Understanding the role of P-type ATPases in regulating pollen fertility and development in pigeonpea.

The P-type ATPase superfamily genes are the cation and phospholipid pumps that transport ions across the membranes by hydrolyzing ATP. They are involved in a diverse range of functions, including fundamental cellular events that occur during the growth of plants, especially in the reproductive organs. The present work has been undertaken to understand and characterize the P-type ATPases in the pigeonpea genome and their potential role in anther development and pollen fertility. A total of 59 P-type ATPases were predicted in the pigeonpea genome. The phylogenetic analysis classified the ATPases into five subfamilies: eleven P1B, eighteen P2A/B, fourteen P3A, fifteen P4, and one P5. Twenty-three pairs of P-type ATPases were tandemly duplicated, resulting in their expansion in the pigeonpea genome during evolution. The orthologs of the reported anther development-related genes were searched in the pigeonpea genome, and the expression profiling studies of specific genes via qRT-PCR in the pre- and post-meiotic anther stages of AKCMS11A (male sterile), AKCMS11B (maintainer) and AKPR303 (fertility restorer) lines of pigeonpea was done. Compared to the restorer and maintainer lines, the down-regulation of CcP-typeATPase22 in the post-meiotic anthers of the male sterile line might have played a role in pollen sterility. Furthermore, the strong expression of CcP-typeATPase2 in the post-meiotic anthers of restorer line and CcP-typeATPase46, CcP-typeATPase51, and CcP-typeATPase52 in the maintainer lines, respectively, compared to the male sterile line, clearly indicates their potential role in developing male reproductive organs in pigeonpea.

Comparative transcriptome analysis of two contrasting genotypes provides new insights into the drought response mechanism in pigeon pea (Cajanus cajan L. Millsp.).

BACKGROUND: Despite plant's ability to adapt and withstand challenging environments, drought poses a severe threat to their growth and development. Although pigeon pea is already quite resistant to drought, the prolonged dehydration induced by the aberrant climate poses a serious threat to their survival and productivity. OBJECTIVE: Comparative physiological and transcriptome analyses of drought-tolerant (CO5) and drought-sensitive (CO1) pigeon pea genotypes subjected to drought stress were carried out in order to understand the molecular basis of drought tolerance in pigeon pea. METHODS: The transcriptomic analysis allowed us to examine how drought affects the gene expression of C. cajan. Using bioinformatics tools, the unigenes were de novo assembled, annotated, and functionally evaluated. Additionally, a homology-based sequence search against the droughtDB database was performed to identify the orthologs of the DEGs. RESULTS: 1102 potential drought-responsive genes were found to be differentially expressed genes (DEGs) between drought-tolerant and drought-sensitive genotypes. These included Abscisic acid insensitive 5 (ABI5), Nuclear transcription factor Y subunit A-7 (NF-YA7), WD40 repeat-containing protein 55 (WDR55), Anthocyanidin reductase (ANR) and Zinc-finger homeodomain protein 6 (ZF-HD6) and were highly expressed in the tolerant genotype. Further, GO analysis revealed that the most enriched classes belonged to biosynthetic and metabolic processes in the biological process category, binding and catalytic activity in the molecular function category and nucleus and protein-containing complex in the cellular component category. Results of KEGG pathway analysis revealed that the DEGs were significantly abundant in signalling pathways such as plant hormone signal transduction and MAPK signalling pathways. Consequently, in our investigation, we have identified and validated by qPCR a group of genes involved in signal reception and propagation, stress-specific TFs, and basal regulatory genes associated with drought response. CONCLUSION: In conclusion, our comprehensive transcriptome dataset enabled the discovery of candidate genes connected to pathways involved in pigeon pea drought response. Our research uncovered a number of unidentified genes and transcription factors that could be used to understand and improve susceptibility to drought.

Whole chloroplast genome-specific non-synonymous SNPs reveal the presence of substantial diversity in the pigeonpea mini-core collection.

To unravel the plastid genome diversity among the cultivated groups of the pigeonpea germplasm, we characterized the SNP occurrence and distribution of 142 pigeonpea mini-core collections based on their reference-based assembly of the chloroplast genome. A total of 8921 SNPs were found, which were again filtered and finally 3871 non-synonymous SNPs were detected and used for diversity estimates. These 3871 SNPs were classified into 12 groups and were present in only 44 of the 125 genes, demonstrating the presence of a precise mechanism for maintaining the whole chloroplast genome throughout evolution. The Acetyl-CoA carboxylase D gene possesses the maximum number of SNPs (12.29%), but the Adenosine Tri-Phosphate synthatase cluster genes (atpA, atpB, atpE, atpF, atpH, and atpI) altogether bear 43.34% of the SNPs making them most diverse. Various diversity estimates, such as the number of effective alleles (1.013), Watterson's estimate (0.19), Tajima's D ( - 3.15), Shannon's information index (0.036), suggest the presence of less diversity in the cultivated gene pool of chloroplast genomes. The genetic relatedness estimates based on pairwise correlations were also in congruence with these diversity descriptors and indicate the prevalence of rare alleles in the accessions. Interestingly, no stratification was observed either through STRUCTURE, PCoA, or phylogenetic analysis, indicating the common origin of the chloroplast in all the accessions used, irrespective of their geographical distribution. Further 6194 Cleaved Amplified Polymorphic Sequences (CAPS) markers for 531 SNPs were developed and validated in a selected set of germplasm. Based on these results, we inferred that all of the cultivated gene pools of pigeonpea have a common origin for the chloroplast genome and they possess less diversity in protein-coding regions, indicating a stable and evolved plastid genome. At the same time, all diversity analysis indicates the occurrence of rare alleles, suggesting the suitability of the mini-core collection in future pigeonpea improvement programs. In addition, the development of chloroplast genome-based CAPS markers would have utility in pigeonpea breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03785-8.

Genome-wide identification and characterization of GRAS gene family in pigeonpea (Cajanus cajan (L.) Millspaugh).

The GRAS proteins are plant-specific transcription factors (TFs) that play a crucial role in various plant physiological processes, including tissue development and stress responses. To date, GRAS family has been comprehensively characterized in Arabidopsis, soybean, rice, chickpea and other plant species. To understand the structural and functional aspects of pigeonpea (C. cajan), we identified 60 putative GRAS (CcGRAS) genes from pigeonpea genome and further analysed their physicochemical properties, subcellular locations, evolutionary classification, exon-intron structures, conserved domains, gene duplication events and cis-promoter regions. Based on the sequence similarity, CcGRAS family was clustered into 9 subfamilies and the genes with a similar structure and motif distribution were clustered in the same group. The gene duplication studies revealed that these genes were derived from tandem and dispersed duplication events. The cis-promoter regulatory analysis of CcGRAS genes indicated the presence of three types of cis-acting elements including light-responsive, hormone-responsive and plant growth and development related. The expression profiling of CcGRAS genes revealed their tissue-specific functions and differential nature. Collectively, this study highlights relevant functional and regulatory elements of GRAS family in pigeonpea creating a significant resource for future functional studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03782-x.

Genome-wide survey, molecular evolution and expression analysis of Auxin Response Factor (ARF) gene family indicating their key role in seed number per pod in pigeonpea (C. cajan L. Millsp.).

Auxin Response Factors (ARF) are a family of transcription factors that mediate auxin signalling and regulate multiple biological processes. Their crucial role in increasing plant biomass/yield influenced this study, where a systematic analysis of ARF gene family was carried out to identify the key proteins controlling embryo/seed developmental pathways in pigeonpea. A genome-wide scan revealed the presence of 12 ARF genes in pigeonpea, distributed across the chromosomes 1, 3, 4, 8 and 11. Domain analysis of ARF proteins showed the presence of B3 DNA binding, AUX response, and IAA domains. Majority of them are of nuclear origin, and do not exhibit the level of genomic expansion as observed in Glycine max (51 members). The duplication events seem to range from 31.6 to 42.3 million years ago (mya). Promoter analysis revealed the presence of multiple cis-acting elements related to stress responses, hormone signalling and other development processes. The expression atlas data highlighted the expression of CcARF8 in hypocotyl, bud and flower whereas, CcARF7 expression was significantly high in pod. The real-time expression of CcARF2, CcARF3 and CcARF18 was highest in genotypes with high seed number indicating their key role in regulating embryo development and determining seed set in pigeonpea.

The chromosome-scale genome assembly of cluster bean provides molecular insight into edible gum (galactomannan) biosynthesis family genes.

Cluster bean (Cyamopsis tetragonoloba (L.) Taub 2n = 14, is commonly known as Guar. Apart from being a vegetable crop, it is an abundant source of a natural hetero-polysaccharide called guar gum or galactomannan. Here, we are reporting a chromosome-scale reference genome assembly of a popular cluster bean cultivar RGC-936, by combining sequencing data from Illumina, 10X Genomics, Oxford Nanopore technologies. An initial assembly of 1580 scaffolds with an N50 value of 7.12 Mb was generated and these scaffolds were anchored to a high density SNP linkage map. Finally, a genome assembly of 550.31 Mb (94% of the estimated genome size of ~ 580 Mb (through flow cytometry) with 58 scaffolds was obtained, including 7 super scaffolds with a very high N50 value of 78.27 Mb. Phylogenetic analysis using single copy orthologs among 12 angiosperms showed that cluster bean shared a common ancestor with other legumes 80.6 MYA. No evidence of recent whole genome duplication event in cluster bean was found in our analysis. Further comparative transcriptomics analyses revealed pod-specific up-regulation of genes encoding enzymes involved in galactomannan biosynthesis. The high-quality chromosome-scale cluster bean genome assembly will facilitate understanding of the molecular basis of galactomannan biosynthesis and aid in genomics-assisted improvement of cluster bean.

A comprehensive review on leguminous galactomannans: structural analysis, functional properties, biosynthesis process and industrial applications.

Galactomannans are neutral hemicellulose biopolymers that strengthen the plant cell walls by interacting with cellulose in the form of storage polysaccharides. They are abundant in nature and are majorly present in the secondary walls of flowering plants. They are primarily extracted from the leguminous seed endosperms and display a wide variation at the structural and abundance level amongst different plant species. Over the last few decades, galactomannans have attracted huge attention due to their unique functional, solution and rheological properties, generally defined by their molar mass and the degree of substitution by galactosyl side chain, which differs between plants. Further, they are nontoxic, originate from renewable sources, fairly inexpensive, and are amenable to both chemical and biochemical modification. Moreover, excellent thickening, stabilizing and gelling abilities of these biopolymers have found extensive use in food, pharmaceutical, biomedical and cosmetic industries. Significant progress has been made to identify and characterize the genes responsible for biosynthesis of galactomannan along with the elucidation of controlling networks by using genetic, bioinformatics and biochemical approaches. This is the first comprehensive coverage on galactomannans which combines detailed structural and physicochemical properties as well as biology associated with the metabolism of galactomannans. It also focuses on different leguminous sources leading to various food and non-food applications of galactomannans.

Structural and functional analysis of CCT family genes in pigeonpea.

BACKGROUND: Pigeonpea (Cajanus cajan L.) is a photoperiod-sensitive short-day plant. Understanding the flowering-related genes is critical to developing photoperiod insensitive cultivars. METHODS: The CCT family genes were identified using 'CCT DOMAIN PROTEIN' as a keyword and localized on the chromosomes using the BLAST search option available at the LIS database. The centromeric positions were identified through BLAST search using the centromeric repeat sequence of C. cajan as a query against the chromosome-wise FASTA files downloaded from the NCBI database. The CCT family genes were classified based on additional domains and/or CCT domains. The orthologous and phylogenetic relationships were inferred using the OrthoFinder and MEGA 10.1 software, respectively. The CCT family genes' expression level in photoperiod-sensitive and insensitive genotypes was compared using RNA-seq data and qRT-PCR analysis. RESULTS: We identified 33 CCT family genes in C. cajan distributed on ten chromosomes and nine genomic scaffolds. They were classified into CMF-type, COL-type, PRR-type, and GTCC- type. The CCT family genes of legumes exhibited an extensive orthologous relationship. Glycine max showed the maximum similarity of CCT family genes with C. cajan. The expression analysis of CCT family genes using photoperiod insensitive (ICP20338) and photoperiod sensitive (MAL3) genotypes of C. cajan demonstrated that CcCCT4 and CcCCT23 are the active CONSTANS in ICP20338. In contrast, only CcCCT23 is active in MAL3. CONCLUSION: The CCT family genes in C. cajan vary considerably in structure and domain types. They are maximally similar to soybean's CCT family genes. The differential photoperiod response of pigeonpea genotypes, ICP20338 and MAL3, is possibly due to the difference in the number and types of active CONSTANS in them.

In silico characterization and homology modeling of cytosolic APX gene predicts novel glycine residue modulating waterlogging stress response in pigeon pea.

Ascorbate peroxidase (APX) is a member of the family of heme-containing peroxidases having a similar structure with Cytochrome c peroxidase (CCP) that effectively scavenge cytosolic and chloroplastic hydrogen peroxide (H2O2) under various stresses. In this study, computational characterization and homology analysis of APX protein from waterlogging tolerant (ICPL 84023) and sensitive (ICP 7035) pigeon pea genotypes were carried out resulting in 100% homology with Glycine max in case of former and 99% in later genotypes respectively with 97.39% alignment coverage among each other. The model structure was further refined by various tools like PROCHECK, ProSA, and Verify3D. The planned model of the APX enzyme was then tested to dock with H2O2along with molecular dynamics (MD) simulation analysis. The docked complex of ICPL 84023 showed the best G-score (23.39 kcal/mol) in comparison to ICP 7035 (16.74 kcal/mol) depicting the higher production of APX for scavenging reactive oxygen species (ROS) production making this genotype more tolerant. The important binding residues in the ICPL 84023-H2O2complex (SER1, THR4, GLU23, and GLY13) have shown less fluctuation than the ICP 7035-H2O2 complex (SER1, THR4, and GLU23). Overall, our results showed that amino acid residue glycine in ICPL 84023 APX gene has a high binding affinity with H2O2 which could be a key factor associated with waterlogging stress tolerance in pigeon pea.

Exploring the edible gum (galactomannan) biosynthesis and its regulation during pod developmental stages in clusterbean using comparative transcriptomic approach.

Galactomannan is a polymer of high economic importance and is extracted from the seed endosperm of clusterbean (C. tetragonoloba). In the present study, we worked to reveal the stage-specific galactomannan biosynthesis and its regulation in clusterbean. Combined electron microscopy and biochemical analysis revealed high protein and gum content in RGC-936, while high oil bodies and low gum content in M-83. A comparative transcriptome study was performed between RGC-936 (high gum) and M-83 (low gum) varieties at three developmental stages viz. 25, 39, and 50 days after flowering (DAF). Total 209,525, 375,595 and 255,401 unigenes were found at 25, 39 and 50 DAF respectively. Differentially expressed genes (DEGs) analysis indicated a total of 5147 shared unigenes between the two genotypes. Overall expression levels of transcripts at 39DAF were higher than 50DAF and 25DAF. Besides, 691 (RGC-936) and 188 (M-83) candidate unigenes that encode for enzymes involved in the biosynthesis of galactomannan were identified and analyzed, and 15 key enzyme genes were experimentally validated by quantitative Real-Time PCR. Transcription factor (TF) WRKY was observed to be co-expressed with key genes of galactomannan biosynthesis at 39DAF. We conclude that WRKY might be a potential biotechnological target (subject to functional validation) for developing high gum content varieties.

Identification and characterization of MADS box gene family in pigeonpea for their role during floral transition.

MADS box genes are class of transcription factors involved in various physiological and developmental processes in plants. To understand their role in floral transition-related pathways, a genome-wide identification was done in Cajanus cajan, identifying 102 members which were classified into two different groups based on their gene structure. The status of all these genes was further analyzed in three wild species i.e. C. scarabaeoides, C. platycarpus and C. cajanifolius which revealed absence of 31-34 MADS box genes in them hinting towards their role in domestication and evolution. We could locate only a single copy of both FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) genes, while three paralogs of SUPPRESSOR OF ACTIVATION OF CONSTANS 1 (SOC1) were found in C. cajan genome. One of those SOC1 paralogs i.e. CcMADS1.5 was found to be missing in all three wild relatives, also forming separate clade in phylogeny. This SOC1 gene was also lacking the characteristic MADS box domain in it. Expression profiling of major MADS box genes involved in flowering was done in different tissues viz shoot apical meristem, vegetative leaf, reproductive meristem, and reproductive bud. Gene-based time tree of FLC and SOC1 gene dictates their divergence from Arabidopsis before 71 and 23 million year ago (mya), respectively. This study provides valuable insights into the functional characteristics, expression pattern, and evolution of MADS box proteins in grain legumes with emphasis on C. cajan, which may help in further characterizing these genes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02605-7.

Identification and characterization of PEBP family genes reveal CcFT8 a probable candidate for photoperiod insensitivity in C. cajan.

Understanding the molecular mechanism underlying photoperiod sensitivity will play a crucial role in extending the cropping area of Cajanus cajan, a photoperiod sensitive major grain legume of India and Africa. In flowering plants, Flowering locus T (FT) gene is involved in the production of florigen molecule which is essential for induction of flowering, influenced largely by the duration of photoperiod. To understand the structural and regulatory nature of this gene, a genome-wide survey was carried out, revealing the presence of 13 PEBP (FT) family genes in C. cajan. Based on the gene expression profiling of 13 PEBP genes across the 30 tissues of C. cajan, CcFT6 and CcFT8 were found to be probable Flowering locus T genes responsible for the production of florigen as both of them showed expression in reproductive leaf. Expression analysis in photoperiod sensitive, MAL3 genotype revealed that CcFT6 is upregulated under SD. However, in photoperiod insensitive genotype (ICP20338) CcFT6 and CcFT8 were upregulated in SD and LD, respectively. Hence, in ICP20338 under SD, flowering induction occurs with the involvement of CcFT6 while under LD, flowering induction seems to be associated with the expression of CcFT8. CcFT6 was found to be expressed only under favourable photoperiodic condition (SD) in both MAL3 and ICP20338 and may be regulated through a photoperiod dependent pathway. The presence of additional florigen producing gene, CcFT8 in ICP20338 which has the ability to flower in a photoperiod independent manner under LD conditions might provide some clues on its photoperiod insensitive nature. This study will provide a detailed characterization of the genes involved in photoperiodic regulation of flowering in C. cajan.

Methods and Tools for Plant Organelle Genome Sequencing, Assembly, and Downstream Analysis.

Organelles play an important role in a eukaryotic cell. Among them, the two organelles, chloroplast and mitochondria, are responsible for the critical function of photosynthesis and aerobic respiration. Organellar genomes are also very important for plant systematic studies. Here we have described the methods for isolation of the mitochondrial and plastid DNA and its subsequent sequencing with the help of NGS technology. We have also discussed in detail the various tools available for assembly, annotation, and visualization of the organelle genome sequence.

Comparative RNA editing profile of mitochondrial transcripts in cytoplasmic male sterile and fertile pigeonpea reveal significant changes at the protein level.

RNA editing is a process which leads to post-transcriptional alteration of the nucleotide sequence of the corresponding mRNA molecule which may or may not lead to changes at the protein level. Apart from its role in providing variability at the transcript and protein levels, sometimes, such changes may lead to abnormal expression of the mitochondrial gene leading to a cytoplasmic male sterile phenotype. Here we report the editing status of 20 major mitochondrial transcripts in both male sterile (AKCMS11) and male fertile (AKPR303) pigeonpea genotypes. The validation of the predicted editing sites was done by mapping RNA-seq reads onto the amplified mitochondrial genes, and 165 and 159 editing sites were observed in bud tissues of the male sterile and fertile plant respectively. Among the resulting amino acid alterations, the most frequent one was the conversion of hydrophilic amino acids to hydrophobic. The alterations thus detected in our study indicates differential editing, but no major change in terms of the abnormal protein structure was detected. However, the above investigation provides an insight into the behaviour of pigeonpea mitochondrial genome in native and alloplasmic state and could hold clues in identification of editing factors and their role in adaptive evolution in pigeonpea.