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Abstract Background A deficiency in phaseolin and phytohemagglutinin is associated with a near doubling of sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris), particularly cysteine, elevated by 70%, and methionine, elevated by 10%. This mostly takes place at the expense of an abundant non-protein amino acid, S-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. Results Profiling of free amino acid in developing seeds of the BAT93 reference genotype revealed a biphasic accumulation of gamma-glutamyl-S-methyl-cysteine, the main soluble form of S-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-S-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, de novo 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. Conclusion 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.

Soybean improvement has entered a new era with the advent of multi-omics strategies and bioinformatics innovations, enabling more precise and efficient breeding practices. This comprehensive review examines the application of multi-omics approaches in soybean-encompassing genomics, transcriptomics, proteomics, metabolomics, epigenomics, and phenomics. We first explore pre-breeding and genomic selection as tools that have laid the groundwork for advanced trait improvement. Subsequently, we dig into the specific contributions of each -omics field, highlighting how bioinformatics tools and resources have facilitated the generation and integration of multifaceted data. The review emphasizes the power of integrating multi-omics datasets to elucidate complex traits and drive the development of superior soybean cultivars. Emerging trends, including novel computational techniques and high-throughput technologies, are discussed in the context of their potential to revolutionize soybean breeding. Finally, we address the challenges associated with multi-omics integration and propose future directions to overcome these hurdles, aiming to accelerate the pace of soybean improvement. This review serves as a crucial resource for researchers and breeders seeking to leverage multi-omics strategies for enhanced soybean productivity and resilience.

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans ( Phaseolus vulgaris ). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O -glycan within these peptides.

The capacity to leverage high resolution mass spectrometry (HRMS) with transient isotope labeling experiments is an untapped opportunity to derive insights on context-specific metabolism, that is difficult to assess quantitatively. Tools are needed to comprehensively mine isotopologue information in an automated, high-throughput way without errors. We describe a tool, Stable Isotope-assisted Metabolomics for Pathway Elucidation (SIMPEL), to simplify analysis and interpretation of isotope-enriched HRMS datasets. The efficacy of SIMPEL is demonstrated through examples of central carbon and lipid metabolism. In the first description, a dual-isotope labeling experiment is paired with SIMPEL and isotopically nonstationary metabolic flux analysis (INST-MFA) to resolve fluxes in central metabolism that would be otherwise challenging to quantify. In the second example, SIMPEL was paired with HRMS-based lipidomics data to describe lipid metabolism based on a single labeling experiment. Available as an R package, SIMPEL extends metabolomics analyses to include isotopologue signatures necessary to quantify metabolic flux., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: There is a common agreement that Alzheimers disease (AD) is inherently complex; otherwise, a general disagreement remains on its etiological underpinning, with numerous alternative hypotheses having been proposed., Objective: To perform a scoping review of original manuscripts describing hypotheses and theories of AD published in the past decades., Results: We reviewed 131 original manuscripts that fulfilled our inclusion criteria out of more than 13,807 references extracted from open databases. Each entry was characterized as having a single or multifactorial focus and assigned to one of 15 theoretical groupings. Impact was tracked using open citation tools., Results: Three stages can be discerned in terms of hypotheses generation, with three quarter of studies proposing a hypothesis characterized as being single-focus. The most important theoretical groupings were the Amyloid group, followed by Metabolism and Mitochondrial dysfunction, then Infections and Cerebrovascular. Lately, evidence towards Genetics and especially Gut/Brain interactions came to the fore., Conclusions: When viewed together, these multi-faceted reports reinforce the notion that AD affects multiple sub-cellular, cellular, anatomical, and physiological systems at the same time but at varying degree between individuals. The challenge of providing a comprehensive view of all systems and their interactions remains, alongside ways to manage this inherent complexity.

Introduction: Although 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., Methods: To 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 Discussion: Successful 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 < 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 His Majesty the King in Right of Canada.)

L-Asparaginase (EC 3.5.1.1) activity has been previously reported to fluctuate with the photoperiod in young pea leaves, with higher activity in the light. The present research sought to investigate this phenomenon in developing leaves of common bean (Phaseolus vulgaris L.). There are two genes coding for K + -dependent asparaginase in this species. Expression of PvASPG1 predominates over PvASPG2 in all tissues. The catalytic efficiency of recombinant PvASPG2 was approximately 2-fold lower than that of PvASPG1. Polyclonal antibodies were raised against a specific peptide present in PvASPG1 to use in immunoblotting. In developing seed, asparaginase protein levels in the seed coat stayed constant, whereas levels in cotyledon were lower and progressively declined. In young leaf, asparagine protein levels showed diurnal variation, increasing at the end of the dark period and slowly decreasing during the light period. This was paralleled by changes in activity levels in leaf extracts. These changes accompanied a transient increase in free asparagine concentration at the beginning of the light period. The present results demonstrated that K + -dependent asparaginase activity reaches a maximum level at the transition from dark to light, anticipating dawn, in young leaves of common bean., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Soybean ( Glycine max (L.) Merr.) is one of the important crops in Canada and has the potential to expand its production further north into the Canadian Prairies. Such expansion, however, requires the search for adapted soybean germplasm useful for the development of productive cultivars with earlier maturity and increased protein concentration. We initiated several research activities to characterize 848 accessions of the soybean collection conserved at Plant Gene Resources of Canada (PGRC) for maturity, oil and protein concentration, and genetic distinctness. The characterization revealed a wide range of variations present in each assessed trait among the PGRC soybean accessions. The trait variabilities allowed for the identification of four core subsets of 35 PGRC soybean accessions, each specifically targeted for early maturity for growing in Saskatoon and Ottawa, and for high oil and protein concentration. The two early maturity core subsets for Saskatoon and Ottawa displayed days to maturity ranging from 103 to 126 days and 94 to 102 days, respectively. The two core subsets for high oil and protein concentration showed the highest oil and protein concentration from 25.0 to 22.7% and from 52.8 to 46.7%, respectively. However, these core subsets did not differ significantly in genetic distinctness (as measured with 19,898 SNP markers across 20 soybean chromosomes) from the whole PGRC soybean collection. These findings are useful, particularly for the management and utilization of the conserved soybean germplasm., Competing Interests: The authors declare no conflict of interest.

In common bean ( Phaseolus vulgaris L.), postharvest seed coat darkening is an undesirable trait that affects crop value. The increased accumulation of proanthocyanidins (PAs) in the seed coat results in darker seeds in many market classes of colored beans after harvest. The precursors of PAs are synthesized in the cytoplasm, and subsequently get glycosylated and then transported to the vacuoles where polymerization occurs. Thus, vacuolar transporters play an important role in the accumulation of PAs. Here, we report that common bean genome contains 59 multidrug and toxic compound extrusion genes ( PvMATE s). Phylogenetic analysis of putative PvMATEs with functionally characterized MATEs from other plant species categorized them into substrate-specific clades. Our data demonstrate that a vacuolar transporter PvMATE8 is expressed at a higher level in the pinto bean cultivar CDC Pintium (regular darkening) compared to 1533-15 (slow darkening). PvMATE8 localizes in the vacuolar membrane and rescues the PA deficient ( tt12 ) mutant phenotype in Arabidopsis thaliana . Analysis of PA monomers in transgenic seeds together with wild-type and mutants suggests a possible feedback regulation of PA biosynthesis and accumulation. Identification of PvMATE8 will help better understand the mechanism of PA accumulation in common bean., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of Nishat S. Islam, Kishor Duwadi, Ling Chen, Aga Pajak, Tim McDowell, Frédéric Marsolais and Sangeeta Dhaubhadel.)

The glutamine amidotransferase gene GAT1&#95;2.1 is a marker of N status in Arabidopsis root, linked to a shoot branching phenotype. The protein has an N-terminal glutamine amidotransferase domain and a C-terminal extension with no recognizable protein domain. A purified, recombinant version of the glutamine amidotransferase domain was catalytically active as a glutaminase, with apparent K m value of 0.66 mM and V max value of 2.6 μkatal per mg. This form complemented an E. coli glutaminase mutant, ΔYneH. Spiking of root metabolite extracts with either the N-terminal or full length form purified from transformed tobacco leaves led to reciprocal changes in glutamine and ammonia concentration. No product derived from amido- 15 N-labeled glutamine was identified. Visualization of GAT1&#95;2.1-YPF transiently expressed in tobacco leaves confirmed its mitochondrial localization. gat1&#95;2.1 exhibited reduced growth as compared with wild-type seedlings on media with glutamine as sole nitrogen source. Results of targeted metabolite profiling pointed to a possible activation of the GABA shunt in the mutant following glutamine treatments, with reduced levels of glutamic acid, 2-oxoglutarate and γ-aminobutyric acid and increased levels of succinic acid. GAT1&#95;2.1 may act as a glutaminase, in concert with Glutamate Dehydrogenase 2, to hydrolyze glutamine and channel 2-oxoglutarate to the TCA cycle under high nitrogen conditions., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Background: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing., Results: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected., Conclusions: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement., (© 2021. The Author(s).)

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.

The endosperm provides nutrients and growth regulators to the embryo during seed development. LEAFY COTYLEDON1 (LEC1) has long been known to be essential for embryo maturation. LEC1 is expressed in both the embryo and the endosperm; however, the functional relevance of the endosperm-expressed LEC1 for seed development is unclear. Here, we provide genetic and transgenic evidence demonstrating that endosperm-expressed LEC1 is necessary and sufficient for embryo maturation. We show that endosperm-synthesized LEC1 is capable of orchestrating full seed maturation in the absence of embryo-expressed LEC1. Inversely, without LEC1 expression in the endosperm, embryo development arrests even in the presence of functional LEC1 alleles in the embryo. We further reveal that LEC1 expression in the endosperm begins at the zygote stage and the LEC1 protein is then trafficked to the embryo to activate processes of seed maturation. Our findings thus establish a key role for endosperm in regulating embryo development.

Background: Abiotic stress, including heat, is one of the major factors that affect alfalfa growth and forage yield. The small RNA, microRNA156 (miR156), regulates multiple traits in alfalfa during abiotic stress. The aim of this study was to explore the role of miR156 in regulating heat response in alfalfa at the protein level., Results: In this study, we compared an empty vector control and miR156 overexpressing (miR156OE) alfalfa plants after exposing them to heat stress (40 °C) for 24 h. We measured physiological parameters of control and miR156OE plants under heat stress, and collected leaf samples for protein analysis. A higher proline and antioxidant contents were detected in miR156OE plants than in controls under heat stress. Protein samples were analyzed by label-free quantification proteomics. Across all samples, a total of 1878 protein groups were detected. Under heat stress, 45 protein groups in the empty vector plants were significantly altered (P < 0.05; |log 2 FC| > 2). Conversely, 105 protein groups were significantly altered when miR156OE alfalfa was subjected to heat stress, of which 91 were unique to miR156OE plants. The identified protein groups unique to miR156OE plants were related to diverse functions including metabolism, photosynthesis, stress-response and plant defenses. Furthermore, we identified transcription factors in miR156OE plants, which belonged to squamosa promoter binding-like protein, MYB, ethylene responsive factors, AP2 domain, ABA response element binding factor and bZIP families of transcription factors., Conclusions: These results suggest a positive role for miR156 in heat stress response in alfalfa. They reveal a miR156-regulated network of mechanisms at the protein level to modulate heat responses in alfalfa.

Pinto bean ( Phaseolus vulgaris ) is one of the leading market classes of dry beans that is most affected by postharvest seed coat darkening. The process of seed darkening poses a challenge for bean producers and vendors as they encounter significant losses in crop value due to decreased consumer preference for darker beans. Here, we identified a novel allele of the P gene, P sd , responsible for the slow darkening seed coat in pintos, and identified trait-specific sequence polymorphisms which are utilized for the development of new gene-specific molecular markers for breeding. These tools can be deployed to help tackle this economically important issue for bean producers., Summary: Postharvest seed coat darkening in pinto bean is an undesirable trait that reduces the market value of the stored crop. Regular darkening (RD) pintos darken faster after harvest and accumulate higher level of proanthocyanidins (PAs) compared to slow darkening (SD) cultivars. Although the markers cosegregating with the SD trait have been known for some time, the SLOW DARKENING ( Sd ) gene identity had not been proven.Here, we identified P sd as a candidate for controlling the trait. Genetic complementation, transcript abundance, metabolite analysis, and inheritance study confirmed that P sd is the Sd gene. P sd is another allele of the P ( Pigment ) gene, whose loss-of-function alleles result in a white seed coat. P sd encodes a bHLH transcription factor with two transcript variants but only one is involved in PA biosynthesis. An additional glutamate residue in the activation domain, and/or an arginine to histidine substitution in the bHLH domain of the P sd -1 transcript in the SD cultivar is likely responsible for the reduced activity of this allele compared to the allele in a RD cultivar, leading to reduced PA accumulation.Overall, we demonstrate that a novel allele of P , P sd , is responsible for the SD phenotype, and describe the development of new, gene-specific, markers that could be utilized in breeding to resolve an economically important issue for bean producers., (© 2020 Her Majesty the Queen in Right of Canada. Plants, People, Planet published by John Wiley & Sons Ltd on behalf of New Phytologist Trust. Reproduced with the permission of Minister of Agriculture and Agri-Food Canada.)

The nitrogen fixing ability of common bean ( Phaseolus vulgaris L.) in association with rhizobia is often characterized as poor compared to other legumes, and nitrogen fertilizers are commonly used in bean production to achieve high yields, which in general inhibits nitrogen fixation. In addition, plants cannot take up all the nitrogen applied to the soil as a fertilizer leading to runoff and groundwater contamination. The overall objective of this work is to reduce use of nitrogen fertilizer in common bean production. This would be a major advance in profitability for the common bean industry in Canada and would significantly improve the ecological footprint of the crop. In the current work, 22 bean genotypes [including recombinant inbred lines (RILs) from the Mist × Sanilac population and a non-nodulating mutant (R99)] were screened for their capacity to fix atmospheric nitrogen under four nitrogen regimes. The genotypes were evaluated in replicated field trials on N-poor soils over three years for the percent nitrogen derived from atmosphere (%Ndfa), yield, and a number of yield-related traits. Bean genotypes differed for all analyzed traits, and the level of nitrogen significantly affected most of the traits, including %Ndfa and yield in all three years. In contrast, application of rhizobia significantly affected only few traits, and the effect was inconsistent among the years. Nitrogen application reduced symbiotic nitrogen fixation (SNF) to various degrees in different bean genotypes. This variation suggests that SNF in common bean can be improved through breeding and selection for the ability of bean genotypes to fix nitrogen in the presence of reduced fertilizer levels. Moreover, genotypes like RIL&#95;38, RIL&#95;119, and RIL&#95;131, being both high yielding and good nitrogen fixers, have potential for simultaneous improvement of both traits. However, breeding advancement might be slow due to an inconsistent correlation between these traits., (Copyright © 2020 Reinprecht, Schram, Marsolais, Smith, Hill and Pauls.)

The suboptimal content of sulfur-containing amino acids methionine and cysteine prevents common bean (Phaseolus vulgaris) from being an excellent source of protein. Nutritional improvements to this significant crop require a better understanding of the biosynthesis of sulfur-containing compounds including the nonproteogenic amino acid S-methylcysteine and the dipeptide γ-glutamyl-S-methylcysteine, which accumulate in seed. In this study, seeds were incubated with isotopically labelled serine, cysteine or methionine and analyzed by reverse phase chromatography-high resolution mass spectrometry to track stable isotopes as they progressed through the sulfur metabolome. We determined that serine and methionine are the sole precursors of free S-methylcysteine in developing seeds, indicating that this compound is likely to be synthesized through the condensation of O-acetylserine and methanethiol. BSAS4;1, a cytosolic β-substituted alanine synthase preferentially expressed in developing seeds, catalyzed the formation of S-methylcysteine in vitro. A higher flux of labelled serine or cysteine was observed in a sequential pathway involving γ-glutamyl-cysteine, homoglutathione and S-methylhomoglutathione, a likely precursor to γ-glutamyl-S-methylcysteine. Preferential incorporation of serine over cysteine supports a subcellular compartmentation of this pathway, likely to be in the chloroplast. The origin of the methyl group in S-methylhomoglutathione was traced to methionine. There was substantial incorporation of carbons from methionine into the β-alanine portion of homoglutathione and S-methylhomoglutathione, suggesting the breakdown of methionine by methionine γ-lyase and conversion of α-ketobutyrate to β-alanine via propanoate metabolism. These findings delineate the biosynthetic pathways of the sulfur metabolome of common bean and provide an insight that will aid future efforts to improve nutritional quality., (© 2019 Her Majesty the Queen in Right of Canada The Plant Journal © 2019 John Wiley & Sons Ltd and Society for Experimental Biology.)

Some grain legumes store sulfur in the form of non-protein amino acids in seed. γ-Glutamyl-S-methylcysteine is found in Phaseolus and several Vigna species. γ-Glutamyl-S-ethenylcysteine, an antinutritional compound, is present in Vicia narbonensis. In P. vulgaris, free S-methylcysteine levels are higher at early stages of seed development followed by a decline. γ-Glutamyl-S-methylcysteine accumulates later, in two phases, with a lag during reserve accumulation. The concentration of total S-methylcysteine, quantified after acid hydrolysis, is positively regulated by sulfate nutrition. The levels of both γ-glutamyl-S-methylcysteine and γ-glutamyl-S-ethenylcysteine are modulated in response to changes in seed protein composition. A model is proposed whereby the majority of γ-glutamyl-S-methylcysteine in P. vulgaris is synthesized via the intermediate S-methylhomoglutathione. Knowledge of the biosynthesis of non-protein sulfur amino acids is required for metabolic engineering approaches, in conjunction with manipulation of the protein sink, to increase the concentration of nutritionally essential methionine and cysteine. This would improve protein quality of some important legume crops., (© Her Majesty the Queen in Right of Canada 2019. Reproduced with the permission of the Minister of Agriculture and Agri-Food.)

Common beans ( Phaseolus vulgaris ) form a relationship with nitrogen-fixing rhizobia and through a process termed symbiotic nitrogen fixation (SNF) which provides them with a source of nitrogen. However, beans are considered poor nitrogen fixers, and modern production practices involve routine use of N fertilizer, which leads to the down-regulation of SNF. High-yielding, conventionally bred bean varieties are developed using conventional production practices and selection criteria, typically not including SNF efficiency, and may have lost this trait over decades of modern breeding. In contrast, heirloom bean genotypes were developed before the advent of modern production practices and may represent an underutilized pool of genetics which could be used to improve SNF. This study compared the SNF capacity under low-N field conditions, of collections of heirloom varieties with and conventionally bred dry bean varieties. The heirloom-conventional panel (HCP) consisted of 42 genotypes from various online seed retailers or from the University of Guelph Bean Breeding program seedbank. The HCP was genotyped using a single nucleotide polymorphism (SNP) array to investigate genetic relatedness within the panel. Field trials were conducted at three locations in ON, Canada from 2014 to 2015 and various agronomic and seed composition traits were measured, including capacity for nitrogen fixation (using the natural abundance method to measure seed N isotope ratios). Significant variation for SNF was found in the panel. However, on average, heirloom genotypes did not fix significantly more nitrogen than conventionally bred varieties. However, five heirloom genotypes fixed >60% of their nitrogen from the atmosphere. Yield (kg ha -1 ) was not significantly different between heirloom and conventional genotypes, suggesting that incorporating heirloom genotypes into a modern breeding program would not negatively impact yield. Nitrogen fixation was significantly higher among Middle American genotypes than among Andean genotypes, confirming previous findings. The best nitrogen fixing line was Coco Sophie, a European heirloom white bean whose genetic makeup is admixed between the Andean and Middle American genepools. Heirloom genotypes represent a useful source of genetics to improve SNF in modern bean breeding.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 γ-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 γ-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 γ-glutamyl- S -methylcysteine may arise from S -methylhomoglutathione. Metabolite profiling during development and in genotypes differing in total S -methylcysteine accumulation showed that γ-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 γ-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-predominant in seed, determined by confocal microscopy of a fusion with the yellow fluorescent protein-and its spatial separation from S -methylhomoglutathione may explain the lack of significant accumulation of S -methylated phytochelatins.

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.

l-asparaginases (EC 3.5.1.1) play an important role in nitrogen mobilization in plants. Here, we investigated the biochemical and biophysical properties of potassium-dependent (PvAspG1) and potassium-independent (PvAspG-T2) l-asparaginases from Phaseolus vulgaris. Our previous studies revealed that PvAspG1 requires potassium for catalytic activation and its crystal structure suggested that Ser-118 in the activation loop plays a critical role in coordinating the metal cation. This amino acid residue is replaced by isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, allosteric effector binding, secondary structure conformation, and pH profile were studied. Introduction of the serine residue conferred potassium activation in PvAspG-T2. Conversely, the PvAspG1-S118I mutant could no longer be activated by potassium. PvAspG1 and the PvAspG-T2-I117S mutant had a similar half-maximal effective concentration (EC 50 ) value for potassium activation, between 0.1 and 0.3 mm. Potassium binding elicited a similar conformational change in PvAspG1 and PvAspG-T2-I117S, as studied by circular dichroism. However, no change in conformation was observed for PvAspG-T2 and PvAspG1-S118I. Analysis of kinetic parameters in function of pH indicated that potassium activation mediated by Ser-118 influences the ionization of specific functional groups in the enzyme-substrate complex. Together, the results indicate that Ser-118 of PvAspG1 is essential and sufficient for potassium activation in plant l-asparaginases. ENZYME: l-Asparaginase (EC 3.5.1.1)., (© 2018 Federation of European Biochemical Societies.)

Acetyl-coenzyme A (acetyl-CoA) is a central metabolite and the acetyl source for protein acetylation, particularly histone acetylation that promotes gene expression. However, the effect of acetyl-CoA levels on histone acetylation status in plants remains unknown. Here, we show that malfunctioned cytosolic acetyl-CoA carboxylase1 (ACC1) in Arabidopsis leads to elevated levels of acetyl-CoA and promotes histone hyperacetylation predominantly at lysine 27 of histone H3 (H3K27). The increase of H3K27 acetylation (H3K27ac) is dependent on adenosine triphosphate (ATP)-citrate lyase which cleaves citrate to acetyl-CoA in the cytoplasm, and requires histone acetyltransferase GCN5. A comprehensive analysis of the transcriptome and metabolome in combination with the genome-wide H3K27ac profiles of acc1 mutants demonstrate the dynamic changes in H3K27ac, gene transcripts and metabolites occurring in the cell by the increased levels of acetyl-CoA. This study suggests that H3K27ac is an important link between cytosolic acetyl-CoA level and gene expression in response to the dynamic metabolic environments in plants.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

During the developmental processes from dry seeds to seedling establishment, the glyoxylate cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the glyoxylate cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the glyoxylate cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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.

Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using L-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.

A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.

The levels of nitric oxide (NO) and reactive oxygen species (ROS), ATP/ADP ratios, reduction levels of ascorbate and glutathione, expression of the genes encoding proteins involved in metabolism of NO and activities of the enzymes involved in fermentation and in metabolism of NO and ROS were studied in the embryos of germinating seeds of two barley (Hordeum vulgare L.) cultivars differing in dormancy level. The level of NO production continuously increased after imbibition while the level of nitrosylated SH-groups in proteins increased. This corresponded to the decrease of free SH-groups in proteins. At early stage of germination (0-48 h post imbibition) the genes encoding class 1 phytoglobin (the protein scavenging NO) and S-nitrosoglutathione reductase (scavenging S-nitrosoglutathione) were markedly expressed. More dormant cultivar exhibited lower ATP/ADP and ascorbate/dehydroascorbate ratios and lower lactate and alcohol dehydrogenase activities, while the production of NO and nitrosylation of proteins was higher as compared to the non-dormant cultivar. The obtained data indicate that at the onset of germination NO is actively generated causing nitrosylation of SH-groups and a switch from respiration to fermentation. After radicle protrusion the metabolism changes in a more reducing type as recorded by ratio of reduced and oxidized glutathione and ascorbate. The turnover of NO by the scavenging systems (phytoglobin, S-nitrosoglutathione reductase and interaction with ROS) might contribute to the maintenance of redox and energy balance of germinating seeds and lead to alleviation of dormancy.

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K + -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the β-subunit of β-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing β-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11-18%, and an increase in free aspartic acid levels, by 25-60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9-13%. Their levels of extractible globulins were reduced by 11-30%. This was accompanied by an increase in oil concentration, by 5-8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation., (© 2015 Scandinavian Plant Physiology Society.)

Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.

It has been hypothesized that the relatively low concentration of sulfur amino acids in legume seeds might be an ecological adaptation to nutrient poor, marginal soils. SARC1 and SMARC1N-PN1 are genetically related lines of common bean (dry bean, Phaseolus vulgaris) differing in seed storage protein composition. In SMARC1N-PN1, the lack of phaseolin and major lectins is compensated by increased levels of sulfur-rich proteins, resulting in an enhanced concentration of cysteine and methionine, mostly at the expense of the abundant non-protein amino acid, S-methylcysteine. To identify potential effects associated with an increased concentration of sulfur amino acids in the protein pool, the response of the two genotypes to low and high sulfur nutrition was evaluated under controlled conditions. Seed yield was increased by the high sulfate treatment in SMARC1N-PN1. The seed concentrations of sulfur, sulfate, and S-methylcysteine were altered by the sulfur treatment in both genotypes. The concentration of total cysteine and extractible globulins was increased specifically in SMARC1N-PN1. Proteomic analysis identified arcelin-like protein 4, lipoxygenase-3, albumin-2, and alpha amylase inhibitor beta chain as having increased levels under high sulfur conditions. Lipoxygenase-3 accumulation was sensitive to sulfur nutrition only in SMARC1N-PN1. Under field conditions, both SARC1 and SMARC1N-PN1 exhibited a slight increase in yield in response to sulfur treatment, typical for common bean.

Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type L-asparaginase from Phaseolus vulgaris (PvAspG1) differing in the type of associated alkali metal ions (K(+), Na(+) or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type L-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to α and β subunits, thus creating the mature heterotetramer or dimer of heterodimers (αβ)2. The αβ subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two β-sheets flanked on each side by a layer of helices. In addition to the `sodium loop' (here referred to as the `stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the `activation loop') in subunit α (residues Val111-Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type L-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K(+) is coordinated) or OFF (when Na(+) is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent L-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.

In higher plants, asparagine (Asn) is a major form of organic nitrogen used for transport and storage. There are two pathways of Asn metabolism, involving asparaginase and Asn aminotransferase. The enzyme serine:glyoxylate aminotransferase encoded by AGT1 has been identified as an asparagine aminotransferase in Arabidopsis. The product of asparagine transamination, alpha-ketosuccinamate, can be hydrolyzed by the enzyme omega-amidase to form oxaloacetate and ammonia. A candidate gene was identified in Arabidopsis based on its sequence similarity with mouse omega-amidase. Recombinant omega-amidase exhibited comparable catalytic activities with alpha-hydroxysuccinamate, alpha-ketosuccinamate and alpha-ketoglutaramate, the product of glutamine transamination. A mutant with a T-DNA inserted in the first exon accumulated alpha-ketosuccinamate and alpha-hydroxysuccinamate as compared with wild-type, both under control conditions and after treatment with Asn. Treatment with Asn led to decreased transcript levels of omega-amidase in root, while transcript levels of AGT1 are increased under these conditions, suggesting that excess Asn may lead to the accumulation of alpha-ketosuccinamate and alpha-hydroxysuccinamate., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

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 ssp1, a recessive, viable mutant that accumulates approximately 15% less protein than wildtype seeds. Molecular analyses revealed that ssp1 is due to the introduction of a premature stop codon in CRU3, 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, ssp1 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 ssp1 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 ssp1 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.

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.

Asparagine (Asn) is a major form of nitrogen transported to sink tissues. Results from a previous study have shown that an Arabidopsis mutant lacking asparaginase activity develops relatively normally, highlighting a possible compensation by other types of asparagine metabolic enzymes. Prior studies with barley and tobacco mutants have associated Asn aminotransferase activity with the photorespiratory enzyme, serine (Ser):glyoxylate aminotransferase. This enzyme is encoded by AGT1 in Arabidopsis thaliana. Recombinant N-terminal His-tagged AGT1 purified from Escherichia coli was characterized with Ser, alanine (Ala) and Asn as amino acid donors and glyoxylate, pyruvate and hydroxypyruvate as organic acid acceptors. The V(max) of AGT1 with Asn was higher than with Ser or Ala by ca. 5- to 20-fold. As a result, the catalytic efficiency (V(max)/K(m)) was slightly higher with Asn than with the two other amino acids. In the roots of 10-day-old seedlings treated for 2h with 20mM Asn, the AGT1 transcript levels were raised by 2-fold. During this treatment, the concentration of Asn in root was raised by ca. 5-fold. These results suggest that AGT1 is involved in Asn metabolism in Arabidopsis., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

The lack of phaseolin and phytohaemagglutinin in common bean (dry bean, Phaseolus vulgaris) is associated with an increase in total cysteine and methionine concentrations by 70% and 10%, respectively, mainly at the expense of an abundant non-protein amino acid, S-methyl-cysteine. Transcripts were profiled between two genetically related lines differing for this trait at four stages of seed development using a high density microarray designed for common bean. Transcripts of multiple sulphur-rich proteins were elevated, several previously identified by proteomics, including legumin, basic 7S globulin, albumin-2, defensin, albumin-1, the Bowman-Birk type proteinase inhibitor, the double-headed trypsin inhibitor, and the Kunitz trypsin inhibitor. A co-ordinated regulation of transcripts coding for sulphate transporters, sulphate assimilatory enzymes, serine acetyltransferases, cystathionine β-lyase, homocysteine S-methyltransferase and methionine gamma-lyase was associated with changes in cysteine and methionine concentrations. Differential gene expression of sulphur-rich proteins preceded that of sulphur metabolic enzymes, suggesting a regulation by demand from the protein sink. Up-regulation of SERAT1;1 and -1;2 expression revealed an activation of cytosolic O-acetylserine biosynthesis. Down-regulation of SERAT2;1 suggested that cysteine and S-methyl-cysteine biosynthesis may be spatially separated in different subcellular compartments. Analysis of free amino acid profiles indicated that enhanced cysteine biosynthesis was correlated with a depletion of O-acetylserine. These results contribute to our understanding of the regulation of sulphur metabolism in developing seed in response to a change in the composition of endogenous proteins.

Ephedrine and pseudoephedrine are phenylpropylamino alkaloids widely used in modern medicine. Some Ephedra species such as E. sinica Stapf (Ephedraceae), a widely used Chinese medicinal plant (Chinese name: Ma Huang), accumulate ephedrine alkaloids as active constituents. Other Ephedra species, such as E. foeminea Forssk. (syn. E. campylopoda C.A. Mey) lack ephedrine alkaloids and their postulated metabolic precursors 1-phenylpropane-1,2-dione and (S)-cathinone. Solid-phase microextraction analysis of freshly picked young E. sinica and E. foeminea stems revealed the presence of increased benzaldehyde levels in E. foeminea, whereas 1-phenylpropane-1,2-dione was detected only in E. sinica. Soluble protein preparations from E. sinica and E. foeminea stems catalyzed the conversion of benzaldehyde and pyruvate to (R)-phenylacetylcarbinol, (S)-phenylacetylcarbinol, (R)-2-hydroxypropiophenone (S)-2-hydroxypropiophenone and 1-phenylpropane-1,2-dione. The activity, termed benzaldehyde carboxyligase (BCL) required the presence of magnesium and thiamine pyrophosphate and was 40 times higher in E. sinica as compared to E. foeminea. The distribution patterns of BCL activity in E. sinica tissues correlates well with the distribution pattern of the ephedrine alkaloids. (S)-Cathinone reductase enzymatic activities generating (1R,2S)-norephedrine and (1S,1R)-norephedrine were significantly higher in E. sinica relative to the levels displayed by E. foeminea. Surprisingly, (1R,2S)-norephedrine N-methyltransferase activity which is a downstream enzyme in ephedrine biosynthesis was significantly higher in E. foeminea than in E. sinica. Our studies further support that benzaldehyde is the metabolic precursor to phenylpropylamino alkaloids in E. sinica., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.

Structural determinants responsible for the substrate preference of the potassium-independent (ASPGA1) and -dependent (ASPGB1) asparaginases from Arabidopsis thaliana have been investigated. Like ASPGA1, ASPGB1 was found to be catalytically active with both L: -Asn and β-Asp-His as substrates, contrary to a previous report. However, ASPGB1 had a 45-fold higher specific activity with Asn as substrate than ASPGA1. A divergent sequence between the two enzymes forms a variable loop at the C-terminal of the alpha subunit. The results of dynamic simulations have previously implicated a movement of the C-terminus in the allosteric transduction of K(+)-binding at the surface of LjNSE1 asparaginase. In the crystal structure of Lupinus luteus asparaginase, most residues in this segment cannot be visualized due to a weak electron density. Exchanging the variable loop in ASPGA1 with that from ASPGB1 increased the affinity for Asn, with a 320-fold reduction in K (m) value. Homology modeling identified a residue specific to ASPGB1, Phe(162), preceding the variable loop, whose side chain is located in proximity to the beta-carboxylate group of the product aspartate, and to Gly(246), a residue participating in an oxyanion hole which stabilizes a negative charge forming on the side chain oxygen of asparagine during catalysis. Replacement with the corresponding leucine from ASPGA1 specifically lowered the V (max) value with Asn as substrate by 8.4-fold.

The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.