Bacterial exopolysaccharide amendment improves the shelf life and functional efficacy of bioinoculant under salinity stress.

AIM: Bacterial exopolysaccharides (EPS) possess numerous properties beneficial for the growth of microbes and plants under hostile conditions. The study aimed to develop a bioformulation with bacterial EPS to enhance the bioinoculant's shelf life and functional efficacy under salinity stress. METHODS AND RESULTS: High EPS-producing and salt-tolerant bacterial strain (Bacillus haynessi SD2) exhibiting auxin-production, phosphate-solubilization, and biofilm-forming ability, was selected. EPS-based bioformulation of SD2 improved the growth of three legumes under salt stress, from which pigeonpea was selected for further experiments. SD2 improved the growth and lowered the accumulation of stress markers in plants under salt stress. Bioformulations with varying EPS concentrations (1% and 2%) were stored for 6 months at 4°C, 30°C, and 37°C to assess their shelf life and functional efficacy. The shelf life and efficacy of EPS-based bioformulation were sustained even after 6 months of storage at high temperature, enhancing pigeonpea growth under stress in both control and natural conditions. However, the efficacy of non EPS-based bioformulation declined following four months of storage. The bioformulation (with 1% EPS) modulated bacterial abundance in the plant's rhizosphere under stress conditions. CONCLUSION: The study brings forth a new strategy for developing next-generation bioformulations with higher shelf life and efficacy for salinity stress management in pigeonpea.

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.

Genome-wide identification and expression profiling of growth­regulating factor (GRF) and GRF­interacting factor (GIF) gene families in chickpea and pigeonpea.

The growth-regulating factor (GRF) and GRF-interacting factor (GIF) families encode plant-specific transcription factors and play vital roles in plant development and stress response processes. Although GRF and GIF genes have been identified in various plant species, there have been no reports of the analysis and identification of the GRF and GIF transcription factor families in chickpea (Cicer arietinum) and pigeonpea (Cajanus cajan). The present study identified seven CaGRFs, eleven CcGRFs, four CaGIFs, and four CcGIFs. The identified proteins were grouped into eight and three clades for GRFs and GIFs, respectively based on their phylogenetic relationships. A comprehensive in-silico analysis was performed to determine chromosomal location, sub-cellular localization, and types of regulatory elements present in the putative promoter region. Synteny analysis revealed that GRF and GIF genes showed diploid-polyploid topology in pigeonpea, but not in chickpea. Tissue-specific expression data at the vegetative and reproductive stages of the plant showed that GRFs and GIFs were strongly expressed in tissues like embryos, pods, and seeds, indicating that GRFs and GIFs play vital roles in plant growth and development. This research characterized GRF and GIF families and hints at their primary roles in the chickpea and pigeonpea growth and developmental process. Our findings provide potential gene resources and vital information on GRF and GIF gene families in chickpea and pigeonpea, which will help further understand the regulatory role of these gene families in plant growth and development.

Transmission of antibiotic resistance through organic amendments in arable land: A 3-year field study with pigeonpea-wheat cropping system.

The worldwide emergence of antimicrobial resistance (AMR) poses a substantial risk to human health and environmental stability. In agriculture, organic amendments (derived from organic sources such as manure, and plant residues) are beneficial in restoring soil properties and providing essential nutrients to crops but raise concerns about harboring antibiotic resistance, which emphasizes the need for vigilant monitoring and strategic interventions in their application. The current study assessed the impact of farming practices (organic and conventional) in a three-year field experiment with pigeonpea-wheat cropping system, focusing on the transmission of AMR using culture-dependent and -independent approaches, and soil nutrient content. Markers for antibiotic resistance genes (ARGs) (aminoglycoside-aacA, ß-lactam-blaTEM, chloramphenicol-cmlA1, macrolide-ermB, sulfonamides-sul1, sul2, and tetracycline-tetO) and integrons (intl1 and intl2) were targeted using qPCR. Manure amendments, particularly FYM1, exhibited a higher abundance of copies of ARGs compared to the rhizospheric soil. Organic farming was associated with higher copies of intl2, sul1, blaTEM, and tetO genes, while conventional farming showed increased copies of sul2 and ermB genes in the rhizosphere. Significant positive correlations were observed among soil nutrient contents, ARGs, and MGEs. The notable prevalence of ARGs linked to manure amendments serves as a cautionary note, demanding responsible management practices.

Evaluation of anti-sickling effects of two varieties of Cajanus cajan (L.) Huth on sickle cell beta thalassemia.

ETHNO-PHARMACOLOGICAL RELEVANCE: Globally, the prevalence of sickle cell disease is on the rise, with developing countries experiencing particularly alarming mortality rate compared to developed nations. The World Health Organization (WHO) and United Nations (UN) have acknowledged sickle cell disease as a significant global public health concern. Unfortunately, a cure for this condition is yet to be discovered, and existing allopathic treatments, while offering relief, come with serious side effects. In recent times, there has been a growing interest in exploring the potential of medicinal plants for treating sickle cell disease due to their content of secondary metabolites that may impact the disease's mechanisms. Cajanus cajan, a crucial grain legume in rain-fed agriculture in semi-arid tropics, has been traditionally used in folk medicine to manage various illnesses and is suggested to possess anti-sickling properties. AIM OF THE STUDY: The present study investigated two varieties of C. cajan for their effectiveness in treating sickle cell beta thalassemia, a variant of sickle cell disease. MATERIALS AND METHODS: The study was divided into four groups consisting of the untreated group (group 1), group treated with standard drug (group 2), group treated with white C. cajan (group 3) and group treated with brown C. cajan (group 4). The effects of the two variety of C. cajan were measured by polymerization test, reversibility test, osmotic fragility test, deoxygenation and beta globin synthesis test. RESULT: The results revealed that both varieties of C. cajan demonstrated a reduction in polymerization rates, reversed sickled red blood cells, increased the oxygen affinity of Hb-S/ß, elevated the Fe2+/Fe3+ ratio, and maintained the membrane stability of red blood cells. Notably, the white variety exhibited superior anti-sickling properties compared to the brown variety. CONCLUSION: This suggests that this significant leguminous crop could be utilized for the treatment and management of sickling disorders, particularly in low-income countries where conventional treatments may be financially inaccessible to patients.

Harnessing the power of native biocontrol agents against wilt disease of Pigeonpea incited by Fusarium udum.

Fusarium wilt, caused by (Fusarium udum Butler), is a significant threat to pigeonpea crops worldwide, leading to substantial yield losses. Traditional approaches like fungicides and resistant cultivars are not practical due to the persistent and evolving nature of the pathogen. Therefore, native biocontrol agents are considered to be more sustainable solution, as they adapt well to local soil and climatic conditions. In this study, five isolates of F. udum infecting pigeonpea were isolated from various cultivars and characterized morphologically and molecularly. The isolate from the ICP 8858 cultivar displayed the highest virulence of 90%. Besides, 100 endophytic bacteria, 100 rhizosphere bacteria and three Trichoderma spp. were isolated and tested against F. udum isolated from ICP 8858 under in vitro conditions. Out of the 200 bacteria tested, nine showed highest inhibition, including Rb-4 (Bacillus sp.), Rb-11 (B. subtilis), Rb-14 (B. megaterium), Rb-18 (B. subtilis), Rb-19 (B. velezensis), Eb-8 (Bacillus sp.), Eb-11 (B. subtilis), Eb-13 (P. aeruginosa), and Eb-21 (P. aeruginosa). Similarly, Trichoderma spp. were identified as T. harzianum, T. asperellum and Trichoderma sp. Notably, Rb-18 (B. subtilis) and Eb-21 (P. aeruginosa) exhibited promising characteristics such as the production of hydrogen cyanide (HCN), cellulase, siderophores, ammonia and nutrient solubilization. Furthermore, treating pigeonpea seedlings with these beneficial microorganisms led to increased levels of key enzymes (POD, PPO, and PAL) associated with resistance to Fusarium wilt, compared to untreated controls. In field trials conducted for four seasons, the application of these potential biocontrol agents as seed treatments on the susceptible ICP2376 cultivar led to the lowest disease incidence. Specifically, treatments T2 (33.33) (P. aeruginosa) and T3 (35.41) (T. harzianium) exhibited the lowest disease incidence, followed by T6 (36.5) (Carbendizim), T1 (36.66) (B. subtilis), T4 (52.91) (T. asperellum) and T5 (53.33) (Trichoderma sp.). Results of this study revealed that, P. aeruginosa (Eb-21), B. subtilis (Rb-18) and T. harzianum can be used for plant growth promotion and management of Fusarium wilt of pigeonpea.

Identification of superior haplotypes for flowering time in pigeonpea through candidate gene-based association study of a diverse minicore collection.

KEY MESSAGE: In current study candidate gene (261 genes) based association mapping on 144 pigeonpea accessions for flowering time and related traits and 29 MTAs producing eight superior haplotypes were identified. In the current study, we have conducted an association analysis for flowering-associated traits in a diverse pigeonpea mini-core collection comprising 144 accessions using the SNP data of 261 flowering-related genes. In total, 13,449 SNPs were detected in the current study, which ranged from 743 (ICP10228) to 1469 (ICP6668) among the individuals. The nucleotide diversity (0.28) and Watterson estimates (0.34) reflected substantial diversity, while Tajima's D (-0.70) indicated the abundance of rare alleles in the collection. A total of 29 marker trait associations (MTAs) were identified, among which 19 were unique to days to first flowering (DOF) and/or days to fifty percent flowering (DFF), 9 to plant height (PH), and 1 to determinate (Det) growth habit using 3 years of phenotypic data. Among these MTAs, six were common to DOF and/or DFF, and four were common to DOF/DFF along with the PH, reflecting their pleiotropic action. These 29 MTAs spanned 25 genes, among which 10 genes clustered in the protein-protein network analysis, indicating their concerted involvement in floral induction. Furthermore, we identified eight haplotypes, four of which regulate late flowering, while the remaining four regulate early flowering using the MTAs. Interestingly, haplotypes conferring late flowering (H001, H002, and H008) were found to be taller, while those involved in early flowering (H003) were shorter in height. The expression pattern of these genes, as inferred from the transcriptome data, also underpinned their involvement in floral induction. The haplotypes identified will be highly useful to the pigeonpea breeding community for haplotype-based breeding.

Pigeon pea crop stage strongly influences plant susceptibility to Helicoverpa armigera (Lepidoptera: Noctuidae).

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae; Hübner) is the major insect pest of pigeon pea [Cajanus cajan; Fabales: Fabaceae; (L.) Millspaugh] worldwide. Research to develop pest management strategies for H. armigera in pigeon pea has focused heavily on developing less susceptible cultivars, with limited practical success. We examined how pigeon pea crop stage influences plant susceptibility to H. armigera using a combination of glasshouse and laboratory experiments. Plant phenology significantly affected oviposition with moths laying more eggs on flowering and podding plants but only a few on vegetative plants. Larval survival was greatest on flowering and vegetative plants, wherein larvae mostly chose to feed inside flowers on flowering plants and on the adaxial surface of expanding leaves on vegetative plants. Larval survival was poor on podding plants despite moths laying many eggs on plants of this stage. When left to feed without restriction on plants for 7 days, larvae feeding on flowering plants were >10 times the weight of larvae feeding on plants of other phenological stages. On whole plants, unrestricted larvae preferred to feed on pigeon pea flowers and on expanding leaves, but in no-choice Petri dish assays H. armigera larvae could feed and survive on all pigeon pea reproductive structures. Our results show that crop stage and the availability of flowers strongly influence pigeon pea susceptibility to H. armigera. An increased understanding of H. armigera-pigeon pea ecology will be useful in guiding the development of resistant varieties and other management tactics.

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.

Longistylin A from Cajanus cajan (L.) Millsp. disturbs glycerophospholipid metabolism and cytokinin biosynthesis of Nocardia seriolae.

ETHNOPHARMACOLOGICAL RELEVANCE: Nocardiosis is an uncommon infectious disease that bears certain similarities to tuberculosis, with a continuous increase in its incidence and a poor prognosis. In traditional Chinese medicine, the leaves of Cajanus cajan (L.) Millsp. are employed to treat wounds, malaria, coughs, and abdominal pain. AIM OF THE STUDY: In this study, we investigated the effects and mechanisms of longistylin A (LGA), a natural stilbene isolated from C. cajan, as a potential antibiotic against nocardiosis. MATERIALS AND METHODS: LGA was isolated from the leaves of C. cajan and assessed using a minimum bactericidal concentration (MBC) determination against Nocardia seriolae. Multi-omics analysis encompassing genes, proteins, and metabolites was conducted to investigate the impact of LGA treatment on N. seriolae. Additionally, quantitative analysis of 40 cytokinins in N. seriolae mycelium was performed to assess the specific effects of LGA treatment on cytokinin levels. Cryo-scanning electron microscopy was utilized to examine morphological changes induced by LGA treatment, particularly in the presence of exogenous trans-zeatin-O-glucoside (tZOG). The therapeutic effect of LGA was investigated by feeding N. seriolae-infected largemouth bass. RESULTS: LGA exhibited significant efficacy against N. seriolae, with MBC value of 2.56 µg/mL. Multi-omics analysis revealed that LGA disrupted glycerophospholipid metabolism and hormone biosynthesis by notably reducing the expression of glycerol-3-phosphate dehydrogenase and calmodulin-like protein. Treatment with LGA markedly disrupted 12 distinct cytokinins in N. seriolae mycelium. Additionally, the addition of exogenous tZOG counteracted the inhibitory effects of LGA on filamentous growth, resulting in mycelial elongation and branching. Furthermore, LGA treatment improved the survival rate of largemouth bass infected with N. seriolae. CONCLUSIONS: We found for the first time that LGA from C. cajan exhibited significant efficacy against N. seriolae by interfering with glycerophospholipid metabolism and cytokinin biosynthesis.

Genetic diversity and population structure of Fusarium udum in India and its correlation with pigeonpea wilt incidence.

In a study conducted in India, 50 Fusarium isolates were collected from pigeonpea growing regions and extensively examined for their cultural and morphological characteristics. These isolates exhibited significant variations in traits including growth rate, mycelial growth patterns, color, zonation, pigmentation, spore size, and septation. Subsequently, 30 isolates were chosen for pathogenicity testing on eight pigeonpea genotypes. Results showed distinct reactions, with four genotypes displaying differential responses (ICP8858, ICP8859, ICP8862, and BDN-2), while ICP9174 and ICP8863 consistently exhibited resistance and ICP2376 and BAHAR remained susceptible to wilt disease. To study the interaction between Fusarium isolates and pigeonpea host differentials (HDs), an additive main effects and multiplicative interaction analysis was conducted. The majority of disease incidence variation (75.54%) was attributed to HD effects, while Fusarium isolate effects accounted for only 1.99%. The interaction between Isolates and HDs (I × HD) contributed 21.95% to the total variation, being smaller than HD but larger than I. Based on HD reactions, isolates were classified into nine variants, showing varying distributions across pigeonpea growing states, with variants 2 and 3 being prevalent in several regions. This diversity underscores the need for location-specific wilt-resistant pigeonpea cultivars. Furthermore, genetic analysis of 23 representative isolates, through internal transcribed spacer region of ribosomal DNA and translation elongation factor 1-α gene sequencing, revealed three major clusters: Fusarium udum, Fusarium solani, and Fusarium equiseti. These findings hold potential for developing location-specific wilt-resistant pigeonpea cultivars and enhancing disease management strategies.

Nano zinc oxide mediated resuscitation of aged Cajanus cajan via modulating aquaporin, cell cycle regulatory genes and hormonal responses.

KEY MESSAGE: Nanoparticle pretreatment improved the health of aged Cajanus cajan seeds viz., regulation of redox status, gene expression, and restoration of hormonal homeostasis. Ageing deteriorates the quality of seeds by lowering their vigor and viability, and terminating with loss of germination. These days, nanotechnology has been seen to revolutionize the agricultural sectors, and particularly nano zinc oxide (nZnO) has gained considerable interests due to its distinctive properties. The aim of the present work was to decipher the possibilities of using nZnO to rejuvenate accelerated aged (AA) seeds of Cajanus cajan. Both chemically (CnZnO) and green (GnZnO; synthesized using Moringa oleifera) fabricated nZnOs were characterized via standard techniques to interpret their purity, size, and shape. Experimental results revealed erratic germination with a decline in viability and membrane stability as outcomes of reactive oxygen intermediate (ROI) buildup in AA seeds. Application of nZnO substantially rebated the accrual of ROI, along with enhanced production of antioxidants, α-amylase activity, total sugar, protein and DNA content. Higher level of zinc was assessed qualitatively/ histologically and quantitatively in nZnO pulsed AA seeds, supporting germination without inducing toxicity. Meantime, augmentation in the gibberellic acid with a simultaneous reduction in the abscisic acid level were noted in nZnO invigorated seeds than that determined in the AA seeds, suggesting possible involvement of ROI in hormonal signalling. Furthermore, nZnO-subjected AA seeds unveiled differential expression of aquaporins and cell cycle regulatory genes. Summarizing, among CnZnO and GnZnO, later one holds better potential for a revival of AA seeds of Cajanus cajan by providing considerable tolerance against ageing-associated deterioration via recouping the cellular redox homeostasis, hormonal signaling, and alteration in expression patterns of aquaporin and cell cycle regulatory genes.

Deciphering miRNA-lncRNA-mRNA interaction through experimental validation of miRNAs, lncRNAs, and miRNA targets on mRNAs in Cajanus cajan.

Pigeon pea (Cajanus cajan) is widely cultivated for its nutritional and medicinal value yet remains an orphan crop as productivity has not been improved because of a lack of genome and non-coding genome information. Non-coding RNAs, like miRNAs and long non-coding RNAs (lncRNAs), are involved in regulation of growth, metabolism, development, and stress response, and have a critical role in post-transcriptional gene regulation (PTGR). We attempted to elucidate the roles of miRNAs and lncRNAs in pigeon pea through experimental validation of computationally predicted miRNAs and lncRNAs and targets of miRNAs on mRNAs. We experimentally validated 20 miRNAs and 11 lncRNAs. We predicted cleavage sites of three miRNA targets: serine/threonine-protein kinase, polygalacturonase, beta-galactosidase. We identified 469 targets of 265 miRNAs and their functional annotations using computational methods. We built a miRNA-mRNA-lncRNA network model, with the miRNAs targeting both mRNAs and lncRNAs, to obtain information on the interplay of these three molecules. A confirmed interaction through experimental validation was established between miRNA, namely cca-miR1535a targeting the mRNA for beta-galactosidase, as well as the lncRNA cca-lnc-020033. Our findings increase knowledge of the non-coding genome of pigeon pea and their roles in PTGR and in improving agronomic traits of this pulse crop.

Genome-wide identification and characterization of DIRIGENT gene family (CcDIR) in pigeonpea (Cajanus cajan L.) provide insights on their spatial expression pattern and relevance to stress response.

This study is a thorough characterization of pigeonpea dirigent gene (CcDIR) family, an important component of the lignin biosynthesis pathway. Genome-wide analysis identified 25 CcDIR genes followed by a range of analytical approaches employed to unravel their structural and functional characteristics. Structural examination revealed a classic single exon and no intron arrangement in CcDIRs contributing to our understanding on evolutionary dynamics. Phylogenetic analysis elucidated evolutionary relationships among CcDIR genes with six DIR sub-families, while motif distribution analysis displayed and highlighted ten conserved protein motifs in CcDIRs. Promoter analyses of all the dirigent genes detected 18 stress responsive cis-acting elements offering insights into transcriptional regulation. While spatial expression analyses across six plant tissues showed preferential expression of CcDIR genes, exposure to salt (CcDIR2 and CcDIR9) and herbivory (CcDIR1, CcDIR2, CcDIR3 and CcDIR11), demonstrated potential roles of specific DIRs in plant defense. Interestingly, increased gene expression during herbivory, also correlated with increased lignin content authenticating the specific response. Furthermore, exogenous application of stress hormones, SA and MeJA on leaves significantly induced the expression of CcDIRs that responded to herbivory. Taken together, these findings contribute to a comprehensive understanding of CcDIR genes impacting development and stress response in the important legume pigeonpea.

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.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Methylglyoxal metabolism is altered during defence response in pigeonpea (Cajanus cajan (L.) Millsp.) against the spotted pod borer (Maruca vitrata).

Pigeonpea (Cajanus cajan ) production can be affected by the spotted pod borer (Maruca vitrata ). Here, we identified biochemical changes in plant parts of pigeonpea after M. vitrata infestation. Two pigeonpea genotypes (AL 1747, moderately resistant; and MN 1, susceptible) were compared for glyoxalase and non-glyoxalase enzyme systems responsible for methylglyoxal (MG) detoxification, γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST) and glutathione content in leaves, flowers and pods under control and insect-infested conditions. MN 1 had major damage due to M. vitrata infestation compared to AL 1747. Lower accumulation of MG in AL 1747 was due to higher activities of enzymes of GSH-dependent (glyoxylase I, glyoxylase II), GSH-independent (glyoxalase III) pathway, and enzyme of non-glyoxalase pathway (methylglyoxal reductase, MGR), which convert MG to lactate. Decreased glyoxylase enzymes and MGR activities in MN 1 resulted in higher accumulation of MG. Higher lactate dehydrogenase (LDH) activity in AL 1747 indicates utilisation of MG detoxification pathway. Higher glutathione content in AL 1747 genotype might be responsible for efficient working of MG detoxification pathway under insect infestation. Higher activity of γ-GCS in AL 1747 maintains the glutathione pool, necessary for the functioning of glyoxylase pathway to carry out the detoxification of MG. Higher activities of GST and GPX in AL 1747 might be responsible for detoxification of toxic products that accumulates following insect infestation, and elevated activities of glyoxylase and non-glyoxylase enzyme systems in AL 1747 after infestation might be responsible for reducing reactive cabanoyl stress. Our investigation will help the future development of resistant cultivars.

Genome-wide identification, characterization, and expression analysis of MIPS family genes in legume species.

BACKGROUND: Evolutionarily conserved in plants, the enzyme D-myo-inositol-3-phosphate synthase (MIPS; EC 5.5.1.4) regulates the initial, rate-limiting reaction in the phytic acid biosynthetic pathway. They are reported to be transcriptional regulators involved in various physiological functions in the plants, growth, and biotic/abiotic stress responses. Even though the genomes of most legumes are fully sequenced and available, an all-inclusive study of the MIPS family members in legumes is still ongoing. RESULTS: We found 24 MIPS genes in ten legumes: Arachis hypogea, Cicer arietinum, Cajanus cajan, Glycine max, Lablab purpureus, Medicago truncatula, Pisum sativum, Phaseolus vulgaris, Trifolium pratense and Vigna unguiculata. The total number of MIPS genes found in each species ranged from two to three. The MIPS genes were classified into five clades based on their evolutionary relationships with Arabidopsis genes. The structural patterns of intron/exon and the protein motifs that were conserved in each gene were highly group-specific. In legumes, MIPS genes were inconsistently distributed across their genomes. A comparison of genomes and gene sequences showed that this family was subjected to purifying selection and the gene expansion in MIPS family in legumes was mainly caused by segmental duplication. Through quantitative PCR, expression patterns of MIPS in response to various abiotic stresses, in the vegetative tissues of various legumes were studied. Expression pattern shows that MIPS genes control the development and differentiation of various organs, and have significant responses to salinity and drought stress. CONCLUSION: The MIPS genes in the genomes of legumes have been identified, characterized and their expression was analysed. The findings pave way for understanding their molecular functions and evolution, and lead to identify the putative MIPS genes associated with different cell and tissue development.

Melatonin-mediated CcARP1 alters F-actin dynamics by phosphorylation of CcADF9 to balance root growth and salt tolerance in pigeon pea.

As a multifunctional hormone-like molecule, melatonin exhibits a pleiotropic role in plant salt stress tolerance. While actin cytoskeleton is essential to plant tolerance to salt stress, it is unclear if and how actin cytoskeleton participates in the melatonin-mediated alleviation of plant salt stress. Here, we report that melatonin alleviates salt stress damage in pigeon pea by activating a kinase-like protein, which interacts with an actin-depolymerizing factor. Cajanus cajan Actin-Depolymerizing Factor 9 (CcADF9) has the function of severing actin filaments and is highly expressed under salt stress. The CcADF9 overexpression lines (CcADF9-OE) showed a reduction of transgenic root length and an increased sensitivity to salt stress. By using CcADF9 as a bait to screen an Y2H library, we identified actin depolymerizing factor-related phosphokinase 1 (ARP1), a novel protein kinase that interacts with CcADF9. CcARP1, induced by melatonin, promotes salt resistance of pigeon pea through phosphorylating CcADF9, inhibiting its severing activity. The CcARP1 overexpression lines (CcARP1-OE) displayed an increased transgenic root length and resistance to salt stress, whereas CcARP1 RNA interference lines (CcARP1-RNAi) presented the opposite phenotype. Altogether, our findings reveal that melatonin-induced CcARP1 maintains F-actin dynamics balance by phosphorylating CcADF9, thereby promoting root growth and enhancing salt tolerance.