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.

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).

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.

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.

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.

Expression of wild rice Porteresia coarctata PcNHX1 antiporter gene (PcNHX1) in tobacco controlled by PcNHX1 promoter (PcNHX1p) confers Na+-specific hypocotyl elongation and stem-specific Na+ accumulation in transgenic tobacco.

Soil salinization is a major abiotic stress condition that affects about half of global agricultural lands. Salinity leads to osmotic shock, ionic imbalance and/or toxicity and build-up of reactive oxygen species. Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electro-neutral exchange of K⁺/Na⁺ for H⁺ and are implicated in cell expansion, development, pH/ion homeostasis and salt tolerance. Porteresia coarctata is a salt secreting halophytic wild rice that thrives in the coastal-riverine interface. P. coarctata NHX1 (PcNHXI) expression is induced by salinity in P. coarctata roots and shows high sequence identity to Oryza sativa NHX1. PcNHX1 confers hygromycin and Li+ sensitivity and Na+ tolerance transport in a yeast strain lacking sodium transport systems. Additionally, transgenic PcNHX1 expressing tobacco seedlings (PcNHX1 promoter) show significant growth advantage under increasing concentrations of NaCl and MS salts. Etiolated PcNHX1 seedlings also exhibit significantly elongated hypocotyl lengths in 100 mM NaCl. PcNHX1 expression in transgenic tobacco roots increases under salinity, similar to expression in P. coarctata roots. Under incremental salinity, transgenic lines show reduction in leaf Na+, stem specific accumulation of Na+ and K+ (unaltered Na+/K+ ratios). PcNHX1 transgenic plants also show enhanced chlorophyll content and reduced malondialdehyde (MDA) production in leaves under salinity. The above data suggests that PcNHX1 overexpression (controlled by PcNHX1p) enhances stem specific accumulation of Na+, thereby protecting leaf tissues from salt induced injury.