Frédéric Marsolais

A single‐repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean

Here we demonstrate that GmMYB176 regulates CHS8 expression and affects isoflavonoid synthesis in soybean. We previously established that CHS8 expression determines the isoflavonoid level in soybean seeds by comparing the transcript profiles of cultivars with different isoflavonoid contents. In the present study, a functional genomic approach was used to identify the factor that regulates CHS8 expression and isoflavonoid synthesis. Candidate genes were cloned, and co-transfection assays were performed in Arabidopsis leaf protoplasts. The results showed that GmMYB176 can trans-activate the CHS8 promoter with maximum activity. Transient expression of GmMYB176 in soybean embryo protoplasts increased endogenous CHS8 transcript levels up to 169-fold after 48 h. GmMYB176 encodes an R1 MYB protein, and is expressed in soybean seed during maturation. Furthermore, GmMYB176 recognizes a 23 bp motif containing a TAGT(T/A)(A/T) sequence within the CHS8 promoter. A subcellular localization study confirmed nuclear localization of GmMYB176. A predicted pST binding site for 14-3-3 protein is required for subcellular localization of GmMYB176. RNAi silencing of GmMYB176 in hairy roots resulted in reduced levels of isoflavonoids, showing that GmMYB176 is necessary for isoflavonoid biosynthesis. However, over-expression of GmMYB176 was not sufficient to increase CHS8 transcript and isoflavonoid levels in hairy roots. We conclude that an R1 MYB transcription factor, GmMYB176, regulates CHS8 expression and isoflavonoid synthesis in soybean.

Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway

A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed.

Biosynthesis of amphetamine analogs in plants

Amphetamine analogs are produced by plants in the genus Ephedra and by Catha edulis, and include the widely used decongestants and appetite suppressants pseudoephedrine and ephedrine. A combination of yeast (Candida utilis or Saccharomyces cerevisiae) fermentation and subsequent chemical modification is used for the commercial production of these compounds. The availability of certain plant biosynthetic genes would facilitate the engineering of yeast strains capable of de novo pseudoephedrine and ephedrine biosynthesis. Chemical synthesis has yielded amphetamine analogs with myriad functional group substitutions and diverse pharmacological properties. The isolation of enzymes with the serendipitous capacity to accept novel substrates could allow the production of substituted amphetamines in synthetic biosystems. Here, we review the biology, biochemistry and biotechnological potential of amphetamine analogs in plants.

Transcriptome Profiling Identifies Candidate Genes Associated with the Accumulation of Distinct Sulfur γ-Glutamyl Dipeptides in Phaseolus vulgaris and Vigna mungo Seeds

Common bean (Phaseolus vulgaris) and black gram (Vigna mungo) accumulate γ-Glutamyl-S-methylcysteine and γ-Glutamyl-methionine in seed, respectively. Transcripts were profiled by 454 pyrosequencing data at a similar developmental stage coinciding with the beginning of the accumulation of these metabolites. Expressed sequence tags were assembled into Unigenes, which were assigned to specific genes in the early release chromosomal assembly of the P. vulgaris genome. Genes involved in multiple sulfur metabolic processes were expressed in both species. Expression of Sultr3 members was predominant in P. vulgaris, whereas expression of Sultr5 members predominated in V. mungo. Expression of the cytosolic SERAT1;1 and -1;2 was approximately fourfold higher in P. vulgaris while expression of the plastidic SERAT2;1 was twofold higher in V. mungo. Among BSAS family members, BSAS4;1, encoding a cytosolic cysteine desulfhydrase, and BSAS1;1, encoding a cytosolic O-acetylserine sulphydrylase were most highly expressed in both species. This was followed by BSAS3;1 encoding a plastidic β-cyanoalanine synthase which was more highly expressed by 10-fold in P. vulgaris. The data identify BSAS3;1 as a candidate enzyme for the biosynthesis of S-methylcysteine through the use of methanethiol as substrate instead of cyanide. Expression of GLC1 would provide a complete sequence leading to the biosynthesis of γ-Glutamyl-S-methylcysteine in plastids. The detection of S-methylhomoglutathione in P. vulgaris suggested that homoglutathione synthetase may accept, to some extent, γ-Glutamyl-S-methylcysteine as substrate, which might lead to the formation of S-methylated phytochelatins. In conclusion, 454 sequencing was effective at revealing differences in the expression of sulfur metabolic genes, providing information on candidate genes for the biosynthesis of distinct sulfur amino acid γ-Glutamyl dipeptides between P. vulgaris and V. mungo.

Higher endogenous methionine in transgenic Arabidopsis seeds affects the composition of storage proteins and lipids

Previous in vitro studies demonstrate that exogenous application of the sulfur-containing amino acid methionine into cultured soybean cotyledons and seedlings reduces the level of methionine-poor storage proteins and elevates those that are methionine-rich. However, the effect of higher endogenous methionine in seeds on the composition of storage products in vivo is not studied yet. We have recently produced transgenic Arabidopsis seeds having significantly higher levels of methionine. In the present work we used these seeds as a model system and profiled them for changes in the abundances of 12S-globulins and 2S-albumins, the two major groups of storage proteins, using 2D-gels and MALDI-MS detection. The findings suggest that higher methionine affects from a certain threshold the accumulation of several subunits of 12S-globulins and 2S-albumins, regardless of their methionine contents, resulting in higher total protein contents. The mRNA abundances of most of the genes encoding these proteins were either correlated or not correlated with the abundances of these proteins, implying that methionine may regulate storage proteins at both transcriptional and post-transcriptional levels. The elevations in total protein contents resulted in reduction of total lipids and altered the fatty acid composition. Altogether, the data provide new insights into the regulatory roles of elevated methionine levels on seed composition.

Physicochemical characterization of a navy bean (Phaseolus vulgaris) protein fraction produced using a solvent-free method

A solvent-free electrostatic separation method was employed to separate navy bean flour (NBF) into protein-rich (PR) and starch-rich (SR) fractions. The physicochemical properties of NBF and separated fractions were compared to proteins (navy bean isolate (NBI) and 7S globulin) prepared using a wet process. Gel electrophoresis confirmed that the protein distribution in the isolated fractions was similar to that of NBF. The protein profile of NBI and 7S globulin was found to be devoid of certain proteins that were found in the NBF and PR fraction. Amino acid analysis revealed that the NBI and 7S globulin had a lower content of sulfur-containing amino acids compared to NBF and the electrostatically isolated fractions. CD and fluorescence spectroscopy confirmed that denaturation of the proteins during the acid precipitation is likely. This novel solvent-free electrostatic separation process preserves the native protein structure found in NBF and improves the recovery of some of the smaller MW proteins.

Comparison of Gene Families: Seed Storage and Other Seed Proteins

Common bean (Phaseolus vulgaris) is an important source of protein and dietary fiber in human diets. Seed proteins, therefore, determine, at least in part, the nutritional value of common bean. From the very beginning of plant molecular biology, in the 1980s, common bean has been a prominent model plant to study seed storage proteins. The recent availability of several sequences for the common bean genome, coupled with seed transcriptomic and proteomic information, enables a comprehensive, in-depth view of seed protein genes in this organism. Comparisons between these sequences highlight interesting variation in lectin gene composition between the two centers of domestication. Alleles conferring storage protein deficiency may be used to improve the levels of essential sulfur amino acids and therefore protein quality. Some of the seed proteins represent anti-nutritionals, including some lectins, trypsin inhibitors, and lipoxygenases, and represent targets to be potentially removed from the genome. Other proteins have potential as bioproducts due to their biological activity against fungi or insects, including defensin D1 and albumin-1.

Determining Sulfur-Limiting Conditions for Studies of Seed Composition in Common Bean ( Phaseolus vulgaris )

Soil fertility and mineral nutrition play an important role in crop yield and grain quality improvement. Two common bean genotypes, SARC1 and SMARC1N-PN1, differ markedly in seed composition. The lack of the 7S globulin phaseolin and major lectins in SMARC1N-PN1 is compensated by increased levels of several sulfur-rich proteins, including the 11S globulin legumin. Conditions were determined that are limiting sulfur nutrition only at the reproductive stage. These conditions will be used to investigate whether the two genotypes respond differently to sulfur nutrition.

Transcriptomics of Legume Seed: Soybean a Model Grain Legume

Grain legumes are the crop plants belonging to family Leguminosae that are cultivated for their seed yield for the purpose of human food or animal feed. They possess high metabolic activity and fluxes in seeds and are an important source of protein in vegetarian diet or when the intake from animal or fish source is not available. Soybean (Glycine max) L. Merr. is the world’s most widely grown grain legume. In addition to a high seed protein content (approximately 40 %), soybean seeds are also a major source of vegetable oil (approximately 20 %) and many beneficial plant natural compounds, such as isoflavonoids. Recently, soybean has gained considerable attention as a major crop for biodiesel production. It has a complex genome with a large genome size (~ 1115 Mb) that has undergone at least two genome duplication events within the last 60 million years (Gill et al. Plant Physiol, 151: 1167–1174, 2009; Schlueter et al. Genome, 47: 868–876, 2004; Shoemaker et al. Genetics, 144: 329–338,1996). The research efforts in the past decades in soybean in the area of genome mapping, molecular breeding, genomics, and whole genome sequencing have generated a vast amount of data and knowledge, providing unique opportunities to the legume community to conduct both basic and applied research in soybean and its close relatives.