RabA2b Overexpression Alters the Plasma-Membrane Proteome and Improves Drought Tolerance in Arabidopsis.

Rab proteins are small GTPases that are important in the regulation of vesicle trafficking. Through data mining, we identified RabA2b to be stress responsive, though little is known about the involvement of RabA in plant responses to abiotic stresses. Analysis of the RabA2b native promoter showed strong activity during osmotic stress, which required the stress hormone Abscisic acid (ABA) and was restricted to the vasculature. Sequence analysis of the promoter region identified predicted binding motifs for several ABA-responsive transcription factors. We cloned RabA2b and overexpressed it in Arabidopsis. The resulting transgenic plants were strikingly drought resistant. The reduced water loss observed in detached leaves of the transgenic plants could not be explained by stomatal aperture or density, which was similar in all the genotypes. Subcellular localization studies detected strong colocalization between RabA2b and the plasma membrane (PM) marker PIP2. Further studies of the PM showed, for the first time, a distinguished alteration in the PM proteome as a result of RabA2b overexpression. Proteomic analysis of isolated PM fractions showed enrichment of stress-coping proteins as well as cell wall/cuticle modifiers in the transgenic lines. Finally, the cuticle permeability of transgenic leaves was significantly reduced compared to the wild type, suggesting that it plays a role in its drought resistant properties. Overall, these data provide new insights into the roles and modes of action of RabA2b during water stresses, and indicate that increased RabA2b mediated PM trafficking can affect the PM proteome and increase drought tolerance.

Elucidating the role of shikimate dehydrogenase in controlling the production of anthocyanins and hydrolysable tannins in the outer peels of pomegranate.

BACKGROUND: The outer peels of pomegranate (Punica granatum L.) possess two groups of polyphenols that have health beneficial properties: anthocyanins (ATs, which also affect peel color); and hydrolysable tannins (HTs). Their biosynthesis intersects at 3-dehydroshikimate (3-DHS) in the shikimate pathway by the activity of shikimate dehydrogenase (SDH), which converts 3-DHS to shikimate (providing the precursor for AT biosynthesis) or to gallic acid (the precursor for HTs biosynthesis) using NADPH or NADP+ as a cofactor. The aim of this study is to gain more knowledge about the factors that regulate the levels of HTs and ATs, and the role of SDH. RESULTS: The results have shown that the levels of ATs and HTs are negatively correlated in the outer fruit peels of 33 pomegranate accessions, in the outer peels of two fruits exposed to sunlight, and in those covered by paper bags. When calli obtained from the outer fruit peel were subjected to light/dark treatment and osmotic stresses (imposed by different sucrose concentrations), it was shown that light with high sucrose promotes the synthesis of ATs, while dark at the same sucrose concentration promotes the synthesis of HTs. To verify the role of SDH, six PgSDHs (PgSDH1, PgSDH3-1,2, PgSDH3a-1,2 and PgSDH4) were identified in pomegranate. The expression of PgSDH1, which presumably contributes to shikimate biosynthesis, was relatively constant at different sucrose concentrations. However, the transcript levels of PgSDH3s and PgSDH4 increased with the accumulation of gallic acid and HTs under osmotic stress, which apparently accumulates to protect the cells from the stress. CONCLUSIONS: The results strongly suggest that the biosynthesis of HTs and ATs competes for the same substrate, 3-DHS, and that SDH activity is regulated not only by the NADPH/NADP+ ratio, but also by the expression of the PgSDHs. Since the outer peel affects the customer's decision regarding fruit consumption, such knowledge could be utilized for the development of new genetic markers for breeding pomegranates having higher levels of both ATs and HTs.

Transgenic tobacco plants having a higher level of methionine are more sensitive to oxidative stress.

Methionine is an essential amino acid the low level of which limits the nutritional quality of plants. We formerly produced transgenic tobacco (Nicotiana tabacum) plants overexpressing CYSTATHIONE γ-SYNTHASE (CGS) (FA plants), methionine's main regulatory enzyme. These plants accumulate significantly higher levels of methionine compared with wild-type (WT) plants. The aim of this study was to gain more knowledge about the effect of higher methionine content on the metabolic profile of vegetative tissue and on the morphological and physiological phenotypes. FA plants exhibit slightly reduced growth, and metabolic profiling analysis shows that they have higher contents of stress-related metabolites. Despite this, FA plants were more sensitive to short- and long-term oxidative stresses. In addition, compared with WT plants and transgenic plants expressing an empty vector, the primary metabolic profile of FA was altered less during oxidative stress. Based on morphological and metabolic phenotypes, we strongly proposed that FA plants having higher levels of methionine suffer from stress under non-stress conditions. This might be one of the reasons for their lesser ability to cope with oxidative stress when it appeared. The observation that their metabolic profiling is much less responsive to stress compared with control plants indicates that the delta changes in metabolite contents between non-stress and stress conditions is important for enabling the plants to cope with stress conditions.

Seed-specific expression of a feedback-insensitive form of CYSTATHIONINE-γ-SYNTHASE in Arabidopsis stimulates metabolic and transcriptomic responses associated with desiccation stress.

With an aim to elucidate novel metabolic and transcriptional interactions associated with methionine (Met) metabolism in seeds, we have produced transgenic Arabidopsis (Arabidopsis thaliana) seeds expressing a feedback-insensitive form of CYSTATHIONINE-γ-SYNTHASE, a key enzyme of Met synthesis. Metabolic profiling of these seeds revealed that, in addition to higher levels of Met, the levels of many other amino acids were elevated. The most pronounced changes were the higher levels of stress-related amino acids (isoleucine, leucine, valine, and proline), sugars, intermediates of the tricarboxylic acid cycle, and polyamines and lower levels of polyols, cysteine, and glutathione. These changes reflect stress responses and an altered mitochondrial energy metabolism. The transgenic seeds also had higher contents of total proteins and starch but lower water contents. In accordance with the metabolic profiles, microarray analysis identified a strong induction of genes involved in defense mechanisms against osmotic and drought conditions, including those mediated by the signaling cascades of ethylene and abscisic acid. These changes imply that stronger desiccation processes occur during seed development. The expression levels of transcripts controlling the levels of Met, sugars, and tricarboxylic acid cycle metabolites were also significantly elevated. Germination assays showed that the transgenic seeds had higher germination rates under salt and osmotic stresses and in the presence of ethylene substrate and abscisic acid. However, under oxidative conditions, the transgenic seeds displayed much lower germination rates. Altogether, the data provide new insights on the factors regulating Met metabolism in Arabidopsis seeds and on the mechanisms by which elevated Met levels affect seed composition and behavior.

Tobacco seeds expressing feedback-insensitive cystathionine gamma-synthase exhibit elevated content of methionine and altered primary metabolic profile.

BACKGROUND: The essential sulfur-containing amino acid methionine plays a vital role in plant metabolism and human nutrition. In this study, we aimed to elucidate the regulatory role of the first committed enzyme in the methionine biosynthesis pathway, cystathionine γ-synthase (CGS), on methionine accumulation in tobacco seeds. We also studied the effect of this manipulation on the seed's metabolism. RESULTS: Two forms of Arabidopsis CGS (AtCGS) were expressed under the control of the seeds-specific promoter of legumin B4: feedback-sensitive F-AtCGS (LF seeds), and feedback-insensitive T-AtCGS (LT seeds). Unexpectedly, the soluble content of methionine was reduced significantly in both sets of transgenic seeds. Amino acids analysis and feeding experiments indicated that although the level of methionine was reduced, the flux through its synthesis had increased. As a result, the level of protein-incorporated methionine had increased significantly in LT seeds by up to 60%, but this was not observed in LF seeds, whose methionine content is tightly regulated. This increase was accompanied by a higher content of other protein-incorporated amino acids, which led to 27% protein content in the seeds although this was statistically insignificantly. In addition, the levels of reducing sugars (representing starch) were slightly but significantly reduced, while that of oil was insignificantly reduced. To assess the impact of the high expression level of T-AtCGS in seeds on other primary metabolites, metabolic profiling using GC-MS was performed. This revealed significant alterations to the primary seed metabolism manifested by a significant increase in eight annotated metabolites (mostly sugars and their oxidized derivatives), while the levels of 12 other metabolites were reduced significantly in LT compared to wild-type seeds. CONCLUSION: Expression of T-AtCGS leads to an increase in the level of total Met, higher contents of total amino acids, and significant changes in the levels of 20 annotated metabolites. The high level of oxidized metabolites, the two stress-associated amino acids, proline and serine, and low level of glutathione suggest oxidative stress that occurs during LT seed development. This study provides information on the metabolic consequence of increased CGS activity in seeds and how it affects the seed's nutritional quality.

Light and sucrose up-regulate the expression level of Arabidopsis cystathionine γ-synthase, the key enzyme of methionine biosynthesis pathway.

The sulfur-containing essential amino acid methionine controls the level of important metabolites and processes in plants. In addition, methionine levels limit the nutritional quality of many crop plants. The level of methionine is regulated mainly by cystathionine γ-synthase (CGS), the first enzyme committed to its biosynthesis. Within our efforts to reveal factors that regulate CGS and methionine content in plants, we have analyzed how light regulates the transcript and protein level of Arabidopsis CGS (AtCGS). The expression of AtCGS is up-regulated in the light and reduced in the dark, independent of the diurnal cycle. Using tobacco plants overexpressing AtCGS, we have found that the light sensitive motives of the AtCGS gene are found within the coding sequence of AtCGS and not in its promoter, terminator or the untranslated regions of the gene. Sucrose can partially mimic the effect of light in dark grown plants while the addition of nitrogen and sulfur sources does not have any effect. The kinetics of the changes in the expression level of AtCGS suggest that its level can be maintained during extended darkness, or even when the sucrose content is reduced, such as during abiotic stresses. The up-regulation of AtCGS by light is in agreement with previous studies showing that other enzymes regulating the level of the carbon/amino skeleton and the sulfur group of Met, are up-regulated by light. The results indicate that light and dark participate in the regulation of the carbon/amino skeleton flux in the synthesis of amino acids of the aspartate family.

Soybean seeds expressing feedback-insensitive cystathionine γ-synthase exhibit a higher content of methionine.

Soybean seeds provide an excellent source of protein for human and livestock nutrition. However, their nutritional quality is hampered by a low concentration of the essential sulfur amino acid, methionine (Met). In order to study factors that regulate Met synthesis in soybean seeds, this study used the Met-insensitive form of Arabidopsis cystathionine γ-synthase (AtD-CGS), which is the first committed enzyme of Met biosynthesis. This gene was expressed under the control of a seed-specific promoter, legumin B4, and used to transform the soybean cultivar Zigongdongdou (ZD). In three transgenic lines that exhibited the highest expression level of AtD-CGS, the level of soluble Met increased significantly in developing green seeds (3.8-7-fold). These seeds also showed high levels of other amino acids. This phenomenon was more prominent in two transgenic lines, ZD24 and ZD91. The total Met content, which including Met incorporated into proteins, significantly increased in the mature dry seeds of these two transgenic lines by 1.8- and 2.3-fold, respectively. This elevation was accompanied by a higher content of other protein-incorporated amino acids, which led to significantly higher total protein content in the seeds of these two lines. However, in a third transgenic line, ZD01, the level of total Met and the level of other amino acids did not increase significantly in the mature dry seeds. This line also showed no significant change in protein levels. This suggests a positive connection between high Met content and the synthesis of other amino acids that enable the synthesis of more seed proteins.

Overexpression of mutated forms of aspartate kinase and cystathionine gamma-synthase in tobacco leaves resulted in the high accumulation of methionine and threonine.

Methionine and threonine are two essential amino acids, the levels of which limit the nutritional quality of plants. Both amino acids diverge from the same branch of the aspartate family biosynthesis pathway; therefore, their biosynthesis pathways compete for the same carbon/amino substrate. To further elucidate the regulation of methionine biosynthesis and seek ways of increasing the levels of these two amino acids, we crossed transgenic tobacco plants overexpressing the bacterial feedback-insensitive aspartate kinase (bAK), containing a significantly higher threonine level, with plants overexpressing Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Plants co-expressing bAK and the full-length AtCGS (F-AtCGS) have significantly higher methionine and threonine levels compared with the levels found in wild-type plants, but the methionine level does not increase beyond that found in plants expressing F-AtCGS alone. This finding can be explained through the feedback inhibition regulation mediated by the methionine metabolite on the transcript level of AtCGS. To test this assumption, plants expressing bAK were crossed with plants expressing two mutated forms of AtCGS in which the domains responsible for the feedback regulation have been deleted. Indeed, significantly higher methionine contents and its metabolites levels accumulated in the newly produced plants, and the levels of threonine were also significantly higher than in the wild-type plants. The transcript level of the two mutated forms of AtCGS significantly increased when there was a high content of threonine in the plants, suggesting that threonine modulates, probably indirectly, the transcript level of AtCGS.