Ajay Kumar Parida (1963-2022), an eminent plant biotechnologist with a passion for mangrove biology.

We remember Dr Ajay Parida, a leading plant biotechnologist, whose premature passing has deprived the Indian plant science community of a committed scientist and an able administrator. Born on 12 December 1963 in Bhagabanpur, Cuttack District (now Jajpur district), Odisha, he passed away in Guwahati on 19 July 2022. A collegial scientist, his down-to-earth and approachable nature, as well as his resourcefulness were instrumental in advancing the cause of Indian science and harnessing frontier biotechnological tools as vehicles of social consciousness. His expertise in quantitative DNA variation and molecular marker analysis, paved the way for subsequent research on mangrove molecular diversity at the M. S. Swaminathan Research Foundation (MSSRF), Chennai. His contributions to mangrove biology, genetics and genomics as well as extremophile plant species in the Indian context over two decades are a benchmark in his field. He also provided commendable leadership in his capacity as Director, Institute of Life Sciences (ILS), Bhubaneshwar during the COVID-19 pandemic.

A conserved SNP variation in the pre-miR396c flanking region in Oryza sativa indica landraces correlates with mature miRNA abundance.

Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group. The miRNA-miRNA* duplex region is completely conserved across eleven Oryza species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the 'A10234287911' allele may promote better processing vis-à-vis the structure conferred by 'G10234287911' allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

Comparing Essentiality of SOS1-Mediated Na+ Exclusion in Salinity Tolerance between Cultivated and Wild Rice Species.

Soil salinity is a major constraint that affects plant growth and development. Rice is a staple food for more than half of the human population but is extremely sensitive to salinity. Among the several known mechanisms, the ability of the plant to exclude cytosolic Na+ is strongly correlated with salinity stress tolerance in different plant species. This exclusion is mediated by the plasma membrane (PM) Na+/H+ antiporter encoded by Salt Overly Sensitive (SOS1) gene and driven by a PM H+-ATPase generated proton gradient. However, it is not clear to what extent this mechanism is operational in wild and cultivated rice species, given the unique rice root anatomy and the existence of the bypass flow for Na+. As wild rice species provide a rich source of genetic diversity for possible introgression of abiotic stress tolerance, we investigated physiological and molecular basis of salinity stress tolerance in Oryza species by using two contrasting pairs of cultivated (Oryza sativa) and wild rice species (Oryza alta and Oryza punctata). Accordingly, dose- and age-dependent Na+ and H+ fluxes were measured using a non-invasive ion selective vibrating microelectrode (the MIFE technique) to measure potential activity of SOS1-encoded Na+/H+ antiporter genes. Consistent with GUS staining data reported in the literature, rice accessions had (~4-6-fold) greater net Na+ efflux in the root elongation zone (EZ) compared to the mature root zone (MZ). Pharmacological experiments showed that Na+ efflux in root EZ is suppressed by more than 90% by amiloride, indicating the possible involvement of Na+/H+ exchanger activity in root EZ. Within each group (cultivated vs. wild) the magnitude of amiloride-sensitive Na+ efflux was higher in tolerant genotypes; however, the activity of Na+/H+ exchanger was 2-3-fold higher in the cultivated rice compared with their wild counterparts. Gene expression levels of SOS1, SOS2 and SOS3 were upregulated under 24 h salinity treatment in all the tested genotypes, with the highest level of SOS1 transcript detected in salt-tolerant wild rice genotype O. alta (~5-6-fold increased transcript level) followed by another wild rice, O. punctata. There was no significant difference in SOS1 expression observed for cultivated rice (IR1-tolerant and IR29-sensitive) under both 0 and 24 h salinity exposure. Our findings suggest that salt-tolerant cultivated rice relies on the cytosolic Na+ exclusion mechanism to deal with salt stress to a greater extent than wild rice, but its operation seems to be regulated at a post-translational rather than transcriptional level.

Morpho-physiological, biochemical and molecular characterization of coastal rice landraces to identify novel genetic sources of salinity tolerance.

Soil salinity is a leading cause for yield losses in rice, affecting nearly 6% of global rice cultivable area. India is host to a rich diversity of coastal rice landraces that are naturally tolerant to salinity and an untapped source to identify novel determinants of salinity tolerance. In the present study, we have assessed the relative salinity tolerance of 43 previously genotyped rice landraces at seedling stage, using thirteen morpho-physiological and biochemical parameters using a hydroponics system. Among 43 rice varieties, 25 were tolerant, 15 were moderately tolerant, 1 was moderately susceptible and 2 sensitive checks were found to be highly susceptible based on standard salinity scoring methods. In addition to previously known saline tolerant genotypes (Pokkali, FL478 and Nona Bokra), the present study has novel genotypes such as Katrangi, Orkyma, Aduisen 1, Orumundakan 1, Hoogla, and Talmugur 2 as potential sources of salinity tolerance through measurement of morpho-physiological and biochemical parameters including Na+, K+ estimations and Na+/K+ ratios. Further, Pallipuram Pokkali may be an important source of the tissue tolerance trait under salinity. Four marker trait associations (RM455-root Na+; RM161-shoot and root Na+/K+ ratios; RM237-salinity tolerance index) accounted for phenotypic variations in the range of 20.97-39.82%. A significant increase in root endodermal and exodermal suberization was observed in selected rice landraces under salinity. For the first time, variation in the number of suberized sclerenchymatous layers as well as passage cells is reported, in addition to expression level changes in suberin biosynthetic genes (CYP86A2, CYP81B1, CYP86A8 and PERL).

Comparative Analysis of Root Na+ Relation under Salinity between Oryza sativa and Oryza coarctata

Na+ toxicity is one of the major physiological constraints imposed by salinity on plant performance. At the same time, Na+ uptake may be beneficial under some circumstances as an easily accessible inorganic ion that can be used for increasing solute concentrations and maintaining cell turgor. Two rice species, Oryza sativa (cultivated rice, salt-sensitive) and Oryza coarctata (wild rice, salt-tolerant), demonstrated different strategies in controlling Na+ uptake. Glasshouse experiments and gene expression analysis suggested that salt-treated wild rice quickly increased xylem Na+ loading for osmotic adjustment but maintained a non-toxic level of stable shoot Na+ concentration by increased activity of a high affinity K+ transporter HKT1;5 (essential for xylem Na+ unloading) and a Na+/H+ exchanger NHX (for sequestering Na+ and K+ into root vacuoles). Cultivated rice prevented Na+ uptake and transport to the shoot at the beginning of salt treatment but failed to maintain it in the long term. While electrophysiological assays revealed greater net Na+ uptake upon salt application in cultivated rice, O. sativa plants showed much stronger activation of the root plasma membrane Na+/H+ Salt Overly Sensitive 1 (SOS1) exchanger. Thus, it appears that wild rice limits passive Na+ entry into root cells while cultivated rice relies heavily on SOS1-mediating Na+ exclusion, with major penalties imposed by the existence of the "futile cycle" at the plasma membrane.

Evolutionary Significance of NHX Family and NHX1 in Salinity Stress Adaptation in the Genus Oryza.

Rice (Oryza sativa), a staple crop for a substantial part of the world's population, is highly sensitive to soil salinity; however, some wild Oryza relatives can survive in highly saline environments. Sodium/hydrogen antiporter (NHX) family members contribute to Na+ homeostasis in plants and play a major role in conferring salinity tolerance. In this study, we analyzed the evolution of NHX family members using phylogeny, conserved domains, tertiary structures, expression patterns, and physiology of cultivated and wild Oryza species to decipher the role of NHXs in salt tolerance in Oryza. Phylogenetic analysis showed that the NHX family can be classified into three subfamilies directly related to their subcellular localization: endomembrane, plasma membrane, and tonoplast (vacuolar subfamily, vNHX1). Phylogenetic and structural analysis showed that vNHX1s have evolved from streptophyte algae (e.g., Klebsormidium nitens) and are abundant and highly conserved in all major land plant lineages, including Oryza. Moreover, we showed that tissue tolerance is a crucial trait conferring tolerance to salinity in wild rice species. Higher Na+ accumulation and reduced Na+ effluxes in leaf mesophyll were observed in the salt-tolerant wild rice species O. alta, O. latifolia, and O. coarctata. Among the key genes affecting tissue tolerance, expression of NHX1 and SOS1/NHX7 exhibited significant correlation with salt tolerance among the rice species and cultivars. This study provides insights into the evolutionary origin of plant NHXs and their role in tissue tolerance of Oryza species and facilitates the inclusion of this trait during the development of salinity-tolerant rice cultivars.

Unravelling the physiological basis of salinity stress tolerance in cultivated and wild rice species.

Wild rice species provide a rich source of genetic diversity for possible introgression of salinity stress tolerance in cultivated rice. We investigated the physiological basis of salinity stress tolerance in Oryza species by using six rice genotypes (Oryza sativa L.) and four wild rice species. Three weeks of salinity treatment significantly (P <0.05) reduced physiological and growth indices of all cultivated and wild rice lines. However, the impact of salinity-induced growth reduction differed substantially among accessions. Salt tolerant accessions showed better control over gas exchange properties, exhibited higher tissue tolerance, and retained higher potassium ion content despite higher sodium ion accumulation in leaves. Wild rice species showed relatively lower and steadier xylem sap sodium ion content over the period of 3weeks analysed, suggesting better control over ionic sodium xylem loading and its delivery to shoots with efficient vacuolar sodium ion sequestration. Contrary to this, saline sensitive genotypes managed to avoid initial Na+ loading but failed to accomplish this in the long term and showed higher sap sodium ion content. Conclusively, our results suggest that wild rice genotypes have more efficient control over xylem sodium ion loading, rely on tissue tolerance mechanisms and allow for a rapid osmotic adjustment by using sodium ions as cheap osmoticum for osmoregulation.

Proto Kranz-like leaf traits and cellular ionic regulation are associated with salinity tolerance in a halophytic wild rice.

Species of wild rice (Oryza spp.) possess a wide range of stress tolerance traits that can be potentially utilized in breeding climate-resilient cultivated rice cultivars (Oryza sativa) thereby aiding global food security. In this study, we conducted a greenhouse trial to evaluate the salinity tolerance of six wild rice species, one cultivated rice cultivar (IR64) and one landrace (Pokkali) using a range of electrophysiological, imaging, and whole-plant physiological techniques. Three wild species (O. latifolia, O. officinalis and O. coarctata) were found to possess superior salinity stress tolerance. The underlying mechanisms, however, were strikingly different. Na+ accumulation in leaves of O. latifolia, O. officinalis and O. coarctata were significantly higher than the tolerant landrace, Pokkali. Na+ accumulation in mesophyll cells was only observed in O. coarctata, suggesting that O. officinalis and O. latifolia avoid Na+ accumulation in mesophyll by allocating Na+ to other parts of the leaf. The finding also suggests that O. coarctata might be able to employ Na+ as osmolyte without affecting its growth. Further study of Na+ allocation in leaves will be helpful to understand the mechanisms of Na+ accumulation in these species. In addition, O. coarctata showed Proto Kranz-like leaf anatomy (enlarged bundle sheath cells and lower numbers of mesophyll cells), and higher expression of C4-related genes (e.g., NADPME, PPDK) and was a clear outlier with respect to salinity tolerance among the studied wild and cultivated Oryza species. The unique phylogenetic relationship of O. coarctata with C4 grasses suggests the potential of this species for breeding rice with high photosynthetic rate under salinity stress in the future.

Reduced apoplastic barriers in tissues of shoot-proximal rhizomes of Oryza coarctata are associated with Na+ sequestration.

Oryza coarctata is the only wild rice species with significant salinity tolerance. The present work examines the role of the substantial rhizomatous tissues of O. coarctata in conferring salinity tolerance. Transition to an erect phenotype (shoot emergence) from prostrate growth of rhizome tissues is characterized by marked lignification and suberization of supporting sclerenchymatous tissue, epidermis, and bundle sheath cells in aerial shoot-proximal nodes and internodes in O. coarctata. With salinity, however, aerial shoot-proximal internodal tissues show reductions in lignification and suberization, most probably related to re-direction of carbon flux towards synthesis of the osmporotectant proline. Concurrent with hypolignification and reduced suberization, the aerial rhizomatous biomass of O. coarctata appears to have evolved mechanisms to store Na+ in these specific tissues under salinity. This was confirmed by histochemical staining, quantitative real-time reverse transcription-PCR expression patterns of genes involved in lignification/suberization, Na+ and K+ contents of internodal tissues, as well as non-invasive microelectrode ion flux measurements of NaCl-induced net Na+, K+, and H+ flux profiles of aerial nodes were determined. In O. coarctata, aerial proximal internodes appear to act as 'traffic controllers', sending required amounts of Na+ and K+ into developing leaves for osmotic adjustment and turgor-driven growth, while more deeply positioned internodes assume a Na+ buffering/storage role.

To exclude or to accumulate? Revealing the role of the sodium HKT1;5 transporter in plant adaptive responses to varying soil salinity.

Arid/semi-arid and coastal agricultural areas of the world are especially vulnerable to climate change-driven soil salinity. Salinity tolerance in plants is a complex trait, with salinity negatively affecting crop yield. Plants adopt a range of mechanisms to combat salinity, with many transporter genes being implicated in Na+-partitioning processes. Within these, the high-affinity K+ (HKT) family of transporters play a critical role in K+ and Na+ homeostasis in plants. Among HKT transporters, Type I transporters are Na+-specific. While Arabidopsis has only one Na + -specific HKT (AtHKT1;1), cereal crops have a multiplicity of Type I and II HKT transporters. AtHKT1; 1 (Arabidopsis thaliana) and HKT1; 5 (cereal crops) 'exclude' Na+ from the xylem into xylem parenchyma in the root, reducing shoot Na+ and hence, confer sodium tolerance. However, more recent data from Arabidopsis and crop species show that AtHKT1;1/HKT1;5 alleles have a strong genetic association with 'shoot sodium accumulation' and concomitant salt tolerance. The review tries to resolve these two seemingly contradictory effects of AtHKT1;1/HKT1;5 operation (shoot exclusion vs shoot accumulation), both conferring salinity tolerance and suggests that contrasting phenotypes are attributable to either hyper-functional or weak AtHKT1;1/HKT1;5 alleles/haplotypes and are under strong selection by soil salinity levels. It also suggests that opposite balancing mechanisms involving xylem ion loading in these contrasting phenotypes exist that require transporters such as SOS1 and CCC. While HKT1; 5 is a crucial but not sole determinant of salinity tolerance, investigation of the adaptive benefit(s) conferred by naturally occurring intermediate HKT1;5 alleles will be important under a climate change scenario.

Revealing the Role of the Calcineurin B-Like Protein-Interacting Protein Kinase 9 (CIPK9) in Rice Adaptive Responses to Salinity, Osmotic Stress, and K+ Deficiency.

In plants, calcineurin B-like (CBL) proteins and their interacting protein kinases (CIPK) form functional complexes that transduce downstream signals to membrane effectors assisting in their adaptation to adverse environmental conditions. This study addresses the issue of the physiological role of CIPK9 in adaptive responses to salinity, osmotic stress, and K+ deficiency in rice plants. Whole-plant physiological studies revealed that Oscipk9 rice mutant lacks a functional CIPK9 gene and displayed a mildly stronger phenotype, both under saline and osmotic stress conditions. The reported difference was attributed to the ability of Oscipk9 to maintain significantly higher stomatal conductance (thus, a greater carbon gain). Oscipk9 plants contained much less K+ in their tissues, implying the role of CIPK9 in K+ acquisition and homeostasis in rice. Oscipk9 roots also showed hypersensitivity to ROS under conditions of low K+ availability suggesting an important role of H2O2 signalling as a component of plant adaptive responses to a low-K environment. The likely mechanistic basis of above physiological responses is discussed.

Clinical effects and safety of ferric carboxymaltose in pregnancy: An Indian real-life experience.

BACKGROUND: There is limited clinical evidence of ferric carboxymaltose injection (FCM) usage in Indian pregnant women. We assessed the efficacy and safety of FCM in Indian pregnant women with moderate-to-severe anemia. METHODS: Single-center, retrospective, observational data collection was conducted at a tertiary care research institute. Data of pregnant women with anemia who received FCM in their second and third trimester was retrieved and analyzed for hematological parameters at baseline and at 4 ± 2 weeks. Neonatal outcomes were also assessed. Adverse events and other safety parameters were noted. RESULTS: Data of 271 patients was retrieved and analyzed for safety and data for 168 patients analyzed for efficacy. A significant increase in hemoglobin was noted with FCM in 4 weeks (1.25 g/dL; p < 0.001). Patients with severe anemia reported an increase in hemoglobin of 4.23 g/dL (p = 0.01). Patients receiving FCM in the second trimester noted a significant increase in hemoglobin of 1.74 g/dL (p < 0.001). A significant increase in hemoglobin was noted as early as 20 days (p < 0.001) and also in patients receiving FCM after 34 weeks (p = 0.002). No adverse fetal or neonatal outcomes were observed. Adverse events noted in 4% of patients with itching and rash being most common. Continuous monitoring of blood pressure, heart rate, and oxygen saturation for 40 min during and after FCM administration reported no deterioration or negative safety signal. CONCLUSION: FCM corrects anemia in all subsets of Indian pregnant women and supports evidence of efficacy and safety. Continuous monitoring of vital parameters during FCM infusions supports its excellent safety.

Sodium sequestration confers salinity tolerance in an ancestral wild rice.

Wild rice Oryza rufipogon, a progenitor of cultivated rice Oryza sativa L., possesses superior salinity tolerance and is a potential donor for breeding salinity tolerance traits in rice. However, a mechanistic basis of salinity tolerance in this donor species has not been established. Here, we examined salinity tolerance from the early vegetative stage to maturity in O. rufipogon in comparison with a salt-susceptible (Koshihikari) and a salt-tolerant (Reiziq) variety of O. sativa. We assessed their phylogeny and agronomical traits, photosynthetic performance, ion contents, as well as gene expression in response to salinity stress. Salt-tolerant O. rufipogon exhibited efficient leaf photosynthesis and less damage to leaf tissues during the course of salinity treatment. In addition, O. rufipogon showed a significantly higher tissue Na+ accumulation that is achieved by vacuolar sequestration compared to the salt tolerant O. sativa indica subspecies. These findings are further supported by the upregulation of genes involved with ion transport and sequestration (e.g. high affinity K+ transporter 1;4 [HKT1;4], Na+ /H+ exchanger 1 [NHX1] and vacuolar H+ -ATPase c [VHA-c]) in salt-tolerant O. rufipogon as well as by the close phylogenetic relationship of key salt-responsive genes in O. rufipogon to these in salt-tolerant wild rice species such as O. coarctata. Thus, the high accumulation of Na+ in the leaves of O. rufipogon acts as a cheap osmoticum to minimize the high energy cost of osmolyte biosynthesis and excessive reactive oxygen species production. These mechanisms demonstrated that O. rufipogon has important traits that can be used for improving salinity tolerance in cultivated rice.

Changes in Expression Level of OsHKT1;5 Alters Activity of Membrane Transporters Involved in K+ and Ca2+ Acquisition and Homeostasis in Salinized Rice Roots.

In rice, the OsHKT1;5 gene has been reported to be a critical determinant of salt tolerance. This gene is harbored by the SKC1 locus, and its role was attributed to Na+ unloading from the xylem. No direct evidence, however, was provided in previous studies. Also, the reported function of SKC1 on the loading and delivery of K+ to the shoot remains to be explained. In this work, we used an electrophysiological approach to compare the kinetics of Na+ uptake by root xylem parenchyma cells using wild type (WT) and NIL(SKC1) plants. Our data showed that Na+ reabsorption was observed in WT, but not NIL(SKC1) plants, thus questioning the functional role of HKT1;5 as a transporter operating in the direct Na+ removal from the xylem. Instead, changes in the expression level of HKT1;5 altered the activity of membrane transporters involved in K+ and Ca2+ acquisition and homeostasis in the rice epidermis and stele, explaining the observed phenotype. We conclude that the role of HKT1;5 in plant salinity tolerance cannot be attributed to merely reducing Na+ concentration in the xylem sap but triggers a complex feedback regulation of activities of other transporters involved in the maintenance of plant ionic homeostasis and signaling under stress conditions.

Homology Modeling Identifies Crucial Amino-Acid Residues That Confer Higher Na+ Transport Capacity of OcHKT1;5 from Oryza coarctata Roxb.

HKT1;5 loci/alleles are important determinants of crop salinity tolerance. HKT1;5s encode plasmalemma-localized Na+ transporters, which move xylem Na+ into xylem parenchyma cells, reducing shoot Na+ accumulation. Allelic variation in rice OsHKT1;5 sequence in specific landraces (Nona Bokra OsHKT1;5-NB/Nipponbare OsHKT1;5-Ni) correlates with variation in salt tolerance. Oryza coarctata, a halophytic wild rice, grows in fluctuating salinity at the seawater-estuarine interface in Indian and Bangladeshi coastal regions. The distinct transport characteristics of the shoots and roots expressing the O. coarctata OcHKT1;5 transporter are reported vis-à-vis OsHKT1;5-Ni. Yeast sodium extrusion-deficient cells expressing OcHKT1;5 are sensitive to increasing Na+ (10-100 mM). Electrophysiological measurements in Xenopus oocytes expressing O. coarctata or rice HKT1;5 transporters indicate that OcHKT1;5, like OsHKT1;5-Ni, is a Na+-selective transporter, but displays 16-fold lower affinity for Na+ and 3.5-fold higher maximal conductance than OsHKT1;5-Ni. For Na+ concentrations >10 mM, OcHKT1;5 conductance is higher than that of OsHKT1;5-Ni, indicating the potential of OcHKT1;5 for increasing domesticated rice salt tolerance. Homology modeling/simulation suggests that four key amino-acid changes in OcHKT1;5 (in loops on the extracellular side; E239K, G207R, G214R, L363V) account for its lower affinity and higher Na+ conductance vis-à-vis OsHKT1;5-Ni. Of these, E239K in OcHKT1;5 confers lower affinity for Na+ transport, as evidenced by Na+ transport assays of reciprocal site-directed mutants for both transporters (OcHKT1;5-K239E, OsHKT1;5-Ni-E270K) in Xenopus oocytes. Both transporters have likely analogous roles in xylem sap desalinization, and differences in xylem sap Na+ concentrations in both species are attributed to differences in Na+ transport affinity/conductance between the transporters.

Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species.

The genome of Asian cultivated rice (Oryza sativa L.) shows the presence of six organelle-specific and one plasma membrane (OsNHX1-7) NHX-type cation proton antiporters. Of these, vacuolar-localized OsNHX1 is extensively characterized. The genus Oryza consists of 27 species and 11 genome-types, with cultivated rice, diploid O. sativa, having an AA-type genome. Oryza NHX1 orthologous regions (gene organization, 5' upstream cis elements, amino acid residues/motifs) from closely related Oryza AA genomes cluster distinctly from NHX1 regions from more ancestral Oryza BB, FF and KKLL genomes. These sequence-specific distinctions also extend to two separate intron retention (IR) events involving Oryza NHX1 transcripts that occur at the 5' and 3' ends of the NHX1 transcripts. We demonstrate that the IR event involving the 5' UTR is present only in more recently evolved Oryza AA genomes while the IR event governing retention of the 13th intron of Oryza NHX1 (terminal intron) is more ancient in origin, also occurring in halophytic wild rice, Oryza coarctata (KKLL). We also report presence of a retro-copy of the OcNHX1 cDNA in the genome of O. coarctata (rOcNHX1). Preferential species and tissue specific up- or down-regulation of the correctly spliced NHX1 transcript/5' UTR/13th intron-retaining splice variants under salinity was observed. The implications of IR on NHX1 mRNA stability and ORF diversity in Oryza spp. is discussed.

Melatonin improves rice salinity stress tolerance by NADPH oxidase-dependent control of the plasma membrane K+ transporters and K+ homeostasis.

This study aimed to reveal the mechanistic basis of the melatonin-mediated amelioration of salinity stress in plants. Electrophysiological experiments revealed that melatonin decreased salt-induced K+ efflux (a critical determinant of plant salt tolerance) in a dose- and time-dependent manner and reduced sensitivity of the plasma membrane K+ -permeable channels to hydroxyl radicals. These beneficial effects of melatonin were abolished by NADPH oxidase blocker DPI. Transcriptome analyses revealed that melatonin induced 585 (448 up- and 137 down-regulated) and 59 (54 up- and 5 down-regulated) differentially expressed genes (DEGs) in the root tip and mature zone, respectively. The most noticeable changes in the root tip were melatonin-induced increase in the expression of several DEGs encoding respiratory burst NADPH oxidases (OsRBOHA and OsRBOHF), calcineurin B-like/calcineurin B-like-interacting protein kinase (OsCBL/OsCIPK), and calcium-dependent protein kinase (OsCDPK) under salt stress. Melatonin also enhanced the expression of potassium transporter genes (OsAKT1, OsHAK1, and OsHAK5). Taken together, these results indicate that melatonin improves salt tolerance in rice by enabling K+ retention in roots, and that the latter process is conferred by melatonin scavenging of hydroxyl radicals and a concurrent OsRBOHF-dependent ROS signalling required to activate stress-responsive genes and increase the expression of K+ uptake transporters in the root tip.

Tissue-Specific Regulation of Na+ and K+ Transporters Explains Genotypic Differences in Salinity Stress Tolerance in Rice.

Rice (Oryza sativa) is a staple food that feeds more than half the world population. As rice is highly sensitive to soil salinity, current trends in soil salinization threaten global food security. To better understand the mechanistic basis of salinity tolerance in rice, three contrasting rice cultivars-Reiziq (tolerant), Doongara (moderately tolerant), and Koshihikari (sensitive)-were examined and the differences in operation of key ion transporters mediating ionic homeostasis in these genotypes were evaluated. Tolerant varieties had reduced Na+ translocation from roots to shoots. Electrophysiological and quantitative reverse transcription PCR experiments showed that tolerant genotypes possessed 2-fold higher net Na+ efflux capacity in the root elongation zone. Interestingly, this efflux was only partially mediated by the plasma membrane Na+/H+ antiporter (OsSOS1), suggesting involvement of some other exclusion mechanisms. No significant difference in Na+ exclusion from the mature root zones was found between cultivars, and the transcriptional changes in the salt overly sensitive signaling pathway genes in the elongation zone were not correlated with the genetic variability in salinity tolerance amongst genotypes. The most important hallmark of differential salinity tolerance was in the ability of the plant to retain K+ in both root zones. This trait was conferred by at least three complementary mechanisms: (1) its superior ability to activate H+-ATPase pump operation, both at transcriptional and functional levels; (2) reduced sensitivity of K+ efflux channels to reactive oxygen species; and (3) smaller upregulation in OsGORK and higher upregulation of OsAKT1 in tolerant cultivars in response to salt stress. These traits should be targeted in breeding programs aimed to improve salinity tolerance in commercial rice cultivars.

Analysis of genetic diversity and population structure using SSR markers and validation of a Cleavage Amplified Polymorphic Sequences (CAPS) marker involving the sodium transporter OsHKT1;5 in saline tolerant rice (Oryza sativa L.) landraces.

Naturally evolved saline tolerant rice landraces found along the coastline of India are a valuable genomic resource to explore the complex, polygenic nature of salinity tolerance. In the present study, a set of 28 genome wide SSR markers, 11 salt responsive genic SSR markers and 8 Saltol QTL linked SSR markers were used to estimate genetic relatedness and population structure within a collection of 47 rice landraces (including a tolerant and 2 sensitive checks) originating from geographically divergent coastal regions of India. All three marker types identified substantial genetic variation among the landraces, as evident from their higher PIC values (0.53 for genomic SSRs, 0.43 for Genic SSRs and 0.59 for Saltol SSRs). The markers RM431, RM484 (Genomic SSRs), OsCAX (D), OsCAX (T) (Genic SSRs) and RM562 (Saltol SSR) were identified as good candidates to be used in breeding programs for improving salinity tolerance in rice. STRUCTURE analysis divided the landraces into five distinct populations, with classification correlating with their geographical locations. Principal coordinate and hierarchical cluster analyses (UPGMA and neighbor joining) are in close agreement with STRUCTURE results. AMOVA analysis indicated a higher magnitude of genetic differentiation within individuals of groups (58%), than among groups (42%). We also report the development and validation of a new Cleavage Amplified Polymorphic Sequence (CAPS) marker (OsHKT1;5V395) that targets a codon in the sodium transporter gene OsHKT1;5 (Saltol/SKC1 locus) that is associated with sodium transport rates in the above rice landraces. The CAPS marker was found to be present in all landraces except in IR29, Kamini, Gheus, Matla 1 and Matla 2. Significant molecular genetic diversity established among the analyzed salt tolerant rice landraces will aid in future association mapping; the CAPS marker, OsHKT1;5V395 can be used to map rice landraces for the presence of the SNP (Single Nucleotide Polymorphism) associated with increased sodium transport rates and concomitant salinity tolerance in rice.