SIMPEL: using stable isotopes to elucidate dynamics of context specific metabolism.

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.

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

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.

Diurnal accumulation of K+-dependent L-asparaginase in leaf of common bean (Phaseolus vulgaris L.).

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.

Variability in Maturity, Oil and Protein Concentration, and Genetic Distinctness among Soybean Accessions Conserved at Plant Gene Resources of Canada.

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.

Global analysis of common bean multidrug and toxic compound extrusion transporters (PvMATEs): PvMATE8 and pinto bean seed coat darkening.

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 (PvMATEs). 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.

Evidence that class I glutamine amidotransferase, GAT1_2.1, acts as a glutaminase in roots of Arabidopsis thaliana.

The glutamine amidotransferase gene GAT1_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 Km value of 0.66 mM and Vmax 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-15N-labeled glutamine was identified. Visualization of GAT1_2.1-YPF transiently expressed in tobacco leaves confirmed its mitochondrial localization. gat1_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_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.

Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing.

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.

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.

LEAFY COTYLEDON1 expression in the endosperm enables embryo maturation in Arabidopsis.

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.

Label-free quantitative proteomic analysis of alfalfa in response to microRNA156 under high temperature.

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; |log2FC| > 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.

Effects of Nitrogen Application on Nitrogen Fixation in Common Bean Production.

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_38, RIL_119, and RIL_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.

Postharvest seed coat darkening in pinto bean (Phaseolus vulgaris) is regulated by Psd , an allele of the basic helix-loop-helix transcription factor P.

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, Psd , 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 Psd as a candidate for controlling the trait. Genetic complementation, transcript abundance, metabolite analysis, and inheritance study confirmed that Psd is the Sd gene. Psd is another allele of the P (Pigment) gene, whose loss-of-function alleles result in a white seed coat. Psd 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 Psd-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, Psd , 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.

Agronomic Performance and Nitrogen Fixation of Heirloom and Conventional Dry Bean Varieties Under Low-Nitrogen Field Conditions.

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.

Distribution and possible biosynthetic pathway of non-protein sulfur amino acids in legumes.

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.

Deciphering S-methylcysteine biosynthesis in common bean by isotopic tracking with mass spectrometry.

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 ß-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 ß-alanine portion of homoglutathione and S-methylhomoglutathione, suggesting the breakdown of methionine by methionine γ-lyase and conversion of α-ketobutyrate to ß-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.

Common Bean (Phaseolus vulgaris L.) Accumulates Most S-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 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.

RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis.

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.

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

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.

Structural basis of potassium activation in plant asparaginases.

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 (EC50 ) 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).