Salinity stress is a major abiotic stress that affects plant growth, resulting in a loss of crop yield, especially rice, which is one of the most salt-sensitive plants in comparison to other cereals.Salt stress affects plants via both osmotic and ionic effects.Osmotic effects result in a reduction of water absorption ability such that the effects are similar to drought stress.Ionic stress causes Na+ toxicity, which disrupts photosynthesis, protein synthesis, and enzyme activity.Numerous reports have shown negative effects of salt stress on rice growth and productivity based on the total chlorophyll content, protein concentration, photosynthetic CO2 fixation, stomatal conductance, transpiration, shoot dry weight, tiller number per plant, spikelets per panicle, and grain yield.Ca2+ is a crucial second messenger consisting of a transient elevation of cytosolic [Ca2+].The Ca2+ signals are transduced and decoded via Ca2+ binding protein, and then the information is relayed to downstream responses.The signals are mainly transduced through kinases mediating the phosphorylation cascade, resulting in downstream response regulation, including changes in gene expression through the regulation of transcription factors.Calcium signaling is used to respond to environmental stimuli, as well as to coordinate growth and development in plants.In the plant calcium signal transduction process, calcium sensors, including calmodulin ,hydroponic nft channel calcineurin B-likeprotein and Ca2+-dependent protein kinase , play important roles in the transduction of various stimuli.CaM is a protein that contains characteristic EF-hand motifs that bind Ca2+ ions with high affinity and specificity.CaM binding to Ca2+ leads to the exposure of hydrophobic regions on the molecule surface and subsequent interactions with target proteins or nucleic acids.
Rice carries 5 CaM-encoding genes: OsCam1–1, OsCam1–2, OsCam1–3, OsCam2 and OsCam3.The expression of OsCam1–1 increases to a great extent in response to NaCl, mannitol and wounding treatment.Several lines of evidence have revealed that calcium sensors are involved with an enhanced abiotic tolerance capacity in plants.Evidence has shown that the constitutive expression of bovine calmodulin in tobacco results in a shortened germination time of transgenic tobacco seeds under salt stress.Arabidopsis over expressing GmCaM4exhibit increased expression of AtMYB2-regulated genes, including proline-synthesizing enzymes, suggesting that this feature confers salt tolerance to the transgenic Arabidopsis by enabling the accumulation of proline.Our previous report have shown that transgenic rice over-expressing OsCam1–1 grow better under salt stress than wild type.Wu H.and colleagues have found that the biphasic Ca2+ signal and enhancement of OsCam1–1 expression in rice cause heat stress-mediated expression of downstream heat shock-related genes, and OsCam1–1 over expression Arabidopsis are more tolerant to heat stress than its wild type.In another report, AtCam3 knockout mutant Arabidopsis showed a clear reduction of thermotolerance after heat treatment at 45 °C, and when AtCam3 was over expressed in mutant and wild type Arabidopsis, the thermotolerant ability was rescued and increased, respectively.Moreover, co-expression of some heat shock protein genes with AtCaM3 suggested that AtCam3 plays a key role in the Ca2+-CaM heat shock transduction pathway.The versatile functions of CaM are interesting, especially the role in the regulation of gene expression.CaM proteins directly modulate transcription factors , and some of these TFs have been verified to play roles in stress signaling pathways; however, the Ca2+ and Ca2+/CaM-regulating TF mechanisms remain incompletely understood and require further investigation.The aim of the present study was to investigate the downstream effects of OsCam1–1 over expression on gene expression regulation in rice under salt stress using a transcriptomic approach and to identify the interacting proteins to elucidate the role of OsCam1–1 in the salt stress response mechanism.
CaM is a multifunctional protein that regulates the activities of numerous target proteins.Genome-wide analysis techniques such as transcriptome profiling are particularly suitable for identifying the downstream components that are potentially regulated by CaM.In our previous report, rice over expressing OsCam1– 1 showed a significantly higher relative growth rate than wild type when grown under salt stress.Here, transcriptome profiling of the 3-week-old rice leaves of transgenic rice over-expressing OsCam1–1and its wild type under normal condition and salt stress conditions for 4 h was conducted.More than 185 million reads from eight libraries from single-end RNA-Seq by Illumina Hi-Seq 2000 were obtained, with a total read of each library between 22 and 25 million reads.The reads were processed by POPE, which provided a total clean read per library of more than 99% of the total reads.At least 93% of the clean reads were mapped to the rice genome reference, Michigan State University rice annotation project’s MSU7 and less than 11% of the clean reads were multiple alignment reads.To compare the transcriptome profiles of the rice, differential gene expression analysis of the transcriptome data using DESeq was carried out, which provided the number of differentially expressed genes summarized in Table 2.Analysis of the wild type identified 12,184 DEGs between the transcriptome profile under normal and salt stress conditions , in which 5842 and 6342 genes were up-regulated and down-regulated, respectively.For transgenic rice over-expressing OsCam1–1, comparisons between normal and salt stress conditions revealed a total of 13,259 DEGs with 6434 and 6825 up-regulated and down-regulated genes, respectively.Furthermore, the transcriptome profiles of the transgenic rice were compared with those of the wild type.Under normal conditions , 2022 DEGs were identified, with 892 and 1130 DEGs expressed at higher or lower levels in the transgenic rice, respectively.Under salt stress, comparisons of transgenic rice with wild type rice revealed 1677 DEGs, with 957 and 720 DEGs expressed at higher or lower levels in the transgenic rice, respectively.The scatter plots showed quantitative overview of the four transcriptome profile comparisons.
OsCam1–1 was found to be highly expressed in transgenic rice under both normal and stress condition, with an average RPKM of 1758.67 and 1644.62, while the average RPKM of wild type under normal and stress conditions was 91.94 and 97.84, respectively.The expression of OsCam1–1 in the wild type was not induced at 4 h after salt stress , in good agreement with a previous study.According to a gene expression study conducted by Chinpongpanich et al., the transcript level of OsCam1–1 determined by qRT-PCR was highly induced at 1 h after 150 mM NaCl treatment and then sharply decreased after 1 h.This result validated the over expression of OsCam1–1 in transgenic rice with an approximately 18-fold change in RPKM compared with wild type.Based on a differential transcriptome analysis, the gene expression levels of those 2022 and 1677 DEGs were thus likely affected by OsCam1–1 over expression.In our previous report, OsCam1–1-overexpressing lines showed a significantly higher relative growth rate than wild type when grown under salt stress.Based on the genes identified herein, among which several were involved in central energy pathways, sucrose and starch levels were determined in the three independent lines under normal and salt stress conditions at day 3 and 5 after treatment.Salt stress led to a significant reduction of the starch level and slightly decreased sucrose levels in both wild type and transgenic rice lines.Noticeably, at day 3, the transgenic lines could maintain the sucrose and starch levels better than the wild type under salt stress conditions.At day 5, the trends observed for sucrose and starch levels in transgenic rice under salt stress conditions were similar to those in wild type.In addition, the photosynthesis rate , stomatal conductance , intercellular carbon dioxide and transpiration rate were examined in the transgenic rice over-expressing OsCam1–1.Under salt stress, Pn, gs and E decreased at both day 3 and day 5, while Ci decreased slightly at day 3 of treatment.Interestingly, transgenic rice had slightly lower Pn values than wild type rice at both day 3 and day 5 and tended to have lower gs and E values at day 5 of salt stress treatment.In contrast, the Ci measurements did not reveal significant difference between the transgenic and wild type.For FV′/FM′, which reflects the maximum efficiency of photosystem II, no change was observed under the given salt stress conditions,nft growing system and the transgenic rice did not exhibit difference either under normal or salt stress conditions compared with the wild type.In a previous study, the northern blot results showed the highest expression levels of OsCam1–1 in transgenic rice line L1 among the three transgenic rice lines.Under both normal and salt stress conditions, the sucrose and starch content correlated with the expression level of OsCam1–1 in those transgenic rice lines.CaM does not possess functional domains other than EF hand motifs, so it functions by binding to and altering the activities of various interacting proteins.To understand how CaM1 mediates Ca2+-signal responses, its specific interacting proteins were identified using a cDNA expression library with 35S-labeled rOsCaM1 protein as the probe.The purity of the prepared 35S-labeled rOsCaM1 protein was examined by SDS-PAGE.To test its specificity, PVDF membrane spotted with various amounts of CaMKII peptide, calcineurin, and BSA was incubated with the probe.The autoradiograph showed that the probe only interacted with CaMKII peptide and calcineurin but not BSA, and the intensity of the signal on the X-ray film was dose-dependent.The results indicated that the 35S-labeled rOsCaM1 protein could specifically bind to well-known target proteins in the presence of Ca2+.After screening the cDNA library, the purified clones from the tertiary screening were titered before performing single-clone excision.As a result, 10 distinct positive cDNA clones were obtained.All unique pBlue script SK plasmids obtained from the single-clone excision were sequenced to determine the cloned cDNA insert sequences.The resulting sequences were BLAST searched against the Rice Genome Annotation Project and the Rice Annotation Project databases.The functions of 8 OsCaM1 targets were identified , which were diverse and potentially involved in various cellular processes, including metabolism, transcription, movement of organelles and vesicles, membrane transport, and signal transduction.Four known CaM-binding proteins previously identified in other plants were obtained from this screening, which included a cyclic nucleotide-gated ion channel , a glutamate decarboxylase, a CaM-binding transcription activator, and a kinesin motor domain-containing protein.The six identified putative novel CaM1-binding proteins comprised a transferase family protein , a response regulator receiver domain-containing protein , a lipin , a myosin heavy chain-containing protein , and two proteins with unknown function, LOC_Os08g34060 and LOC_Os02g13060.Interaction of eight putative target proteins with OsCaM1 was confirmed by protein blot analysis.
The mitogen-activated protein kinase cascade is a highly conserved central regulator of diverse cellular processes.CaM plays role in the MAPK/MPK cascade by binding to mitogen-activated protein kinase and/or mitogen-activated protein kinase phosphatase.A rice MAPK, BWMK1 encoded by an HT salt-responsive DEG, could phosphorylate the OsEREBP1 transcription factor for binding to the GCC box element , which is a basic component of several pathogenesis-related gene promoters.Inositol 1,3,4-trisphosphate 5/6-kinaseencoded by two HT salt-responsive DEGs, phosphorylates inositol 1,3,4-trisphosphate to form inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6, tetrakisphosphate, which are ultimately converted to inositol hexaphosphate and play roles in plant growth and development.In rice, the T-DNA mutant of an IPTK gene showed reduced osmolyte accumulation and growth under drought conditions, and some genes involved in osmotic adjustment and reactive oxygen species scavenging were down-regulated.In addition, over expression of DSM3resulted in a decrease in inositol trisphosphate , and the phenotypes were similar to the mutant under salt and drought stress conditions.These findings suggested that DSM3 might play a role in fine-tune balancing the inositol phosphate level when plants are exposed to stress or during development.Diacylglycerol kinaseen coded by an HT salt-responsive DEG, catalyzes the conversion of diacylglycerolto phosphatidic acid, and PA plays a role in the stress signaling pathway, including the MAPK/MPK cascade.A report has shown that the expression of OsBIDK1 encoding rice DGK is induced by benzothiadiazole and fungal infection.Moreover,transgenic tobacco constitutively expressing OsBIDK1 was more tolerant to plant pathogenic virus and fungi.These findings suggest that several genes in the signaling process might be enhanced by OsCam1–1 under salt stress.Interestingly, a universal stress protein gene was identified as an HT salt-responsive DEG.Evidence has shown that the expression of tomato USPis induced by drought, salt, oxidative stress and ABA, and over expression of spUSP improves tomato drought tolerance via interactions with annexin, leading to the accumulation of ABA.In addition, a xylanase inhibitor protein gene , was highly expressed and induced by salt stress and OsCam1–1 over expression.A previous report has shown that OsXIP can be induced by methyl jasmonate and wounding, so it was suggested that OsXIP may play a role in pathogen defense.As many OsCam1–1 and/or salt stress affecting DEGs involve both biotic and abiotic stresses, OsCam1–1 may be a component that mediates the crosstalk of biotic stress and abiotic stress responses.