HK1165486B

HK1165486B - Transgenic plants comprising constructs encoding phosphoenolpyruvate carboxykinase and/or pyruvate ortho phos phate dikinase

Info

Publication number: HK1165486B
Application number: HK12106079.7A
Authority: HK
Inventors: Julian Michael Hibberd; Lucy Elisabeth Taylor; Anna Elizabeth Leiss
Original assignee: British American Tobacco(Investments) Limited
Priority date: 2009-02-27
Filing date: 2010-02-25
Publication date: 2015-11-13

Description

Transgenic plants comprising a construct encoding phosphoenolpyruvate carboxykinase and/or pyruvate orthophosphate dikinase

The present invention relates to genetic constructs useful for making transgenic plants. The construct has the ability to cause nitrogen remobilisation during leaf senescence, so that nitrogen can be transferred from the leaves to other parts of the plant. The invention provides plant cells transformed with such constructs and the transgenic plants themselves. The invention also relates to methods of producing transgenic plants and methods of increasing the rate of nitrogen remobilisation (nitrogen remobilisation) in senescent plants. The invention also relates to harvested plant leaves (e.g. tobacco leaves) that have been transformed with the genetic construct and to smoking articles comprising such harvested plant leaves.

Leaf senescence is a stage of plant development during which cells undergo unique metabolic and structural changes before cell death. Physiological and genetic studies indicate that aging is a highly regulated process. The progression of leaf senescence is evident by loss of chlorophyll and consequent yellowing caused by chloroplast disintegration. For example, the reduced chlorophyll levels characteristic of this developmental stage can be measured by solvent extraction and spectrophotometry or by a chlorophyll content meter. A decrease in chlorophyll levels compared to earlier chlorophyll levels recorded for the same plant (preferably grown under constant conditions) is indicative of senescence.

Molecular studies have shown that senescence is associated with changes in gene expression. The level of mRNA encoding a protein involved in photosynthesis decreases during senescence, while the level of mRNA encoding a gene encoding a protein thought to be involved in senescence increases. Senescence is an extremely organized process, regulated by a gene called senescence-associated gene (SAG). Leaf senescence involves degradation of proteins, nucleic acids and membranes, and subsequent transport of nutrients resulting from this degradation to other parts of the plant, such as developing seeds, leaves or storage organs. One problem with plant senescence is that many of the useful minerals and nutrients present in senescent leaves will remain in the leaves and, as the leaves die, will be virtually lost. For example, the nitrogen (which may be in the form of an amino group on an amino acid) present in senescent leaves would be wasted if not left from dying leaves.

Thus, enhancing nitrogen remobilisation in plants, particularly as they age, can have important applications in crop production. First, nitrogen remobilized from the leaves can be transported to younger leaves and developing seeds. Thus, an increase in the efficiency of nitrogen removal from senescent leaves could potentially increase the nitrogen supply to plant seeds and younger parts, thereby increasing crop yield and nitrogen utilization efficiency. This is clearly a valuable goal when the world population increases but the increase in crop yield is insufficient to meet demand. One potential target crop is Brassica napus (oilseed rape), which is nitrogen inefficient due to poor nitrogen remobilization by vegetative tissues. Another crop of interest is wheat, as the potential benefit of increasing grain protein content is very great. The kernel protein content not only affects the nutritional value of wheat, but also determines the kernel utilization rate and thus the market value. For example, increased grain protein content causes bread volume to increase. Also, the ability to increase nitrogen remobilisation may be very useful in the tobacco industry, as residual nitrogen in tobacco leaves is known to contribute to the formation of nitrosamines.

The enzymes phosphoenolpyruvate carboxykinase (PEPCK or PCK) [ EC 4.1.1.49] and pyruvate orthophosphate dikinase (PPDK) [ EC 2.7.9.1] are known. PPDK is present in both prokaryotes and eukaryotes and is conserved between bacteria and higher plants in terms of sequence and tertiary structure (Pocalyyko et al, 1990, Biochemistry, 29, 10757-10765). This enzyme catalyzes the reversible phosphorylation of pyruvate to phosphoenolpyruvate (PEP) in the following reaction (Carroll et al, 1990, Federation of European biochemical Societies, 274, 178-: pyruvate + Pi + ATP ═ PEP + PPi + AMP. In both C3 and C4 plants, the PPDK gene has a unique structure in which both transcripts are produced from the same gene. The longer transcript encodes a chloroplast protein, with the first exon encoding the chloroplast transit peptide, while the shorter transcript is transcribed from an independent promoter within the first intron of the longer transcript, and therefore lacks the first exon encoding the chloroplast transit peptide. This shorter transcript produced the cytoplasmic isoform of PPDK. This gene structure has been reported in maize, rice, C3 and C4 Flaveria (Flaveria) varieties and arabidopsis thaliana (arabidopsis thaliana).

Phosphoenolpyruvate Carboxykinase (PCK) catalyzes the reversible reaction between ADP, carbon dioxide and phosphoenolpyruvate to form ATP and oxaloacetate in the following reaction: oxaloacetate + ATP ═ PEP + ADP + CO₂. Studies have shown that PCK is present in the cytosol of cells of various plant tissues. These tissues include developing seeds, algal filaments and roots. In plants, PCK occurs in tissues where nitrogen-containing compounds are highly metabolized.

The inventors have constructed a number of genetic constructs in which the genes encoding the enzymes PCK and/or PPDK, alone or in combination, are placed under the control of a promoter in order to determine which, if any, overexpression of these genes has an effect on nitrogen remobilisation in senescent leaves.

According to a first aspect of the present invention there is provided a genetic construct comprising a senescence-specific promoter operably linked to at least one coding sequence encoding at least one polypeptide having Phosphoenolpyruvate Carboxykinase (PCK) activity and/or pyruvate orthophosphate dikinase (PPDK) activity.

According to a second aspect of the present invention there is provided a genetic construct comprising a promoter operably linked to at least one coding sequence encoding at least one polypeptide having Phosphoenolpyruvate Carboxykinase (PCK) activity and pyruvate orthophosphate dikinase (PPDK) activity.

The inventors believe that during senescence, both the PCK and PPDK enzymes may play a role in interconversion of various amino acids during nitrogen remobilisation of the leaves. Although the inventors do not wish to be bound by the hypothesis, a putative biochemical pathway illustrating how PCK and PPDK may affect nitrogen remobilisation is illustrated in fig. 18. Thus, the inventors believe that stimulating overexpression of these two enzymes in plants, either separately or simultaneously, during senescence may play a role in nitrogen remobilization.

As a result of their studies, the present inventors unexpectedly found that the constructs of the present invention, which encode PCK and/or PPDK, cause an increase in the rate of remobilisation of senescent leaf nitrogen. The present inventors hypothesize that nitrogen may be transported as an amino acid from senescent leaves to younger parts of the plant, such as the plant seeds. Furthermore, the inventors have found that when these enzymes are overexpressed in senescent leaves, the amount of vegetative plant growth is increased (which corresponds to an increase in crop yield). The inventors therefore believe that the constructs of the invention may be used to prepare transgenic plants which may be capable of exhibiting an increased rate of nitrogen remobilisation and/or an increased growth rate of senescent leaves.

The promoter in the genetic construct of the first or second aspect may be capable of inducing RNA polymerase to bind to at least one coding region encoding at least one polypeptide having PCK and/or PPDK activity and initiate transcription.

The promoter present in the construct of the second aspect may be constitutive, non-constitutive or tissue-specific. Examples of suitable promoters include the cauliflower mosaic virus 35S promoter (complete promoter or truncated promoter), the rubisco promoter, the pea plastocyanin promoter, the nopaline synthase promoter, the chlorophyll r/b binding promoter, the high molecular weight glutenin promoter, the alpha, beta-gliadin promoter, the hordein promoter or the patatin promoter.

The promoter present in the construct of the second aspect may be a senescence-specific promoter.

The "senescence specific promoter" (SAG) may be a promoter involved in controlling the expression of senescence-associated genes. Thus, the promoter can substantially exclusively limit the expression of a coding sequence (i.e., gene) to which it is operably linked in older tissues. Thus, a senescence-specific promoter may be one that is capable of preferentially promoting gene expression in a plant tissue in a developmentally regulated manner such that expression of the 3' protein coding region occurs substantially only when the plant tissue is undergoing senescence. It will be appreciated that senescence tends to occur in older parts of the plant (e.g. older leaves) but not in younger parts of the plant, such as seeds.

One example of a plant known to express various senescence-associated genes is Arabidopsis (Arabidopsis). Thus, the promoter in the construct of the first or second aspect may be isolated from a senescence-associated gene of Arabidopsis. Gepstein et al (The plant journal, 2003, 36, 629-642) used Arabidopsis as a model for detailed study of SAG and its promoter. The genetic construct may comprise a promoter from any of the SAGs disclosed in this paper. For example, a suitable promoter may be selected from SAG12, SAG13, SAG101, SAG21, and SAG18, or a functional variant or functional fragment thereof.

Preferred promoters are the SAG12 and SAG13 promoters. In one embodiment, the promoter is the SAG12 promoter known to the skilled person or a functional variant or fragment thereof (Gan and Amasino, 1997, Plant Physiology, 113: 313-319). The DNA sequence encoding the SAG12 promoter is referred to herein as SEQ ID No.16, see below:

TCGAGACCCGATTGTTATTTTTAGACTGAGACAAAAAAGTAGAATCGTTGATTGTTAAAATTTA

AAATTAGTTTCATTACGTTTCGATAAAAAAATGATTAGTTTATCATAGCTTAATTATAGCATTG

ATTTCTAAATTTGTTTTTTGACCACCCTTTTTTCTCTCTTTGGTGTTTTCTTAACATTAGAAGA

ACCCATAACAATGTACGTTCAAATTAATTAAAAACAATATTTCCAAGTTTTATATACGAAACTT

GTTTTTTTTAATGAAAACAGTTGAATAGTTGATTATGAATTAGTTAGATCAATACTCAATATAT

GATCAATGATGTATATATATGAACTCAGTTGTTATACAAGAAATGAAAATGCTATTTAAATACA

GATCATGAAGTGTTAAAAAGTGTCAGAATATGACATGAAGCGTTTTGTCCTACCGGGTATTCGA

GTTATAGGTTTGGATCTCTCAAGAATATTTTGGGCCATACTAGTTATATTTGGGCTTAAGCGTT

TTGCAAAGAGACGAGGAAGAAAGATTGGGTCAAGTTAACAAAACAGAGACACTCGTATTAGTTG

GTACTTTGGTAGCAAGTCGATTTATTTGCCAGTAAAAACTTGGTACACAACTGACAACTCGTAT

CGTTATTAGTTTGTACTTGGTACCTTTGGTTCAAGAAAAAGTTGATATAGTTAAATCAGTTGTG

TTCATGAGGTGATTGTGATTTAATTTGTTGACTAGGGCGATTCCTTCACATCACAATAACAAAG

TTTTATAGATTTTTTTTTTATAACATTTTTGCCACGCTTCGTAAAGTTTGGTATTTACACCGCA

TTTTTCCCTGTACAAGAATTCATATATTATTTATTTATATACTCCAGTTGACAATTATAAGTTT

ATAACGTTTTTACAATTATTTAAATACCATGTGAAGATCCAAGAATATGTCTTACTTCTTCTTT

GTGTAAGAAAACTAACTATATCACTATAATAAAATAATTCTAATCATTATATTTGTAAATATGC

AGTTATTTGTCAATTTTGAATTTAGTATTTTAGACGTTATCACTTCAGCCAAATATGATTTGGA

TTTAAGTCCAAAATGCAATTTCGTACGTATCCCTCTTGTCGTCTAATGATTATTTCAATATTTC

TTATATTATCCCTAACTACAGAGCTACATTTATATTGTATTCTAATGACAGGGAAACCTTCATA

GAGATTCAGATAGATGAAATTGGTGGGAAACATCATTGAACAGGAAACTTTTAGCAAATCATAT

CGATTTATCTACAAAAGAATACGTAGCGTAATGAAGTCCACTTGTTGTGAATGACTATGATTTG

ATCAAATTAGTTAATTTTGTCGAATCATTTTTCTTTTTGATTTGATTAAGCTTTTAACTTGCAC

GAATGGTTCTCTTGTGAATAAACAGAATCTTTGAATTCAAACTATTTGATTAGTGAAAAGACAA

AAGAAGATTCCTTGTTTTTATGTGATTAGTGATTTTGATGCATGAAAGGTACCTACGTACTACA

AGAAAAATAAACATGTACGTAACTACGTATCAGCATGTAAAAGTATTTTTTTCCAAATAATTTA

TACTCATGATAGATTTTTTTTTTTTGAAATGTCAATTAAAAATGCTTTCTTAAATATTAATTTT

AATTAATTAAATAAGGAAATATATTTATGCAAAACATCATCAACACATATCCAACTTCGAAAAT

CTCTATAGTACACAAGTAGAGAAATTAAATTTTACTAGATACAAACTTCCTAATCATCAAATAT

AAATGTTTACAAAACTAATTAAACCCACCACTAAAATTAACTAAAAATCCGAGCAAAGTGAGTG

AACAAGACTTGATTTCAGGTTGATGTAGGACTAAAATGACTACGTATCAAACATCAACGATCAT

TTAGTTATGTATGAATGAATGTAGTCATTACTTGTAAAACAAAAATGCTTTGATTTGGATCAAT

CACTTCATGTGAACATTAGCAATTACATCAACCTTATTTTCACTATAAAACCCCATCTCAGTAC

CCTTCTGAAGTAATCAAATTAAGAGCAAAAGTCATTTAACTTAGG

SEQ ID NO：16

thus, the promoter in the construct of the invention may comprise a nucleotide sequence substantially as shown in SEQ ID No.16 or a functional variant or fragment thereof. The SAG12 promoter sequence may be obtained from arabidopsis thaliana, see US 5,689,042. This promoter sequence may be present in each genetic construct of the invention, as shown in figure 3. In embodiments where the promoter is SAG12, it will be understood that the promoter may comprise the amino acid sequence of SEQ ID No: 16, bases 1-2093. However, functional variants or functional fragments of promoters may also be used in the genetic constructs of the invention.

A "functional variant or functional fragment of a promoter" may be a derivative or part of a promoter that is functionally sufficient to drive expression of any coding region to which it is operably linked. For example, in embodiments where the promoter is based on SAG12, the skilled artisan will appreciate that the promoter can be substituted for SEQ ID No: 16, or perhaps only a portion of the SAG12 promoter may be needed to still drive gene expression from the construct.

Functional variants and functional fragments of a promoter can be readily identified by assessing whether the transcriptase binds to a putative promoter region and then causes the coding region to be transcribed into a polypeptide having PCK and/or PPDK activity. Alternatively, such functional variants and fragments can be detected by subjecting the promoter to mutagenesis while bound to the coding region and assessing whether gene expression can occur.

During senescence, the genetic construct of the first aspect may be capable of causing expression of at least one polypeptide having PCK activity and/or PPDK activity. Thus, the genetic construct may comprise at least one coding sequence encoding (i) a Phosphoenolpyruvate Carboxykinase (PCK) or a functional variant or fragment thereof, and/or (ii) a pyruvate orthophosphate dikinase (PPDK) or a functional variant or fragment thereof. As described in the examples, the inventors have developed a series of genetic constructs based on polypeptides having PCK and/or PPDK activity, which are shown in FIG. 3.

In a first embodiment of the genetic construct of the first aspect, the promoter may induce expression of a coding sequence encoding a polypeptide having PCK activity. This is referred to herein as the "PCK construct" and is shown in figure 3. Thus, in a first embodiment, the genetic construct may comprise a senescence-specific promoter and a coding sequence encoding Phosphoenolpyruvate Carboxykinase (PCK) or a functional variant or fragment thereof. The genetic construct may not encode a polypeptide having PPDK activity.

In a second embodiment of the construct of the first aspect, the promoter may induce expression of a coding sequence encoding a polypeptide having PPDK activity. This is referred to herein as the "PPDK construct" and is shown in FIG. 3. In a second embodiment, the genetic construct may comprise a senescence-specific promoter and a coding sequence encoding pyruvate orthophosphate dikinase (PPDK) or a functional variant or fragment thereof. The genetic construct may not encode a polypeptide having PCK activity.

In a third embodiment of the construct of the first aspect, the promoter induces expression of a single coding sequence encoding a polypeptide having both PCK and PPDK activity. This is referred to as "PCK/PPDK construct 1". In a third embodiment, the genetic construct may comprise a senescence-specific promoter and a coding sequence encoding (i) Phosphoenolpyruvate Carboxykinase (PCK) or a functional variant or fragment thereof and (ii) pyruvate orthophosphate dikinase (PPDK) or a functional variant or fragment thereof. The construct of the third embodiment may encode a single transcript having dual activity (i.e., both PCK and PPDK enzymatic activity). The PCK coding region may be located 3' to the PPDK coding region. However, it is preferred that the PCK coding region is located 5' to the PPDK coding region.

In a fourth embodiment of the construct of the first aspect, the promoter can induce expression of: (i) a first coding sequence encoding a first polypeptide having PCK activity, and (ii) a second coding sequence encoding a second polypeptide having PPDK activity. This is referred to as "PCK/PPDK construct 2". Thus, in a fourth embodiment, the genetic construct may comprise at least one senescence-specific promoter and (i) a first coding sequence encoding PCK, or a functional variant or fragment thereof, and (ii) a second coding sequence encoding PPDK, or a functional variant or fragment thereof, i.e., encoding two transcripts, one enzyme and one transcript.

As described in example 6, the inventors found that overexpression of PCK or PPDK in a host cell (e.g., by transformation with a "PCK construct" or a "PPDK construct") resulted in enhanced nitrogen remobilization in senescent leaves. Furthermore, they found that a single construct of PCK and PPDK resulted in increased vegetative growth.

As described in example 8, the inventors found that simultaneous overexpression of both PCK and PPDK in a host cell (e.g., by transformation with both the "PCK construct" and the "PPDK construct") was unexpectedly effective in inducing nitrogen remobilization in senescent leaves. Thus, nitrogen can be transported out of the senescent leaves (e.g., as a transport amino acid). Suitable transport amino acids may be glutamine and/or asparagine. In addition, simultaneous overexpression of PCK and PPDK during senescence may also increase growth rate, which may lead to increased vegetative growth. Thus, the construct of the first aspect may comprise a coding sequence encoding both PCK and PPDK, or a functional variant or fragment thereof.

It will be appreciated that the construct of the second aspect comprises a coding sequence which encodes both PCK and PPDK, or a functional variant or fragment thereof. The two enzymes may be encoded as a single polypeptide having dual activity, or as two polypeptides, one having PCK activity and the other having PPDK activity.

Phosphoenolpyruvate Carboxykinase (PCK) or a functional variant or fragment thereof and pyruvate orthophosphate dikinase (PPDK) or a functional variant or fragment thereof, respectively, may be derived from any suitable source, such as a plant. The coding sequence for each enzyme may be derived from a suitable plant source, for example from arabidopsis thaliana. Thus, a coding sequence encoding a polypeptide having PCK activity may be derived from arabidopsis thaliana. In addition, the coding sequence encoding a polypeptide having PPDK activity may be derived from Arabidopsis (Arabidopsis spp.), Zea spp, Flaveria spp or Leuconostoc (Cleome spp). The coding sequence encoding a polypeptide having PPDK activity may be derived from Arabidopsis thaliana, maize (Zea mays), Flaveriatrinervia, Flaveria bidentis, Flaveria brown or Cleome gynandra.

It is believed that there are three genes encoding PCK in Arabidopsis thaliana. Provided herein below is a genomic DNA sequence (including introns and exons) encoding one embodiment of an arabidopsis enolpyruvate Phosphate Carboxykinase (PCK) as SEQ ID No: 17:

ATGTCGGCCGGTAACGGAAATGCTACTAACGGTGACGGAGGGTTTAGTTTCCCTAAAGGACCGG

TGATGCCGAAGATAACGACCGGAGCAGCAAAGAGAGGTAGCGGAGTCTGCCACGACGATAGTGG

TCCGACGGTGAATGCCACAACCATCGATGAGCTTCATTCGTTACAGAAGAAACGTTCTGCTCCT

ACCACACCGATCAACCAAAACGCCGCCGCTGCTTTTGCCGCCGTCTCCGAGGAGGAGCGTCAGA

AGATTCAGCTTCAATCTATCAGGTCCTTATAATAACTTCACATATACAGATTATTCATACGTTA

CTTTTGTTTATAACATACTTTATATCGAATTAAGGAAGATTATTGCGTTTTCGTGTCCGATCAT

TTTCATGGAAAAAGTGTCTTTTAGCTAAATATATGGTGTAGTATTAAATATTTCTGACGTGATA

TACACTAAACTTGAAAATTTTCAATTACTATTTCTTCCTTTAATTCGGCAATATAATTTGTTTT

TGTTTATTTTTGGATTAGACATTTATGGACAAGTTAATGCGCTATTGTGACTATTACCAGAAAA

TAATACTTTAATGTACATGACACGTGTTTAAAACGACACGTGGAAACTAATTTTGATTAATTGT

GAAACAGTGCATCGTTAGCATCGTTAACGAGAGAGTCAGGACCAAAGGTGGTGAGAGGAGATCC

GGCGGAGAAGAAGACCGATGGTTCAACTACTCCGGCGTACGCTCACGGCCAACATCATTCTATC

TTTTCTCCGGCTACTGGTGCTGTCAGTGATAGCTCCTTGAAGTTTACTCACGTCCTCTACAATC

TTTCGCCTGCAGGTCAACAAATAAACCTAGAATCCGAATCTGAATATTGATAAATGTTTCTGCA

ACGAGTTTGATAGATTTGGTTTGTGATTTTGTTGTTTGTAGAGCTTTATGAGCAAGCTATTAAG

TATGAGAAAGGTTCGTTTATCACTTCTAATGGAGCTTTGGCGACGCTTTCTGGTGCTAAGACTG

GTCGTGCTCCCAGAGATAAGCGTGTTGTTAGAGATGCTACTACTGAGGATGAGCTTTGGTGGGG

AAAGTGAGTATTCCTAATCTCGATTTTGATTGATGGAGTTTTTGGGTTTATGCTCTGTTTTCGT

TTATTGATTTTGGAGTTTGATTTTGATTTTAGGGGTTCGCCGAATATCGAAATGGATGAACATA

CTTTCATGGTGAACAGAGAAAGAGCTGTTGATTACTTGAATTCCTTGGAAAAGGTATTAAATTT

TGAAAACTTTAATCAATGTTGTTGAGTGTAGAACTTTTGATCTAAGTTTATGAAATTTCTGTTG

TTGTTGGGGTTTTTAGGTCTTTGTCAATGACCAATACTTAAACTGGGATCCAGAGAACAGAATC

AAAGTCAGGATTGTCTCAGCTAGAGCTTACCATTCATTGTTTATGCACAACATGTAAGTAAAAT

CATTATTGACTCCTTGTATGTCAATCCATTATTGTGGGTGAAAGAAAACAACAAATTAGTAACT

GGGGAGGGTGTCAGGTGTATCCGACCAACTCAGGAGGAGCTTGAGAGCTTTGGTACTCCGGATT

TTACTATATACAATGCTGGGCAGTTTCCATGTAATCGTTACACTCATTACATGACTTCGTCCAC

TAGCGTAGACCTTAATCTGGCTAGGAGGGAAATGGTTATACTTGGTACTCAGTATGCTGGGGAA

ATGAAGAAGGGTCTTTTCAGTGTGATGCATTACCTTATGCCTAAGCGTCGTATTCTCTCCCTTC

ATTCTGGATGCAATATGGGAAAAGATGGAGATGTTGCTCTCTTCTTTGGACTTTCAGGTATAGT

AGAGACAGTACCAACTATGGTGTTGGGTGATGATGGAAGGAACGATAAATCAAATGATACAATA

CAATTACTGCTGAACTGACTTGAGAACTGCTTGCCTCTTTGTTGAGTTTAGCGGGTGAATTGAG

ATTGATGATTGTGTTTTTTGTTTTCTATGAATGATGATTTTAGGTACCGGGAAGACAACGCTGT

CTACTGATCACAACAGGTATCTTATTGGAGATGATGAGCATTGTTGGACTGAGACTGGTGTTTC

GAACATTGAGGGTGGGTGCTATGCTAAGTGTGTTGATCTTTCGAGGGAGAAGGAGCCTGATATC

TGGAACGCTATCAAGTTTGGAACAGGTAGAAAGACAGTACGTTGGAATTGTTTTTGAGAAAAAA

ACATAAAGCAGTGATATAACAATAAGATTCTGATCTTGTTGCAGTTTTGGAAAATGTTGTGTTT

GATGAGCACACCAGAGAAGTGGATTACTCTGATAAATCTGTTACAGGTAAAACAATTGTTATTT

CTTTCATTCTCTTCGTCCTCACAATTAACAGAATGATCATTTTCGATTCTCTTTGGTTGCAGAG

AACACACGTGCTGCCTACCCAATTGAGTTCATTCCAAATGCGAAAATACCTTGTGTTGGTCCAC

ACCCGACAAATGTGATACTTCTGGCTTGTGATGCCTTTGGTGTTCTCCCACCTGTGAGCAAGCT

GAATCTGGCACAAACCATGTACCACTTCATCAGTGGTTACACTGCTCTGGTAAGGCCAAAGTAA

AAGTCTTTATTTTGCACATCGTCTTCATAAATTTCAAAAGCATAACCAAAGATGTGCAACATAT

ATAGGTTGCTGGCACAGAGGATGGTATCAAGGAGCCAACAGCAACATTCTCAGCTTGCTTTGGT

GCAGCTTTCATAATGTTGCATCCCACAAAGTATGCAGCTATGTTAGCTGAGAAGATGAAGTCAC

AAGGTGCTACTGGTTGGCTCGTCAACACTGGTTGGTCTGGTGGCAGGTATATATGTCCTTCTAT

GGAAATCGATACAACAAAACGCTGCCTTGTAACACATGTTTGTAGGCTATTAACATGATCTGTA

ATGTTTTATTTCCTGCAGTTATGGTGTTGGAAACAGAATCAAGCTGGCATACACTAGAAAGATC

ATCGATGCAATCCATTCGGGCAGTCTCTTGAAGGCAAACTACAAGAAAACCGAAATCTTTGGAT

TTGAAATCCCAACTGAGATCGAAGGGATACCTTCAGAGATCTTGGACCCCGTCAACTCCGTAAG

TTTCTGCAAATCTGTATAATGTAATTGCTTAAGTGATGATGAACAATTTTTTGTTGATTTGGGT

TTAATGAAAATGCAGTGGTCTGATAAGAAGGCACACAAAGATACTCTGGTGAAACTGGGAGGTC

TGTTCAAGAAGAACTTCGAGGTTTTTGCTAACCATAAGATTGGTGTGATGGTAAGCTTACGGAG

GAGATTCTCGCTGCTGGTCCTATCTTTTAG

SEQ ID No：17

the cDNA sequence (exon only) encoding arabidopsis Phosphoenolpyruvate Carboxykinase (PCK) is provided herein below as SEQ ID No: 18:

ATGTCGGCCGGTAACGGAAATGCTACTAACGGTGACGGAGGGTTTAGTTTCCCTAAAGGA

CCGGTGATGCCGAAGATAACGACCGGAGCAGCAAAGAGAGGTAGCGGAGTCTGCCACGAC

GATAGTGGTCCGACGGTGAATGCCACAACCATCGATGAGCTTCATTCGTTACAGAAGAAA

CGTTCTGCTCCTACCACACCGATCAACCAAAACGCCGCCGCTGCTTTTGCCGCCGTCTCC

GAGGAGGAGCGTCAGAAGATTCAGCTTCAATCTATCAGTGCATCGTTAGCATCGTTAACG

AGAGAGTCAGGACCAAAGGTGGTGAGAGGAGATCCGGCGGAGAAGAAGACCGATGGTTCA

ACTACTCCGGCGTACGCTCACGGCCAACATCATTCTATCTTTTCTCCGGCTACTGGTGCT

GTCAGTGATAGCTCCTTGAAGTTTACTCACGTCCTCTACAATCTTTCGCCTGCAGAGCTT

TATGAGCAAGCTATTAAGTATGAGAAAGGTTCGTTTATCACTTCTAATGGAGCTTTGGCG

ACGCTTTCTGGTGCTAAGACTGGTCGTGCTCCCAGAGATAAGCGTGTTGTTAGAGATGCT

ACTACTGAGGATGAGCTTTGGTGGGGAAAGGGTTCGCCGAATATCGAAATGGATGAACAT

ACTTTCATGGTGAACAGAGAAAGAGCTGTTGATTACTTGAATTCCTTGGAAAAGGTCTTT

GTCAATGACCAATACTTAAACTGGGATCCAGAGAACAGAATCAAAGTCAGGATTGTCTCA

GCTAGAGCTTACCATTCATTGTTTATGCACAACATGTGTATCCGACCAACTCAGGAGGAG

CTTGAGAGCTTTGGTACTCCGGATTTTACTATATACAATGCTGGGCAGTTTCCATGTAAT

CGTTACACTCATTACATGACTTCGTCCACTAGCGTAGACCTTAATCTGGCTAGGAGGGAA

ATGGTTATACTTGGTACTCAGTATGCTGGGGAAATGAAGAAGGGTCTTTTCAGTGTGATG

CATTACCTTATGCCTAAGCGTCGTATTCTCTCCCTTCATTCTGGATGCAATATGGGAAAA

GATGGAGATGTTGCTCTCTTCTTTGGACTTTCAGGTACCGGGAAGACAACGCTGTCTACT

GATCACAACAGGTATCTTATTGGAGATGATGAGCATTGTTGGACTGAGACTGGTGTTTCG

AACATTGAGGGTGGGTGCTATGCTAAGTGTGTTGATCTTTCGAGGGAGAAGGAGCCTGAT

ATCTGGAACGCTATCAAGTTTGGAACAGTTTTGGAAAATGTTGTGTTTGATGAGCACACC

AGAGAAGTGGATTACTCTGATAAATCTGTTACAGAGAACACACGTGCTGCCTACCCAATT

GAGTTCATTCCAAATGCGAAAATACCTTGTGTTGGTCCACACCCGACAAATGTGATACTT

CTGGCTTGTGATGCCTTTGGTGTTCTCCCACCTGTGAGCAAGCTGAATCTGGCACAAACC

ATGTACCACTTCATCAGTGGTTACACTGCTCTGGTTGCTGGCACAGAGGATGGTATCAAG

GAGCCAACAGCAACATTCTCAGCTTGCTTTGGTGCAGCTTTCATAATGTTGCATCCCACA

AAGTATGCAGCTATGTTAGCTGAGAAGATGAAGTCACAAGGTGCTACTGGTTGGCTCGTC

AACACTGGTTGGTCTGGTGGCAGTTATGGTGTTGGAAACAGAATCAAGCTGGCATACACT

AGAAAGATCATCGATGCAATCCATTCGGGCAGTCTCTTGAAGGCAAACTACAAGAAAACC

GAAATCTTTGGATTTGAAATCCCAACTGAGATCGAAGGGATACCTTCAGAGATCTTGGAC

CCCGTCAACTCCTGGTCTGATAAGAAGGCACACAAAGATACTCTGGTGAAACTGGGAGGT

CTGTTCAAGAAGAACTTCGAGGTTTTTGCTAACCATAAGATTGGTGTGATGGTAAGCTTA

CGGAGGAGATTCTCGCTGCTGGTCCTATCTTTTAG

SEQ ID No：18

thus, a coding sequence encoding a polypeptide having PCK activity may comprise a sequence substantially as set forth in SEQ ID No:17 or SEQ ID No.18 or a functional variant or fragment thereof.

The polypeptide sequence of arabidopsis PCK is provided herein as SEQ ID No: 19:

MSAGNGNATNGDGGFSFPKGPVMPKITTGAAKRGSGVCHDDSGPTVNATTIDELHSLQKK

RSAPTTPINQNAAAAFAAVSEEERQKIQLQSISASLASLTRESGPKVVRGDPAEKKTDGS

TTPAYAHGQHHSIFSPATGAVSDSSLKFTHVLYNLSPAELYEQAIKYEKGSFITSNGALA

TLSGAKTGRAPRDKRVVRDATTEDELWWGKGSPNIEMDEHTFMVNRERAVDYLNSLEKVF

VNDQYLNWDPENRIKVRIVSARAYHSLFMHNMCIRPTQEELESFGTPDFTIYNAGQFPCN

RYTHYMTSSTSVDLNLARREMVILGTQYAGEMKKGLFSVMHYLMPKRRILSLHSGCNMGK

DGDVALFFGLSGTGKTTLSTDHNRYLIGDDEHCWTETGVSNIEGGCYAKCVDLSREKEPD

IWNAIKFGTVLENVVFDEHTREVDYSDKSVTENTRAAYPIEFIPNAKIPCVGPHPTNVIL

LACDAFGVLPPVSKLNLAQTMYHFISGYTALVAGTEDGIKEPTATFSACFGAAFIMLHPT

KYAAMLAEKMKSQGATGWLVNTGWSGGSYGVGNRIKLAYTRKIIDAIHSGSLLKANYKKT

EIFGFEIPTEIEGIPSEILDPVNSWSDKKAHKDTLVKLGGLFKKNFEVFANHKIGVMVSL

RRRFSLLVLSF

SEQ ID No：19

thus, a polypeptide having PCK activity may comprise an amino acid sequence substantially as set forth in SEQ ID No:19 or a functional variant or fragment thereof.

Arabidopsis thaliana is believed to have at least two forms of PPDK, the chloroplast form and the cytosolic form, encoded by the same gene, wherein a minor splice change at the 5' end of the gene produces both forms. Genomic DNA sequences (including introns and exons) encoding two forms of arabidopsis pyruvate orthophosphate dikinase (PPDK) are provided herein as follows as SEQ ID nos: 20:

ATGACAAGTATGATCGTGAAGACAACGCCGGAGCTCTTCAAAGGAAATGGAGTGTTCCGTACGG

ATCATCTCGGAGAAAACCGAATGGTTAGTCGATCAAACCGGCTAGGTGATGGATCAAACCGTTT

CCCTAGAACCGGTACAATCCATTGCCAACGGTTAAGCATAGCAAAGACCGGTTTGCATCGTGAG

ACGAAGGCTCGAGCCATACTTAGCCCTGTGTCCGATCCGGCCGCTTCCATAGCCCAAAAGGTAA

GCCTTTCCATTTCAATCATTCTGGTGTATTTTCACCATAAAATTTTATACACTTTTTTATTACG

TTTTGTTTTATGATTCTGACGTGAGATTCTTGAGAGAAACTATCACCGATCATTGGGTCGAACC

ATCTAGCAGCTCAATTATTATCGGTTATAACCCTACCGGTTATAGAATACAAAACAGGTTACGC

CATTGTGACATTTGCTTTGTGATCTTGTGAGACGATTAATTATTTGATGTTGATTGGTTTCGTT

ACTCTTGTTTAAACAATCGAACGGTTCAAACTAATACACACATGTGATGTGAGATCATTTCGGT

AGTAATACCAAATAGCGTCTGGCCTAAATTATGAAAGTACTATTTTGAATTAAATTATTGTGGA

AACATGAACTTATTTAAATTCAAGTATTTTCGAAATTTGTAATAAAAAAAAACTTTTCCTCTAG

ATTCATTAGCCCTACTTTTCGTAGAAACAACTTTAATGTATTCAAAGACCACTTTGCTGCTTAA

GTCAGACTCTTGTGCCACTTGGTAGATCCACCAATGCCACGTTTTGTTATTGTGCCAAAGAATA

CGTGAATATGTCCAAACGGCAATCAAATTCTTGGCGTAAAACACAAAAATTATGATACTAGTTT

AAATCCACAATTCACCTTCACCATAAAGAATTCATGTATTAGAGATGGTATGACAAGAACTGGT

TGAATTTGATGACATTTGTTTGCTATTGTTTTGGTTAAGTAAAAGTTTTGTTAAAAAGGAAAAT

AGCATCGGTAGTGGCAGATAGCAAGTGTGTGAGTGAGATCAGATATGGTTGACACATCTATGAC

GAGTCATCGCAACGAAACTTCTTTAATTTTGGTCAATTATATTACAATTTAGCATTTCGAGGTT

GGAATTTTGGAATGATCTCTTGATAAGATAATAATGTATTTTTGATGACGTATCCATCAAAACT

ATAAATGATTTATATTAAATATGAAATTTCGACTGTATACAAGTTTTTATATTATAAAATTATT

CGATGTACATATGATCATAATAACTTTACTATATATATAGATACGTATATGTGTTCTTAAACTT

GCACAAACATTTCTGCAATCTAAACCTCAATCAAAACAAACAAACAAAAAACCATGATGCAGCG

AGTATTCACCTTTGGAAAAGGAAGAAGCGAAGGCAACAAGGGCATGAAGTCCTTGGTATGTTAC

CAATACCATCATCATGATCATATCAATTCATTAATAATTTAGTGTTTGCTATTTTCAAGAACCA

TTTATCAAAAATGTTAATTGTTGTTGTGTATGAAGTTGGGAGGGAAAGGAGCCAACCTGGCGGA

GATGGCTAGCATAGGCTTGTCGGTGCCGCCGGGGCTAACCATATCGACGGAGGCTTGTCAGCAG

TATCAGATCGCCGGCAAAAAGCTTCCAGAAGGTTTATGGGAAGAGATCTTAGAAGGTCTTAGCT

TCATCGAACGTGACATTGGAGCTTCCCTCGCTGATCCCTCCAAGCCACTCCTCCTCTCTGTTCG

CTCCGGCGCCGCCGTAAGTTAATTATAACTTTTTTTCTTGACTATTTTTATTTTAAGGATTTTT

TCTAATGTTAAATTTCTGTTTTTTTTTCTTTCTATGTTTTCTTTAATCTTTTGAAGATTTTTTG

ACGCAGATTTTGACTTGTTAGATTTCTTTTATTGAAGTTGAGATCCAAATATTTTTTTGGTTAT

TTTGCCATTTGGCCGTTTTTGGAAGAGTTTAAAATGTACTAGATAGAAAATGAATAAGTTTTGT

GGCTATTGAAAGACCTAATGATTTTGGTATTCAAACTATAACGTAGAAAATGAAGATCTTTCGT

TTATCTATTTTTAAAACAGAACTACATTGACTTGTCTTTGATCGATATTTTGCATTGTAGATCT

CAATGCCTGGTATGATGGACACTGTACTTAACCTTGGCTTGAACGACCAAGTCGTCGTTGGTCT

GGCCGCAAAAAGCGGAGAGCGTTTTGCTTACGATTCGTTCCGGCGTTTTCTTGATATGTTTGGT

GATGTTGTAAGTCCTCTGTTTTTCAATACTATTTCAGGTAACTTGCATGACAAGAAAATTCTTT

GACCTACCTTATAATTGTTTTCTTGATCAATAAAAGGTGATGGGAATTCCACACGCCAAGTTTG

AAGAGAAGTTAGAGAGAATGAAGGAGAGGAAAGGAGTTAAAAATGACACTGACTTAAGCGCGGC

TGATCTCAAGGAATTGGTTGAGCAGTACAAGAGTGTTTACTTAGAGGCCAAGGGTCAAGAGTTT

CCTTCAGGTTTGTTTTGATTCCTACTTGAGGTCAAGTGATAAAAATTAGTTATTAGTTACAAAT

GTTTAAACGGGGTTAATTGCAGATCCAAAGAAGCAATTGGAGCTAGCGATTGAAGCGGTATTCG

ATTCTTGGGATAGCCCGAGAGCGAACAAGTACAGAAGTATTAACCAGATAACTGGATTGAAAGG

AACCGCGGTTAACATTCAGTGTATGGTGTTTGGAAACATGGGGGACACTTCAGGGACTGGTGTT

CTCTTCACTAGGAACCCTAGCACAGGAGAGAAGAAGCTTTATGGCGAGTTTCTAGTTAATGCTC

AGGTTTGGCATCTATCACAATGTGTGAATCTCATATCAACAAGTAAGCCCATACTCATTAAATA

TTGGTTTTGGGACAGGGAGAGGATGTGGTTGCAGGGATAAGAACACCAGAAGATTTGGATACAA

TGAAGAGATTTATGCCTGAGGCTTACGCTGAACTTGTTGAGAACTGCAACATCTTAGAAAGACA

TTACAAAGACATGATGGTTGATACACATAAACAATACTTCAATTAGTCCTCATCAACAATTCTT

TAGTAATTTAAACAAAATCTCAAATGTGTATTGCAGGATATTGAATTCACAGTACAAGAAGAGA

GATTGTGGATGCTGCAATGCAGAGCGGGTAAGCGAACGGGTAAAGGCGCCGTGAAGATAGCAGT

TGATATGGTAGGTGAAGGGCTTGTTGAGAAATCTTCTGCTATCAAAATGGTGGAGCCTCAACAT

CTTGATCAACTACTTCACCCACAGGTACAAACTCAAATATTCATCTTCTTCTTTTTTCATAGTC

ATAAACTTGATGTTGAAACCAAAATTCGAAACTTACTGGTAATGATTGGTTCACTTGAACAAGA

ACTAATGGGTTTAAGACGTTTAGGGTTTAGGAGTAAAAGCAGAGATGATTGTCTGACACGTAAC

CGATGAATAGGGTTTGGAAATTTTGATTCAGAGGTCAATGAAGGTTTTTTTTTTTTTTTTTTAT

TGATGGATTAGTTTCATGATCCATCGGGGTATCGTGAAAAAGTGGTGGCCAAAGGCTTACCTGC

GTCACCAGGAGCGGCGGTTGGACAGGTTGTGTTCACGGCGGAGGAAGCCGAAGCTTGGCATTCT

CAGGGTAAAACTGTGATTCTGGTTCGAACTGAGACAAGCCCTGACGATGTGGGAGGTATGCACG

CAGCGGAAGGTATATTGACGGCTAGAGGAGGAATGACGTCACACGCGGCTGTTGTTGCTCGCGG

TTGGGGAAAATGTTGCATTGCTGGTTGTTCCGAGATTCGTGTCGACGAGAACCACAAGGTTTTT

GGATTCGATTTTAGAAACTTGTCATATAAGTTAGGGGAAGATTGTTTCTAAAGTTAGGGTTTAA

AAATTTTCAGGTTCTATTGATTGGAGATTTGACGATTAATGAAGGCGAATGGATCTCAATGAAC

GGATCAACCGGTGAGGTTATATTAGGGAAACAAGCATTGGCTCCTCCGGCTTTAAGTCCAGATT

TGGAGACTTTCATGTCCTGGGCTGATGCAATCAGACGTCTCAAGGTGTTTATGAGTTTCTGTTC

CTTTAACTTGTTTGATATTTTTAAACTTTCTAACTCAAATGTTCGATGACCGATAAGGTTATGG

CGAATGCGGATACACCTGAAGACGCCATTGCAGCTAGGAAAAACGGAGCTCAAGGAATCGGGCT

TTGTAGGACAGAGCATATGGTAACTCCTCCTCTGTACTTGATTTCATGTTTTTGATGATTTAGA

TTGTTTGTATCCAAATGTTTAATGTTGTCTTTGGTTTGGTTTTTAAGTTCTTTGGAGCAGATAG

GATTAAAGCAGTGAGAAAGATGATAATGGCGGTAACAACAGAGCAAAGGAAAGCTTCTCTCGAC

ATCTTGCTTCCTTACCAACGTTCGGATTTCGAAGGGATCTTCCGTGCTATGGATGGTAAATGTT

TTGAGTCGTCTCTCTAAAATGTATCACAACTTAAAACATGCCTAAACCTTTTTATTTTTCTAGG

TTTACCGGTAACAATCCGTTTGTTAGACCCTCCGCTTCACGAGTTTCTCCCGGAAGGCGACTTG

GACAACATTGTACATGAGCTAGCTGAAGAAACTGGTGTGAAAGAAGATGAAGTCTTGTCACGGA

TAGAGAAACTCTCTGAAGTGAATCCAATGCTTGGTTTCCGCGGTTGCAGGTTTCTTACTCTCTT

TGTTTCTCTCTGTCTCTTTGCACCTGAAGAACAATCTGATGATCGGTAAACTTGTACGTTATAG

GCTCGGAATATCGTATCCAGAGCTAACGGAGATGCAAGCGCGTGCAATTTTTGAAGCTGCAGCG

TCAATGCAGGACCAAGGTGTTACTGTCATTCCTGAGATTATGGTTCCACTTGTAGGAACTCCTC

AGGAATTGGGTCACCAAGTTGATGTAATTCGTAAAGTTGCAAAGAAAGTATTTGCTGAGAAGGG

TCATACCGTGAGCTACAAGGTTGGGACAATGATTGAGATCCCTCGAGCCGCGCTCATTGCAGAT

GAGGTAAATGTAACAAGACACAAAATGTGTTTTAGGCACTTGAAACCATGTTGCTATTTGCTAA

GTAGGAACCTTTTTCTTTTGACAGATTGCGAAAGAGGCGGAGTTTTTCTCGTTCGGGACAAACG

ACTTGACGCAGATGACGTTTGGATACAGTAGAGACGATGTCGGCAAGTTTCTACCGATTTACCT

CGCCAAAGGAATCTTACAGCACGACCCTTTTGAGGTATAATGACTACCATTTCGTTTGCTCTCT

ATCCATAGGATAAAATCTTGATAGCCATTTTTTTGTGTTTGGACCAGGTTCTTGATCAGCAAGG

TGTAGGGCAATTGATCAAGATGGCGACAGAAAAAGGACGAGCAGCTAGGCCTAGCCTCAAGGTT

GGGATATGTGGAGAACATGGAGGAGATCCATCTTCTGTGGGATTCTTTGCTGAAGCAGGACTTG

ACTATGTCTCTTGTTCTCCTTTCAGGTAATTGATTAATTTCCAAACCAATAAACACTTTTTTTA

CAACACTATTGTATAACTCAGATTGATGTAATTTTGGGATTTCTGTTGTTGTTGTTGTTGTTGT

TGTTGTTGCAGGGTTCCAATTGCAAGGCTTGCAGCTGCTCAAGTAGTTGTTGCATGA

SEQ ID No：20

the cDNA sequence encoding the cytosolic form of arabidopsis pyruvate orthophosphate dikinase (PPDK) is provided herein as SEQ ID No: 21:

ATGATGCAGCGAGTATTCACCTTTGGAAAAGGAAGAAGCGAAGGCAACAAGGGCATGAAG

TCCTTGTTGGGAGGGAAAGGAGCCAACCTGGCGGAGATGGCTAGCATAGGCTTGTCGGTG

CCGCCGGGGCTAACCATATCGACGGAGGCTTGTCAGCAGTATCAGATCGCCGGCAAAAAG

CTTCCAGAAGGTTTATGGGAAGAGATCTTAGAAGGTCTTAGCTTCATCGAACGTGACATT

GGAGCTTCCCTCGCTGATCCCTCCAAGCCACTCCTCCTCTCTGTTCGCTCCGGCGCCGCC

ATCTCAATGCCTGGTATGATGGACACTGTACTTAACCTTGGCTTGAACGACCAAGTCGTC

GTTGGTCTGGCCGCAAAAAGCGGAGAGCGTTTTGCTTACGATTCGTTCCGGCGTTTTCTT

GATATGTTTGGTGATGTTGTGATGGGAATTCCACACGCCAAGTTTGAAGAGAAGTTAGAG

AGAATGAAGGAGAGGAAAGGAGTTAAAAATGACACTGACTTAAGCGCGGCTGATCTCAAG

GAATTGGTTGAGCAGTACAAGAGTGTTTACTTAGAGGCCAAGGGTCAAGAGTTTCCTTCA

GATCCAAAGAAGCAATTGGAGCTAGCGATTGAAGCGGTATTCGATTCTTGGGATAGCCCG

AGAGCGAACAAGTACAGAAGTATTAACCAGATAACTGGATTGAAAGGAACCGCGGTTAAC

ATTCAGTGTATGGTGTTTGGAAACATGGGGGACACTTCAGGGACTGGTGTTCTCTTCACT

AGGAACCCTAGCACAGGAGAGAAGAAGCTTTATGGCGAGTTTCTAGTTAATGCTCAGGTT

TGGCATCTATCACAATGTGTGAATCTCATATCAACAAGGATAAGAACACCAGAAGATTTG

GATACAATGAAGAGATTTATGCCTGAGGCTTACGCTGAACTTGTTGAGAACTGCAACATC

TTAGAAAGACATTACAAAGACATGATGGATATTGAATTCACAGTACAAGAAGAGAGATTG

TGGATGCTGCAATGCAGAGCGGGTAAGCGAACGGGTAAAGGCGCCGTGAAGATAGCAGTT

GATATGGTAGGTGAAGGGCTTGTTGAGAAATCTTCTGCTATCAAAATGGTGGAGCCTCAA

CATCTTGATCAACTACTTCACCCACAGTTTCATGATCCATCGGGGTATCGTGAAAAAGTG

GTGGCCAAAGGCTTACCTGCGTCACCAGGAGCGGCGGTTGGACAGGTTGTGTTCACGGCG

GAGGAAGCCGAAGCTTGGCATTCTCAGGGTAAAACTGTGATTCTGGTTCGAACTGAGACA

AGCCCTGACGATGTGGGAGGTATGCACGCAGCGGAAGGTATATTGACGGCTAGAGGAGGA

ATGACGTCACACGCGGCTGTTGTTGCTCGCGGTTGGGGAAAATGTTGCATTGCTGGTTGT

TCCGAGATTCGTGTCGACGAGAACCACAAGGTTCTATTGATTGGAGATTTGACGATTAAT

GAAGGCGAATGGATCTCAATGAACGGATCAACCGGTGAGGTTATATTAGGGAAACAAGCA

TTGGCTCCTCCGGCTTTAAGTCCAGATTTGGAGACTTTCATGTCCTGGGCTGATGCAATC

AGACGTCTCAAGGTTATGGCGAATGCGGATACACCTGAAGACGCCATTGCAGCTAGGAAA

AACGGAGCTCAAGGAATCGGGCTTTGTAGGACAGAGCATATGATTGTTTGTATCCAAATG

TTTAATGTTGTCTTTGGTTTGGTTTTTAAGTTCTTTGGAGCAGATAGGATTAAAGCAGTG

AGAAAGATGATAATGGCGGTAACAACAGAGCAAAGGAAAGCTTCTCTCGACATCTTGCTT

CCTTACCAACGTTCGGATTTCGAAGGGATCTTCCGTGCTATGGATGGTTTACCGGTAACA

ATCCGTTTGTTAGACCCTCCGCTTCACGAGTTTCTCCCGGAAGGCGACTTGGACAACATT

GTACATGAGCTAGCTGAAGAAACTGGTGTGAAAGAAGATGAAGTCTTGTCACGGATAGAG

AAACTCTCTGAAGTGAATCCAATGCTTGGTTTCCGCGGTTGCAGGCTCGGAATATCGTAT

CCAGAGCTAACGGAGATGCAAGCGCGTGCAATTTTTGAAGCTGCAGCGTCAATGCAGGAC

CAAGGTGTTACTGTCATTCCTGAGATTATGGTTCCACTTGTAGGAACTCCTCAGGAATTG

GGTCACCAAGTTGATGTAATTCGTAAAGTTGCAAAGAAAGTATTTGCTGAGAAGGGTCAT

ACCGTGAGCTACAAGGTTGGGACAATGATTGAGATCCCTCGAGCCGCGCTCATTGCAGAT

GAGATTGCGAAAGAGGCGGAGTTTTTCTCGTTCGGGACAAACGACTTGACGCAGATGACG

TTTGGATACAGTAGAGACGATGTCGGCAAGTTTCTACCGATTTACCTCGCCAAAGGAATC

TTACAGCACGACCCTTTTGAGGTTCTTGATCAGCAAGGTGTAGGGCAATTGATCAAGATG

GCGACAGAAAAAGGACGAGCAGCTAGGCCTAGCCTCAAGGTTGGGATATGTGGAGAACAT

GGAGGAGATCCATCTTCTGTGGGATTCTTTGCTGAAGCAGGACTTGACTATGTCTCTTGT

TCTCCTTTCAGGGTTCCAATTGCAAGGCTTGCAGCTGCTCAAGTAGTTGTTGCATGA

SEQ ID No：21

the cDNA sequence encoding the chloroplast form of arabidopsis pyruvate orthophosphate dikinase (PPDK) is provided herein as SEQ ID No: 22:

ATGACAAGTATGATCGTGAAGACAACGCCGGAGCTCTTCAAAGGAAATGGAGTGTTCCGT

ACGGATCATCTCGGAGAAAACCGAATGGTTAGTCGATCAAACCGGCTAGGTGATGGATCA

AACCGTTTCCCTAGAACCGGTACAATCCATTGCCAACGGTTAAGCATAGCAAAGACCGGT

TTGCATCGTGAGACGAAGGCTCGAGCCATACTTAGCCCTGTGTCCGATCCGGCCGCTTCC

ATAGCCCAAAAGCGAGTATTCACCTTTGGAAAAGGAAGAAGCGAAGGCAACAAGGGCATG

AAGTCCTTGTTGGGAGGGAAAGGAGCCAACCTGGCGGAGATGGCTAGCATAGGCTTGTCG

GTGCCGCCGGGGCTAACCATATCGACGGAGGCTTGTCAGCAGTATCAGATCGCCGGCAAA

AAGCTTCCAGAAGGTTTATGGGAAGAGATCTTAGAAGGTCTTAGCTTCATCGAACGTGAC

ATTGGAGCTTCCCTCGCTGATCCCTCCAAGCCACTCCTCCTCTCTGTTCGCTCCGGCGCC

GCCATCTCAATGCCTGGTATGATGGACACTGTACTTAACCTTGGCTTGAACGACCAAGTC

GTCGTTGGTCTGGCCGCAAAAAGCGGAGAGCGTTTTGCTTACGATTCGTTCCGGCGTTTT

CTTGATATGTTTGGTGATGTTGTGATGGGAATTCCACACGCCAAGTTTGAAGAGAAGTTA

GAGAGAATGAAGGAGAGGAAAGGAGTTAAAAATGACACTGACTTAAGCGCGGCTGATCTC

AAGGAATTGGTTGAGCAGTACAAGAGTGTTTACTTAGAGGCCAAGGGTCAAGAGTTTCCT

TCAGATCCAAAGAAGCAATTGGAGCTAGCGATTGAAGCGGTATTCGATTCTTGGGATAGC

CCGAGAGCGAACAAGTACAGAAGTATTAACCAGATAACTGGATTGAAAGGAACCGCGGTT

AACATTCAGTGTATGGTGTTTGGAAACATGGGGGACACTTCAGGGACTGGTGTTCTCTTC

ACTAGGAACCCTAGCACAGGAGAGAAGAAGCTTTATGGCGAGTTTCTAGTTAATGCTCAG

GTTTGGCATCTATCACAATGTGTGAATCTCATATCAACAAGGATAAGAACACCAGAAGAT

TTGGATACAATGAAGAGATTTATGCCTGAGGCTTACGCTGAACTTGTTGAGAACTGCAAC

ATCTTAGAAAGACATTACAAAGACATGATGGATATTGAATTCACAGTACAAGAAGAGAGA

TTGTGGATGCTGCAATGCAGAGCGGGTAAGCGAACGGGTAAAGGCGCCGTGAAGATAGCA

GTTGATATGGTAGGTGAAGGGCTTGTTGAGAAATCTTCTGCTATCAAAATGGTGGAGCCT

CAACATCTTGATCAACTACTTCACCCACAGTTTCATGATCCATCGGGGTATCGTGAAAAA

GTGGTGGCCAAAGGCTTACCTGCGTCACCAGGAGCGGCGGTTGGACAGGTTGTGTTCACG

GCGGAGGAAGCCGAAGCTTGGCATTCTCAGGGTAAAACTGTGATTCTGGTTCGAACTGAG

ACAAGCCCTGACGATGTGGGAGGTATGCACGCAGCGGAAGGTATATTGACGGCTAGAGGA

GGAATGACGTCACACGCGGCTGTTGTTGCTCGCGGTTGGGGAAAATGTTGCATTGCTGGT

TGTTCCGAGATTCGTGTCGACGAGAACCACAAGGTTCTATTGATTGGAGATTTGACGATT

AATGAAGGCGAATGGATCTCAATGAACGGATCAACCGGTGAGGTTATATTAGGGAAACAA

GCATTGGCTCCTCCGGCTTTAAGTCCAGATTTGGAGACTTTCATGTCCTGGGCTGATGCA

ATCAGACGTCTCAAGGTTATGGCGAATGCGGATACACCTGAAGACGCCATTGCAGCTAGG

AAAAACGGAGCTCAAGGAATCGGGCTTTGTAGGACAGAGCATATGATTGTTTGTATCCAA

ATGTTTAATGTTGTCTTTGGTTTGGTTTTTAAGTTCTTTGGAGCAGATAGGATTAAAGCA

GTGAGAAAGATGATAATGGCGGTAACAACAGAGCAAAGGAAAGCTTCTCTCGACATCTTG

CTTCCTTACCAACGTTCGGATTTCGAAGGGATCTTCCGTGCTATGGATGGTTTACCGGTA

ACAATCCGTTTGTTAGACCCTCCGCTTCACGAGTTTCTCCCGGAAGGCGACTTGGACAAC

ATTGTACATGAGCTAGCTGAAGAAACTGGTGTGAAAGAAGATGAAGTCTTGTCACGGATA

GAGAAACTCTCTGAAGTGAATCCAATGCTTGGTTTCCGCGGTTGCAGGCTCGGAATATCG

TATCCAGAGCTAACGGAGATGCAAGCGCGTGCAATTTTTGAAGCTGCAGCGTCAATGCAG

GACCAAGGTGTTACTGTCATTCCTGAGATTATGGTTCCACTTGTAGGAACTCCTCAGGAA

TTGGGTCACCAAGTTGATGTAATTCGTAAAGTTGCAAAGAAAGTATTTGCTGAGAAGGGT

CATACCGTGAGCTACAAGGTTGGGACAATGATTGAGATCCCTCGAGCCGCGCTCATTGCA

GATGAGATTGCGAAAGAGGCGGAGTTTTTCTCGTTCGGGACAAACGACTTGACGCAGATG

ACGTTTGGATACAGTAGAGACGATGTCGGCAAGTTTCTACCGATTTACCTCGCCAAAGGA

ATCTTACAGCACGACCCTTTTGAGGTTCTTGATCAGCAAGGTGTAGGGCAATTGATCAAG

ATGGCGACAGAAAAAGGACGAGCAGCTAGGCCTAGCCTCAAGGTTGGGATATGTGGAGAA

CATGGAGGAGATCCATCTTCTGTGGGATTCTTTGCTGAAGCAGGACTTGACTATGTCTCT

TGTTCTCCTTTCAGGGTTCCAATTGCAAGGCTTGCAGCTGCTCAAGTAGTTGTTGCATGA

SEQ ID No：22

thus, a coding sequence encoding a polypeptide having PPDK activity may comprise a sequence substantially as set forth in SEQ ID No: 20. a nucleic acid sequence as shown in SEQ ID No.21 or SEQ ID No.22 or a functional variant or fragment thereof.

The polypeptide sequence of the cytoplasmic form of arabidopsis PPDK is provided herein as seq id No: 23:

MMQRVFTFGKGRSEGNKGMKSLLGGKGANLAEMASIGLSVPPGLTISTEACQQYQIAGKK

LPEGLWEEILEGLSFIERDIGASLADPSKPLLLSVRSGAAISMPGMMDTVLNLGLNDQVV

VGLAAKSGERFAYDSFRRFLDMFGDVVMGIPHAKFEEKLERMKERKGVKNDTDLSAADLK

ELVEQYKSVYLEAKGQEFPSDPKKQLELAIEAVFDSWDSPRANKYRSINQITGLKGTAVN

IQCMVFGNMGDTSGTGVLFTRNPSTGEKKLYGEFLVNAQVWHLSQCVNLISTRIRTPEDL

DTMKRFMPEAYAELVENCNILERHYKDMMDIEFTVQEERLWMLQCRAGKRTGKGAVKIAV

DMVGEGLVEKSSAIKMVEPQHLDQLLHPQFHDPSGYREKVVAKGLPASPGAAVGQVVFTA

EEAEAWHSQGKTVILVRTETSPDDVGGMHAAEGILTARGGMTSHAAVVARGWGKCCIAGC

SEIRVDENHKVLLIGDLTINEGEWISMNGSTGEVILGKQALAPPALSPDLETFMSWADAI

RRLKVMANADTPEDAIAARKNGAQGIGLCRTEHMIVCIQMFNVVFGLVFKFFGADRIKAV

RKMIMAVTTEQRKASLDILLPYQRSDFEGIFRAMDGLPVTIRLLDPPLHEFLPEGDLDNI

VHELAEETGVKEDEVLSRIEKLSEVNPMLGFRGCRLGISYPELTEMQARAIFEAAASMQD

QGVTVIPEIMVPLVGTPQELGHQVDVIRKVAKKVFAEKGHTVSYKVGTMIEIPRAALIAD

EIAKEAEFFSFGTNDLTQMTFGYSRDDVGKFLPIYLAKGILQHDPFEVLDQQGVGQLIKM

ATEKGRAARPSLKVGICGEHGGDPSSVGFFAEAGLDYVSCSPFRVPIARLAAAQVVVA

SEQ ID No：23

the polypeptide sequence of the chloroplast form of arabidopsis PPDK is provided herein as SEQ ID No: 24:

MTSMIVKTTPELFKGNGVFRTDHLGENRMVSRSNRLGDGSNRFPRTGTIHCQRLSIAKTG

LHRETKARAILSPVSDPAASIAQKRVFTFGKGRSEGNKGMKSLLGGKGANLAEMASIGLS

VPPGLTISTEACQQYQIAGKKLPEGLWEEILEGLSFIERDIGASLADPSKPLLLSVRSGA

AISMPGMMDTVLNLGLNDQVVVGLAAKSGERFAYDSFRRFLDMFGDVVMGIPHAKFEEKL

ERMKERKGVKNDTDLSAADLKELVEQYKSVYLEAKGQEFPSDPKKQLELAIEAVFDSWDS

PRANKYRSINQITGLKGTAVNIQCMVFGNMGDTSGTGVLFTRNPSTGEKKLYGEFLVNAQ

VWHLSQCVNLISTRIRTPEDLDTMKRFMPEAYAELVENCNILERHYKDMMDIEFTVQEER

LWMLQCRAGKRTGKGAVKIAVDMVGEGLVEKSSAIKMVEPQHLDQLLHPQFHDPSGYREK

VVAKGLPASPGAAVGQVVFTAEEAEAWHSQGKTVILVRTETSPDDVGGMHAAEGILTARG

GMTSHAAVVARGWGKCCIAGCSEIRVDENHKVLLIGDLTINEGEWISMNGSTGEVILGKQ

ALAPPALSPDLETFMSWADAIRRLKVMANADTPEDAIAARKNGAQGIGLCRTEHMIVCIQ

MFNVVFGLVFKFFGADRIKAVRKMIMAVTTEQRKASLDILLPYQRSDFEGIFRAMDGLPV

TIRLLDPPLHEFLPEGDLDNIVHELAEETGVKEDEVLSRIEKLSEVNPMLGFRGCRLGIS

YPELTEMQARAIFEAAASMQDQGVTVIPEIMVPLVGTPQELGHQVDVIRKVAKKVFAEKG

HTVSYKVGTMIEIPRAALIADEIAKEAEFFSFGTNDLTQMTFGYSRDDVGKFLPIYLAKG

ILQHDPFEVLDQQGVGQLIKMATEKGRAARPSLKVGICGEHGGDPSSVGFFAEAGLDYVS

CSPFRVPIARLAAAQVVVA

SEQ ID No：24

thus, a polypeptide having PPDK activity may comprise a sequence substantially as set forth in SEQ ID No:23 or SEQ ID No.24 or a functional variant or fragment thereof. Since the greatest PPDK abundance was observed almost exclusively in cell lines containing genomic DNA encoding PPDK, it seems that introns may have a positive effect on PPDK expression. Thus, in one embodiment, the construct may comprise cDNA of a gene encoding PPDK and/or PCK.

The genetic construct of the invention may be in the form of an expression cassette, which may be suitable for expression of at least one coding sequence in a host cell. The genetic constructs of the invention need not be integrated into a vector for introduction into a host cell. For example, the genetic construct, which may be a nucleic acid molecule, may be incorporated into a liposome or viral particle. Alternatively, purified nucleic acid molecules (e.g., histone-free DNA or naked DNA) can be inserted directly into host cells by suitable methods (e.g., direct endocytic uptake). The genetic construct may be introduced directly into the cells of a host subject (e.g., a plant) by transfection, infection, microinjection, cell fusion, protoplast fusion, or ballistic bombardment (ballistical bombedment). Alternatively, the genetic constructs of the invention can be introduced directly into host cells using a gene gun. Alternatively, the genetic construct may be contained within a recombinant vector for expression in a suitable host cell.

Accordingly, in a third aspect, there is provided a recombinant vector comprising the genetic construct of the first or second aspect.

The recombinant vector may be a plasmid, a cosmid, or a phage. Such recombinant vectors are highly useful for transforming host cells with the genetic constructs of the present invention and replicating the expression cassettes therein. The skilled artisan will appreciate that the genetic constructs of the invention may be used in combination with many types of backbone vectors for expression purposes. The backbone vector may be a binary vector, such as one that is replicable in both E.coli (E.coli) and Agrobacterium tumefaciens (Agrobacterium tumefaciens). For example, a suitable vector may be a pBIN plasmid, such as pBIN 19. However, a preferred backbone vector is based on pBINPLUS (F.A. van Engelen et al, Transgenic Research (1995)4, 288-290) and BNP1380000001 with the SAG12 promoter.

In addition to a promoter (e.g., a senescence-associated promoter), the recombinant vector can include a variety of other functional elements and at least one coding sequence (encoding PCK and/or PPDK). For example, a recombinant vector can be designed such that the vector is autonomously replicating in the cytosol of the host cell. In such cases, elements that induce or regulate DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed such that it is integrated into the genome of the host cell. In this case, DNA sequences are envisaged which facilitate integration of the target, for example by homologous recombination.

The recombinant vector may also comprise DNA encoding a gene that can be used as a selectable marker in a cloning procedure, i.e.cells that have been transfected or transformed can be selected, and cells comprising a vector incorporating heterologous DNA can be selected. The vector may also comprise DNA which is involved in regulating the expression of the coding sequence or which is used to target the expressed polypeptide to a component of the host cell, for example the chloroplast. Thus, the vector of the third aspect may comprise at least one further element selected from: a selectable marker gene (e.g., an antibiotic resistance gene), a polypeptide termination signal, and a protein targeting sequence (e.g., a chloroplast transit peptide).

Examples of suitable marker genes include antibiotic resistance genes, such as genes conferring resistance to kanamycin, geneticin (G418), and hygromycin (npt-II, hyg-B); herbicide resistance genes, for example genes conferring resistance to glufosinate and sulfonamides (bar and suI; EP-A-242246, EP-A-0249637, respectively); and selection markers such as beta-glucuronidase (GB2197653), luciferase and Green Fluorescent Protein (GFP). The marker gene may be under the control of a second promoter (which may not be a senescence-associated promoter) which allows expression in cells (which may or may not be in seeds) allowing selection of cells or tissues containing the marker at any stage of plant development. Suitable second promoters are the promoter of the nopaline synthase gene of Agrobacterium and the promoter derived from the gene encoding the 35S cauliflower mosaic virus (CaMV) transcript. However, any other suitable second promoter may be employed.

Various embodiments of the genetic constructs of the present invention can be made using the cloning method described in FIG. 1, which can be summarized as follows. Genomic and cDNA forms of the genes encoding PCK and PPDK can be amplified by PCR from genomic or cDNA templates using appropriate primers. The PCR product can be checked by agarose gel electrophoresis. The PCR product can then be ligated with a suitable vector for cloning purposes, such as the pCR4Blunt-TOPO vector (Invitrogen). The vector containing the PCR product can be grown in a suitable host, such as E.coli. Coli colonies were then screened by PCR using the appropriate primers and inserts from plasmids with the correct restriction endonuclease digest pattern were sequenced using the appropriate primers.

Coli colonies carrying TOPO-cDNA (PCK or PPDK) or TOPO-gDNA (PCK or PPDK) can be cultured to produce appropriate amounts of the various plasmids, which can then be purified. The plasmid may then be digested to release a DNA fragment encoding PPDK or PCK, which fragment may then be cloned into a vector comprising a suitable promoter (e.g., a SAG promoter), such as a pBNP plasmid. The resulting PPDK constructs were designated BNP-PPDKcDNA and BNP-PPDKgDNA, and the resulting PCK constructs were designated pALBNP1(cDNA) and pALBNP2 (gDNA).

An embodiment of the vector of the third aspect may be substantially as shown in figure 3.

In a fourth aspect, there is provided a method of increasing the concentration of PCK and/or PPDK in a leaf of a test plant above the corresponding concentration of PCK and/or PPDK in a wild type plant cultivated under the same conditions, the method comprising altering plant metabolism of the test plant such that the level of PCK and/or PPDK in the plant leaf is increased after the onset of leaf senescence.

In a fifth aspect, there is provided a method of reducing the nitrogen concentration in leaves of a test plant to below that of a corresponding nitrogen concentration in a wild type plant cultured under the same conditions, the method comprising altering plant metabolism in the test plant such that the level of PCK and/or PPDK in the plant leaves is increased after the onset of leaf senescence.

In a sixth aspect, there is provided a method of increasing the growth rate of a test plant compared to the corresponding growth rate of a wild type plant cultured under the same conditions, the method comprising altering plant metabolism in the test plant such that the level of PCK and/or PPDK in the plant leaves is increased after the onset of leaf senescence.

Methods for determining nitrogen levels in plant leaves and plant growth rates are given in the examples. The method of the fourth, fifth or sixth aspect may comprise transforming a test plant cell with the genetic construct of the first or second aspect or the vector of the third aspect. The genetic construct or vector may be introduced into the host cell by any suitable method.

In a seventh aspect, there is provided a cell comprising the genetic construct of the first or second aspects or the recombinant vector of the third aspect.

The cell may be a plant cell. Since the inventors have observed that overexpression of both PCK and/or PPDK in a host cell is unexpectedly effective in inducing nitrogen remobilisation in senescent leaves, the cell of the seventh aspect may comprise one or more constructs of the first or second aspect or one or more vectors of the third aspect such that both PCK and/or PPDK are overexpressed.

For example, a host cell may be transformed with only the first embodiment of the genetic construct of the first aspect (i.e., the PCK construct). Alternatively, the host cell may be transformed with only the second embodiment of the genetic construct of the second aspect (i.e., the PPDK construct). In another embodiment, the host cell may be transformed with the first and second embodiments of the genetic construct of the first aspect such that both PCK and PPDK are expressed in the host cell. Alternatively, the host cell may be transformed with the third or fourth embodiment of the construct of the first aspect (i.e., PCK/PPDK construct 1 or 2) such that both PCK and PPDK are expressed in the host cell. It is also envisaged that the host cell may be transformed with the construct of the second aspect encoding both PCK and PPDK.

Cells can be transformed with the genetic constructs or vectors of the invention using known techniques. Suitable methods for introducing the genetic construct into the host cell may include the use of disarmed Ti-plasmid vectors carried by Agrobacterium by methods known in the art (e.g.as described in EP-A-0116718 and EP-A-0270822). Another method may be to transform plant protoplasts, which involves first removing the cell wall and introducing nucleic acids, followed by reforming the cell wall. The transformed cells can then be grown into plants.

In an eighth aspect, there is provided a transgenic plant comprising the genetic construct of the first or second aspects or the vector of the third aspect.

The transgenic plant of the eighth aspect may comprise a Brassicaceae (Brassicaceae), such as Brassica (Brassica spp.). The plant may be brassica napus (oilseed rape).

Other examples of transgenic plants of the eighth aspect include poaceae (Poales), such as Triticeae spp. The plant may be of the genus Triticum spp. Increasing the grain protein content of wheat can result in an increase in the volume of a food product (e.g., bread) comprising wheat.

Other examples of suitable transgenic plants of the eighth aspect include Solanaceae (Solanaceae) plants including, for example, stramonium, eggplant, solanum lycopersicum (manddrake), belladonna (deadly lightshade, belladonna), capsicum (capsicum, paprika, chilipeper), potato and tobacco. An example of a suitable genus of solanaceae is the genus Nicotiana (Nicotiana). The tobacco belonging to a proper variety can be called tobacco plant, and is called tobacco for short. Various methods of transforming plants with the genetic construct of the first or second aspect or the vector of the third aspect are known and may be used in the present invention.

For example, tobacco can be transformed as follows. Tobacco (Nicotiana tabacum) was transformed using a leaf disc co-cultivation method essentially as described by Horsch et al (Science 227: 1229-1231, 1985). The youngest two expanded leaves can be picked from 7-week-old tobacco plants, which can be 8% Domestoses^TMAfter surface sterilization for 10 minutes, the mixture was washed 6 times with sterile distilled water. Leaf disks can be cut with a No.6 corkscrew and placed in Agrobacterium suspension containing the appropriate binary vector (according to the invention) for about 2 minutes. The leaf discs can be gently blotted dry between two sheets of sterile filter paper. 10 leaf discs can be placed on LS 3% sucrose + 2. mu.M BAP + 0.2. mu. MNAA plates, which are then incubated in the growth chamber for 2 days. Leaf discs can be transferred to plates of LS + 3% sucrose + 2. mu.M BAP + 0.2. mu.M NAA supplemented with 500g/l of Kalfuron and 100g/l of kanamycin. After 2 weeks, leaf disks can be transferred to new plates of the above medium. After a further 2 weeks, leaf disks can be transferred to plates containing LS + 3% sucrose + 0.5. mu.M BAP supplemented with 500mg/l of Kalfuron and 100mg/l of kanamycin. Leaf disks were transferred to fresh medium every two weeks. When shoots appear, shoots can be cut off and transferred to jars supplemented with 500mg/l of kevlar LS + 3% sucrose. After about 4 weeks, the sprouts in the jars can be transferred to LS + 3% sucrose +250mg/l Kalfuron. After 3-4 weeks, the plants can be harvestedTransfer to LS + 3% sucrose (no antibiotics) and rooting. Once the plants were rooted, they were transferred to soil in the greenhouse.

In a ninth aspect, a plant propagation product obtained from the transgenic plant of the eighth aspect is provided.

A "plant propagation product" may be any plant material taken from a plant from which other plants may be produced. The plant propagation product may suitably be a seed.

In a tenth aspect of the invention, there is provided a method of producing a transgenic plant which remobilizes nitrogen at a higher rate than a corresponding wild-type plant cultured under the same conditions, comprising the steps of:

i) transforming a plant cell with the genetic construct of the first or second aspect or the vector of the third aspect; and

ii) regenerating a plant from the transformed cell.

In an eleventh aspect, there is provided a method of producing a transgenic plant having a faster growth rate than a corresponding wild type plant cultured under the same conditions, the method comprising the steps of:

ii) regenerating a plant from the transformed cell.

Preferably and advantageously, the method of the invention does not impair the health or fitness of the resulting test plant. Preferably the method comprises transforming a test plant, preferably a plant leaf, with the genetic construct of the first or second aspect or the vector of the third aspect. The inventors observed that overexpression of both PCK and PPDK in the host cell was effective in inducing nitrogen remobilisation in senescent leaves. Thus, it is preferred that the methods of the tenth and eleventh aspects comprise transforming a test plant with one or more constructs of the invention such that both PCK and PPDK are overexpressed. For example, a test plant may be transformed with a first embodiment of the genetic construct of the first aspect of the invention (i.e., the PCK construct) and then transformed with a second embodiment of the construct of the first aspect (i.e., the PPDK construct). Thus, transformation of these two constructs results in overexpression of both enzymes. Alternatively, a test plant may be transformed with the third or fourth embodiment of the constructs of the first aspect of the invention, each of which encodes PCK and PPDK. Alternatively, a test plant may be transformed with a construct of the second aspect of the invention which encodes both PCK and PPDK.

The inventors observed that plant leaves of test plants that have been transformed with constructs encoding both PCK and PPDK show an increased nitrogen remobilisation at the onset of senescence, so that the nitrogen concentration in the leaves decreases. Furthermore, the inventors observed an increase in vegetative growth rate. Although the inventors are not bound by the hypothesis, the inventors believe that a decrease in nitrogen concentration in the leaves may induce an increase in growth rate and thus crop yield.

In a twelfth aspect of the invention there is provided a harvested leaf containing a lower level of nitrogen than the corresponding level of nitrogen in a harvested leaf taken from a wild type plant cultured under the same conditions, wherein the leaf is harvested from the transgenic plant of the eighth aspect or produced by the method of the tenth or eleventh aspect.

In a thirteenth aspect of the invention, there is provided a smoking article comprising nitrogen-reduced tobacco obtained from a mutant tobacco plant, the mutant being capable of reducing the nitrogen concentration in senescent leaves.

The nitrogen-reduced tobacco can include tobacco in which the nitrogen concentration is less than the corresponding concentration in a wild-type plant cultured under the same conditions. Such a smoking article may comprise tobacco obtained from a mutant tobacco plant which may be transformed with the genetic construct of the first or second aspect of the invention or the vector of the third aspect.

The term "smoking article" as used herein may include smokeable products such as cigarettes, cigars and cigarillos, whether based on tobacco, tobacco derivatives, expanded cut tobacco, reconstituted tobacco or tobacco substitutes, and heat-not-burn products.

It will be appreciated that the present invention provides any nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid sequence or nucleic acid sequence (including functional variants or functional fragments thereof) of any of the sequences referred to herein. The terms "substantial amino acid/polynucleotide/polypeptide sequence", "functional variant" and "functional fragment" may be a sequence having at least 40% sequence identity to the amino acid/polynucleotide/polypeptide sequence of any one of the sequences mentioned herein, for example 40% identity to the gene defined in SEQ ID No.17 (which encodes an embodiment of the PCK enzyme), or 40% identity to the polypeptide defined in SEQ ID No.19 (i.e. an embodiment of the PCK enzyme), or 40% identity to the gene defined in SEQ ID No.21 (which encodes an embodiment of the PPDK enzyme), or 40% identity to the polypeptide defined in SEQ ID No.23 (i.e. an embodiment of the PPDK enzyme).

Also envisaged are amino acid/polynucleotide/polypeptide sequences having the following sequence identities with any of the sequences mentioned: greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity. Preferably the amino acid/polynucleotide/polypeptide sequence is at least 85% identical to any of the sequences mentioned, more preferably at least 90% identical, even more preferably at least 92% identical, even more preferably at least 95% identical, even more preferably at least 97% identical, even more preferably at least 98% identical and most preferably at least 99% identical to any of the sequences mentioned herein.

The skilled artisan will understand how to calculate the percent identity between 2 amino acids per polynucleotide/polypeptide sequence. To calculate the percent identity between 2 amino acids/polynucleotide/polypeptide sequences, an alignment of the 2 sequences must first be prepared, followed by calculation of the sequence identity value. The percent identity of the 2 sequences can take on different values according to: (i) methods for aligning sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (performed in different programs), or structural alignments from 3D comparisons; and (ii) parameters employed by the alignment method, such as local to global alignments, pairwise scoring matrices employed (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as functional types and constants.

If the alignment is complete, there are many different methods of calculating the percent identity between the 2 sequences. For example, the same number may be divided by: (i) the length of the shortest sequence; (ii) length of comparison; (iii) the average length of the sequence; (iv) the number of non-vacancy positions; or (iv) the number of equivalent positions outside the overhang. Furthermore, it is understood that percent identity is also strongly length dependent. Thus, the shorter the sequence pair, the higher the sequence identity that can be expected to occur occasionally.

Thus, it is understood that the exact alignment of protein or DNA sequences is a complex process. The commonly used multiplex alignment program ClustalW (Thompson et al, 1994, Nucleic acids Research, 22, 4673-. Suitable parameters for ClustalW may be as follows: for DNA alignment: gap development penalty 15.0, gap extension penalty 6.66, matrix identity. For protein alignment: gap development penalty of 10.0, gap extension penalty of 0.2, matrix Gonnet. For DNA and protein alignments: ENDGAP ═ -1 and gapist ═ 4. It will be clear to those skilled in the art that these and other parameters may need to be varied for optimal sequence alignment.

Preferably, a calculation of percent identity between 2 amino acids/polynucleotide/polypeptide sequences is then calculated from such alignments as (N/T) × 100, where N is the number of positions in the sequence having identical residues and T is the total number of positions compared including gaps but not overhangs. Thus, the most preferred method for calculating percent identity between 2 sequences comprises (i) applying the ClustalW program to prepare sequence alignments using a suitable set of parameters (e.g., those described above); and (ii) substituting the value of N and the value of T into the following equation: sequence identity is (N/T) × 100.

Alternative methods for identifying similar sequences are known to those skilled in the art. For example, a substantially similar nucleotide sequence may be encoded by a sequence that hybridizes under stringent conditions to the sequence set forth in SEQ ID nos.16, 17, 18, 20, 21, 22, or the complement thereof. By stringent conditions, it is meant that the nucleotides hybridize to filter-bound DNA or RNA in 3 XSSC/sodium citrate (SSC) at about 45 ℃ followed by at least one wash in 0.2 XSSC/0.1% SDS at about 20-65 ℃. Alternatively, a substantially similar polypeptide may differ from the sequence set forth in SEQ ID nos.19, 23 or 24 by at least 1 but less than 5, 10, 20, 50 or 100 amino acids.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence can be altered or altered to provide a functional variant thereof without substantially affecting the sequence of the protein encoded thereby. Suitable nucleotide variants are those having a sequence that is altered by substitution of a different codon encoding the same amino acid within the sequence to produce a silent change (silent change). Other suitable variants are those having a homologous nucleotide sequence but comprising all or part of the sequence altered by substitution of a different codon encoding an amino acid having a side chain with biophysical properties similar to the amino acid it was substituted for to produce a conservative change. For example, small, nonpolar hydrophobic amino acids, including glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated that amino acids may be substituted by amino acids having similar biophysical properties, the nucleotide sequences encoding these amino acids being known to the skilled person.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects, in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the present invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the accompanying drawings, in which:

FIG. 1 shows the protocol for cloning the genetic constructs BNP-PPDKcDNA and BNP-PPDKgDNA, in which: (a) PCR amplification of cDNA and genomic DNA forms of the arabidopsis PPDK cytoplasmic isoform; (b) inserting a cloning vector pCR4 Blunt-TOPO; (c) the cloning vector (left) was digested with AvrII and BamHI restriction endonucleases to release PPDK, and the vector pBNP containing the target SAG12 (right); (d) PPDK was ligated to pBNP and agarose gel showed DNA fragments generated by restriction endonuclease digestion of the constructs with AvrII and BamHI; and (e) introducing the construct into the arabidopsis thaliana ecotype Columbia 0 by agrobacterium-mediated transformation;

FIG. 2a shows the plasmid pCR4BLUNT-TOPO used to construct the expression vector of the present invention, and FIG. 2b is a summary of the insertion of cDNA or gDNA encoding PPDK into pCR4BLUNT-TOPO using AvrII and BamHI digestions;

FIG. 3a shows plasmid pBNP containing SAG12 promoter, FIG. 3b is a list of vectors of the present embodiment generated by introducing PPDK insert (cDNA or gDNA) into pBNP using AvrII and BamHI digestion, and introducing PCK insert (cDNA or gDNA) into pBNP using XbaI and SacI digestion;

FIG. 4 shows selection of transgenic SAG12-PPDK Arabidopsis cell line by Western blotting. The top panel shows SAG12-PPDK cDNA cell line, the bottom panel shows SAG12-PPDKgDNA cell line;

FIG. 5 shows the quantitative determination of PPDK abundance of wild type, Δ PPDK and 5 independent SAG12-PPDKgDNA lines from week five;

FIG. 6 shows the selection of SAG12-PPDK (cDNA and gDNA) in (b) K326 and (c) Burley21 tobacco lines;

FIG. 7 shows PPDK overexpression in K326 tobacco mature leaves;

FIG. 8 shows photographs of rosette of different cell lines of Arabidopsis thaliana, i.e.wild type,. DELTA.PPDK and SAG12-PPDKgDNA, as a function of time;

FIG. 9 shows Arabidopsis thaliana reproductive tissue mass at week 9;

FIG. 10 shows total plant fresh mass (i.e., rosette plus reproductive tissue) of wild type and SAG12-PPDgDNA plants from week 3 to week 9 after sowing;

FIG. 11 Nitrogen content in leaves of wild type,. DELTA.PPDK and SAG12-PPDKgDNA plants at week 7;

FIG. 12 shows nitrogen content of single seeds of wild type, Δ PPDK and SAG12-PPDK plants;

FIG. 13 shows total free amino acid content in leaves of wild type,. DELTA.PPDK and SAG12-PPDKgDNA Arabidopsis cell line;

figure 14 shows seed quality for different tobacco cell lines, with zero copies as wild type, G4(gDNA), G10, G8 and C10(cDNA) for the SAG12-PPDK line;

figure 15 shows seed nitrogen content of different tobacco cell lines, zero copy wild type, G4(gDNA), G10, G8 and C10(cDNA) are of the SAG12-PPDK line;

FIG. 16 shows the Western blot results of the PCK/PPDK double insert. All lines showed higher levels of PCK associated with SAG12 promoter activity, especially at week 7;

FIG. 17 shows that expression of both PCK and PPDK in transformed plants results in increased rosette mass compared to control 7; and

FIG. 18 shows a hypothetical biochemical pathway demonstrating nitrogen remobilisation by transport of PCK-and PPDK-dependent formation of the amino acids asparagine and glutamine.

Examples

Example 1-production of the SAG12 promoter Arabidopsis PPDK plant transformation construct

As shown in FIG. 3, a series of SAG12PPDK and PCK expression vectors were prepared and then analyzed for their ability to enhance the expression of PPDK and PCK in transformed plants. In one embodiment, the construct may encode PCK, or a functional variant or fragment thereof, and not PPDK. In another embodiment, the construct may encode PPDK, or a functional variant or fragment thereof, without encoding PCK. However, in yet another embodiment, the construct may encode PCK or a functional variant or fragment thereof and PPDK or a functional variant or fragment thereof. First, genomic DNA of the Arabidopsis thaliana PPDK gene was isolated as follows.

Isolation of genomic DNA

Genomic DNA (gDNA) of Arabidopsis thaliana ecotype Columbia 0 was extracted from leaves using DNeasy plant Mini Kit (Qiagen) according to the recommended protocol. Genomic DNA was used as a template for the PCR reaction described below using the primer sequences summarized in Table 1.

Table 1: primer sequences

Primer and method for producing the same	Sequence of	SEQ ID No.
			AtCytFWD-AvrII	AAT CCT AGG ATG ATG CAG CGA GTA TTC ACC	1
AtCytREV-BamHI	AAT GGA TCC TCA TGC AAC AAC TAC TTG AGC AGC	2
			AtPPDKexon15FWD	CCT CGC CAA AGG AAT CTT AC	7
AtPPDKSeqF1	CTT GGC TTG AAC GAC CAA GTC	3
			AtPPDKSeqF2	GGT TGC AGG GAT AGG AAC ACC	4
AtPPDKSeqR1	CGT CTG ATT GCA TCA GCC CAG	5
			AtPPDKSeqR2	CCT GAG GAG TTC CTA CAA GTG	6
BNP-1271F	TGC CTG CTT GCC GAA TAT C	10
			BNP-1291TV	CAG AAA AGC GGC CAT TTT CCA CCA	12
BNP-1334R	CCG GCC CAC AGT CGA TGA	11
			BNP-nostREV	CAA GAC CGG CAA CAG GAT TCA	8
BNP-SAG12FWD	ACC CCA TCT CAG TAC CCT TCT G	9
			NtCyc-184F	CTC AAC CTT CCA CCG TGT GAT	13
NtCyc-267T	TCT ACG GTG CCA AAT TCG CCG A	15
			NtCyc-316R	ACC GGT GTG CTT CCT CTT GAA	14
AtPCK-XbaI-FOR	ATTTCTAGAATGTCGGCCGGTAACGGAAATG	25
			AtPCK-SacI-REV	ATTGAGCTCCTAAAAGATAGGACCAGCAGCG	26

Isolation of RNA and Synthesis of cDNA

Total RNA was extracted from 7-day-old Arabidopsis cotyledons. RNA extraction was performed on an ice bath using an RNase-free apparatus, and the solution was prepared with water treated with diethylpyrocarbonate (DEPC, Sigma-Aldrich). 1ml of DEPC was added per 1 liter of water and the mixture was stirred overnight in a fume hood and then autoclaved. RNA was extracted using TriPure Isolation Reagent (TriPure Isolation Reagent) (Roche). 200mg of tissue was ground in liquid nitrogen with a mortar and pestle, 1ml of TriPure isolation reagent was added, and the procedure was followed as recommended. The RNA pellet was resuspended in 20. mu.l of RNA Secure (Ambion) preheated to 60 ℃ for 10 min. The samples were centrifuged at 13,000rpm for 5 minutes at4 ℃ and the supernatant was transferred to a clean 1.5ml microcentrifuge tube to remove contaminating debris.

The amount and purity of RNA was determined spectrophotometrically by reading at 260 and 280nm using an Eppendorf Biophotometer spectrophotometer (Maniatis et al, 1982, Molecular cloning: a Laboratory Manual. Cold Spring Harbor Laboratory). The RNA was also examined by agarose gel electrophoresis using 1.5% (w/v) agarose (Melford Laboratories) in 0.5 XTBE. Samples were suspended in 1x RNA sample buffer and run at 80V using 0.5x TBE as the running buffer. The 4 working concentration RNA sample buffer contained 0.002% (w/v) ethidium bromide (Sigma-Aldrich), 2 XTBE (2 XTRU-Tris-borate-EDTA buffer (2 XTBE) containing 180mM Tris-HCl, 180mM boric acid and 8mM EDTA (pH 8.3), 13% Ficoll 400(Ficoll 400) (Sigma-Aldrich), 0.01% bromophenol blue and 7M urea (Ficher Scientific).

RNA reverse transcription was performed to synthesize cDNA using 2. mu.g of RNA, 1. mu.g of oligo dT (15) primer (Roche), 1 XMoloney murine leukemia virus (MMLV) buffer (Promega), 0.4mM dNTPs (Bioline), 40 units of recombinant RNase ribonuclease inhibitor (Promega), 200 units of MMLV reverse transcriptase (Promega) and nuclease-free water to a final volume of 25. mu.l. The samples were incubated at 42 ℃ for 1 hour.

Amplification of Arabidopsis PPDK

The genomic and cDNA forms of the gene encoding the cytoplasmic isoform of PPDK were amplified by PCR from genomic or cDNA templates using the forward primer containing the AvrII restriction site (AtCytFWD-AvrII, SEQ ID No.1) and the reverse primer containing the BamHI restriction site (AtCytREV-BamHI, SEQ ID No.2) shown in Table 1. The PCR reaction mixture contained 1 XHF buffer (NEB), 2mM magnesium chloride (MgCl)₂NEB), 0.5mM dNTPs (Bioline), 100ng template (cDNA or genomic DNA), 0.5. mu.M of each primer and 1 unit Phusion high Fidelity DNA Polymerase (Phusionhigh-Fidelity DNA Polymerase) (NEB). Thermal cycling was performed using a Techne thermal Cycler (Techne thermal Cycler) with an initial denaturation step at 98 ℃ for 30 seconds, followed by 30 cycles of 98 ℃ for 10 seconds, 55 ℃ for 30 seconds and 72 ℃ for 2 minutes and a final extension step at 72 ℃ for 5 minutes.

Once the cDNA and gDNA of arabidopsis PPDK were isolated/prepared, they were used to generate various constructs using the protocol outlined in fig. 1. The cDNA and genomic DNA forms of the cytoplasmic isoform of arabidopsis PPDK were fused to the senescence-induced SAG12 promoter in the pBNP vector, thereby overexpressing PPDK during senescence.

(a) PCR amplification of cDNA and genomic DNA versions of the cytoplasmic isoform of Arabidopsis PPDK using primers containing AvrII and BamHI restriction sites yielded 2.4 and 4.3kb products, respectively.

(b) Inserted into a cloning vector pCR4 Blunt-TOPO. The PPDK insert was sequenced in its entirety and found to be identical to the expected sequence.

(c) The cloning vector and the target vector pBNP were subjected to restriction endonuclease digestion with AvrII and BamHI.

(d) To BNP, agarose gels show the DNA fragments generated by restriction endonuclease digestion of the constructs with AvrII and BamHI. The expected band sizes for the cDNA construct were 14.8kb and 2.4kb, and for the gDNA construct 14.8kb and 4.4 kb.

(e) The construct was introduced into the arabidopsis thaliana ecotype Columbia 0 by agrobacterium-mediated transformation. The constructs were sequenced across the ligation sites. The gene nptII encoding neomycin phosphotransferase confers kanamycin resistance to plants. LB and RB: the left and right borders of the T-DNA, respectively; nos Pro: a nopaline synthase promoter; nos. t: a nopaline synthase terminator; SAG12 Pro: the promoter of the Arabidopsis SAG12 gene.

The generation of the construct will be described in detail below with reference to FIG. 1:

cloning into pCR4Blunt-TOPO vector

1% (w/v) agarose, 0.5. mu.g ml was used^-1The PCR products were examined by agarose gel electrophoresis in 0.5XTBE using ethidium bromide (Sigma-Aldrich). Samples were suspended in 1xDNA sample buffer (6 Xworking concentration DNA sample buffer containing 50mM Tris (hydroxymethyl) aminomethane (Tris-HCl, Melford Laboratories), 60% glycerol, and 0.25% (w/V) bromophenol blue (Sigma-Aldrich) using 0.5XTBE as running buffer and run at 80V.

According to the recommended protocol, a 2.6kb PPDK cDNA band and a 4.4kb PPDK genomic DNA band were purified using QIAQuick PCR purification kit (Qiagen) and eluted using 30. mu.l molecular biological water (BDH Laboratory Supplies). The PCR product was ligated (Blunt-ended) into pCR4Blunt-TOPO vector (Invitrogen) according to the recommended protocol. The cloning reaction (2. mu.l) was transformed into 50. mu.l of sub-cloning efficiency DH5a E.coli (sub-cloning efficiency DH5a E.coli) (Invitrogen) according to the recommended method. In the package 10g l^-1Tryptone (BD) for bacteria, 5g l^-1Yeast extract (Oxoid) for bacteria and 85mMLuria-Bertani (LB) agar of sodium chloride (Fisher Scientific) transformed E.coli cells were grown overnight at 37 deg.C in Luria Bertani broth (LB broth.) pH was adjusted to 7.0 with 10M sodium hydroxide before autoclaving by adding 1.5% (w/v) agar (BD) to a solution containing 50. mu.g ml^-1LB agar was prepared in LB medium with kanamycin (Melford Laboratories).

Using 1 XNH₄Buffer (Bioline), 2.5mM MgCl₂(Bioline), 0.5mM dNTPs (Bioline), 0.3. mu.M of each primer (AtCytFWD-AvrII and AtCytREV-BamHI), SEQ ID Nos 1 and 2, and 0.5 unit BioTaq NA polymerase (Bioline), and E.coli colonies were screened by PCR. Thermal cycling was performed with an initial denaturation step at 95 ℃ for 5 minutes, followed by 30 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 4 minutes and 30 seconds, and a final extension step at 72 ℃ for 5 minutes. The PCR products were examined using 1% (w/v) agarose gel electrophoresis as described above. The colonies containing the desired insert were allowed to grow in 5ml medium containing 50. mu.g ml in a shaking incubator at 37 ℃^-1Kanamycin was grown overnight in LB broth. Plasmid DNA was extracted using the QIAPrep Spin Miniprep kit (Qiagen) according to the recommended protocol. The DNA was digested with 10 units BamHI (NEB1) in1 XBamHI buffer for 1 hour at 37 ℃. Digests were examined using 1% (w/v) agarose gel electrophoresis as described above. The insert fragments in the plasmid with the correct restriction endonuclease digestion profile were sequenced using the primers AtCytFWD-AvrII (SEQ ID No.1), AtPPDKSeqF1(SEQ ID No.3), AtPPDKSeqF2(SEQ ID No.4), AtPPDKSeqR1(SEQ ID No.5), AtPPDKSeqR2(SEQ ID No.6) and AtCytREV-BamHI (SEQ ID No.2) shown in Table 1 using a 3730DNA analyzer (applied biosystems). The sequence was analyzed by BioEdit (Ibis biosciences).

These constructs were called TOPO-PPDKcDNA and TOPO-PPDKgDNA, as shown in FIG. 2a and FIG. 2 b.

Cloning of the pBNP construct

Ligation of PPDK to BNP1380000001 binary under the control of the senescence-inducible promoter SAG12In a carrier. The backbone plasmid BNP1380000001 containing SAG12 is based on pBINPLUS (F.A. van Engelen et al, Transgenic Research (1995)4, 288-290) as shown in FIG. 3 a. First, 25ml of E.coli colonies carrying TOPO-cDNA or TOPO-gDNA were used to inoculate 25ml of E.coli colonies containing 50. mu.g ml^-1LB broth of kanamycin. These cultures were incubated overnight in a shaking 37 ℃ incubator and Plasmid DNA was extracted using a Plasmid Midi Kit (Qiagen) according to the recommended protocol. Using plasmid Midi kit, from 100ml containing 50. mu.g ml^-1The pBNP1380000001 vector was purified from kanamycin cultures.

These plasmids were then digested by the restriction enzymes AvrII and BamHI. Digestion reactions containing 2. mu.g DNA (BNP) or 4. mu.g DNA (TOPO-PPDKcDNA and TOPO-PPDKgDNA), 1 Xbuffer 2, 10 units of AvrII and 10 units of BamHI were incubated overnight at 37 ℃. Crystal violet sample buffer (250. mu.M crystal violet (Hopkin and Williams) and 30% (w/V) sucrose (Fisher Scientific)) was used and 0.5XTBE containing 25. mu.M crystal violet was used as the running buffer, electrophoresis was performed at 50V for about 2 hours using 0.8% (w/V) agarose, 0.5XTBE and 25. mu.M crystal violet (Hopkin and Williams), and the samples were separated by crystal violet agarose gel electrophoresis (Rand, 1996, crystal violet can be used to observe DNA bands during gel electrophoresis and improve cloning efficiency. Gel strip extraction using QIAQuick gel extraction kit (Qiagen) according to the recommended protocol was used to extract the 14.4kb BNP, 2.6kb PPDK cDNA and 4.4kb PPDK genomic DNA fragments. The fragments were examined and quantified relative to Hyperladder I (Bioline) using 1% agarose gel electrophoresis using ethidium bromide.

Since the genomic DNA fragment (4.4kb) and TOPO backbone (3.9kb) were similar in size, the gel extracted genomic DNA fragment was phosphatase treated to prevent ligation with any contaminating TOPO backbone. Shrimp alkaline phosphatase (SAP, 1 unit, Roche) was added to 1 Xdephosphorylated buffer (Roche) containing 1. mu.g of gel-extracted genomic DNA fragments, and the reaction was incubated at 37 ℃ for 30 minutes and then inactivated at 65 ℃ for 10 minutes. Ligation was performed using 10: 1 molar ratio of cDNA or genomic DNA fragments to digested BNP, 1 Xligation buffer (NEB), and 1 unit of T4DNA ligase (NEB). The ligation reaction was incubated overnight at 16 ℃ according to the recommended protocol, and 2. mu.l of DH5_ E.coli (library-efficiency DH5_ E.coli) (Invitrogen) were used to transform the library efficiency.

The transformed E.coli was transferred to a medium containing 50. mu.g ml^-1Kanamycin in LB agar and incubated at 37 ℃ overnight. Using 1 XNH₄Buffer, 2.5mM MgCl₂0.5mM dNTPs, 0.3. mu.M of each primer shown in Table 1 (AtPPDKexon15FWD (SEQ ID No.7) and BNPnosTREV (SEQ ID No.8) and 0.5 units BioTaq DNA polymerase, screening of E.coli colonies by PCR, thermal cycling was performed with an initial denaturation step of 95 ℃ for 5 minutes, followed by 30 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 3 minutes and a final extension step of 72 ℃ for 5 minutes, PCR products were examined as above using 1% (w/v) agarose gel electrophoresis, and colonies containing the desired insert were subjected to 5ml of a medium containing 50. mu.g ml of the desired insert in a shaking incubator at 37 ℃^-1Kanamycin was grown overnight in LB broth. Plasmid DNA was extracted using the QIAPrep Spin Miniprep kit according to the recommended protocol. The DNA was digested with 10 units BamHI and 10 units StuI in1 XBamHI buffer at 37 ℃ for 1 hour and then subjected to 1% (w/v) agarose gel electrophoresis as described above. One colony resulting from the ligation of cDNA and genomic DNA, respectively, with the correct restriction endonuclease digestion pattern was selected and sequenced using the primer BNP-SAG12FWD (SEQ ID No.9) shown in Table 1 above.

These constructs were termed BNP-PPDKcDNA and BNP-PPDKgDNA, as shown in FIGS. 3a and 3 b.

Example 2 transformation of Arabidopsis thaliana with BNP-PPDKcDNA and BNP-PPDKgDNA

Agrobacterium tumefaciens strain GV3101-R was transformed by electroporation using the following plasmids: BNP-PPDKcDNA and BNP-PPDKgDNA are shown in FIG. 3, the preparation of which is described in example 1.

From a content of 25mg l^-1Electrotransformation competent (Electrocompetent) Agrobacterium bacteria were prepared in cultures of LB medium of rifampicin (Sigma-Aldrich), grown at 30 ℃ and with an optical density of 0.4-0.6 measured at 600nm using an Eppendorf Biophorometer spectrophotometer. After centrifugation of 500ml of the culture at 4000g for 15 minutes and removal of the supernatant, the cells were resuspended in 500ml of cold 10% glycerol (Fisher Scientific). Centrifugation and resuspension were repeated using 250ml of glycerol, followed by 10ml of glycerol and finally 2ml of glycerol. Cells were aliquoted into 50 μ l aliquots, snap frozen in liquid nitrogen, and stored at-80 ℃. Electroporation was performed using a BioRad Gene Pulser. Plasmid DNA (200ng) was added to 50. mu.l of the above Agrobacterium cells in a pre-cooled electroporation Cuvette (Gene Pulser Cuvette, BioRad) and the cells were incubated for 5 minutes on an ice bath. The cuvette was electroporated with a pulse of 2.5mV, an impedance of 400ohm and a capacitance of 25 μ F and 1ml SOC (Super Optimal Broth, Catalite reproduction containing 20g/l bactotryptone, 5g/l bactotrypsin extract, 85mM sodium chloride and 250mM potassium chloride). Autoclaved after adjusting to pH 7.0 with 10M sodium hydroxide. Sterile magnesium chloride was added to a final concentration of 10mM and sterile glucose (Fisher Scientific) was added to a final concentration of 20mM before use. After incubating the cells at 30 ℃ for 2 hours in a shaking incubator, the cells were transferred to a medium containing 50. mu.g ml^-1Kanamycin and 50. mu.g ml^-1Rifampicin in LB agar.

After 2 days of incubation at 30 ℃, colonies were screened by PCR followed by restriction enzyme digestion. Colonies positive for PCR screening and having the expected restriction digest spectrum were inoculated into 5ml of a medium containing 50. mu.l of the medium^-1Kanamycin and 50. mu.l ml^-1Rifampicin in LB medium and incubated overnight at 30 ℃ in a shaking incubator. The next day, 600. mu.l of this culture was used to inoculate 500ml of the above LB medium, and used for inflorescence dipping (floral dipping) after incubation at 30 ℃ for 30 hours in a shaking incubator.

4-week-old Arabidopsis plants were used for inflorescence dipping. All constructs were transformed into the wild-type ecotype Columbia 0. The Agrobacterium culture prepared above was centrifuged at 5,000g for 15 minutes at4 ℃ and the cell pellet resuspended in 250ml sterile 5% sucrose (Fisher scientific) solution (w/v) and 0.05% Silwett L-77(OSi Specialties). While gently agitating, the plants were immersed in the cell suspension for about 10 seconds and then covered with a film (clinfilm) to avoid direct light for 24 hours. After this time, the plants were returned to normal growth protocol and the seeds were harvested.

Selection of transgenic Arabidopsis thaliana

According to Harrison et al ((2006), Plant Methods, 2, 19), at 50. mu.g ml^-1Seeds of transformed plants were selected in kanamycin (Dufeca Biochemie). Antibiotic resistant T1 plants were grown to seed and self-pollinated. Seeds were selected as described above, and the T2 line with the 3: 1 resistance: non-resistance ratio was selected as carrying a single copy of the transgene. These plants were again self-pollinated and selected as above. The T2 line that produced 100% resistant progeny (T3) was selected as the homozygous line and used for all experiments. Plants of generation T2 that were homozygous for the transgene and contained a single copy of the transgene were selected using mendelian genetics principles. For plants transformed with SAG12-PPDKcDNA and SAG12-PPDKgDNA, 5 independent single insert homozygous lines were selected each.

Example 3 transformation of tobacco with BNP-PPDKcDNA and BNP-PPDKgDNA.

Transformation of competent Agrobacterium tumefaciens strain LBA4404 was transformed by electroporation as described in example 2 using plasmids BNP, BNP-PPDKcDNA and BNPPPDKgDNA. After electroporation, 1ml of LB medium was added to the electroporated cells, which were then incubated at 28 ℃ for 2 hours in a shaking incubator and transferred to a medium containing 50. mu.g ml^-1Kanamycin and 100. mu.g ml^-1Spectinomycin (Sigma-Aldrich) in LB agar. After 2 days of incubation at 28 ℃ one colony was used to inoculate 50ml of a culture containing50μg ml^-1Kanamycin and 100. mu.g ml^-1LB culture solution of spectinomycin. The culture was incubated at 28 ℃ for 3 days in a shaking incubator. Plasmid DNA was extracted and analyzed by restriction enzyme digestion, and 50. mu.l of the culture was used to inoculate 50ml of a culture containing 50. mu.g ml^-1Kanamycin and 100. mu.g ml^-1Spectinomycin in LB culture solution. The culture was incubated overnight at 28 ℃ in a shaking incubator.

Tobacco cultivars Burley21 and K326 were grown from seeds, the youngest leaves were cut from 8 week old plants, sterilized in 8% (v/v) Domestos concentrated bleach (Domestos thickbleach) (Domests) for 10 minutes, and rinsed in sterile distilled water. The leaf discs were drilled using a No.6 corkscrew, then placed in 25ml of Agrobacterium culture for 2 minutes. The leaf discs were then placed face down on MS medium containing 2.2. mu.M 6-Benzylaminopurine (BAP) and 0.27. mu. M a-naphthaleneacetic acid (NAA). These media were placed in a 22 ℃ growth chamber for 2 days. The leaf discs were then transferred to a container containing 500. mu.g ml^-1Kalfuron (Clarofan) (Roussel laboratories) (non co-incubation control) or 500. mu.g ml^-1Kalfuron and 100. mu.g ml^-1Kanamycin in the selective MS medium described above. Leaf discs were transferred to fresh selective MS medium as described above every 14 days for 6 weeks. Then, callus and shoot mass were taken out from the leaf disk, and 500. mu.g ml of BAP containing 0.5. mu.M was put^-1Kalfuron and 100. mu.g ml^-1Kanamycin on LS medium. After 2 weeks shoots were transferred to 500. mu.g ml containing 0.5. mu.M BAP^-1In 150ml jars of kevlar and LS medium without kanamycin.

After the subsequent 3 weeks, the dominant shoots were transferred to a medium containing 250. mu.g ml^-1Kevlar and LS medium without BAP or kanamycin. After a further 3 weeks, shoots were further washed by transferring shoot tips to LS medium without antibiotics or BAP. When sufficient roots were grown, the plants were transferred to soil in the greenhouse.

Selection of transgenic tobacco

Quantitative PCR (Q-PCR) was used to determine the transgene copy number in T0 and T1 plants. Single insert T0 and homozygous T1 plants were selected for analysis, if possible. For the detection of the transgene, the primers BNP-1271F (SEQ ID No.10) and BNP-1334R (SEQ ID No.11) shown in Table 1 were used, and the Vic/TAMARA-labeled probe BNP1291TV (SEQ ID No.12) annealed to the nptII transgene was used. For internal quantification, primers NtCyc-184F (SEQ ID No.13) and NtCyc-316R (SEQ ID No.14) were used, and a FAM/TAMARA labeled probe NtCyc-267T (SEQ ID No.15) annealed to the endogenous gene encoding the cyclophilin was used. Quantification was performed by comparing the Ct method (comprehensive Ct method,. DELTA.Ct method, Bubner and Baldwin (2004), Plant Cell Reports, 23, 263-charge 271). The reaction mixture contained 1x Universal Master Mix (ABI), 0.9 μ M of each primer, 0.2 μ M of each probe (separate reactions were used for BNP and NtCyc primers and probes), and about 500ng of genomic DNA template, which was extracted from leaf tissue using DNeasy plant mini kit (Qiagen) according to the recommended protocol. An initial denaturation step at 95 ℃ for 10 minutes followed by thermal cycling at 95 ℃ for 15 seconds and 60 ℃ for 40 cycles of 1 minute was performed in a 7900HT Fast Real-Time PCR System (7900HT Fast Real-Time PCR System, Applied Biosystems). The data were analyzed using the SDS2.2 software provided (Applied Biosystems).

After regeneration in tissue culture of transformed T0 generations of K326 and Burley21 tobacco, quantitative PCR (Q-PCR) was used to select plants carrying a single copy of the transgene. Single insert plants were selected to reduce the likelihood of transgene silencing. Q-PCR was performed using oligonucleotides complementary to the nptII transgene encoding neomycin phosphotransferase. This gene is present between the left and right borders of the T-DNA in the genome of the plant transferred to the constructs pBNP, BNP-PPDKcDNA and BNP-PPDKgDNA. Plants transformed with pBNP were used only as an empty vector control to ensure that regeneration by tissue culture was consistent between plants transformed with the empty vector and plants transformed with the vector containing the PPDK coding sequence. These plants were discarded after regeneration and Q-PCR checks to confirm that successful transformation had occurred.

For each construct (SAG12-PPDKcDNA and SAG12-PPDKgDNA) and each cultivar (K326 and Burley 21), 6 plants were selected. It is not possible to select only single insert plants, so if 6 single insert plants are not available, then the plant with the lowest copy number possible is selected. These plants were self-pollinated and the seeds were harvested.

For each selected T0 plant, 14 progeny were grown and Q-PCR was repeated to select homozygous plants. The use of homozygous plants should ensure stability of the transgene in the progeny. For each T0 parent, 4 progeny carrying the transgene were selected. However, it is not always possible to select homozygous progeny for each T0 parent. If the parental copy number is higher than 2, plants with lower copy numbers are selected to reduce the likelihood of transgene silencing and also to simplify selection of the T2 generation homozygotes (if necessary). Thus, for each construct and each cultivar, 4 biological replicates (siblings) from 5 independent lines with low transgene copy number were selected for all other experiments.

Example 4 detection and quantitative determination of PPDK protein in transformed Arabidopsis plants Protein extraction

Arabidopsis leaf tissue (100mg) was ground in a 1.5ml microcentrifuge tube using a mini-pestle (micropestle) under liquid nitrogen, and 400. mu.l of extraction buffer (potassium phosphate buffer (plus protease inhibitor cocktail (PIC, Sigma)) was added, protein concentration was determined by Bradford assay using Bio-Rad protein assay reagent (Bio-Rad) according to the recommended protocol (Jones et al, 1989, Journal of Chemical Ecology, 15, 979-.

Polyacrylamide gel electrophoresis

Proteins were separated by polyacrylamide gel electrophoresis (PAGE) in a separation gel containing 10% (v/v) acrylamide (37.5: 1 acryloyl: bisacryloyl, Severn Biotech Ltd), 50% (v/v) immunoblot separation buffer (see section 2.10), 0.05% (w/v) ammonium peroxodisulfate (APS, AnalaR) and 0.05% (v/v) N, N, N 'N' tetramethylethylenediamine (TEMED, Severn Biotech Ltd). The layering gel contained 5% of the above acrylamide, 50% (v/v) immunoblot layering buffer, 0.06% (w/v) APS and 0.1% (v/v) TEMED. Mu.g of extracted protein was electrophoresed at 70mA current for 1 hour 30 minutes using 1 Ximmunoblot sample buffer (66mM Tris-HCl, 10% (v/v) glycerol, 0.7mM SDS, 0.7M β -mercaptoethanol (BDH laboratories Supplies) and 0.05% (w/v) bromophenol blue and immunoblot running buffer (25mM Tris-HCl, 0.29mM glycine (Fisher Scientific) and 3.5mM SDS).

Duplicate gels were run simultaneously. The first was used for immunoblot analysis (described below) and the second was stained with GelCode Blue Safe protein dye (ThermoScientific) according to the recommended protocol. The dyed Gel was dried using a Gel Drying Film (Promega).

Immunoblot analysis

The proteins in the gel used for the immunoblot analysis were transferred onto Protan BA83 nitrocellulose membrane (Schleicher and Schuell) at 15V for 1 hour on Semi-Dry Blotter (Bio-Ra d) using Protean Extra-Thick blotting Paper (Blot Paper) (Bio-Rad) and immunoblot transfer buffer (48mM Tris-HCl, 39mM glycine, 1.3mM SDS and 20% (V/V) methanol (Fisher Scientific)). After blot confirmation of protein transfer, ponceau dye (0.5% (w/v) ponceau s (fluka) and 1% (w/v) glacial acetic acid (FisherScientific)) was coated on the membrane, and the membrane was then rinsed in distilled water.

Blocking buffer for PPDK immunoblot analysis was freshly prepared daily using phosphate buffered saline (PBS: 1.5mM potassium dihydrogen orthophosphate (KH2PO4, AnalaR), 8.1mM disodium hydrogen orthophosphate, 2.7mM potassium chloride, and 137mM sodium chloride) containing 1% (w/v) dry skim milk powder (Marvel) and 0.1% (v/v) polyoxyethylene sorbitan monolaurate (Tween 20(TWEEN 20), Sigma-Aldrich). Adjusted to pH 7.4 using hydrochloric acid. The membrane was incubated in blocking buffer for 1 hour at room temperature on a shaker. Primary hybridization was performed using a 1: 10,000 dilution of rabbit anti-PPDK antibody (Chris Chastatin, Minnesota State University) followed by 3 washes in blocking buffer for 5 minutes each. A second hybridization was performed using a 1: 1000 dilution of donkey anti-rabbit biotinylated intact antibody (GE Healthcare), followed by 3 washes as above. A third hybridization was performed using a 1: 1000 dilution of streptavidin-biotinylated horseradish peroxidase complex (GE Healthcare), followed by 3 washes in PBS containing 0.1% (v/v) Tween 20 for 5 minutes each.

The membrane was then rinsed 3 times in distilled water. Detection was performed using WesternLighting chemiluminescent Reagent (Western Lighting cheminescence Reagent) (Enhanced Luminol, Perkin Elmer) according to the recommended protocol.

Referring to FIG. 4, the selection of transgenic SAG12-PPDK Arabidopsis cell line by Western blot is shown. Proteins were extracted from leaf tissues of 8-week old Arabidopsis wild type,. DELTA.PPDK SAG12-PPDKcDNA and SAG12-PPDKgDNA plants and immunoblot analysis was performed to select strains expressing higher levels of PPDK. Recombinant maize PPDK (30. mu.g) was used as a positive control. PPDK was not detected in wild type or PPDK mutant plants (Δ PPDK). SAG12-PPDKgDNA plants expressed higher levels of PPDK compared to SAG 12-PPDKcDNA. All subsequent analyses were performed on 5 SAG12-PPDKgDNA lines.

PPDK could not be detected in wild type and Δ PPDK plants, low levels of PPDK could be detected in some SAG12-PPDKcDNA plants, and PPDK could be detected in all SAG12-PPDKgDNA lines. Much higher amounts of PPDK were detected in these 3 lines compared to wild type or SAG12-PPDKcDNA lines. Thus, the presence of an intron appeared to have a positive effect on the PPDK expression level, selecting the SAG12-PPDKgDNA line for all other experiments.

Wild type, Δ PPDK and SAG12-PPDKgDNA plants were immunoblotted against PPDK to determine the onset and extent of PPDK accumulation in SAG12-PPDKgDNA plants.

Referring to fig. 5, a quantitative determination of PPDK abundance of wild type, Δ PPDK and 5 independent SAG12-PPDKgDNA lines from week five is shown. PPDK abundance was calculated as a percentage of wild type for each time point from week 5 to week 9. Data are shown as the average of these 5 time points. Error bars represent 1 SEM. SAG12-PPDKgDNA lines were arranged in order of increasing PPDK abundance. ANOVA was used to test for differences between genotypes (F-4.775, df-6, p-0.001). Lines significantly different from the wild type are G12.3(p 0.005) and G16.1(p 0.004).

Protein was extracted from leaf tissue harvested at weekly intervals from 3 to 9 weeks after seeding. A small amount of PPDK was detected in wild type plants from week five, confirming that the observed increase in transcripts resulted in increased protein abundance. In SAG12-PPDKgDNA plants, PPDK accumulation was also found to begin at week 5, but the PPDK abundance was higher than that of wild type. In Δ PPDK plants, no PPDK protein was detected at any stage.

The method allowing quantitative determination of PPDK in leaf protein extracts was also optimized. SDS-PAGE and immunoblot analyses were performed on varying amounts of recombinant maize PPDK protein. And (5) calculating the intensity of the strip by using AlphaEase imaging software, and drawing a standard curve. Regression lines were calculated using SigmaPlot software, which was then used to calculate the amount of PPDK in plant leaf samples. Since immunoblot detection levels can vary, it is necessary to include at least 3 standards for each immunoblot for true comparison between different immunoblots. This technique for quantitative determination of PPDK abundance in different extracts was used to select and compare different transgenic lines.

PPDK protein abundances of at least 4 biological replicates of each line at each time point were calculated with reference to known standards of recombinant maize PPDK protein. The analysis showed that from the fifth week onwards, SAG12-PPDKgDNA had a higher abundance of PPDK than wild-type. For each SAG12-PPDKgDNA line, PPDK abundance was calculated as a percentage of wild type at each time point from the fifth week and the mean values derived from these values quantified PPDK accumulation in each SAG12-PPDKgDNA line during senescence. This allowed sequencing of SAG12-PPDKgDNA lines according to increasing PPDK abundance, as shown in FIG. 5.

Since proteins accumulated in large amounts in transgenic plants can undergo inactivation, increased protein abundance does not necessarily mean increased enzyme activity. In the dark, PPDK undergoes reversible phosphorylation, resulting in its inactivation. Antibodies raised against the phosphorylated form of PPDK (phospho-PPDK) allow for the specific detection of the inactive form of PPDK, whereas the previously used antibodies (anti-PPDK) allow for the detection of PPDK irrespective of the phosphorylation state (Chastatin et al, 2002, plantaphysiology, 128, 1368-containing 1378). For immunoblots that were initially probed with anti-PPDK antibody and then stripped and re-probed, anti-phospho PPDK antibody was used to detect inactive PPDK over the time course from 3 weeks to 9 weeks after seeding.

Surprisingly, only very low levels of inactive PPDK (phosphorylated PPDK) were detectable on multiple immunoblots in wild type plants and only late in senescence. In SAG12-PPDKgDNA plants, higher levels of PPDK were detected depending on the time point at which it was inactivated. However, despite the high abundance of total PPDK, very little or no inactive PPDK was detected at a time point shortly after the PPDK abundance began to increase. These results indicate that although some PPDK inactivation may occur in SAG12-PPDKgDNA plants, enzymatically active PPDK of increased abundance is present in SAG12-PPDK plants at least early in senescence.

Example 5 detection and quantitative determination of PPDK protein in transformed tobacco plants

Tobacco leaf tissue (100mg) was ground in a 1.5ml microcentrifuge tube using a mini-pestle under liquid nitrogen, and 400. mu.l of extraction buffer (Overcoat buffer (PIC)) was added. Protein concentrations were determined by Bradford assay using Bio-Rad protein assay reagents (Bio-Rad) according to the recommended protocol (Jones et al, 1989). The proteins were separated by polyacrylamide gel electrophoresis (PAGE) as described in example 4. The protein was analyzed by immunoblotting and the PPDK protein was quantitatively determined as described in example 4.

Leaf proteins extracted from K326 and Burley 21T1 tobacco leaves, which were senescence-induced by peeling and incubation at 30 ℃ in the dark, were subjected to immunoblotting against PPDK. Senescence was induced in this manner, so that before the plants reached maturity, the transgenic line with the highest PPDK abundance could be identified and the other lines discarded. In K326 plants, a high abundance of PPDK was detected in 3 SAG12-PPDKgDNA lines (G4, G8 and G10) and in1 SAG12-PPDKcDNA line (C10). In Burley21 plants, PPDK was abundant in 4 SAG12-PPDKgDNA strains (G3, G7, G15 and G23), but PPDK was not present in as high abundance as the K326 strain. Each of the 4 strains of K326 and Burley21 with the highest PPDK abundance was used for all further analyses. Since the highest PPDK abundance was observed almost exclusively in the SAG12-PPDKgDNA strain, it appears that the intron has a positive effect on PPDK expression.

Referring to FIG. 6, the selection of SAG12-PPDK K326 and Burley21 tobacco lines is shown as follows:

(b) PPDK immunoblots of transgenic K326 strains were selected. Senescence was induced in green leaves of 6-week-old plants by peeling and incubation for 3 days at 30 ℃ in the dark. Proteins were extracted and immunoblot analysis was performed to select strains expressing higher levels of PPDK. Recombinant maize PPDK (50. mu.g) was used as a positive control. One representative immunoblot was shown with one progeny of each parental T0 plant. Plants of parental lines SAG12-PPDKgDNA G4, G8 and G10 and SAG12-PPDKcDNA line C10 were used for all subsequent analyses.

(c) PPDK immunoblotting of the transgenic Burley21 line was selected as was done for K326 in part (b). Plants of parental line SAG12-PPDKgDNA G3, G7, G15 and G23 were used for all subsequent analyses.

Immunoblots against PPDK were performed on wild type, zero copy (negative isolate), SAG12-PPDKgDNA K326, SAG12-PPDKcDNA K326 and SAG12-PPDKgDNA Burley21 tobacco to determine the onset of PPDK expression and to quantify the extent of PPDK overexpression in transformed plants. Proteins were extracted from K326 wild type of 3 month old plants and transformed plant leaves 1 (oldest) to 8 (younger) and immunoblotted against PPDK.

Referring to FIG. 7, there is shown the over-expression of PPDK in K326 tobacco mature leaves. PPDK abundance was calculated from immunoblots. Data are shown as the average of 10 biological replicates for zero copy plants and 4 biological replicates for each SAG12-PPDK strain. Error bars represent 1 SEM. SAG12-PPDK strains were arranged in order of increasing PPDK abundance. Differences between genotypes were examined using ANOVA (F ═ 6.995, df ═ 4, and p ═ 0.001). Lines significantly different from zero copy plants are G10(p 0.006), G8(p 0.003) and C10(p 0.000).

PPDK is more abundant in older leaves of wild type plants, indicating that PPDK is naturally up-regulated during tobacco and arabidopsis senescence. PPDK abundance in older leaves was also higher in transformed K326 plants and much higher than that of wild type plants. The abundance of PPDK was also higher in younger leaves of the transformed lines with the highest PPDK abundance. Immunoblots were performed on proteins extracted from zero copy plants (negative segregants) and the 'mature' leaves of transformed plants. Mature leaves are leaves at harvest stage and are leaves late in senescence. PPDK abundance was determined quantitatively. PPDK was more abundant in mature leaves of all 4 independent transformed lines compared to zero copy plants. For K326 tobacco, PPDK overexpression using the SAG12 promoter was successful during senescence.

Example 6 phenotypic analysis of transformed Arabidopsis plants

Analysis of growth of Arabidopsis plants

Referring to FIG. 8, it is clearly shown that over-expression of PPDKgDNA in Arabidopsis thaliana results in plants with larger rosette leaf plexus. Wild type, Δ PPDK and SAG12-PPDKgDNA plants were photographed 3, 5, 7 and 9 weeks after sowing. SAG12-PPDKgDNA plants were larger than wild type. There was no discernable difference between PPDK plants and wild type plants.

The fresh mass of rosette and reproductive tissue was measured to an accuracy of 0.1mg using a Mettler Toledo AB104-S balance. The dry mass of rosette tissue was determined to the nearest 0.1mg from samples freeze-dried overnight using an Edwards Super Modulyo freeze-dryer.

Referring to FIG. 9, the reproductive tissue mass at week 9 is shown. Dose response to PPDK was observed, with lines with higher abundance of PPDK having higher reproductive tissue quality. The total reproductive tissue (stem, cauline leaves, flowers and siliques) was weighed. Values are the average of 3 biological replicates. Error bars represent 1 SEM. Transgenic SAG12-PPDKgDNA lines were arranged in order of increasing abundance of PPDK. ANOVA was used to test for differences between genotypes (F ═ 8.062, df ═ 6, p ═ 0.001). All SAG12-PPDKgDNA lines differed significantly from the wild type (g9.4p ═ 0.031, g5.4p ═ 0.000, g19.2p ═ 0.000, g12.3p ═ 0.001, and g16.1p ═ 0.001). Lines with higher PPDK abundance tend to have higher reproductive tissue quality, although this trend is not absolute. The percentage of reproductive tissue in total fresh mass of the plants was also calculated, but when the ANOVA test was applied, there was no significant difference from wild type, Δ PPDK or SAG12-PPDKgDNA plants (F ═ 0.544, df ═ 6, p ═ 0.767, data not shown).

However, the proportion of reproductive tissue as a percentage of the total mass of the plant did not change, indicating that the plant as a whole was larger and that the resource allocation between vegetative and reproductive tissue did not change. The total fresh and reproductive tissue mass of the plants was also determined in Δ PPDK plants and was not significantly different from the wild type. The rosette dry mass was also determined and for the total fresh mass and reproductive tissue mass of the plants, SAG12-PPDKgDNA plants were found to have significantly higher mass compared to the wild type.

Referring to FIG. 10, the total fresh mass of plants (rosette plus reproductive tissue) of wild type and SAG12-PPDgDNA plants from week 3 to week 9 after sowing is shown. Data are expressed as the average of 3 biological replicates of wild type and 15 biological replicates of SAG12-PPDKgDNA (3 plants each of 5 independent lines). Error bars represent 1 SEM. The total mass of wild type and SAG12-PPDKgDNA plants was compared at various time points using the student's t-test (student's t-test). Rosette mass was significantly higher in SAG12-PPDKgDNA plants at week 9 (p ═ 0.032).

There was no significant difference before PPDK overexpression started at week 5 due to the SAG12 promoter. Rosette stem mass was increased for all 5 independent SAG12-PPDKgDNA strains. With respect to reproductive tissue quality, a dose response to PPDK abundance was observed, with higher quality lines with higher PPDK content. Rosette stem quality was also determined in Δ PPDK plants, and was not significantly different from wild type plants.

Determination of leaf surface area of Arabidopsis thaliana

The surface area of the rosette was determined by taking a single sample of mature leaves (passage 10 leaves), measuring leaf mass and total rosette mass as described above, and taking a picture of the leaves placed beside the ruler. The surface area of the leaves was measured using ImageJ software (National Institutes of Health) and the surface area of the entire rosette was calculated therefrom.

After the onset of PPDK overexpression, rosette surface area increased in SAG12-PPDKgDNA plants. Although the surface area of the SAG12-PPDKgDNA plants was significantly larger at the sixth and seventh weeks, the surface area was not significantly different from the wild type at weeks 8 and 9. This large reduction in surface area was probably due to increased remobilisation of nutrients in SAG12-PPDKgDNA plants compared to wild type. The surface area of the Δ PPDK plants was not significantly different from that of the wild type plants.

Determination of chlorophyll concentration

The chlorophyll content in relative units was determined using a CCM-200 portable chlorophyll apparatus (Opti-Sciences).

Of Arabidopsis thalianaFluorescence measurement

FV/FM ratios were determined using a Hansatech FMS2 fluorimeter. Leaves were dark adapted overnight prior to assay using leaf clips provided by the manufacturer. To determine whether the onset of senescence was altered in SAG12-PPDKgDNA or Δ PPDK plants relative to wild type plants, the FV/FM ratio was determined. FV/FM is an estimate of the quantum efficiency of photosystem II, which decreases when photoinhibition occurs. It is a useful measure of the onset of senescence, since a decrease in photosynthesis occurs early in senescence, before a decrease in chlorophyll content is detected. FV/FM was determined within the time course of 4 weeks (when the leaves became large enough to determine FV/FM) to 9 weeks after sowing. In wild type, SAG12-PPDKgDNA and Δ PPDK plants, FV/FM was maximal at week 7 and subsequently declined, indicating that the timing of senescence initiation was the same in all 3 genotypes. However, FV/FM was significantly higher at week 8 in SAG12-PPDKgDNA plants compared to wild type, indicating prolonged photosynthetic activity late in senescence.

Nitrogen content

Tissues for nitrogen analysis were freeze dried using an Edwards Super Modulyo freeze dryer. Arabidopsis thaliana leaf tissue (25mg) or tobacco leaf tissue (100mg) was packed in nitrogen-free weighing paper (Elementar Ahalysensysteme GmbH). The Nitrogen content as a percentage of dry weight was determined using the recommended settings using a Rapid N III Nitrogen Analyzer (Rapid N III Nitrogen Analyzer) (Elementar analysis GmbH). Aspartic acid (Sigma-Aldrich) was used as standard. Leaf nitrogen content of Arabidopsis wild type, SAG12-PPDKgDNA and Δ PPDK plants was determined over a time course of 3-9 weeks after sowing.

Referring to FIG. 11, nitrogen content of leaves of wild type, Δ PPDK and SAG12-PPDKgDNA plants at week 7 is shown. Data are presented as the average of 8 biological replicates of wild type and Δ PPDK and 4 biological replicates of each of the SAG12-PPDKgDNA strain. Error bars are 1 SEM. SAG12-PPDKgDNA lines were arranged in order of increasing abundance of PPDK. ANOVA was used to test for differences between genotypes (F-6.047, df-6, p-0.000). Δ PPDK plants and all SAG12-PPDKgDNA lines differ significantly from the wild type (Δ PPDK p 0.004, g9.4p 0.000, g5.4p 0.000, g19.2p 0.001, g12.3p 0.007, and g16.1p 0.006).

Compared to wild type, in all 5 independent SAG12-PPDKgDNA lines, there was significantly lower folate after week 7, which supports the hypothesis that increasing PPDK abundance could increase the efficiency of nitrogen remobilization during senescence. PPDK expression and activity peaked at week 6 in SAG12-PPDKgDNA plants, indicating a delay between the increase in PPDK abundance and the measurable decrease in leaf nitrogen, probably due to the time it takes to convert protein amino acids into transport amino acids (asparagine and glutamine) and transport them out of the leaves.

Analysis of Arabidopsis seeds

Single seed mass and nitrogen content were determined in seeds of wild type, SAG12-PPDKgDNA and Δ PPDK Arabidopsis plants.

Referring to FIG. 12, nitrogen content of single seeds of wild type, Δ PPDK and SAG12-PPDK plants is shown. SAG12-PPDKgDNA plants have a significant increase in seed nitrogen content. Data are presented as the average of 8 biological replicates of wild type and Δ PPDK and 4 biological replicates of each SAG12-PPDKgDNA line. Error bars are 1 SEM. SAG12-PPDKgDNA lines were arranged in order of increasing abundance of PPDK. ANOVA was used to test for differences between genotypes (F-6.704, df-6, p-0.000). Lines significantly different from the wild type are G19.2(p ═ 0.000), G12.3(p ═ 0.005) and G16.1(p ═ 0.002). Lines with higher PPDK abundance tend to have higher seed nitrogen quality. Thus, the individual seed mass was increased in all 5 independent SAG12-PPDKgDNA lines compared to the wild type, and a dose response to PPDK abundance was observed, with higher seed mass in plants with higher PPDK abundance. The total seed harvested from each plant was also weighed, with no significant difference between wild type and SAG12-PPDKgDNA plants, so increasing the seed size of SAG12-PPDK plants did not harm the seed assembly.

Arabidopsis thalianaFree amino acid content of leaf tissue

Leaf tissue (100mg) was ground in a 1.5ml microcentrifuge tube under liquid nitrogen using a micro-pestle (Eppendorf) and 300. mu.l sterile deionized water was added. The samples were centrifuged at 13,000rpm for 5 minutes at4 ℃. Protein concentrations were determined by Bradford assay using Bio-Rad protein assay reagents (Bio-Rad) according to the recommended protocol (Jones et al, 1989). Samples for amino acid analysis were prepared according to the recommended protocol using the ezfast amino acid sample test kit (Phenomenex). The samples were resuspended in 50% sterile distilled water containing 10mM ammonium formate (supplied by BDH laboratories) and 50% super grade methanol (Romil).

The samples were then subjected to liquid chromatography-mass spectrometry (LC-MS) using Q Trap LC/MS/MS (Applied Biosystems/MDS SCIEX) with an EZfast 250x 3.0mM AAA-MS column (Phenomenex) and using 50% (v/v) sterile distilled water containing 10mM ammonium formate and 50% (v/v) super methanol as mobile phases. The mass spectrometer was used in positive ion mode with the conditions recommended in the ezfast kit (pheronex). The results were analyzed using the supplied Analyst software (Applied Biosystems/MDS SCIEX).

Total free amino acid content was determined in leaves of wild type, SAG12-PPDKgDNA and Δ PPDK Arabidopsis plants over the time course from week 3 to week 9 after sowing.

Referring to FIG. 13, the increase in total free amino acid content in PPDK-overexpressing Arabidopsis leaves is shown. Data are presented as the average of 6 biological replicates of wild type and Δ PPDK and 4 biological replicates of each SAG12-PPDKgDNA line. Error bars are 1 SEM. SAG12-PPDKgDNA lines were arranged in order of increasing abundance of PPDK. All 5 SAG12-PPDKgDNA lines had higher total amino acid content compared to the wild type, but varied widely with insignificant differences when tested using ANOVA (F1.314, df 6, p 0.289).

At week 7, the same time when leaf nitrogen content became significantly lower than wild type nitrogen content and one week after PPDK was most abundant in the leaves of SAG12-PPDKgDNA plants, total free amino acid content was significantly higher in SAG12-PPDKgDNA plants compared to wild type plants. The increase occurred in all 5 independent SAG12-PPDKgDNA lines, although the variation was large and the difference between each line and the wild type was not significant. This increase indicates that at this point in time, the production of amino acids proceeds at a greater rate than the amino acid export that can occur, and thus amino acids accumulate in the leaves. Total free amino acids were not significantly different in Δ PPDK plants compared to wild type.

Transport amino acids (glutamine and asparagine) were also determined, expressed as a percentage of total free amino acids. At weeks 7 and 8, the transported amino acid content in SAG12-PPDKgDNA plants was significantly higher than that of wild type plants. The increase occurred in all 5 independent SAG12-PPDKgDNA lines, although the variation was large and the difference between each line and the wild type was not significant. Thus, in addition to the increase in total free amino acid content in SAG12-PPDKgDNA plants, the amount of transported amino acids was also increased in proportion to the total amount. This again suggests that at these time points glutamine and asparagine production exceeded leaf export capacity, supporting the hypothesis that increased PPDK abundance during senescence increases the efficiency of amino acid interconversion leading to the formation of transport amino acids. In Δ PPDK plants, no significant difference from wild type was observed.

Example 7 phenotypic analysis of transformed tobacco plants

Analysis of tobacco leaf nitrogen content

The leaf nitrogen content in the negative isolate and in the mature leaves of SAG12-PPDK K326 tobacco was determined. Mature leaves are leaves that have been prepared for harvest for tobacco production. For some of the 4 independent SAG12-PPDK lines, the leaf nitrogen content of SAG12-PPDK plants was lower than that of negative segregant plants.

Mature leaves are used to determine leaf nitrogen content because the leaves are at a stage where they can be harvested for tobacco production. However, the fact that the difference in leaf nitrogen content in mature leaves is small does not necessarily mean that nitrogen remobilisation does not increase. During the time course of the senescence onset of SAG12-PPDKgDNA Arabidopsis plants, leaf nitrogen content was significantly lower than wild type, but by late senescence, the difference was much smaller. Therefore, the difference in leaf nitrogen content in tobacco is likely to occur at an earlier stage of aging, and it is likely that the difference is diminished by the time the leaf nitrogen content is measured.

Analysis of amino acid content in tobacco leaves

The amino acid content of K326 mature leaves of tobacco, which induced senescence by peeling and incubation at 30 ℃ for 3 days in the absence of light, was determined. This allows a comparison between the same leaves before and after induction of senescence. Induction of senescence by leaf stripping and light-shielded incubation showed the greatest overlap of gene expression patterns with age-related senescence.

In K326 tobacco, the total amino acid content increased after induction of senescence in the negative segregant plants and the SAG12-PPDK plants. The increase was calculated as a percentage of the total amino acid content before induction of senescence. For SAG12-PPDK line C10, the increase was significantly less than that of the negative segregant plants, but none of the other SAG12-PPDK lines differed significantly from the negative segregant plants. Thus, after induction of dark induced senescence, over-expression of PPDK during senescence appeared to have little effect on the total amino acid content. After senescence induction, the transported amino acid (glutamine and asparagine) content in K326 tobacco also increased, but this increase occurred in both the negative isolate and the SAG12-PPDK strain, and there was no significant difference in the degree of increase between genotypes after senescence induction.

Analysis of tobacco seeds

Single seed mass of K326 tobacco was determined and was significantly higher in SAG12-PPDKgDNA and SAG12-PPDKcDNA plants compared to negatively segregating plants for all 4 independent transgenic lines. Seed mass was also determined for each seed pod and was significantly higher in SAG12-PPDKgDNA and SAG12-PPDKcDNA plants.

Referring to FIG. 14, there is shown an increase in seed size in SAG12-PPDK K326 tobacco. The mass of each seed of the K326 zero copy (plants negative segregant for SAG12-PPDK insert) and SAG12-PPDK plants was calculated by photographing, counting and weighing about 1000 seeds. Data are expressed as the average of 10 biological replicates of zero copy plants and the average of 4 biological replicates of each SAG12-PPDK line. Error bars are 1 SEM. SAG12-PPDK lines were arranged in order of increasing PPDK abundance in mature leaves. The seed mass of SAG12-PPDK plants was greater. ANOVA was used to test for differences between genotypes (F-4.870, df-4, p-0.006). Lines significantly different from zero-copy plants are G8(p 0.005) and C10(p 0.002).

The percent nitrogen content of the seeds was higher for all 4 independent SAG12-PPDKgDNA and SAG12-PPDKcDNA strains, but the difference was not significant. However, nitrogen quality per seed was significantly higher in SAG12-PPDKgDNA and SAG12-PPDKcDNA plants.

Referring to FIG. 15, it is clearly shown that SAG12-PPDK K326 tobacco has an increased seed nitrogen content. Nitrogen mass of single seeds of K326 zero copy and SAG12-PPDK plants was calculated from seed mass and seed nitrogen content data. Data are expressed as the average of 10 biological replicates of zero copy plants and the average of 4 biological replicates of each SAG12-PPDK line. Error bars are 1 SEM. SAG12-PPDK lines were arranged in order of increasing PPDK abundance in mature leaves. Single seeds in SAG12-PPDK plants have higher nitrogen quality. ANOVA was used to test for differences between genotypes (F-7.807, df-4, p-0.001). Lines significantly different from zero copy plants are G8(p 0.000) and C10(p 0.000).

This indicates that the nitrogen supply to the seeds is increased in SAG12-PPDKgDNA and SAG12-PPDKcDNA K326 tobacco plants. In K326 plants, a dose response to PPDK was observed. Plants with higher abundance of PPDK in mature leaves have higher seed quality per seed, higher seed quality per seed pod and increased nitrogen quality per seed. This strongly supports a role in nitrogen remobilisation of PPDK, as an increase in PPDK content appears to be associated with an increase in nitrogen supply to the seeds.

Overall, both the seed mass (fig. 14) and nitrogen mass/seed (fig. 15) were increased in SAG12-PPDK K326 tobacco, indicating that nitrogen remobilisation was increased by over-expression of PPDK. Thus, for Arabidopsis SAG12-PPDKgDNA plants, an increase in the content of trans-amino acids in older leaves can be expected. However, the amino acid content of naturally senescent leaves was determined in Arabidopsis, whereas in tobacco, senescence was induced by leaf stripping and incubation in the dark. Since the processes occurring in dark induction and age-related senescence are different, the processes occurring in tobacco leaves are not necessarily similar to those occurring in Arabidopsis leaves.

Example 8 Generation of Arabidopsis plants overexpressing PCK and PPDK

Overexpression of arabidopsis PCK (at4g37870.1) during senescence was achieved by fusing the coding region and genomic clone of PCK to the promoter of senescence-associated gene 12(SAG12) within the BNP1380000001 binary vector, which was transformed into arabidopsis thaliana as described in examples 1 and 2.

The At PCK coding sequence was first isolated from arabidopsis cDNA and the genomic sequence from arabidopsis genomic DNA using PCR as described in example 1 for PPDK. On the other hand, PCR primers were designed to facilitate subsequent ligation of the gene to the BNP1380000001 vector (see fig. 3a), including the start and stop codons of the arabidopsis thaliana (At) PCK gene, and also as shown in table 1, including an XbaI restriction site in the forward primer AtPCK-Xba IFOR (SEQ ID No.25) and a SacI restriction site in the reaction primer AtPCK-SacIREV (SEQ ID No. 26).

cDNA and genomic DNA templates for each PCR reaction were prepared as in example 1. The PCR reaction mixture contained 1 XHF buffer (NEB), 2mM magnesium chloride (NEB), 0.5mM dNTPs (Bioline), 100ng template (cDNA or genomic DNA), 0.5. mu.M of each primer and 1 unit Phusion high fidelity DNA polymerase (NEB). Thermal cycling was performed using a Techne thermal cycler as follows: an initial denaturation step at 98 ℃ for 30 seconds, followed by 35 following cycles: an extension time of 10 seconds at 98 ℃, 30 seconds at 60 ℃ and 2 minutes 30 seconds for coding region 72 ℃ and 4 minutes 30 seconds for genomic clones. The final step included a final extension at 72 ℃ for 10 minutes.

These PCR products produced a 2kb band of coding sequence (cDNA) and a 3.5kb band of PCK genomic sequence, and were subjected to PEG precipitation. The amplified DNA was ligated (Blunt-ended) into pCR4Blunt-TOPO vector (Invitrogen) according to the recommended protocol as described for PPDK in example 1. The plasmids were then transformed into Library efficient (Library Efficiency) DH 5. alpha. E.coli cells. Kanamycin (50. mu.g ml)^-1) Used as selective antibiotic. For both the coding region and the genomic clone, positive colonies were selected by colony PCR using both the primers AtPCK-Xba IFOR (SEQ ID No.25) and AtPCK-Sac IREV (SEQ ID No. 26). Positive colonies were subjected to shaking culture at 37 ℃ in a 5ml shaking culture tank containing 50. mu.g ml^-1Kanamycin was grown overnight in LB broth.

Plasmid DNA was isolated using the QIAprep Spin Miniprep kit (Qiagen Ltd), and the insert size was analyzed by sequential restriction digestion. This was done by digesting 1. mu.g of DNA with 10 units of XbaI and 1 XbaI buffer at 37 ℃ for 2 hours, then adding 10 units of SacI and 1 XSacI buffer at 37 ℃ overnight. Plasmid DNA containing the correct insert was sequenced using AtPCK-XbaIFOR/AtPCK-Sac IREV. Sequences were analyzed using BioEdit and the amplified AtPCK coding and genomic sequences were verified using BLASTX.

To ligate the AtPCK coding region and the genomic clone to the pBNP vector shown in FIG. 3a, 25ml of E.coli colonies containing 50. mu.g ml were inoculated with E.coli colonies representing the two plasmids respectively^-1LB broth of kanamycin. After shaking the culture overnight at 37 ℃, the plasmid DNA was purified using QIAfilter plasmid midi kit (Qiagen Ltd). In the same manner but containing 50. mu.g ml from 100ml^-1Kanamycin in culturepBNP vector was generated. All purified plasmid DNA was subjected to the same sequential enzymatic digestion as described above. For plasmids containing coding regions and genomic clones, 3. mu.g of DNA was digested, and for the pBNP vector, 1. mu.g of DNA was digested. Samples were separated by crystal violet gel electrophoresis and the product was purified using the Qiaquick gel extraction kit (qiagen ltd). The 14.4kb pBNP vector product was treated with alkaline phosphatase to prevent self-ligation, and the coding region and genomic clone insert were ligated to the pBNP vector using XbaI/SacI digestion. Library efficiency DH 5. alpha. E.coli cells were transformed with 2. mu.l ligation reactions and positive colonies were screened by PCR using AtPCK-Xba IFOR and AtPCK-Sac IREV.

Plasmid DNA was extracted from colonies containing the desired insert using the QIAprep Spin Miniprep kit (Qiagen Ltd), and the DNA was subsequently digested with 10 units XbaI, 15 units SacI, and 1 XbaI buffer at 37 ℃ for 2 hours. Two isolated colonies each of the coding sequence and genomic sequence insert (which had the correct expected restriction endonuclease digestion profile) in the pBNP vector were selected and then sequenced using the BNP-SAG12FWD primer (SEQ ID No. 9). Sequences were analyzed using the BioEdit program. The resulting constructs were designated pALBNP1 (coding sequence) and pALBNP2 (genomic sequence) as shown in FIG. 3 b.

5 homozygous single insert lines were generated and confirmed PCK overexpression by immunoblotting as described in example 3 using PCK-specific antibodies. Polyclonal antisera were generated in rabbits using synthetic peptides designed against the PCK amino acid sequence. The sequences were chemically synthesized (i) DEHCWTETGVSNIEG (SEQ ID No.27), and (ii) CVDLSREKEPDIWNA (SEQ ID No.28), then conjugated to keyhole limpet hemocyanin, followed by co-injection into rabbits. The results obtained using the antibody are shown in FIG. 16, which shows that the transformed plants have high levels of PCK protein.

The SAG12-PCK-PPDK cell line was generated by crossing single SAG12-PCK transformants (3)1 and (19)4 with a strong SAG12-PPDK overexpressing strain. Analysis of plants with high levels of both PCK and PPDK showed improved rosette quality, as shown in fig. 17.

Finally, while the inventors do not wish to be bound by the hypothesis, fig. 18 represents a putative biochemical pathway, demonstrating how PCK and PPDK may affect nitrogen remobilization.

PCK/PPDK double construct

The inventors have observed that the single construct of PCK and PPDK shown in figure 3, when transformed into plants, results in an increased rate of nitrogen remobilisation in senescent leaves, and that the amount of vegetative plant growth is also increased when these enzymes are overexpressed in senescent leaves (this corresponds to an increased crop yield). The inventors therefore decided to make two dual constructs in which both the genes encoding PCK and PPDK were inserted into pBNP130000001 under the control of the SAG12 promoter.

The first double construct was prepared by ligating the gDNA encoding PCK downstream (i.e., 3' to) of the gDNA fragment encoding PPDK of BNP-PPDKgDNA using an XbaI/SacI digestion. Thus, the SAG12 promoter in the plasmid was responsible for the expression of both the PPDK gene and the PCK gene.

A second double construct was prepared by ligating the gDNA encoding PPDK directly downstream of the PCK-encoding gDNA fragment of pALBNP2 using an AvrII/BamHI digest. SAG12 is also responsible for the expression of both the PPDK gene and the PCK gene.

Claims

1. A genetic construct comprising a senescence-specific promoter operably linked to at least one coding sequence encoding at least one polypeptide having Phosphoenolpyruvate Carboxykinase (PCK) activity and/or cytosolic pyruvate orthophosphate dikinase (PPDK) activity.

2. The genetic construct of claim 1, wherein the promoter is isolated from a senescence-associated gene of Arabidopsis.

3. The genetic construct of claim 1 or 2, wherein the promoter is selected from the group consisting of: SAG12, SAG13, SAG101, SAG21 and SAG 18.

4. The genetic construct of claim 1 or 2, wherein the promoter is the SAG12 promoter.

5. A genetic construct according to claim 1 or 2, wherein the promoter is the nucleotide sequence shown as SEQ ID No. 16.

6. The genetic construct of claim 1, wherein the at least one coding sequence encodes (i) Phosphoenolpyruvate Carboxykinase (PCK), and/or (ii) cytosolic pyruvate orthophosphate dikinase (PPDK).

7. A genetic construct according to any one of claims 1, 2 or 6, wherein the coding sequence encoding a polypeptide having PCK activity is derived from Arabidopsis thaliana.

8. The genetic construct of any one of claims 1, 2, or 6, wherein the coding sequence encoding the polypeptide having cytoplasmic PPDK activity is derived from Arabidopsis, Zea, Helianthus, or Brassica.

9. A genetic construct according to any one of claims 1, 2 or 6, wherein the coding sequence encoding a polypeptide having PCK activity is a nucleic acid sequence as shown in SEQ ID No.17 or SEQ ID No. 18.

10. A genetic construct according to any one of claims 1, 2 or 6, wherein the polypeptide having PCK activity is an amino acid sequence as shown in SEQ ID No. 19.

11. A genetic construct according to any one of claims 1, 2 or 6, wherein the coding sequence encoding a polypeptide having cytoplasmic PPDK activity is a nucleic acid sequence as shown in SEQ ID No. 20 or SEQ ID No. 21.

12. A genetic construct according to any one of claims 1, 2 or 6, wherein the polypeptide having cytoplasmic PPDK activity is the amino acid sequence shown in SEQ ID No. 23.

13. The genetic construct of claim 1, wherein the construct encodes PCK and not PPDK.

14. The genetic construct of claim 1, wherein the construct encodes PPDK but not PCK.

15. The genetic construct of any one of claims 1, 2 or 6, wherein the construct encodes PCK and PPDK.

16. A recombinant vector comprising the genetic construct of any one of claims 1-15.

17. A recombinant vector, wherein the recombinant vector is any one of BNP-PPDK cDNA, BNP-PPDKgDNA, pALBNP1, and pALBNP2, wherein the BNP-PPDK cDNA and the BNP-PPDKgDNA are generated by usingAvrII andBamHI digestion PPDK cDNA and PPDK gDNA were separately introduced into pBNP to construct; and pALBNP1 and pALBNP2 by usingXbaI andSaci digestion PCK cDNA and PCK gDNA were separately introduced into pBNP and constructed.

18. A method of increasing the concentration of PCK and/or PPDK in the leaves of an arabidopsis thaliana or solanaceae test plant to above the corresponding concentration of PCK and/or PPDK in a wild type plant cultured under the same conditions, the method comprising altering plant metabolism of the test plant such that the level of PCK and/or cytoplasmic PPDK in the plant leaves is increased after the onset of leaf senescence, wherein the method comprises transforming the test plant cells with the genetic construct of any one of claims 1 to 15 or the vector of claim 16 or claim 17.

19. A method of reducing the nitrogen concentration in leaves of an arabidopsis or solanaceae test plant to below that of the corresponding nitrogen concentration in wild type plants cultured under the same conditions, the method comprising altering plant metabolism of the test plant such that the level of PCK and/or cytosolic PPDK in plant leaves is increased following the onset of leaf senescence, wherein the method comprises transforming a test plant cell with the genetic construct of any one of claims 1-15 or the vector of claim 16 or claim 17.

20. A method of increasing the growth rate of an arabidopsis thaliana or a test plant of the solanaceae family compared to the corresponding growth rate of a wild type plant cultured under the same conditions, the method comprising altering plant metabolism of the test plant such that the level of PCK and/or cytoplasmic PPDK in plant leaves is increased after the onset of leaf senescence, wherein the method comprises transforming a test plant cell with the genetic construct of any one of claims 1-15 or the vector of claim 16 or claim 17.

21. The method of any one of claims 18-20, wherein the method comprises increasing the concentration of PCK and cytoplasmic PPDK in a plant leaf to be higher than the corresponding concentration of PCK and cytoplasmic PPDK in a wild-type plant cultured under the same conditions.

22. A method of producing a transgenic arabidopsis thaliana or solanaceae plant that remobilizes nitrogen at a higher rate than a corresponding wild-type plant grown under the same conditions, comprising the steps of:

i) transforming an arabidopsis thaliana or solanaceae plant cell with the genetic construct of any one of claims 1-15 or the vector of claim 16 or 17; and

ii) regenerating a plant from the transformed cell.

23. A method of producing a transgenic arabidopsis thaliana or solanaceae plant that grows faster than a corresponding wild-type plant grown under the same conditions, comprising the steps of:

ii) regenerating a plant from the transformed cell.

24. A smoking article comprising nitrogen-reduced tobacco obtained from a transgenic tobacco plant capable of reducing nitrogen concentration in senescent leaves, wherein the smoking article is made from tobacco obtained from a transgenic tobacco plant that has been transformed with the genetic construct of any one of claims 1-15 or the vector of claim 16 or claim 17.