Comparative phylogenomic and structural analysis of canonical secretory PLA2 and novel PLA2-like family in plants.

Plant secretory phospholipase A2 (sPLA2) is a family of lipolytic enzymes involved in the sn-2 hydrolysis of phospholipid carboxyester bonds, characterized by the presence of a conserved PA2c domain. PLA2 produces free fatty acids and lysophospholipids, which regulate several physiological functions, including lipid metabolism, plant growth and development, signal transduction, and response to various environmental stresses. In the present work, we have performed a comparative analysis of PA2c domain-containing genes across plants, focusing on gene distribution, phylogenetic analysis, tissue-specific expression, and homology modeling. Our data revealed the widespread occurrence of multiple sPLA2 in most land plants and documented single sPLA2 in multiple algal groups, indicating an ancestral origin of sPLA2. We described a novel PA2c-containing gene family present in all plant lineages and lacking secretory peptide, which we termed PLA2-like. Phylogenetic analysis revealed two independent clades in canonical sPLA2 genes referred to as α and ß clades, whereas PLA2-like genes clustered independently as a third clade. Further, we have explored clade-specific gene expressions showing that while all three clades were expressed in vegetative and reproductive tissues, only sPLA2-ß and PLA2-like members were expressed in the pollen and pollen tube. To get insight into the conservation of the gene regulatory network of sPLA2 and PLA2-like genes, we have analyzed the occurrence of various cis-acting promoter elements across the plant kingdom. The comparative 3D structure analysis revealed conserved and unique features within the PA2c domain for the three clades. Overall, this study will help to understand the evolutionary significance of the PA2c family and lay the foundation for future sPLA2 and PLA2-like characterization in plants.

With no lysine kinases: the key regulatory networks and phytohormone cross talk in plant growth, development and stress response.

With No Lysine kinases (WNKs) are a distinct family of Serine/Threonine protein kinase with unique arrangement of catalytic residues in kinase domain. In WNK, an essential catalytic lysine requisite for attaching ATP and phosphorylation reaction is located in subdomain I, instead of subdomain II, which is essentially a typical feature of other Ser/Thr kinases. WNKs are identified in diverse organisms including multicellular and unicellular organisms. Mammalian WNKs are well characterized at structural and functional level, while plant WNKs are not explored much except few recent studies. Plant WNKs role in various physiological processes viz. ion maintenance, osmotic stress, pH homeostasis, circadian rhythms, regulation of flowering time, proliferation and organ development, and abiotic stresses are known, but the mechanisms involved are unclear. Plant WNKs are known to be involved in enhanced drought and salt stress response via ABA-signaling pathway, but the complete signaling cascade is yet to be elucidated. The current review will discuss the interplay between WNKs and growth regulators and their cross talks in plant growth and development. We have also highlighted the link between the stress phytohormones and WNK members in regulating abiotic stress responses in plants. The present review will provide an overall known mechanism on the involvement of WNKs in plant growth and development and abiotic stress response and highlight its role/applications in the development of stress-tolerant plants.

Molecular insights into the role of plant transporters in salt stress response.

Salt stress disturbs the cellular osmotic and ionic balance, which then creates a negative impact on plant growth and development. The Na+ and Cl- ions can enter into plant cells through various membrane transporters, including specific and non-specific Na+ , K+ , and Ca2+ transporters. Therefore, it is important to understand Na+ and K+ transport mechanisms in plants along with the isolation of genes, their characterization, the structural features, and their post-translation regulation under salt stress. This review summarizes the molecular insights of plant ion transporters, including non-selective cation transporters, cyclic nucleotide-gated cation transporters, glutamate-like receptors, membrane intrinsic proteins, cation proton antiporters, and sodium proton antiporter families. Further, we discussed the K+ transporter families such as high-affinity K+ transporters, HAK/KUP/KT transporters, shaker type K+ transporters, and K+ efflux antiporters. Besides the ion transport process, we have shed light on available literature on epigenetic regulation of transport processes under salt stress. Recent advancements of salt stress sensing mechanisms and various salt sensors within signaling transduction pathways are discussed. Further, we have compiled salt-stress signaling pathways, and their crosstalk with phytohormones.

Overexpression of rice OsWNK9 promotes arsenite tolerance in transgenic Arabidopsis plants.

Protein kinases are involved in the transfer of phosphate group to serine, threonine, and tyrosine residues of a target protein. With No Lysine (WNK) kinase is a member of the serine/threonine protein kinase family, which has conserved catalytic lysine (K) residue in subdomain I instead of being in subdomain II.The WNKs family members in plants are stress inducible and have been validated for their role in abiotic stress tolerance. In the present study, we have characterized Arabidopsis overexpressed lines of OsWNK9 regulated by the constitutive promoter under arsenite stress. Moreover, we have performed In silico expression analysis of OsWNK9 under nutrient deficiency and heavy metal stress. Three independent transgenic Arabidopsis (OsWNK9-OX T11, T12,andT13) lines showed tolerance to arsenite stress compared to wild-type (WT) plants. Under arsenite stress, transgenic lines T11, T12 and T13 showed 56.46, 57.8 and 51.66 % increased biomass respectively, as compared to WT plants. All three ArabidopsisOsWNK9-OX lines exhibited higher proline content, increased antioxidant enzyme activities and lower hydrogen peroxide levels under arsenite stress. Besides, the total antioxidant capacity in terms of DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging percentage was increased by 8-15 % in three independent OsWNK9-OX lines compared with those of WT plants. Protein-protein interaction analysis of OsWNK9 predicted interaction partners with protein kinase and oxidative stress-responsive protein. Co-expression analysis of OsWNK9 in phosphate deficiency and arsenate stress condition predicted various proteins including membrane transporter and transcription factors. Taken together, our results, for the first time, provide evidence that OsWNK9 could positively mediate arsenite stress tolerance in plants.

Selection of reference genes for quantitative real-time PCR analysis in halophytic plant Rhizophora apiculata.

Rhizophora apiculata is a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in R. apiculata physiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), ß-tubulin (ß-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively ranked EF1α followed by ACT as the most stable candidate reference genes in R. apiculata physiological tissues. Moreover, ß-TUB and 18S were ranked as moderately stable candidate reference genes, while GAPDH and rbcL were least stable reference genes. Under salt stress, EF1α was comprehensively recommended top-ranked candidate reference gene followed by ACT and 18S. In order to validate the identified most stable candidate reference genes, EF1α, ACT, 18S and UBQ were used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level of NHX varied according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR in R. apiculata physiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes in R. apiculata.

Expression of OsWNK9 in Arabidopsis conferred tolerance to salt and drought stress.

With No Lysine (WNK) kinase belongs to ser/thr protein kinase group in which conserved catalytic lysine (K) residue of subdomain II is shifted to subdomain I. In this study, we cloned full-length coding region of WNK9 from Oryza sativa (OsWNK9) and performed in silico studies to confirm the presence of all kinase signature regulatory elements. The transcript analysis revealed that OsWNK9 was strongly down regulated under salinity, drought and ABA stress in shoots. Constitutive expression of OsWNK9 in Arabidopsis thaliana imparted increased tolerance to salt, drought, and ABA stress. Transgenic lines showed healthy phenotypes such as green leaves, achieved higher fresh weight and longer roots under salt, drought and ABA stress as compared to wild-type (WT). Transgenic plants showed better seed germination, higher chlorophyll retention and less water loss under salt and drought stress compared to WT. Promoter/gene expression studies revealed that OsWNK9 were expressed throughout plant tissues with higher expression in roots. Subcellular localization studies of OsWNK9 showed their presence in the nucleus. The transcript analysis of abiotic stress marker genes and ABA dependent genes showed they were highly expressed in transgenic lines compared to WT in response to salt and drought stress. The endogenous ABA level under salt and drought stress in transgenic lines was higher than WT. The results indicated that OsWNK9 may regulate salt and drought response in ABA dependent manner.

Evaluation of multilocus marker efficacy for delineating mangrove species of West Coast India.

The plant DNA barcoding is a complex and requires more than one marker(s) as compared to animal barcoding. Mangroves are diverse estuarine ecosystem prevalent in the tropical and subtropical zone, but anthropogenic activity turned them into the vulnerable ecosystem. There is a need to build a molecular reference library of mangrove plant species based on molecular barcode marker along with morphological characteristics. In this study, we tested the core plant barcode (rbcL and matK) and four promising complementary barcodes (ITS2, psbK-psbI, rpoC1 and atpF-atpH) in 14 mangroves species belonging to 5 families from West Coast India. Data analysis was performed based on barcode gap analysis, intra- and inter-specific genetic distance, Automated Barcode Gap Discovery (ABGD), TaxonDNA (BM, BCM), Poisson Tree Processes (PTP) and General Mixed Yule-coalescent (GMYC). matK+ITS2 marker based on GMYC method resolved 57.14% of mangroves species and TaxonDNA, ABGD, and PTP discriminated 42.85% of mangrove species. With a single locus analysis, ITS2 exhibited the higher discriminatory power (87.82%) and combinations of matK + ITS2 provided the highest discrimination success (89.74%) rate except for Avicennia genus. Further, we explored 3 additional markers (psbK-psbI, rpoC1, and atpF-atpH) for Avicennia genera (A. alba, A. officinalis and A. marina) and atpF-atpH locus was able to discriminate three species of Avicennia genera. Our analysis underscored the efficacy of matK + ITS2 markers along with atpF-atpH as the best combination for mangrove identification in West Coast India regions.

Assessment of mangroves from Goa, west coast India using DNA barcode.

Mangroves are salt-tolerant forest ecosystems of tropical and subtropical intertidal regions. They are among most productive, diverse, biologically important ecosystem and inclined toward threatened system. Identification of mangrove species is of critical importance in conserving and utilizing biodiversity, which apparently hindered by a lack of taxonomic expertise. In recent years, DNA barcoding using plastid markers rbcL and matK has been suggested as an effective method to enrich traditional taxonomic expertise for rapid species identification and biodiversity inventories. In the present study, we performed assessment of available 14 mangrove species of Goa, west coast India based on core DNA barcode markers, rbcL and matK. PCR amplification success rate, intra- and inter-specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in rbcL (97.7 %) and matK (95.5 %) region. The two candidate chloroplast barcoding regions (rbcL, matK) yielded barcode gaps. Our results clearly demonstrated that matK locus assigned highest correct identification rates (72.09 %) based on TaxonDNA Best Match criteria. The concatenated rbcL + matK loci were able to adequately discriminate all mangrove genera and species to some extent except those in Rhizophora, Sonneratia and Avicennia. Our study provides the first endorsement of the species resolution among mangroves using plastid genes with few exceptions. Our future work will be focused on evaluation of other barcode markers to delineate complete resolution of mangrove species and identification of putative hybrids.

Genome-wide identification and expression analysis of WNK kinase gene family in rice.

Eukaryotic protein kinases represent one of the largest gene families involved in diverse regulatory functions. WNK (With No Lysine) kinases are members of ser/thr protein kinase family, which lack conserved catalytic lysine (K) residue at protein kinase subdomain II and is replaced by either asparagine, serine or glycine residues. They are involved in regulation of flowering time, circadian rhythms and abiotic stresses in Arabidopsis thaliana. In the present study, we have identified 9 members of WNK in rice, showed resemblance to Arabidopsis and human WNK and clustered into five main clades phylogenetically. The predicted genes structure, bonafide conserved signature motif and domains strongly support their identity, as members of WNK kinase family. We have analyzed their chromosomal distribution, physio-chemical properties, subcellular localizations and cis-elements in the promoter regions in silico. Further, transcript analysis of OsWNK by qRT-PCR revealed their differential regulation in tissue specific and abiotic stresses libraries. In conclusion, the identification of nine OsWNK and transcript level expression pattern under abiotic stress using qRT-PCR in rice will significantly contribute towards the understanding of WNK genes in monocots and thus provide a set up for functional genomics studies of WNK protein kinases.