), or

iii) is encoded by a nucleotide sequence which is at least 70%, preferably 80%, more preferably 90%, even more preferably 95% identical with SEQ ID No. 93; or

iv) is encoded by a nucleotide sequence which hybridizes under stringent conditions with SEQ ID No. 93.

In one embodiment the GIF used in accordance with the present invention may be the GIF designated Os03g52320 wherein Os03g523201) comprises the amino acid shown herein as:
MQQQHLMQMNQGMMGGYASPTTVTTDLIQQYLDENKQLILAILDNQNNGKVEECA RNQAKLQHNLMYLAAIADSQPPQTAAMSQYPSNLMMQSGARYMPQQSAQMMAP QSLMAARSSMMYAQPALSPLQQQQQQQAAAAHGQLGMGSGGTTSGFSILHGEAS MGGGGGGGGAGNSMMNAGVFSDFGRGGGGGGKEGSTSLSVDVRGANSGAQSG DGEYLKGTEEEGS (SEQ ID No. 100) or an amino acid sequence having at least 80% identity therewith; or

ii) is encoded by the nucleotide sequence:

ATGCAGCAGCAACACCTGATGCAGATGAACCAGGGCATGATGGGGGGATATGC TTCCCCTACCACCGTCACCACTGATCTCATTCAGCAGTATCTGGATGAGAACAA GCAGCTGATCCTGGCCATCCTTGACAACCAGAACAATGGGAAGGTGGAAGAGT GCGCTCGGAACCAAGCTAAGCTCCAGCACAATCTCATGTACCTCGCCGCCATC GCCGACAGCCAGCCGCCGCAGACGGCCGCCATGTCCCAGTATCCGTCGAACC TGATGATGCAGTCCGGGGCGAGGTACATGCCGCAGCAGTCGGCGCAGATGAT GGCGCCGCAGTCGCTGATGGCGGCGAGGTCTTCGATGATGTACGCGCAGCCG GCGCTGTCGCCGCTCCAGCAGCAGCAGCAGCAGCAGGCGGCGGCGGCGCAC GGGCAGCTGGGCATGGGCTCGGGGGGCACCACCAGCGGGTTCAGCATCCTCC ACGGCGAGGCCAGCATGGGCGGCGGCGGCGGCGGCGGTGGCGCCGGTAACA GCATGATGAACGCCGGCGTGTTCTCCGACTTCGGACGCGGCGGCGGCGGCGG CGGCAAGGAGGGGTCCACCTCGCTGTCCGTCGACGTCCGGGGCGCCAACTCC GGCGCCCAGAGCGGCGACGGGGAGTACCTCAAGGGCACCGAGGAGGAAGGC AGCTAG (SEQ ID No. 94), or

iii) is encoded by a nucleotide sequence which is at least 70%, preferably 80%, more preferably 90%, even more preferably 95% identical with SEQ ID No. 94; or

iv) is encoded by a nucleotide sequence which hybridizes under stringent conditions with SEQ ID No. 94.

Furthermore, the inventors have demonstrated that overexpression of GIF1, GIF2, and GIF3 promotes cell proliferation and leaf size and that GIF2 and GIF3 proteins are functional equivalents of GIF1 (se FIG. 43 in combination with FIG. 9).
Previously Horiguchi et al. (2005) have shown that overexpression of the GIF1/AN3 gene stimulates cell proliferation as well, leading to enlarged leaves by about 20%.
These results suggest that all of the GIF genes function redundantly as positive regulators of cell proliferation, thereby determining plant organ size.
Therefore the use of any GIF gene in accordance with the present invention is contemplated herein.
In addition combinations of GIF genes are also contemplated herein.
In one aspect, preferably the sequence is in an isolated form. The term “isolated” means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.
In one aspect, preferably the sequence is in a purified form. The term “purified” means that the sequence is in a relatively pure state—e.g. at least about 90% pure, or at least about 95% pure or at least about 98% pure.
The terms “nucleotide sequence” or “nucleic acid” as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.
The terms “nucleotide sequence” or “nucleic acid” in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for the present invention.
In a preferred embodiment, the nucleotide sequence when relating to and when encompassed by the per se scope of the present invention does not include the native nucleotide sequence according to the present invention when in its natural environment and when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, this preferred embodiment shall be called the “non-native nucleotide sequence” or “non-native nucleic acid”
Typically, the nucleotide sequence or nucleic acid encompassed by scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence or nucleic acid could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers M H et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).
Due to degeneracy in the genetic code, nucleotide sequences may be readily produced in which the triplet codon usage, for some or all of the amino acids encoded by the original nucleotide sequence, has been changed thereby producing a nucleotide sequence with low homology to the original nucleotide sequence but which encodes the same, or a variant, amino acid sequence as encoded by the original nucleotide sequence. For example, for most amino acids the degeneracy of the genetic code is at the third position in the triplet codon (wobble position) (for reference see Stryer, Lubert, Biochemistry, Third Edition, Freeman Press, ISBN 0-7167-1920-7) therefore, a nucleotide sequence in which all triplet codons have been “wobbled” in the third position would be about 66% identical to the original nucleotide sequence. However, the amended nucleotide sequence would encode for the same, or a variant, primary amino acid sequence as the original nucleotide sequence.
Therefore, the present invention in some embodiments further relates to any nucleotide sequence that has alternative triplet codon usage for at least one amino acid encoding triplet codon, but which encodes the same, or a variant, polypeptide sequence as the polypeptide sequence encoded by the original nucleotide sequence.
Furthermore, specific organisms typically have a bias as to which triplet codons are used to encode amino acids. Preferred codon usage tables are widely available, and can be used to prepare codon optimised genes. Such codon optimisation techniques are routinely used to optimise expression of transgenes in a heterologous host.
The present invention also encompasses the use of sequences which have identity or similarity with the sequences according to the present invention.
Here, the term “identity” means an entity having a certain identity with the amino acid sequences and the nucleotide sequences. Identity means the percentage of amino acids or bases that are the same in one sequence when compared with another sequence.
Here, the term “similarity” means an entity having similar chemical properties/functions. Hence the term similarity takes into account conservative changes.
In the present context, a sequence which has a certain percentage identity or similarity is taken to include a sequence which may be at least 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a sequence of the present invention (the subject sequence). Typically, the sequences will comprise the same sequences that code for the active sites etc. as the subject sequence.
Identity or similarity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. The available computer programs can calculate % identity and % similarity between two or more sequences.
% identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.
However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap.
This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is −12 for a gap and −4 for each extension.
Calculation of maximum % identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12 p 387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).
However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
Although the final % identity can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Alternatively, percentage identity may be calculated using the multiple alignment feature in DNASISTM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene 73(1), 237-244).
Generally percentage identity is calculated over at least 50, preferably at least 100, preferably at least 200 contiguous bases or residues. Preferably the percentage identity is calculated using the full length sequence.
Once the software has produced an optimal alignment, it is possible to calculate % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based on the properties of their side chain alone. However it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl Biosci. 9: 745-756) (Taylor W. R. (1986) “The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids.













SET


SUB-SET







Hydrophobic
Phe, Trp, Tyr, His,
Aromatic
Phe, Trp,



Lys, Met, Ile, Leu, 

Tyr, His



Val, Ala, Gly, Cys
Aliphatic
Ile, Leu, Val


Polar
Trp, Tyr, His, Lys,
Charged
His, Lys,



Arg, Glu, Asp, Cys, 

Arg, Glu, Asp



Ser, Thr, Asn, Gln
Positively
His, Lys,




Charged
Arg




Negatively
Glu, Asp




Charged



Small
Val, Cys, Ala, Gly, 
Tiny
Ala, Gly, Ser



Ser, Thr, Asn, Asp









The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present invention.
The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
Polynucleotides which are not 100% identical to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
Variants and strain/species orthologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated procedures are readily available in the art.
Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
The term “hybridisation” as used herein shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
Preferably, the hybridisation is determined under stringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015 M Na3citrate pH 7.0}).
Suitably, the hybridisation may be determined under high stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na3citrate pH 7.0}).
The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
A skilled person will understand that the modified GRF3-orthologue may be obtainable from any plant. In a preferred embodiment the GRF3-orthologue is obtainable, preferably obtained, from one or more of the plants selected from the group consisting of: Arabidopsis thaliana, Oryza sativa, Zea mays, Glycine max, Medicago truncatula, Populus trichocarpa, Prunus persica, Carica papaya, Triticum aestivum, Sorghum bicolor, Gossypium hirstutum, sugar cane (Saccharum spp.), Panicum virgatum, Helianthis annus, Beta vulgaris, and Brassica species.
In an even more preferred embodiment the GRF3-orthologue is obtainable, preferably obtained, from one or more of the plants selected from the group consisting of: Arabidopsis thaliana, Oryza sativa, Zea mays, Glycine max, Medicago truncatula, Populus trichocarpa, Prunus persica, Carica papaya, 
The nucleic acid, vector or construct according to the present invention may be transformed in to any (host) plant.
The plant, plant cell or plant tissue according to the present invention may be a monocotyledonous (monocot) plant or a dicotyledonous (dicot) plant.
In one embodiment the plant, plant cell or plant tissue according to the present invention may be a dicot.
A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, onion, leek, millet, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, sugarcane grass, false oat grass, fescue, Bermuda grass, brome, heath grass, meadow grasses (e.g. naturally mixed grassland swards, orchard grass, rye grass, Timothy-grass) or Festuca species
A dicot plant which may be selected from the families including, but not limited to Asteraceae, Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, spinach, water melon, squash, oilseed rapeseed (including canola), cabbage, broccoli, kale, turnip, rutabaga (swede), tomato, potato, capsicum, tobacco, cotton, legumes sugar beet, okra, apple, rose, strawberry, alfalfa (lucerne), birdsfoot trefoil, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, coffee, cocoa, apricots, apples, pears, peach, grape vine or citrus species.
Also included are biofuel and bioenergy crops such as sugar cane, oilseed rape/oil-seed rape, linseed, jatropha, oil-palm, copra and willow, eucalyptus, poplar, poplar hybrids. Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (e.g. forage grass species or forage maize), grazing or fodder (pasture grasses, clover, alsike clover, red clover, subterranean clover, white clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed), rubber plants, and crops for amenity purposes (e.g. turf grasses for sports and amenity surfaces), ornamentals for public and private gardens (e.g. species of Angelonia, Begonia, Catharanthus, Euphorbia, Gazania, Impatiens, Nicotiana, Pelargonium, Petunia, Rosa, Verbena, and Viola) and flowers of any plants for the cut-flower market (such as tulips, roses, daffodils, lilies, stallions, gerbera, carnations, chrysanthemums, irises, gladioli, alstromerias, marigold, sweet pea, freesia, anemone poppy).
Preferably, the plant, plant cell or plant tissue, or host plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use for other non-food/feed use. Preferred plants are corn (maize), millet, wheat, Durum wheat, rice, oilseed rape (or canola), sorghum, sugar cane, soybean, sunflower, potato, tomato, barley, rye, oats, pea, bean, field bean, sugar beet, oil-palm, groundnut, peanut, cassava, alfalfa, clover, copra, raisin, coffee, cotton, lettuce, banana, broccoli or other vegetable brassicas.
In one embodiment the plant, pant cell or plant tissue, or host plant is Brassica, suitably Brassica oleracea (e.g. broccoli or other vegetable Brassicas).
The plant may be a tree such as eucalyptus, poplar, or conifer such as Picea species (e.g. spruce) or Pinus species (pines), a hardwood tree species such as teak, a plantation tree such as rubber (Hevea), palm tree (date- or oil-palm) or jatropha or an orchard fruit tree (such as plum, peach, apple and pear).
EXAMPLES
While the invention disclosed herein is described in general above, and those skilled in the art based on that disclosure would be enabled to practice this invention, including its best mode, the following examples are provided to further support this written description and enabling disclosure. The details of these examples are, however, non-limiting. For an understanding of the scope of this invention, reference should be had to the appended claims and their equivalents.
Transgenes
See Table 1 for a list of binary plasmids used.










TABLE 1





Vector
Construct
Arabidopsis Chromosome: start-enda
Purpose







pJP123
35S:miR396b
5: 13628907-13629319
Overexpression of





miR396b





stem-loop


pRER31
GRF3
2: 15274101-15270081
Genomic GRF3




CGC AAC CGT TCA AGA AAG CCT GTG 





GAA ACT CCA (SEQ ID NO: 122)



pRER32
rGRF3
2: 15274101-15270081
Genomic mutant 




CGC AAC CGT TCT AGA AAA CCA GTA 
GRF3




GAG ACT CCA (SEQ ID NO: 123)



pRER35
rGRF2
4: 17729683-17725302
Genomic mutant 




CGT CAT CGT TCT AGA AAA CCG GTC 
GRF2




GAA CTC CAA (SEQ ID NO: 124)



pJD16
35S:GIF1
5: 10647830-10649620
Overexpression of





AtGIF1





aThe nucleotides annealing with miR396 are 8-19, 21-27 of GRF3 construct; 8-11, 13-17, 19, 22-23, 25-27 of rGRF3 construct and 8-11, 13-17, 19, 22-23, 25-27 of rGRF2. Underlined, mutagenized residues are 12, 18, 21 and 24 of rGRF3 construct and 12, 18, 21 and 24 of rGRF2 construct. The upstream and downstream codons are 1-6, 28-33 of GRF3 construct; 1-6, 28-33 of rGFR3 construct and 1-6, 28-33 of rGRF2 construct.






Expression Analysis

First, 0.5-1.0 μg of total RNA was treated with RQ1 RNase-free DNase (Promega). Then, first-strand cDNA synthesis was carried out using SuperScript III reverse transcriptase (Invitrogen). PCR reactions were performed in a Mastercycler ep realplex thermal cycler (Eppendorf) using SYBR Green I (Roche) to monitor double-stranded (ds)DNA synthesis. Quantitative (q)PCR of each gene was carried out for at least three biological replicates, with technical duplicates for each biological replicate. The relative transcript level was determined for each sample, normalized using PROTEIN PHOSPHATASE 2A cDNA level (Czechowski et al., 2005). Primer sequences are given in Table 2:







TABLE 2







Relevant Locus IDs and oligonucleotide primers used in RT-qPCR.










Gene
Locus ID
Forward primer
Reverse primer





AtGRF3
AT2G36400
GTCTTCGCTGGCCACAAGTATT
TGTTGCTGTTGTAGTGGTGGCT




SEQ ID NO: 104
SEQ ID NO: 105


 


AtGRF2
AT4G37740
CACATCAACAGAGGCCGTCATcg
AACCGGAGATTCCTTGGGTTGTAAG




SEQ ID NO: 106
SEQ ID NO: 107


 


AtGIF1
AT5G28640
TTGGACGAAAACAAATCGTTGA
CTGTTGCTGTTGAGTCGCTTGT




SEQ ID NO: 108
SEQ ID NO: 109









Small RNA Analysis

RNA was extracted using TRIzol reagent (Invitrogen). Total RNA was resolved on 17% polyacrylamide gels under denaturing conditions (7 M urea). Blots were hybridized using either radioactively labelled or digoxigenin end-labelled locked nucleic acid (LNA) oligonucleotide probes designed against miR396 (Exiqon, Denmark).
Alternatively, miR396 levels were determined by stem-loop RT-qPCR, as described previously (Chen et al., 2005). The sequences of the oligonucleotides used were: retrotranscription stem-loop oligo,
5′GTCTCCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGGAGACMAAAGTTC3′ (SEQ ID NO: 110);
PCR forward primer, 5′GGCGGTTCCACAGCTTTCTT3′ (SEQ ID NO: 111); and PCR reverse primer,
5′TGGTGCAGGGTCCGAGGTATT3′ (SEQ ID NO: 112).
Microarray Analyses
Total RNA was extracted from the aerial part of seedlings grown on plates for 10 days using the RNeasy plant mini kit (QIAGEN). Microarray analyses using the Affymetrix ATH1 platform were performed on two biological replicates as described (Schmid et al., 2005). Differentially expressed genes were identified using per-gene variance, calculated using logit-T (Lemon et al., 2003). The corresponding fold change of the transcripts was obtained by expression estimates using gcRMA (www.bioconductor.org), a modification of the robust multi-array analysis (RMA) algorithm (Irizarry et al., 2003). The expression of gene groups was assessed by gene set enrichment analysis using GSEA-P 2.0 (Subramanian et al., 2007; Subramanian et al., 2005).
Microscopic Observations
Tissue was fixed in FAA and embedded in paraffin. Sections 10 μm thick were stained with Toluidine Blue.
To obtain paradermal views of palisade cells, leaves were fixed with FAA and cleared with chloral hydrate solution as described (Horiguchi et al., 2005). Palisade leaf cells were observed using differential interference contrast (DIC) microscopy.
In Situ Hybridization
DIG-labelled sense and antisense probes were synthesized by transcription with T7 or SP6 RNA polymerase with the DIG RNA labelling kit (SP6/T7) (Roche) using cloned cDNAs of GRF2 and HISTONE H4 as templates. For the miR396 probe, LNA oligonucleotides (Exiqon) were end labelled with the DIG oligonucleotide 3′-end labelling kit (Roche). Shoot apices from 15-day-old plants grown in short photoperiods were dissected and fixed in FAA. Paraffin-embedded material was sectioned to 8 μm thickness. Hybridization and detection were performed as previously described (Palatnik et al., 2003).
GUS Assays
To visualize the activity of the reporters, transgenic plants were subjected to GUS staining, according to Donnelly et al. (Donnelly et al., 1999). Stained tissue was paraffin embedded, sectioned and mounted in Canada balsam.
Accession Numbers
A list of relevant AGI locus identifiers is provided in Table 2. The accession number for the microarray experiments is GSE11250.







TABLE 3







GRF expression in 35S:miR396b plants


compared to that in wild type, as estimated by


Affymetrix microarray










Description
Relative expression*







GRF1
0.81



GRF2
0.58



GRF3
0.73



GRF4
WA



GRF5
0.89



GRF6
A



GRF7
0.57



GRF8
A



GRF9
NP







*Fold change relative to wild type, normalized with gcRMA. The average of two biological replicates for each genotype is shown.



A, a gene termed ‘absent’ by MAS 5.0 software (Affymetrix);



NP, not present in ATH1 arrays;



WA, wrongly annotated in ATH1 arrays.






Example #1
A miR396 Resistant Version of GRF3 Increases Plant Size and Biomass Accumulation
The GRF family of transcription factors comprises nine members in Arabidopsis (Kim et al., 2003). Seven of them, including GRF3, have a target site for miR396 (Jones-Rhoades and Bartel, 2004). GRF loss-of-function or overexpression of miR396 have been shown to reduce cell number in Arabidopsis leaves (Horiguchi et al., 2005) (Kim et al., 2003) (Kim and Kende, 2004; Liu et al., 2009).
To study the importance of miR396 in restricting GRF3 expression, two GRF3 genomic fragments were introduced into Arabidopsis thaliana plants. One of them contained the wild-type GRF3 gene (FIG. 1, top panel), while the second harbored a modified GRF3 sequence in which the miRNA-targeting motif was altered through synonymous mutations that prevent miR396 targeting (named rGRF3, FIG. 1, middle panel).
The wild-type sequence of GRF3 contains a region complementary to miR396 with a high interaction energy (ΔG=−33.9 kcal/mol). In contrast, the modified GRF3 sequence (rGRF3), which includes five changes from the wild-type sequence (A→U, G→A, U→A, a G→A and a A→G modifications) does not have a clear miR396 interacting site, as the interaction energy is reduced from −33.9 kcal/mol to −14.4 kcal/mol. The complete sequence of GRF3 is detailed in FIGS. 21 and 22. The complete sequence of rGRF3 is detailed in FIG. 35. The full sequence and a map of the binary vector used (named RER32, see Table 1) can be found in FIGS. 40 and 42, respectively.
Transgenic Arabidopsis plants expressing rGRF3 had bigger leaves and rosettes than wild-type or transgenic plants expressing the miR396-regulated GRF3 sequence (FIGS. 3, 5, 13 and 17). They also accumulate more biomass, as judged by the fresh and dry weight of leaves and rosettes (FIG. 6). In general, it was observed that rGRF3 nearly doubled the size and weight of the first leaf with respect to wild-type plants (FIG. 3). The FFD domain of rGRF3 increased the activity of the protein (FIG. 54).
Plants expressing rGRF3 also had a thicker stem with higher dry weight and growth speed than wild-type plants (FIG. 12). It was observed that the stem diameter increased 20% in rGRF3 plants with respect to wild type (FIG. 12).
Materials and Methods
The Arabidopsis thaliana Columbia (Col-0) accession was used as a wild type. All transgenics are in the Col-0 background. Plants were grown in long photoperiods (16 hr light/8 hr dark) or in short photoperiods (8 hr light/16 hr dark) at 23° C. See Table 1 for a list of binary plasmids generated and details on how transgenic plants were prepared. The miRNA target motif in AtGRF3 was altered introducing synonymous mutations in a cloned AtGRF3 wild type genomic fragment using the QuikChange® Site Directed Mutagenesis Kit (Stratagene).
All constructs were cloned in the binary vector pCHF3 (Jarvis et al., 1998). T-DNA constructs were introduced into Agrobacterium tumefaciens strain ASE and Arabidopsis transgenic plants were obtained by floral-dip.
Leaf area was measured by first taking a photograph of detached fully-expanded leaves, and then measuring the foliar area with the NIH software ImageJ.
To determine biomass accumulation, complete rosettes or individual leafs were weighed to measure fresh weight. Then, tissue was dried at 60° C. during 2 days and dry weight was measured. To determine stem growth, elongation was measured starting with 5 cm long stems during 10 days until full extension was reached. Maximum elongation speed was calculated from the elongation plot. Stem biomass accumulation was estimated by measuring the dry weight of 10 cm long fully elongated stem segments. Finally stem diameter was measured in the lower part of the stem, 0.5 cm above the rosette.
The FFD motif in AtGRF3 was altered introducing mutations in a cloned AtGRF3 cDNA using the QuikChange® Site Directed Mutagenesis Kit (Stratagene). The rGRF3 cDNA native sequence “TTC TTT GAC GAT TGG” (SEQ ID NO: 113) coding for FFDDW (amino acids 1-5 of SEQ ID NO: 127) was mutagenized to “GCT GCT GAC GAT GCT” (SEQ ID NO: 115) coding for AADDA (SEQ ID NO: 116), replacing all aromatic amino acids for alanines in the FFD motif. The wt (rGRF3 (FFD)) and mutagenized (rGRF3(AAD)) genes were placed under the AtGRF3 promoter
Conclusions


    
    
        Transgenic Arabidopsis plants transformed with the miR396-resistant version of GRF3 (named rGRF3) show a striking increase in leaf size and biomass accumulation in comparison to wild-type plants or transgenic plants expressing a GRF3 sequence with a miR396 binding site.
        rGRF3 promotes growth of other tissues as well, such as the stems.
        The FFD domain increases the activity of rGRF3.
    
    


Example #2
Overexpression of GIF1 Enhances the Effect of rGRF3
The GRF family of transcription factors comprises nine members in Arabidopsis (Kim et al., 2003). Seven of them have a target site for miR396 (Jones-Rhoades and Bartel, 2004). Mutations in different GRFs or overexpression of miR396 have been shown to reduce cell number in Arabidopsis leaves (Horiguchi et al., 2005; Kim and Kende, 2004; Rodriguez et al., 2010). The GRFs interact with GRF-INTERACTING FACTORs (GIFs), a small gene family composed by three members (GIF1, GIF2 and GIF3) encoding proteins with homology to the human SYT transcriptional co-activator (Kim and Kende, 2004). Inactivation of GIF1, also known as ANGUSTIFOLIA 3 (AN3), produces narrower leaves as a result of a reduction in cell proliferation in a similar way to GRF-deficient plants (Horiguchi et al., 2005).
Transgenic plants overexpressing GIF1 (FIGS. 1 and 2) from the 35S viral promoter (named 35S:GIF1) were prepared. The full sequence and a map of the binary vector used (named JD16, see Table 1) can be found in FIGS. 39 and 41, respectively. These plants were similar to wild-type plants. Later, 35S:GIF1 was crossed to plants expressing rGRF3 (GRF3 insensitive to miR396, described in example #1). The resulting plants co-overexpressing rGRF3 and GIF1 (named rGRF3×35S:GIF1) were analyzed in more detail (FIGS. 1 and 2).
Upon analysing the biomass productivity of these plants it was found that rGRF3 in combination with GIF1 overexpression produce plants with larger leaves and accumulate more than double fresh and dry weight than wild-type plants (FIGS. 3 and 13). The performance of rGRF3×GIF was better than rGRF3 alone.
Materials and Methods
The Arabidopsis thaliana Columbia (Col-0) accession was used as a wild type. All transgenics are in the Col-0 background. Plants were grown in long photoperiods (16 hr light/8 hr dark) or in short photoperiods (8 hr light/16 hr dark) at 23° C. See Table 1 for a list of binary plasmids generated and details on how transgenic plants were prepared. The miRNA target motif in AtGRF3 was altered introducing synonymous mutations in a cloned AtGRF3 wild type genomic fragment using the QuikChange® Site Directed Mutagenesis Kit (Stratagene). All constructs were cloned in the binary vector pCHF3 (Jarvis et al., 1998). T-DNA constructs were introduced into Agrobacterium tumefaciens strain ASE and Arabidopsis transgenic plants were obtained by floral-dip.
For expression analysis by RT-PCR, RNA was prepared from apices of 20-day-old plants grown in short photoperiods, including developing leaves smaller than 3 mm. 0.5 to 1.0 μg of total RNA was treated with RQ1 RNase-free Dnase (Promega). Then, first-strand cDNA synthesis was carried out using SuperScriptTM III Reverse Transcriptase (Invitrogen). PCR reactions were performed in a Mastercycler® ep realplex thermal cycler (Eppendorf) using SYBRGreen I (Roche) to monitor dsDNA synthesis. qPCR for each gene was done on at least 3 biological replicates with technical duplicates for each biological replicate. The relative transcript level was determined for each sample, normalized using PROTEIN PHOSPHATASE 2A cDNA level (Czechowski et al., 2005).
Leaf area and fresh and dry weight measurements were made as in Example#1.
Conclusions


    
    
        The rGRF3 performance in plant productivity can be enhanced by co-overexpression of GIF1.
    
    


Example #3
Delayed Leaf Senescence and Increased Drought Resistance of rGRF3 Plants
As shown in Example #1, rGRF3 plants produces bigger leaves than wild-type plants, accumulating more biomass. This effect is enhanced by co-overexpression of rGRF3 and GIF1 (rGRF3×35S:GIF1) (see example #2 for further details). In addition, the inventors observed that rGRF3 and rGRF3/35S:GIF1 stay green for a longer period of time than wild-type plants (FIG. 4).
To test whether there this delay in leaf senescence in rGRF3 and rGRF3×35S: GIF1 transgenic plants, a dark-induced senescence experiment was performed. Incubation of detached leaves in the dark induces senescence and this process can be followed by measuring the decrease in the maximum efficiency of photosystem II (PSII) photochemistry (Fv/Fm) as described previously (Baker, 2008; Schommer et al., 2008). To do this, the fifth leaf of wild-type, rGRF3, 35S:GIF1 and rGRF3×35S:GIF1 were collected and kept in the dark, and Fv/Fm was measured every day. As detailed in FIG. 4, there is no difference between wild-type and 35S:GIF1 plants. However, senescence in rGRF3 leaves starts 2 days after the wild-type leaves. Interestingly, leaves that co-overexpress high levels of both rGRF3 and GIF1 showed an even larger delay in Fv/Fm decay, indicating that overexpression of GIF1 enhances even further the senescence delay of rGRF3 plants.
Furthermore, the performance of the transgenic under water deprivation (FIG. 14) was assayed. 25 days-old plant of 35S:miR396, wild-type, rGRF3, 35S:GIF1 and rGRF3×35S:GIF1 were deprived of water for 2 weeks. Then, the plants were irrigated once a week. MiR396 over-expressers, wild-type and 35S:GIF1 were severely affected in their growth by the end of the water deprivation and subsequently to it (FIG. 14). In contrast, both rGRF3 and rGRF3×35S:GIF1 lines recovered and developed well following the water deprivation (FIG. 14).
Materials and Methods
To study leaf senescence, fifth-fully expanded leaves were detached and stored in darkness. Dark-induced senescence was followed by measuring Maximal Photochemical Efficiency (Fv/Fm) of Photosystem II, as described (Baker, 2008). In the water deprivation assays, plants were grown in long photoperiods (16 hour light/8 hour dark) at 23° C. When the plants were 25 day-old, they were deprived of water for two weeks. After that, the plants were irrigated once a week. Pictures were taken when the plants were 50 day-old.
Conclusions


    
    
        rGRF3 plants have a delay in leaf senescence. This effect is further enhanced by the co-overexpression of GIF1.
        rGRF3 plants are more tolerant to water deprivation.
    
    


Example #4
Expression from Tissue Specific Promoters Improves rGRF3 Performance in Plant Productivity
The GRF family of transcription factors comprises nine members in Arabidopsis (Kim et al., 2003). Seven of them, including GRF3, have a target site for miR396 (Jones-Rhoades and Bartel, 2004). MiR396 is expressed at low levels in the meristem and leaf primordia, and then it steadily accumulates with the development of the leaf, in concert with the retreat of cell proliferation (Rodriguez et al., 2010). It is shown in Examples #1 and #2 that the abolishment of miR396-repression of GRF3 in Arabidopsis generates plants with a significant increase in biomass accumulation and a delay in senescence.
To study if it is possible to improve further performance of rGRF3 this miR396 resistant version of GRF3 was expressed from tissue specific promoters. The promoters of AS1 (ASYMMETRIC LEAVES 1) and ANT (AINTEGUMENTA), which are known to be specifically expressed in the proliferative stages of leaf development, were selected (FIG. 50).
Transgenic Arabidopsis plants transformed with the vectors AS1:rGRF3 and ANT:rGRF3 had bigger leaves than wild-type plants and even than plants expressing the rGRF3 from the native GRF3 promoter (FIGS. 18 and 51). These plants also had thicker stems (FIG. 19).
Interestingly, the expression of rGRF3 from ANT and AS1 promoters had only a minor effect on leaf senescence, and less than that observed in rGRF3 plants expressing plants from the endogenous promoter (FIGS. 20 and 54).
Expression of rGRF3 from the ANT and AS1 promoters shows similar apical dominance (FIG. 52) to wild-type plants.
Materials and Methods
The Arabidopsis thaliana Columbia (Col-0) accession was used as a wild type. All transgenics are in the Col-0 background. Plants were grown in long photoperiods (16 hr light/8 hr dark) or in short photoperiods (8 hr light/16 hr dark) at 23° C. See Table 1 for a list of binary plasmids generated and details on how transgenics plants were prepared. The miRNA target motif in AtGRF3 was altered introducing synonymous mutations in a cloned AtGRF3 wild type genomic fragment using the QuikChange® Site Directed Mutagenesis Kit (Stratagene).
Leaf area was measured by first taking a photograph of detached fully expanded leaves, and then measuring the foliar area with the NIH software ImageJ. Finally stem diameter was measured in the lower part of the stem, 0.5 cm above the rosette.
Senescence phenotype was analyzed by dark-induced senescence experiments on fully expanded leaves #5. Pictures were taken just after the full expanded leaves were detached from the rosette (Day 1) and after they were incubated 6 days in darkness (Day 6). Chlorophyll degradation is an indicator of senescence (Schommer et al., 2008).
Conclusions


    
    
        Expression of rGRF3 form tissue specific promoters can improve its performance in plant productivity.
        Expression of rGRF3 from tissue specific promoters can uncouple the different functions of GRF3, such as the control of leaf size and senescence.
    
    


Example #5
rGRF3 Outperforms rGRF2 in Increasing Plant Size and Biomass Accumulation
As was previously showed, high levels of miR396 reduce considerably leaf (Rodriguez et al., 2010). On the other hand, plants expressing a miR396 resistant version of GRF2 (rGRF2) accumulate high levels of GRF2 that cause a slight decrease of leaf size (Rodriguez et al., 2010). It has been shown in Examples #1 and #2 that rGRF3 plants also accumulate more biomass than wild-type plants. This example shows that rGRF3 significantly outperforms rGRF2 in increasing plant size and biomass accumulation.
To compare biomass accumulation in rGRF2 and rGRF3 lines, we measured fresh and dry weight of 40 day-old rosettes of 35S:miR396, wild-type, rGRF2 and rGRF3 plants (FIG. 6). Plants with high levels of miR396 had a reduction of plant biomass of 25%. rGRF2 plants have only a minor increase in biomass accumulation that was not statistically significant (FIG. 6). rGRF3 rossettes accumulated nearly 40% more biomass compared to wild-type plants, which is statistically significant (FIG. 6).
Another remarkable difference between rGRF2 and rGRF3 plants was observed when comparing leaf morphology. Leaves of rGRF2 plants have downward “rolling” shape, while leaves of rGRF3 plants are bigger than wild-type leaves with no major change in leaf morphology (FIG. 10). In this way, rGRF3 produced plants with bigger leaves without affecting leaf morphology.
To analyze the correlation between biomass accumulation and GRF levels in rGRF2 and rGRF3 plants, one independent line of each rGRF transgenic line was selected. Then, GRF2 and GRF3 mRNA levels were measured by RT-PCR and the dry weight of 1 month-old rosettes of rGRF2 and rGRF3 plants. It was observed that a 25-fold increase in GRF2 mRNA levels in rGRF2 plants produced a biomass increase of only 30% (FIG. 11). On the contrary, only a 2.5 fold increase in GRF3 mRNA levels in rGRF3 plants resulted in almost twice as much biomass accumulation compared with wild type Col-0 (FIG. 11).
As a further comparison the effect of rGRF2 or rGRF3 expression with wild-type (FIG. 55) was compared. Leaf area in rGRF3 expressing plants was almost double that of wild-type and increased compared to rGRF2 (FIG. 55). When rGRF2 was placed under the control of the GRF3 promoter the increase in leaf area was not as significant as in rGRF3-expressing plants, showing that the differential activity of rGRF3 and rGRF2 is caused by their different primary sequences and not promoter strength and/or expression levels (FIG. 55).
Materials and Methods
The Arabidopsis thaliana Columbia (Col-0) accession was used as a wild-type. All transgenics are in the Col-0 background. Plants were grown in long photoperiods (16 hr light/8 hr dark) or in short photoperiods (8 hr light/16 hr dark) at 23° C. See Table 1 for a list of binary plasmids generated and details on how transgenics plants were prepared. The miRNA target motif in AtGRF3 or AtGRF2 was altered introducing synonymous mutations in a cloned AtGRF3 wild type genomic fragment using the QuikChange® Site Directed Mutagenesis Kit (Stratagene).
All constructs were cloned in the binary vector pCHF3 (Jarvis et al., 1998). T-DNA constructs were introduced into Agrobacterium tumefaciens strain ASE and Arabidopsis transgenics plants were obtained by floral-dip.
To determine biomass accumulation, complete rosettes were weighed to measure fresh weight. Then, tissue was dried at 60° C. during 2 days and dry weight was measured.
For expression analysis by RT-PCR, RNA was prepared from apices of 20-day-old plants grown in short photoperiods, including developing leaves smaller than 3 mm. 0.5 to 1.0 μg of total RNA was treated with RQ1 RNase-free Dnase (Promega).
Then, first-strand cDNA synthesis was carried out using SuperScriptTM III Reverse Transcriptase (Invitrogen). PCR reactions were performed in a Mastercycler® ep realplex thermal cycler (Eppendorf) using SYBRGreen I (Roche) to monitor dsDNA synthesis. qPCR for each gene was done on at least 3 biological replicates with technical duplicates for each biological replicate. The relative transcript level was determined for each sample, normalized using PROTEIN PHOSPHATASE 2A cDNA level (Czechowski et al., 2005).
Conclusions


    
    
        High levels of rGRF2 are required to slightly increase plant biomass (e.g., 25 times more GRF2 caused 30% biomass increase).
        Moderate increases of GRF3 expression in rGRF3 plants caused a high increase in biomass accumulation (e.g., 2.5 times more GRF3 caused 85% biomass increase).
        High levels of rGRF2 affect leaf development.
        Expression of rGRF3 in plants leads to approximately 2 times increase in leaf area compared to wild-type
        Increased leaf area in rGRF3 compared to rGRF2 is dependent on the primary sequence of the genes and not a result of promoter strength
    
    


Example #6
Arabidopsis GRF3 and GIF1 Homologues are Found in Crop Plants: GRF Family in Arabidopsis thaliana and Other Plant Species
The GROWTH-REGULATING FACTOR (GRF) family of transcription factors is a plant specific family of proteins defined by the presence of two highly conserved protein motifs, the QLQ and WRC (Kim et al., 2003). The QLQ domain is involved in protein-protein interactions with GRF-INTERACTING FACTORS proteins, and the WRC domain contains a functional nuclear localization signal and a DNA-binding motif consisting of three conserved cysteines and one histidine (Kim and Kende, 2004). The GRF family of transcription factors comprises nine members in Arabidopsis (Kim et al., 2003) (FIGS. 21 and 22), 12 in Oryza sativa (Choi et al., 2004) (FIGS. 23 and 24) and 14 in Zea mays (Zhang et al., 2008) (FIGS. 25 and 26). Besides, GRFs can be found in many other plant species (Zhang et al., 2011) (See selected examples from Glycine max, Medicago truncatula, Prunus persica, Carica papaya and Populus trichocarpa in FIGS. 27 to 34).
At least two other conserved regions can be found in GRF coding sequences. First, at the nucleotide level, only a subgroup of the GRFs from each species contains a miR396-target site. For example, only 7 of the nine GRFs found in Arabidopsis are miR396 targets (FIGS. 7 and 8) (Jones-Rhoades and Bartel, 2004).
Second, only a subgroup of the GRFs of each species contains the FFD conserved motif (FIG. 8). For example, in Arabidopsis only GRF3 and GRF4 have the FFD motif. Furthermore, GRFs containing the miR396-targeting motif and the FFD motif, and with high homology to Arabidopsis GRF3 can be found in rice, maize and many other plant species (FIGS. 7, 8, 22, 24, 26 31-34, 38).
GRFs Expression Patterns in Arabidopsis thaliana and Zea mays 
GRF3 expression pattern was analyzed by RT-qPCR in developing leaves (FIG. 15, left). The fifth rosette leaf was collected at three-day intervals, starting from the day that it first became visible ( ̃1 mm) to the naked eye, which was 16 days after sowing (DAS). Next, the level of GRF3 was determined by RT-qPCR. It was observed that this transcription factor was expressed during the early stages of leaf development (FIG. 15, left). An expression atlas of Arabidopsis development (Schmid et al., 2005) indicates that mitosis specific genes are expressed in proliferating tissues (FIG. 15, right). Consistent with a role of the GRFs as positive regulators of cell proliferation during organ growth, their expression profile is very similar to that of the mitosis specific genes (shown for GRF3 in FIG. 15, right).
To confirm the functional equivalency between Arabidopsis and Zea mays GRFs their expression patterns during maize leaf development were analysed using the Maize eFP browser (Li et al., 2010; Winter et al., 2007). As detailed in FIG. 16, maize GRFs, in the same way as Arabidopsis GRFs, are coexpressed with mitosis specific genes.
GRF-INTERACTING FACTORS in Arabidopsis and Crop Plants
As described in example #2, rGRF3 performance in plant productivity can be greatly enhanced by cooverexpression of GRF-INTERACTING FACTOR 1. This gene belongs to a small gene family composed by three members (GIF1, GIF2 and GIF3) in Arabidopsis. Also, GIF1 homologs are readily found in other plant species, such as rice (FIG. 9). The three GIFs in Arabidopsis are highly redundant, as mutants in GIF1 can be complemented by the overexpression of GIF2 or GIF3 (FIG. 43) (Lee et al., 2009). These results suggest that the enhancement of the rGRF3 phenotype by overexpression of GIF1 is also achieved by co-overexpression of GIF2 and GIF3.
Materials and Methods
RNA was prepared from apices of 20-day-old plants grown in short photoperiods, including developing leaves smaller than 3 mm. 0.5 to 1.0 μg of total RNA was treated with RQ1 RNase-free Dnase (Promega). Then, first-strand cDNA synthesis was carried out using SuperScriptTM III Reverse Transcriptase (Invitrogen). PCR reactions were performed in a Mastercycler® ep realplex thermal cycler (Eppendorf) using SYBRGreen I (Roche) to monitor dsDNA synthesis. qPCR for each gene was done on at least 3 biological replicates with technical duplicates for each biological replicate. The relative transcript level was determined for each sample, normalized using PROTEIN PHOSPHATASE 2A cDNA level. Primer sequences are given in Table 2.
GRFs sequences from Arabidopsis thaliana, Oryza sativa and Zea maize were obtained from Genebank using the accession numbers provided in the literature (Choi et al., 2004; Kim et al., 2003; Zhang et al., 2008). Pairwise sequence alignments and calculations of percentage of identity and similarity were performed with NEEDLE using the Needleman-Wunche alignment algorithm (Rice et al., 2000). Multiple sequence alignments of protein sequences were performed using MCOFFE (Moretti et al., 2007). The PHYLIP package version 3.67 (Felsenstein, 1989) was used to perform 100 bootstrap replicas of a neighbor joining (NJ) tree based on a JTT distance matrix. Trees were visualized using TreeView 1.6.6. (Page, 1996).
Conclusions


    
    
        GRFs in general and homologs (orthologues) of GRF3 in particular exist in many plant species.
        GIFs also exist in many plant species.
        According to its function as a positive regulator of cell proliferation, GRF3 is co-expressed with mitosis genes during leaf development in Arabidopsis. As expected for functional equivalent genes, Zea mays GRFs expression also co-expressed with mitosis genes during leaf development.
        The enhancement of the rGRF3 phenotype by overexpression of GIF1 might also be achieved by homologs (orthologues) from Arabidopsis and crop plants.
    
    


Example #7
Introduction of rGRF3 and rGRF3+GIF into Brassica oleracea 
Materials and Methods
Plant Material
A genetically uniform doubled haploid Brassica oleracea genotype, DH 1012 (Sparrow et al., 2004) was used in this study. This genotype is derived from a cross between a rapid cycling B. oleracea alboglabra (A12) and a B. oleracea Italica Green Duke (GD33).
Bacterial Strains
Transformations were carried out using the Agrobacterium tumefaciens strain AGL1 harbouring the appropriate plasmids pBRACT114 rGRF3 and pBRACT114 rGRF3:GIF1 and (see FIG. 44) containing the neomycin phosphotransferase (nptII) selectable marker gene driven by the 35S promoter and the gene(s) of interest (namely rGRF3 driven by its own promoter; or the combined construct which contained both rGRF3 driven by its own promoter, and additionally GIF driven by the 35S promoter, respectively).
The cloning procedure used to make the transformation vector pBRACT114-rGRF3 GIF1 is described below. pBRACT114-rGRF3 GIF1 contains both the rGRF3 gene driven by its native promoter and the coding region of GIF1 over-expressed by the CaMV 35S promoter.
Digestion of  ̃1.7 μg of pGRF3:GRF3r DNA in a 20 μl total volume reaction with PvuII (Invitrogen) in the appropriate buffer was performed at 37° C. for 1 hour in a water bath. A 4950 bp fragment containing the rGRF3 native promoter, coding region, 3′UTR and terminator was isolated by gel extraction.
The Brassica transformation vector pBRACT114 (www.bract.org) is based on pGreen (Hellens et al., 2000) and is GatewayTM (Invitrogen) compatible. Approximately 1 μg of pBract114 was digested with restriction enzyme StuI (Roche) in the appropriate buffer for 1 hour at 37° C. The linearised vector was dephosphorylated by incubation at 37° C. for a further hour with shrimp alkaline phosphatase (SAP). The SAP was denatured by heating to 65° C. for 15 minutes.
An overnight ligation reaction was performed at 14° C. and contained the rGRF3 fragment and the linear pBRACT114 at a 3:1 ratio respectively. Five units of T4 ligase (Invitrogen) were used in the 10 μl blunt end ligation. To 50 μl of ccdB competent E. coli cells (Invitrogen) 2 μl of the ligation reaction was added and transformation by heat shock. The cells were grown in 250 μl of SOC medium for 1 hour at 37° C. and shaken at 200 rpm. 20 μl and 100 μl of the culture was spread onto plates of solid LB medium (Sambrook and Russel, 2001) containing appropriate selection and incubated overnight at 37° C.
E. coli colonies were screened by direct colony PCR to ensure that they contained pBRACT114 with the insert in the desired orientation. Twelve PCR positive single colonies were transferred to 10 ml of liquid LB media containing the appropriate selection and incubated at 37° C. shaken 220 rpm overnight. Plasmid DNA was isolated using a mini-prep kit (Qiagen). The integrity of the construct known as pBRACT114-rGRF3 was confirmed by enzyme digestion and sequencing of the insertion sites.
Phase two of the cloning process to create pBRACT-rGRF3 GIF1 used the GatewayTM (Invitrogen) system to recombine the coding region of GIF1 downstream of the CaMV 35S promoter. The coding region of GIF1 was amplified by PCR using high fidelity PlatinumTM polymerase (Invitrogen) and Topo T/A cloned into the GatewayTM entry vector pCR8/GW/Topo® TA (Invitrogen). To 50 μl of chemically competent E. coli DH5-α cells (Invitrogen) 2 μl of the Topo reaction was added and transformation by heat shock. The cells were grown in 250 μl of SOC medium for 1 hour at 37° C. and shaken at 200 rpm. 20 μl and 100 μl of the culture was spread onto plates of solid LB medium (Sambrook and Russel, 2001) containing appropriate selection and incubated overnight at 37° C.
E. coli colonies were screened by direct colony PCR to ensure that they contained pCR8 with the GIF1 amplicon in the desired orientation. Six PCR positive single colonies were transferred to 10 ml of liquid LB medium containing the appropriate selection and incubated at 37° C. shaken 220 rpm overnight. Plasmid DNA was isolated using a plasmid mini-prep kit (Qiagen). The entry vector pCR8-GIF1 was checked by enzyme digestion. Sequencing of the entire GIF1 coding region was performed to ensure its integrity.
A GatewayTM LR recombination reaction was performed to insert the GIF1 coding region into pBRACT114-rGRF3 between the gateway sites downstream of the CaMV 35S promoter. The 10 μl LR reaction contained  ̃100 ng of pBRACT114-rGRF3+35 ng of pCR8-GIF1 with 2 μl Gateway® LR ClonaseTM II enzyme MixTM (Invitrogen) in TE buffer. The LR reaction was incubated at room temperature overnight. A proteinase K treatment was performed a 37° C. for 10 minutes. To 50 μl of chemically competent E. coli DH5-α cells (Invitrogen) 1 μl of the LR reaction was added and transformation by heat shock. The cells were grown in 250 μl of SOC medium for 1 hour at 37° C. and shaken at 200 rpm. 20 μl and 100 μl of the culture was spread onto plates of solid LB medium (Sambrook and Russel, 2001) containing appropriate selection and incubated overnight at 37° C.
Twelve single colonies were transferred to 10 ml of liquid LB media containing the appropriate selection and incubated at 37° C. shaken 220 rpm overnight. Plasmid DNA was isolated using a mini-prep kit (Qiagen). The integrity of the construct known as pBRACT114-rGRF3 GIF1 was confirmed by enzyme digestion and sequencing of the GIF1 insertion sites.
The plasmid pBRACT-rGRF3 GIF1 along with its helper plasmid pSoup (Hellens et al., 2000) was transformed into Agrobacterium tumefaciens strain AGL1 by electroporation. The plasmid pGRF3:rGRF3 was also transformed by electroporation into A. tumefaciens. Briefly, 100 ng of plasmid DNA was added to 40 μl of electro-competent A. tumefaciens cells in a pre-chilled electroporation cuvette with 2 mm electrode separation. The cells were electroporated in a GenePulser (Biorad) with the following settings 2.50 kV, 25 uFD and 400 Ohms. Immediately 300 μl of liquid LB medium was added to recover the cells, these were grown at room temperature, shaken at 180 rpm for 6 hours. The A. tumefaciens cultures were spread onto solid LB medium (Sambrook and Russel, 2001) containing appropriate selection and incubated at 28° C. for 48 hours. Single colonies were selected and used to inoculate 10 ml of liquid LB media containing the appropriate antibiotics and incubated at 28° C., shaken at 200 rpm for 48 hours. Glycerol stocks and standard inoculums were prepared and stored at −80° C. The plasmids were checked once again, by enzyme digestion, prior to embarking on the Brassica transformation experiments.
The A. tumefaciens was streaked onto solid LB medium (Sambrook and Russel, 2001) containing appropriate selection (and incubated at 28° C. for 48 hours. A single colony was transferred to 10 ml of liquid LB media containing the appropriate selection and transferred to a 28° C. shaker for 48 hours. A 50 μl aliquot of the resulting bacterial suspension was transferred to 10 ml of MGL liquid medium with selection and grown over night in a 28° C. shaker. Overnight cultures were spun down at 3,000 rpm for 5 minutes at R.T. before being re suspended in liquid MS medium. Suspensions of O.D650=0.3 were used for inoculations (dilutions made using liquid MS medium).
Plant Transformation
Seeds were surface sterilised in 100% ethanol for 2 minutes, 15% sodium hypochlorite plus 0.1% Tween-20 for 15 minutes and rinsed three times for 10 minutes in sterile distilled water. Seeds were germinated on full strength MS (Murashige and Skoog, 1962) plant salt base, containing 3% sucrose and 0.8% phytagar (Difco) at pH 5.6. Prior to pouring, filter-sterilised vitamins were added to the medium; myo-Inositol (100 mg/l), Thiamine-HCL (10 mg/l), Pyridoxine (1 mg/l) and Nicotinic acid (1 mg/l). Seeds were sown at a density of 15 seed per 90 mm petri dish and transferred to a 10° C. cold room overnight before being transferred to a 23° C. culture room under 16 hour day length with 70 μmol m−2 sec−1 illumination.
Based on the transformation protocol developed for Brassica napus (Moloney et al. 1989), and further developed by BRACT (www.bract.org), cotyledonary petioles excised from 4-day-old seedlings were dipped into an overnight suspension of Agrobacterium. Explants were maintained, 10 explants per plate, on co-cultivation medium (germination medium supplemented with 2 mg/l 6-benzylaminopurine); with the petioles embedded and ensuring the cotyledonary lamella were clear of the medium. Cultures were maintained in growth rooms at 23° C. with 16 hour day length, under scattered light of 40 μmol m−2 sec−1 for 72 hours. After 72 hours explants were transferred to selection medium (co-cultivation medium supplemented with 160 mg/l timentin (or appropriate Agrobacterium eliminating antibiotic) and 15 mg/l kanamycin as the selection agent. Controls were established on kanamycin-free medium, as explants that had, and had not, been inoculated with Agrobacterium. 
Shoot Isolation and Plant Regeneration
Regenerating green shoots were excised and transferred to Gamborgs B5 medium (Gamborg et al. 1968), containing 1% sucrose, 0.8% Phytagar, 160 mg/l timentin and 50 mg/l kanamycin. Where dense multiple shoots were isolated, further sub-culturing was made after shoot elongation to ensure a main stem was isolated thus reducing the likelihood of escapes and the frequency of multi-stemmed plants when transferred to the glasshouse. Shoots were maintained on Gamborgs B5 medium until roots developed. Plantlets were then transferred to sterile peat pots (Jiffy No. 7) to allow further root development, before being transferred to the glasshouse.
Plant Maintenance and Seed Production
Transgenic plants were maintained in a containment lit glasshouse (of 16-hour photoperiod, +18/12° C. day/night) and self-pollinated, to generate the T1 seed. Plants were covered with clear, perforated ‘bread-bags’ (Cryovac (UK) Ltd) as soon as they came into flower to prevent cross-pollination. The background genotype DH1012 is a self-compatible genotype and daily shaking of the ‘bread-bag’ was carried out to facilitate pollination. Pods were allowed to develop on the plant until fully swollen and were harvested when pods had dried and turned brown. Harvested pods were threshed when dry, and seed stored in the John Innes Centre seed store (+1.5° C., 7-10 relative humidity).
Molecular Analysis
Leaf tissue from putative transgenic shoots (in vitro) was used for initial DNA extractions to PCR test for presence of the transgenes.
Copy Number Analysis by Multiplexed Real Time PCR
The copy number of the transgene was measured using multiplexed real time PCR (TaqMan) assays, carried out by ‘iDNA genetics’ (www.idnagenetics.com). The nptII target gene was detected using a Fam labelled, Tamra quenched probe, and simultaneously an internal positive control gene was detected using a Vic labelled, Tamra quenched probe. The reactions were carried out using 5-20 ng of genomic DNA from each sample, in a 20 μl reaction volume, with each sample assayed twice. The cycle threshold (Cts) for the Fam and Vic signals were found for each tube, and the average DeltaCt (CtFam−CtVIC) calculated for each sample. The samples were ranked by DeltaCt (where high delta Ct relates to samples with low numbers of copies, and low DeltaCt to high numbers of copies). Plant samples were classified with respect to reference samples (of known copy number).
Preliminary investigations show that enhanced growth and improved plant productivity is obtained in Brassica plants comprising the AtrGRF3 or AtrGRF3:GIF1
FIG. 49 shows data comparing Brassica oleracea plants transformed with Arabidopsis rGRF3 and control plants (without the At rGRF3). Transforming Brassica oleracea plants with At rGRF3 significantly improved growth and productivity of the plants. For example, at flowering the stem width 10 cm above soil level and the maximum stem width at flowering were both significantly greater in Brassica oleracea plants transformed with At rGRF3 compared with control plants. These results were significant using either the t-test (p<0.01) or regression analysis (p=0.008).
FIG. 56 shows data for Brassica oleracea plants transformed with Arabidopsis rGRF3 (rGRF3) and a control of regeneration (TC). The widest stem width at flowering is increased in rGRF3 when compared to the control (FIG. 56). The figure also shows that the 10 cm stem weight is increased in rGRF3 when compared to the control (FIG. 56).
Root growth of transgenic Brassica oleracea plants expressing Arabidopsis rGRF3 was measured. To do this, wild-type and transgenic plants were grown in vertical MS plates. Root length was measured in at least 10 plants for each genotype from 4 to 7 days after sowing (FIG. 57, left). From the slope of these lines, the root growth rate was estimated (FIG. 57, right).
Conclusions


    
    
        Transgenic Brassica oleracea plants expressing Arabidopsis rGRF3 and rGRF3:GIF1 show enhanced growth and improved plant productivity.
        Transgenic Brassica oleracea plants transformed with the miR396-resistant version of GRF3 (named rGRF3) show a striking increase in root growth.
    
    


Example #8
Expression in Arabidopsis of GRF3 Orthologues from Soybean and Rice Also Increases Plant Biomass
In Example #6, GRFs from other species than Arabidopsis were described. To test if these GRFs behave in a similar way to Arabidopsis rGRF3, selected sequences were introduced into Arabidopsis. The GRFs with the highest homology to At-rGRF3 and containing a FFD motif and a miR396 target site were selected from rice (FIG. 37) and soybean (FIG. 36). The GRF3 from soybean and rice were uncoupled from miR396 control by introducing mutations in the miRNA binding site as described previously for Arabidopsis GRF3.
A vector expressing these sequences from the Arabidopsis GRF3 promoter was prepared and then, Arabidopsis transgenic plants were obtained. In a similar way to plants expressing At-rGRF3, transgenic Arabidopsis plants expressing Os-rGRF4 and Gm-rGRF had bigger leaves than wild-type plants (FIG. 46). These transgenic plants expressing the soybean and rice rGRF3 orthologues also had a delay in leaf senescence (not shown).
Materials and Methods
The Arabidopsis thaliana Columbia (Col-0) accession was used as a wild type control. All transgenics are in the Col-0 background. Plants were grown in long photoperiods (16 hr light/8 hr dark) or in short photoperiods (8 hr light/16 hr dark) at 23° C. See Table 1 for a list of binary plasmids generated and details on how transgenics plants were prepared. The miRNA target motif in OsGRF4 and Gm-GRF was altered introducing mutations using the QuikChange® Site Directed Mutagenesis Kit (Stratagene) as described previously for Arabidopsis GRF3. The mutated miR396 motif in Os-GRF4 and Gm-GRF is shown FIG. 37 and FIG. 36 respectively.
All constructs were cloned in the binary vector pCHF3 (Jarvis et al., 1998). T-DNA constructs were introduced into Agrobacterium tumefaciens strain ASE and Arabidopsis transgenics plants were obtained by floral-dip.
Leaf area was measured by first taking a photograph of detached fully expanded leaves, and then measuring the foliar area with the NIH software ImageJ (as described in Example #1 and other examples above).
Conclusions
rGRF3 orthologues from species other than Arabidopsis (e.g. at least rice and soybean) species can also increase plant size and biomass accumulation.
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## Claims

1. A method of producing a plant with increased productivity compared to a wild-type plant, comprising transforming a plant with a construct comprising an isolated nucleic acid encoding a growth regulatory factor (AtGRF-3) of SEQ ID NO: 20, which nucleic acid comprises a modification in the miRNA396 target site as set forth in SEQ ID NO: 86, operably linked with a native AtGRF3 promoter and a terminator.

2. The method of claim 1 wherein the increased productivity is selected from the group consisting of increased yield, increased biomass, increased stress resistance, increased drought tolerance, delayed leaf senescence, increased seed production, increased seed yield, increased root growth and increased root elongation speed and combinations thereof, compared to the wild-type plant.

3. The method according to claim 2 wherein the increased biomass compared to the wild-type plant comprises one or more of the following selected from the group consisting of: increased overall plant biomass, increased fresh weight, increased leaf area or size, increased root length, increased stem growth, increased stem biomass, increased stem diameter, and increased stem width at flowering compared to the wild-type plant.

4. The method according to claim 2 wherein the increased productivity compared to the wild-type plant occurs without leaf shape changes.

5. The method according to claim 2 wherein the increased biomass compared to the wild-type plant is uncoupled from delayed leaf senescence by using tissue specific promoters.

6. The method of claim 1 wherein the method further comprises overexpressing a nucleic acid encoding a GRF-interacting factor (GIF), wherein the GIF comprises SEQ ID NO: 95.

7. The method of claim 1, wherein the nucleic acid encoding said growth regulatory factor (atGRF-3) comprises SEQ ID NO: 81.

8. The method of claim 6, wherein the nucleic acid encoding said growth regulatory factor (atGRF-3 comprises SEQ ID NO: 81.

9. The method of claim 7, wherein the increased productivity compared to the wild-type plant is selected from the group consisting of increased yield, increased biomass, increased stress resistance, increased drought tolerance, delayed leaf senescence, increased seed production, increased seed yield, increased root growth, increased root elongation speed and combinations thereof.

10. The method of claim 8, wherein the increased productivity compared to the wild-type plant is selected from the group consisting of increased yield, increased biomass, increased stress resistance, increased drought tolerance, delayed leaf senescence, increased seed production, increased seed yield, increased root growth, increased root elongation speed and combinations thereof.