Transcriptome unveiled the gene expression patterns of root architecture in drought-tolerant and sensitive wheat genotypes.

Drought is a big challenge for agricultural production. Root attributes are the important target traits for breeding high-yielding sustainable wheat varieties against ever changing climatic conditions. However, the transcriptomic of wheat concerning root architecture remained obscure. Here, we explored RNA-Seq based transcriptome to dissect putative genes involved in root system variations in naturally occurring six genotypes (drought-tolerant and sensitive) of wheat. Global RNA-Seq based root transcriptome analysis revealed single nucleotide polymorphisms (SNPs) variations and differentially expressed genes. Putative 56 SNPs were identified related to 15 genes involved in root architecture. Enrichment of these genes using GO terms demonstrated that differentially expressed genes (DEGs) are divided into sub-categories implicated in molecular functions, cellular components and biological processes. The KEGG analysis of DEGs in each comparison of genotype include metabolic, biosynthesis of secondary metabolites, microbial metabolism in diverse environments and biosynthesis of antibiotics. A deeper insight into DEGs unveiled various pathways involved in drought response and positive gravitropism. These genes belong to various transcription factor families such as DOF, C3H, MYB, and NAC involved in root developmental and stress-related pathways. Local White and UZ-11-CWA-8, which are drought-tolerant genotypes, harbor over-representation of most of DEGs or transcription factors. Notably, a microtubule-associated protein MAPRE1 belonging to RP/EB family recruited in positive gravitropism was enriched. Real-time PCR analysis revealed expression of MAPRE1 and PAL genes is consistent with RNA-seq data. The presented data and genetic resources seem valuable for providing genes involved in the root system architecture of drought-tolerant and susceptible genotypes.

Genome-wide investigation and functional analysis of RNA editing sites in wheat.

Wheat is an important cereal and half of the world population consumed it. Wheat faces environmental stresses and different techniques (CRISPR, gene silencing, GWAS, etc.) were used to enhance its production but RNA editing (RESs) is not fully explored in wheat. RNA editing has a special role in controlling environmental stresses. The genome-wide identification and functional characterization of RESs in different types of wheat genotypes was done. We employed six wheat genotypes by RNA-seq analyses to achieve RESs. The findings revealed that RNA editing events occurred on all chromosomes equally. RNA editing sites were distributed randomly and 10-12 types of RESs were detected in wheat genotypes. Higher number of RESs were detected in drought-tolerant genotypes. A-to-I RNA editing (2952, 2977, 1916, 2576, 3422, and 3459) sites were also identified in six wheat genotypes. Most of the genes were found to be engaged in molecular processes after a Gene Ontology analysis. PPR (pentatricopeptide repeat), OZ1 (organelle zinc-finger), and MORF/RIP gene expression levels in wheat were also examined. Normal growth conditions diverge gene expression of these three different gene families, implying that normal growth conditions for various genotypes can modify RNA editing events and have an impact on gene expression levels. While the expression of PPR genes was not change. We used Variant Effect Predictor (VEP) to annotate RNA editing sites, and Local White had the highest RESs in the CDS region of the protein. These findings will be useful for prediction of RESs in other crops and will be helpful in drought tolerance development in wheat.

Evolution of Deeper Rooting 1-like homoeologs in wheat entails the C-terminus mutations as well as gain and loss of auxin response elements.

Root growth angle (RGA) in response to gravity controlled by auxin is a pertinent target trait for obtainment of higher yield in cereals. But molecular basis of this root architecture trait remain obscure in wheat and barley. We selected four cultivars two each for wheat and barley to unveil the molecular genetic mechanism of Deeper Rooting 1-like gene which controls RGA in rice leading to higher yield under drought imposition. Morphological analyses revealed a deeper and vertically oriented root growth in "NARC 2009" variety of wheat than "Galaxy" and two other barley cultivars "Scarlet" and "ISR42-8". Three new homoeologs designated as TaANDRO1-like, TaBNDRO1-like and TaDNDRO1-like corresponding to A, B and D genomes of wheat could be isolated from "NARC 2009". Due to frameshift and intronization/exonization events the gene structures of these paralogs exhibit variations in size. DRO1-like genes with five distinct domains prevail in diverse plant phyla from mosses to angiosperms but in lower plants their differentiation from LAZY, NGR and TAC1 (root and shoot angle genes) is enigmatic. Instead of IGT as denominator motif of this family, a new C-terminus motif WxxTD in the V-domain is proposed as family specific motif. The EAR-like motif IVLEM at the C-terminus of the TaADRO1-like and TaDDRO1-like that diverged to KLHTLIPNK in TaBDRO1-like and HvDRO1-like is the hallmark of these proteins. Split-YFP and yeast two hybrid assays complemented the interaction of TaDRO1-like with TOPLESS-a repressor of auxin regulated root promoting genes in plants-through IVLEM/KLHTLIPNK motif. Quantitative RT-PCR revealed abundance of DRO1-like RNA in root tips and spikelets while transcript signals were barely detectable in shoot and leaf tissues. Interestingly, wheat exhibited stronger expression of TaBDRO1-like than barley (HvDRO1-like), but TaBDRO1-like was the least expressing among three paralogs. The underlying cause of this expression divergence seems to be the presence of AuxRE motif TGTCTC and core TGTC with a coupling AuxRE-like motif ATTTTCTT proximal to the transcriptional start site in TaBDRO1-like and HvDRO1-like promoters. This is evident from binding of ARF1 to TGTCTC and TGTC motifs of TaBDRO1-like as revealed by yeast one-hybrid assay. Thus, evolution of DRO1-like wheat homoeologs might incorporate the C-terminus mutations as well as gain and loss of AuxREs and other cis-regulatory elements during expression divergence. Since root architecture is an important target trait for wheat crop improvement, therefore DRO1-like genes have potential applications in plant breeding for enhancement of plant productivity by the use of modern genome editing approaches.