Collagen's 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.

Cystathionine ␥-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.The sulfur-containing amino acid Met is an important essential amino acid in animal nutrition. Apart from its role as a protein constituent and its central function in initiating mRNA translation, Met indirectly regulates a variety of cellular processes as the precursor of S-adenosyl-Met (SAM), the primary biological methyl group donor. SAM is also the precursor of plant metabolites such as ethylene, polyamines, vitamin B1, and the Fe-chelator mugineic acid (Anderson, 1990;Ma et al., 1995;Sun, 1998). In addition, Met also serves as a donor for secondary metabolites through S-methyl-Met (SMM; Mudd and Datko, 1990). As can be expected of its cellular importance, Met biosynthesis is subject to complex regulatory control whose mechanism is only now being gradually clarified. Two main elements of this complex regulation have recently been elucidated in plants. In the first, the Met level is controlled by competition between its first specific enzyme, cystathionine ␥-synthase (CGS), and Thr synthase, for their common substrate, O-phosphohomo-Ser. Evidence of this competition and its role in Met synthesis was recently obtained by analyzing a mto2-1 mutant of Arabidopsis. This mutant, in which the gene encoding Thr synthase is impaired, demonstrated a approximately 22-fold higher accumulation of soluble Met in rosette leaves than wild-type Arabidopsis (Bartlem et al., 2000).A second regulatory mechanism of Met synthesis in plants occurs at the level...

SummaryWith 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 γ -synthase (AtCGS), the enzyme that controls the synthesis of the first intermediate metabolite in the methionine pathway. The At CGS 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 biosynthesis pathways of the essential amino acids methionine and threonine diverge from O-phosphohomoserine, an intermediate metabolite in the aspartate family of amino acids. Thus, the enzymes cystathionine-gamma-synthase (CGS) in the methionine pathway and threonine synthase (TS), the last enzyme in the threonine pathway, compete for this common substrate. To study this branching point, we overexpressed TS in sense and antisense orientation in Arabidopsis plants with the aim to study its effect on the level of threonine but more importantly on the methionine content. Positive correlation was found not only between TS expression level and threonine content, but also between TS/threonine and CGS expression level. Plants expressing the sense orientation of TS showed a higher level of threonine, increased expression level of CGS, and a significantly higher level of S-methylmethionine, the transport form of methionine. By contrast, plants expressing the antisense form of TS showed lower levels of threonine and of CGS expression level. In these antisense plants, the methionine level increased up to 47-fold compared to wild-type plants. To study further the effect of threonine on CGS expression level, wild-type plants were irrigated with threonine and control plants were irrigated with methionine or water. While threonine increased the expression level of CGS but reduced that of TS, methionine reduced the expression level of CGS but increased that of TS. This data demonstrate that both methionine and threonine affect the two enzymes at the branching point, thus controlling not only their own level, but also the level of each other. This mechanism probably aids in keeping the levels of these two essential amino acids sufficiently high to support plant growth.

With the general aim of elevating the content of the essential amino acid methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a beta-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether soluble methionine content limited the accumulation of the 15 kDa zein::3HA, methionine was first added to the growth medium of the different transgenic plants and the level of the alien protein was determined. Results demonstrated that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco plants. Next, the endogenous levels of methionine were elevated in the transgenic tobacco and alfalfa plants by crossing them with plants expressing the Arabidopsis cystathionine gamma-synthase (AtCGS) having significantly higher levels of soluble methionine in their leaves. Compared with plants expressing only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes showed significantly enhanced levels of this protein concurrently with a reduction in the soluble methionine content, thus implying that soluble methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also occurred in tobacco, but were considerably less pronounced. The results demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a species-specific manner and that soluble methionine plays a major role in the accumulation of the 15 kDa zein in some plant species but less so in others.