The rice landrace Horkuch, endemic to the southern saline coast of Bangladesh, is known to have salt tolerance traits and can therefore contribute to a high yielding recipient for breeding purposes. In this study, we reciprocally crossed Horkuch with high yielding but salt sensitive IR29 to detect the complement of genes that were responsible for conferring salt tolerance versus sensitivity at the seedling developmental stage. We looked at tolerant and sensitive F₃families from individual F₂segregating plants and analyzed them for differential gene expressions using RNAseq. In general, we observed higher numbers of genes differentially expressed in leaves compared to root tissues. This included both upregulation and downregulation of gene expression across our experimental factors. Gene expression decreased in sensitive leaf after stress exposure where tolerant plants showed the opposite trend. In root, tolerant plants expression decreased at higher time points of stress exposure. We also observed a strong maternal cytoplasmic effect on gene expression and this was most evident in roots where there was upregulation in functional enrichments related to phosphorylation, electron carriers, transporter and cation transmembrane activities. Stress groups (tolerant and sensitive) response in F₃families were distinctive in both cytoplasmic backgrounds and involved uniquely upregulated genes in tolerant progenies including membrane sensor proteins, enzymes involved with signaling pathways, such as those producing trehalose and G-protein coupled receptor proteins, photosynthesis-related enzymes and golgi body recycling as well as prolamin precursor proteins involved in refolding of proteins. On the other hand, sensitivity was found to be associated with differential upregulation of only a few redox proteins and higher number of apoptosis related genes compared to the tolerant response. Overall, our highly replicated experimental design was powerful and allowed the detection of relatively subtle differential expression. Our future goal is to correlate these expression differences with QTLs in this population, which would help identify the relative importance of specific genetic loci and provide a direct avenue for combining higher levels of salt tolerance with better agronomic traits in rice.

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5ʹ UTR with CDS and the other of 2.3 kb, including 5ʹ UTR, CDS and the 3ʹ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5ʹ and 3ʹ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P 1.9 kb> and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P<0.01) compared respectively to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

Background: Plant growth-promoting endophytic fungi (PGPEF) that are associated with halophytes have the potential to boost crop salinity tolerance and productivity. This in turn has the potential of enabling and improving cultivation practices in coastal lands affected by salt stress. Methods: Endophytic fungi from the wild halophytic rice Oryza coarctata were isolated, characterized, identified, and studied for their effects on all developing stages of rice plant growth and their yields both with and without salt stress. Key results: In this study, three different fungal endophytes were isolated from the halophytic wild rice Oryza coarctata. Two isolates were identified as Talaromyces adpressus (OPCRE2) and Talaromyces argentinensis (OPCRh1) by ITS region sequencing. The remaining isolate NPCRE2 was confirmed as a novel strain named Aspergillus welwitschiae Ocstreb1 (AwOcstreb1) by whole genome sequencing. These endophytes showed various plant growth-promoting (PGP) abilities in vitro (e.g., IAA, ACC-deaminase and siderophore production, phosphate, and zinc solubilization as well as nitrogen fixation), where AwOcstreb1 was significantly more efficient compared to the other two isolates at high salinity (900 mm). Independent application of these fungi in commercial rice (Oryza sativa) showed significant elevation in plant growth, especially in the case of the AwOcstreb1 inoculants, which had enhanced metabolite and chlorophyll content at the seedling stage in both no-salt control and 100-mm salt-stressed plants. At the same time, AwOcstreb1-treated plants had a significantly lower level of H₂O₂, electrolyte leakage, and Na⁺/K⁺ ratio under saline conditions. Higher expression (1.6 folds) of the SOS1 (salt overly sensitive 1) gene was also observed in these plants under salinity stress. This strain also improved percent fertility, tillering, panicle number, and filled grain number in both no-salt control and 45-mm salt-stressed inoculated plants at the reproductive stage. Consequently, the differences in their yield was 125.16% and 203.96% (p < 0.05) in colonized plants in normal and saline conditions, respectively, compared to uninoculated controls. Conclusions: We propose that AwOcstreb1 is a potential candidate for an eco-friendly biofertilizer formula to improve the cultivation and yield of rice or any other crop in the highly saline coastal regions of Bangladesh.

Plants need to maintain a low Na⁺/K⁺ ratio for their survival and growth when there is high sodium concentration in soil. Under these circumstances, the high affinity K⁺ transporter (HKT) and its homologs are known to perform a critical role with HKT1;5 as a major player in maintaining Na⁺ concentration. Preferential expression of HKT1;5 in roots compared to shoots was observed in rice and rice-like genotypes from real time PCR, microarray, and RNAseq experiments and data. Its expression trend was generally higher under increasing salt stress in sensitive IR29, tolerant Pokkali, both glycophytes; as well as the distant wild rice halophyte, Porteresia coarctata, indicative of its importance during salt stress. These results were supported by a low Na⁺/K⁺ ratio in Pokkali, but a much lower one in P. coarctata. HKT1;5 has functional variability among salt sensitive and tolerant varieties and multiple sequence alignment of sequences of HKT1;5 from Oryza species and P. coarctata showed 4 major amino acid substitutions (140 P/A/T/I, 184 H/R, D332H, V395L), with similarity amongst the tolerant genotypes and the halophyte but in variance with sensitive ones. The best predicted 3D structure of HKT1;5 was generated using Ktrab potassium transporter as template. Among the four substitutions, conserved presence of aspartate (332) and valine (395) in opposite faces of the membrane along the Na⁺/K⁺ channel was observed only for the tolerant and halophytic genotypes. A model based on above, as well as molecular dynamics simulation study showed that valine is unable to generate strong hydrophobic network with its surroundings in comparison to leucine due to reduced side chain length. The resultant alteration in pore rigidity increases the likelihood of Na⁺ transport from xylem sap to parenchyma and further to soil. The model also proposes that the presence of aspartate at the 332 position possibly leads to frequent polar interactions with the extracellular loop polar residues which may shift the loop away from the opening of the constriction at the pore and therefore permit easy efflux of the Na⁺. These two substitutions of the HKT1;5 transporter probably help tolerant varieties maintain better Na⁺/K⁺ ratio for survival under salt stress.