Differential Gene Expression of Brachypodium distachyon Roots Colonized by Gluconacetobacter diazotrophicus and the Role of BdCESA8 in the Colonization.

Alternatives to synthetic nitrogen fertilizer are needed to reduce the costs of crop production and offset environmental damage. Nitrogen-fixing bacterium Gluconacetobacter diazotrophicus has been proposed as a possible biofertilizer for monocot crop production. However, the colonization of G. diazotrophicus in most monocot crops is limited and deep understanding of the response of host plants to G. diazotrophicus colonization is still lacking. In this study, the molecular response of the monocot plant model Brachypodium distachyon was studied during G. diazotrophicus root colonization. The gene expression profiles of B. distachyon root tissues colonized by G. diazotrophicus were generated via next-generation RNA sequencing, and investigated through gene ontology and metabolic pathway analysis. The RNA sequencing results indicated that Brachypodium is actively involved in G. diazotrophicus colonization via cell wall synthesis. Jasmonic acid, ethylene, gibberellin biosynthesis. nitrogen assimilation, and primary and secondary metabolite pathways are also modulated to accommodate and control the extent of G. diazotrophicus colonization. Cellulose synthesis is significantly downregulated during colonization. The loss of function mutant for Brachypodium cellulose synthase 8 (BdCESA8) showed decreased cellulose content in xylem and increased resistance to G. diazotrophicus colonization. This result suggested that the cellulose synthesis of the secondary cell wall is involved in G. diazotrophicus colonization. The results of this study provide insights for future research in regard to gene manipulation for efficient colonization of nitrogen-fixing bacteria in Brachypodium and monocot crops.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Analysis of a novel mutant allele of GSL8 reveals its key roles in cytokinesis and symplastic trafficking in Arabidopsis.

BACKGROUND: Plant cell walls are mainly composed of polysaccharides such as cellulose and callose. Callose exists at a very low level in the cell wall; however, it plays critical roles at different stages of plant development as well as in defence against unfavorable conditions. Callose is accumulated at the cell plate, at plasmodesmata and in male and female gametophytes. Despite the important roles of callose in plants, the mechanisms of its synthesis and regulatory properties are not well understood. RESULTS: CALLOSE SYNTHASE (CALS) genes, also known as GLUCAN SYNTHASE-LIKE (GSL), comprise a family of 12 members in Arabidopsis thaliana. Here, we describe a new allele of GSL8 (named essp8) that exhibits pleiotropic seedling defects. Reduction of callose deposition at the cell plates and plasmodesmata in essp8 leads to ectopic endomitosis and an increase in the size exclusion limit of plasmodesmata during early seedling development. Movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development, are dysregulated in the primary root of essp8. This observation provides evidence for a molecular mechanism explaining the gsl8 root phenotype. We demonstrated that GSL8 interacts with PLASMODESMATA-LOCALIZED PROTEIN 5, a ß-1,3-glucanase, and GSL10. We propose that they all might be part of a putative callose synthase complex, allowing a concerted regulation of callose deposition at plasmodesmata. CONCLUSION: Analysis of a novel mutant allele of GSL8 reveals that GSL8 is a key player in early seedling development in Arabidopsis. GSL8 is required for maintaining the basic ploidy level and regulating the symplastic trafficking. Callose deposition at plasmodesmata is highly regulated and occurs through interaction of different components, likely to be incorporated into a callose biosynthesis complex. We are providing new evidence supporting an earlier hypothesis that GSL8 might have regulatory roles apart from its enzymatic function in plasmodesmata regulation.

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.

Cytosolic acetyl-CoA promotes histone acetylation predominantly at H3K27 in Arabidopsis.

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.

Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis.

Disruption of gene silencing by Polycomb protein complexes leads to homeotic transformations and altered developmental-phase identity in plants. Here we define short genomic fragments, known as Polycomb response elements (PREs), that direct Polycomb repressive complex 2 (PRC2) placement at developmental genes regulated by silencing in Arabidopsis thaliana. We identify transcription factor families that bind to these PREs, colocalize with PRC2 on chromatin, physically interact with and recruit PRC2, and are required for PRC2-mediated gene silencing in vivo. Two of the cis sequence motifs enriched in the PREs are cognate binding sites for the identified transcription factors and are necessary and sufficient for PRE activity. Thus PRC2 recruitment in Arabidopsis relies in large part on binding of trans-acting factors to cis-localized DNA sequence motifs.

Transcriptomic evidence for the control of soybean root isoflavonoid content by regulation of overlapping phenylpropanoid pathways.

BACKGROUND: Isoflavonoids are a class of specialized metabolites found predominantly in legumes. They play a role in signaling for symbiosis with nitrogen-fixing bacteria and inhibiting pathogen infection. RESULTS: A transcriptomic approach using soybean cultivars with high (Conrad and AC Colombe) and low (AC Glengarry and Pagoda) root isoflavonoid content was used to find elements that underlie this variation. Two genes, encoding the flavonoid-metabolizing enzymes, flavonoid 3'-hydroxylase (GmF3'H) and dihydroflavonol 4-reductase (GmDFR), had lower expression levels in high isoflavonoid cultivars. These enzymes compete with isoflavonoid biosynthetic enzymes for the important branch-point substrate naringenin and its derivatives. Differentially expressed genes, between the two sets of cultivars, encode transcription factors, transporters and enzymatic families of interest, such as oxidoreductases, hydrolases and transferases. In addition, genes annotated with stress and disease response were upregulated in high isoflavonoid cultivars. CONCLUSIONS: Coordinated regulation of genes involved in flavonoid metabolism could redirect flux into the isoflavonoid branch of the phenylpropanoid pathway, by reducing competition for the flavanone substrate. These candidate genes could help identify mechanisms to overcome the endogenous bottleneck to isoflavonoid production, facilitate biosynthesis in heterologous systems, and enhance crop resistance against pathogenic infections.

Lotus japonicus NF-YA1 Plays an Essential Role During Nodule Differentiation and Targets Members of the SHI/STY Gene Family.

Legume plants engage in intimate relationships with rhizobial bacteria to form nitrogen-fixing nodules, root-derived organs that accommodate the microsymbiont. Members of the Nuclear Factor Y (NF-Y) gene family, which have undergone significant expansion and functional diversification during plant evolution, are essential for this symbiotic liaison. Acting in a partially redundant manner, NF-Y proteins were shown, previously, to regulate bacterial infection, including selection of a superior rhizobial strain, and to mediate nodule structure formation. However, the exact mechanism by which these transcriptional factors exert their symbiotic functions has remained elusive. By carrying out detailed functional analyses of Lotus japonicus mutants, we demonstrate that LjNF-YA1 becomes indispensable downstream from the initial cortical cell divisions but prior to nodule differentiation, including cell enlargement and vascular bundle formation. Three affiliates of the SHORT INTERNODES/STYLISH transcription factor gene family, called STY1, STY2, and STY3, are demonstrated to be among likely direct targets of LjNF-YA1, and our results point to their involvement in nodule formation.

Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa.

BACKGROUND: Medicago sativa (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group. These plants (miR156OE) showed enhanced biomass yield, reduced internodal length, increased shoot branching and trichome density, and a delay in flowering time. Transcripts of three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6, MsSPL12, and MsSPL13) were found to be targeted for cleavage by MsmiR156 in alfalfa. RESULTS: To further illustrate the molecular mechanisms underlying the effects of miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next Generation RNA Sequencing with Illumina HiSeq. More than 1.11 billion clean reads were obtained from our available sequenced samples. A total of 160,472 transcripts were generated using Trinity de novo assembly and 4,985 significantly differentially expressed genes were detected in miR156OE plants A11a and A17 using the Medicago truncatula genome as reference. A total of 17 genes (including upregulated, downregulated, and unchanged) were selected for quantitative real-time PCR (qRT-PCR) validation, which showed that gene expression levels were largely consistent between qRT-PCR and RNA-Seq data. In addition to the established SPL genes MsSPL6, MsSPL12 and MsSPL13, four new SPLs; MsSPL2, MsSPL3, MsSPL4 and MsSPL9 were also down-regulated significantly in both miR156OE plants. These seven SPL genes belong to genes phylogeny clades VI, IV, VIII, V and VII, which have been reported to be targeted by miR156 in Arabidopsis thaliana. The gene ontology terms characterized electron transporter, starch synthase activity, sucrose transport, sucrose-phosphate synthase activity, chitin binding, sexual reproduction, flavonoid biosynthesis and lignin catabolism correlate well to the phenotypes of miR156OE alfalfa plants. CONCLUSIONS: This is the first report of changes in global gene expression in response to miR156 overexpression in alfalfa. The discovered miR156-targeted SPL genes belonging to different clades indicate miR156 plays fundamental and multifunctional roles in regulating alfalfa plant development.

New BAR tools for mining expression data and exploring Cis-elements in Arabidopsis thaliana.

Identifying sets of genes that are specifically expressed in certain tissues or in response to an environmental stimulus is useful for designing reporter constructs, generating gene expression markers, or for understanding gene regulatory networks. We have developed an easy-to-use online tool for defining a desired expression profile (a modification of our Expression Angler program), which can then be used to identify genes exhibiting patterns of expression that match this profile as closely as possible. Further, we have developed another online tool, Cistome, for predicting or exploring cis-elements in the promoters of sets of co-expressed genes identified by such a method, or by other methods. We present two use cases for these tools, which are freely available on the Bio-Analytic Resource at http://BAR.utoronto.ca.

Next-generation mapping of genetic mutations using bulk population sequencing.

Next-generation sequencing platforms have made it possible to very rapidly map genetic mutations in Arabidopsis using whole-genome resequencing against pooled members of an F2 mapping population. In the case of recessive mutations, all individuals expressing the phenotype will be homozygous for the mutant genome at the locus responsible for the phenotype, while all other loci segregate roughly equally for both parental lines due to recombination. Importantly, genomic regions flanking the recessive mutation will be in linkage disequilibrium and therefore also be homozygous due to genetic hitchhiking. This information can be exploited to quickly and effectively identify the causal mutation. To this end, sequence data generated from members of the pooled population exhibiting the mutant phenotype are first aligned to the reference genome. Polymorphisms between the mutant and mapping line are then identified and used to determine the homozygous, nonrecombinant region harboring the mutation. Polymorphisms in the identified region are filtered to provide a short list of markers potentially responsible for the phenotype of interest, which is followed by validation at the bench. Although the focus of recent studies has been on the mapping of point mutations exhibiting recessive phenotypes, the techniques employed can be extended to incorporate more complicated scenarios such as dominant mutations and those caused by insertions or deletions in genomic sequence. This chapter describes detailed procedures for performing next-generation mapping against an Arabidopsis mutant and discusses how different mutations might be approached.

The root transcriptome for North American ginseng assembled and profiled across seasonal development.

BACKGROUND: Ginseng including North American ginseng (Panax quinquefolius L.) is one of the most widely used medicinal plants. Its success is thought to be due to a diverse collection of ginsenosides that serve as its major bioactive compounds. However, few genomic resources exist and the details concerning its various biosynthetic pathways remain poorly understood. As the root is the primary tissue harvested commercially for ginsenosides, next generation sequencing was applied to the characterization and assembly of the root transcriptome throughout seasonal development. Transcripts showing homology to ginsenoside biosynthesis enzymes were profiled in greater detail. RESULTS: RNA extracts from root samples from seven development stages of North American ginseng were subjected to 454 sequencing, filtered for quality and used in the de novo assembly of a collective root reference transcriptome consisting of 41,623 transcripts. Annotation efforts using a number of public databases resulted in detailed annotation information for 34,801 (84%) transcripts. In addition, 3,955 genes were assigned to metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes. Among our results, we found all of the known enzymes involved in the ginsenoside backbone biosynthesis and used co-expression analysis to identify a number of candidate sequences involved in the latter stages ginsenoside biosynthesis pathway. Transcript profiles suggest ginsenoside biosynthesis occurs at distinct stages of development. CONCLUSIONS: The assembly generated provides a comprehensive annotated reference for future transcriptomic study of North American ginseng. A collection of putative ginsenoside biosynthesis genes were identified and candidate genes predicted from the lesser understood downstream stages of biosynthesis. Transcript expression profiles across seasonal development suggest a primary dammarane-type ginsenoside biosynthesis occurs just prior to plant senescence, with secondary ginsenoside production occurring throughout development. Data from the study provide a valuable resource for conducting future ginsenoside biosynthesis research in this important medicinal plant.

Forward chemical genetic screens in Arabidopsis identify genes that influence sensitivity to the phytotoxic compound sulfamethoxazole.

BACKGROUND: The sulfanilamide family comprises a clinically important group of antimicrobial compounds which also display bioactivity in plants. While there is evidence that sulfanilamides inhibit folate biosynthesis in both bacteria and plants, the complete network of plant responses to these compounds remains to be characterized. As such, we initiated two forward genetic screens in Arabidopsis in order to identify mutants that exhibit altered sensitivity to sulfanilamide compounds. These screens were based on the growth phenotype of seedlings germinated in the presence of the compound sulfamethoxazole (Smex). RESULTS: We identified a mutant with reduced sensitivity to Smex, and subsequent mapping indicated that a gene encoding 5-oxoprolinase was responsible for this phenotype. A mutation causing enhanced sensitivity to Smex was mapped to a gene lacking any functional annotation. CONCLUSIONS: The genes identified through our forward genetic screens represent novel mediators of Arabidopsis responses to sulfanilamides and suggest that these responses extend beyond the perturbation of folate biosynthesis.

Transcripts of sulphur metabolic genes are co-ordinately regulated in developing seeds of common bean lacking phaseolin and major lectins.

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 ß-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.

Next-generation mapping of Arabidopsis genes.

Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F2 population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F2 population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F2 lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.

LEAFY target genes reveal floral regulatory logic, cis motifs, and a link to biotic stimulus response.

The transition from vegetative growth to flower formation is critical for the survival of flowering plants. The plant-specific transcription factor LEAFY (LFY) has central, evolutionarily conserved roles in this process, both in the formation of the first flower and later in floral patterning. We performed genome-wide binding and expression studies to elucidate the molecular mechanisms by which LFY executes these roles. Our study reveals that LFY directs an elaborate regulatory network in control of floral homeotic gene expression. LFY also controls the expression of genes that regulate the response to external stimuli in Arabidopsis. Thus, our findings support a key role for LFY in the coordination of reproductive stage development and disease response programs in plants that may ensure optimal allocation of plant resources for reproductive fitness. Finally, motif analyses reveal a possible mechanism for stage-specific LFY recruitment and suggest a role for LFY in overcoming polycomb repression.

C-terminal motif prediction in eukaryotic proteomes using comparative genomics and statistical over-representation across protein families.

BACKGROUND: The carboxy termini of proteins are a frequent site of activity for a variety of biologically important functions, ranging from post-translational modification to protein targeting. Several short peptide motifs involved in protein sorting roles and dependent upon their proximity to the C-terminus for proper function have already been characterized. As a limited number of such motifs have been identified, the potential exists for genome-wide statistical analysis and comparative genomics to reveal novel peptide signatures functioning in a C-terminal dependent manner. We have applied a novel methodology to the prediction of C-terminal-anchored peptide motifs involving a simple z-statistic and several techniques for improving the signal-to-noise ratio. RESULTS: We examined the statistical over-representation of position-specific C-terminal tripeptides in 7 eukaryotic proteomes. Sequence randomization models and simple-sequence masking were applied to the successful reduction of background noise. Similarly, as C-terminal homology among members of large protein families may artificially inflate tripeptide counts in an irrelevant and obfuscating manner, gene-family clustering was performed prior to the analysis in order to assess tripeptide over-representation across protein families as opposed to across all proteins. Finally, comparative genomics was used to identify tripeptides significantly occurring in multiple species. This approach has been able to predict, to our knowledge, all C-terminally anchored targeting motifs present in the literature. These include the PTS1 peroxisomal targeting signal (SKL*), the ER-retention signal (K/HDEL*), the ER-retrieval signal for membrane bound proteins (KKxx*), the prenylation signal (CC*) and the CaaX box prenylation motif. In addition to a high statistical over-representation of these known motifs, a collection of significant tripeptides with a high propensity for biological function exists between species, among kingdoms and across eukaryotes. Motifs of note include a serine-acidic peptide (DSD*) as well as several lysine enriched motifs found in nearly all eukaryotic genomes examined. CONCLUSION: We have successfully generated a high confidence representation of eukaryotic motifs anchored at the C-terminus. A high incidence of true-positives in our results suggests that several previously unidentified tripeptide patterns are strong candidates for representing novel peptide motifs of a widely employed nature in the C-terminal biology of eukaryotes. Our application of comparative genomics, statistical over-representation and the adjustment for protein family homology has generated several hypotheses concerning the C-terminal topology as it pertains to sorting and potential protein interaction signals. This approach to background reduction could be expanded for application to protein motif prediction in the protein interior. A parallel N-terminal analysis is presented as supplementary data.