HK1162585B - Transgenic plants - Google Patents

Description

Transgenic plants

The present invention relates to genetic constructs useful for making transgenic plants. The constructs may have the ability to cause a plant to accumulate threonine in its leaves, particularly during leaf senescence. The invention extends to plant cells transformed with the constructs, as well as to the transgenic plants themselves. The invention also relates to methods for producing transgenic plants and to methods for increasing threonine concentration in senescent plants. The invention also relates to harvested plant leaves, such as tobacco leaves, transformed with the gene construct and to smoking articles comprising the harvested plant leaves.

One of the main goals in enhancing the taste of flue-cured tobacco is the production of the amino acid threonine. Accumulation of high levels of threonine in the leaves of mutant tobacco plants can produce significant taste and aroma benefits. However, threonine production is generally tightly regulated as is the production of other amino acids of the aspartate family, namely methionine, lysine and isoleucine. Therefore, there is a need to alter the biosynthetic pathway for threonine production to obtain taste and aroma benefits.

As shown in fig. 1 and 2, Aspartokinase (AK) is the first enzyme in the plant biosynthetic pathway that converts aspartate to amino acids including threonine. Endogenous aspartokinase is regulated by feedback inhibition from lysine and threonine. Therefore, there is a need for an aspartokinase that does not undergo feedback inhibition. However, achieving this need requires not forcing plants into methionine starvation or consuming levels of aspartate to the extent that other pathways dependent thereon are restricted.

It has been shown in the past that transgenic plants comprising a feedback insensitive aspartate kinase can overproduce threonine compared to wild type plants, grow poorly and show catastrophic health damage if the plants are homozygous for the mutation. Poor growth and health damage are any adverse changes in plant growth noted by farmers. It is clear that any such change is not desirable.

The inventors of the present invention therefore sought to provide a transgenic plant which can accumulate threonine in leaves by overcoming the above-mentioned feedback inhibition loop, but ideally without any health damage. In view of this, the present inventors have developed genetic constructs in which a gene encoding a threonine insensitive Aspartate Kinase (AK) enzyme is placed under the control of a promoter to determine what, if any, the overexpression of the gene has an effect on threonine levels in senescent leaves.

Leaf senescence is a stage of plant development in which cells undergo distinct metabolic and structural changes before they die. Physiological and genetic studies indicate that aging is a highly regulated process. The progression of leaves through senescence is visibly characterized by loss of chlorophyll and consequent yellowing, which is caused by chloroplast disintegration. The reduction in chlorophyll levels in leaves, characteristic of the developmental stage, can be detected, for example, by solvent extraction and spectrophotometric measurements or by a chlorophyll content meter. Reduced leaf chlorophyll levels compared to recorded earlier leaf chlorophyll levels of the same plants (preferably grown under constant conditions) showed senescence.

Molecular studies indicate that senescence is associated with changes in gene expression. The level of mRNA encoding proteins involved in photosynthesis decreases during senescence, while the level of mRNA encoding genes believed to be involved in senescence increases. Senescence is a highly organized process regulated by a gene known as senescence-associated gene (SAG). Leaf senescence involves the degradation of proteins, nucleic acids and membranes, and the subsequent transport of nutrients resulting from this degradation to other regions 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 are retained in the leaves and are therefore significantly lost when the leaves die. For example, threonine, as well as many other amino acids present in senescent leaves, would be wasted if not removed from dying leaves.

As described in example 2, the experiments carried out by the present inventors involved the use of a gene construct expressing threonine insensitive AK at a specific site in plants, i.e. using the leaf-specific pea plastocyanin promoter. However, the inventors found that such transgenic plants had pale leaves, thickened, fragile and banded. Internodes shorten and show browning as maturity increases, and shoots either do not develop or are misshapen. Thus, such transgenic plants cannot overcome health damage.

Thus, the present inventors developed a series of genetic constructs in which feedback insensitive Aspartate Kinase (AK) activity is expressed only after a plant has entered senescence (controlled by the SAG12 promoter), thus allowing normal development. The inventors observed that the constructs developed, which allow transgenic plants transformed with these constructs to grow normally to maturity (i.e. to senescence) before feedback insensitive Aspartate Kinase (AK) initiation, surprisingly were able to overcome health damage.

Thus, according to a first aspect of the present invention there is provided a genetic construct comprising a senescence-specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity.

The present inventors used a senescence-specific promoter linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity to form the construct of the first aspect, which was then used to transform plants. As a result of their investigation, the inventors surprisingly found that the construct according to the invention leads to an increase in threonine levels in senescent leaves. Furthermore, the time limitation of transgene expression controlled by senescence-specific promoters overcomes the negative effects of health damage previously seen in previous attempts to produce threonine-accumulating plants. As shown in the examples, the resulting transgenic plants produce threonine at a higher level during leaf senescence than the wild type. Threonine accumulation was demonstrated to occur in the lamina and the elevated levels are believed to positively contribute to the taste of tobacco lamina comprising the constructs and smoking articles made with the lamina.

The promoter in the genetic construct of the first aspect may be capable of inducing RNA polymerase to bind to and initiate transcription of the coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity.

A "senescence specific promoter" (SAG) can be any promoter involved in controlling the expression of senescence-associated genes. Thus, the promoter is substantially specific for limiting expression of the coding sequence (i.e., gene) to which it is operably linked in senescent tissues. Thus, a senescence-specific promoter may be a promoter that is capable of preferentially driving gene expression in a developmentally regulated manner in plant tissue, such that expression of the 3' protein coding region occurs substantially only when the plant tissue undergoes senescence. It will be appreciated that senescence tends to occur in older parts of the plant, such as older leaves, rather than in newer 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 present in the construct according to the first aspect may be isolated from a senescence-associated gene in Arabidopsis. Gepstein et al (The Plant Journal, 2003, 36, 629-642) performed detailed studies of SAG and its promoter using Arabidopsis thaliana as a model. Thus, the gene construct may comprise a promoter from any of the SAGs disclosed in this paper. For example, suitable promoters 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 those skilled in the art, or a functional variant or functional fragment thereof (Gan & Amasino, 1997, Plant Physiology, 113: 313-319). The DNA sequence encoding the SAG12 promoter is shown in FIG. 6 and is referred to herein as SEQ ID No.1, as follows:

TCGAGACCCGATTGTTATTTTTAGACTGAGACAAAAAAGTAGAATCGTTGATTGTTAAAATTTAAAATTAGTTTCATTACGTTTCGATAAAAAAATGATTAGTTTATCATAGCTTAATTATAGCATTGATTTCTAAATTTGTTTTTTGACCACCCTTTTTTCTCTCTTTGGTGTTTTCTTAACATTAGAAGAACCCATAACAATGTACGTTCAAATTAATTAAAAACAATATTTCCAAGTTTTATATACGAAACTTGTTTTTTTTAATGAAAACAGTTGAATAGTTGATTATGAATTAGTTAGATCAATACTCAATATATGATCAATGATGTATATATATGAACTCAGTTGTTATACAAGAAATGAAAATGCTATTTAAATACAGATCATGAAGTGTTAAAAAGTGTCAGAATATGACATGAAGCGTTTTGTCCTACCGGGTATTCGAGTTATAGGTTTGGATCTCTCAAGAATATTTTGGGCCATACTAGTTATATTTGGGCTTAAGCGTTTTGCAAAGAGACGAGGAAGAAAGATTGGGTCAAGTTAACAAAACAGAGACACTCGTATTAGTTGGTACTTTGGTAGCAAGTCGATTTATTTGCCAGTAAAAACTTGGTACACAACTGACAACTCGTATCGTTATTAGTTTGTACTTGGTACCTTTGGTTCAAGAAAAAGTTGATATAGTTAAATCAGTTGTGTTCATGAGGTGATTGTGATTTAATTTGTTGACTAGGGCGATTCCTTCACATCACAATAACAAAGTTTTATAGATTTTTTTTTTATAACATTTTTGCCACGCTTCGTAAAGTTTGGTATTTACACCGCATTTTTCCCTGTACAAGAATTCATATATTATTTATTTATATACTCCAGTTGACAATTATAAGTTTATAACGTTTTTACAATTATTTAAATACCATGTGAAGATCCAAGAATATGTCTTACTTCTTCTTTGTGTAAGAAAACTAACTATATCACTATAATAAAATAATTCTAATCATTATATTTGTAAATATGCAGTTATTTGTCAATTTTGAATTTAGTATTTTAGACGTTATCACTTCAGCCAAATATGATTTGGATTTAAGTCCAAAATGCAATTTCGTACGTATCCCTCTTGTCGTCTAATGATTATTTCAATATTTCTTATATTATCCCTAACTACAGAGCTACATTTATATTGTATTCTAATGACAGGGAAACCTTCATAGAGATTCAGATAGATGAAATTGGTGGGAAACATCATTGAACAGGAAACTTTTAGCAAATCATATCGATTTATCTACAAAAGAATACGTAGCGTAATGAAGTCCACTTGTTGTGAATGACTATGATTTGATCAAATTAGTTAATTTTGTCGAATCATTTTTCTTTTTGATTTGATTAAGCTTTTAACTTGCACGAATGGTTCTCTTGTGAATAAACAGAATCTTTGAATTCAAACTATTTGATTAGTGAAAAGACAAAAGAAGATTCCTTGTTTTTATGTGATTAGTGATTTTGATGCATGAAAGGTACCTACGTACTACAAGAAAAATAAACATGTACGTAACTACGTATCAGCATGTAAAAGTATTTTTTTCCAAATAATTTATACTCATGATAGATTTTTTTTTTTTGAAATGTCAATTAAAAATGCTTTCTTAAATATTAATTTTAATTAATTAAATAAGGAAATATATTTATGCAAAACATCATCAACACATATCCAACTTCGAAAATCTCTATAGTACACAAGTAGAGAAATTAAATTTTACTAGATACAAACTTCCTAATCATCAAATATAAATGTTTACAAAACTAATTAAACCCACCACTAAAATTAACTAAAAATCCGAGCAAAGTGAGTGAACAAGACTTGATTTCAGGTTGATGTAGGACTAAAATGACTACGTATCAAACATCAACGATCATTTAGTTATGTATGAATGAATGTAGTCATTACTTGTAAAACAAAAATGCTTTGATTTGGATCAATCACTTCATGTGAACATTAGCAATTACATCAACCTTATTTTCACTATAAAACCCCATCTCAGTACCCTTCTGAAGTAATCAAATTAAGAGCAAAAGTCATTTAACTTAGG

SEQ ID NO：1

thus, the promoter in the construct of the invention may comprise a nucleotide sequence substantially as shown in SEQ ID No.1, or a functional variant or functional fragment thereof. The SAG12 promoter sequence may be from Arabidopsis thaliana (Arabidopsis thaliana), as described in U.S. Pat. No.5,689,042. In embodiments where the promoter is SAG12, it will be appreciated that the promoter may comprise SEQ ID No: 1, each of bases 1-2093. However, functional variants or functional fragments of the promoters described may also be used in the gene constructs of the invention.

A "functional variant or functional fragment of a promoter" may be a derivative or a portion of said 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, one skilled in the art would understand that SEQ ID No: 1 may be modified or only some part of the SAG12 promoter may be required, whereby it will still promote gene expression of a polypeptide having threonine insensitive aspartate kinase activity in the construct. Similar modifications can be used for the nucleotide sequence of any other known SAG promoter, such as SAG13, SAG101, SAG21, and SAG 18.

Functional variants or functional fragments of a promoter can be readily identified by: assessing whether the transcriptase binds to a putative promoter region, which then results in transcription of the coding sequence into a polypeptide having threonine insensitive aspartate kinase activity. Alternatively, such functional variants and fragments can be detected by mutagenesis on the promoter when binding the coding region and assessing whether gene expression occurs.

The gene construct of the first aspect may cause expression of a polypeptide having threonine insensitive aspartate kinase activity during senescence. The promoter can induce the expression of a coding sequence that encodes a polypeptide that exhibits threonine insensitive aspartate kinase activity. Thus, the genetic construct may comprise at least one coding sequence encoding a threonine insensitive Aspartate Kinase (AK), or a functional variant or fragment thereof. Thus, in a first embodiment, the genetic construct may comprise a senescence-specific promoter and a coding sequence encoding a threonine insensitive Aspartate Kinase (AK), or a functional variant or fragment thereof.

As described in examples 3-4, by transforming plants with the constructs of the present invention, the present inventors found that threonine insensitive aspartate kinase expression in host cells causes a significant increase in threonine levels. Furthermore, advantageously, the inventors found that the construct did not have any adverse effect on the health of the transformed plant.

As shown in fig. 1 and 2, in plants, the amino acids lysine, threonine, methionine and isoleucine are synthesized from aspartic acid. Multiple feedback suppression loops have been found in this path. The first enzyme in this pathway, Aspartate Kinase (AK) (EC 2.7.2.4), catalyzes phosphorylation of aspartate to form 3-aspartyl phosphate with hydrolysis of ATP. It is believed that higher plants typically have at least 2 or 3 AK isozymes. AK activity is subject to negative feedback from the end products, the amino acids lysine and threonine. At least one of the AK isozymes is feedback-inhibited by threonine, and the other by lysine and S-adenosylmethionine. In barley, AK activity can be divided into 3 isozyme peaks, one of which is inhibited by threonine and the other two by lysine. Other feedback loops act on other enzymes of the biosynthetic pathway, such as dihydrodipicolinate synthase (DHPS) and homoserine desaturase (HSD).

As shown in fig. 1 and 2, a homoserine desaturase (HSD) (EC 1.1.1.3), also known as homoserine dehydrogenase (HSDH), catalyzes the first reaction associated with threonine, methionine and isoleucine biosynthesis only, i.e., the conversion of 3-aspartate semialdehyde to homoserine. Higher plants typically have at least two forms of HSDH: one threonine-sensitive and one threonine-insensitive form. Purification characteristics of HSDH and cDNA clones it has been demonstrated that the HSDH isozyme ligates AK to a single protein and is therefore referred to as "AK: HSDH".

Elevated levels of leaf threonine (Thr) depend on overcoming negative feedback control in the synthetic pathway. For example, transgenic tobacco expressing lysine insensitive AK from e. In these transformants, bacterial AK was expressed under the control of 35S in tobacco, targeting the cytoplasm or chloroplast. The endogenous activity of AK is still completely affected by lysine and threonine inhibition. The activity of transgenic AK targeting chloroplasts was higher. Higher threonine levels are found in plants expressing the chloroplast form. However, poor plant growth was noted and the homozygous plants showed a loss of health including upper leaf shrinkage, delayed flowering and partial sterility.

Work in Arabidopsis (Paris et al, 2003, The Journal of biological chemistry, volume 278, No.7, pp5361-5366) suggests that The regulatory domain of The AK: HSDH enzyme comprises two homologous subdomains, characterized by The common loop-alpha helix-loop-beta sheet motif. Site-directed mutagenesis was used to define the threonine binding site. It was found that each regulatory domain of the aspartokinase-homoserine dehydrogenase monomer has two non-equivalent threonine binding sites, partially consisting of Gln⁴⁴³And Gln⁵²⁴And (4) forming. Threonine binding to Gln⁴⁴³Inhibiting AK activity and promoting second threonine binding to Gln⁵²⁴Resulting in HSDH inhibition.

FIG. 3 shows a hypothetical model for threonine inhibition of AK-HSDH, where the active catalytic domains of AK and HSDH are represented by squares and the inhibited catalytic domains are represented by triangles. Threonine (Thr) binding to the first subdomain triggers (i) a conformational change in the other subdomain, and (ii) a conformational change in the AK catalytic domain that results in AK inhibition.

Conformational change of the second subdomain will induce second threonine binding, resulting in conformational change of the HSDH catalytic domain and HSDH inhibition.

Mutation of the glutamine residue to alanine negates threonine inhibition of the enzyme. The mutation does not affect the kinetics of HSDH activity, only its sensitivity to threonine; AK kinetics were only slightly altered. Unfortunately, however, HSDH transgenic plants that have been introduced and expressed feedback insensitive AK from Arabidopsis also cause health damage.

In contrast, the genetic constructs of the present invention cause a polypeptide having threonine insensitive aspartate activity to be expressed during senescence without showing any adverse effect on transgenic plant health. Thus, the genetic construct of the first aspect may encode a threonine insensitive Aspartate Kinase (AK), or a threonine insensitive bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH), or a functional variant or functional fragment thereof.

The threonine insensitive AK or bifunctional AK-HSDH, or functional variants or fragments thereof, may be derived from any suitable source, such as a plant. The coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity may be derived from Arabidopsis (Arabidopsis spp.), Zea spp., Chrysanthemum (Flaveria spp.), or Brassica (Cleome spp.). The coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity may be derived from south-mimetic blue, maize, flaveriatriervia, Flaveria didentis, Flaveria brown or plumbum. Preferably, the coding sequence for the enzyme may be derived from arabidopsis, e.g. arabidopsis thaliana.

A particularly preferred threonine insensitive enzyme is mutated AK-HSDH, wherein at least one threonine is presentThe acid binding site has been altered. Preferably, part of the compound is Gln⁴⁴³And Gln⁵²⁴One or both of the constituent threonine binding sites have been mutated. For example, Arabidopsis AK-HSDH may be in Gln⁴⁴³And/or Gln⁵²⁴And (4) mutation. Preferably, Arabidopsis AK-HSDH is in Gln⁴⁴³And Gln⁵²⁴And (4) mutation. HSDH gene is shown in figure 7, wherein the mutated bases are underlined.

Thus, provided herein is a DNA sequence encoding one embodiment of an arabidopsis threonine insensitive aspartate kinase (i.e., the Gln443Ala single mutation) as set forth in SEQ ID No: 2, as follows:

ATGGCGACTCTGAAGCCGTCATTTACTGTTTCTCCGCCGAATAGTAATCCGATTAGATTTGGAAGTTTTCCGCCGCAATGCTTTCTCCGTGTTCCGAAACCGCGGCGACTTATATTGCCTAGGTTTCGGAAGACGACTGGTGGTGGCGGCGGCTTGATTCGATGTGAGCTTCCAGATTTTCATCTATCAGCAACAGCAACTACTGTATCAGGTGTATCGACGGTGAATTTAGTGGATCAAGTTCAGATTCCTAAAGGTGAAATGTGGAGTGTTCACAAGTTTGGTGGGACTTGTGTGGGAAACTCTCAGAGGATCAGAAATGTAGCAGAGGTTATAATCAATGATAATTCCGAAAGAAAACTTGTGGTTGTCTCGGCGATGTCGAAGGTTACGGACATGATGTATGACTTAATCCGCAAGGCACAATCACGAGATGATTCTTATTTATCCGCGTTGGAAGCTGTCTTGGAAAAGCATCGTTTAACAGCTCGTGACCTTCTCGATGGAGATGATCTCGCTAGTTTCTTGTCACATTTGCATAATGATATTAGTAATCTTAAAGCAATGCTTCGTGCTATATACATAGCTGGCCATGCATCAGAGTCGTTTTCAGATTTTGTTGCAGGACATGGGGAGCTTTGGTCTGCTCAGATGCTATCATATGTTGTCAGAAAGACTGGGCTTGAGTGCAAGTGGATGGATACTAGAGACGTGCTCATTGTTAATCCCACCAGCTCTAATCAGGTTGATCCTGATTTTGGTGAATCTGAGAAGAGACTCGATAAATGGTTCTCCTTAAATCCGTCGAAAATTATTATTGCGACTGGGTTTATTGCTAGCACTCCGCAAAATATTCCAACAACTTTGAAAAGAGATGGGAGTGATTTCTCAGCAGCTATTATGGGTGCTTTATTGAGAGCTCGTCAAGTAACCATTTGGACAGATGTTGATGGTGTATACAGTGCGGATCCTCGTAAAGTTAATGAGGCAGTGATACTCCAGACACTTTCTTATCAAGAGGCCTGGGAAATGTCTTATTTTGGAGCAAATGTGTTACATCCTCGCACCATCATTCCTGTGATGCGATATAATATTCCGATTGTGATTAGAAATATTTTCAATCTCTCTGCACCGGGAACAATAATCTGTCAACCTCCTGAAGATGATTATGACCTTAAACTGACAACTCCTGTCAAAGGGTTTGCAACTATTGACAATTTGGCCCTCATAAATGTTGAAGGTACTGGAATGGCTGGTGTACCCGGTACTGCAAGTGACATTTTTGGCTGTGTAAAAGATGTTGGAGCTAATGTGATTATGATATCAGCTGCTAGCAGTGAGCATTCTGTGTGCTTTGCTGTGCCTGAGAAGGAAGTAAACGCAGTCTCTGAGGCATTGCGGTCGAGATTTAGTGAAGCTTTACAAGCGGGACGTCTTTCTCAGATTGAGGTGATACCAAACTGTAGCATCTTAGCTGCAGTCGGCCAGAAAATGGCTAGTACACCTGGAGTTAGTTGTACACTTTTCAGTGCTTTGGCGAAGGCTAATATTAATGTCCGAGCTATATCTCARGGTTGTTCTGAGTACAATGTTACTGTCGTTATTAAACGTGAAGATAGCGTTAAGGCGTTAAGAGCTGTACACTCGAGGTTTTTCTTGTCAAGAACAACATTAGCAATGGGAATCGTAGGACCGGGCTTGATTGGTGCAACATTACTTGACCAGCTGCGGGATCAGGCTGCTGTTCTCAAACAAGAATTTAACATTGATCTGCGTGTTTTGGGAATCACTGGTTCAAAGAAGATGTTATTGAGTGACATTGGTATTGATTTGTCGAGATGGAGAGAACTTCTAAACGAGAAGGGAACAGAGGCGGATTTGGATAAATTCACTCAACAAGTGCATGGAAATCATTTTATCCCCAACTCTGTAGTGGTTGATTGTACAGCAGACTCTGCTATTGCAAGCCGTTACTATGATTGGTTACGAAAGGGAATTCATGTCATTACCCCAAATAAAAAGGCTAACTCAGGTCCCCTCGATCAGTACTTGAAACTGAGAGATCTTCAAAGGAAATCCTACACTCATTACTTCTACGAAGCCACTGTTGGAGCTGGTCTTCCAATTATCAGCACTTTACGTGGTCTCCTTGAGACAGGAGATAAGATACTACGCATAGAGGGCATTTGCAGTGGAACTTTGAGTTATCTATTCAACAATTTTGTTGGAGATCGAAGTTTCAGCGAGGTTGTCACTGAAGCAAAGAACGCAGGTTTCACTGAGCCTGATCCAAGAGATGATTTATCTGGAACTGATGTTGCAAGGAAGGTGATTATCCTCGCTCGAGAATCTGGACTGAAATTGGACCTCGCTGATCTCCCCATTAGAAGTCTCGTACCAGAACCTCTAAAAGGATGCACTTCTGTTGAAGAATTCATGGAGAAACTCCCACAGTACGATGGAGACCTAGCAAAAGAAAGGCTAGATGCTGAAAACTCTGGGGAAGTTCTGAGATATGTTGGAGTGGTGGACGCTGTTAACCAAAAGGGAACAGTTGAACTTCGAAGATACAAGAAAGAACATCCATTTGCGCAGCTCGCAGGTTCAGACAACATAATAGCCTTCACAACGACAAGGTACAAGGATCATCCACTTATAGTCCGAGGACCTGGAGCTGGTGCTCAAGTCACGGCCGGTGGTATATTCAGCGACATACTAAGGCTTGCATCTTATCTCGGTGCACCGTCTTAA

SEQ ID No：2

in SEQ ID No: 2 (i.e. Q443A), the highlighted GCT corresponds to the alanine-encoding mutant Gln⁴⁴³While highlighted CAR corresponds to wild-type Gln⁵²⁴Wherein R may be G or A.

Provided herein is a DNA sequence encoding another embodiment of an arabidopsis threonine insensitive aspartate kinase (i.e., a Gln524Ala single mutation) as SEQ ID No: 3, as follows:

ATGGCGACTCTGAAGCCGTCATTTACTGTTTCTCCGCCGAATAGTAATCCGATTAGATTTGGAAGTTTTCCGCCGCAATGCTTTCTCCGTGTTCCGAAACCGCGGCGACTTATATTGCCTAGGTTTCGGAAGACGACTGGTGGTGGCGGCGGCTTGATTCGATGTGAGCTTCCAGATTTTCATCTATCAGCAACAGCAACTACTGTATCAGGTGTATCGACGGTGAATTTAGTGGATCAAGTTCAGATTCCTAAAGGTGAAATGTGGAGTGTTCACAAGTTTGGTGGGACTTGTGTGGGAAACTCTCAGAGGATCAGAAATGTAGCAGAGGTTATAATCAATGATAATTCCGAAAGAAAACTTGTGGTTGTCTCGGCGATGTCGAAGGTTACGGACATGATGTATGACTTAATCCGCAAGGCACAATCACGAGATGATTCTTATTTATCCGCGTTGGAAGCTGTCTTGGAAAAGCATCGTTTAACAGCTCGTGACCTTCTCGATGGAGATGATCTCGCTAGTTTCTTGTCACATTTGCATAATGATATTAGTAATCTTAAAGCAATGCTTCGTGCTATATACATAGCTGGCCATGCATCAGAGTCGTTTTCAGATTTTGTTGCAGGACATGGGGAGCTTTGGTCTGCTCAGATGCTATCATATGTTGTCAGAAAGACTGGGCTTGAGTGCAAGTGGATGGATACTAGAGACGTGCTCATTGTTAATCCCACCAGCTCTAATCAGGTTGATCCTGATTTTGGTGAATCTGAGAAGAGACTCGATAAATGGTTCTCCTTAAATCCGTCGAAAATTATTATTGCGACTGGGTTTATTGCTAGCACTCCGCAAAATATTCCAACAACTTTGAAAAGAGATGGGAGTGATTTCTCAGCAGCTATTATGGGTGCTTTATTGAGAGCTCGTCAAGTAACCATTTGGACAGATGTTGATGGTGTATACAGTGCGGATCCTCGTAAAGTTAATGAGGCAGTGATACTCCAGACACTTTCTTATCAAGAGGCCTGGGAAATGTCTTATTTTGGAGCAAATGTGTTACATCCTCGCACCATCATTCCTGTGATGCGATATAATATTCCGATTGTGATTAGAAATATTTTCAATCTCTCTGCACCGGGAACAATAATCTGTCAACCTCCTGAAGATGATTATGACCTTAAACTGACAACTCCTGTCAAAGGGTTTGCAACTATTGACAATTTGGCCCTCATAAATGTTGAAGGTACTGGAATGGCTGGTGTACCCGGTACTGCAAGTGACATTTTTGGCTGTGTAAAAGATGTTGGAGCTAATGTGATTATGATATCACARGCTAGCAGTGAGCATTCTGTGTGCTTTGCTGTGCCTGAGAAGGAAGTAAACGCAGTCTCTGAGGCATTGCGGTCGAGATTTAGTGAAGCTTTACAAGCGGGACGTCTTTCTCAGATTGAGGTGATACCAAACTGTAGCATCTTAGCTGCAGTCGGCCAGAAAATGGCTAGTACACCTGGAGTTAGTTGTACACTTTTCAGTGCTTTGGCGAAGGCTAATATTAATGTCCGAGCTATATCTGCTGGTTGTTCTGAGTACAATGTTACTGTCGTTATTAAACGTGAAGATAGCGTTAAGGCGTTAAGAGCTGTACACTCGAGGTTTTTCTTGTCAAGAACAACATTAGCAATGGGAATCGTAGGACCGGGCTTGATTGGTGCAACATTACTTGACCAGCTGCGGGATCAGGCTGCTGTTCTCAAACAAGAATTTAACATTGATCTGCGTGTTTTGGGAATCACTGGTTCAAAGAAGATGTTATTGAGTGACATTGGTATTGATTTGTCGAGATGGAGAGAACTTCTAAACGAGAAGGGAACAGAGGCGGATTTGGATAAATTCACTCAACAAGTGCATGGAAATCATTTTATCCCCAACTCTGTAGTGGTTGATTGTACAGCAGACTCTGCTATTGCAAGCCGTTACTATGATTGGTTACGAAAGGGAATTCATGTCATTACCCCAAATAAAAAGGCTAACTCAGGTCCCCTCGATCAGTACTTGAAACTGAGAGATCTTCAAAGGAAATCCTACACTCATTACTTCTACGAAGCCACTGTTGGAGCTGGTCTTCCAATTATCAGCACTTTACGTGGTCTCCTTGAGACAGGAGATAAGATACTACGCATAGAGGGCATTTGCAGTGGAACTTTGAGTTATCTATTCAACAATTTTGTTGGAGATCGAAGTTTCAGCGAGGTTGTCACTGAAGCAAAGAACGCAGGTTTCACTGAGCCTGATCCAAGAGATGATTTATCTGGAACTGATGTTGCAAGGAAGGTGATTATCCTCGCTCGAGAATCTGGACTGAAATTGGACCTCGCTGATCTCCCCATTAGAAGTCTCGTACCAGAACCTCTAAAAGGATGCACTTCTGTTGAAGAATTCATGGAGAAACTCCCACAGTACGATGGAGACCTAGCAAAAGAAAGGCTAGATGCTGAAAACTCTGGGGAAGTTCTGAGATATGTTGGAGTGGTGGACGCTGTTAACCAAAAGGGAACAGTTGAACTTCGAAGATACAAGAAAGAACATCCATTTGCGCAGCTCGCAGGTTCAGACAACATAATAGCCTTCACAACGACAAGGTACAAGGATCATCCACTTATAGTCCGAGGACCTGGAGCTGGTGCTCAAGTCACGGCCGGTGGTATATTCAGCGACATACTAAGGCTTGCATCTTATCTCGGTGCACCGTCTTAA

SEQ ID No：3

in SEQ ID No: 3 (i.e., Q524A), GCT highlighted corresponds to the alanine-encoding mutant Gln⁵²⁴While highlighted CAR corresponds to wild-type Gln⁴⁴³Wherein R may be G or A.

Provided herein is a DNA sequence encoding another embodiment of an arabidopsis threonine insensitive aspartate kinase (i.e., Gln443 Ala; Gln524Ala double mutation) as SEQ ID No: 4, as follows:

ATGGCGACTCTGAAGCCGTCATTTACTGTTTCTCCGCCGAATAGTAATCCGATTAGATTTGGAAGTTTTCCGCCGCAATGCTTTCTCCGTGTTCCGAAACCGCGGCGACTTATATTGCCTAGGTTTCGGAAGACGACTGGTGGTGGCGGCGGCTTGATTCGATGTGAGCTTCCAGATTTTCATCTATCAGCAACAGCAACTACTGTATCAGGTGTATCGACGGTGAATTTAGTGGATCAAGTTCAGATTCCTAAAGGTGAAATGTGGAGTGTTCACAAGTTTGGTGGGACTTGTGTGGGAAACTCTCAGAGGATCAGAAATGTAGCAGAGGTTATAATCAATGATAATTCCGAAAGAAAACTTGTGGTTGTCTCGGCGATGTCGAAGGTTACGGACATGATGTATGACTTAATCCGCAAGGCACAATCACGAGATGATTCTTATTTATCCGCGTTGGAAGCTGTCTTGGAAAAGCATCGTTTAACAGCTCGTGACCTTCTCGATGGAGATGATCTCGCTAGTTTCTTGTCACATTTGCATAATGATATTAGTAATCTTAAAGCAATGCTTCGTGCTATATACATAGCTGGCCATGCATCAGAGTCGTTTTCAGATTTTGTTGCAGGACATGGGGAGCTTTGGTCTGCTCAGATGCTATCATATGTTGTCAGAAAGACTGGGCTTGAGTGCAAGTGGATGGATACTAGAGACGTGCTCATTGTTAATCCCACCAGCTCTAATCAGGTTGATCCTGATTTTGGTGAATCTGAGAAGAGACTCGATAAATGGTTCTCCTTAAATCCGTCGAAAATTATTATTGCGACTGGGTTTATTGCTAGCACTCCGCAAAATATTCCAACAACTTTGAAAAGAGATGGGAGTGATTTCTCAGCAGCTATTATGGGTGCTTTATTGAGAGCTCGTCAAGTAACCATTTGGACAGATGTTGATGGTGTATACAGTGCGGATCCTCGTAAAGTTAATGAGGCAGTGATACTCCAGACACTTTCTTATCAAGAGGCCTGGGAAATGTCTTATTTTGGAGCAAATGTGTTACATCCTCGCACCATCATTCCTGTGATGCGATATAATATTCCGATTGTGATTAGAAATATTTTCAATCTCTCTGCACCGGGAACAATAATCTGTCAACCTCCTGAAGATGATTATGACCTTAAACTGACAACTCCTGTCAAAGGGTTTGCAACTATTGACAATTTGGCCCTCATAAATGTTGAAGGTACTGGAATGGCTGGTGTACCCGGTACTGCAAGTGACATTTTTGGCTGTGTAAAAGATGTTGGAGCTAATGTGATTATGATATCAGCTGCTAGCAGTGAGCATTCTGTGTGCTTTGCTGTGCCTGAGAAGGAAGTAAACGCAGTCTCTGAGGCATTGCGGTCGAGATTTAGTGAAGCTTTACAAGCGGGACGTCTTTCTCAGATTGAGGTGATACCAAACTGTAGCATCTTAGCTGCAGTCGGCCAGAAAATGGCTAGTACACCTGGAGTTAGTTGTACACTTTTCAGTGCTTTGGCGAAGGCTAATATTAATGTCCGAGCTATATCTGCTGGTTGTTCTGAGTACAATGTTACTGTCGTTATTAAACGTGAAGATAGCGTTAAGGCGTTAAGAGCTGTACACTCGAGGTTTTTCTTGTCAAGAACAACATTAGCAATGGGAATCGTAGGACCGGGCTTGATTGGTGCAACATTACTTGACCAGCTGCGGGATCAGGCTGCTGTTCTCAAACAAGAATTTAACATTGATCTGCGTGTTTTGGGAATCACTGGTTCAAAGAAGATGTTATTGAGTGACATTGGTATTGATTTGTCGAGATGGAGAGAACTTCTAAACGAGAAGGGAACAGAGGCGGATTTGGATAAATTCACTCAACAAGTGCATGGAAATCATTTTATCCCCAACTCTGTAGTGGTTGATTGTACAGCAGACTCTGCTATTGCAAGCCGTTACTATGATTGGTTACGAAAGGGAATTCATGTCATTACCCCAAATAAAAAGGCTAACTCAGGTCCCCTCGATCAGTACTTGAAACTGAGAGATCTTCAAAGGAAATCCTACACTCATTACTTCTACGAAGCCACTGTTGGAGCTGGTCTTCCAATTATCAGCACTTTACGTGGTCTCCTTGAGACAGGAGATAAGATACTACGCATAGAGGGCATTTGCAGTGGAACTTTGAGTTATCTATTCAACAATTTTGTTGGAGATCGAAGTTTCAGCGAGGTTGTCACTGAAGCAAAGAACGCAGGTTTCACTGAGCCTGATCCAAGAGATGATTTATCTGGAACTGATGTTGCAAGGAAGGTGATTATCCTCGCTCGAGAATCTGGACTGAAATTGGACCTCGCTGATCTCCCCATTAGAAGTCTCGTACCAGAACCTCTAAAAGGATGCACTTCTGTTGAAGAATTCATGGAGAAACTCCCACAGTACGATGGAGACCTAGCAAAAGAAAGGCTAGATGCTGAAAACTCTGGGGAAGTTCTGAGATATGTTGGAGTGGTGGACGCTGTTAACCAAAAGGGAACAGTTGAACTTCGAAGATACAAGAAAGAACATCCATTTGCGCAGCTCGCAGGTTCAGACAACATAATAGCCTTCACAACGACAAGGTACAAGGATCATCCACTTATAGTCCGAGGACCTGGAGCTGGTGCTCAAGTCACGGCCGGTGGTATATTCAGCGACATACTAAGGCTTGCATCTTATCTCGGTGCACCGTCTTAA

SEQ ID No：4

in SEQ ID No: 4 (i.e., Q443A; Q524A), the highlighted GCT corresponds to the mutant Gln, both encoding alanine⁴⁴³And Gln⁵²⁴。

Thus, a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity may comprise a sequence substantially as set forth in SEQ ID No: 2. 3 or 4, or a functional variant or fragment thereof.

Provided herein is a polypeptide sequence of one embodiment of a threonine insensitive aspartate kinase (i.e., Gln443Ala single mutation) as set forth in SEQ ID No: 5, as follows:

MATLKPSFTVSPPNSNPIRFGSFPPQCFLRVPKPRRLILPRFRKTTGGGGGLIRCELPDFHLSATATTVSGVSTVNLVDQVQIPKGEMWSVHKFGGTCVGNSQRIRNVAEVIINDNSERKLVVVSAMSKVTDMMYDLIRKAQSRDDSYLSALEAVLEKHRLTARDLLDGDDLASFLSHLHNDISNLKAMLRAIYIAGHASESFSDFVAGHGELWSAQMLSYVVRKTGLECKWMDTRDVLIVNPTSSNQVDPDFGESEKRLDKWFSLNPSKIIIATGFIASTPQNIPTTLKRDGSDFSAAIMGALLRARQVTIWTDVDGVYSADPRKVNEAVILQTLSYQEAWEMSYFGANVLHPRTIIPVMRYNIPIVIRNIFNLSAPGTIICQPPEDDYDLKLTTPVKGFATIDNLALINVEGTGMAGVPGTASDIFGCVKDVGANVIMISAASSEHSVCFAVPEKEVNAVSEALRSRFSEALQAGRLSQIEVIPNCSILAAVGQKMASTPGVSCTLFSALAKANINVRAISQGCSEYNVTVVIKREDSVKALRAVHSRFFLSRTTLAMGIVGPGLIGATLLDQLRDQAAVLKQEFNIDLRVLGITGSKKMLLSDIGIDLSRWRELLNEKGTEADLDKFTQQVHGNHFIPNSVVVDCTADSAIASRYYDWLRKGIHVITPNKKANSGPLDQYLKLRDLQRKSYTHYFYEATVGAGLPIISTLRGLLETGDKILRIEGICSGTLSYLFNNFVGDRSFSEVVTEAKNAGFTEPDPRDDLSGTDVARKVIILARESGLKLDLADLPIRSLVPEPLKGCTSVEEFMEKLPQYDGDLAKERLDAENSGEVLRYVGVVDAVNQKGTVELRRYKKEHPFAQLAGSDNIIAFTTTRYKDHPLIVRGPGAGAQVTAGGIFSDILRLASYLGAPS

SEQ ID No：5

in SEQ ID No: in 5, the mutant alanine (A) at position 443 and the wild-type glutamine (Q) at position 524 are highlighted.

Provided herein is a polypeptide sequence of another embodiment of a threonine insensitive aspartate kinase (i.e., a single mutation of Gln524 Ala) as SEQ ID No: 6, as follows:

MATLKPSFTVSPPNSNPIRFGSFPPQCFLRVPKPRRLILPRFRKTTGGGGGLIRCELPDFHLSATATTVSGVSTVNLVDQVQI PKGEMWSVHKFGGTCVGNSQRIRNVAEVIINDNSERKLVVVSAMSKVTDMMYDLIRKAQSRDDSYLSALEAVLEKHRLTARDLLDGDDLASFLSHLHNDISNLKAMLRAIYIAGHASESFSDFVAGHGELWSAQMLSYVVRKTGLECKWMDTRDVLIVNPTSSNQVDPDFGESEKRLDKWFSLNPSKIIIATGFIASTPQNIPTTLKRDGSDFSAAIMGALLRARQVTIWTDVDGVYSADPRKVNEAVILQTLSYQEAWEMSYFGANVLHPRTIIPVMRYNIPIVIRNIFNLSAPGTIICQPPEDDYDLKLTTPVKGFATIDNLALINVEGTGMAGVPGTASDIFGCVKDVGANVIMISQASSEHSVCFAVPEKEVNAVSEALRSRFSEALQAGRLSQIEVIPNCSILAAVGQKMASTPGVSCTLFSALAKANINVRAISAGCSEYNVTVVIKREDSVKALRAVHSRFFLSRTTLAMGIVGPGLIGATLLDQLRDQAAVLKQEFNIDLRVLGITGSKKMLLSDIGIDLSRWRELLNEKGTEADLDKFTQQVHGNHFIPNSVVVDCTADSAIASRYYDWLRKGIHVITPNKKANSGPLDQYLKLRDLQRKSYTHYFYEATVGAGLPIISTLRGLLETGDKILRIEGICSGTLSYLFNNFVGDRSFSEVVTEAKNAGFTEPDPRDDLSGTDVARKVIILARESGLKLDLADLPIRSLVPEPLKGCTSVEEFMEKLPQYDGDLAKERLDAENSGEVLRYVGVVDAVNQKGTVELRRYKKEHPFAQLAGSDNIIAFTTTRYKDHPLIVRGPGAGAQVTAGGIFSDILRLASYLGAPS

SEQ ID No：6

in SEQ ID No: in 6, the mutant alanine at position 524 (A) and the wild-type glutamine at position 443 (Q) are highlighted.

The polypeptide sequence of another embodiment of the threonine insensitive aspartate kinase (i.e., Gln443 Ala; Gln524Ala double mutation) is provided herein as SEQ ID No: 7, as follows:

MATLKPSFTVSPPNSNPIRFGSFPPQCFLRVPKPRRLILPRFRKTTGGGGGLIRCELPDFHLSATATTVSGVSTVNLVDQVQIPKGEMWSVHKFGGTCVGNSQRIRNVAEVIINDNSERKLVVVSAMSKVTDMMYDLIRKAQSRDDSYLSALEAVLEKHRLTARDLLDGDDLASFLSHLHNDISNLKAMLRAIYIAGHASESFSDFVAGHGELWSAQMLSYVVRKTGLECKWMDTRDVLIVNPTSSNQVDPDFGESEKRLDKWFSLNPSKIIIATGFIASTPQNIPTTLKRDGSDFSAAIMGALLRARQVTIWTDVDGVYSADPRKVNEAVILQTLSYQEAWEMSYFGANVLHPRTIIPVMRYNIPIVIRNIFNLSAPGTIICQPPEDDYDLKLTTPVKGFATIDNLALINVEGTGMAGVPGTASDIFGCVKDVGANVIMISAASSEHSVCFAVPEKEVNAVSEALRSRFSEALQAGRLSQIEVIPNCSILAAVGQKMASTPGVSCTLFSALAKANINVRAISAGCSEYNVTVVIKREDSVKALRAVHSRFFLSRTTLAMGIVGPGLIGATLLDQLRDQAAVLKQEFNIDLRVLGITGSKKMLLSDIGIDLSRWRELLNEKGTEADLDKFTQQVHGNHFIPNSVVVDCTADSAIASRYYDWLRKGIHVITPNKKANSGPLDQYLKLRDLQRKSYTHYFYEATVGAGLPIISTLRGLLETGDKILRIEGICSGTLSYLFNNFVGDRSFSEVVTEAKNAGFTEPDPRDDLSGTDVARKVIILARESGLKLDLADLPIRSLVPEPLKGCTSVEEFMEKLPQYDGDLAKERLDAENSGEVLRYVGVVDAVNQKGTVELRRYKKEHPFAQLAGSDNIIAFTTTRYKDHPLIVRGPGAGAQVTAGGIFSDILRLASYLGAPS

SEQ ID No：7

in SEQ ID No: in 7, the mutant alanine (A) at positions 443 and 524 are highlighted.

Thus, a polypeptide sequence having threonine insensitive aspartate kinase activity may comprise an amino acid sequence substantially as set forth in SEQ ID No: 5. 6 or 7, or a functional variant or fragment thereof.

The genetic constructs of the invention may be in the form of expression cassettes, which may be suitable for expression of the coding sequence in a host cell. The gene construct of the present invention can be introduced into a host cell without being integrated into a vector. For example, the genetic construct, which may be a nucleic acid molecule, may be incorporated in a liposome or a viral particle. Alternatively, the purified nucleic acid molecule (e.g., histone-free DNA, or naked DNA) may be inserted directly into the host cell by a suitable method, e.g., direct endocytic uptake. The genetic construct may be introduced directly into a cell of a host object (e.g., a plant) by transfection, infection, microinjection, cell fusion, protoplast fusion, or microprojectile bombardment. Alternatively, the gene constructs of the invention may be introduced directly into a host cell using a gene gun. Alternatively, the genetic construct may be comprised in a recombinant vector for expression in a suitable host cell.

Thus, in a second aspect, there is provided a recombinant vector comprising a gene construct according to the first aspect.

The recombinant vector may be a plasmid, cosmid, or phage. Such recombinant vectors are highly beneficial for transforming a host cell with the genetic construct of the first aspect and replicating the expression cassette therein. It will be appreciated by those skilled in the art that the genetic constructs of the present invention may be used in conjunction with a variety of types of backbone vectors for expression purposes. The backbone vector may be a binary vector, such as a vector replicable in both E.coli and Agrobacterium (Agrobacterium tumefaciens). For example, a suitable vector may be a pBIN plasmid, such as pBIN 19. However, a preferred backbone vector is BNP1380000001, which is based on pBINPLUS (F.A. VanEngelen et al, Transgenic Research (1995)4, 288-290) and which comprises the SAG12 promoter. One embodiment of such a vector is shown in figure 16.

The recombinant vector may include a variety of other functional elements in addition to a promoter (e.g., a senescence-associated promoter), as well as at least one coding sequence (encoding a mutant AK-HSDH). For example, a recombinant vector can be designed to replicate autonomously in the cytosol of a 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 so that it integrates into the genome of the host cell. In this case, DNA sequences are considered which are susceptible to targeted integration (e.g. by homologous recombination).

The recombinant vector may also comprise DNA encoding a gene that can be used as a selectable marker during cloning, i.e., cells that have been transfected or transformed may be selected, as well as cells comprising a vector incorporating heterologous DNA. Alternatively, the selectable marker gene may be used in a different vector in conjunction with the vector containing the gene of interest. The vector may also contain DNA involved in regulating the expression of the coding sequence, or in targeting the expressed polypeptide to a part of the host cell, such as the chloroplast. Thus, the vector of the second aspect may comprise at least one further element selected from: selectable marker genes (e.g., antibiotic resistance genes); a polypeptide termination signal; and protein targeting sequences (e.g., chloroplast transit peptides).

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, such as genes conferring resistance to phosphinothricin and sulfonamide based herbicides (bar and suI; EP-A-242246, EP-A-0249637, respectively); and screenable markers such as beta-glucuronidase (GB2197653), luciferase and Green Fluorescent Protein (GFP).

The marker gene may be controlled by a second promoter (which may or may not be a senescence-associated promoter) which allows expression in cells, which may or may not be in seeds, thereby allowing selection of cells or tissues containing the marker at any stage of plant development. Suitable second promoters are the promoter of the Agrobacterium nopaline synthase gene, and promoters derived from genes encoding 35S cauliflower mosaic virus (CaMV) transcripts. However, any other suitable second promoter may be used.

Various embodiments of the genetic constructs of the present invention can be prepared using suitable cloning procedures, as described in example 2, and can be summarized as follows. The gene encoding wild-type AK-HSDH can be amplified by PCR from genomic or cDNA templates using appropriate primers. Suitable primers for amplifying the wild-type AK-HSDH gene may be SEQ ID No: 8 and/or SEQ ID No: 9. the PCR product can be detected using agarose gel electrophoresis. Site-directed mutagenesis can then be performed using appropriate primer pairs to mutate the wild-type codon at positions 443 and/or 524 to generate Gln443Ala and Gln524Ala single or double mutations. For example, for altering Gln⁴⁴³Suitable primers for codons can be SEQ ID No: 10 and/or SEQ ID No: 11. for altering Gln⁵²⁴Suitable primers for codons can be SEQ ID No: 12And/or SEQ ID No: 13.

the PCR product encoding either or both of the single mutants may then be ligated into a suitable vector for cloning purposes, such as the vector commercially available from Invitrogen under the trade name pCR4Blunt-TOPO vector. The vector containing the PCR product can then be grown in a suitable host, e.g., e. Coli colonies can then be screened by PCR using appropriate primers, and plasmid inserts showing the correct restriction enzyme digestion pattern can be sequenced using appropriate primers.

Coli colonies carrying TOPO-cDNA (AK-HSDH) can be cultured to produce appropriate quantities of plasmid, and then purified. The plasmid may then be digested to release the DNA fragment encoding the mutant AK-HSDH, which may then be cloned into a vector, such as the pBNP plasmid, containing a suitable promoter, such as the SAG promoter (preferably, SAG 12). The resulting plasmid was designated pBNP 138-0453-001. An embodiment of a plasmid according to the second aspect may be substantially as shown in figure 15.

In a third aspect, there is provided a method of producing a transgenic plant that accumulates a level of threonine in leaves that is higher than that of a corresponding wild-type plant grown under the same conditions, the method comprising:

(i) transforming a plant cell with the gene construct of the first aspect or the vector of the second aspect, and

(ii) regenerating a plant from the transformed cell.

The method for determining the threonine level in the leaves of plants and the plant growth rate is described in example 1. The method of the third aspect may comprise transforming a test plant cell with a genetic construct according to the first aspect or a vector according to the second aspect. The genetic construct or vector may be introduced into the host cell using any suitable method.

In a fourth aspect, there is provided a cell comprising a gene construct according to the first aspect or a recombinant vector according to the second aspect.

The cell may be a plant cell. Since the inventors have observed that expression of a threonine insensitive aspartate kinase under the control of a senescence-specific promoter in a host cell is surprisingly effective in increasing threonine concentration in senescent leaves without compromising health, the cell of the fourth aspect may comprise one or more constructs of the first aspect, or one or more vectors of the second aspect.

The cells may be transformed with the genetic constructs or vectors according to the invention using known techniques. Suitable methods for introducing the genetic construct into the host cell may include the use of Agrobacterium-carried disarmed Ti-plasmid vectors by methods known in the art, for example 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 the nucleic acid, followed by rebuilding the cell wall. The transformed cells can then be grown into plants.

In a fifth aspect, there is provided a transgenic plant comprising a genetic construct according to the first aspect or a vector according to the second aspect.

The transgenic plant according to the fifth aspect may comprise a brassicaceae family, such as Brassica spp. The plant may be Brassica napus (Brassica napus).

Other embodiments of the transgenic plant according to the fifth aspect include the poaceae family, such as triticeae. The plant may be a Triticum spp. Increasing the grain protein content in wheat can result in an increase in the volume of a food product comprising such wheat, e.g., bread.

Further examples of suitable transgenic plants according to the fifth aspect include plants of the solanaceae family, which include, for example, stramonium, eggplant, mandragora, belladonna (belladonna), capsicum (paprika, pepper), potato and tobacco. An example of a suitable genus of the solanaceae family is the genus Nicotiana (Nicotiana). Suitable species of the genus nicotiana can be referred to as the tobacco plant, or simply tobacco. Various methods for transforming plants with the gene construct of the first aspect or the vector of the second aspect are known and can be used in the present invention.

For example, tobacco can be transformed as follows. Tobacco was transformed using a leaf disc co-cultivation method essentially as described by Horsch et al (Science 227: 1229-1231, 1985). The two most tender expanded leaves are obtained from 7 week old tobacco plants, available in 8% Domestoses^TMThe surface was sterilized for 10 minutes and washed 6 times with sterile distilled water. Leaf disks can be cut using a No. 6 cork punch and placed in agrobacterium suspension containing the appropriate binary vector (according to the invention) for about 2 minutes. The leaf discs were gently blotted between two layers of sterile filter paper. 10 leaf discs can be placed on LS 3% sucrose + 2. mu.M BAP + 0.2. mu.M NAA plates, which can then be cultured in a growth chamber for 2 days.

Leaf disks can be transferred to LS + 3% sucrose + 2. mu.M BAP + 0.2. mu.M NAA plates supplemented with 500g/l of Kalfuron and 100g/l of kanamycin. After two weeks the leaf discs can be transferred to fresh plates of the above medium. After a further two weeks, the leaf discs 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. The leaf discs were transferred to fresh medium every two weeks. When seedlings appeared, they were removed and transferred to LS + 3% sucrose bottles supplemented with 500mg/l of kevlar. After about 4 weeks the seedlings in the bottles can be transferred to LS + 3% sucrose +250mg/l Kalfuron. After a further 3-4 weeks, the plants can be transferred to LS + 3% sucrose (without antibiotics) and rooted. Once rooted, the plants can be transferred to greenhouse soil.

In a sixth aspect, there is provided a plant propagation product obtainable from a transgenic plant according to the fifth aspect.

A "plant propagation product" may be any plant material obtained from a plant from which more plants may be produced. Suitably, the plant propagation product may be a seed.

The invention also comprises harvested leaves from a transgenic plant of the invention, wherein the harvested leaves comprise a higher level of threonine than harvested leaves of a corresponding wild-type plant grown under the same conditions.

Thus, in a seventh aspect, there is provided a harvested leaf comprising a threonine level higher than the corresponding threonine level in a harvested leaf obtained from a wild-type plant grown under the same conditions, wherein the leaf is harvested from a transgenic plant according to the fifth aspect or produced by a method according to the third aspect.

An eighth aspect of the invention provides a smoking article comprising threonine-enriched tobacco obtained from a mutant tobacco plant, which mutant is capable of overproducing threonine in senescent leaves.

Advantageously and preferably, said mutant tobacco plant may be transformed with a genetic construct or vector of the invention. The smoking article may be a cigarette, cigar, cigarillo, or cigarette, or the like.

Threonine-enriched tobacco can include tobacco having a threonine concentration that is higher than the corresponding concentration of a wild-type plant grown under the same conditions. Such a smoking article may comprise tobacco obtained from a mutant tobacco plant which may be transformed with the gene construct according to the first aspect of the invention or the vector according to the second aspect. The threonine-enriched tobacco has improved taste and aroma.

It will be appreciated that the present invention provides a method of increasing the level of threonine in the leaves of a plant to levels above that of the corresponding wild type without compromising plant health, which comprises altering plant metabolism to achieve an increase in threonine production following the onset of leaf senescence.

Thus, in a ninth aspect of the invention, there is provided a method of increasing the level of threonine in the leaves of a plant to levels above that of the corresponding wild type without compromising plant health, which method comprises altering plant metabolism to achieve an increase in threonine production following the onset of leaf senescence.

Preferably and advantageously, the method according to the invention does not impair the health or fitness of the plants produced. Preferably, the method comprises transforming a test plant, and preferably its leaves, with the gene construct of the first aspect or the vector of the second aspect.

In addition to measuring threonine levels in the transformed plants of the invention and showing increased threonine concentrations in senescent leaves, the present inventors also measured the concentrations of other amino acids, including glutamine, glutamic acid, aspartic acid and histidine, in the transgenic plants as described in example 4. As shown in fig. 10-14, the concentration of each of the amino acids was comparable to or even higher than the control, strongly suggesting that the health of the transgenic plants was not compromised. Thus, in general, the constructs of the invention may be suitable for increasing threonine levels in senescent leaves without negatively affecting the health of the transgenic plants.

It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises the essential amino acid or nucleic acid sequence of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "primary 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, e.g. to the amino acid sequence identified as SEQ ID No: 1 (i.e., the SAG12 promoter) or the gene identified as SEQ ID No.2, 3 or 4 (which encodes various embodiments of an AK-HSDH enzyme) 40% identical, or the polypeptide identified as SEQ ID No.5, 6 or 7 (i.e., various embodiments of a mutant AK-HSDH enzyme) 40% identical, and so forth.

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

One skilled in the art would know how to calculate the percent identity between two amino acids/polynucleotide/polypeptide sequences. To calculate the percent identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, and then a sequence identity value calculated. The percent identity of two sequences can have different values depending on: (i) methods for aligning sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (performed in different programs), or structural alignment for 3D comparison; and (ii) parameters used by the alignment method, such as local to global alignments, the pair-scoring matrices used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as functional forms and constants.

After alignment, there are a number of different methods to calculate the percent identity between two sequences. For example, some 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 sites; or (v) the number of identical sites except for the overhang. Furthermore, it should be appreciated that percent uniformity is also strongly dependent on length. Thus, the shorter a pair of sequences, the higher the sequence identity that can be expected to occur by chance.

Thus, it will be appreciated that precise alignment of protein or DNA sequences is a complex process. The general multiplex alignment program ClustalW (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-. Suitable parameters for ClustalW may be the following: for DNA alignment: gap open penalty 15.0, gap stretch penalty 6.66, matrix identity. For protein alignment: gap opening penalty of 10.0, gap stretch penalty of 0.2, matrix Gonnet. For DNA to protein alignment: ENDGAP ═ -1 and gapist ═ 4. One skilled in the art will appreciate that these and other parameters may have to be varied in order to optimize sequence alignment.

Preferably, the percent identity calculation between two amino acid/polynucleotide/polypeptide sequences can be calculated from, for example, an alignment (N/T) × 100, where N is the number of sites that the sequences share the same bases and T is the total number of sites compared, including gaps but not overhangs. Thus, the most preferred method for calculating percent identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, e.g., those described above; and (ii) inserting the values of N and 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 will be encoded by a sequence that hybridizes under stringent conditions to the sequence shown in SEQ ID Nos 1, 2, 3 or 4, or the complement thereof. By stringent conditions, we mean that the nucleotide hybridizes to the DNA or RNA bound to the filter paper at about 45 ℃ in 3 XSSC/sodium citrate (SSC) and then washed at least once at about 20-65 ℃ in 0.2 XSSC/0.1% SDS. Alternatively, the substantially similar polypeptide may be identical to SEQ ID No: 5. 6 or 7 differ 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 of the nucleic acid sequences described herein may be varied or altered without substantially affecting the sequence of the protein encoded thereby, thereby providing a functional variant thereof. Suitable nucleotide variants are those having a sequence that is altered by a different codon substitution of the same amino acid in the coding sequence, thereby producing a silent alteration. Other suitable variants have homologous nucleotide sequences, but comprise sequences altered in whole or in part by substitution with a different codon that encodes an amino acid having a side chain with biophysical properties similar to the amino acid being substituted, thereby resulting in conservative alterations. For example, small nonpolar, hydrophobic amino acids include 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 for amino acids having similar biophysical properties, and that the nucleotide sequences encoding these amino acids are known to those skilled in the art.

All of the elements 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 elements and/or steps are mutually exclusive.

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

FIG. 1 shows a part of the plant biosynthetic pathway for aspartate conversion to other amino acids. The related enzymes are Aspartokinase (AK) and homoserine dehydrogenase (HSDH);

FIG. 2 shows the plant biosynthetic pathway for the conversion of aspartate to other amino acids, including threonine. The first enzyme activity in the pathway is Aspartate Kinase (AK). Plants can produce bifunctional enzyme aspartokinase: homoserine dehydrogenase (AK-HSDH). The biosynthetic pathway is tightly regulated, including positive and negative feedback of the final product;

FIG. 3 shows a model for allosteric modulation of aspartokinase-homoserine dehydrogenase by threonine in Arabidopsis (after Paris et al (2003));

FIG. 4 shows schematically a mutant bifunctional aspartokinase homoserine desaturase;

FIG. 5 shows the threonine levels detected in leaf discs in example 2;

FIG. 6 shows the sequence of the SAG12 promoter;

FIG. 7 shows the sequence of a mutant aspartokinase homoserine desaturase. The mutant bases required to overcome threonine feedback inhibition are shaded and underlined;

FIG. 8 shows threonine content in leaf disks obtained from transgenic plants in which threonine insensitive AK-HSDH is controlled by a senescence-specific promoter, as described in example 3;

FIG. 9 is a bar graph showing threonine content in baked leaves grown in a field trial;

FIG. 10 is a bar graph showing the glutamine content in baked leaves grown in a field trial;

FIG. 11 is a bar graph showing the glutamic acid content in the roasted leaves grown in the field trial;

FIG. 12 is a bar graph showing the level of asparagine in field test grown roasted leaves;

FIG. 13 is a bar graph showing the aspartic acid content in field test grown roasted leaves;

FIG. 14 is a bar graph showing histidine content in field trial grown roasted leaves;

FIG. 15 is a plasmid map of one embodiment of a vector according to the present invention; and

FIG. 16 is a plasmid map of a backbone vector used in accordance with the present invention.

Examples

As described in example 2, the present inventors have developed transgenic plants in which an altered feedback insensitive aspartate kinase (AK: HSDH) is expressed under the control of a leaf-specific promoter. However, the present inventors found that local expression of modified AK HSDH in leaves resulted in health damage in transgenic plants. Thus, as described in examples 3-4, the present inventors used a senescence-specific promoter (SAG12) linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity (AK: HSDH). The resulting transgenic plants produce threonine at levels higher than wild type levels during leaf senescence without compromising the health of the plants.

Example 1 testing of threonine levels in leaves

Threonine levels were tested on green or yellow leaves. Leaf discs were taken and used for analysis, thus measuring the threonine content (i.e. threonine content/leaf area) on a per disc basis or correlating with the protein content (i.e. threonine content/mg protein) in the supernatant. The leaf discs were mashed with a quantity of water and centrifuged to pellet the insoluble leaf residue. The supernatant from the process was then treated with the phenomenex ezfaast Kit. This is a special kit for derivatizing amino acids in extracts so that they can be quantified using standard liquid chromatography/mass spectrometry (lc/ms) equipment.

Calibration was performed using an external standard for each amino acid to be quantified, normalizing the efficiency of the derivatization step between samples by including an internal standard in the process. The chromatogram is evaluated by peak area and related to concentration using an integration algorithm in the liquid chromatography/mass spectrometry software. If no peaks can be identified as determined by the software or the operator, this is said to be "below detection limit". This was found to be the case for some empty vector controls and some segregating null plants in the progeny.

Example 2 feedback insensitive AK HSDH with operably linked leaf specific promoter Transgenic plants of (3)

The present inventors performed site-directed mutagenesis on a bifunctional AK HSDH wild-type sequence from Arabidopsis thaliana (At4g 19710). Thus, the wild-type sequence was first isolated by PCR from a leaf-specific cDNA library from Arabidopsis thaliana using the following primers:

at4g19710 forward ATGGCGACTCTGAAGCCGTCATTTAC (SEQ ID NO: 8);

at4g19710 reverse TTAAGACGGTGCACCGAGATAAGATGC (SEQ ID No: 9).

The wild-type sequence is altered in one of three ways: (i) an AK domain only mutation, (ii) HSDH domain mutation, or (iii) both domains mutated to not accept regulation by threonine binding. The specific mutations are in Gln443 and Gln524, both in the enzyme regulatory domain, both mutated to alanine by site-directed mutagenesis (Paris et al (2003), "Mechanism of control of Arabidopsis thaliana aspartate kinase-homeoserine dehydrogenase by threonine",J.Biol.Chem 278: 5361-: 1285-1293.

Site-directed mutagenesis kit (catalog #200518) was used for this step. To change the codon encoding Glu443, the following primers were used in the site-directed mutagenesis reaction:

gln443 Forward GTGATTATGATATCAGCTGCTAGCAGTGAGCATTCTG

(SEQ ID No: 10); and

gln443 reverse CAGAATGCTCACTGCTAGCAGCTGATATCATAATTCAC

(SEQ ID No：11).

For Gln524, the following primer pairs were used:

gln524 forward GTCCGAGCTATATCTGCTGGTTGTTCTGAGTACAATG;

(SEQ ID No: 12); and

gln524 reverse CATTGATCTCAGAACAACCAGCAGATATAGCTCGGAC

(SEQ ID No：13).

Transformation of tobacco plants, e.g. wild type mimetics, with these 3 mutant sequences, respectivelyThe same applies to Arabidopsis AK HSDH. In all cases, the gene of interest was expressed under the control of the leaf-specific pea plastocyanin promoter. Plants of all populations were generated by agrobacterium-mediated transformation and grown under greenhouse conditions in Cambridge, UK. Kit using EZfaast amino acidFree amino acids were extracted and derivatized in each sample. Quantification was performed by liquid chromatography/mass spectrometry.

The results are shown in FIG. 5 and Table 1. In fig. 5, the series of plant names with only AK domain mutations was initiated with AK, the series of plant names with only HSDH domain mutations was initiated with HSDH, the series of plant names with both AK and HSDH domains mutated was initiated with AK/HSDH, the wild type plant name was initiated with WT, and the transgenic plant name containing an empty vector control was initiated with EV.

The inventors achieved increased leaf threonine levels in all populations transformed with arabidopsis sequences, including populations transformed with non-mutated arabidopsis sequences. The highest level of leaf threonine was found in the population transformed with the sequence mutated in the AK domain. This is in highest agreement with the population showing a severe health risk. Although the present inventors have used a leaf-specific promoter in an attempt to reduce the effects of fertility changes, the effect is sufficient that the whole plant is still affected by down-regulating metabolic consequences of the feedback control of the enzyme.

However, in all plants showing increased threonine there is a correlation with altered growth habit. Leaves are pale, thickened, fragile and banded. Internodes shorten and show browning as maturation increases. Shoots either do not develop or are malformed. In conclusion, the site-directed mutagenesis successfully provided for a leaf threonine elevation. However, if agriculturally viable high leaf threonine plants are to be obtained from this approach, there is still a need to overcome the health damage that results from the release of feedback control over aspartokinase.

TABLE 1

Example 3 threonine insensitivity comprising operably linked to a senescence-specific promoter Transgenic plants of AK-HSDH

The cultivar K326 of tobacco plants was used to provide leaf disks for co-cultivation with Agrobacterium (Agrobacterium tumefaciens) previously transformed (by electroporation) under the control of the senescence-specific promoter SAG12 (whose sequence is shown in FIG. 6, i.e., SEQ ID No: 1) using a binary vector carrying the gene of interest (i.e., mutated AK: HSDH, whose sequence is shown in FIG. 7, i.e., SEQ ID Nos 2, 3 and 4). A control population was also cultured in which the binary vector contained the promoter but no AK: HSDH gene. These leaf disks were then treated by tissue culture methods to provide plantlets (Horsch et al Science 227: 1229-1231, 1985). Each plantlet is generated from a single transformation event in which DNA is transferred from the bacterium and integrated into the plant genomic DNA. These plants were transferred to the greenhouse and grown to maturity. Mature leaves were isolated from the plants and placed in polyethylene bags in the dark at 35 ℃ for 72 hours. After this time the leaves were used to provide leaf discs which were assessed for threonine content as described in example 1 above.

The results are shown in FIG. 8, which shows that high levels of threonine are obtained in some plants. Advantageously, these plants grow normally. Thus, these transgenic plants did not show the health damage observed in the plants described in example 2.

Example 4 conversion of HSDH to threonine insensitive AK comprising ligation to the SAG12 promoter Field test of genetic plants

Selected from SAG 12: the plant cell line of the aspartokinase (pBNP 138-0253-001) experimental population was planted in the field of North Carolina in 2008. The leaves were baked dry and analyzed for the presence of selected free amino acids. Confirmed by liquid chromatography/mass spectrometry analysis and by internal and external standard calibration. When the analyte is outside the range for calibration, it is labeled "> S" in Table 2 and is indicated in the figure by a star over the relevant bar.

The sample list is divided into "AK" numbers, which are K326 background lines altered with the genetic constructs of the invention, and 3 representative controls; unaltered K326, unaltered KV1, unaltered NC71, all grown and baked at the same time under the same conditions.

TABLE 2

Referring to fig. 9, a bar graph showing the threonine concentrations produced by 5 test plants (AK1-AK5) compared to 3 controls (K326, KV1, and NC7) is shown. As can be seen, all 5 test plants produced significantly more threonine than any control. Furthermore, none of the 5 test plants suffered from damage to their growth health. These data strongly support the observation that test plants transformed with the constructs according to the invention produce leaves with elevated (at least 2 to 3 fold) free threonine under commercial field conditions and that this is maintained during the roasting process. Furthermore, the test plants were non-segregating lines, which showed no yield loss under these field conditions.

The inventors also evaluated the concentration of various other amino acids (GLN: glutamine; GLU: glutamic acid; ASN: asparagine; ASP: aspartic acid; HIS: histidine) in the flue-cured leaves produced from the field trial plants, and the data are shown in FIGS. 10-14.

As can be seen in fig. 10, the concentration of glutamine for cell line AK3 was comparable to that in the 3 control cell lines. As shown in FIG. 11, the glutamic acid content of AK1-3 was the same as the control, while the concentration of AK4-5 was slightly higher. Fig. 12 shows the concentration of each asparagine.

Referring to FIG. 13, it can be seen that the concentration of aspartic acid was approximately the same in the test cell line AK1-AK3 as in the control, whereas its concentration appeared higher in AK 4-5. Finally, as shown in fig. 14, histidine concentrations were consistently about the same, except that the test cell line AK4 showed elevated levels.

In summary, figures 10-14 clearly show that the concentrations of each of these amino acids are comparable to or higher than the control, indicating that the health of the transgenic plants is not compromised.

Claims (10)

1. A genetic construct comprising a senescence-specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity, wherein the coding sequence consists of the nucleic acid sequence shown in any one of SEQ ID nos. 2, 3 or 4, or wherein the polypeptide consists of the amino acid sequence shown in any one of SEQ ID nos. 5,6 or 7.

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

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

4. The genetic construct of claim 3, wherein the senescence-specific promoter is SAG 12.

5. The genetic construct according to claim 3, wherein the promoter consists of the nucleotide sequence shown as SEQ ID No. 1.

6. The genetic construct of claim 1, wherein the coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity is derived from Arabidopsis spp.

7. A vector comprising the genetic construct of any one of claims 1-6.

8. A method for producing a transgenic solanaceous plant that accumulates in the leaf a level of threonine that is higher than that of a corresponding solanaceous wild-type plant grown under the same conditions, said method comprising:

(i) transforming a cell of a solanaceous plant with the genetic construct according to any one of claims 1-6 or the vector according to claim 7; and

(ii) regenerating a plant from the transformed cell.

9. A smoking article comprising threonine-enriched tobacco obtained from a mutant tobacco plant capable of overproducing threonine in senescent leaves, wherein the mutant tobacco plant has been transformed with the genetic construct of any one of claims 1-6 or the vector of claim 7.

10. A method for increasing the level of threonine in leaves of a solanaceous plant above the corresponding wild type level without compromising plant health, comprising altering plant metabolism to achieve an increase in threonine production following the onset of leaf senescence, wherein the method comprises transforming a solanaceous plant with the construct of any one of claims 1-6 or the vector of claim 7.