Comparative RNA-Seq analysis of the root revealed transcriptional regulation system for aluminum tolerance in contrasting indica rice of North East India.

Expression pattern of aluminum (Al) tolerance genes is one of the major determinants of Al avoidance/tolerance within plant cultivars. We have performed transcriptome analysis of two contrasting (Al-tolerant, Disang; Al-sensitive, Joymati) cultivars of India's North Eastern region, an indica rice diversity hotspot, on exposure to excess Al3+ treatment in acidic condition. Co-expression analysis and SNPs enrichment analysis proposed the role of both trans-acting and cis-acting polymorphisms in Al signaling in the Al-tolerant cultivar. We proposed ten major genes, including arginine decarboxylase, phytase, and beta-glucosidase aggregating factor as candidates responsible for Al tolerance based on transcriptome analysis. Al3+ stress led to changes in the alternative splicing profile of the Al-tolerant cultivar. These studies demonstrated the transcriptional variations affiliated to Al avoidance/tolerance in contrasting indica rice of North East India and provided us with several candidate genes responsible for Al tolerance.

A single-population GWAS identified AtMATE expression level polymorphism caused by promoter variants is associated with variation in aluminum tolerance in a local Arabidopsis population.

Organic acids (OA) are released from roots in response to aluminum (Al), conferring an Al tolerance to plants that is regulated by OA transporters such as ALMT (Al-activated malate transporter) and multi-drug and toxic compound extrusion (MATE). We have previously reported that the expression level polymorphism (ELP) of AtALMT1 is strongly associated with variation in Al tolerance among natural accessions of Arabidopsis. However, although AtMATE is also expressed following Al exposure and contributes to Al tolerance, whether AtMATE contributes to the variation of Al tolerance and the molecular mechanisms of ELP remains unclear. Here, we dissected the natural variation in AtMATE expression level in response to Al at the root using diverse natural accessions of Arabidopsis. Phylogenetic analysis revealed that more than half of accessions belonging to the Central Asia (CA) population show markedly low AtMATE expression levels, while the majority of European populations show high expression levels. The accessions of the CA population with low AtMATE expression also show significantly weakened Al tolerance. A single-population genome-wide association study (GWAS) of AtMATE expression in the CA population identified a retrotransposon insertion in the AtMATE promoter region associated with low gene expression levels. This may affect the transcriptional regulation of AtMATE by disrupting the effect of a cis-regulatory element located upstream of the insertion site, which includes AtSTOP1 (sensitive to proton rhizotoxicity 1) transcription factor-binding sites revealed by chromatin immunoprecipitation-qPCR analysis. Furthermore, the GWAS performed without the accessions expressing low levels of AtMATE, excluding the effect of AtMATE promoter polymorphism, identified several candidate genes potentially associated with AtMATE expression.

Genome-Wide Association Study and Genomic Prediction Elucidate the Distinct Genetic Architecture of Aluminum and Proton Tolerance in Arabidopsis thaliana.

Under acid soil conditions, Al stress and proton stress can occur, reducing root growth and function. However, these stressors are distinct, and tolerance to each is governed by multiple physiological processes. To better understand the genes that underlie these coincidental but experimentally separable stresses, a genome-wide association study (GWAS) and genomic prediction (GP) models were created for approximately 200 diverse Arabidopsis thaliana accessions. GWAS and genomic prediction identified 140/160 SNPs associated with Al and proton tolerance, respectively, which explained approximately 70% of the variance observed. Reverse genetics of the genes in loci identified novel Al and proton tolerance genes, including TON1-RECRUITING MOTIF 28 (AtTRM28) and THIOREDOXIN H-TYPE 1 (AtTRX1), as well as genes known to be associated with tolerance, such as the Al-activated malate transporter, AtALMT1. Additionally, variation in Al tolerance was partially explained by expression level polymorphisms of AtALMT1 and AtTRX1 caused by cis-regulatory allelic variation. These results suggest that we successfully identified the loci that regulate Al and proton tolerance. Furthermore, very small numbers of loci were shared by Al and proton tolerance as determined by the GWAS. There were substantial differences between the phenotype predicted by genomic prediction and the observed phenotype for Al tolerance. This suggested that the GWAS-undetectable genetic factors (e.g., rare-allele mutations) contributing to the variation of tolerance were more important for Al tolerance than for proton tolerance. This study provides important new insights into the genetic architecture that produces variation in the tolerance of acid soil.

Transcriptome Analysis and Identification of a Transcriptional Regulatory Network in the Response to H2O2.

Hydrogen peroxide (H2O2) is a common signal molecule initiating transcriptional responses to all the known biotic and abiotic stresses of land plants. However, the degree of involvement of H2O2 in these stress responses has not yet been well studied. Here we identify time-dependent transcriptome profiles stimulated by H2O2 application in Arabidopsis (Arabidopsis thaliana) seedlings. Promoter prediction based on transcriptome data suggests strong crosstalk among high light, heat, and wounding stress responses in terms of environmental stresses and between the abscisic acid (ABA) and salicylic acid (SA) responses in terms of phytohormone signaling. Quantitative analysis revealed that ABA accumulation is induced by H2O2 but SA is not, suggesting that the implied crosstalk with ABA is achieved through ABA accumulation while the crosstalk with SA is different. We identified potential direct regulatory pairs between regulator transcription factor (TF) proteins and their regulated TF genes based on the time-course transcriptome analysis for the H2O2 response, in vivo regulation of the regulated TF by the regulator TF identified by expression analysis of mutants and overexpressors, and in vitro binding of the regulator TF protein to the target TF promoter. These analyses enabled the establishment of part of the transcriptional regulatory network for the H2O2 response composed of 15 regulatory pairs of TFs, including five pairs previously reported. This regulatory network is suggested to be involved in a wide range of biotic and abiotic stress responses in Arabidopsis.

Comparative genome-wide analysis of WRKY transcription factors in two Asian legume crops: Adzuki bean and Mung bean.

The seminal participation of WRKY transcription factors in plant development, metabolism and in the governance of defense mechanism implicated their gaining importance for genomic and functional studies. The recent release of draft genome sequences of two legume crops, Adzuki bean (Vigna angularis) and Mung bean (Vigna radiata) has paved the way for characterization of WRKY gene family in these crops. We found 84 WRKY genes in Adzuki bean (VaWRKY) and 85 WRKY genes in Mung bean (VrWRKY). Based on the phylogenetic analysis, VaWRKY genes were classified into three groups with 15 members in Group I, 56 members in Group II, and 13 members in Group III, which was comparable to VrWRKY distribution in Mung bean, 16, 56 and 13 members in Group I, II and III, respectively. The few tandem and segmental duplication events suggested that recent duplication plays no prominent role in the expansion VaWRKY and VrWRKY genes. The illustration of gene-structure and their encoded protein-domains further revealed the nature of WRKY proteins. Moreover, the identification of abiotic or biotic stress-responsive cis-regulatory elements in the promoter regions of some WRKY genes provides fundamental insights for their further implementation in stress-tolerance and genetic improvement of agronomic traits.

Prediction of bipartite transcriptional regulatory elements using transcriptome data of Arabidopsis.

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of '4 bases-a spacer with a flexible length-4 bases'. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein-DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly.

Identification of Arabidopsis genic and non-genic promoters by paired-end sequencing of TSS tags.

Information about transcription start sites (TSSs) provides baseline data for the analysis of promoter architecture. In this paper we used paired- and single-end deep sequencing to analyze Arabidopsis TSS tags from several libraries prepared from roots, shoots, flowers and etiolated seedlings. The clustering of approximately 33 million mapped TSS tags led to the identification of 324 461 promoters that covered 79.7% (21 672/27 206) of protein-coding genes in the Arabidopsis genome. In addition we identified intragenic, antisense and orphan promoters that were not associated with any gene models. Of these, intragenic promoters exhibited unique characteristics regarding dinucleotide sequences at TSSs and core promoter element composition, suggesting that these promoters use different mechanisms of transcriptional initiation. An analysis of base composition with regard to promoter position revealed a low GC content throughout the promoter region and several local strand biases that were evident for TATA-type promoters, but not for Coreless-type promoters. Most observed strand biases coincided with strand biases of single nucleotide polymorphism rate. Our analysis also revealed that transcription of a gene is supported by an average of 2.7 genic promoters, among which one specific promoter, designated as a top promoter, substantially determines the expression level of the gene.

Plant Promoter Database (PPDB).

ppdb ( http://ppdb.agr.gifu-u.ac.jp ) is a web-based plant promoter database that provides promoter information of each gene in genomes of Arabidopsis, rice, poplar, and Physcomitrella patens. In this database, recognition of a promoter structure is achieved by annotating genome sequences with our sequence lists of bioinformatically identified octamers for core promoter structure (TATA boxes, Initiators, Y Patches, GA and CA Elements) and regulatory element groups (REGs), together with information of transcription start sites (TSSs) that have been experimentally identified. Our promoter elements are octamer sequences that show strongly biased localization profiles in the promoter region, extracted by the local distribution of short sequence (LDSS) analysis. In addition, REGs are linked with the information of the PLACE database and also with their physiological roles that are predicted using large-scale gene expression data.

Transcriptomic variation among six Arabidopsis thaliana accessions identified several novel genes controlling aluminium tolerance.

Differences in the expression levels of aluminium (Al) tolerance genes are a known determinant of Al tolerance among plant varieties. We combined transcriptomic analysis of six Arabidopsis thaliana accessions with contrasting Al tolerance and a reverse genetic approach to identify Al-tolerance genes responsible for differences in Al tolerance between accession groups. Gene expression variation increased in the signal transduction process under Al stress and in growth-related processes in the absence of stress. Co-expression analysis and promoter single nucleotide polymorphism searching suggested that both trans-acting polymorphisms of Al signal transduction pathway and cis-acting polymorphisms in the promoter sequences caused the variations in gene expression associated with Al tolerance. Compared with the wild type, Al sensitivity increased in T-DNA knockout (KO) lines for five genes, including TARGET OF AVRB OPERATION1 (TAO1) and an unannotated gene (At5g22530). These were identified from 53 Al-inducible genes showing significantly higher expression in tolerant accessions than in sensitive accessions. These results indicate that the difference in transcriptional signalling is partly associated with the natural variation in Al tolerance in Arabidopsis. Our study also demonstrates the feasibility of comparative transcriptome analysis by using natural genetic variation for the identification of genes responsible for Al stress tolerance.

Joint genetic and network analyses identify loci associated with root growth under NaCl stress in Arabidopsis thaliana.

Plants have evolved a series of tolerance mechanisms to saline stress, which perturbs physiological processes throughout the plant. To identify genetic mechanisms associated with salinity tolerance, we performed linkage analysis and genome-wide association study (GWAS) on maintenance of root growth of Arabidopsis thaliana in hydroponic culture with weak and severe NaCl toxicity. The top 200 single-nucleotide polymorphisms (SNPs) determined by GWAS could cumulatively explain approximately 70% of the variation observed at each stress level. The most significant SNPs were linked to the genes of ATP-binding cassette B10 and vacuolar proton ATPase A2. Several known salinity tolerance genes such as potassium channel KAT1 and calcium sensor SOS3 were also linked to SNPs in the top 200. In parallel, we constructed a gene co-expression network to independently verify that particular groups of genes work together to a common purpose. We identify molecular mechanisms to confer salt tolerance from both predictable and novel physiological sources and validate the utility of combined genetic and network analysis. Additionally, our study indicates that the genetic architecture of salt tolerance is responsive to the severity of stress. These gene datasets are a significant information resource for a following exploration of gene function.