Search

Abstract In Arabidopsis, non-expressor of pathogenesis related genes-1, NPR1 has been shown to be a positive regulator of the salicylic acid controlled systemic acquired resistance pathway and modulates the cross talk between SA and JA signaling. Transgenic plants expressing AtNPR1 constitutively exhibited resistance against pathogens as well as herbivory. In the present study, tobacco transgenic plants expressing AtNPR1 were studied further for their response to infection by the sedentary endoparasitic root knot nematode, Meloidogyne incognita. Transgenic plants showed enhanced resistance against the root-knot nematode infection. Prominent differences in the shoot and root weights of wild type and transgenic plants were observed post-inoculation with M. incognita. This was associated with a decrease in the number of root galls and egg masses in transgenic plants compared to WT. The transgenic plants also showed constitutive and induced expression of some PR protein genes, when challenged with M. incognita.

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., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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., (© 2024 Scandinavian Plant Physiology Society.)

Heat stress substantially reduces tomato (Solanum lycopersicum) growth and yield globally, thereby jeopardizing food security. DnaJ proteins, constituents of the heat shock protein system, protect cells from diverse environmental stresses as HSP-70 molecular co-chaperones. In this study, we demonstrated that AdDjSKI, a serine-rich DnaJ III protein induced by pathogens, plays an important role in stabilizing photosystem II (PSII) in response to heat stress. Our results revealed that transplastomic tomato plants expressing the AdDjSKI gene exhibited increased levels of total soluble proteins, improved growth and chlorophyll content, reduced malondialdehyde (MDA) accumulation, and diminished PSII photoinhibition under elevated temperatures when compared with wild-type (WT) plants. Intriguingly, these transplastomic plants maintained higher levels of D1 protein under elevated temperatures compared with the WT plants, suggesting that overexpression of AdDjSKI in plastids is crucial for PSII protection, likely due to its chaperone activity. Furthermore, the transplastomic plants displayed lower accumulation of superoxide radical (O 2 •─ ) and H 2 O 2 , in comparison with the WT plants, plausibly attributed to higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. This also coincides with an enhanced expression of corresponding genes, including SlCuZnSOD, SlFeSOD, SlAPX2, and SltAPX, under heat stress. Taken together, our findings reveal that chloroplastic expression of AdDjSKI in tomatoes plays a critical role in fruit yield, primarily through a combination of delayed senescence and stabilizing PSII under heat stress., (© 2024 Scandinavian Plant Physiology Society.)

Peanut Arachis hypogaea is a segmental allotetraploid in the section Arachis of the genus Arachis along with the Section Rhizomataceae. Section Arachis has several diploid species along with Arachis hypogaea and A. monticola . The section Rhizomataceae comprises polyploid species. Several species in the genus are highly tolerant to biotic and abiotic stresses and provide excellent sets of genotypes for studies on differential gene expression. Though there were several studies in this direction, more studies are needed to identify more and more gene combinations. Next generation RNA-seq based differential gene expression study is a powerful tool to identify the genes and regulatory pathways involved in stress tolerance. Transcriptomic and proteomic study of peanut plants under biotic stresses reveals a number of differentially expressed genes such as R genes (NBS-LRR, LRR-RLK, protein kinases, MAP kinases), pathogenesis related proteins (PR1, PR2, PR5, PR10) and defense related genes (defensin, F-box, glutathione S-transferase) that are the most consistently expressed genes throughout the studies reported so far. In most of the studies on biotic stress induction, the differentially expressed genes involved in the process with enriched pathways showed plant-pathogen interactions, phenylpropanoid biosynthesis, defense and signal transduction. Differential gene expression studies in response to abiotic stresses, reported the most commonly expressed genes are transcription factors (MYB, WRKY, NAC, bZIP, bHLH, AP2/ERF), LEA proteins, chitinase, aquaporins, F-box, cytochrome p450 and ROS scavenging enzymes. These differentially expressed genes are in enriched pathways of transcription regulation, starch and sucrose metabolism, signal transduction and biosynthesis of unsaturated fatty acids. These identified differentially expressed genes provide a better understanding of the resistance/tolerance mechanism, and the genes for manipulating biotic and abiotic stress tolerance in peanut and other crop plants. There are a number of differentially expressed genes during biotic and abiotic stresses were successfully characterized in peanut or model plants (tobacco or Arabidopsis ) by genetic manipulation to develop stress tolerance plants, which have been detailed out in this review and more concerted studies are needed to identify more and more gene/gene combinations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kumar and Kirti.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Splicing of pre-mRNA is an essential part of eukaryotic gene expression. Serine-/arginine-rich (SR) proteins are highly conserved RNA-binding proteins present in all metazoans and plants. SR proteins are involved in constitutive and alternative splicing, thereby regulating the transcriptome and proteome diversity in the organism. In addition to their role in splicing, SR proteins are also involved in mRNA export, nonsense-mediated mRNA decay, mRNA stability, and translation. Due to their pivotal roles in mRNA metabolism, SR proteins play essential roles in normal growth and development. Hence, any misregulation of this set of proteins causes developmental defects in both plants and animals. SR proteins from the animal kingdom are extensively studied for their canonical and noncanonical functions. Compared with the animal kingdom, plant genomes harbor more SR protein-encoding genes and greater diversity of SR proteins, which are probably evolved for plant-specific functions. Evidence from both plants and animals confirms the essential role of SR proteins as regulators of gene expression influencing cellular processes, developmental stages, and disease conditions. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing., (© 2022 Wiley Periodicals LLC.)

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 F v /F m ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H 2 O 2 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gautam, Meena, Rampuria, Shukla and Kirti.)

The target of rapamycin (TOR) protein phosphorylates its downstream effector p70kDa ribosomal protein S6 kinases (S6K1) for ribosome biogenesis and translation initiation in eukaryotes. However, the molecular mechanism of TOR-S6K1-ribosomal protein (RP) signaling is not well understood in plants. In the present study, we report the transcriptional upregulation of ribosomal protein large and small subunit ( RPL and RPS ) genes in the previously established TOR overexpressing transgenic lines of rice (in Oryza sativa ssp. indica , variety BPT-5204, TR-2.24 and TR-15.1) and of Arabidopsis thaliana (in Col 0 ecotype, ATR-1.4.27 and ATR-3.7.32). The mRNA levels of RP genes from this study were compared with those previously available in transcriptomic datasets on the expression of RP s in relation to TOR inhibitor and in the TOR -RNAi lines of Arabidopsis thaliana . We further analyzed TOR activity, i.e., S6K1 phosphorylation in SALK lines of Arabidopsis with mutation in rpl6 , rpl18 , rpl23 , rpl24 and rps28C, where the rpl18 mutant showed inactivation of S6K1 phosphorylation. We also predicted similar putative Ser/Thr phosphorylation sites for ribosomal S6 kinases (RSKs) in the RPs of Oryza sativa ssp. indica and Arabidopsis thaliana. The findings of this study indicate that the TOR pathway is possibly interlinked in a cyclic manner via the phosphorylation of S6K1 as a modulatory step for the regulation of RP function to switch 'on'/'off' the translational regulation for balanced plant growth.

Main Conclusion: Manipulation of autophagic pathway represents a tremendous opportunity for designing climate-smart crops with improved yield and better adaptability to changing environment. For exploiting autophagy to its full potential, identification and comprehensive characterization of adapters/receptor complex and elucidation of its regulatory network in crop plants is highly warranted.  Autophagy is a major intracellular trafficking pathway in eukaryotes involved in vacuolar degradation of cytoplasmic constituents, mis-folded proteins, and defective organelles. Under optimum conditions, autophagy operates at a basal level to maintain cellular homeostasis, but under stressed conditions, it is induced further to provide temporal stress relief. Our understanding of this highly dynamic process has evolved exponentially in the past few years with special reference to several plant-specific roles of autophagy. Here, we review the most recent advances in the field of autophagy in plants and discuss its potential implications in designing crops with improved stress and disease-tolerance, enhanced yield potential, and improved capabilities for producing metabolites of high economic value. We also assess the current knowledge gaps and the possible strategies to develop a robust module for biotechnological application of autophagy to enhance bioeconomy and sustainability of agriculture., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Sheath Blight (SB) disease in rice is caused by the infection from the fungal pathogen Rhizoctonia solani (R. solani). SB is one of the most severe rice diseases that can cause up to 50% yield losses in rice. Naturally occurring rice varieties resistant to SB have not been reported yet. We have performed a Time-Series RNA-Seq analysis on a widely cultivated rice variety BPT-5204 for identifying transcriptome level response signatures during R. solani infection at 1st, 2nd and 5th day post infection (dpi). In total, 428, 3225 and 1225 genes were differentially expressed in the treated rice plants on 1, 2 and 5 dpi, respectively. GO and KEGG enrichment analysis identified significant processes and pathways differentially altered in the rice plants during the fungal infection. Machine learning and network based integrative approach was used to construct rice Transcriptional Regulatory Networks (TRNs) for the three time points. TRN analysis identified SUB1B, MYB30 and CCA1 as important regulatory hub transcription factors in rice during R. solani infection. Jasmonic acid, salicylic acid, ethylene biogenesis and signaling were induced on infection. SAR was up regulated, while photosynthesis and carbon fixation processes were significantly down regulated. Involvement of MAPK, CYPs, peroxidase, PAL, chitinase genes were also observed in response to the fungal infection. The integrative analysis identified seven putative SB resistance genes differentially regulated in rice during R. solani infection., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2022 Drishtee Barua et al.)

Our group has previously identified the activation of a GRAS transcription factor (TF) gene in the gain-of-function mutant population developed through activation tagging in rice (in an indica rice variety, BPT 5204) that was screened for water use efficiency. This family of GRAS transcription factors has been well known for their diverse roles in gibberellin signaling, light responses, root development, gametogenesis etc. Recent studies indicated their role in biotic and abiotic responses as well. Although this family of TFs received significant attention, not many genes were identified specifically for their roles in mediating stress tolerance in rice. Only OsGRAS23 (here named as OsGRAS22 ) was reported to code for a TF that induced drought tolerance in rice. In the present study, we have analyzed the expression patterns of rice GRAS TF genes under abiotic (NaCl and ABA treatments) and biotic (leaf samples infected with pathogens, Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight and Rhizoctonia solani that causes sheath blight) stress conditions. In addition, their expression patterns were also analyzed in 13 different developmental stages. We studied their spatio-temporal regulation and correlated them with the in-silico studies. Fully annotated genomic sequences available in rice database have enabled us to study the protein properties, ligand interactions, domain analysis and presence of cis -regulatory elements through the bioinformatic approach. Most of the genes were induced immediately after the onset of stress particularly in the roots of ABA treated plants. OsGRAS39 was found to be a highly expressive gene under sheath blight infection and both abiotic stress treatments while OsGRAS8 , OsSHR1 and OsSLR1 were also responsive. Our earlier activation tagging based functional characterization followed by the genome-wide characterization of the GRAS gene family members in the present study clearly show that they are highly appropriate candidate genes for manipulating stress tolerance in rice and other crop plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dutta, Saha, Moin and Kirti.)

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.

Our previous study demonstrated that the expression of GhNAC4, a NAC transcription factor from cotton, was induced by abiotic stresses and abscisic acid (ABA). In the present study, we investigated the molecular mechanisms underlying ABA and stress response of GhNAC4. Overexpression of GhNAC4 in transgenic tobacco conferred tolerance to salinity and drought treatments with associated enhanced expression of several stress-responsive marker genes. GhNAC4 is a protein that is translocated to the nucleus where it exhibits transcriptional activation property and also forms homo-dimers. In this study, we also investigated the domains essential for the biochemical functions of GhNAC4. We developed transgenic tobacco plants overexpressing the GhNAC4 NAC-domain and the transcriptional regulatory (TR) domain separately. NAC-domain transgenics showed hypersensitivity to exogenous ABA while TR-domain transgenics exhibited reduced sensitivity. Abiotic stress assays indicated that transgenic plants expressing both the domains separately were more tolerant than wild type plants with the NAC-domain transgenics showing increased tolerance as compared to TR-domain transgenics. Expression analysis revealed that various stress-responsive genes were upregulated in both NAC-domain and TR-domain transgenics under salinity and drought treatments. These results suggest that the stress tolerance ability of GhNAC4 is associated with both the component domains while the ABA responsiveness is largely associated with N-terminal NAC-domain., (Copyright © 2020 Elsevier B.V. All rights reserved.)

CBL interacting protein kinases play important roles in adaptation to stress conditions. In the present study, we isolated a CBL-interacting protein kinase homolog (AdCIPK5) from a wild peanut (Arachis diogoi) with similarity to AtCIPK5 of Arabidopsis. Expression analyses in leaves of the wild peanut showed AdCIPK5 induction by exogenous signaling molecules including salicylic acid, abscisic acid and ethylene or abiotic stress factors like salt, PEG and sorbitol. The recombinant AdCIPK5-GFP protein was found to be localized to the nucleus, plasma membrane and cytoplasm. We overexpressed AdCIPK5 in tobacco plants and checked their level of tolerance to biotic and abiotic stresses. While wild type and transgenic plants displayed no significant differences to the treatment with the phytopathogen, Phytophthora parasitica pv nicotianae, the expression of AdCIPK5 increased salt and osmotic tolerance in transgenic plants. Analysis of different physiological parameters revealed that the transgenic plants maintained higher chlorophyll content and catalase activity with lower levels of H 2 O 2 and MDA content during the abiotic stress conditions. AdCIPK5 overexpression also contributed to the maintenance of a higher the K + /Na + ratio under salt stress. The enhanced tolerance of transgenic plants was associated with elevated expression of stress-related marker genes; NtERD10C, NtERD10D, NtNCED1, NtSus1, NtCAT and NtSOS1. Taken together, these results indicate that AdCIPK5 is a positive regulator of salt and osmotic stress tolerance.

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 T 0 and T 1 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.

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 T 1 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).

Manifestation of male sterility in plants is an important requirement for hybrid seed production. Tapetum cell layer of anther is a primary target for genetic manipulation for male sterility. In our previous report, the targeted expression of Arachis cysteine protease in tapetum led to premature degeneration of tapetal layer that resulted in complete male sterility in transgenic tobacco plants. To correlate cysteine protease mediated cell death of tapetum, transmission electron microscopy (TEM) and proteomic pattern of anthers of cysteine protease induced male sterile plant were compared with the untransformed control plant. TEM study revealed the abnormal growth of tapetal cells exhibiting excessive vacuolization that synchronized with irregular exine wall formation of the microspores. In anther proteome, a total 250 protein spots were detected that were reproducible and exhibited similar distribution pattern. Further, anther proteome of male sterile plant showed the significant upregulation (≥ 1.5) of 56 protein spots. Using Mass spectroscopy (MALDI TOF/TOF), we have identified 14 protein spots that were involved in several processes such as energy metabolism, protein synthesis, plastid protein, lipid metabolism, and cell wall assembly. Upregulation of patatin-like protein-2 homolog, carboxylesterase 17 and dicer like protein-4 in male sterile anthers that have been demonstrated to induce cell death, suggesting that cysteine protease mediated premature tapetal cell death might involve the lipid peroxidation pathway in coordination with gene silencing mechanism., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest.

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 T 1 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 T 2 transgenic eggplants under salt stress, compared to non-transformed plants. Furthermore, T 2 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 T 2 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.

Aldehyde dehydrogenase (ALDH) carries out oxidation of toxic aldehydes using NAD + /NADP + as cofactors. In the present study, we performed a genome-wide identification and expression analysis of genes in the ALDH gene family in Brassica rapa. A total of 23 ALDH genes in the superfamily have been identified according to the classification of ALDH Gene Nomenclature Committee (AGNC). They were distributed unevenly across all 10 chromosomes. All the 23 Brassica rapa ALDH (BrALDH) genes exhibited varied expression patterns during treatments with abiotic stress inducers and hormonal treatments. The relative expression profiles of ALDH genes in B. rapa showed that they are predominantly expressed in leaves and stem suggesting their function in the vegetative tissues. BrALDH7B2 showed a strong response to abiotic stress and hormonal treatments as compared to other ALDH genes; therefore, it was overexpressed in heterologous hosts, E. coli and yeast to study its possible function under abiotic stress conditions. Over-expression of BrALDH7B2 in heterologous systems, E. coli and yeast cells conferred significant tolerance to abiotic stress treatments. Results from this work demonstrate that BrALDH genes are a promising and untapped genetic resource for crop improvement and could be deployed further in the development of drought and salinity tolerance in B. rapa and other economically important crops.

The target of rapamycin (TOR) protein regulates growth and development in photosynthetic and non-photosynthetic eukaryotes. Although the TOR regulatory networks are involved in nutrient and energy signalling, and transcriptional and translational control of multiple signalling pathways, the molecular mechanism of TOR regulation of plant abiotic stress responses is still unclear. The TOR-mediated transcriptional regulation of genes encoding ribosomal proteins (RP) is a necessity under stress conditions for balanced growth and productivity in plants. The activation of SnRKs (sucrose non-fermenting-related kinases) and the inactivation of TOR signalling in abiotic stresses is in line with the accumulation of ABA and transcriptional activation of stress responsive genes. Autophagy is induced under abiotic stress conditions, which results in degradation of proteins and the release of amino acids, which might possibly induce phosphorylation of TOR and, hence, its activation. TOR signalling also has a role in regulating ABA biosynthesis for transcriptional regulation of stress-related genes. The switch between activation and inactivation of TOR by its phosphorylation and de-phosphorylation maintains balanced growth in response to stresses. In the present review, we discuss the important signalling pathways that are regulated by TOR and try to assess the relationship between TOR signalling and tolerance to abiotic stresses in plants. The review also discusses possible cross-talk between TOR and RP genes in response to abiotic stresses., (© 2018 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

One of the important and direct ways of investigating the function of a gene is to characterize the phenotypic consequences associated with loss or gain-of-function of the corresponding gene. These mutagenesis strategies have been successfully deployed in Arabidopsis, and subsequently extended to crop species including rice. Researchers have made vast advancements in the area of rice genomics and functional genomics, as it is a diploid plant with a relatively smaller genome size unlike other cereals. The advent of rice genome research and the annotation of high-quality genome sequencing along with the developments in databases and computer searches have enabled the functional characterization of unknown genes in rice. Further, with the improvements in the efficiency of regeneration and transformation protocols, it has now become feasible to produce sizable mutant populations in indica rice varieties also. In this review, various mutagenesis methods, the current status of the mutant resources, limitations and strengths of insertional mutagenesis approaches and also results obtained with suitable screens for stress tolerance in rice are discussed. In addition, targeted genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or Cas9/single-guide RNA system and its potential applications in generating transgene-free rice plants through genome engineering as an efficient alternative to classical transgenic technology are also discussed.

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.

Powdery mildew (PM, caused by Golovinomyces orontii) is one of the major diseases on sunflower that causes severe yield losses in the tropics. Sources of resistance to PM are reported in an exotic accession and some wild Helianthus species. The present study aims at quantitative proteomic analysis of susceptible, resistant, and immune genotypes of sunflower in response to PM infection at 3, 7, 10 days post infection. The majority of differentially expressed proteins in the resistant genotype belonged to oxidative stress (catalase, ATP-sulfurylase, and formate dehydrogenase), defense (HSP-70, heat shock transcription factors), and photosynthesis (LHCB3). In case of immune genotype, 50% of proteins are related to photosynthesis, which play a key role in plant immunity, whereas a few similar proteins are also expressed in the susceptible genotype, but in their reduced abundance besides being inadequate in timing of expression probably leading to its susceptibility to PM. KEGG enrichment analysis shows that carbon metabolism (6-phosphogluconate dehydrogenase, pyruvate dehydrogenase, glutamine synthetase), photosynthesis, and plant-pathogen protein pathways are key pathways governing the resistance. The transcriptional expression of eight of nine differentially expressed proteins are in agreement with the expression of proteins at the corresponding time. The present study provides information on the key proteins that are upregulated in resistant and immune genotypes which restrict the disease progression and constitutes the first quantitative proteomic data of sunflower-PM infection process., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A gene encoding a serine-rich DnaJIII protein called AdDjSKI that has a 4Fe-4S cluster domain was found to be differentially upregulated in the wild peanut, Arachis diogoi in its resistance responses against the late leaf spot causing fungal pathogen Phaeoisariopsis personata when compared with the cultivated peanut, Arachis hypogaea. AdDjSKI is induced in multiple stress conditions in A. diogoi. Recombinant E. coli cells expressing AdDjSKI showed better growth kinetics when compared with vector control cells under salinity, osmotic, acidic and alkaline stress conditions. Overexpression of this type three J-protein potentiates not only abiotic stress tolerance in Nicotiana tabacum var. Samsun, but also enhances its disease resistance against the phytopathogenic fungi Phytophthora parasitica pv nicotianae and Sclerotinia sclerotiorum. In the present study we show transcriptional upregulation of APX, Mn-SOD and HSP70 under heat stress and increased transcripts of PR genes in response to fungal infection. This transmembrane-domain-containing J protein displays punctate localization in chloroplasts. AdDjSKI appears to ensure proper folding of proteins associated with the photosynthetic machinery under stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Annexins belong to a plasma membrane binding (in a calcium dependent manner), multi-gene family of proteins, which play ameliorating roles in biotic and abiotic stresses. The expression of annexin AnnBj2 of Indian mustard is tissue specific with higher expression in roots and under treatments with sodium chloride and abscisic acid (ABA) at seedling stage. The effect of constitutive expression of AnnBj2 in mustard was analyzed in detail. AnnBj2 OE (over expression) plants exhibited insensitivity to ABA, glucose and sodium chloride. The insensitivity/tolerance of the transgenic plants was associated with enhanced total chlorophylls, relative water content, proline, calcium and potassium with reduced thiobarbituric acid reactive substances and sodium ion accumulation. The altered ABA insensitivity of AnnBj2 OE lines is linked to downregulation of ABI4 and ABI5 transcription factors and upregulation of ABA catabolic gene CYP707A2. Furthermore, we found that overexpression of AnnBj2 upregulated the expression of ABA-dependent RAB18 and ABA-independent DREB2B stress marker genes suggesting that the tolerance phenotype exhibited by AnnBj2 OE lines is probably controlled by both ABA-dependent and -independent mechanisms., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Our previous findings on the screening of a large-pool of activation tagged rice plants grown under limited water conditions revealed the activation of Ribosomal Protein Large (RPL) subunit genes, RPL6 and RPL23A in two mutants that exhibited high water-use efficiency (WUE) with the genes getting activated by the integrated 4x enhancers (Moin et al., 2016a). In continuation of these findings, we have comprehensively characterized the Ribosomal Protein (RP) gene family including both small (RPS) and large (RPL) subunits, which have been identified to be encoded by at least 70 representative genes; RP-genes exist as multiple expressed copies with high nucleotide and amino acid sequence similarity. The differential expression of all the representative genes in rice was performed under limited water and drought conditions at progressive time intervals in the present study. More than 50% of the RP genes were upregulated in both shoot and root tissues. Some of them exhibited an overlap in upregulation under both the treatments indicating that they might have a common role in inducing tolerance under limited water and drought conditions. Among the genes that became significantly upregulated in both the tissues and under both the treatments are RPL6, 7, 23A, 24 , and 31 and RPS4, 10 and 18a . To further validate the role of RP genes in WUE and inducing tolerance to other stresses, we have raised transgenic plants overexpressing RPL23A in rice. The high expression lines of RPL23A exhibited low Δ 13 C, increased quantum efficiency along with suitable growth and yield parameters with respect to negative control under the conditions of limited water availability. The constitutive expression of RPL23A was also associated with transcriptional upregulation of many other RPL and RPS genes. The seedlings of RPL23A high expression lines also showed a significant increase in fresh weight, root length, proline and chlorophyll contents under simulated drought and salt stresses. Taken together, our findings provide a secure basis for the RPL gene family expression as a potential resource for exploring abiotic stress tolerant properties in rice.

Usable pollination control systems have proven to be effective system for the development of hybrid crop varieties, which are important for optimal performance over varied environments and years. They also act as a biocontainment to check horizontal transgene flow. In the last two decades, many genetic manipulations involving genes controlling the production of cytotoxic products, conditional male sterility, altering metabolic processes, post-transcriptional gene silencing, RNA editing and chloroplast engineering methods have been used to develop a proper pollination control system. In this review article, we outline the approaches used for generating male sterile plants using an effective pollination control system to highlight the recent progress that occurred in this area. Furthermore, we propose possible future directions for biotechnological improvements that will allow the farmers to buy hybrid seed once for many generations in a cost-effective manner.

Ribosomal proteins (RPs) are indispensable in ribosome biogenesis and protein synthesis, and play a crucial role in diverse developmental processes. Our previous studies on Ribosomal Protein Large subunit (RPL) genes provided insights into their stress responsive roles in rice. In the present study, we have explored the developmental and stress regulated expression patterns of Ribosomal Protein Small (RPS) subunit genes for their differential expression in a spatiotemporal and stress dependent manner. We have also performed an in silico analysis of gene structure, cis -elements in upstream regulatory regions, protein properties and phylogeny. Expression studies of the 34 RPS genes in 13 different tissues of rice covering major growth and developmental stages revealed that their expression was substantially elevated, mostly in shoots and leaves indicating their possible involvement in the development of vegetative organs. The majority of the RPS genes have manifested significant expression under all abiotic stress treatments with ABA, PEG, NaCl, and H 2 O 2 . Infection with important rice pathogens, Xanthomonas oryzae pv. oryzae ( Xoo ) and Rhizoctonia solani also induced the up-regulation of several of the RPS genes. RPS4, 13a, 18a , and 4a have shown higher transcript levels under all the abiotic stresses, whereas, RPS4 is up-regulated in both the biotic stress treatments. The information obtained from the present investigation would be useful in appreciating the possible stress-regulatory attributes of the genes coding for rice ribosomal small subunit proteins apart from their functions as house-keeping proteins. A detailed functional analysis of independent genes is required to study their roles in stress tolerance and generating stress- tolerant crops.

Expression analysis of genes associated with development at different growth stages such as shoot apical meristem (SAM), root apical meristem (RAM), shoot and root tissues 10 DAG, flowers and grains of 2 high expression transgenic lines of rice ectopically expressing AtTOR revealed the involvement of AtTOR in transcriptional regulation of these genes. We have observed that in the SAM of these 2 selected lines, TR-2.24 and TR-15.1, OsFON1 and OsFON4 (orthologs of AtCLV1 and AtCLV3, respectively), OsKNOX2, OsKNOX3 and OsWOX3 became upregulated. The upregulation of OsFON1 and OsFON4 is likely to be involved in the maintenance of effective meristem size of the inflorescence and phyllotaxis. The grains and spikes of transgenic plants exhibited enhanced transcript levels of OsMADS1, OsMADS6, and OsMADS29 further implicating the role of TOR in modulating the expression of the genes in rice grain formation and development. Moreover, the upregulation of auxin transporter, PIN1c in RAM and roots derived from seedlings 10 DAG showed the involvement of TOR in root development. The seeds of 2 high expression lines also showed increased expression of OSE2 and GAMYB transcription factors involved in seed development. In summary, the present study, by heterologous expression of AtTOR in rice, demonstrated the involvement of TOR in regulating genes involved in various growth and developmental stages of rice plant and also in photosynthesis, productivity related functions and water-use efficiency.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor with three distinct NF-YA, NF-YB and NF-YC subunits. It plays important roles in plant growth, development and stress responses. We have reported earlier on development of gain-of-function mutants in an indica rice cultivar, BPT-5204. Now, we screened 927 seeds from 70 Ac/Ds plants for salinity tolerance and identified one activation-tagged salt tolerant DS plant (DS-16, T 3 generation) that showed enhanced expression of a novel 'histone-like transcription factor' belonging to rice NF-Y subfamily C and was named as OsNF-YC13. Localization studies using GFP-fusion showed that the protein is localized to nucleus and cytoplasm. Real time expression analysis confirmed upregulation of transcript levels of OsNF-YC13 during salt treatment in a tissue specific manner. Biochemical and physiological characterization of the DS-16 revealed enhanced K + /Na + ratio, proline content, chlorophyll content, enzymes with antioxidant activity etc. DS-16 also showed transcriptional up-regulation of genes that are involved in salinity tolerance. In-silico analysis of OsNF-YC13 promoter region evidenced the presence of various key stress-responsive cis-regulatory elements. OsNF-YC13 subunit alone does not appear to have the capacity for direct transcription activation, but appears to interact with the B- subunits in the process of transactivation.

The epitome of any genome research is to identify all the existing genes in a genome and investigate their roles. Various techniques have been applied to unveil the functions either by silencing or over-expressing the genes by targeted expression or random mutagenesis. Rice is the most appropriate model crop for generating a mutant resource for functional genomic studies because of the availability of high-quality genome sequence and relatively smaller genome size. Rice has syntenic relationships with members of other cereals. Hence, characterization of functionally unknown genes in rice will possibly provide key genetic insights and can lead to comparative genomics involving other cereals. The current review attempts to discuss the available gain-of-function mutagenesis techniques for functional genomics, emphasizing the contemporary approach, activation tagging and alterations to this method for the enhancement of yield and productivity of rice., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The target of Rapamycin (TOR) present in all eukaryotes is a multifunctional protein, regulating growth, development, protein translation, ribosome biogenesis, nutrient, and energy signaling. In the present study, ectopic expression of TOR gene of Arabidopsis thaliana in a widely cultivated indica rice resulted in enhanced plant growth under water-limiting conditions conferring agronomically important water-use efficiency (WUE) trait. The AtTOR high expression lines of rice exhibited profuse tillering, increased panicle length, increased plant height, high photosynthetic efficiency, chlorophyll content and low ∆ 13 C. Δ 13 C, which is inversely related to high WUE, was as low as 17‰ in two AtTOR high expression lines. These lines were also insensitive to the ABA-mediated inhibition of seed germination. The significant upregulation of 15 stress-specific genes in high expression lines indicates their contribution to abiotic stress tolerance. The constitutive expression of AtTOR is also associated with significant transcriptional upregulation of putative TOR complex-1 components, OsRaptor and OsLST8. Glucose-mediated transcriptional activation of AtTOR gene enhanced lateral root formation. Taken together, our findings indicate that TOR, in addition to its multiple cellular functions, also plays an important role in response to abiotic stress and potentially enhances WUE and yield related attributes.

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 Δ 13 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., (© 2016 John Wiley & Sons Ltd.)

Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2-3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in rice and other crops, which may be achieved by overexpressing and raising independent transgenic plants carrying the genes that became up-regulated significantly and instantaneously.

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.

Fertility restoration in male sterile plants is an essential requirement for their utilization in hybrid seed production. In an earlier investigation, we have demonstrated that the targeted expression of a cysteine protease in tapetal cell layer resulted in complete male sterility in tobacco transgenic plants. In the present investigation, we have used a cystatin gene, which encodes for a cysteine protease inhibitor, from a wild peanut, Arachis diogoi and developed a plant gene based restoration system for cysteine protease induced male sterile transgenic tobacco plants. We confirmed the interaction between the cysteine protease and a cystatin of the wild peanut, A. diogoi through in silico modeling and yeast two-hybrid assay. Pollen from primary transgenic tobacco plants expressing cystatin gene under the tapetum specific promoter- TA29 restored fertility on cysteine protease induced male sterile tobacco plants developed earlier. This has confirmed the in vivo interaction of cysteine protease and cystatin in the tapetal cells, and the inactivation of cysteine protease and modulation of its negative effects on pollen fertility. Both the cysteine protease and cystatin genes are of plant origin in contrast to the analogous barnase-barstar system that deploys genes of prokaryotic origin. Because of the deployment of genes of plant origin, this system might not face biosafety problems in developing hybrids in food crops., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Abiotic stress results in massive loss of crop productivity throughout the world. Because of our limited knowledge of the plant defense mechanisms, it is very difficult to exploit the plant genetic resources for manipulation of traits that could benefit multiple stress tolerance in plants. To achieve this, we need a deeper understanding of the plant gene regulatory mechanisms involved in stress responses. Understanding the roles of different members of plant gene families involved in different stress responses, would be a step in this direction. Arabidopsis, which served as a model system for the plant research, is also the most suitable system for the functional characterization of plant gene families. Annexin family in Arabidopsis also is one gene family which has not been fully explored. Eight annexin genes have been reported in the genome of Arabidopsis thaliana. Expression studies of different Arabidopsis annexins revealed their differential regulation under various abiotic stress conditions. AnnAt8 (At5g12380), a member of this family has been shown to exhibit ~433 and ~175 fold increase in transcript levels under NaCl and dehydration stress respectively. To characterize Annexin8 (AnnAt8) further, we have generated transgenic Arabidopsis and tobacco plants constitutively expressing AnnAt8, which were evaluated under different abiotic stress conditions. AnnAt8 overexpressing transgenic plants exhibited higher seed germination rates, better plant growth, and higher chlorophyll retention when compared to wild type plants under abiotic stress treatments. Under stress conditions transgenic plants showed comparatively higher levels of proline and lower levels of malondialdehyde compared to the wild-type plants. Real-Time PCR analyses revealed that the expression of several stress-regulated genes was altered in AnnAt8 over-expressing transgenic tobacco plants, and the enhanced tolerance exhibited by the transgenic plants can be correlated with altered expressions of these stress-regulated genes. Our findings suggest a role for AnnAt8 in enhancing abiotic stress tolerance at different stages of plant growth and development.

Late embryogenesis abundant (LEA) proteins are a group of hydrophilic proteins, which accumulate in plants under varied stress conditions like drought, salinity, extreme temperatures and oxidative stress suggesting their role in the protection of plants against these stresses. A transcript derived fragment (TDF) corresponding to LEA gene, which got differentially expressed in wild peanut, Arachis diogoi against the late leaf spot pathogen, Phaeoisariopsis personata was used in this study. We have cloned its full length cDNA by RACE-PCR, which was designated as AdLEA. AdLEA belongs to the atypical Group 5C of LEA protein family as confirmed by sequence analysis. Group 5C LEA protein subfamily contains Pfam LEA_2 domain and is highly hydrophobic. In native conditions, expression of AdLEA was upregulated considerably upon hormonal and abiotic stress treatments emphasizing its role in abiotic stress tolerance. Subcellular localization studies showed that AdLEA protein is distributed in both nucleus and cytosol. Ectopic expression of AdLEA in tobacco resulted in enhanced tolerance of plants to dehydration, salinity and oxidative stress with the transgenic plants showing higher chlorophyll content and reduced lipid peroxidation as compared to wild type plants. Overexpressed AdLEA tobacco plants maintained better photosynthetic efficiency under drought conditions as demonstrated by chlorophyll fluorescence measurements. These plants showed enhanced transcript accumulation of some stress-responsive genes. Our study also elucidates that ROS levels were significantly reduced in leaves and stomatal guard cells of transgenic plants upon stress treatments. These results suggest that AdLEA confers multiple stress tolerance to plants, which make it a potential gene for genetic modification in plants.

A novel zinc-binding alcohol dehydrogenase 2 (AdZADH2) was significantly upregulated in a wild peanut, Arachis diogoi treated with conidia of late leaf spot (LLS) pathogen, Phaeoisariopsis personata. This upregulation was not observed in a comparative analysis of cultivated peanut, which is highly susceptible to LLS. This zinc-binding alcohol dehydrogenase possessed a Rossmann fold containing NADB domain in addition to the MDR domain present in all previously characterized plant ADH genes/proteins. Transient over-expression of AdZADH2 under an estradiol inducible promoter (XVE) resulted in hypersensitive response (HR)-like cell death in tobacco leaf. However, the same level of cell death was not observed when the domains were transiently expressed individually. Cell death observed in tobacco was associated with overexpression of cell death related proteins, antioxidative enzymes such as SOD, CAT and APX and pathogenesis-related (PR) proteins. In A. diogoi, AdZADH2 expression was significantly upregulated in response to the plant signaling hormones salicylic acid, methyl jasmonate, and sodium nitroprusside.

This study aimed to validate the physiological importance of Arabidopsis thaliana alternative oxidase 1a (AtAOX1a) in alleviating oxidative stress using Saccharomyces cerevisiae as a model organism. The AOX1a transformant (pYES2AtAOX1a) showed cyanide resistant and salicylhydroxamic acid (SHAM)-sensitive respiration, indicating functional expression of AtAOX1a in S. cerevisiae. After exposure to oxidative stress, pYES2AtAOX1a showed better survival and a decrease in reactive oxygen species (ROS) when compared to S. cerevisiae with empty vector (pYES2). Furthermore, pYES2AtAOX1a sustained growth by regulating GPX2 and/or TSA2, and cellular NAD (+)/NADH ratio. Thus, the expression of AtAOX1a in S. cerevisiae enhances its respiratory tolerance which, in turn, maintains cellular redox homeostasis and protects from oxidative damage.

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.

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.