Whilst the OsCaM group contains five members that share a highest degree of amino acid sequence identity (≥ 97%) to known typical CaMs from other plants, previous report phylogenetically classified the highly conserved OsCML proteins into four groups. The OsCML proteins are small proteins of 145 to 250 amino acid residues with an approximately 44% to 85% amino acid identity to typical plant CaMs. A large family of six Cam and 50 CML genes have also been annotated in the A.thaliana genome, suggesting the existence of an extensive set of CaM and CML proteins in each plant species.
Even though rice is considered an important crop and a model for other monocots and especially cereal crops, almost all of OsCam and OsCML genes have not been characterized. From the publicly available microarray data, all OsCam genes were found highly expressed in almost all organs examined (Figure1A). One of the defining characteristics of CaMs in plants is the presence of multiple CaM isoforms. Even though they are ubiquitously expressed, the different isoforms can display differential responses to individual stimuli in any given plant tissue[5, 11, 19–22], suggesting that each of the OsCam genes may have distinct physiological functions depending on where and how the expression of each gene is up-regulated in addition to the different biochemical properties that might be affected by the slight differences in their primary structures[21, 23].
In contrast, several highly conserved OsCML genes (groups 2–5) genes tend to be highly expressed in some organs/tissues and their levels of expression are modulated during different stages of development (Figure1A), indicating that they are developmentally regulated. It is conceivable that CaM proteins have many targets and are ubiquitously involved in numerous cellular processes while the highly conserved CML proteins have more specialized targets and are involved in more specific processes of the cell. Interestingly, each subgroup of the highly diverged group of OsCML (group 6a to 6e) genes had one member whose expression is found at a noticeably higher level than the other members. Members in the same subgroups have the same number and configuration of EF-hand motifs. It is speculated that these highly expressed genes may be the predominant gene representing each subgroup that has essential functions in the cell.
When the microarray data was closely examined for the transcript expression levels of the OsCam1-1 and nine OsCML genes (Figure1B), ubiquitous expression at relatively high levels of OsCam1-1, OsCML7, and 13 was observed, which suggests that they may have important functions during the regular growth and development processes of rice plants. The increasing transcript expression levels of OsCML1, 4, and 8 with maturation of the inflorescence until they reached similar levels as those in the leaf and root tissues in the early developing seed, suggest that they may have important roles during the maturation of the inflorescence and the early seed development, especially OsCML1, which expression level peaked during those stages.
The transcript expression levels in the ‘KDML105’ rice in the leaf blades/sheaths of the three-week old ‘KDML105’ rice seedlings, as evaluated by rt-RT-PCR (Figure2A) agree well with the microarray data in the IR64 cultivar (Figure1A). When the transcript expression levels of OsCam1-1 and OsCML s were examined in different organs (Figure2B), their profiles could be divided into three groups as those with the highest expression level in (i) the root, or (ii) in the flower and the seed, or (iii) those genes with similar expression levels among the organs examined. Relatively high transcript levels of OsCML3 and OsCML11 in the flower and the seed, and OsCML4, and 5 in the root suggest their functional significance in those respective organs. The OsCam1-1 and the other five OsCML genes (OsCML1, 7, 8, 9 and 13) had more or less similar expression levels among the different organs examined. From the microarray data of the IR64 rice cultivar, four of these genes (OsCam1-1, OsCML7, 9 and 13), were expressed at more or less constant levels in different developmental stages. However, expression patterns among different organs/tissues that are not consistent with those analyzed from the microarray database may also result from variation in the rice varieties examined.
Up-regulated expression of a gene in response to a stress signal may reflect the function of the corresponding gene product, especially in signal cascades. In a large gene family, investigating expression patterns of their members could point to genes or isoforms that potentially function under the conditions of interest. In this study, the transcript expression analysis of OsCam1-1 and nine OsCML genes in the KDML105 rice cultivar by rt-RT-PCR revealed that expression levels of OsCam1-1, OsCML4, 5, 8, and 11 were increased under osmotic stress (20% (w/v) PEG) (Figure3B), which is consistent with the GSE6901 data for the IR64 cultivar from the DNA microarray database (Figure3A) and suggests that these genes may function in the mechanisms of Ca2+-mediated responses to osmotic stress. Similarly, expression of these genes was also found to increase in the KDML105 rice cultivar under salt stress (150 mM NaCl) along with OsCML1 (Figure3C), suggesting that OsCam1-1, OsCML1, 4, 5, 8, and 11 may function in the mechanisms of Ca2+-mediated responses to salt stress. The up-regulated transcript expression levels under salt stress of OsCML4, 8 and 11 were also consistent with the GSE6901 data for the IR64 cultivar from the DNA microarray database (Figure3A). Whether the discrepancy in the transcript expression patterns of OsCam1-1, OsCML1, and 5 is due to the different timings of expression being monitored or differences in the rice varieties examined remains to be evaluated but, overall, up-regulation of almost the same set of the highly conserved OsCML genes under osmotic and salt stresses was observed, confirming that conclusion that genes responsive to osmotic stress overlap to a high extent with those that are responsive to salt stress.
Several of these genes not only exhibited up-regulated transcript expression levels by osmotic stress and salt stress, but also exhibited different patterns of up-regulation in terms of timing and levels of expression within a tissue type and exhibited differential expression in different tissues/organs. Together, differential temporal and spatial expression patterns of these OsCam1-1 and OsCML genes suggest that each individual gene product may possess specific roles during Ca2+-mediated responses to osmotic stress and salt stress. Comparison of the transcript expression patterns of these genes under osmotic or salt stress in the KDML105 rice cultivar, as determined by rt-RT-PCR in this study, with the publicly available RNA-Seq data and the other microarray data sets, revealed that OsCML4 and OsCML8 consistently exhibited higher expression levels under osmotic and salt stresses suggesting their significant functions in Ca2+-mediated responses to these stimuli. In addition, the early induction within 1 h of OsCam1-1 and OsCML5 under osmotic and salt stresses, and of OsCML1 under salt stress suggests their importance in conveying the stress signals early in the transduction cascades of Ca2+ signaling. However, caution must be taken in interpreting these results because the changes in levels of transcripts generally do not coincide with the changes in the levels of the proteins they encode and, given the time it takes to express a protein, may not necessarily reflect their involvement in the responses of the cell to stress. Nonetheless, a gene of which transcript expression is early induced would be given special attention as a candidate for further investigation into its possible involvement in response to a particular stress.
Several cis-acting elements in response to abiotic stresses in the 5′ upstream regions of the OsCam1-1 and OsCML genes were detected (Figure4). DREs, important promoter elements that are responsive to drought, high salt, and cold, were located in the putative promoters of OsCam1-1, OsCML4, 5, 7, and 11. Consistent with this is that the transcript expression level of all these genes was shown by rt-RT-PCR to increase in the KDML105 rice cultivar under osmotic and salt stresses with OsCML7 exhibiting a slight increase in its expression level and the others significantly much higher level of up-regulation. DREs specifically interact with the transcription factors DREBs and regulate expression of many stress-inducible genes. In rice, the DRE binding protein 1 (Os DREB1) functions in the cold stress response, whereas Os DREB2 functions in the heat and osmotic stress responses. The results here indicate that these DREs are potentially responsible for the osmotic stress-induced expression of these genes and interesting candidates for further characterization.
In the 5′ upstream region of the OsCam1-1 promoter, the putative DRE motif (ACCGAC) was located at −1062. Induction of the GUS activity level in the three independent OsCam::gus transgenic rice plants (Figure5) suggested that induction of OsCam1-1 expression under salt stress is, at least, partly due to the activity of its promoter. Its induction appears to be dependent on the severity of the salt stress (concentration of NaCl), with no significant induction in the plants treated with 100 mM NaCl while a monophasic and biphasic induction were observed in the plants treated with 150 mM and 300 mM NaCl, respectively, in all three independent transgenic lines. A biphasic induction of OsCam1-1 expression by salt stress in the KDML105 rice cultivar, as determined by rt-RT-PCR has been reported previously, whilst heat shock (HS) induced biphasic [Ca2+cyt signal in rice root cells and the HS-induced expression of OsCam1-1 strongly oscillated. The complex responses of the OsCam1-1 gene to salt stress suggest that OsCam1-1 is a significant player in the Ca2+ signal transduction network under salt stress. However, caution must be taken in interpreting these results, especially on timings of the induction because of the possible differences in mRNA and protein stability between the gus gene and the OsCam1-1 gene.