Long non-coding RNA-mediated epigenetic response for abiotic stress tolerance in plants.

Plants perceive environmental fluctuations as stress and confront several stresses throughout their life cycle individually or in combination. Plants have evolved their sensing and signaling mechanisms to perceive and respond to a variety of stresses. Epigenetic regulation plays a critical role in the regulation of genes, spatiotemporal expression of genes under stress conditions and imparts a stress memory to encounter future stress responses. It is quintessential to integrate our understanding of genetics and epigenetics to maintain plant fitness, achieve desired genetic gains with no trade-offs, and durable long-term stress tolerance. The long non-coding RNA >200 nts having no coding potential (or very low) play several roles in epigenetic memory, contributing to the regulation of gene expression and the maintenance of cellular identity which include chromatin remodeling, imprinting (dosage compensation), stable silencing, facilitating nuclear organization, regulation of enhancer-promoter interactions, response to environmental signals and epigenetic switching. The lncRNAs are involved in a myriad of stress responses by activation or repression of target genes and hence are potential candidates for deploying in climate-resilient breeding programs. This review puts forward the significant roles of long non-coding RNA as an epigenetic response during abiotic stresses in plants and the prospects of deploying lncRNAs for designing climate-resilient plants.

Genome-wide characterization of the soybean DOMAIN OF UNKNOWN FUNCTION 679 membrane protein gene family highlights their potential involvement in growth and stress response.

The DMP (DUF679 membrane proteins) family is a plant-specific gene family that encodes membrane proteins. The DMP family genes are suggested to be involved in various programmed cell death processes and gamete fusion during double fertilization in Arabidopsis. However, their functional relevance in other crops remains unknown. This study identified 14 genes from the DMP family in soybean (Glycine max) and characterized their physiochemical properties, subcellular location, gene structure, and promoter regions using bioinformatics tools. Additionally, their tissue-specific and stress-responsive expressions were analyzed using publicly available transcriptome data. Phylogenetic analysis of 198 DMPs from monocots and dicots revealed six clades, with clade-I encoding senescence-related AtDMP1/2 orthologues and clade-II including pollen-specific AtDMP8/9 orthologues. The largest clade, clade-III, predominantly included monocot DMPs, while monocot- and dicot-specific DMPs were assembled in clade-IV and clade-VI, respectively. Evolutionary analysis suggests that soybean GmDMPs underwent purifying selection during evolution. Using 68 transcriptome datasets, expression profiling revealed expression in diverse tissues and distinct responses to abiotic and biotic stresses. The genes Glyma.09G237500 and Glyma.18G098300 showed pistil-abundant expression by qPCR, suggesting they could be potential targets for female organ-mediated haploid induction. Furthermore, cis-acting regulatory elements primarily related to stress-, hormone-, and light-induced pathways regulate GmDMPs, which is consistent with their divergent expression and suggests involvement in growth and stress responses. Overall, our study provides a comprehensive report on the soybean GmDMP family and a framework for further biological functional analysis of DMP genes in soybean or other crops.

De Novo Mining and Validating Novel Microsatellite Markers to Assess Genetic Diversity in Maruca vitrata (F.), a Legume Pod Borer.

Maruca vitrata (Fabricius) is an invasive insect pest capable of causing enormous economic losses to a broad spectrum of leguminous crops. Microsatellites are valuable molecular markers for population genetic studies; however, an inadequate number of M. vitrata microsatellite loci are available to carry out population association studies. Thus, we utilized this insect's public domain databases for mining expressed sequence tags (EST)-derived microsatellite markers. In total, 234 microsatellite markers were identified from 10053 unigenes. We discovered that trinucleotide repeats were the most predominant microsatellite motifs (61.53%), followed by dinucleotide repeats (23.50%) and tetranucleotide repeats (14.95%). Based on the analysis, twenty-five markers were selected for validation in M. vitrata populations collected from various regions of India. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) ranged from 2 to 5; 0.00 to 0.80; and 0.10 to 0.69, respectively. The polymorphic loci showed polymorphism information content (PIC), ranging from 0.09 to 0.72. Based on the genetic distance matrix, the unrooted neighbor-joining dendrogram differentiated the selected populations into two discrete groups. The SSR markers developed and validated in this study will be helpful in population-level investigations of M. vitrata to understand the gene flow, demography, dispersal patterns, biotype differentiation, and host dynamics.

Comparative transcriptome analysis, unfolding the pathways regulating the seed-size trait in cultivated lentil (Lens culinaris Medik.).

Market class, cooking time, quality, and milled grain yield are largely influenced by the seed size and shape of the lentil (Lens culinaris Medik.); thus, they are considered to be important quality traits. To unfold the pathways regulating seed size in lentils, a transcriptomic approach was performed using large-seeded (L4602) and small-seeded (L830) genotypes. The study has generated nearly 375 million high-quality reads, of which 98.70% were properly aligned to the reference genome. Among biological replicates, very high similarity in fragments per kilobase of exon per million mapped fragments values (R > 0.9) showed the consistency of RNA-seq results. Various differentially expressed genes associated mainly with the hormone signaling and cell division pathways, transcription factors, kinases, etc. were identified as having a role in cell expansion and seed growth. A total of 106,996 unigenes were used for differential expression (DE) analysis. String analysis identified various modules having certain key proteins like Ser/Thr protein kinase, seed storage protein, DNA-binding protein, microtubule-associated protein, etc. In addition, some growth and cell division-related micro-RNAs like miR3457 (cell wall formation), miR1440 (cell proliferation and cell cycles), and miR1533 (biosynthesis of plant hormones) were identified as having a role in seed size determination. Using RNA-seq data, 5254 EST-SSR primers were generated as a source for future studies aiming for the identification of linked markers. In silico validation using Genevestigator® was done for the Ser/Thr protein kinase, ethylene response factor, and Myb transcription factor genes. It is of interest that the xyloglucan endotransglucosylase gene was found differentially regulated, suggesting their role during seed development; however, at maturity, no significant differences were recorded for various cell wall parameters including cellulose, lignin, and xylose content. This is the first report on lentils that has unfolded the key seed size regulating pathways and unveiled a theoretical way for the development of lentil genotypes having customized seed sizes.

Identification and characterization of three nearly identical linalool/nerolidol synthase from Acorus calamus.

Acorus calamus is a perennial aromatic medicinal plant from the Acorusaceae family, known for its pharmaceutical and medicinal value. A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of volatile organic compounds (VOCs) in rhizomes and leaves of A. calamus essential oil. Here, we performed VOC profiling and transcriptome-based identification and functional characterization of terpene synthase (TPS) genes. A total of 110 VOCs were detected from the rhizomes and leaves of A. calamus, and some VOCs showed significant differences between them. The further transcriptome-based analysis led to the identification of six putative TPSs genes. In phylogenetic analysis, three TPSs belonged to the TPS-g clade, one to each of the TPS-a, TPS-c, and TPS-e clades. The heterologous E. coli-based expression of recombinant TPSs identified three genes (AcTPS3, AcTPS4, and AcTPS5) as bifunctional linalool/nerolidol synthase. The correlation of TPS gene expression and VOC metabolite profiles supported the function of these genes in A. calamus. Our findings provide a roadmap for future efforts to enhance the molecular mechanisms of terpene biosynthesis and our understanding of Acorus-insect interactions.

Correction to: Identification of novel QTLs for late leaf spot resistance and validation of a major rust QTL in peanut (Arachis hypogaea L.).

[This corrects the article DOI: 10.1007/s13205-020-02446-4.].

Identification of novel QTLs for late leaf spot resistance and validation of a major rust QTL in peanut (Arachis hypogaea L.).

Co-occurrence of two devastating foliar-fungal diseases of peanut, viz., late leaf spot (LLS), and rust may cause heavy yield loss besides adversely affecting the quality of kernel and fodder. This study reports the mapping of seven novel stress-related candidate EST-SSRs in a region having major QTLs for LLS and rust diseases using an F2 mapping population (GJG17 × GPBD4) consisting of 328 individuals. The parental polymorphism using 1311 SSRs revealed 84 SSRs (6.4%) as polymorphic and of these 70 SSRs could be mapped on 14 linkage groups (LG). QTL analysis has identified a common QTL (LLSQTL1/RustQTL) for LLS and rust diseases in the map interval of 1.41 cM on A03 chromosome, explaining 47.45% and 70.52% phenotypic variations, respectively. Another major QTL for LLS (LLSQTL1), explaining a 29.06% phenotypic variation was also found on LG_A03. A major rust QTL has been validated which was found harboring R-gene and resistance-related genes having a role in inducing hypersensitive response (HR). Further, 23 linked SSRs including seven novel EST-SSRs were also validated in 177 diverse Indian groundnut genotypes. Twelve genotypes resistant to both LLS and rust were found carrying the common (rust and LLS) QTL region, LLS QTL region, and surrounding regions. These identified and validated candidate EST-SSR markers would be of great use for the peanut breeding groups working for the improvement of foliar-fungal disease resistance.

Characterization of terpene synthase genes potentially involved in black fig fly (Silba adipata) interactions with Ficus carica.

The black fig fly (Silba adipata) is one of the major pests of figs worldwide. This study investigated the effect of pollination on black fig fly infestation and volatile emission during fruit development of facultative parthenocarpic Ficus carica. The results from in-field oviposition preference of black fig fly, olfactory analysis, and fruit volatile profiles indicate that the black fig fly gave a strong preference to unpollinated figs that showed higher emissions of volatile organic compounds. Terpenes are known to be important compounds determining many insect-plant interactions, so we report a transcriptome-based identification and functional characterization of a terpene synthase (TPS) gene family in F. carica. The protein expression in Escherichia coli of eight terpene synthases (TPSs) revealed that three were monoterpene synthases belonging to the TPS-b clade, with FcTPS6 catalyzing the formation of 1,8-cineole while the other two converted GPP into linalool. Four sesquiterpene synthases from the TPS-a clade catalyze the formation of germacrene D (FcTPS1), E-ß-caryophyllene (FcTPS2), cadinene (FcTPS3) and δ-elemene (FcTPS5) while one sesquiterpene synthase FcTPS4 from the TPS-b clade showed nerolidol synthase activity. Most of the enzymatic products closely matched the volatile terpenes emitted from fig fruits and all the genes were expressed during fruit development. This study provides new insights into fig-insect interactions and understanding the molecular mechanisms of terpene biosynthesis and could provide the foundations for sustainable pest management strategies.

Unraveling the mechanisms of resistance to Sclerotium rolfsii in peanut (Arachis hypogaea L.) using comparative RNA-Seq analysis of resistant and susceptible genotypes.

Stem rot, a devastating fungal disease of peanut, is caused by Sclerotium rolfsii. RNA-sequencing approaches have been used to unravel the mechanisms of resistance to stem rot in peanut over the course of fungal infection in resistant (NRCG-CS85) and susceptible (TG37A) genotypes under control conditions and during the course of infection. Out of about 290 million reads, nearly 251 million (92.22%) high-quality reads were obtained and aligned to the Arachis duranensis and Arachis ipaensis genomes with the average mapping of 78.91% and 78.61%, respectively. In total, about 48.6% of genes were commonly regulated, while approximately 21.8% and 29.6% of uniquely regulated genes from A. duranensis and A. ipaensis genomes, respectively, were identified. Several annotated transcripts, such as receptor-like kinases, jasmonic acid pathway enzymes, and transcription factors (TFs), including WRKY, Zinc finger protein, and C2-H2 zinc finger, showed higher expression in resistant genotypes upon infection. These transcripts have a known role in channelizing the downstream of pathogen perception. The higher expression of WRKY transcripts might have induced the systemic acquired resistance (SAR) by the activation of the jasmonic acid defense signaling pathway. Furthermore, a set of 30 transcripts involved in the defense mechanisms were validated with quantitative real-time PCR. This study suggested PAMP-triggered immunity as a probable mechanism of resistance, while the jasmonic acid signaling pathway was identified as a possible defense mechanism in peanut. The information generated is of immense importance in developing more effective ways to combat the stem rot disease in peanut.

Analysis of apocarotenoid volatiles during the development of Ficus carica fruits and characterization of carotenoid cleavage dioxygenase genes.

In plants the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including ß-ionone, 6-methyl-5-hepten-2-ol, and α-ionone; these compounds are important in herbivore-plant communication. Combined chemical, biochemical, and molecular studies were conducted to evaluate the differential accumulation of carotenoids and volatile apocarotenoids during the development of pollinated and parthenocarpic fig fruits. Pollinated fig fruits showed less emission of apocarotenoid volatiles than the parthenocarpic figs, while in the case of carotenoid pigments, pollinated figs manifested higher accumulation. The apocarotenoids, 6-methyl-5-hepten-2-ol and ß-cyclogeraniol, showed a marked increase after the two weeks of hand-pollination in pollinated and parthenocarpic figs; but afterwards these volatile levels decreased during further fruit development. In addition, we report a transcriptome-based identification and functional characterization of the carotenoid cleavage dioxygenase (FcCCD) genes. These genes were overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant FcCCD1A enzyme showed specificity for the 9,10 (9',10') double bond position of cyclic carotenoids to generate α-ionone and ß-ionone, while FcCCD1B cleaved lycopene and an acyclic moiety of δ-carotene, producing 6-methyl-5-hepten-2-one. The qRT-PCR analysis of FcCCD genes revealed differential gene expression during fig fruit development. Our results suggest a role for the FcCCD1genes in apocarotenoid biosynthesis in fig fruits.

Regulation of antioxidant mechanisms by AtDREB1A improves soil-moisture deficit stress tolerance in transgenic peanut (Arachis hypogaea L.).

The present study evaluated the soil-moisture deficit stress tolerance of AtDREB1A transgenic peanut lines during reproductive stages using lysimetric system under controlled glasshouse conditions. The antioxidant activities of AtDREB1A transgenic lines were measured by biochemical assays. The transgenic peanut lines recorded significantly lower accumulation of malondialdehyde and hydrogen peroxide than the wild-type. Whereas, specific activity of catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and ascorbic acid were found to be significantly higher in transgenic lines than in the wild-type line under drought stress. The results showed that the transgenic lines expressed lower oxidative damage than wild-type and could protect themselves from the elevated levels of reactive oxygen species under drought stress. This could be attributed to the regulation of various stress-inducible genes by AtDREB1A transcription factor. Improved photosynthetic and growth parameters were also recorded in transgenic lines over wild-type under drought stress. Improved physio-biochemical mechanisms in transgenic peanut lines might have resulted in improved growth-related traits as significant correlations were observed between physio-biochemical parameters and growth-related traits under drought stress. The potential target genes of AtDREB1A transcription factor in transgenic peanut lines during drought stress were identified, which helped in understanding the molecular mechanisms of DREB-regulated stress responses. The transgenic line D6 reported the best physio-biochemical mechanisms and growth-related parameters under drought stress over other transgenic lines and wild-type, suggesting it may be used to develop high yielding and terminal drought-tolerant peanut varieties.

Insights into the Indian Peanut Genotypes for ahFAD2 Gene Polymorphism Regulating Its Oleic and Linoleic Acid Fluxes.

In peanut (Arachis hypogaea L.), the customization of fatty acid profile is an evolving area to fulfill the nutritional needs in the modern market. A total of 174 peanut genotypes, including 167 Indian cultivars, 6 advanced breeding lines and "SunOleic95R"-a double mutant line, were investigated using AS-PCRs, CAPS and gene sequencing for the ahFAD2 allele polymorphism, along with its fatty acid compositions. Of these, 80 genotypes were found having substitution (448G>A) mutation only in ahFAD2A gene, while none recorded 1-bp insertion (441_442insA) mutation in ahFAD2B gene. Moreover, 22 wild peanut accessions found lacking both the mutations. Among botanical types, the ahFAD2A mutation was more frequent in ssp. hypogaea (89%) than in ssp. fastigiata (17%). This single allele mutation, found affecting not only oleic to linoleic acid fluxes, but also the composition of other fatty acids in the genotypes studied. Repeated use of a few selected genotypes in the Indian varietal development programs were also eminently reflected in its ahFAD2 allele polymorphism. Absence of known mutations in the wild-relatives indicated the possible origin of these mutations, after the allotetraploidization of cultivated peanut. The SNP analysis of both ahFAD2A and ahFAD2B genes, revealed haplotype diversity of 1.05% and 0.95%, while Ka/Ks ratio of 0.36 and 0.39, respectively, indicating strong purifying selection pressure on these genes. Cluster analysis, using ahFAD2 gene SNPs, showed presence of both mutant and non-mutant genotypes in the same cluster, which might be due the presence of ahFAD2 gene families. This investigation provided insights into the large number of Indian peanut genotypes, covering various aspects related to O/L flux regulation and ahFAD2 gene polymorphism.

De novo Transcriptome Sequencing to Dissect Candidate Genes Associated with Pearl Millet-Downy Mildew (Sclerospora graminicola Sacc.) Interaction.

Understanding the plant-pathogen interactions is of utmost importance to design strategies for minimizing the economic deficits caused by pathogens in crops. With an aim to identify genes underlying resistance to downy mildew, a major disease responsible for productivity loss in pearl millet, transcriptome analysis was performed in downy mildew resistant and susceptible genotypes upon infection and control on 454 Roche NGS platform. A total of ~685 Mb data was obtained with 1 575 290 raw reads. The raw reads were pre-processed into high-quality (HQ) reads making to ~82% with an average of 427 bases. The assembly was optimized using four assemblers viz. Newbler, MIRA, CLC and Trinity, out of which MIRA with a total of 14.10 Mb and 90118 transcripts proved to be the best for assembling reads. Differential expression analysis depicted 1396 and 936 and 1000 and 1591 transcripts up and down regulated in resistant inoculated/resistant control and susceptible inoculated/susceptible control respectively with a common of 3644 transcripts. The pathways for secondary metabolism, specifically the phenylpropanoid pathway was up-regulated in resistant genotype. Transcripts up-regulated as a part of defense response included classes of R genes, PR proteins, HR induced proteins and plant hormonal signaling transduction proteins. The transcripts for skp1 protein, purothionin, V type proton ATPase were found to have the highest expression in resistant genotype. Ten transcripts, selected on the basis of their involvement in defense mechanism were validated with qRT-PCR and showed positive co-relation with transcriptome data. Transcriptome analysis evoked potentials of hypersensitive response and systemic acquired resistance as possible mechanism operating in defense mechanism in pearl millet against downy mildew infection.

Novel and Stress Relevant EST Derived SSR Markers Developed and Validated in Peanut.

With the aim to increase the number of functional markers in resource poor crop like cultivated peanut (Arachis hypogaea), large numbers of available expressed sequence tags (ESTs) in the public databases, were employed for the development of novel EST derived simple sequence repeat (SSR) markers. From 16424 unigenes, 2784 (16.95%) SSRs containing unigenes having 3373 SSR motifs were identified. Of these, 2027 (72.81%) sequences were annotated and 4124 gene ontology terms were assigned. Among different SSR motif-classes, tri-nucleotide repeats (33.86%) were the most abundant followed by di-nucleotide repeats (27.51%) while AG/CT (20.7%) and AAG/CTT (13.25%) were the most abundant repeat-motifs. A total of 2456 EST-SSR novel primer pairs were designed, of which 366 unigenes having relevance to various stresses and other functions, were PCR validated using a set of 11 diverse peanut genotypes. Of these, 340 (92.62%) primer pairs yielded clear and scorable PCR products and 39 (10.66%) primer pairs exhibited polymorphisms. Overall, the number of alleles per marker ranged from 1-12 with an average of 3.77 and the PIC ranged from 0.028 to 0.375 with an average of 0.325. The identified EST-SSRs not only enriched the existing molecular markers kitty, but would also facilitate the targeted research in marker-trait association for various stresses, inter-specific studies and genetic diversity analysis in peanut.