Rachel Amir

The N-Terminal Region of Arabidopsis Cystathionine γ-Synthase Plays an Important Regulatory Role in Methionine Metabolism

Cystathionine γ-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.

Cystathionine γ-synthase and threonine synthase operate in concert to regulate carbon flow towards methionine in plants

The sulfur-containing amino acid methionine is a nutritionally important essential amino acid and is the precursor of several metabolites that regulate plant growth and responses to the environment. Methionine production is largely regulated by cystathionine gamma-synthase, the first specific enzyme for its synthesis. This enzyme competes in a complex manner with threonine synthase, the last enzyme in threonine biosynthesis, for their common substrate O-phosphohomoserine. New genetic and molecular data suggest that methionine synthesis and catabolism are coordinately regulated by novel post-transcriptional and post-translational mechanisms that are associated with a regulatory part within the N-terminal part of cystathionine gamma-synthase.

Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality

Humans, as well as farm animals, cannot synthesize a number of essential amino acids, which are critical for their survival. Hence, these organisms must obtain these essential amino acids from their diets. Cereal and legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limiting levels of some of these essential amino acids, particularly Lys and Met. Extensive efforts were made to fortify crop plants with these essential amino acids using traditional breeding and mutagenesis. However, aside from some results obtained with maize, none of these approaches was successful. Therefore, additional efforts using genetic engineering approaches concentrated on increasing the synthesis and reducing the catabolism of these essential amino acids and also on the expression of recombinant proteins enriched in them. In the present review, we discuss the basic biological aspects associated with the synthesis and accumulation of these amino acids in plants and also describe recent developments associated with the fortification of crop plants with essential amino acids by genetic engineering approaches.

Improving the levels of essential amino acids and sulfur metabolites in plants

Abstract Plants represent the major source of food for humans, either directly or indirectly through their use as livestock feeds. Plant foods are not nutritionally balanced because they contain low proportions of a number of essential metabolites, such as vitamins and amino acids, which humans and a significant proportion of their livestock cannot produce on their own. Among the essential amino acids needed in human diets, Lys, Met, Thr and Trp are considered as the most important because they are present in only low levels in plant foods. In the present review, we discuss approaches to improve the levels of the essential amino acids Lys and Met, as well as of sulfur metabolites, in plants using metabolic engineering approaches. We also focus on specific examples for which a deeper understanding of the regulation of metabolic networks in plants is needed for tailor-made improvements of amino acid metabolism with minimal interference in plant growth and productivity.

Environmental Conditions Affect the Color, Taste, and Antioxidant Capacity of 11 Pomegranate Accessions' Fruits

The well-established health beneficial value of pomegranate juice is leading to increased demand for pomegranate products and to the expansion of pomegranate orchards worldwide. The current study describes differences in the chemical composition of major ingredients of the arils and peels of 11 accessions grown in Mediterranean and desert climates in Israel. In most of the accessions, the levels of antioxidant activity and content of total phenolics, total anthocyanins, total soluble solids, glucose, fructose, and acidity were higher in the aril juice of fruit grown in the Mediterranean climate compared to those grown in the desert climate. However, the peels of fruit grown in the desert climate exhibited higher antioxidant activity, and the levels of total phenolics, including the two hydrolyzable tannins, punicalagin and punicalin, were higher compared to those in the peels of fruit grown in the Mediterranean climate. The results indicate that environmental conditions significantly affect pomegranate fruit quality and health beneficial compounds.

Production of Bioactive, Post-Translationally Modified, Heterotrimeric, Human Recombinant Type-I Collagen in Transgenic Tobacco †

Collagens biocompatibility, biodegradability and low immunogenicity render it advantageous for extensive application in pharmaceutical or biotechnological disciplines. However, typical collagen extraction from animal or cadaver sources harbors risks including allergenicity and potential sample contamination with pathogens. In this work, two human genes encoding recombinant heterotrimeric collagen type I (rhCOL1) were successfully coexpressed in tobacco plants with the human prolyl-4-hydroxylase (P4H) and lysyl hydroxylase 3 (LH3) enzymes, responsible for key posttranslational modifications of collagen. Plants coexpressing all five vacuole-targeted proteins generated intact procollagen yields of approximately 2% of the extracted total soluble proteins. Plant-extracted rhCOL1 formed thermally stable triple helical structures and demonstrated biofunctionality similar to human tissue-derived collagen supporting binding and proliferation of adult peripheral blood-derived endothelial progenitor-like cells. Through a simple, safe and scalable method of rhCOL1 production and purification from tobacco plants, this work broadens the potential applications of human recombinant collagen in regenerative medicine.

Overexpression of mutated forms of aspartate kinase and cystathionine γ-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.

Current understanding of the factors regulating methionine content in vegetative tissues of higher plants.

Methionine is a nutritionally essential, sulfur-containing amino acid found in low levels in plants, which often limits its value as a source of dietary protein to humans and animals. Methionine is also a fundamental metabolite in plant cells since, through its first metabolite, S-adenosylmethionine (SAM), it controls the level of several key metabolites, such as ethylene, polyamines and biotin. SAM is also the primary methyl group donor that regulates different processes in plants. Despite its nutritional and regulatory significance, the factors regulating methionine content in plants are not fully known. In this review, we summarize the current knowledge and recent progress made in our understanding of the methionine metabolism. The enzymes and substrates that regulate methionine synthesis were described, as well as the influences of the catabolic pathways of methionine on its content. The current effort to tailor an improvement of methionine content in vegetative tissues with minimal interference in plant growth and productivity is described as well. The accumulated knowledge has provided new insights into the control of methionine level in plants and, in some cases, has resulted in significant improvements in the nutritional value of plants.

Color, sugars and organic acids composition in aril juices and peel homogenates prepared from different pomegranate accessions.

The current study describes differences in pomegranate fruit size and aril weight of 29 accessions grown in Israel. The contents of sugars and organic acids in their aril juices and peel homogenates, as well as color parameters, were determined. While the levels of total soluble solids (TSS) and soluble sugars in the aril juices differ only slightly, those of titratable acidity (TA) and citric acid changed significantly, suggesting that they are the main contributors to juice taste. In general, significant positive correlations were found between TA values and the red color parameters, and these two parameters, as well as TSS, appeared to be higher in the juices of accessions harvested late in the season. Peel homogenates exhibited lower levels of TSS, TA, soluble sugars and organic acids than aril juices. Some red color parameters, TA and citric acid were found to correlate significantly between the aril juices and peel homogenates.

The Regulation of Essential Amino Acid Synthesis and Accumulation in Plants

Although amino acids are critical for all forms of life, only proteogenic amino acids that humans and animals cannot synthesize de novo and therefore must acquire in their diets are classified as essential. Nine amino acids-lysine, methionine, threonine, phenylalanine, tryptophan, valine, isoleucine, leucine, and histidine-fit this definition. Despite their nutritional importance, several of these amino acids are present in limiting quantities in many of the worlds major crops. In recent years, a combination of reverse genetic and biochemical approaches has been used to define the genes encoding the enzymes responsible for synthesizing, degrading, and regulating these amino acids. In this review, we describe recent advances in our understanding of the metabolism of the essential amino acids, discuss approaches for enhancing their levels in plants, and appraise efforts toward their biofortification in crop plants.