Risultati finali
The goal of our project was the nutritional improvement of potato, specifically the enhancement of lysine, cysteine and methionine content. For this purpose molecular breeding tools were combined with classical breeding techniques.
Potato is the most important non-cereal food crop. Transformation systems for potato relying on recovery of transgenic plants from different organs were developed in the past. As part of the process a selectable marker, generally an antibiotic resistance gene is co-introduced with the gene of interest. Recently, a large number of transgenic crops have been produced; however, there is still a public concern with respect to the safety of antibiotic resistant transgenic plants in agricultural application. To avoid this problem efficiency of marker-free transformation was tested in different potato varieties. Trangenic plants were isolated from Bintje and Hopehely. The frequency of transformation, however, was much lower than in Desiree (2%) used as a control. Thus we concluded that efficiency of transformation highly depends on cultivars.
The marker-free transformation system was applied for increasing the cysteine content of the Hungarian potato variety, White Lady. Two transgenic lines expressing the serine acetyltransferase (SAT) gene from the constitutive CaMV35S promoter were isolated. RP-HPLC analysis showed increased cysteine and glutathione contents in leaves.
A co-transformation experiment was carried out to enhance S flux towards methionine synthesis and storage. Double transgenic plants expressing the potato S-transporter, StST1, from the root-specific RIAGS promoter and the methionine-rich storage protein gene zein from the constitutive CaMV35S promoter were isolated. It was shown that these plants grew better under S-limited conditions, in vitro, than the non-transformed controls. Immunoprecipitation revealed that the selected transgenic lines exhibited zein expression at protein level.
In order to simultaneously increase lysine synthesis and storage in Desiree a feedback insensitive isoform of dihydrodipicolonate synthase (DHDPS) involved in lysine sythesis and the modified chymotrypsine inhibitor KDEL, as a lysine storage protein gene, were inserted into a binary vector suitable for cloning of multiple genes. Co-insertion of the genes was verified by PCR analysis and expression of the genes was tested on RNA gel blots. Interestingly, much more KDEL than DHDPS mRNA was detected in each line tested. Further studies are needed to detect KDEL and DHDPS expression at protein level.
Morphological studies on transgenic lines showed that increases in amino acid levels can lead to morphological alterations and fertility problems. However, the intensity of these changes was variable in the different transgenic lines even if they were obtained with the same construct. Thus it is feasible that transgenic lines with no phenotypical alterations but with a moderate increase in amino acid content can be selected in the future. An alternative solution might be the utilisation of storage proteins to decrease the level of free-amino acids that may serve as signal molecules for other biosynthetic pathways.
In contrast, SAT-expressing White Lady lines increased in cysteine content had no morphological alterations. Since these lines were obtained by marker-free transformation they are suitable for further breeding either by molecular or classical approaches.
Desiree lines with increased S-transport and zein production are valuable tools of basic studies on relation of S-transport, S-assimilation and storage.
Sulphur rich amino acids (cysteine and methionine) as well as lysine are amino acids, which are not produced by monogastric animals and need therefore to be supplied by their diet. Corn seeds, which are extensively, used as animal feed, have a limited amount in those sulphur rich amino acids. Cysteine synthesis involves production of the carbone backbone from serine through the enzyme serine acetyltransferase (SAT), and addition of sulphur, which is provided through the action of the enzyme adenosyl phosphosulfate reductase (APR). As a mean to over-produce cysteine production in maize, a gene encoding Adenosine 5' Phosphosulfate Reductase from Lemna minor, has been introduced into maize plants.
Serine acetyl transferase (SAT) is a key enzyme in the cysteine biochemical pathway in activating L-serine by transfer of an acetyl group to generate O-acetyl-serine, an intermediate in cysteine production. A bacterial, cysteine insensitive SAT has been cloned behind a constitutively expressed promoter and introduced into maize. The over-produced cysteine could then be trapped into sulphur-rich proteins, i.e. the sunflower 2S seed albumin (SSA) protein. Expression of the SSA alone did not lead to increases in total cysteine and methionine levels, probably due to a limitation in the cysteine, methionine synthesis. Therefore an approach combining both the SAT enzyme and the SSA protein has been undertaken.
Among the proteinogenic amino acids cysteine and methionine display plenty of essential direct or indirect functions in cellular metabolism. Met is an essential amino acid required in the diet of non-ruminant animals. Major crops, such as cereals and legumes, are low in Met. The aim of this work was to improve the nutritional quality of crops by either expressing or down-regulating target genes in the whole plant or phloem specific manner, respectively. Target genes of interest such as cystathionine gamma-synthase (CgS) and homoserine kinase (HSK) were expressed in companion cells under the control of a cell specific promoter or in whole plants by using the 35S promoter to manipulate the flux towards end product amino acids such as cysteine and methionine or the cysteine homeostasis was misbalanced by down-regulation of O-acetylserine(thiol)lyase (OASTL).
Results obtained during the project indicate that first; OASTL activity does not only regulate cysteine de novo synthesis but also its homeostasis, second; the provision of the carbon backbone in form of O-phosphohomoserine in the whole plants is probably only successful if TS activity is down regulated, thirdly; potato CgS catalyses a near-equilibrium reaction and, more importantly, does not display features of a pathway regulating enzyme and supports data obtained for the HSK over-expressing plants, fourthly; preliminary data suggest that expression of HSK in plastids had no effect on amino acids of the aspartate family while several plants with the cytosolicly expressed HSK contained higher levels of methionine, threonine and isoleucine. Thus, the cell type specific expression has a much stronger effect on methionine and threonine levels as if expressed under a ubiquitous promoter and has to be investigated in more detail in the future.
Two sulfate transporter genes were cloned as part of this project. ZmST1;1 (Zea mays: ZmST1;1, AF355602) and StST1;1 (Solanum tuberosum: StST1, AF309643) from maize and potato respectively are members of group 1, generally considered to be high affinity transporters, principally responsible for uptake in the roots. Expression of these was analysed in respect to sulfur nutrition. A transcriptional repression at high sulfur availability was noted. A strategy to enhance sulfur flux into plants is the over-expression of the transporter responsible for initial sulfate uptake: StST1;1 in potato and ZmST1;1 in maize. This was attempted using the pRCg2promoter in maize and the pRIAGS promoter in potato. Both of these promoters are expressed in roots as shown with reporter genes.
The use of these promoters circumvents endogenous controls of transcription as seen in these species. A wheat TtST1;1 genes was expressed in maize (this could be distinguished from endogenous maize ST expression whilst the native StST1;1 genes was over-expressed in potato (expression could still be distinguished using appropriate primer combinations centred on the cloning sites). Confirmation of expression of the transgenes was obtained by RT-PCR. To further enhance sulfur accumulation, a suitable sink was required. Double transformants were produced containing both the zein sulfur-rich storage protein and the StST1;1 transporter. These various transgenic lines will be analysed in the future.
Lysine levels have been increased in potato tubers by introduction of a bacterial dihydrodipicolinate synthase (bacterial DHPS; DapA) driven by the CaMV 35S promoter or the potato tuber-specific gbss promoter. Introduction of a mutant bacterial aspartate kinase (LysC) led to potato plants with increased levels of threonine and a small increase of methionine. The bacterial enzymes are feedback insensitive for the end product amino acids unlike the plant enzymes. In the Opti-2 project, plants with elevated levels of lysine, methionine, or threonine were used for further characterisation of the influence of the transgenes on gene expression of other genes via transcriptomics and proteomics methodologies.
A dedicated potato microarray was used to study the influence of the transgene in general and to find new genes involved in the regulation of the aspartate pathway or genes differentially expressed as a result of the accumulation of the aminoacids themselves. Proteomics tools such as two-dimensional gel electrophoresis, protein gel patterns comparison and LC-QTOF MS/MS protein sequencing were used in addition to analyse differences in protein expression between transgenic and wild type potato tubers.
The analyses showed that the effect of increasing the amino acid level on the expression of other genes in tubers is very low. The differences between various harvests of the same plants or between different potato cultivars or different tuber tissues are much higher than between wt and transgenics. Only by using special search programs for comparable expression patterns, a small set of (regulatory) genes could be traced that show an increased expression in the transgenic plants compared to the control plants. These genes might be involved in (the regulation of) essential amino acid biosynthesis, and will be studied in more detail in the future.
The data from mRNA analysis suggest the developmental regulation of 14-3-3 isoform expressions in potato plants. To test 14-3-3 protein function transgenic potato plants under-expressing 14-3-3 isoforms were analysed. The transgenic plants showed a decrease in tuber number and an increase in tuber size; an increase in the fresh weight of tubers was detected.
The repressed plants showed significant increases in nitrate reductase (NR) activity. The increase in NR activity resulted in a significant decrease in nitrate level and increase in protein content. The level of sucrose phosphate synthase and starch synthase activities is also significantly increased in all the 14-3-3 under-expressed transgenes, and remarkably, the increase in enzyme activity is accompanied by respective changes in sucrose and starch levels in the tubers. In opposite the key enzymes of tyrosine metabolism are decreased upon 14-3-3 repression and resulted in catecholamine content reduction. The decrease in catecholamine content in transgenic tubers might be also responsible for starch increase in these plants.
The significant increase in ethylene content, which follows methionine level, was also found. The substantial increase of ethylene level in the repressed forms might explain the significant shortening of the vegetation period of the analyzed transgenic plants.
Lysine belongs to the family of amino acids, which is not produced by monogastric animals and needs therefore to be supplied by their diet. Corn seeds, which are extensively, used as animal feed, have a limited amount in Lysine. Lysine, present at high levels is toxic to the plant cell and is therefore tightly regulated at its level of synthesis and/or degradation. DHPS is a key enzyme involved in lysine biosynthesis and is subjected to lysine feedback inhibition. Lysine keto-glutarate reductase (LKR) is the first enzyme participating in Lysine degradation. A mean to increase Lysine content in maize plants, is to over-express lysine insensitive versions of the DHPS gene (bacterial or mutated plant genes), and/or to decrease the production of LKR either by an anti-sense or RNAi approach aimed at down-regulating Lysine keto-glutarate reductase gene or by knocking out this gene via transposon insertion. Finally the over-produced lysine may be trapped into lysine-rich storage proteins, like a modified lysine-rich gamma Zein.
An increase in lysine (and methionine) was observed in mutated Arabidopsis DHPS over-expressing maize plants. LKR KO mutant lines were identified and introgressed into commercial type maize lines. The transposon insertions are located within the first intron of the gene. An increase in total lysine amounts was observed for those lines as it has been observed in Arabidopsis by G. Galili within this project. A commercial type maize hybrid has been produced and is in the process of being analysed.
Introduction of a proprietary modified lysine-rich gamma Zein (Biogemma Patent) gene into maize leads to increased total lysine content in the maize seeds. A depressed kernel phenotype has also been observed for the highest expressing line. It needs to be verified that this phenotype is linked to the presence of the modified lyine-rich gamma Zein.
Over-expression of a lysine insensitive DHPS gene in maize leads to increased total lysine amounts. A combination between this approach and the modified lysine-rich gamma Zein may have synergistical effects and further boost lysine concentration in maize seeds.
Aspartate semialdehyde (ASA) is a common precursor for lysine, threonine and methionine synthesis in plants. Dihydrodipicolinate synthase (DHDPS, commitment to lysine synthesis) and homoserine dehydrogenase (HSDH, leads to threonine and methionine) are sharing ASA as a common substrate. Thus ASA is expected to be the key point in controlling the partitioning of the carbon skeleton into these two branches of the pathway. Genetic and molecular studies we have performed on Arabidopsis thaliana revealed that two dhdps genes, 2 akthr genes encoding bifunctional AK-HSDH enzymes and a mhsdh gene could be involved in the control of this partitioning.
In addition, 3 aklys genes encoding monofunctional aspartate kinase (AK) have to be considered too as AK activity is determining the rate of ASA synthesis. Arabidopsis thaliana knockout mutants in dhdps1, dhdps2, aklys2, aklys3, akthr1 and mhsdh were isolated in order to determine the respective role of these enzymes in amino acid synthesis. The only significant modification in the content of amino acids we recorded in the analyses of all the lines concerned an increase of threonine content in the dhdps2 knockout line (up to five fold the wild type level in reproductive organs) while the level of lysine was only weakly affected. In the dhdps1 knockout mutant, lysine was decreased but threonine did not accumulate. The global AK specific activity of aklys2, aklys3 and akthr1 knockouts was comparable to the activity of the wild type plants.
The reduction of the HSDH activity in the akthr1 knockout did not impair the synthesis of threonine and methionine. We showed that the use of a DHDPS inhibitor allowed us to obtain wild type plants in which no DHDPS activity was left. This also led to a drastic accumulation of free threonine. In this study, we demonstrated that a molecular or chemically obtained limitation of the DHDPS activity leads to threonine overproduction and that any manipulation of the HSDH activity we tried did not affect significantly the synthesis of aspartate derived amino acids.
Over-expression of enzymes involved in sulfur assimilation, as for example APS reductase or methionine S-methyltransferase, is one strategy to increase the content of reduced sulfur containing compounds (e.g. glutathione or methionine) in edible plant tissues. Data obtained during the project show that overexpression of APS reductase in major European crops like maize or potato plants, does not lead to increased glutathione levels. These results are in contrast to results obtained from the model plant Arabidopsis.
Preliminary results from plants over-expressing methionine S-methyltransferase show in a few transgenic lines an increase in the content of S-methylmethionine, a major transport form of reduced sulfur in a number of plant species. Preliminary results as well as transgenic lines generated during the project are exchanged between partners. The transgenic plants mentioned above are being combined with additional traits, e.g. plants with increased sink strength for sulfur containing amino acids.
In the current state of progress, the future industrial applicability and the commercial value of these transgenic lines cannot yet be estimated. Results obtained contribute to a better understanding of the field and will be made publicly available by publications in scientific journals so that the scientific community targeting plant sulfur metabolism will benefit from this project.
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