The present invention relates generally to methods for generating plants having changed architecture and to plants so generated and parts of these plants. More particularly, the present invention relates to a method for modifying a plant so as to produce a plant exhibiting an altered phenotype. Plants and parts of plants, such as flowering and reproductive parts including seeds, also form part of the present invention. The ability to modify the phenotype of a plant may be useful for producing plants with more highly desired characteristics.
Manipulation of plant architecture has been one of the greatest mainstays of plant improvement—perhaps second only to the discoveries of the nutritional requirements of plants. With the advent of the ‘gene revolution’, there are countless new opportunities for selective modification of plant architecture
Plants are complex structures which can be described in many different ways depending on the requirements of the application, e.g. for bio-mechanics, hydraulic architecture or micrometeorology, or for simulation of plant growth. There is a general agreement that plants can be regarded as a collection of components having specific morphological characteristics, organized at several scales (White, 1979; Barthelemy, 1991). Plant architecture is a term applied to the organization of plant components in space, which can change with time. At a given time, plant architecture can be defined by topological and geometric information. Topology deals with the physical connections between plant components, while geometry includes the shape, size, orientation and spatial location of the components.
Plant architecture is defined as the three dimensional organization of the plant body. For the parts that are above ground, this includes the spatial arrangement of leaves and other photosynthetic organs and floral organs on stems and branching pattern. Plant architecture is even today the best means of identifying a plant species and has been the only criterion for systematic and taxonomic classification for a long time (Reinhardt and Kuhlemeier, 2002). Since the leaves collect solar energy and are surfaces for gas exchange, their arrangement in plant canopies is crucial for light interception and photosynthesis. Interception of light by the plants is dependent on the plant architecture. In both natural and agricultural systems, thus plant fitness and yield are affected by plant architecture. The plants to maximize canopy light interception have evolved different adaptive traits and plant architecture is one of the major adaptive traits. To maximize light interception the modified plant architecture include modification of size, shape, angle of leaf, plant height, branches and tillers. Some plants can modify their canopy architecture transiently to maximize light interception.
Plant architecture is a genetically controlled trait and therefore it s heritable, nevertheless environmental factors, both abiotic and biotic, can modify canopy architecture. Abiotic factors that affect canopy architecture include soil moisture content, nutrient availability, temperature and light. While biotic factors include herbivores, pathogens and competition with other plants.
Plant architecture is of major agronomic importance, strongly influencing the suitability of plants for cultivation and yield. In agriculture the yield improving plant architecture can increase potential of crops. Dwarf cultivars have been developed with modified canopy architectures capable of better light interception in different crops (Coyne, 1980). One of the greatest successes of the green revolution, which led to major increase in productivity was based on the modification of plant architecture where the selection of dwarf wheat varieties with short and sturdy stems helped the plants to resist damage from wind and rain resulting in higher yield (Peng et al., 1999).
Abiotic factors that can affect plant architecture include resources for plant growth such as soil moisture, temperature and light, under sufficient supply of these resources plants attain growth rate close to their genetic potential with maximum fitness and express typical architectures. However under scarce supply of these resources plants undergo physiological and growth changes leading to modified architecture for increasing their fitness.
Plants continuously form new leaves that are arranged in regular patterns this is called phyllotaxis. Inhibition of auxin transport, either by a mutation in the auxin transport protein PIN1 or by chemical inhibitors of auxin transport, specifically abolishes organ formation at the shoot apical meristem (SAM), whereas stem growth and meristem perpetuation are not affected resulting in the formation of pinelike stalks (Okada et al., 1991; Reinhardt et al., 2000). P-glycoproteins PGPs are plasma membrane anion transporters PGPs in Arabidopsis transport the hormone auxin, which controls cell elongation, plant shape, root branching and fruit development. pgp mutants examined thus far have reduced auxin transport and are dwarfs that have varying degrees of tropic responses (Murphy et al., 2000; Noh et al., 2001). AVP1, a pyrophosphate-driven proton pump, is important in the establishment and maintenance of auxin gradients required for root growth and development. Plants that overexpress AVP1 (AVP1OX) have greater shoot & root mass & surface area. AVP1 is highly conserved across the plant kingdom, with similar effects of overexpression being observed in Arabidopsis, tomato and rice (Gaxiola et al., 2001; Drozdowicz et al., 200).
TWD is an immunophilin-like protein with a putative plasma membrane GPI anchor. TWD interacts with many proteins within the plant, including PGPs. pgp1 pgp19 double mutants resemble twd mutants, indicating that TWD mediates interactions between PGPs and other proteins. twd mutants are dwarfs, and all anatomical features have hypemutation resulting in shorter plants with organs that twist, notably the stems, leaves, and flowers, resulting delightfully unusual looking plants (Kamphausen et al., 2002; Geisler et al., 2003).
Recent evidence has implicated Trehalose-6-Phospate synthase (TPS) genes as important modulators of plant development and inflorescence architecture. In one example, trehalose appears to modulate inflorescence branching in maize (Satoh-Nagasawa et al., 2006). Inflorescence branching in maize is controlled by the RAMOSA genes, and one of the genes (RAMOSA3) encodes a trehalose biosynthetic gene that functions through the regulation of the transcription factor RAMOSA1 (Satoh-Nagasawa et al., 2006). Chary et al., 2008 have provided evidence indicating that class II TPS gene functions in the control of cell morphology in addition to functioning as a broad modifier of whole plant developmental phenotypes. They identified a cell shape phenotype-1 (csp-1) mutant that has a dramatic cellular effect in the leaf epidermis, resulting in loss of pavement cell lobes. In addition, csp-1 was shown to impact the cell morphology of trichomes, resulting in an altered pattern of branching. The mutant shows a range of developmental defects that include reduced stature, altered stem branching, and pronounced leaf serrations.
Gamma-Amino butyric acid (GABA) is a four-carbon non-protein amino acid conserved from bacteria to plants and vertebrates. GABA is a significant component of the free amino acid pool. GABA has an amino group on the g-carbon rather than on the a-carbon, and exists in an unbound form. It is highly soluble in water: structurally it is a flexible molecule that can assume several conformations in solution, including a cyclic structure that is similar to prolinel. GABA is zwitterionic (carries both a positive and negative charge) at physiological pH values (pK values of 4.03 and 10.56).
It was discovered in plants more than half a century ago, but interest in GABA shifted to animals when it was revealed that GABA occurs at high levels in the brain, playing a major role in neurotransmission. Thereafter, research on GABA in vertebrates focused mainly on its role as a signaling molecule, particularly in neurotransmission. In plants and in animals, GABA is mainly metabolized via a short pathway composed of three enzymes, called the GABA shunt because it bypasses two steps of the tricarboxylic acid (TCA) cycle. The pathway is composed of the cytosolic enzyme glutamate decarboxylase (GAD) and the mitochondrial enzymes GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). The regulation of this conserved metabolic pathway seems to have particular characteristics in plants.
The pathway that converts glutamate to succinate via GABA is called the GABA shunt. The first step of this shunt is the direct and irreversible a-decarboxylation of glutamate by glutamate decarboxylase (GAD, EC 4.1.1.15). In vitro GAD activity has been characterized in crude extracts from many plant species and tissues (Brown & Shelp, 1989). GAD is specific for L-glutamate, pyridoxal 5′-phosphate-dependent, inhibited by reagents known to react with sulfhydryl groups, possesses a calmodulin-binding domain, and exhibits a sharp acidic pH optimum of ˜5.8. GAD genes from Petunia (Baum et al., 1993), tomato (Gallego et al., 1995), tobacco (Yu & Oh, 1998) and Arabidopsis (Zik et al., 1998) have been identified. The second enzyme involved in the GABA shunt, GABA transaminase (GABA-T; EC 2.6.1.19), catalyzes the reversible conversion of GABA to succinic semialdehyde using either pyruvate or a-ketoglutarate as amino acceptors. In crude extracts, in vitro GABA-T activity appears to prefer pyruvate to a-ketoglutarate. However, distinct pyruvate-dependent and aketoglutarate-dependent activities are present in crude extracts of tobacco leaf, and these can be separated from each other by ion exchange chromatography (Van Cauwenberghe & Shelp). Both activities exhibit a broad pH optimum from 8 to 10. The Michaelis constants (Km) of a pyruvate-specific mitochondrial GABA-T from tobacco, purified ˜1000-fold, are 1.2 mM for GABA and 0.24 mM for pyruvate (Van Cauwenberghe & Shelp).
The last step of the GABA shunt is catalysed by succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.16), irreversibly oxidizing succinic semialdehyde to succinate. The partially purified plant enzyme has an alkaline pH optimum of ˜9; activity is up to 20-times greater with NAD than with NADP (Shelp et al., 1995).
Indeed, interest in the GABA shunt in plants emerged mainly from experimental observations that GABA is largely and rapidly produced in response to biotic and abiotic stresses. The GABA shunt has since been associated with various physiological responses, including the regulation of cytosolic pH, carbon fluxes into the TCA cycle, nitrogen metabolism, deterrence of insects, protection against oxidative stress, osmoregulation and signaling.
This is for the first time a method employing the glutamate decarboxylase gene to change the morphological architecture in plants has been demonstrated. Attempts have been made in this direction using genes like P-glycoproteins, auxin transporters, plant harmones and proton pumps. No attempt has been made till date to use genes involved in the GABA shunt pathway, specifically glutamate decarboxylase to change the architecture of the plants. Previous attempts directed at two glutamate decarboxylase genes from rice OsGAD1 and OsGAD2, which were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Regenerated rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility (Akama & Takaiwa, 2007).
The present invention relates of a method of changing the plant architecture (in both monocotyledons and dicotyledons) via Agrobacterium-mediated transformation with a glutamate decarboxylase gene. Further more the present invention relates to a method of plant modification to express genes, related to plant architecture and to the plants produced using this method.
Compositions and methods for altering the architecture of the plants by manipulation of GAD gene family in transgenic plants are provided.
The present invention provides nucleotides sequences of GAD gene. The nucleotide sequence and polypeptides of the invention include GAD gene, protein and functional fragments or variants thereof.
The methods of the invention comprise introducing into a plant a nucleotide sequence and expressing the corresponding polypeptide within the plant. The sequences of the invention can be used to alter plant architecture, carbon and nitrogen partitioning, enhanced biomass and or improved harvestable yield in plants. The methods of the invention find use in improving biomass and harvestable yield of the plants.
Additionally provided are transformed plants, plant tissues, plant cells, seeds, and leaves. Such transformed plants, tissues, cells, seeds, and leaves comprise stably incorporated in their genomes at least one copy of a nucleotide sequence of the invention.
One embodiment of the invention is a method for plant characteristics, the method comprising:
a. introducing into a plant cell a recombinant expression cassette comprising a nucleotide sequence whose expression, alone or in combination with additional polynucleotides, functions as an effector of nitrogen use efficiency within the plant;
b. culturing the plant cell under plant forming conditions to produce a plant; and,
c. inducing expression of the nucleotide sequence to alter the architecture of the plant.
SEQ ID 1 shows the nucleic acid sequence of Oryza saliva glutamate decarboxylase gene. The start and stop codons are in italic.
SEQ ID 0.2 shows amino acid sequence of Oryza saliva glutamate decarboxylase gene. The asterisk denotes the stop codon.
The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description of the invention should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein may be made by those of ordinary skill in the art without departing from the spirit or scope of the present invention.
This invention relates to a purified and isolated DNA sequence having characteristics of glutamate decarboxylase.
According to the present invention, the purified and isolated DNA sequence usually consists of a glutamate decarboxylase nucleotide sequence or a fragment thereof.
Included in the present invention are as well complementary sequences of the above-mentioned sequences or fragment, which can be produced by any means.
Encompassed by this present invention variants of the above mentioned sequences, that is nucleotide sequences that vary from the reference sequence by conservative nucleotide substitutions, whereby one or more nucleotides are substituted by another with same characteristics.
According to the present invention, the above mentioned nucleotide sequences could be located at both the 5′ and the 3′ ends of the sequence containing the promoter and the gene of interest in the expression vector.
Included in the present invention are the use of above mentioned sequences in altering the architecture of the plants produced thereof. “plant architecture” means that after introduction of DNA sequence under suitable conditions into a host plant, the sequence is capable of enhancing the leaf size, internodal distance, stem thickness, biomass and the harvestable yield in the plants as compared to control plants where the plants are not transfected with the said DNA sequence.
The following definitions are used in order to help in understanding the invention.
“Chromosome” is organized structure of DNA and proteins found inside the cell.
“Chromatin” is the complex of DNA and protein, found inside the nuclei of eukaryotic cells, which makes up the chromosome.
“DNA” or Deoxyribonucleic Acid, contain genetic informations. It is made up of different nucleotides.
A “gene” is a deoxyribonucleotide (DNA) sequence coding for a given mature protein. “gene” shall not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or enhancers.
“Promoter” is a nucleic acid sequence that controls expression of a gene.
“Enhancer” referes to the sequence of gene that acts to initiate the transcription of the gene independent of the position or orientation of the gene.
The definition of “vector” herein refers to a DNA molecule into which foreign fragments of DNA may be inserted. Vectors, usually derived from plasmids, functions like a “molecular carrier”, which will carry fragments of DNA into a host cell.
“Plasmid” are small circles of DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome.
“Transcription” refers the synthesis of RNA from a DNA template.
“Translation” means the synthesis of a polypeptide from messenger RNA.
“Orinetation” refers to the order of nucleotides in the DNA sequence.
“Gene amplification” refers to the repeated replication of a certain gene without proportional increase in the copy number of other genes.
“Transformation” means the introduction of a foreign genetic material (DNA) into plant cells by any means of trasnfer. Different method of transformation includes bombardment with gene gun (biolistic), electroporation, Agrobacterium mediated transformation etc.
“Transformed plant” refers to the plant in which the foreign DNA has been introduced into the said plant. This DNA will be a part of the host chromosome.
“Stable gene expression” means preparation of stable transformed plant that permanently express the gene of interest depends on the stable integration of plasmid into the host chromosome.
While the invention is broadly as defined above, it will be appreciated by those persons skilled in the art that it is not limited thereto and that it also includes embodiments of which the following description gives examples.
The GAD gene is cloned downstream of a 35S cauliflower mosaic virus promoter and terminated with a NOS terminator, all operably linked.
Oryza saliva (cv Rasi) was used for preparation of nucleic acids. After germination of the seeds, they were grown in hydroponic solution in a culture room. The seedlings were treated with 150 mM NaCl for 7-16 h.
The RNA was extracted from the whole seedlings. An EST library of the salt stressed RASI cDNA was constructed. An EST showing identity to glutamate decarboxylase was identified from the EST library.
GABA accumulates in higher plants following the onset of a variety of stresses such as acidification, oxygen deficiency, low temperature, heat shock, mechanical stimulation, pathogen attack, drought and salt stress. Glutamate decarboxylase, the gene in the GABA shunt has been isolated from the salt stressed library of O. sativa.
The Glutamate decarboxylase gene has been cloned into a cloning vector and also into plant transformation vectors (biolistic and binary) under a constitutive promoter. The cDNA encoding the complete coding sequence of glutamate decarboxylase gene was amplified from the indica rice (cv. RASI) cDNA using the following pairs of primers tagged with BglII and EcoRI restriction enzyme sites (underlined nucleotide sequences)
Using the following PCR conditions 94° C. for 1 min; 94° C. for 30 sec; 75° C. for 3 min (cycled for five times); 94° C. for 30 sec; 68° C. for 3 min (cycled for 30 times) with a final extension of 68° C. for 7 min.
The amplified cDNA consists of 1479 base pairs of nucleotides and encodes for a mature glutamate decarboxylase enzyme.
The amplified fragment was cloned into pGEMT easy vector. The gene was restriction digested at BamHI and EcoRI sites and ligated into a biolistic vector pV1. This biolistic vector was excised at BglII and EcoRI restriction sites (BglII and BamHI enzymes are isoschizomers) to confirm the presence of the gene. The gene was also confirmed by sequencing. The resultant vector (pV1-GAD) has the GAD gene (1.479 kb) driven by 35S Cauliflower mosaic virus (35S CaMV) promoter and NOS terminator along with the ampicillin resistance gene as a selectable marker.
The gene cassette, GAD gene driven by the CaMV promoter and terminated by the NOS terminator from pV1-GD was restriction digested at HindIII and BamHI sites. This gene cassette was ligated into pCAMBIA 1390 pNG15 which was restriction digested at HindIII and BamHI sites. The resultant vector (pAPTV 1390-GAD) has the GAD gene (1.479 kb) driven by 35S cauliflower mosaic virus (35S CaMV) promoter and terminated by NOS terminator along with the nptII (Kanamycin resistance) gene and hph gene (Hygromycin resistance) as selectable markers (
The Glutamate decarboxylase gene has been transformed via Agrobacterium into tobacco (model plant) and rice (crop plant) to arrive at the proof of concept for the identified gene.
Detailed Steps Involved in Agrobacterium Mediated Transformation of Tobacco Leaf Explants with a Binary Vector Harboring Gad Gene:
Leaf samples of transgenic GAD tobacco plant were collected and genomic. DNA was extracted.
1. PCR with Hygromycin Forward (Hyg F) & Hygromycin Reverse (Hyg R) primers:
3 U/μl
The amplified product was visualized on 0.8% agarose gel as shown in
2. PCR with Gene specific primers GAD Forward (GD F) & GAD Reverse (GD R):
3 U/μl
The amplified product was visualized on 0.8% agarose gel as shown in
3. PCR with GD F & Nos MR:
3 U/μl
The amplified product was visualized on 0.8% agarose gel as shown in
Primer sequences used in different PCR reactions are listed below:
The confirmation of the expression of the introduced GAD gene involved steps like RNA extraction, cDNA synthesis and Reverse Transcription PCR.
RNA of transgenic GAD tobacco plants along with the control plant (wild type) was isolated.
cDNA synthesis of transgenic GAD tobacco plants along with the wild type was done.
Total RNA: 4 ul (1 ug)
Oligo dT's: 0.5 ul
0.1% DEPC/nuclease free water: 6.5 ul
Total: 11 ul
5× reaction buffer: 4 ul
dNTP's (10 mM): 2 ul
RNase inhibitor (20 U/ul): 0.5 ul
0.1% DEPC/nuclease free water: 2 ul
Total: 8.5 ul
The cDNA samples from GAD transgenic tobacco and wild type plant were analyzed by PCR with Gene specific primers to check for the expression of the introduced GAD gene in tobacco:
PCR of cDNA with Gene Specific Primers:
3 U/μl
PCR Conditions (Eppendorf Machine):
The amplified product was visualized on 0.8% agarose gel as shown in
Evidence that Plants with Altered GAD Gene have an Altered Plant Architecture in to Generation
The experiments were performed with the wild type and T0 transgenic tobacco plants. Seedlings were cultivated in a green house in pots containing mixture of field soil. Plants were irrigated with normal water, without any external application of fertilizers. The FYM mixed in the soil served as the only source of nutrition to the plants.
The size of the leaf was measured in the transgenic plants and the wild type plants (plants with out the introduced glutamate decarboxylase gene). The leaf size of the T0 transgenic plants was larger when compared with the leaves of the control plants. The leaf size increased at least 20% more than the control while the highest increase in leaf size was 160% over the wild type plants (Table 1 and
Phenotype of the transgenic plants was studied in the T1 generation to evaluate the changes in the plant architecture during the adult plant stage encompassing the whole life cycle of the plant.
The three transgenic event D1A, E2 and H1 were selected for evaluating the change in plant architecture in pot culture in the green house. The experiments were performed with the wild type and transgenic tobacco. The T1 seeds were germinated on moist filter paper discs supplemented with hygromycin (50 mg/L); the positive seedlings that germinated and grew on this were selected and placed on soil in big pots (11 inch diameter) along with the wild type seedlings. Seedlings were cultivated in a green house in pots containing mixture of field soil and farmyard manure (FYM). Plants were irrigated with normal water or saline water 200 mM NaCl. The experiments were performed with three replications with four genotypes (wild type and D1A, E2 and H1 transgenic tobacco) as indicated in Table 1.
The phenotypic characters were observed and parameters contributing plant architecture like plant height, internodal distance, number of branches, number of leaves, leaf area, stem thickness (girth), total biomass, grain yield etc were recorded.
The height of the plant was measured in the transgenic plants and the wild type plants (plants with out the introduced glutamate decarboxylase gene). The plant height was measured using scale from the soil level to the tip of the plant including the inflorescence and the branches. The transgenic plants from the three events showed higher plant height as compared to the wild type plants (
The distance between two internodes on the stem was measured in the transgenic plants and the wild type plants (plants with out the introduced glutamate decarboxylase gene). The internodal distance was measured between the 5th & 6th leaf and 6th & 7th leaf. The leaf was counted from the top with the fully expanded leaf considered to be leaf number-1. The distance was measured using a thread and then measuring the thread length on a scale and expressed in cms. The transgenic plant (H1) showed at least 44% increase in internodal distance as compared to the wild type (
The number of leaves on each plant was counted in the transgenic plants and the wild type plants (plants with out the introduced glutamate decarboxylase gene). The transgenics exhibited 35% higher number of leaves when compared to wild type (
The thickness of the stem was measured in the transgenic plants and the wild type plants (plants with out the introduced glutamate decarboxylase gene). Girth of the stem was measured at a height of 5-6 cms above from the soil level. A thread was used to circle the stem at the appropriate height and then the length of the thread was measured on a scale and expressed in cms. The transgenics definitely had a thicker stem when compared to the wild type plants (
The size of the leaf was measured in the transgenic plants and the wild type plants (plants with out the introduced glutamate decarboxylase gene). The leaf was measured vertically from the node to the tip of the leaf and was considered as the length of the leaf. The transgenic plants possessed 27%-37% longer leaves than the wild type plants (
The biomass generated was measured in the transgenic plants and the wild type plants (plants with out the introduced glutamate decarboxylase gene). Plant biomass was estimated as the total plant dry weight. The total biomass from the transgenics was significantly higher (22%-88%) as compared to the wild types (
The total grain yield was significantly higher (up to 50% more) in the transgenics than the wild type (
To summarize the GAD transgenic plants from all the three events tested showed a positive altered phenotype or plant architecture. The GAD transgenic plants performed better than the wild type plants for the different agronomic and physiological status of the plants thus indicating the role of GAD gene for the altered plant architecture contributing towards the superior performance of the transgenic plants.
Number | Date | Country | Kind |
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1698/CHE/2008 | Jul 2008 | IN | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/IB2009/006227 | 7/7/2009 | WO | 00 | 4/11/2011 |