Enhancing drought tolerance in chilli pepper through AdDjSKI-mediated modulation of ABA sensitivity, photosynthetic preservation, and ROS scavenging.

Drought stress threatens the productivity of numerous crops, including chilli pepper (Capsicum annuum). DnaJ proteins are known to play a protective role against a wide range of abiotic stresses. This study investigates the regulatory mechanism of the chloroplast-targeted chaperone protein AdDjSKI, derived from wild peanut (Arachis diogoi), in enhancing drought tolerance in chilli peppers. Overexpressing AdDjSKI in chilli plants increased chlorophyll content, reflected in the maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm) compared with untransformed control (UC) plants. This enhancement coincided with the upregulated expression of PSII-related genes. Our subsequent investigations revealed that transgenic chilli pepper plants expressing AdDjSKI showed reduced accumulation of superoxide and hydrogen peroxide and, consequently, lower malondialdehyde levels and decreased relative electrolyte leakage percentage compared with UC plants. The mitigation of ROS-mediated oxidative damage was facilitated by heightened activities of antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, coinciding with the upregulation of the expression of associated antioxidant genes. Additionally, our observations revealed that the ectopic expression of the AdDjSKI protein in chilli pepper plants resulted in diminished ABA sensitivity, consequently promoting seed germination in comparison with UC plants under different concentrations of ABA. All of these collectively contributed to enhancing drought tolerance in transgenic chilli plants with improved root systems when compared with UC plants. Overall, our study highlights AdDjSKI as a promising biotechnological solution for enhancing drought tolerance in chilli peppers, addressing the growing global demand for this economically valuable crop.

Male sterility in plants: an overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production.

KEY MESSAGE: The male sterility system in plants has traditionally been utilized for hybrid seed production. In last three decades, genetic manipulation for male sterility has revolutionized this area of research related to hybrid seed production technology. Here, we have surveyed some of the natural cytoplasmic male sterility (CMS) systems that existed/ were developed in different crop plants for developing male sterility-fertility restoration systems used in hybrid seed production and highlighted some of the recent biotechnological advancements in the development of genetically engineered systems that occurred in this area. We have indicated the possible future directions toward the development of engineered male sterility systems. Cytoplasmic male sterility (CMS) is an important trait that is naturally prevalent in many plant species, which has been used in the development of hybrid varieties. This is associated with the use of appropriate genes for fertility restoration provided by the restorer line that restores fertility on the corresponding CMS line. The development of hybrids based on a CMS system has been demonstrated in several different crops. However, there are examples of species, which do not have usable cytoplasmic male sterility and fertility restoration systems (Cytoplasmic Genetic Male Sterility Systems-CGMS) for hybrid variety development. In such plants, it is necessary to develop usable male sterile lines through genetic engineering with the use of heterologous expression of suitable genes that control the development of male gametophyte and fertile male gamete formation. They can also be developed through gene editing using the recently developed CRISPR-Cas technology to knock out suitable genes that are responsible for the development of male gametes. The present review aims at providing an insight into the development of various technologies for successful production of hybrid varieties and is intended to provide only essential information on male sterility systems starting from naturally occurring ones to the genetically engineered systems obtained through different means.

Gain-of-function mutagenesis through activation tagging identifies XPB2 and SEN1 helicase genes as potential targets for drought stress tolerance in rice.

KEY MESSAGE: XPB2 and SEN1 helicases were identified through activation tagging as potential candidate genes in rice for inducing high water-use efficiency (WUE) and maintaining sustainable yield under drought stress. As a follow-up on the high-water-use-efficiency screening and physiological analyses of the activation-tagged gain-of-function mutant lines that were developed in an indica rice variety, BPT-5204 (Moin et al. in Plant Cell Environ 39:2440-2459, 2016a, https://doi.org/10.1111/pce.12796 ), we have identified two gain-of-function mutant lines (XM3 and SM4), which evidenced the activation of two helicases, ATP-dependent DNA helicase (XPB2) and RNA helicase (SEN1), respectively. We performed the transcript profiling of XPB2 and SEN1 upon exposure to various stress conditions and found their significant upregulation, particularly in ABA and PEG treatments. Extensive morpho-physiological and biochemical analyses based on 24 metrics were performed under dehydration stress (PEG) and phytohormone (ABA) treatments for the wild-type and the two mutant lines. Principal component analysis (PCA) performed on the dataset captured 72.73% of the cumulative variance using the parameters influencing the first two principal components. The tagged mutants exhibited reduced leaf wilting, improved revival efficiency, constant amylose:amylopectin ratio, high chlorophyll and proline contents, profuse tillering, high quantum efficiency and yield-related traits with respect to their controls. These observations were further validated under greenhouse conditions by the periodic withdrawal of water at the pot level. Germination of the seeds of these mutant lines indicated their insensitivity to high ABA concentration. The associated upregulation of stress-specific genes further suggests that their drought tolerance might be because of the coordinated expression of several stress-responsive genes in these two mutants. Altogether, our results provided a firm basis for SEN1 and XPB2 as potential candidates for manipulation of drought tolerance and improving rice performance and yield under limited water conditions.

Expressing class I wheat NHX (TaNHX2) gene in eggplant (Solanum melongena L.) improves plant performance under saline condition.

Brinjal or eggplant (Solanum melongena L.) is an important solanaceous edible crop, and salt stress adversely affects its growth, development, and overall productivity. To cope with excess salinity, vacuolar Na+/H+ antiporters provide the best mechanism for ionic homeostasis in plants under salt stress. We generated transgenic eggplants by introducing wheat TaNHX2 gene that encodes a vacuolar Na+/H+ antiporter in to the eggplant genome via Agrobacterium-mediated transformation using pBin438 vector that harbors double35S:TaNHX2 to confer salinity tolerance. Polymerase chain reaction and southern hybridization confirmed the presence and integration of TaNHX2 gene in T1 transgenic plants. Southern positive transgenic eggplants showed varied levels of TaNHX2 transcripts as evident by RT-PCR and qRT-PCR. Stress-inducible expression of TaNHX2 significantly improved growth performance and Na+ and K+ contents from leaf and roots tissues of T2 transgenic eggplants under salt stress, compared to non-transformed plants. Furthermore, T2 transgenic eggplants displayed the stable leaf relative water content and chlorophyll content, proline accumulation, improved photosynthetic efficiency, transpiration rate, and stomatal conductivity than the non-transformed plants under salinity stress (200 mM NaCl). Data showed that the T2 transgenic lines revealed that reduction in MDA content, hydrogen peroxide, and oxygen radical production associated with the significant increase of antioxidant enzyme activity in transgenic eggplants than non-transformed plants under salt stress (200 mM NaCl). This study suggested that the TaNHX2 gene plays an important regulatory role in conferring salinity tolerance of transgenic eggplant and thus may serve as a useful candidate gene for improving salinity tolerance in other vegetable crops.

Targeted expression of a cysteine protease (AdCP) in tapetum induces male sterility in Indian mustard, Brassica juncea.

The development of male sterile plants is a prerequisite to developing hybrid varieties to harness the benefits of hybrid vigor in crops and enhancing crop productivity for sustainable agriculture. In plants, cysteine proteases have been known for their multifaceted roles during programmed cell death, and in ubiquitin- and proteasome-mediated proteolysis. Here, we showed that Arachis diogoi cysteine protease (AdCP) expressed under the TA-29 promoter induced complete male sterility in Indian mustard, Brassica juncea. The herbicide resistance gene bar was used for the selection of transgenic plants. Mustard transgenic plants exhibited male sterile phenotype and failed to produce functional pollen grains. Irregularly shaped aborted pollen grains with groove-like structures were observed in male sterile plants during scanning electron microscopy analysis. The T1 progeny plants obtained from the seed of primary transgenic male sterile plants crossed with the wild-type plants exhibited segregation of the progeny into male sterile and fertile plants with normal seed development. Further, male sterile plants exhibited higher transcript levels of AdCP in anther tissues, which is consistent with its expression under the tapetum-specific promoter. Our results clearly suggest that the targeted expression of AdCP provides a potential tool for developing male sterile lines in crop plants by the malfunction of tapetal cells leading to male sterility as shown earlier in tobacco transgenic plants (Shukla et al. 2014, Funct Integr Genomics 14:307-317).

Improved salinity tolerance and growth performance in transgenic sunflower plants via ectopic expression of a wheat antiporter gene (TaNHX2).

Sunflower (Helianthus annuus. L) is one of the principal oil seed crops affected by the salinity stress, which limits the oil content and crop yield of sunflower plants. The acclimatization of plants to abiotic stresses such as salinity tolerance is mainly mediated by the vacuolar Na+/H+ antiporters (NHX) by tagging Na+ into vacuoles from the cytosol. We show here that the over-expression of wheat TaNHX2 gene in transgenic sunflower conferred improved salinity stress tolerance and growth performance. Transgenic sunflower plants were produced by infecting the embryonic axis ex-plants with Agrobacterium tumefaciens strain EHA105 containing a pBin438-TaNHX2 binary vector that carried a wheat antiporter (TaNHX2) gene under the control of a double CaMV 35S promoter with NPT II gene as a selectable marker. PCR analysis of T0 and T1 transgenic plants confirmed the integration of TaNHX2 in sunflower genome. Stable integration and expression of TaNHX2 in sunflower genome was further verified by Southern hybridization and semi-quantitative RT-PCR analyses. As compared to the non-transformed plants, TaNHX2 expressing transgenic plants showed better growth performance and accumulated higher Na+, K+ contents in leaves and roots under salt stress (200 mM NaCl). Transgenic sunflower plants displayed improved protection against cell damage exhibiting stable relative water content, chlorophyll content, increased proline accumulation and improved reactive oxygen species (ROS) scavenging because of higher activities of the antioxidant enzymes like superoxide dismutase and ascorbate peroxidase, along with decreased production of hydrogen peroxide, free oxygen radical and malondialdehyde (MDA) under salt stress (200 mM NaCl). Taken together, our findings suggest that TaNHX2 expression in sunflower plants contributed towards improving growth performance under sodium chloride stress.

Heterologous expression of Brassica juncea annexin, AnnBj2 confers salt tolerance and ABA insensitivity in transgenic tobacco seedlings.

Annexins are multifunctional proteins with roles in plant development and alleviation of stress tolerance. In the present communication, we report on the effect of heterologous expression of Brassica juncea annexin, AnnBj2 in tobacco. Transgenic tobacco plants expressing AnnBj2 exhibited salt-tolerant and abscisic acid (ABA)-insensitive phenotype at the seedling stage. Biochemical analysis showed that AnnBj2 transgenic plants retained higher chlorophyll and proline content, and lower malondialdehyde (MDA) levels compared to the null line under salt stress. They exhibited better water retention capacity compared to the null segregant (NS) line. AnnBj2 overexpression altered the transcript levels of several stress-related marker genes involved in reactive oxygen species (ROS) scavenging and abiotic stress signaling. Taken together, these results suggest a positive role for AnnBj2 in salt stress response upon heterologous expression in tobacco.

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water-use efficiency and abiotic stress tolerance in plants.

We have generated 3900 enhancer-based activation-tagged plants, in addition to 1030 stable Dissociator-enhancer plants in a widely cultivated indica rice variety, BPT-5204. Of them, 3000 were screened for water-use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield-related attributes under water-limiting conditions that identified 200 activation-tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ13 C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up-regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating-tagging. The high up-regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis-regulatory elements in their promoter regions along with the high up-regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance.

Co-overexpression of Brassica juncea NPR1 (BjNPR1) and Trigonella foenum-graecum defensin (Tfgd) in transgenic peanut provides comprehensive but varied protection against Aspergillus flavus and Cercospora arachidicola.

KEY MESSAGE: Coexpression of two antifungal genes ( NPR1 and defensin ) in transgenic peanut results in the development of resistance to two major fungal pathogens, Aspergillus flavus and Cercospora arachidicola. Fungal diseases have been one of the principal causes of crop losses with no exception to peanut (Arachis hypogeae L.), a major oilseed crop in Asia and Africa. To address this problem, breeding for fungal disease resistance has been successful to some extent against specific pathogens. However, combating more than one fungal pathogen via breeding is a major limitation in peanut. In the present study, we demonstrated the potential use of co-overexpression of two genes, NPR1 and defensin isolated from Brassica juncea and Trigonella foenum-graecum respectively; that offered resistance towards Aspergillus flavus in peanut. The transgenic plants not only resisted the mycelial growth but also did not accumulate aflatoxin in the seeds. Resistance was also demonstrated against another pathogen, Cercospora arachidicola at varied levels; the transgenic plants showed both reduction in the number of spots and delay in the onset of disease. PCR, Southern and Western blot analysis confirmed stable integration and expression of the transgenes in the transgenic plants. The combinatorial use of the two pathogen resistance genes presents a novel approach to mitigate two important fungal pathogens of peanut.

Characterization of a vacuolar processing enzyme expressed in Arachis diogoi in resistance responses against late leaf spot pathogen, Phaeoisariopsis personata.

Vacuolar processing enzymes are cysteine proteases responsible for maturation of vacuolar proteins. They have been shown to possess caspase-1-like activity, mediate cell death and display increased activity during pathogen infections. A transcript derived fragment corresponding to VPE was found to be up-regulated in a cDNA-AFLP analysis of host responses of a wild peanut, Arachis diogoi upon challenge from the late leaf spot pathogen Phaeoisariopsis personata, which was subsequently validated by q-PCR in a time course analysis, where susceptible peanut did not show its upregulation. In transient conditional and constitutive expression studies in tobacco leaves using agroinfiltration, we have observed that expression of AdVPE was associated with hypersensitive response (HR) like cell death. AdVPE expression was found to be high at 24 h post estradiol application and this was associated with the enhanced co-expression of molecular markers of HR cell death genes and genes for pathogenesis related proteins indicating that AdVPE positively regulates defense responses and its estradiol induced expression is sufficient for HR-like cell death in tobacco. We found that AdVPE expression was very strongly induced in response to sodium nitroprusside, which indicates its involvement in stress signaling. Induced expression of AdVPE in response to jasmonic acid and ethylene also indicates its involvement in an interconnected network of signaling. Transgenic tobacco plants ectopically expressing AdVPE exhibited enhanced resistance against Phytophthora parasitica var. nicotianae, Alternaria alternata var.  nicotianae and Rhizoctonia solani. To our knowledge, this is the first report on the heterologous expression of a pathogen induced VPE enhancing resistance to fungal pathogens with cell death phenomenon under transient expression.

Comparative proteomics reveals differential induction of both biotic and abiotic stress response associated proteins in rice during Xanthomonas oryzae pv. oryzae infection.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice and brutally affects the yield up to 50 % of total production. Here, we report a comparative proteomics analysis of total foliar protein isolated from infected rice leaves of susceptible Pusa Basmati 1 (PB1) and resistant Oryza longistaminata genotypes. Two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) approaches identified 29 protein spots encoding unique proteins from both the genotypes. Identified proteins belonged to a large number of biological and molecular functions related to biotic and abiotic stress proteins which are potentially involved during Xoo infection. Biotic and abiotic stress-related proteins were induced during Xoo infection, indicating the activation of common stress pathway during bacterial blight infection. Candidate genes conferring tolerance against bacterial blight, which include germin-like protein, putative r40c1, cyclin-dependent kinase C, Ent-isokaur-15-ene synthase and glutathione-dependent dehydroascorbate reductase 1 (GSH-DHAR1), were also induced, with germin-like proteins induced only in the resistant rice genotype O. longistaminata. Energy, metabolism and hypothetical proteins were common among both the genotypes. Further, host defence/stress-related proteins were mostly expressed in resistant genotype O. longistaminata, indicating possible co-evolution of the pathogen and the wild rice, O. longistaminata.

A synchronized symphony: Intersecting roles of ubiquitin proteasome system and autophagy in cellular degradation.

Eukaryotic cells have evolved dynamic quality control pathways and recycling mechanisms for cellular homeostasis. We discuss here, the two major systems for quality control, the ubiquitin-proteasome system (UPS) and autophagy that regulate cellular protein and organelle turnover and ensure efficient nutrient management, cellular integrity and long-term wellbeing of the plant. Both the pathways rely on ubiquitination signal to identify the targets for proteasomal and autophagic degradation, yet they use distinct degradation machinery to process these cargoes. Nonetheless, both UPS and autophagy operate together as an interrelated quality control mechanism where they communicate with each other at multiple nodes to coordinate and/or compensate the recycling mechanism particularly under development and environmental cues. Here, we provide an update on the cellular machinery of autophagy and UPS, unravel the nodes of their crosstalk and particularly highlight the factors responsible for their differential deployment towards protein, macromolecular complexes and organelles.

CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.).

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon-intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

Ectopic expression of DnaJ type-I protein homolog of Vigna aconitifolia (VaDJI) confers ABA insensitivity and multiple stress tolerance in transgenic tobacco plants.

Reduced crop productivity results from altered plant physiological processes caused by dysfunctional proteins due to environmental stressors. In this study, a novel DnaJ Type-I encoding gene, VaDJI having a zinc finger motif in its C-terminal domain was found to be induced early upon treatment with heat stress (within 5 min) in a heat tolerant genotype of Vigna aconitifolia RMO-40. VaDJI is induced by multiple stresses. In tobacco, ectopic expression of VaDJI reduced ABA sensitivity during seed germination and the early stages of seedling growth of transgenic tobacco plants. Concomitantly, it also improved the ability of transgenic tobacco plants to withstand drought stress by modulating the photosynthetic efficiency, with the transgenic plants having higher Fv/Fm ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H2O2 and lower MDA levels and higher chlorophyll content during drought stress, which attenuated cell damage and reduced oxidative damage. An analysis using the qRT-PCR study demonstrated that VaDJI overexpression is associated with the expression of some ROS-detoxification-related genes and stress-marker genes that are often induced during drought stress responses. These findings suggest a hypothesis whereby VaDJI positively influences drought stress tolerance and ABA signalling in transgenic tobacco, and suggests that it is a potential gene for genetic improvement of drought and heat stress tolerance in crop plants.

Novel role for a serine/arginine-rich splicing factor, AdRSZ21 in plant defense and HR-like cell death.

A splicing factor gene belonging to the serine/arginine (SR)-rich protein family was cloned from Arachis diogoi, a wild relative of peanut in a study on differential gene expression and was designated as AdRSZ21. AdRSZ21 exhibits a RNA recognition motif (RRM), a CCHC type zinc finger domain (Zinc Knuckle, ZnK) and a C-terminal RS domain that is rich in arginine and serine. Multiple sequence alignment of AdRSZ21 with putative orthologs from diverse taxa including lower plants and monocots showed that the RRM and ZnK domains are evolutionarily conserved. Phylogenetic studies revealed that AdRSZ21 belongs to the RSZ subfamily and is closely related to the Arabidopsis ortholog AtRSZ22. Transient constitutive and conditional heterologous expression of AdRSZ21 resulted in HR-like cell death in tobacco leaves. The presence of a functional RRM domain, but not ZnK domain was essential for AdRSZ21 induced HR-like cell death phenotype. On the other hand, expression of AdRSZ21 with mutated ZnK domain lead to accelerated cell death. The cell death induced by AdRSZ21 was found to be associated with specific upregulation of patatin-like protein gene and other defense related gene transcripts suggesting a role for AdRSZ21 in plant defense and HR-like cell death.

Increased resistance to late leaf spot disease in transgenic peanut using a combination of PR genes.

Peanut (Arachis hypogaea L.) is the sixth most important oil seed crop in the world. Yield loss due to Cercospora leaf spot (early and late leaf spots) is a serious problem in cultivating this crop. Non-availability of resistant genes within crossable germplasms of peanut necessitates the use of a genetic engineering strategy to develop genetic resistance against various biotic stresses. The pathogenesis-related (PR) proteins are a group of plant proteins that are toxic to invading fungal pathogens, but are present in trace amounts in plants. The PR proteins, PR-5 and defensins, are potent antifungal proteins. A double gene construct with SniOLP (Solanum nigrum osmotin-like protein) and Rs-AFP2 (Raphanus sativus antifungal protein-2) genes under separate constitutive 35S promoters was used to transform peanut plants. Transgenic peanut plants expressing the SniOLP and Rs-AFP2 genes showed enhanced disease resistance to late leaf spot based on a reduction in number and size of lesions on leaves and delay in the onset of Phaeoisariopsis personata leaf spot disease. PCR, RT-PCR, and Southern hybridization analyses confirmed stable integration and expression of these genes in peanut transgenics. The results demonstrate the potential of SniOLP and Rs-AFP2 genes in developing late leaf spot disease resistance in transgenic peanut.

Mungbean plants expressing BjNPR1 exhibit enhanced resistance against the seedling rot pathogen, Rhizoctonia solani.

Mungbean, Vigna radiata (L.) Wilczek is an important pulse crop that is widely cultivated in semi- arid tropics. The crop is attacked by various soil-borne pathogens like Rhizoctonia solani, which causes dry rot disease and seriously affects its productivity. Earlier we characterized the non-expressor of pathogenesis related gene-1(BjNPR1) of mustard, Brassica juncea, the counterpart of AtNPR1 of Arabidopsis thaliana. Here, we transformed mungbean with BjNPR1 via Agrobacterium tumefaciens. Because of the recalcitrant nature of mungbean, the effect of some factors like Agrobacterium tumefaciens strains (GV2260 and LBA4404), pH, L: -cysteine and tobacco leaf extract was tested in transformation. The transgenic status of 15 plants was confirmed by PCR using primers for nptII. The independent integration of T-DNA in transgenic plants was analyzed by Southern hybridization with an nptII probe and the expression of BjNPR1 was confirmed by RT-PCR. Some of the T(0) plants were selected for detached leaf anti-fungal bioassay using the fungus Rhizoctonia solani, which showed moderate to high level of resistance depending on the level of expression of BjNPR1. The seedling bioassay of transgenic T(2) plants indicated resistance against dry rot disease caused by R. solani.

Identification and characterization of annexin gene family in rice.

Plant annexins are Ca(2+)-dependent phospholipid-binding proteins and are encoded by multigene families. They are implicated in the regulation of plant development as well as protection from drought and other stresses. They are well characterized in Arabidopsis, however no such characterization of rice annexin gene family has been reported thus far. With the availability of the rice genome sequence information, we have identified ten members of the rice annexin gene family. At the protein level, they share 16-64% identity with predicted molecular masses ranging from 32 to 40 kDa. Phylogenetic analysis of rice annexins together with annexins from other monocots led to their classification into five different orthologous groups and share similar motif patterns in their protein sequences. Expression analysis by real-time RT-PCR revealed differential temporal and spatial regulation of these genes. The rice annexin genes are also found to be regulated in seedling stage by various abiotic stressors including salinity, drought, heat and cold. Additionally, in silico analysis of the putative upstream sequences was analyzed for the presence of stress-responsive cis-elements. These results provide a basis for further functional characterization of specific rice annexin genes at the tissue/developmental level and in response to abiotic stresses.

Expression of chitinase genes of Metarhizium anisopliae isolates in lepidopteran pests and on synthetic media.

Pathogenecity of the well characterized entomopathogenic fungus Metarhizium anisopliae used for biocontrol of a wide range of insect pests secretes hydrolytic enzymes that degrade the host cuticle. The chitinolytic activity of high and low virulent isolates of M. anisopliae was assayed on minimal medium (MM) + colloidal chitin and MM supplemented with insect cuticles. Ex- pression pattern of four chitinase genes (chitinase (chi), chi 1, chi 2, chi 3) was profiled during pathogenic stages of the entomopathogen under in vitro and in vivo conditions. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that chitinase cDNAs were expressed during the germination of fungus under nutrient-deprived conditions. RT-PCR analysis performed for the four chitinase genes on the two insect hosts Spodoptera litura and Helicoverpa armigera at six developmental stages of the pathogen displayed up-regulation in S. litura at mycosed and conidiated condition while with H. armigera there was expression only after 48 h of incubation. Differential expression of chi, chi 1 and chi 2 genes in vitro (nitrogen rich and nitrogen limiting media) and in vivo (live insect hosts S. litura and H. armigera) implicate the role of substrate differences in pathogenesis.

Identifying Signal-Crosstalk Mechanism in Maize Plants during Combined Salinity and Boron Stress Using Integrative Systems Biology Approaches.

Combined stress has been seen as a major threat to world agriculture production. Maize is one of the leading cereal crops of the world due to its wide spectrum of growth conditions and is moderately sensitive to salt stress. A saline soil environment is a major factor that hinders its growth and overall yield and causes an increase in the concentration of micronutrients like boron, leading to excess over the requirement of the plant. Boron toxicity combined with salinity has been reported to be a serious threat to the yield and quality of maize. The response signatures of the maize plants to the combined effect of salinity and boron stress have not been studied well. We carried out an integrative systems-level analysis of the publicly available transcriptomic data generated on tolerant maize (Lluteño maize from the Atacama Desert, Chile) landrace under combined salt and boron stress. We identified significant biological processes that are differentially regulated in combined salt and boron stress in the leaves and roots of maize, respectively. Protein-protein interaction network analysis identified important roles of aldehyde dehydrogenase (ALDH), galactinol synthase 2 (GOLS2) proteins of leaf and proteolipid membrane potential regulator (pmpm4), metallothionein lea protein group 3 (mlg3), and cold regulated 410 (COR410) proteins of root in salt tolerance and regulating boron toxicity in maize. Identification of transcription factors coupled with regulatory network analysis using machine learning approach identified a few heat shock factors (HSFs) and NAC (NAM (no apical meristem, Petunia), ATAF1-2 (Arabidopsis thaliana activating factor), and CUC2 (cup-shaped cotyledon, Arabidopsis)) family transcription factors (TFs) to play crucial roles in salt tolerance, maintaining reactive oxygen species (ROS) levels and minimizing oxidative damage to the cells. These findings will provide new ways to design targeted functional validation experiments for developing multistress-resistant maize crops.