Insights into the role of SUMO in regulating drought stress responses in pigeonpea (Cajanus cajan).

KEY MESSAGE: We have identified and analyzed 28 SUMO-pathway proteins from pigeonpea. Enhanced transcripts of pathway genes and increased SUMO conjugation under drought signifies the role of SUMO in regulating stress. Being a protein-rich and nutrient-dense legume crop, pigeonpea (Cajanus cajan) holds a vital position in a vegetarian meal. It is a resilient crop capable of striving in harsh climates and provides a means of subsistence to small-holding farmers. Nevertheless, extremes of water scarcity and drought conditions, especially during seedling and reproductive stages, remains a major issue severely impacting the growth and overall productivity of pigeonpea. Small ubiquitin-like modifier (SUMO), a post-translational modification system, plays a pivotal role in fortifying plants against stressful conditions by rapid reprogramming of molecular events. In this study, we have scanned the entire pigeonpea genome and identified 28 candidates corresponding to SUMO machinery components of pigeonpea. qRT-PCR analysis of different SUMO machinery genes validated their presence under natural conditions. The analysis of the promoters of identified SUMO machinery genes revealed the presence of abiotic stress-related cis-regulatory elements highlighting the potential involvement of the genes in abiotic stress responses. The transcript level analysis of selected SUMO machinery genes and global SUMO status of pigeonpea proteins in response to drought stress suggests an integral role of SUMO in regulating drought stress conditions in pigeonpea. Collectively, the work puts forward a detailed in silico analysis of pigeonpea SUMO machinery candidates and highlights the essential role of SUMOylation in drought stress responses. Being the first report on a pulse crop, the study will serve as a resource for devising strategies for counteracting drought stress in pigeonpea that can be further extended to other pulse crops.

Identification and expression analysis of SBP-Box-like (SPL) gene family disclose their contribution to abiotic stress and flower budding in pigeon pea (Cajanus cajan).

The SPL gene family (for Squamosa Promoter-binding like Proteins) represents specific transcription factors that have significant roles in abiotic stress tolerance, development and the growth processes of different plants, including initiation of the leaf, branching and development of shoot and fruits. The SPL gene family has been studied in different plant species; however, its role is not yet fully explored in pigeon pea (Cajanus cajan ). In the present study, 11 members of the CcSPL gene family were identified in C. cajan . The identified SPLs were classified into nine groups based on a phylogenetic analysis involving SPL protein sequences from C. cajan , Arabidopsis thaliana , Cicer arietinum , Glycine max , Phaseolus vulgaris , Vigna unguiculata and Arachis hypogaea . Further, the identification of gene structure, motif analysis, domain analysis and presence of cis -regulatory elements in the SPL family members were studied. Based on RNA-sequencing data, gene expression analysis was performed, revealing that CcSPL2.1, 3 and 13A were significantly upregulated for salt-tolerance and CcSPL14 and 15 were upregulated in a salt-susceptible cultivar. Real-time qPCR validation indicated that CcSPL3, 4, 6 and 13A were upregulated under salt stress conditions. Therefore, molecular docking was performed against the proteins of two highly expressed genes (CcSPL3 and CcSPL14 ) with three ligands: abscisic acid, gibberellic acid and indole-3-acetic acid. Afterward, their binding affinity was obtained and three-dimensional structures were predicted. In the future, our study may open avenues for harnessing CcSPL genes in pigeon pea for enhanced abiotic stress resistance and developmental traits.

Methyl jasmonate reduces cadmium toxicity by enhancing phenol and flavonoid metabolism and activating the antioxidant defense system in pigeon pea (Cajanus cajan).

Cadmium (Cd) is absorbed by plant roots from soil along with essential nutrients and affects plant growth and productivity. Methyl jasmonate (Me-JA) play important roles to mitigate Cd toxicity in plants. We have investigated the role of Me-JA to ameliorate Cd toxicity in Pigeon pea (Cajanus cajan). Plant root growth, biomass, cellular antioxidant defense system and expression of key regulatory genes in molecular and signaling process have been analyzed. Two Cajanus cajan varieties AL-882 and PAU-881 were grown at 25 °C, 16/8h light/dark conditions in three biological replicates at 5 mM Cd concentration, three concentration of Me-JA (0, 10 nM, 100 nM) and two concentrations in combination of Me-JA + Cd (10 nM Me-JA +5 mM Cd, 100 nM Me-JA +5 mM Cd). The seedlings were exposed to Cd stress consequently plants showed decrease in primary root growth (60.71%, in AL-882 and 8.33%, in PAU-881), shoot and root biomass and antioxidant enzymes activities. Me-JA treatment resulted in increased primary root growth (63.64%, in AL-882) and overall plant biomass. Oxidative stress generated due to Cd stress was counter balanced by Me-JA treatment. Me-JA reduced H2O2 free radicals formation and enhanced antioxidant enzyme activities and phenolic content in stressed seedlings. Me-JA treatment increased expression of CALM, IP3, CDPK2, MPKs (involved in calcium and kinase signaling pathways) and reduced expression of metal transporters (IRT1 and HMA3) genes. This reduction in metal transporters gene expression is a probable reason for low toxicity effect of Cd in root after Me-JA treatment which has potential implications in reducing the risk of Cd in the food chain.

Physico-functional and nutritional characteristics of germinated pigeon pea (Cajanus cajan) flour as a functional food ingredient.

The study investigated the effect of germination on pigeon pea flour's physico-functional (pH, color, water and oil absorption capacities, swelling and foaming capacities and bulk densities) and proximate, total polyphenols and antioxidant activity. The physico-functional and proximate parameters were determined using standard protocols. The color analysis showed that germination significantly increased the flour samples' lightness (L*) (70.7; p = 0.009) by almost 1.5-fold. Germination resulted in almost 1.1 times higher oil absorption capacity than the control (219.9%; p = 0.022). The foaming capacity of the germinated samples significantly (p = 0.015) increased by 6.4%. Germination significantly reduced the loose bulk density (0.54 vs 0.63; p = 0.012) but significantly increased the tapped bulk density (0.84 vs 0.77; p = 0.002). The germinated samples recorded significantly (1.62%; p = 0.010) lower crude fat, about 1.2 times lower than the raw flour. Germination significantly increased the flour's total ash (4.2% vs 3.6%; p = 0.003) and crude protein (11.6% vs 9.4%; p = 0.047) content. Germinated pigeon pea flour will perform better in formulating baked products, aerated foods and food extenders than non-germinated pigeon pea flour. Hence, the germination of pigeon peas should be encouraged because it harnesses the functional and proximate attributes measured.

Genome-wide identification and characterization of ABC transporter superfamily in the legume Cajanus cajan.

Plant ATP-binding cassette (ABC) protein family is the largest multifunctional highly conserved protein superfamily that transports diverse substrates across biological membranes by the hydrolysis of ATP and is also the part of the several other biological processes like cellular detoxification, growth and development, stress biology, and signaling processes. In the agriculturally important legume crop Cajanus cajan, a genome-wide identification and characterization of the ABC gene family was carried out. A total of 159 ABC genes were identified that belong to eight canonical classes CcABCA to CcABCG and CcABCI based on the phylogenetic analysis. The number of genes was highest in CcABCG followed by CcABCC and CcABCB class. A total of 85 CcABC genes were found on 11 chromosomes and 74 were found on scaffold. Tandem duplication was the major driver of CcABC gene family expansion. The dN/dS ratio revealed the purifying selection. The phylogenetic analysis revealed class-specific eight superclades which reflect their functional importance. The largest clade was found to be CcABCG which reflects their functional significance. CcABC proteins were mainly basic in nature and found to be localized in the plasma membrane. The secondary structure prediction revealed the dominance of α-helix. The canonical transmembrane and nucleotide binding domain, signature motif LSSGQ, Walker A, Walker B region, and Q loop were also identified. A class-specific exon-intron pattern was also observed. In addition to core elements, different cis-acting regulatory elements like stress, hormone, and cellular responsive were also identified. Expression profiling of CcABC genes at various developmental stages of different anatomical tissues was performed and it was noticed that CcABCF3, CcABCF4, CcABCF5, CcABCG66, and CcABCI3 had the highest expression. The results of the current study endow us with the further functional analysis of Cajanus ABC in the future.

Genome-wide identification and expression analysis of MYB gene family in Cajanus cajan and CcMYB107 improves plant drought tolerance.

MYB transcription factor (TF) is one of the largest superfamilies that play a vital role in multiple plant biological processes. However, the MYB family has not been comprehensively identified and functionally verified in Cajanus cajan, which is the sixth most important legume crop. Here, 170 CcR2R3-MYBs were identified and divided into 43 functional subgroups. Segmental and tandem duplications and alternative splicing events were found and promoted the expansion of the CcR2R3-MYB gene family. Functional prediction results showed that CcR2R3-MYBs were mainly involved in secondary metabolism, cell fate and identity, developmental processes, and responses to abiotic stress. Cis-acting element analysis of promoters revealed that stress response elements were widespread in the above four functional branches, further suggesting CcR2R3-MYBs were extensively involved in abiotic stress response. The transcriptome data and qRT-PCR results indicated that most of the CcR2R3-MYB genes responded to various stresses, of which the expression of CcMYB107 was significantly induced by drought stress. Overexpression of CcMYB107 enhanced antioxidant enzyme activity and increased proline and lignin accumulation, thus improving the drought resistance of C. cajan. Furthermore, Overexpression of CcMYB107 up-regulated the expression of stress-related genes and lignin biosynthesis genes after drought stress. Our findings established a strong foundation for the investigation of biological function of CcR2R3-MYB TFs in C. cajan.

Genomic survey of high-throughput RNA-Seq data implicates involvement of long intergenic non-coding RNAs (lincRNAs) in cytoplasmic male-sterility and fertility restoration in pigeon pea.

BACKGROUND: Long-intergenic non-coding RNAs (lincRNAs) originate from intergenic regions and have no coding potential. LincRNAs have emerged as key players in the regulation of various biological processes in plant development. Cytoplasmic male-sterility (CMS) in association with restorer-of-fertility (Rf) systems makes it a highly reliable tool for exploring heterosis for producing commercial hybrid seeds. To date, there have been no reports of lincRNAs during pollen development in CMS and fertility restorer lines in pigeon pea. OBJECTIVE: Identification of lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines. METHODS: We employed a computational approach to identify lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines using RNA-Seq data. RESULTS: We predicted a total of 2145 potential lincRNAs of which 966 were observed to be differentially expressed between the sterile and fertile pollen. We identified, 927 cis-regulated and 383 trans-regulated target genes of the lincRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the target genes revealed that these genes were specifically enriched in pathways like pollen and pollen tube development, oxidative phosphorylation, etc. We detected 23 lincRNAs that were co-expressed with 17 pollen-related genes with known functions. Fifty-nine lincRNAs were predicted to be endogenous target mimics (eTMs) for 25 miRNAs, and found to be associated with pollen development. The, lincRNA regulatory networks revealed that different lincRNA-miRNA-mRNA networks might be associated with CMS and fertility restoration. CONCLUSION: Thus, this study provides valuable information by highlighting the functions of lincRNAs as regulators during pollen development in pigeon pea and utilization in hybrid seed production.

Molecular mechanism of naringenin regulation on flavonoid biosynthesis to improve the salt tolerance in pigeon pea (Cajanus cajan (Linn.) Millsp.).

Flavonoids are important secondary metabolites in the plant growth and development process. As a medicinal plant, pigeon pea is rich in secondary metabolites. As a flavonoid, there are few studies on the regulation mechanism of naringenin in plant stress resistance. In our study, we found that naringenin can increase the pigeon pea's ability to tolerate salt and influence the changes that occur in flavonoids including naringenin, genistein and biochanin A. We analyzed the transcriptome data after 1 mM naringenin treatment, and identified a total of 13083 differentially expressed genes. By analyzing the metabolic pathways of these differentially expressed genes, we found that these differentially expressed genes were enriched in the metabolic pathways of phenylpropanoid biosynthesis, starch and sucrose metabolism and so on. We focused on the analysis of flavonoid biosynthesis related pathways. Among them, the expression levels of enzyme genes CcIFS, CcCHI and CcCHS in the flavonoid biosynthesis pathway had considerably higher expression levels. By counting the number of transcription factors and the binding sites on the promoter of the enzyme gene, we screened the transcription factors CcMYB62 and CcbHLH35 related to flavonoid metabolism. Among them, CcMYB62 has a higher expression level than the others. The hairy root transgene showed that CcMYB62 could induce the upregulation of CcCHI, and promote the accumulation of naringenin, genistein and biochanin A. Our study revealed the molecular mechanism of naringenin regulating flavonoid biosynthesis under salt stress in pigeon pea, and provided an idea for the role of flavonoids in plant resistance to abiotic stresses.

Cocultivation of pigeon pea hairy root cultures and Aspergillus for the enhanced production of cajaninstilbene acid.

Pigeon pea hairy root cultures (PPHRCs) have been proven to be a promising alternative for the production of health-beneficial phenolic compounds, such as the most important health-promoting compound, i.e., cajaninstilbene acid (CSA). In this study, PPHRCs were cocultured with live Aspergillus fungi for further improving phenolic productivity via biological elicitation. Aspergillus oryzae CGMCC 3.951 (AO 3.951) was found to be the optimal fungus that could achieve the maximum increment of CSA (10.73-fold increase) in 42-day-old PPHRCs under the inoculum size of mycelia 0.50% and cocultivation time 36 h. More precisely, the contents of CSA in hairy roots and culture media after fungal elicitation increased by 9.87- and 62.18-fold over control, respectively. Meanwhile, the contents of flavonoid glycosides decreased, while aglycone yields increased upon AO 3.951 elicitation. Moreover, AO 3.951 could trigger the oxidative stress and pathogen defense response thus activating the expression of biosynthesis- and ABC transporter-related genes, which contributed to the intracellular accumulation and extracellular secretion of phenolic compounds (especially CSA) in PPHRCs. And PAL2, 4CL2, STS1, and I3'H were likely to be the potential key enzyme genes regulating the biosynthesis of CSA, and ABCB11X1-1, ABCB11, and ABCG24X2 were closely related to the transmembrane transport of CSA. Overall, the cocultivation approach could make PPHRCs more commercially attractive for the production of high-value phenolic compounds such as CSA and flavonoid aglycones in nutraceutical/medicinal fields. And the elucidation of crucial biosynthesis and transport genes was important for systematic metabolic engineering aimed at increasing CSA productivity. KEY POINTS: • Cocultivation of PPHRCs and live fungi was to enhance CSA production and secretion. • PPHRCs augmented CSA productivity 10.73-fold when cocultured with AO 3.951 mycelia. • Several biosynthesis and transport genes related to CSA production were clarified.

Cajanus platycarpus Flavonoid 3'5' Hydroxylase_2 (CpF3'5'H_2) Confers Resistance to Helicoverpa armigera by Modulating Total Polyphenols and Flavonoids in Transgenic Tobacco.

Pod borer Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3'5' hydroxylase_2 (F3'5'H_2) from a pigeonpea wild relative Cajanus platycarpus, possessing a robust chemical resistance response to H. armigera. Though F3'5'H_2 displayed a dynamic expression pattern in both C. platycarpus (Cp) and the cultivated pigeonpea, Cajanus cajan (Cc) during continued herbivory, CpF3'5'H_2 showed a 4.6-fold increase vis a vis 3-fold in CcF3'5'H_2. Despite similar gene copy numbers in the two Cajanus spp., interesting genic and promoter sequence changes highlighted the stress responsiveness of CpF3'5'H_2. The relevance of CpF3'5'H_2 in H. armigera resistance was further validated in CpF3'5'H_2-overexpressed transgenic tobacco based on reduced leaf damage and increased larval mortality through an in vitro bioassay. As exciting maiden clues, CpF3'5'H_2 deterred herbivory in transgenic tobacco by increasing total flavonoids, polyphenols and reactive oxygen species (ROS) scavenging capacity. To the best of our knowledge, this is a maiden attempt ascertaining the role of F3'5'H_2 gene in the management of H. armigera. These interesting leads suggest the potential of this pivotal branch-point gene in biotic stress management programs.

Cajanus cajan (L) Millsp. seed extract ameliorates scopolamine-induced amnesia through increase in antioxidant defense mechanisms and cholinergic neurotransmission.

Decline in cholinergic function and oxidative/nitrosative stress play a central role in Alzheimer's disease (AD). Previous quantitative HPLC profiling analysis has revealed the presence of Pinostrobin, formononetin, vitexin and other neuroprotective flavonoids in Cajanus cajan seed extract. This study was designed to investigate the protective action of Cajanus cajan ethanol seed extract (CC) on learning and memory functions using scopolamine mouse model of amnesia. Materials and methods: Adult mice were pretreated with CC (50, 100, or 200mg/kg, p.o) or vehicle (10ml/kg, p.o) for 16 days consecutively. Scopolamine, a competitive muscarinic cholinergic receptor antagonist (1mg/kg, i.p.) was given an hour after CC pretreatment from days 3 to 16.  The mice were subjected to behavioural tests from day 11 (open field test (OFT)/ Y-maze test (YMT) and Morris water maze task (MWM) from days 12-16. Animals were euthanized 1h after behavioral test on day 16 and discrete brain regions isolated for markers of oxidative stress and cholinergic signaling. Molecular docking analysis was undertaken to predict the possible mechanism(s) of CC-induced anti-amnesic action.  pre-administration of CC significantly reversed working memory and learning deficits caused by scopolamine in YMT and MWM tests, respectively. Moreover, CC prevented scopolamine-induced oxidative and nitrosative stress radicals in the hippocampus evidenced in significant increase in glutathione (GSH) level, superoxide dismutase (SOD) and catalase (CAT) activities with a marked decrease in malondialdehyde (MDA) production, as well as significant inhibition of hippocampal scopolamine-induced increase in acetylcholinesterase activity by CC. The molecular docking analysis showed that out of the 19 compounds, the following had the highest binding affinity; Pinostrobin (-8.7 Kcal/mol), friedeline (-7.5kCal/mol), and lupeol (-8.2 Kcal/mol), respectively, to neuronal muscarinic M1 acetylcholine receptor, α7 nicotinic acetylcholine receptor and amyloid beta peptide binding pockets, which further supports the ability of CC to enhance neuronal cholinergic signaling and possible inhibition of amyloid beta aggregation. This study showed that Cajanus cajan seeds extract improved working memory and learning through enhancement of cholinergic signaling, antioxidant capacity and reduction in amyloidogenesis.

Cajanus cajan shows multiple novel adaptations in response to regular mechanical stress.

Cajanus cajan is one of the least studied crop plants regarding its responses to stress conditions. Regular mechanical stress suppresses plant physiology and growth at the cellular and systemic levels. In the current study, we have explored morphological, physiological, and anatomical adaptations of C. cajan seedlings to regular mechanical stress. Young seedlings of C. cajan were given mechanical stress in the form of touch for fifteen days and observed for various changes. Touch stimuli caused an immediate release of oxidative burst, suppressed plant growth, increased compactness of the stem tissue, and altered the chlorophyll a/b ratio. We have also identified two novel phenotypes; regular touch stimuli affected the nyctinasty movements of the leaves and also affected the root nodule development. We have identified and studied the expression of four putative touch responsive calcium binding genes, TCH gene homologs, in C. cajan using Arabidopsis TCH gene sequences. At an early time point, the expression of two TCH gene homologs (CcTCH1-1 and CcTCH2-2) were found to be upregulated. This study unravels the novel adaptation displayed by C. cajan in response to mechanical stress that can be used as a phenotypic marker for future studies in this plant.

Insulin sensitizer and antihyperlipidemic effects of Cajanus cajan (L.) millsp. root in methylglyoxal-induced diabetic rats.

Cajanus cajan (L.) Millsp., known as pigeon pea, is one of the major grain legume crops of the tropical world. It recognizes as an ethnomedicine to possess various functions, such as helping in healing wound and cancer therapy. We investigated whether 95% ethanol extracts from C. cajan root (EECR) protect against methylglyoxal (MGO)-induced insulin resistance (IR) and hyperlipidemia in male Wistar rats and explored its possible mechanisms. The hypoglycemic potential of EECR was evaluated using α-amylase, α-glucosidase activities, and advanced glycation end products (AGEs) formation. For in vivo study, the rats were divided into six groups and orally supplemented with MGO except for Group 1 (controls). Group 2 was supplemented with MGO only, Group 3: MGO + metformin, Group 4: MGO + Low dose-EECR (L-EECR; 10 mg/kg bw), Group 5: MGO + Middle dose-EECR (M-EECR; 50 mg/kg bw), and Group 6: MGO + High dose-EECR (H-EECR; 100 mg/kg bw). EECR possessed good inhibition of α-glucosidase, α-amylase activities, and AGEs formation (IC50 = 0.12, 0.32, and 0.50 mg/mL), respectively. MGO significantly increased serum levels of blood glucose (GLU), glycosylated hemoglobin, homeostasis model assessment of IR, AGEs, lipid biochemical values, and atherogenic index, whereas EECR decreased these levels in a dose-dependent manner. EECR can also act as an insulin sensitizer, which significantly decreased (47%, P < 0.05) the blood GLU levels after intraperitoneal injection of insulin in the insulin tolerance tests. The hypoglycemic and antihyperlipidemic mechanisms of EECR are likely through several possible pathways including the inhibition of carbohydrate-hydrolyzing enzymes (α-glucosidase and α-amylase) and the enhancement of MGO-trapping effects on inhibition of AGEs formation.

Genome-wide transcriptome analysis and characterization of the cytochrome P450 flavonoid biosynthesis genes in pigeon pea (Cajanus cajan).

MAIN CONCLUSION: 226 CcCYP450 genes were identified at the genomic level and were classified into 45 clades based on phylogenetic analysis. CcCYP75B165 gene was found that might play important roles in the biosynthesis of flavonoids in pigeon pea, and was significantly induced by methyl jasmonate (MeJA). The cytochrome P450 mono-oxygenase (CYP450) superfamily plays a key role in the flavonoid biosynthesis pathway and resists different kinds of stresses. Several CYP450 genes have been identified to be involved in the biosynthesis of crop protection agents. However, the CcCYP450 genes from pigeon pea have not been identified. Here, 226 CcCYP450 genes were identified at the genomic level by analysing the gene structure, distribution on chromosomes, gene duplication, and conserved motifs and were classified into 45 clades based on phylogenetic analysis. RNA-seq analysis revealed clear details of CcCYP450 genes that varied with time of MeJA (methyl jasmonate) induction. Among them, six CcCYP450 subfamily genes were found that might play important roles in the biosynthesis of flavonoids in pigeon pea. The overexpression of CcCYP75B165 in pigeon pea significantly induced the accumulation of genistin and downregulated the contents of cajaninstilbene acid, apigenin, isovitexin, and genistein and the expression of flavonoid synthase genes. This study provides theoretical guidance and plant genetic resources for cultivating new pigeon pea varieties with high flavonoid contents and exploring the molecular mechanisms of the biosynthesis of flavonoids under MeJA treatment.

Genome-wide exploration of sugar transporter (sweet) family proteins in Fabaceae for Sustainable protein and carbon source.

Sugar transporter proteins (STPs) are membrane proteins required for sugar transport throughout cellular membranes. They plays an imperative role in sugar transmission across the plant and determinants of crop yield. However, the analysis of these important STPs Sugars Will Eventually be Exported Transporters (SWEET) family in legumes is still not well-documented and remains unclear. Therefore, the in-silico analysis of STPs has been performed to unravel their cellular, molecular, and structural composition in legume species. This study conducted a systematic search for STPs in Cajanus cajan using the Blastp algorithm to understand its molecular basis. Here, we performed a comprehensive analysis of 155 identified SWEET proteins across 12 legumes species, namely (Cajanus cajan, Glycine max, Vigna radiate, Vigna angularis, Medicago truncatula, Lupinus angustifolius, Glycine soja, Spatholobus suberectus, Cicer arietinum, Arachis ipaensis, Arachis hypogaea, Arachis duranensis). The amino acid composition and motif analysis revealed that SWEET proteins are rich in essential amino acids such as leucine, valine, isoleucine, phenylalanine, and serine while less profuse in glutamine, tryptophan, cysteine, and histidine. A total of four main conserved motifs of SWEET proteins are also highly abundant in these amino acids. The present study deciphered the details on primary physicochemical properties, secondary, tertiary structure, and phylogenetic analysis of SWEETs protein. Majorities of SWEET proteins (72.26%) are in stable form with an average instability index of 36.5%, and it comprises a higher fraction of positively charged amino acid Arg + Lys residues. Secondary structure analysis shown that these proteins are richer in alpha-helix (40%) than extended strand (30%) and random coil (25%), respectively. Furthermore, to infer their mechanism at a structural and functional level which play an essential roles in growth, development, and stress responses. This study will be useful to examine photosynthetic productivity, embryo sugar content, seed quality, and yield enhancement in Fabaceae for a sustainable source of essential amino acids and carbon source.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

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.

Genome-wide analysis and characterization of R2R3-MYB family in pigeon pea (Cajanus cajan) and their functional identification in phenylpropanoids biosynthesis.

MAIN CONCLUSION: Thirty CcMYB were identified to involve in flavonoid and lignin biosynthesis in pigeon pea genome. A comprehensive analysis of gene structure, phylogenetic relationships, distribution on chromosomes, gene duplication, and expression patterns was performed. MYB transcription factor is one of the largest gene families in plants and plays critical roles in plant growth and development, as well as resistance to biotic and abiotic stress. However, the function of MYB genes in pigeon pea (Cajanus cajan) remains largely unknown. Here, 30 R2R3-MYB which involved flavonoid and lignin biosynthesis were identified in the pigeon pea genome and were classified into five groups based on phylogenetic analysis. Simultaneously, another 122 key enzyme genes from biosynthetic pathways of flavonoid and lignin were identified and all of them were mapped on 11 chromosomes with the co-linearity relationship. Among these genes, the intron/exon organization and motif compositions were conserved and they have undergone a strong purifying selection and tandem duplications during evolution. Expression profile analysis demonstrated most of these genes were expressed in different tissues and responded significantly to MeJA, RNA-seq analysis revealed clear details of genes varied with time of induction. Ten key genes from the phenylpropanoid pathway were selected to further verify whether they responded to induction under different abiotic stress conditions (UV-B, cold, heat, salt, drought, and GA3). This study elaborates on potential regulatory relationships between R2R3-MYB genes and some key genes involved in flavonoid and lignin biosynthesis under MeJA treatment, as well as adding to the understanding of improving abiotic stress tolerance and regulating the secondary metabolism in woody crops. A simplified discussion model for the different regulation networks involved with flavonoid and lignin biosynthesis in pigeon pea is proposed.

Investigation of bacterial diversity in Cajanus cajan-planted gangue soil via high-throughput sequencing.

The ecological restoration of coal gangue can be achieved by planting Cajanus cajan (pigeon pea) because of its developed root system. The close relationships soil microorganisms have with plants are crucial for improving soil composition; the soil composition affects nutrient absorption. The microbial composition and function of soil planted with C. cajan in reclaimed land were compared with soil that was not planted with C. cajan (the control). Results showed that the dominant microflora in the soil significantly changed after planting C. cajan. Before planting, the dominant microflora included members of the phyla Sulfobacteria and Acidobacteria. After planting, the dominant microflora contained bacteria from phyla and classes that included Actinobacteria, Acidimicubia, Thermoleophilia, and Anaerolineae. Additionally, there were significant differences in the bacterial composition of each layer in soils planted with C. cajan. Principal component analysis revealed that the interpretation degrees of the results for PC2 and PC3 axes were 10.46% and 3.87%, respectively. The dominant microflora were Vicinamibacterales, Nocardioides, and Arthrobacter in the surface soil; Actinophytocola and Sphingomonas in the deep soil; and Sulfobacillus and Acidimicrobium in the mixed-layer soil. Function prediction analysis using the bioinformatics software package PICRUSt revealed that the abundance of operational taxonomic units corresponding to sigma 54-specific transcriptional regulators, serine threonine protein kinase, and histidine kinase increased by 111.2%, 56.8%, and 47.4%, respectively, after planting C. cajan. This study provides a reference for interactions among microorganisms in reclaimed soils for guiding the development and restoration of waste coal gangue hills.