Genome-wide association study for salinity tolerance at the flowering stage in a panel of rice accessions from Thailand.

BACKGROUND: Salt stress, a major plant environmental stress, is a critical constraint for rice productivity. Dissecting the genetic loci controlling salt tolerance in rice for improving productivity, especially at the flowering stage, remains challenging. Here, we conducted a genome-wide association study (GWAS) of salt tolerance based on exome sequencing of the Thai rice accessions. RESULTS: Photosynthetic parameters and cell membrane stability under salt stress at the flowering stage; and yield-related traits of 104 Thai rice (Oryza sativa L.) accessions belonging to the indica subspecies were evaluated. The rice accessions were subjected to exome sequencing, resulting in 112,565 single nucleotide polymorphisms (SNPs) called with a minor allele frequency of at least 5%. LD decay analysis of the panel indicates that the average LD for SNPs at 20 kb distance from each other was 0.34 (r2), which decayed to its half value (~ 0.17) at around 80 kb. By GWAS performed using mixed linear model, two hundred loci containing 448 SNPs on exons were identified based on the salt susceptibility index of the net photosynthetic rate at day 6 after salt stress; and the number of panicles, filled grains and unfilled grains per plant. One hundred and forty six genes, which accounted for 73% of the identified loci, co-localized with the previously reported salt quantitative trait loci (QTLs). The top four regions that contained a high number of significant SNPs were found on chromosome 8, 12, 1 and 2. While many are novel, their annotation is consistent with potential involvement in plant salt tolerance and in related agronomic traits. These significant SNPs greatly help narrow down the region within these QTLs where the likely underlying candidate genes can be identified. CONCLUSIONS: Insight into the contribution of potential genes controlling salt tolerance from this GWAS provides further understanding of salt tolerance mechanisms of rice at the flowering stage, which can help improve yield productivity under salinity via gene cloning and genomic selection.

Downstream components of the calmodulin signaling pathway in the rice salt stress response revealed by transcriptome profiling and target identification.

BACKGROUND: Calmodulin (CaM) is an important calcium sensor protein that transduces Ca2+ signals in plant stress signaling pathways. A previous study has revealed that transgenic rice over-expressing the calmodulin gene OsCam1-1 (LOC_Os03g20370) is more tolerant to salt stress than wild type. To elucidate the role of OsCam1-1 in the salt stress response mechanism, downstream components of the OsCam1-1-mediated response were identified and investigated by transcriptome profiling and target identification. RESULTS: Transcriptome profiling of transgenic 'Khao Dawk Mali 105' rice over-expressing OsCam1-1 and wild type rice showed that overexpression of OsCam1-1 widely affected the expression of genes involved in several cellular processes under salt stress, including signaling, hormone-mediated regulation, transcription, lipid metabolism, carbohydrate metabolism, secondary metabolism, photosynthesis, glycolysis, tricarboxylic acid (TCA) cycle and glyoxylate cycle. Under salt stress, the photosynthesis rate in the transgenic rice was slightly lower than in wild type, while sucrose and starch contents were higher, suggesting that energy and carbon metabolism were affected by OsCam1-1 overexpression. Additionally, four known and six novel CaM-interacting proteins were identified by cDNA expression library screening with the recombinant OsCaM1. GO terms enriched in their associated proteins that matched those of the differentially expressed genes affected by OsCam1-1 overexpression revealed various downstream cellular processes that could potentially be regulated by OsCaM1 through their actions. CONCLUSIONS: The diverse cellular processes affected by OsCam1-1 overexpression and possessed by the identified CaM1-interacting proteins corroborate the notion that CaM signal transduction pathways compose a complex network of downstream components involved in several cellular processes. These findings suggest that under salt stress, CaM activity elevates metabolic enzymes involved in central energy pathways, which promote or at least maintain the production of energy under the limitation of photosynthesis.

C-terminal extension of calmodulin-like 3 protein from Oryza sativa L.: interaction with a high mobility group target protein.

A large number of calmodulin-like (CML) proteins are present in plants, but there is little detailed information on the functions of these proteins in rice (Oryza sativa L.). Here, the CML3 protein from rice (OsCML3) and its truncated form lacking the C-terminal extension (OsCML3m) were found to exhibit a Ca2+-binding property and subsequent conformational change, but the ability to bind the CaM kinase II peptide was only observed for OsCML3m. Changes in their secondary structure upon Ca2+-binding measured by circular dichroism revealed that OsCML3m had a higher helical content than OsCML3. Moreover, OsCML3 was mainly localized in the plasma membrane, whereas OsCML3m was found in the nucleus. The rice high mobility group B1 (OsHMGB1) protein was identified as one of the putative OsCML3 target proteins. Bimolecular fluorescence complementation analysis revealed that OsHMGB1 bound OsCML3, OsCML3m or OsCML3s (cysteine to serine mutation at the prenylation site) in the nucleus presumably through the methionine and phenylalanine-rich hydrophobic patches, confirming that OsHMGB1 is a target protein in planta. The effect of OsCML3 or OsCML3m on the DNA-binding ability of OsHMGB1 was measured using an electrophoretic mobility shift assay. OsCML3m decreased the level of OsHMGB1 binding to pUC19 double-stranded DNA whereas OsCML3 did not. Taken together, OsCML3 probably provides a mechanism for manipulating the DNA-binding ability of OsHMGB1 in the nucleus and its C-terminal extension provides an intracellular Ca2+ regulatory switch.

Expression analysis of calmodulin and calmodulin-like genes from rice, Oryza sativa L.

BACKGROUND: In plants, a large family of calmodulin (CaM) and CaM-like (CML) proteins transduce the increase in cytosolic Ca2+ concentrations by binding to and altering the activities of target proteins, and thereby affecting the physiological responses to a vast array of stimuli. Here, transcript expression analysis of Cam and CML gene family members in rice (Oryza sativa L.) was extensively examined. RESULTS: Cam and CML genes in rice exhibited differential expression patterns in tissues/organs. Under osmotic stress and salt stress, expression of OsCam1-1, OsCML4, 5, 8, and 11 was induced with different kinetics and magnitude. OsCML4 and 8 mRNA levels significantly increased by 3 h after treatment and remained elevated for at least 24 h while expression of OsCam1-1, OsCML5 and 11 was up-regulated as early as 1-3 h before rapidly returning to normal levels. Several cis-acting elements in response to abiotic stresses, including DREs (important promoter elements responsive to drought, high salt, and cold stress), were detected in the 5' upstream regions of these genes. The observed induction of the GUS activity of transgenic rice plants via the OsCam1-1 promoter appeared to be biphasic and dependent on the severity of salt stress. CONCLUSIONS: Large OsCam and OsCML gene family members likely play differential roles as signal transducers in regulating various developmental processes and represent important nodes in the signal transduction and transcriptional regulation networks in abiotic stresss responses mediated by the complex Ca2+ signals in plants, which are rich in both spatial and temporal information.

Biophysical characterization of calmodulin and calmodulin-like proteins from rice, Oryza sativa L.

Calmodulin (CaM) transduces the increase in cytosolic Ca(2+) concentrations by binding to and altering the activities of target proteins, thereby affecting the physiological responses to the vast array of stimuli. Here, we examined the purified recombinant proteins encoded by three Cam and eight Cam-like (CML) genes from rice. With the exception of one OsCML, all recombinant proteins could be purified by Ca(2+)-dependent hydrophobic chromatography and exhibited an electrophoretic mobility shift when incubated with Ca(2+). The three CaMs all bound CaM kinase II peptide, but none of the eight CMLs did, suggesting a possible differential target binding between the CaM and CML proteins. In addition, their conformational changes upon Ca(2+)-binding were evaluated by circular dichroism spectroscopy and fluorescence spectroscopy using 8-Anilino-1-naphthalene-sulfonic acid. Taken together, OsCMLs were found exhibiting a spectrum of both structural and functional characteristics that ranged from typical to atypical of CaMs. From structural comparison, the OsCMLs have overall main-chain conformation nearly identical to OsCaMs, but with distinct distribution of some charged and hydrophobic amino acids on their target-binding site. These results suggest that genetic polymorphism has promoted the functional diversity of the OsCML family, whose members possess modes of actions probably different from, though maybe overlapping with, those of OsCaMs.