Enhanced levels of methionine and cysteine in transgenic alfalfa (Medicago sativa L.) plants over-expressing the Arabidopsis cystathionine gamma-synthase gene

With the aim of increasing the methionine level in alfalfa (Medicago sativa L.) and thus improving its nutritional quality, we produced transgenic alfalfa plants that expressed the Arabidopsis cystathionine gamma-synthase (AtCGS), the enzyme that controls the synthesis of the first intermediate metabolite in the methionine pathway. The AtCGS cDNA was driven by the Arabidopsis rubisco small subunit promoter to obtain expression in leaves. Thirty transgenic plants were examined for the transgene protein expression, and four lines with a high expression level were selected for further work. In these lines, the contents of methionine, S-methylmethionine (SMM), and methionine incorporated into the water-soluble protein fraction increased up to 32-fold, 19-fold, and 2.2-fold, respectively, compared with that in wild-type plants. Notably, in these four transgenic lines, the levels of free cysteine (the sulphur donor for methionine synthesis), glutathione (the cysteine storage and transport form), and protein-bound cysteine increased up to 2.6-fold, 5.5-fold, and 2.3-fold, respectively, relative to that in wild-type plants. As the transgenic alfalfa plants over-expressing AtCGS had significantly higher levels of both soluble and protein-bound methionine and cysteine, they may represent a model and target system for improving the nutritional quality of forage crops.     

The N-terminal region of Arabidopsis cystathionine gamma-synthase plays an important regulatory role in methionine metabolism

Cystathionine gamma-synthase (CGS) is a key enzyme of Met biosynthesis in bacteria and plants. Aligning the amino acid sequences revealed that the plant enzyme has an extended N-terminal region that is not found in the bacterial enzyme. However, this region is not essential for the catalytic activity of this enzyme, as deduced from the complementation test of an Escherichia coli CGS mutant. To determine the function of this N-terminal region, we overexpressed full-length Arabidopsis CGS and its truncated version that lacks the N-terminal region in transgenic tobacco (Nicotiana tabacum) plants. Transgenic plants expressing both types of CGS had a significant higher level of Met, S-methyl-Met, and Met content in their proteins. However, although plants expressing full-length CGS showed the same phenotype and developmental pattern as wild-type plants, those expressing the truncated CGS showed a severely abnormal phenotype. These abnormal plants also emitted high levels of Met catabolic products, dimethyl sulfide and carbon disulfide. The level of ethylene, the Met-derived hormone, was 40 times higher than in wild-type plants. Since the alien CGS was expressed at comparable levels in both types of transgenic plants, we further suggest that post-translational modification(s) occurs in this N-terminal region, which regulate CGS and/or Met metabolism. More specifically, since the absence of the N-terminal region leads to an impaired Met metabolism, the results further suggest that this region plays a role in protecting plants from a high level of Met catabolic products such as ethylene.     

Increased sucrose level and altered nitrogen metabolism in Arabidopsis thaliana transgenic plants expressing antisense chloroplastic fructose-1,6-bisphosphatase

The pea chloroplastic fructose-1,6-bisphosphatase (FBPase) antisense construct reduced the endogenous level of expression of the corresponding Arabidopsis thaliana gene. The reduction of foliar FBPase activity in the transformants T(2) and T(3) generation ranged from 20% to 42%, and correlated with lower levels of FBPase protein. FBPase antisense plants displayed different phenotypes with a clear increase in leaf fresh weight. Measurements of photosynthesis revealed a higher carbon-assimilation rate. Decreased FBPase activity boosted the foliar carbohydrate contents, with a shift in the sucrose:starch ratio, which reached a maximum of 0.99 when the activity loss was 41%. Nitrate reductase activity decreased simultaneously with an increase in glutamine synthetase activity, which could be explained in terms of ammonium assimilation regulation by sugar content. These results suggest the role of FBPase as a key enzyme in CO(2) assimilation, and also in co-ordinating carbon and nitrogen metabolism.     

Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A

Transgenic rice (Oryza sativa) overexpressing Arabidopsis phytochrome A (phyA) was cultivated up to the T₃ generation in paddy to elucidate the role of phyA in determining the plant architecture and the productivity of sunlight-grown rice plants. PhyA is light-labile and controls plant growth in response to the far-red light-dependent high-irradiance response as well as the very low fluence response. The Arabidopsis phyA gene linked to the rice rbcS promoter was transformed into embryogenic rice calli, and the calli were regenerated to whole plants. Compared to wild-type seedlings, the rbcS::PHYA transgenic seedlings contained more phyA when grown in the dark, and at least 10-fold more phyA when exposed to white light. When grown in paddy, the phyA transgenic plants in general exhibited reduced plant height (dwarfing), larger grain size, higher chlorophyll content, smaller tiller number, and low grain fertility compared to wild-type plants. The heading stage was not significantly changed. However, it is likely that a certain level of phyA is a prerequisite for induction of such changes. It is suggested that phyA overproduction in rice could be a useful tool to improve rice grain productivity by the larger grain size that increases grain yield and the dwarfing that tolerates lodging-associated damage.     

Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

In vivo analysis of various substrates utilized by cystathionine gamma-synthase and O-acetylhomoserine sulfhydrylase in methionine biosynthesis

To gain insight into the evolution of the methionine biosynthesis pathway, in vivo complementation tests were performed. The substrate specificity of three enzymes that intrinsically use different homoserine-esterified substrates and have different sulfur assimilation pathways was examined: two cystathionine gamma-synthases (the Escherichia coli enzyme that naturally utilizes O-succinylhomoserine [OSH]) and the Arabidopsis thaliana enzyme that naturally exploits O-phosphohomoserine [OPH]. Both of these act through the transsulfuration pathway. The third enzyme investigated was O-acetylhomoserine (OAH) sulfhydrylase of Leptospira meyeri, representing the enzyme that utilizes OAH and operates through the direct sulfhydrylation pathway. All the three enzymes were able to utilize OSH and OAH as substrates, with different degrees of efficiency, but only the plant enzyme was able to utilize OPH as a substrate. In addition to their inherent activity in the transsulfuration pathway, the two cystathionine gamma-synthases were also capable of acting in the direct sulfhydrylation pathway. Based on the phylogenic tree and the results of the complementation tests, we suggest that the ancestral gene was able to act as OAH or OSH sulfhydrylase. In some bacteria and plants, this ancient enzyme most probably evolved into a cystathionine gamma-synthase, thereby maintaining the ability to utilize various homoserine-esterified substrates, as well as various sulfur sources, and thus keeping the multisubstrate specificity of its ancestor. In some organisms, this ancestral gene probably underwent a duplication event, which resulted in a cystathionine gamma-synthase and a separate OAH or OSH sulfhydrylase. This led to the development of two parallel pathways of methionine biosynthesis, transsulfuration and direct sulfhydrylation, in these organisms. Although both pathways exist in several organisms, most seem to favor a single specific pathway for methionine biosynthesis in vivo.     

The first exon coding region of cystathionine gamma-synthase gene is necessary and sufficient for downregulation of its own mRNA accumulation in transgenic Arabidopsis thaliana.

Expression of the gene for cystathionine gamma-synthase (CGS), which catalyzes the key step of methionine biosynthesis, is feedback regulated at the level of mRNA stability. The first exon polypeptide of CGS is suggested to be involved in this regulation and amino acid sequence alterations caused by mto1 mutations in that region lead to an overaccumulation of CGS mRNA [Chiba et al. (1999) Science 286: 1371-1374]. Transgenic Arabidopsis thaliana harboring chimeric constructs in which wild-type or mto1 mutant CGS exon 1 are fused in-frame to reporter genes and driven by the cauliflower mosaic virus 35S RNA promoter were constructed. Studies with these transgenic lines demonstrated that the coding region of CGS exon 1 is necessary and sufficient for downregulation of its own mRNA accumulation in response to methionine application and that this region acts in cis in this process.     

Regulation of one-carbon metabolism in Arabidopsis: the N-terminal regulatory domain of cystathionine gamma-synthase is cleaved in response to folate starvation

In all organisms, control of folate homeostasis is of vital importance to sustain the demand for one-carbon (C1) units that are essential in major metabolic pathways. In this study we induced folate deficiency in Arabidopsis (Arabidopsis thaliana) cells by using two antifolate inhibitors. This treatment triggered a rapid and important decrease in the pool of folates with significant modification in the distribution of C1-substituted folate coenzymes, suggesting an adaptive response to favor a preferential shuttling of the flux of C1 units to the synthesis of nucleotides over the synthesis of methionine (Met). Metabolic profiling of folate-deficient cells indicated important perturbation of the activated methyl cycle because of the impairment of Met synthases that are deprived of their substrate 5-methyl-tetrahydrofolate. Intriguingly, S-adenosyl-Met and Met pools declined during the initial period of folate starvation but were further restored to typical levels. Reestablishment of Met and S-adenosyl-Met homeostasis was concomitant with a previously unknown posttranslational modification that consists in the removal of 92 amino acids at the N terminus of cystathionine gamma-synthase (CGS), the first specific enzyme for Met synthesis. Rescue experiments and analysis of different stresses indicated that CGS processing is specifically associated with perturbation of the folates pool. Also, CGS processing involves chloroplastic serine-type proteases that are expressed in various plant species subjected to folate starvation. We suggest that a metabolic effector, to date unidentified, can modulate CGS activity in vivo through an interaction with the N-terminal domain of the enzyme and that removal of this domain can suppress this regulation.     

The creation of transgenic tobacco plants expressing fragments of the ARGOS and NtEXPA4 genes in antisense orientation

Transgenic tobacco plants expressing the fragments of the ARGOS and NtEXPA4 genes in antisense orientation have been created. Eleven lines of transgenic plants were investigated and five of them were characterized by a decrease in the sizes of the leaves and flowers as compared to control. Stem sizes decreased when only the NtEXPA4 gene fragment was used. The organ size of the experimental plants decreased because of a reduction in the level of both cell division and cell expansion. Two lines of transgenic tobacco plants expressing the part of the ARGOS gene in antisense orientation were characterized by a reduction in the level of the NtEXPA1 and NtEXPA4 gene expression.     

Over-expression of a cDNA for human ornithine decarboxylase in transgenic rice plants alters the polyamine pool in a tissue-specific manner

We investigated how over-expression of a cDNA for human ornithine decarboxylase (odc) affects the polyamine pools in transgenic rice. We further investigated tissue-specific expression patterns and product accumulation levels of the transgene driven by either constitutive or seed-specific promoters. Our results indicate that: (1) whereas the expression of a heterologous arginine decarboxylase (adc) cDNA in rice resulted in increased putrescine and spermine levels only in seeds, plants engineered to express odc cDNA exhibited significant changes in the levels of all three major polyamines in seeds and also in vegetative tissues (leaves and roots); (2) there was no linear correlation between odc mRNA levels, ODC enzyme activity and polyamine accumulation, suggesting that control of the polyamine pathway in plants is more complex than in mammalian systems; (3) ODC activity and polyamine changes varied in different tissues, indicating that the pathway is regulated in a tissue-specific manner. Our results suggest that ODC rather than ADC is responsible for the regulation of putrescine synthesis in plants.