Interaction between long noncoding RNA (lnc663) and microRNA (miR1128) regulates PDAT-like gene activity in bread wheat (Triticum aestivum L.).

Amylose, a starch subcomponent, can bind lipids within its helical groove and form an amylose-lipid complex, known as resistant starch type 5 (RS-5). RS contributes to lower glycaemic index of grain with health benefits. Unfortunately, genes involved in lipid biosynthesis in wheat grain remain elusive. Our study aims to characterize the lipid biosynthesis gene and its post-transcriptional regulation using the parent bread wheat variety 'C 306' and its EMS-induced mutant line 'TAC 75' varying in amylose content. Quantitative analyses of starch-bound lipids showed that 'TAC 75' has significantly higher lipid content in grains than 'C 306' variety. Furthermore, expression analyses revealed the higher expression of wheat phospholipid: diacylglycerol acyltransferase-like (PDAT-like) in the 'TAC 75' compared to the 'C 306'. Overexpression and ectopic expression of TaPDAT in yeast and tobacco leaf confirmed its ability to accumulate lipids in vivo. Enzyme activity assay showed that TaPDAT catalyzes the triacylglycerol synthesis by acylating 1,2-diacylglycerol. Interestingly, the long non-coding RNA, lnc663, was upregulated with the TaPDAT gene, while the miRNA, miR1128, downregulated in the 'TAC 75', indicating a regulatory relationship. The GFP reporter assay confirmed that the lnc663 acts as a positive regulator, and the miR1128 as a negative regulator of the TaPDAT gene, which controls lipid accumulation in wheat grain. Our findings outline TaPDAT-mediated biosynthesis of lipid accumulation and reveal the molecular mechanism of the lnc663 and miR1128 mediated regulation of the TaPDAT gene in wheat grain.

Rethinking underutilized cereal crops: pan-omics integration and green system biology.

MAIN CONCLUSION: Due to harsh lifestyle changes, in the present era, nutritional security is needed along with food security so it is necessary to include underutilized cereal crops (UCCs) in our daily diet to counteract the rising danger of human metabolic illness. We can attain both the goal of zero hunger and nutritional security by developing improved UCCs using advanced pan-omics (genomics, transcriptomics, proteomics, metabolomics, nutrigenomics, phenomics and ionomics) practices. Plant sciences research progressed profoundly since the last few decades with the introduction of advanced technologies and approaches, addressing issues of food demand of the growing population, nutritional security challenges and climate change. However, throughout the expansion and popularization of commonly consumed major cereal crops such as wheat and rice, other cereal crops such as millet, rye, sorghum, and others were impeded, despite their potential medicinal and nutraceutical qualities. Undoubtedly neglected underutilized cereal crops (UCCs) also have the capability to withstand diverse climate change. To relieve the burden of major crops, it is necessary to introduce the new crops in our diet in the way of UCCs. Introgression of agronomically and nutritionally important traits by pan-omics approaches in UCCs could be a defining moment for the population's well-being on the globe. This review discusses the importance of underutilized cereal crops, as well as the application of contemporary omics techniques and advanced bioinformatics tools that could open up new avenues for future study and be valuable assets in the development and usage of UCCs in the perspective of green system biology. The increased and improved use of UCCs is dependent on number of factors that necessitate a concerted research effort in agricultural sciences. The emergence of functional genomics with molecular genetics might gear toward the reawakening of interest in underutilized cereals crops. The need of this era is to focus on potential UCCs in advanced agriculture and breeding programmes. Hence, targeting the UCCs, might provide a bright future for better health and scientific rationale for its use.

Comparative transcriptome analyses revealed key genes involved in high amylopectin biosynthesis in wheat.

High amylopectin starch is an important modified starch for food processing industries. Despite a thorough understanding of starch biosynthesis pathway, the regulatory mechanism responsible for amylopectin biosynthesis is not well explored. The present study utilized transcriptome sequencing approach to understand the molecular basis of high amylopectin content in three high amylopectin mutant wheat lines ('TAC 6', 'TAC 358', and 'TAC 846') along with parent variety 'C 306'. Differential scanning calorimetry (DSC) of high amylopectin starch identified a high thermal transition temperature and scanning electron microscopy (SEM) revealed more spherical starch granules in mutant lines compared to parent variety. A set of 4455 differentially expressed genes (DEGs) were identified at two-fold compared to the parent variety in high amylopectin wheat mutants. At ten-fold, 279 genes, including two starch branching genes (SBEIIa and SBEIIb), were up-regulated and only 30 genes, including the starch debranching enzyme (DBE), were down-regulated. Among the genes, different isoforms of sucrose non-fermenting-1-related protein kinase-1 (TaSnRK1α2-3B and TaSnRK1α2-3D) and its regulatory subunit, sucrose non-fermenting-4 (SNF-4-2A, SNF-4-2B, and SNF-4-5D), were found to be highly up-regulated. Further, expression of the DEGs related to starch biosynthesis pathway and TaSnRK1α2 and SNF-4 was performed using qRT-PCR. High expression of TaSnRK1α2, SNF-4, and SBEII isoforms suggests their probable role in high amylopectin starch biosynthesis in grain endosperm. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03364-3.

Genetic dissection of quantitative traits loci identifies new genes for gelatinization parameters of starch and amylose-lipid complex (Resistant starch 5) in bread wheat.

Starch is a major component of cereal grains such as wheat. Physicochemical and functional properties of starch affect end-use food quality and nutrients. To improve cultivars that preserve superior starch quality, the genetic foundation of the wheat starch and amylose-lipid complex (ALc, Resistant starch type 5) gelatinization are needed. This genome-wide association (GWA) mapping used 192 wheat genotypes (previously reported) to generate SNPs using an enhanced version of sequencing termed ddRAD on the Illumina Hi-seq X platform and 3696 high-quality influential SNPs were filtered out. The heterozygosity and Fst ranges in five subpopulations were 0.31-0.40 and 0.18-0.30 respectively. Nucleotide diversity and PIC ranged from 0.21 (6A) to 0.32 (2A) and 0.29 (6A) to 0.39 (4D) respectively. The Shannon waiver index was 1.7 and the whole-genome LD decay was 22 Mb at r2 = 0.38. Following FDR, 23 and 8 SNPs showed association with starch properties in the year 2017 and 2018, respectively while 93 and 20 SNPs were associated with ALc gelatinization in the year 2017 and 2018 respectively. The identified potential new genes (GSK3-alpha, RING-type domain-containing protein, Tetratricopeptide repeat, Hexosyltransferase, GLP, SNF1, and WRKY transcription factor) within LD range (∼16 Kb to ∼15 Mb), BLUP value, and cis and trans-position of SNPs network provide valuable information for the future wheat breeding strategy for the improvement of the starch quality trait.

Genome-wide analysis of RING-type E3 ligase family identifies potential candidates regulating high amylose starch biosynthesis in wheat (Triticum aestivum L.).

In ubiquitin-mediated post-translational modifications, RING finger families are emerged as important E3 ligases in regulating biological processes. Amylose and amylopectin are two major constituents of starch in wheat seed endosperm. Studies have been found the beneficial effects of high amylose or resistant starch on health. The ubiquitin-mediated post-translational regulation of key enzymes for amylose/amylopectin biosynthesis (GBSSI and SBEII) is still unknown. In this study, the genome-wide analysis identified 1272 RING domains in 1255 proteins in wheat, which is not reported earlier. The identified RING domains classified into four groups-RING-H2, RING-HC, RING-v, RING-G, based on the amino acid residues (Cys, His) at metal ligand positions and the number of residues between them with the predominance of RING-H2 type. A total of 1238 RING protein genes were found to be distributed across all 21 wheat chromosomes. Among them, 1080 RING protein genes were identified to show whole genome/segmental duplication within the hexaploid wheat genome. In silico expression analysis using transcriptome data revealed 698 RING protein genes, having a possible role in seed development. Based on differential gene expression and correlation analysis of 36 RING protein genes in diverse (high and low) amylose mutants and parent, 10 potential RING protein genes found to be involved in high amylose biosynthesis and significantly associated with two starch biosynthesis genes; GBSSI and SBEIIa. Characterization of mutant lines using next-generation sequencing method identified unique mutations in 698 RING protein genes. This study signifies the putative role of RING-type E3 ligases in amylose biosynthesis and this information will be helpful for further functional validation and its role in other biological processes in wheat.

Understanding the regulatory relationship of abscisic acid and bZIP transcription factors towards amylose biosynthesis in wheat.

Starch is biosynthesized during seed development and this process is regulated by many bZIP proteins in bread wheat. Abscisic acid (ABA), an important phyto-hormone involved in various physiological processes mediated by bZIPs in plants including seed development. The 'Group A' TabZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis, rice and other cereal crops but their role in regulation of amylose biosynthesis in wheat is limited. In this study 83 'Group A' TabZIPs were characterized by gene expression analysis in wheat amylose mutants. A set of 17 TabZIPs was selected on the basis of differential expression (> 2 fold) in low and high amylose mutants from RNA-seq data and validated by qRT PCR. Based on qRT PCR and correlation analysis out of the 17 TabZIPs six differentially expressed candidate TabZIPs were identified, involving in high amylose biosynthesis. The TabZIP175.2, identified as upregulated in all high amylose lines and TabZIP90.2, TabZIP129.1, TabZIP132.2, TabZIP143 and TabZIP159.2 were found downregulated in all low amylose lines, after exogenous supply of ABA. Proximal promoter analysis of starch pathway genes revealed the presence of ABA-responsive elements (ABREs) that are putative binding sites for bZIPs. Collectively, these findings indicated the involvement of putative six candidate TabZIPs as transcriptional regulators of amylose related genes via an ABA-dependent pathway in wheat. This study could help the investigators to make an informed decision to edit wheat genome for high/low amylose content using gene-editing technologies.

Development and characterization of bZIP transcription factor based SSRs in wheat.

Assessment of existing diversity is the key for germplasm conservation and crop improvement. Wheat (Triticum aestivum L.) is among the most important cereal crop and consumed by two billion world's populations. DNA-based markers are predominantly used for diversity characterization because they are easy to develop and not influenced by environment. Among them microsatellites (simple sequence repeats, SSRs) are most suitable due to their genome-wide distribution, hypervariability and reproducibility for their applications in diversity, genetic improvement, and molecular breeding. bZIP transcription factors play major roles in plants in light and stress signalling, seed development, and defence. A total of 846 SSRs were identified from 370 wheat cDNA sequences and a sub-set of 35 polymorphic TabZIPMS (TriticumaestivumbZIP MicroSatellites) was used for diversity and genetic structure analysis of 92 Indian wheat varieties and related species. 114 SSR variants ranging from 2 to 5 per SSR locus were detected for 35 SSRs in the varieties. Average polymorphic information content (PIC) and observed heterozygosity was found to be 0.135 and 0.838, respectively. Thirty-four SSRs showed cross-transferability into different related species. Combined Bayesian model and Jaccard's similarity based genetic clustering analysis revealed two clusters of 80 bread wheat varieties and one separate cluster of related species. In this study, a total 35 novel bZIP-derived SSRs were identified in a set 370 bZIP genes and shown high polymorphism and cross-species transferability in wheat. The findings provide resources for future utilization in genetic resource conservation, trait introgression, breeding and varietal development.

Identification and characterization of long non-coding RNAs regulating resistant starch biosynthesis in bread wheat (Triticum aestivum L.).

Resistant starch (RS) also known as healthy starch has shown several health benefits. Enhancing the RS through genetic modification approaches has huge commercial importance. Regulatory RNA like long non-coding RNA (lncRNA) plays an important role in gene regulation. In this study, we mined 63 transcriptome datasets of wheat belonging to 35 genotypes representing two seed developmental stages. Contrasting expression of a subset of lncRNAs in RS mutant lines compared to parent wheat variety 'C 306' signifies their probable role in RS biosynthesis. Further, lncRNA- TCONS_00130663 showed strong positive correlation (r2 = 1) with LYPL gene and strong negative correlation with SBEIIb (r2 = -0.94). We found TCONS_00130663 as positive regulator of LYPL gene through interaction with miR1128. Based on relative expression, in silico interaction and DSC analysis we hypothesize the dual role of TCONS_00130663 in RS type 2 and type 5. The study provides a useful resource for functional mechanism of lncRNAs.

Novel intron length polymorphic (ILP) markers from starch biosynthesis genes reveal genetic relationships in Indian wheat varieties and related species.

Introns experience lesser selection pressure, thus are liable for higher polymorphism. Intron Length Polymorphic (ILP) markers designed from exon-flanking introns exploits this polymorphic potential and have been proved to be a robust co-dominant marker in eukaryotes. Wheat is among the most consumed cereal crop by majority of the word population. It is a rich source of calories in the form of stored starch. In the current study, starch biosynthesis genes were mined for development of ILP markers and their subsequent utilization for genetic characterization of popular Indian wheat varieties and transferability to wild relatives. Sixty-one markers generated 122 alleles and showed 77-88.5% transferability (mean PIC: 0.36) to the related species. A subset of markers showed clear genetic distinctions (Avg. genetic dissimilarity = 0.42) among Indian wheat varieties, signifying the importance of novel ILPs. 'Kenphad25' showed maximum genetic dissimilarity with 'K 8962' (0.82), while maximum genetic similarity was observed between 'Safed Lerma' and 'RAJ 4037' (0.1). This is the first report of ILP markers in wheat and will be a useful genomic resource for future germplasm conservation and molecular breeding studies.

Applications and challenges for efficient exploration of omics interventions for the enhancement of nutritional quality in rice (Oryza sativa L.).

Rice nutritional quality is one of the major concerns along with productivity enhancement to feed the continuously growing population. To address wide-spread malnutrition influencing global health, novel high yielding rice cultivars with better nutritional quality need to be bred. No doubt, the conventional breeding approaches have helped to decrease the gap between demand and supply for yield and nutrition; however, to meet today's demands more advanced approaches need to be employed. This review discusses approaches for the improvement of nutritional quality of rice and gauges the availability of omics resources. Recent omics advances providing numerous tools and techniques for the efficient exploration of genetic resources as well as for the understanding of molecular mechanism involved in the trait development have been discussed. Understanding of genes or loci governing different traits has been found to be effective in accelerating the crop breeding programs. In this regard, approaches like QTL (quantitative trait loci) mapping, genome-wide association study and genomic selection are discussed in light of their utilization for rice nutritional quality improvements. Efficient integration of different omics approaches is recognized as a promising way to achieve the desired improvements in rice cultivars. Therefore, advances in omics branches like transcriptomics, proteomics, ionomics, and metabolomics being efficiently explored for rice improvement programs are also addressed. This article provides a catalog of genes, loci, mutants, online resources and computational approaches for rice improvement. The information provided here will be helpful for pursuing present progress and directing rice research program for better future.

Pivotal role of bZIPs in amylose biosynthesis by genome survey and transcriptome analysis in wheat (Triticum aestivum L.) mutants.

Starch makes up 70% of the wheat grain, and is an important source of calories for humans, however, the overconsumption of wheat starch may contribute to nutrition-associated health problems. The challenge is to develop resistant starch including high amylose wheat varieties with health benefits. Adapting advance genomic approaches in EMS-induced mutant lines differing in amylose content, basic leucine zipper (bZIP) regulatory factors that may play role in controlling amylose biosynthesis were identified in wheat. bZIP transcription factors are key regulators of starch biosynthesis genes in rice and maize, but their role in regulating these genes in wheat is poorly understood. A genome-wide survey identified 370 wheat bZIPs, clustered in 11 groups, showing variations in amino acids composition and predicted physicochemical properties. Three approaches namely, whole transcriptome sequencing, qRT-PCR, and correlation analysis in contrasting high and low amylose mutants and their parent line identified 24 candidate bZIP (positive and negative regulators), suggesting bZIPs role in high amylose biosynthesis. bZIPs positive role in high amylose biosynthesis is not known. In silico interactome studies of candidate wheat bZIP homologs in Arabidopsis and rice identified their putative functional role. The identified bZIPs are involved in stress-related pathways, flower and seed development, and starch biosynthesis. An in-depth analysis of molecular mechanism of novel candidate bZIPs may help in raising and improving high amylose wheat varieties.