Parinita Agarwal

NAC proteins are a large family of plant-specific transcription factors which regulate both ABA-dependent and -independent gene expression. These transcription factors participate in biotic and abiotic stress-response through intricate regulation at transcriptional, post-transcriptional and post-translational levels. In the present study, AlNAC4 transcription factor was isolated from a salt excreting halophyte Aeluropus lagopoides. The AlNAC4 has an open reading frame of 936 bp, encoding a protein of 312 amino acid, with an estimated molecular mass of 34.9 kDa. The AlNAC4 showed close homology to monocot NACs in the phylogenetic tree. In silico analysis revealed that AlNAC4 possess the characteristic A-E subdomains within the NAC domain. The AlNAC4 showed sixteen post-translational phosphorylation sites. The AlNAC4 transcript was significantly upregulated with dehydration and H₂O₂ treatments, showing its role in osmotic and oxidative stress, respectively. The recombinant protein showed binding to mono as well as tandem repeats of NAC recognition sequence (NACRS) of the erd1 promoter. This is the first report mentioning that overexpression of AlNAC4 improved oxidative stress tolerance in tobacco transgenics. The transgenics maintained ROS homeostasis during H₂O₂ treatment. The transgenics showed regulation of stress-responsive genes including CAT, SOD, LEA5, PLC3, ERD10B, THT1 and transcription factors like AP2, ZFP during oxidative stress. Key Message: The AlNAC4 transcription factor from recretohalophyte Aeluropus showed regulation with abiotic stresses and binding to NACRS elements of erd1 promoter. The AlNAC4 tobacco transgenics showed improved growth with oxidative stress.

Plants, being sessile, have developed intricate signalling network to specifically respond to the diverse environmental stress. The crop performance and yield is limited due to exposure of multiple stresses simultaneously. The coordinated crosstalk between different signalling components, including phytohormones and transcription factor(s) play a crucial role towards increased stress resistance in crops. Transcription factors play an important role in regulating the downstream processes during stress. The plant-specific WRKY TFs form one of the largest TF family and are involved in diverse plant processes, involving growth, development and stress signalling through auto and cross regulation with different genes and TFs. The salicylic acid -inducible JcWRKY TF confers salinity tolerance in transgenic tobacco, as was evident by increased chlorophyll content and seed germination potential. The transgenic plants showed increased soluble sugar, membrane stability, reduced electrolyte leakage and generation of reactive oxygen species (H2O2 and O2•−) as compared to the wild type (WT). Furthermore, the low SA treatment along with salinity improved the tolerance potential of the transgenics by maintaining ROS homeostasis and high K+/Na+ ratio. The transcript expression of SA biosynthetic gene ICS1 and antioxidative enzymes (CAT and SOD) showed upregulation during stress. Thus, the present study reflects that JcWRKY might be working in co-ordination with SA and salt stress signalling to orchestrate the different biochemical and molecular pathways to manoeuvre stress tolerance of the transgenic plants.

Plants in ecosystems are simultaneously exposed to abiotic and biotic stresses, which restrict plant growth and development. The complex responses to these stresses are largely regulated by plant hormones, which in turn, orchestrate the different biochemical and molecular pathways to manoeuvre stress tolerance. The PR-10 protein family is reported to be involved in defence regulation, stress response and plant growth and development. The JcPR-10a overexpression resulted in increased number of shoot buds in tobacco (Nicotiana tabacum), which could be due to high cytokinin to auxin ratio in the transgenics. The docking analysis shows the binding of three BAP molecules at the active sites of JcPR-10a protein. JcPR-10a transgenics showed enhanced salt tolerance, as was evident by increased germination rate, shoot and root length, relative water content, proline, soluble sugar and amino acid content under salinity. Interestingly, the transgenics also showed enhanced endogenous cytokinin level as compared to WT, which, further increased with salinity. Exposure of gradual salinity resulted in increased stomatal conductance, water use efficiency, photosynthesis rate and reduced transpiration rate. Furthermore, the transgenics also showed enhanced resistance against Macrophomina fungus. Thus, JcPR-10a might be working in co-ordination with cytokinin signalling in mitigating the stress induced damage by regulating different stress signalling pathways, leading to enhanced stress tolerance.

The endomembrane system plays an important role during cellular adaptation of the plants with the extracellular environment. The small GTP-binding protein Rab7 located at the vacuolar membrane regulates the vesicle fusion with the vacuole and thereby helps in recycling of the molecules. This is the first report on isolation and characterization of AlRab7 gene from the halophyte plant, Aeluropus that extrudes NaCl through salt glands and grows luxuriantly throughout the year at the Gujarat coast, India. The AlRab7 encodes a protein with 206 amino acids, and a highly conserved effector-binding domain and four nucleotide-binding domains. The in silico analysis predicts the presence of the prenylation site for Rab geranylgeranyltransferase 2 and the Rab escort protein site. The C-terminal two cysteine residues in –XCC sequence are present for membrane attachment. Transcript expression of the AlRab7 gene was differentially regulated by different environmental stimuli such as dehydration, salinity, and hormone abscisic acid (ABA). The recombinant Escherichia coli cells showed improved growth in Luria Bertani medium supplemented with NaCl, KCl, mannitol, ABA, and indole-3-acetic acid. A novel Rab7 interacting partner AlRabring7 was identified by yeast two-hybrid screening.

The WRKY family of transcription factors (TFs) play an intricate role in regulating the stress signaling pathways by autoregulation or may be by cross regulation through interaction with other proteins. Although WRKY TFs are considered to be plant specific, however, their presence has been reported from unicellular algae, slime mould, and gymnosperms. We have isolated the JcWRKY cDNA from an important biofuel crop Jatropha curcas growing in the wastelands of India. The JcWRKY gene has an ORF of 693 bp and encodes a 230 amino acids protein with estimated molecular mass of 25.25 kDa. JcWRKY shows close homology to FaWRKY1 and St-WRKY1. The JcWRKY contains seven potential phosphorylation sites, which might be involved in regulating its function. The transcript analysis revealed that the JcWRKY transcript gets upregulated in response to salinity, dehydration, salicylic acid (SA), methyl jasmonate (MeJa), and collar rot fungus Macrophomina. However, maximum expression is observed under SA, highlighting its role in enhancing systemic acquired resistance for disease tolerance. The JcWRKY recombinant protein showed binding to W-box of pathogenesis related-1 (PR-1) and iso1 (encoding isoamylase1) promoters. Overexpression of JcWRKY in Escherichia coli enhanced the growth of cells in NaCl, KCl, mannitol, sorbitol, SA, and MeJa treatments, indicating that it protects and promotes growth under ionic, osmotic, and chemical stresses. The enhancement in growth can be due to the regulation of stress responsive genes. Therefore, it can be used as an important gene for enhancing abiotic and biotic resistance in plants and to facilitate faster growth of E. coli cells under stress conditions for efficient expression.

Pathogenesis related-10 proteins are small proteins with cytosolic localization, conserved three dimensional structures and single intron at 185 bp position. These proteins have a broad spectrum of roles significantly in biotic and abiotic stresses. The RNase activity, ligand binding activity, posttranslational modification (phosphorylation) and phytohormone signaling provide some information into the mechanism of the regulation of PR-10 proteins, however the presence of isoforms makes it difficult to decipher its exact mode of function. The involvement of phosphorylation/dephosphorylation events in its activation is interesting and provides unique and unbiased insights into the complexity of its regulation. Studies on upstream region of different PR-10 genes indicate the presence of cis-acting elements for WRKY, RAVI, bZ1P, ERF, SEBF and Pti4 transcription factors indicating their role in regulating PR-10 promoter. In this review, we discuss in detail the structure and mechanism of regulation of PR-10 proteins.

The pathogenesis-related proteins have a broad spectrum of roles, ranging from seed germination, development to resistance. The PR-10 is a multigene family differing from other PR proteins in being intracellular, small and acidic with similar 3D structures. We have isolated JcPR-10a cDNA with an ORF of 483 bp from J. curcas, an important biofuel crop grown in the wastelands of India. JcPR-10a gets clustered with dicots in phylogenetic tree. The genomic organisation analysis of JcPR-10a revealed the presence of an intron at conserved 185 bp position. Transcript expression of JcPR-10a was upregulated in response to different stimuli such as NaCl, salicylic acid, methyl jasmonate and M. phaseolina. In response to SA and Macrophomina the transcript was found increased at 48 h, however, in case of NaCl and MeJa a strong induction was observed at 12 h which decreased at 48 h. We first time report the transcript up regulation of PR-10 gene by Macrophomina, a pathogen causing collar rot in Jatropha. The recombinant E. coli cells showed better growth in LB medium supplemented with NaCl, whereas growth of recombinant cells was inhibited in LB medium supplemented with KCl, mannitol, sorbitol, methyl jasmonate and salicylic acid. The JcPR-10a protein was overexpressed in E. coli cells, and was purified to homogeneity, the purified protein exhibited RNase and DNase activity. Furthermore, the protein also showed antifungal activity against Macrophomina, indicating that JcPR-10a can serve as an important candidate to engineer stress tolerance in Jatropha as well as other plants susceptible to collar rot by Macrophomina.