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.

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.

The pigeon pea CcCIPK14-CcCBL1 pair positively modulates drought tolerance by enhancing flavonoid biosynthesis.

Calcineurin B-like (CBL)-interacting protein kinases (CIPKs) play a central role in Ca2+ signalling and promote drought tolerance in plants. The CIPK gene family in pigeon pea (Cajanus cajan L.), a major food crop affected by drought, has not previously been characterised. Here, we identified 28 CIPK genes in the pigeon pea genome. Five CcCIPK genes were strongly upregulated in roots upon drought treatment and were selected for further characterisation. Overexpression of CcCIPK13 and CcCIPK14 increased survival rates by two- to three-fold relative to controls after 14 days of drought. Furthermore, the three major flavonoids, genistin, genistein and apigenin, were significantly upregulated in the same transgenic plants. Using CcCIPK14 as bait, we performed a yeast two-hybrid screen and identified six interactors, including CcCBL1. CcCIPK14 exhibited autophosphorylation and phosphorylation of CcCBL1 in vitro. CcCBL1-overexpressed plants displayed higher survival rates upon drought stress as well as higher expression of flavonoid biosynthetic genes and flavonoid content. CcCIPK14-overexpressed plants in which CcCBL1 transcript levels were reduced by RNA interference had lower survival rates, which indicated CcCBL1 in the same pathway as CcCIPK14. Together, our results demonstrate a role for the CcCIPK14-CcCBL1 complex in drought stress tolerance through the regulation of flavonoid biosynthesis in pigeon pea.

Identification of microRNAs and their gene targets in cytoplasmic male sterile and fertile maintainer lines of pigeonpea.

MAIN CONCLUSION: Comparative analysis of genome-wide miRNAs and their gene targets between cytoplasmic male sterile (CMS) and fertile lines of pigeonpea suggests a possible role of miRNA-regulated pathways in reproductive development. Exploitation of hybrid vigor using CMS technology has delivered nearly 50% yield gain in pigeonpea. Among various sterility-inducing cytoplasms (A1-A9) reported so far in pigeonpea, A2 and A4 are the two major sources that facilitate hybrid seed production. Recent evidence suggests involvement of micro RNA in vast array of biological processes including plant reproductive development. In pigeonpea, information about the miRNAs is insufficient. In view of this, we sequenced six small RNA libraries of CMS line UPAS 120A and isogenic fertile line UPAS 120B using Illumina technology. Results revealed 316 miRNAs including 248 known and 68 novel types. A total of 637 gene targets were predicted for known miRNAs, while 324 genes were associated with novel miRNAs. Degradome analysis revealed 77 gene targets of predicted miRNAs, which included a variety of transcription factors playing key roles in plant reproduction such as F-box family proteins, apetala 2, auxin response factors, ethylene-responsive factors, homeodomain-leucine zipper proteins etc. Differential expression of both known and novel miRNAs implied roles for both conserved as well as species-specific players. We also obtained several miRNA families such as miR156, miR159, miR167 that are known to influence crucial aspects of plant fertility. Gene ontology and pathway level analyses of the target genes showed their possible implications for crucial events during male reproductive development such as tapetal degeneration, pollen wall formation, retrograde signaling etc. To the best of our knowledge, present study is first to combine deep sequencing of small RNA and degradome for elucidating the role of miRNAs in flower and male reproductive development in pigeonpea.

Characterization of microRNA genes from Pigeonpea (Cajanus cajan L.) and understanding their involvement in drought stress.

MicroRNAs (miRNA) are non-coding 20-24 nucleotide long RNAs regulating gene expression. In this study, we have characterized and analysed expression of miRNAs in Pigeonpea by using bioinformatics and experimental tools. We identified 116 miRNAs belonging to 32 phylogenetic families. Further, transcription start sites of miRNA genes revealed abundance and unique arrangement of adenine at +1 and thymine at -1 position. Promoter analysis exhibited presence of 19 most prevalent motifs which comprises majorly of TATA box and MYC domains. In total, 252 miRNA-targets were identified and found to be involved in various developmental processes and stress responses. Moreover, genome-wide localization studies demonstrated clustering of cca-miRNA 395 and 169 genes. The tandem and segmental duplication events were observed suggesting miRNA genes have been originated parallelly with protein coding genes. The expression analysis revealed induction of cca-miR169a, 398a and 408 miRNAs under drought stress highlighting their involvement. Conversely, down-regulation of their putative targets (NFYA, SOD, and UCLA, respectively) confirmed regulatory role of miRNAs in their expression emphasising the negative relationship between these miRNAs and targets in Pigeonpea. This study reports vast repertoire of miRNA genes which further can be experimentally characterized to elucidate their functions in various biological processes and can be recommended for Pigeonpea improvement programs.

Transcriptome profiling of differentially expressed genes in cytoplasmic male-sterile line and its fertility restorer line in pigeon pea (Cajanus cajan L.).

BACKGROUND: Pigeon pea (Cajanus cajan L.) is the sixth major legume crop widely cultivated in the Indian sub-continent, Africa, and South-east Asia. Cytoplasmic male-sterility (CMS) is the incompetence of flowering plants to produce viable pollens during anther development. CMS has been extensively utilized for commercial hybrid seeds production in pigeon pea. However, the molecular basis governing CMS in pigeon pea remains unclear and undetermined. In this study transcriptome analysis for exploring differentially expressed genes (DEGs) between cytoplasmic male-sterile line (AKCMS11) and its fertility restorer line (AKPR303) was performed using Illumina paired-end sequencing. RESULTS: A total of 3167 DEGs were identified, of which 1432 were up-regulated and 1390 were down-regulated in AKCMS11 in comparison to AKPR303. By querying, all the 3167 DEGs against TAIR database, 34 pigeon pea homologous genes were identified, few involved in pollen development (EMS1, MS1, ARF17) and encoding MYB and bHLH transcription factors with lower expression in the sterile buds, implying their possible role in pollen sterility. Many of these DEGs implicated in carbon metabolism, tricarboxylic acid cycle (TCA), oxidative phosphorylation and elimination of reactive oxygen species (ROS) showed reduced expression in the AKCMS11 (sterile) buds. CONCLUSION: The comparative transcriptome findings suggest the potential role of these DEGs in pollen development or abortion, pointing towards their involvement in cytoplasmic male-sterility in pigeon pea. The candidate DEGs identified in this investigation will be highly significant for further research, as they could lend a comprehensive basis in unravelling the molecular mechanism governing CMS in pigeon pea.

Interactive effects of polyamines and arbuscular mycorrhiza in modulating plant biomass, N2 fixation, ureide, and trehalose metabolism in Cajanus cajan (L.) Millsp. genotypes under nickel stress.

Nickel (Ni) is an essential micronutrient but considered toxic for plant growth when present in excess in the soil. Polyamines (PAs) and arbuscular mycorrhiza (AM) play key roles in alleviating metal toxicity in plants. Present study compared the roles of AM and PAs in improving rhizobial symbiosis, ureide, and trehalose (Tre) metabolism under Ni stress in Cajanus cajan (pigeon pea) genotypes (Pusa 2001, AL 201). The results documented significant negative impacts of Ni on plant biomass, especially roots, more in AL 201 than Pusa 2001. Symbiotic efficiency with Rhizobium and AM declined under Ni stress, resulting in reduced AM colonization, N2 fixation, and ureide biosynthesis. This decline was proportionate to increased Ni uptake in roots and nodules. Put-reduced Ni uptake improved plant growth and functional efficiency of nodules and ureides synthesis, with higher positive effects than other PAs. However, AM inoculations were most effective in enhancing nodulation, nitrogen fixing potential, and Tre synthesis under Ni toxicity. Combined applications of AM with respective PAs, especially +Put+AM, were highly beneficial in alleviating Ni-induced nodule senescence by arresting leghemoglobin degradation and improving functional efficiency of nodules by boosting Tre metabolism, especially in Pusa 2001. The study suggested use of Put along with AM as a promising approach in imparting Ni tolerance to pigeon pea plants.

Identification, characterization and expression profiles of Fusarium udum stress-responsive WRKY transcription factors in Cajanus cajan under the influence of NaCl stress and Pseudomonas fluorescens OKC.

The WRKY gene family has never been identified in pigeonpea (Cajanus cajan). Therefore, objective of the present study was to identify the WRKY gene family in pigeonpea and characterize the Fusarium udum stress-responsive WRKY genes under normal, NaCl-stressed and Pseudomonas fluorescens OKC (a plant growth-promoting bacterial strain) treated conditions. The aim was to characterize the Fusarium udum stress-responsive WRKY genes under some commonly occurring field conditions. We identified 97 genes in the WRKY family of pigeonpea, using computational prediction method. The gene family was then classified into three groups through phylogenetic analysis of the homologous genes from the representative plant species. Among the 97 identified WRKY genes 35 were further classified as pathogen stress responsive genes. Functional validation of the 35 WRKY genes was done through generating transcriptional profiles of the genes from root tissues of pigeonpea plants under the influence of P. fluorescens OKC after 24 h of stress application (biotic: Fusarium udum, abiotic: NaCl). The entire experiment was conducted in two pigeonpea cultivars Asha (resistant to F. udum) and Bahar (susceptible to F. udum) and the results were concluded on the basis of transcriptional regulation of the WRKY genes in both the pigeonpea cultivars. The results revealed that among the 35 tentatively identified biotic stress responsive CcWRKY genes, 26 were highly F. udum responsive, 17 were better NaCl responsive compared to F. udum and 11 were dual responsive to both F. udum and NaCl. Application of OKC was able to enhance transcript accumulation of the individual CcWRKY genes to both the stresses when applied individually but not in combined challenge of the two stresses. The results thus indicated that CcWRKY genes play a vital role in the defense signaling against F. udum and some of the F. udum responsive CcWRKYs (at least 11 in pigeonpea) are also responsive to abiotic stresses such as NaCl. Further, plant beneficial microbes such as P. fluorescens OKC also help pegionpea to defend itself against the two stresses (F. udum and NaCl) through enhanced expression of the stress responsive CcWRKY genes when the stresses are applied individually.

Assessing C-N balance and soil rejuvenation capacity of vermicompost application in a degraded landscape: A study in an alluvial river basin with Cajanus cajan.

The value and potential of organic-dependent agriculture has gradually become realized worldwide despite scarcity of data on its ability to rejuvenate land quality. In this work, a popular cultivar of Cajanus cajan was grown in a nutrient-fatigued area with vermicompost (VC) and farmyard manure (FYM) to partially replace chemical fertilization (CF) (reduced by 10 and 50%) over a four-year period. C-N mineralization kinetic equations were applied for the first time to assess the efficacy of vermicompost-based nutrient schemes on C-N balance in arable soil. The results suggested that 10 and 50% replacement of chemical fertilization with vermicompost was helpful to significantly improveC- and N-based mineralization in the cultivated soil. In addition, the gains of humified C (humic and fulvic acid) content (such as 4-and 10-fold increases) were achieved under CF50/VC50 and CF90/VC10 treatments, respectively. In the CF50/VC50 treatment, there was apparent improvement in soil organic C storage (SOCstr) (42.6-57.4%), water holding capacity, and microbial health along with reduction in bulk density (6.10-13.0%). Moreover, improvement in soil quality was strongly evident in terms of crop production with the application of CF50/VC50. Overall, this study successfully validated that vermicompost can be used effectively to achieve sustainable agricultural growth by efficiently arresting land degradation.

Tissue specific disruption of photosynthetic electron transport rate in pigeonpea (Cajanus cajan L.) under elevated temperature.

Nutritional rich pigeonpea (Cajanus cajan [L.]), a perennial shrub member of family Fabaceae is the sixth most important grain legume of the world. Continuous rise of temperature and current global climate scenario limits plant growth and performance but photosynthetic machineries are adversely affected. The aim of this study was the analysis of tissue specific photosynthetic pigments and photosynthetic electron transport rate under elevated temperature. Two different stages of both leaf (young and mature), and pod (young and mature) were chosen, and photosynthetic pigment and J-I-P tests were measured. Leaves and pods were detached and incubated in normal temperature (25°C) for 24 h in two different condition one light irradiance (100 µmol m-2 s-1) and another darkness as control, and treated with high temperature 45°C for 24 h and repeated previous measurements were taken. Tissue specific photosynthetic pigments variation were observed; J-I-P parameters clearly revealed that elevated temperature has greater influence on photosystem II (PSII) electron transport than photosystem I (PSI), and significant changes were observed in pods than leaves. Young tissues were adversely affected by elevated temperature. In addition, the J-I-P tests and energy pipeline model indicated that PSI electron transport rate of leaves and pods appeared to be more thermo-tolerance than those in PSII. Only a minor drop in pigments pool and photosynthetic performance was observed after 24 h of darkness. O-J-I-P transients can be used as a sensitive, nondestructive method for measuring heat stress damage and a special tool for investigating action sites of high temperature stress. Findings of this study will contribute to basic understanding of photosynthetic performance, and to screen potential thermo-tolerant genotypes of pigeonpea to sustain in either current scenario of climate change or/and erratic future climatic conditions.

Joint effects of Si and mycorrhiza on the antioxidant metabolism of two pigeonpea genotypes under As (III) and (V) stress.

Arsenic (As) is the most hazardous soil contaminant, which inactivates metabolic enzymes and restrains plant growth. To withstand As stress conditions, use of some alleviative tools, such as arbuscular mycorrhizal (AM) fungi and silicon (Si), has gained importance. Therefore, the present study evaluated comparative and interactive effects of Si and arbuscular mycorrhiza-Rhizophagus irregularis on phytotoxicity of arsenate (As V) and arsenite (As III) on plant growth, ROS generation, and antioxidant defense responses in pigeonpea genotypes (Tolerant-Pusa 2002; Sensitive-Pusa 991). Roots of As III treated plants accumulated significantly higher total As than As V supplemented plants, more in Pusa 991 than Pusa 2002, which corresponded to proportionately decreased plant growth, root to biomass ratio, and oxidative burst. Although Si nutrition and AM inoculations improved plant growth by significantly reducing As uptake and the resultant oxidative burst, AM was relatively more efficient in upregulating enzymatic and non-enzymatic antioxidant defense responses as well as ascorbate-glutathione pathway when compared with Si. Pusa 2002 was more receptive to Si nourishment due to its ability to establish more efficient mycorrhizal symbiosis, which led to higher Si uptake and lower As concentrations. Moreover, +Si+AM bestowed better metalloid resistance by further reducing ROS and strengthening antioxidants. Results demonstrated that the genotype with more efficient AM symbiosis in As-contaminated soils could accrue higher benefits of Si fertilization in terms of metalloid tolerance in pigeonpea.

Molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea (Cajanus cajan (L.) Millsp.).

KEY MESSAGE: We report molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea. We have also developed PCR-based markers amenable to low-cost genotyping to identify fertility restorer lines. Commercial hybrids in pigeonpea are based on A4 cytoplasmic male sterility (CMS) system, and their fertility restoration is one of the key prerequisites for breeding. In this context, an effort has been made to understand the genetics and identify quantitative trait loci (QTL) associated with restoration of fertility (Rf). One F2 population was developed by crossing CMS line (ICPA 2039) with fertility restorer line (ICPL 87119). Genetic analysis has shown involvement of two dominant genes in regulation of restoration of fertility. In parallel, the genotyping-by-sequencing (GBS) approach has generated ~ 33 Gb data on the F2 population. GBS data have provided 2457 single nucleotide polymorphism (SNPs) segregating across the mapping population. Based on these genotyping data, a genetic map has been developed with 306 SNPs covering a total length 981.9 cM. Further QTL analysis has provided the region flanked by S8_7664779 and S8_6474381 on CcLG08 harboured major QTL explained up to 28.5% phenotypic variation. Subsequently, sequence information within the major QTLs was compared between the maintainer and the restorer lines. From this sequence information, we have developed two PCR-based markers for identification of restorer lines from non-restorer lines and validated them on parental lines of hybrids as well as on another F2 mapping population. The results obtained in this study are expected to enhance the efficiency of selection for the identification of restorer lines in hybrid breeding and may reduce traditional time-consuming phenotyping activities.

Characterization of CcSTOP1; a C2H2-type transcription factor regulates Al tolerance gene in pigeonpea.

MAIN CONCLUSION: Al-responsive citrate-transporting CcMATE1 function and its regulation by CcSTOP1 were analyzed using NtSTOP1 -KD tobacco- and pigeonpea hairy roots, respectively, CcSTOP1 binding sequence of CcMATE1 showed similarity with AtALMT1 promoter. The molecular mechanisms of Aluminum (Al) tolerance in pigeonpea (Cajanus cajan) were characterized to provide information for molecular breeding. Al-inducible citrate excretion was associated with the expression of MULTIDRUGS AND TOXIC COMPOUNDS EXCLUSION (CcMATE1), which encodes a citrate transporter. Ectopic expression of CcMATE1-conferred Al tolerance to hairy roots of transgenic tobacco with the STOP1 regulation system knocked down. This gain-of-function approach clearly showed CcMATE1 was involved in Al detoxification. The expression of CcMATE1 and another Al-tolerance gene, ALUMINUM SENSITIVE 3 (CcALS3), was regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (CcSTOP1) according to loss-of-function analysis of pigeonpea hairy roots in which CcSTOP1 was suppressed. An in vitro binding assay showed that the Al-responsive CcMATE1 promoter contained the GGNVS consensus bound by CcSTOP1. Mutation of GGNVS inactivated the Al-inducible expression of CcMATE1 in pigeonpea hairy roots. This indicated that CcSTOP1 binding to the promoter is critical for CcMATE1 expression. The STOP1 binding sites of both the CcMATE1 and AtALMT1 promoters contained GGNVS and a flanking 3' sequence. The GGNVS region was identical in both CcMATE1 and AtALMT1. By contrast, the 3' flanking sequence with binding affinity to STOP1 did not show similarity. Putative STOP1 binding sites with similar structures were also found in Al-inducible MATE and ALMT1 promoters in other plant species. The characterized Al-responsive CcSTOP1 and CcMATE1 genes will help in pigeonpea breeding in acid soil tolerance.

Gene expression atlas of pigeonpea and its application to gain insights into genes associated with pollen fertility implicated in seed formation.

Pigeonpea (Cajanus cajan) is an important grain legume of the semi-arid tropics, mainly used for its protein rich seeds. To link the genome sequence information with agronomic traits resulting from specific developmental processes, a Cajanus cajan gene expression atlas (CcGEA) was developed using the Asha genotype. Thirty tissues/organs representing developmental stages from germination to senescence were used to generate 590.84 million paired-end RNA-Seq data. The CcGEA revealed a compendium of 28 793 genes with differential, specific, spatio-temporal and constitutive expression during various stages of development in different tissues. As an example to demonstrate the application of the CcGEA, a network of 28 flower-related genes analysed for cis-regulatory elements and splicing variants has been identified. In addition, expression analysis of these candidate genes in male sterile and male fertile genotypes suggested their critical role in normal pollen development leading to seed formation. Gene network analysis also identified two regulatory genes, a pollen-specific SF3 and a sucrose-proton symporter, that could have implications for improvement of agronomic traits such as seed production and yield. In conclusion, the CcGEA provides a valuable resource for pigeonpea to identify candidate genes involved in specific developmental processes and to understand the well-orchestrated growth and developmental process in this resilient crop.

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants.

The nitrogen phosphotransferase system (PTS(Ntr)) consists of EI(Ntr), NPr, and EIIA(Ntr). The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI(Ntr) and NPr to EIIA(Ntr). Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI(Ntr) and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA(Ntr)) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-ß-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA(Ntr) in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI(Ntr) with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS(Ntr) in symbiosis was also observed when related PTS(Ntr) mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS(Ntr) in addition to the negative role of unphosphorylated EIIA(Ntr).

Bee diversity assemblage on pigeon pea, Cajanus cajan along habitat gradient.

The regional bee diversity was investigated on pigeon pea in three agroclimatic zones of Punjab state in northern India. Of the total nine species recorded, population of Megachilids, in particular, was significantly higher in all the three zones. Appraisal of diversity metrics revealed highest bee community diversity in Sub-mountain Undulating zone, followed by Central Plain zone, while least diversity was noted in Western zone, which highlighted the effect of habitat on species richness and abundance. Diurnal variations were evident with the most diverse bee communities recorded at 11:00 hr, followed by at 14:00 and 08:00 hr, with the lowest community diversity at 17:00 hr. The relative abundance of bee species was highly in favour of non-Apis species than Apis species. The results obtained can be used in the application of risk management through planned plant protection measures, study of plant-pollinator interactions along with conservation and augmentation of bee species.

Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions.

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.

Delayed flowering is associated with lack of photosynthetic acclimation in Pigeon pea (Cajanus cajan L.) grown under elevated CO2.

In the present study, we investigated the likely consequences of future atmospheric CO2 concentrations [CO2] on growth, physiology and reproductive phenology of Pigeonpea. A short duration Pigeonpea cultivar (ICPL 15011) was grown without N fertilizer from emergence to final harvest in CO2 enriched atmosphere (open top chambers; 550µmolmol(-1)) for two seasons. CO2 enrichment improved both net photosynthetic rates (Asat) and foliar carbohydrate content by 36 and 43%, respectively, which further reflected in dry biomass after harvest, showing an increment of 29% over the control plants. Greater carboxylation rates of Rubisco (Vcmax) and photosynthetic electron transport rates (Jmax) in elevated CO2 grown plants measured during different growth periods, clearly demonstrated lack of photosynthetic acclimation. Further, chlorophyll a fluorescence measurements as indicated by Fv/Fm and ΔF/Fm' ratios justified enhanced photosystem II efficiency. Mass and number of root nodules were significantly high in elevated CO2 grown plants showing 58% increase in nodule mass ratio (NMR) which directly correlated with Pn. Growth under high CO2 showed significant ontogenic changes including delayed flowering. In conclusion, our data demonstrate that the lack of photosynthetic acclimation and increased carbohydrate-nitrogen reserves modulate the vegetative and reproductive growth patterns in Pigeonpea grown under elevated CO2.

Study the effect of insecticide dimethoate on photosynthetic pigments and photosynthetic activity of pigeon pea: Laser-induced chlorophyll fluorescence spectroscopy.

Pigeon pea is one of the most important legume crops in India and dimethoate is a widely used insecticide in various crop plants. We studied the effect of dimethoate on growth and photosynthetic activity of pigeon pea plants over a short and long term exposure. Plant growth parameters, photosynthetic pigment content and chlorophyll fluorescence response of pigeon pea (Cajanus cajan L.) plants treated with various concentrations of the insecticide dimethoate (10, 20, 40 and 80 ppm) have been compared for 30 days at regular intervals of 10 days each. Laser induced chlorophyll fluorescence spectra and fluorescence-induction kinetics (FIK) curve of dimethoate treated pigeon pea plants were recorded after 10, 20 and 30 days of treatment. Fluorescence intensity ratio at the two fluorescence maxima (F685/F730) was calculated by evaluating curve-fitted parameters. The variable chlorophyll fluorescence decrease ratio (Rfd) was determined from the FIK curves. Our study revealed that after 10 days of treatment, 10 ppm of dimethoate showed stimulatory response whereas 20, 40 and 80 ppm of dimethoate showed inhibitory response for growth and photosynthetic activity of pigeon pea plants, but after 20 and 30 days of treatment all the tested concentrations of dimethoate became inhibitory. This study clearly shows that dimethoate is highly toxic to the pigeon pea plant, even at very low concentration (10 ppm), if used for a prolonged duration. Our study may thus be helpful in determining the optimal dose of dimethoate in agricultural practices.

NAC transcription factor genes: genome-wide identification, phylogenetic, motif and cis-regulatory element analysis in pigeonpea (Cajanus cajan (L.) Millsp.).

The NAC (NAM, ATAF and CUC) proteins are plant-specific transcription factors implicated in development and stress responses. In the present study 88 pigeonpea NAC genes were identified from the recently published draft genome of pigeonpea by using homology based and de novo prediction programmes. These sequences were further subjected to phylogenetic, motif and promoter analyses. In motif analysis, highly conserved motifs were identified in the NAC domain and also in the C-terminal region of the NAC proteins. A phylogenetic reconstruction using pigeonpea, Arabidopsis and soybean NAC genes revealed 33 putative stress-responsive pigeonpea NAC genes. Several stress-responsive cis-elements were identified through in silico analysis of the promoters of these putative stress-responsive genes. This analysis is the first report of NAC gene family in pigeonpea and will be useful for the identification and selection of candidate genes associated with stress tolerance.