Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins

<p>Abstract</p> <p>Background</p> <p>A deficiency in phaseolin and phytohemagglutinin is associated with a near doubling of sulfur amino acid content in genetically related lines of common bean (<it>Phaseolus vulgaris</it>), particularly cysteine, elevated by 70%, and methionine, elevated by 10%. This mostly takes place at the expense of an abundant non-protein amino acid, <it>S</it>-methyl-cysteine. The deficiency in phaseolin and phytohemagglutinin is mainly compensated by increased levels of the 11S globulin legumin and residual lectins. Legumin, albumin-2, defensin and albumin-1 were previously identified as contributing to the increased sulfur amino acid content in the mutant line, on the basis of similarity to proteins from other legumes.</p> <p>Results</p> <p>Profiling of free amino acid in developing seeds of the BAT93 reference genotype revealed a biphasic accumulation of gamma-glutamyl-<it>S</it>-methyl-cysteine, the main soluble form of <it>S</it>-methyl-cysteine, with a lag phase occurring during storage protein accumulation. A collection of 30,147 expressed sequence tags (ESTs) was generated from four developmental stages, corresponding to distinct phases of gamma-glutamyl-<it>S</it>-methyl-cysteine accumulation, and covering the transitions to reserve accumulation and dessication. Analysis of gene ontology categories indicated the occurrence of multiple sulfur metabolic pathways, including all enzymatic activities responsible for sulfate assimilation, <it>de novo </it>cysteine and methionine biosynthesis. Integration of genomic and proteomic data enabled the identification and isolation of cDNAs coding for legumin, albumin-2, defensin D1 and albumin-1A and -B induced in the absence of phaseolin and phytohemagglutinin. Their deduced amino acid sequences have a higher content of cysteine than methionine, providing an explanation for the preferential increase of cysteine in the mutant line.</p> <p>Conclusion</p> <p>The EST collection provides a foundation to further investigate sulfur metabolism and the differential accumulation of sulfur amino acids in seed of common bean. Identification of sulfur-rich proteins whose levels are elevated in seed lacking phaseolin and phytohemagglutinin and sulfur metabolic genes may assist the improvement of protein quality.</p

Evaluation of beneficial and inhibitory effects of nitrate on nodulation and nitrogen fixation in common bean (Phaseolus vulgaris)

AbstractThe effects of applied nitrate on symbiotic nitrogen fixation in legumes are complex. Both inhibition and promotion of nodulation by nitrate have been observed in a dose‐dependent manner. The objectives of this study were to determine the effects of nitrate at different concentrations on root nodulation in different genotypes in common bean (Phaseolus vulgaris). Six genotypes were inoculated with the same rhizobial strain and grown hydroponically in growth pouches in a growth chamber and exposed to six nitrate concentrations, including 0, 2.5, 5, 10, 15, and 20 mM for 4 weeks. The tested genotypes included three recombinant inbred lines (RILs, 25, 46, and 70) that differed in their responses to nitrogen (based on observations of one field growing season), their parents (Mist and Sanilac—registered varieties), which are different in N‐fixing abilities, and one nonnodulating mutant (R99). Our results showed that small amounts of nitrate (2.5 and 5 mM) promoted nodule formation and increased nodule biomass, compared with plants in the 0 nitrate control treatment. In contrast, nitrate concentrations over 10 mM inhibited nodulation, resulting in reductions in nodule number and nodule biomass. Nodulation was completely inhibited by 15‐mM nitrate in all the genotypes. Regression analyses indicated that 5‐mM nitrate is the optimum concentration for promoting nodulation as measured by the total number of nodules formed, the number of effective nodules formed, and the nodule biomass formed. In contrast, nitrogen fixation was inhibited by all levels of nitrate. No genotypic differences were observed in nodulation among the three RILs and their parental cultivars, but all were significantly different than R99, a nonnodulating mutant

CRISPR/Cas9-mediated lipoxygenase gene-editing in yellow pea leads to major changes in fatty acid and flavor profiles

IntroductionAlthough pulses are nutritious foods containing high amounts of protein, fiber and phytochemicals, their consumption and use in the food industry have been limited due to the formation of unappealing flavors/aromas described as beany, green, and grassy. Lipoxygenase (LOX) enzymes are prevalent among pulse seeds, and their activity can lead to the formation of specific volatile organic compounds (VOCs) from certain polyunsaturated fatty acids (PUFAs). As a widespread issue in legumes, including soybean, these VOCs have been linked to certain unappealing taste perception of foods containing processed pulse seeds.MethodsTo address this problem in pea and as proof of principle to promote the wider use of pulses, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) construct was designed to create null alleles (knockouts) of PsLOX2 which had been implicated in the generation of VOCs in peas.Results and discussionSuccessful CRISPR/Cas9-mediated LOX gene editing of stable transgenic pea lines (TGP) was confirmed by DNA sequencing of the wild type (WT) and TGP pslox2 mutant lines. These lines were also assessed for LOX activity, PUFA levels, and VOCs. Compared to WT peas, the TGP lines showed a significant reduction (p &lt; 0.05) in LOX activity and in the concentration of key VOCs, including hexanal, 2-hexenal, heptanal, (E)-2-heptenal, (E,E)-2,4-heptadienal, 1-octen-3-ol, octanal, (E)-2-octenal (E,E)-2,4-nonadienal and furan-2-pentyl. The content of two essential PUFAs, linoleic and α-linolenic acids, the known substrates of LOX in plants, was higher in TGP flours, indicating the efficacy of the CRISPR-mediated gene editing in minimizing their oxidation and the further modification of PUFAs and their products. The collection of VOCs from the headspace of ground pea seeds, using a portable eNose also distinguished the TGP and WT lines. Multiple regression analysis showed that LOX activity correlated with the two VOCs, heptanal and (E,E)-2,4-heptadienal in pea flours. Partial Least Squares Regression (PLS-R) plot for selected PUFAs, VOCs, and sensor responses in WT and TGP lines showed distinct clusters for WT and TGP lines. Together this data demonstrates the utility of CRISPR mediated mutagenesis of PsLOX2 to quickly improve aroma and fatty acid (FA) profiles of pea seeds of an elite Canadian variety

Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids

Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98% of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified

Slow darkening of pinto bean seed coat is associated with significant metabolite and transcript differences related to proanthocyanidin biosynthesis

Abstract Background Postharvest seed coat darkening in pinto bean is an undesirable trait resulting in a loss in the economic value of the crop. The extent of darkening varies between the bean cultivars and their storage conditions. Results Metabolite analysis revealed that the majority of flavonoids including proanthocyanidin monomer catechin accumulated at higher level in a regular darkening (RD) pinto line CDC Pintium than in a slow darkening (SD) line 1533–15. A transcriptome analysis was conducted to compare gene expression between CDC Pintium and 1533–15 and identify the gene (s) that may play a role in slow darkening processes in 1533–15 pinto. RNAseq against total RNA from RD and SD cultivars found several phenylpropanoid genes, metabolite transporter genes and genes involved in gene regulation or modification to be differentially expressed between CDC Pintium and 1533–15. Conclusion RNAseq analysis and metabolite data of seed coat tissue from CDC Pintium and 1533–15 revealed that the whole proanthocyanidin biosynthetic pathway was downregulated in 1533–15. Additionally, genes that encode for putative transporter proteins were also downregulated in 1533–15 suggesting both synthesis and accumulation of proanthocyanidin is reduced in SD pintos

Common Bean (<i>Phaseolus vulgaris</i> L.) Accumulates Most <i>S</i>-Methylcysteine as Its γ-Glutamyl Dipeptide

The common bean (Phaseolus vulgaris) constitutes an excellent source of vegetable dietary protein. However, there are sub-optimal levels of the essential amino acids, methionine and cysteine. On the other hand, P. vulgaris accumulates large amounts of the &#947;-glutamyl dipeptide of S-methylcysteine, and lower levels of free S-methylcysteine and S-methylhomoglutathione. Past results suggest two distinct metabolite pools. Free S-methylcysteine levels are high at the beginning of seed development and decline at mid-maturation, while there is a biphasic accumulation of &#947;-glutamyl-S-methylcysteine, at early cotyledon and maturation stages. A possible model involves the formation of S-methylcysteine by cysteine synthase from O-acetylserine and methanethiol, whereas the majority of &#947;-glutamyl-S-methylcysteine may arise from S-methylhomoglutathione. Metabolite profiling during development and in genotypes differing in total S-methylcysteine accumulation showed that &#947;-glutamyl-S-methylcysteine accounts for most of the total S-methylcysteine in mature seed. Profiling of transcripts for candidate biosynthetic genes indicated that BSAS4;1 expression is correlated with both the developmental timing and levels of free S-methylcysteine accumulated, while homoglutathione synthetase (hGS) expression was correlated with the levels of &#947;-glutamyl-S-methylcysteine. Analysis of S-methylated phytochelatins by liquid chromatography and high resolution tandem mass spectrometry revealed only small amounts of homophytochelatin-2 with a single S-methylcysteine. The mitochondrial localization of phytochelatin synthase 2&#8212;predominant in seed, determined by confocal microscopy of a fusion with the yellow fluorescent protein&#8212;and its spatial separation from S-methylhomoglutathione may explain the lack of significant accumulation of S-methylated phytochelatins

Patterns of Genetic Variation in a Soybean Germplasm Collection as Characterized with Genotyping-by-Sequencing

Genomic characterization is playing an increasing role in plant germplasm conservation and utilization, as it can provide higher resolution with genome-wide SNP markers than before to identify and analyze genetic variation. A genotyping-by-sequencing technique was applied to genotype 541 soybean accessions conserved at Plant Gene Resources of Canada and 30 soybean cultivars and breeding lines developed by the Ottawa soybean breeding program of Agriculture and Agri-Food Canada. The sequencing generated an average of 952,074 raw sequence reads per sample. SNP calling identified 43,891 SNPs across 20 soybean chromosomes and 69 scaffolds with variable levels of missing values. Based on 19,898 SNPs with up to 50% missing values, three distinct genetic groups were found in the assayed samples. These groups were a mixture of the samples that originated from different countries and the samples of known maturity groups. The samples that originated from Canada were clustered into all three distinct groups, but 30 Ottawa breeding lines fell into two groups only. Based on the average pairwise dissimilarity estimates, 40 samples with the most genetic distinctness were identified from three genetic groups with diverse sample origin and known maturity. Additionally, 40 samples with the highest genetic redundancy were detected and they consisted of different sample origins and maturity groups, largely from one genetic group. Moreover, some genetically duplicated samples were identified, but the overall level of genetic duplication was relatively low in the collection. These findings are useful for soybean germplasm management and utilization

Characterization of a Cruciferin Deficient Mutant of Arabidopsis and Its Utility for Overexpression of Foreign Proteins in Plants

<div><p>Plant seeds naturally accumulate storage reserves (proteins, carbohydrates, lipids) that are mobilized during germination to provide energy and raw materials to support early seedling growth. Seeds have been exploited as bioreactors for the production to foreign materials, but stable, high level expression has been elusive, in part due to the intrinsic bias for producing the natural reserves in their typical proportions. To identify mutants governing seed filling, we screened a population of mutagenized Arabidopsis plants for a mutant that failed to fill its seeds. Here we report the identification of <i>ssp1</i>, a recessive, viable mutant that accumulates approximately 15% less protein than wildtype seeds. Molecular analyses revealed that <i>ssp1</i> is due to the introduction of a premature stop codon in <i>CRU3</i>, one of the major cruciferin genes. Unlike many other reserve mutants or transgenic lines in which seed storage protein levels are reduced by antisense/RNAi technologies, <i>ssp1</i> exhibits low level compensation by other reserves, and represents a mutant background that might prove useful for high level expression of foreign proteins. To test this hypothesis, we used a bean phytohemagglutinin (PHA) gene as a reporter and compared PHA expression levels in single copy insertion lines in <i>ssp1</i> vs. wildtype. These near isogenic lines allow reporter protein levels to be compared without the confounding and sometimes unknown influences of transgene copy number and position effects on gene expression. The <i>ssp1</i> lines consistently accumulated more PHA than the backcrossed counterparts, with increases ranging from 12% to 126%. This proof of principle study suggests that similar strategies in crop plants may improve the yield of foreign proteins of agronomic and economic interest.</p></div

Immunoblotting validates <i>ssp1</i> as an empty container.

<p>Duplicate one microgram aliquots of total seed protein from tester and backcross homozygotes were subjected to SDS-PAGE and immunoblotting with an anti-PHA antibody. Reporter lines 1, 3, 5, and 7 are shown in duplicate as testers (T) and the corresponding backcrosses (B). Aliquots of purified PHA were included as standards. The autoradiographs were scanned by densitometry and quantified by employing Image J software. The bar graphs represent the average PHA values relative to 30 ng of purified PHA. T-test statistical analyses revealed that lines 1, 3, and 5 have p-values less than 0.05, while the line 7 p-value was greater than 0.05. An independent experiment on the previous generation of these tester/backcross lines revealed similar enrichment trends and p-values.</p