3.2 Tissue and cellular specificity of starch synthesis-related gene expression
Tissue and cellular specificity of starch synthesis-related gene expression was studied by histochemical staining of GUS activity in transgenic rice with the promoter::GUS chimeric genes. As reported previously (Morita et al., 2015), CRCT expression in the leaf blade and leaf sheath was mostly found in vascular bundles, particularly around the phloem (Figure 2a, g). This pattern of expression was not affected by the extension of staining time or sucrose treatment to increase sensitivity of detection (Figure S1). According to the Rice XPro expression database, the expression of CRCT in the vascular tissues of root isolated by laser microdissection was greatly higher than that in other tissues of root (Figure S2). These findings confirm that the expression of CRCT is basically limited to vascular tissues.
In leaf blade, the expressions of OsGPT2 and OsBEI were also predominantly observed in the phloem (Figures 2b, e). The expressions of other genes were detected in both the mesophyll cell and vascular bundles, whereas the expressions of OsAGPS1 andOsPho1 in the vascular bundle were lower than those in the mesophyll cell (Figures 2c-e). The cell-specific expression of starch synthesis genes in the leaf sheath was largely similar to that in the leaf blades, although the leaf sheath contained storage parenchyma cells, where the accumulation of starch occurred (Figures 2g-l). In the leaf sheath, OsGPT2 was expressed in cells other than vascular bundle (Figure 2h). The expression of OsAGPL1 in the vascular bundle was lower than that in the mesophyll and storage parenchyma cells (Figure 2i). The expression of OsBEI was observed in the entire vascular bundle and not limited to the phloem (Figure 2k).
The expression levels of these starch synthesis-related genes were also analyzed in other organs (Figure S3, S4 and S5). Both CRCT andOsGPT2 were predominantly expressed in the vascular bundle of the culm (Figure S3). As observed with CRCT , the expression ofOsGPT2 and OsBEI were highly detected in the non-elongation internode (Figure S3). OsAGPL1, OsAGPS1 and OsPho1 were expressed in anther, although the expression levels were rather low judging from the staining intensities (Figure S5). Unexpectedly, the tissue- and cell- specific expression patterns ofCRCT was rather similar to those of OsGPT2 andOsBEI , despite there being no correlation between CRCTexpression level and OsGPT2 and OsBEI expression levels among rice cultivars.
The organ-specific expression of starch synthesis-related genes was also analyzed by qRT-PCR (Figure S6). Overall, the starch synthesis-related genes selected in this study were mostly expressed in the vegetative organs such as the leaf blade, leaf sheath and culm, though as an exception, OsBEI was also expressed in seed. Among the tested organs, CRCT expression was highest in the leaf blade. In contrast, the expression of all other starch synthesis-related genes was highest in the culm. In addition, the expression levels ofOsAGPL1 , OsAGPS1 and OsPho1 were higher in the leaf sheath than in the leaf blade. This result is inconsistent with our previous report showing a high level of CRCT expression in the leaf sheath (Morita et al., 2015). Previously, the expression ofCRCT in the leaf sheath was analyzed at the seedling stage, and it was higher than that in the leaf blade. Thus, it is thought thatCRCT expression in the leaf blade exceeds in the leaf sheath during plant maturation.
The expression of CRCT in the leaf blade and leaf sheath was further compared during diurnal changes in matured plants in the vegetative stage by qRT-PCR and western blot analyses (Figure 3). As reported previously (Morita et al., 2015), the transcript level ofCRCT in the leaf blade was higher during the light period than in the dark period as assessed by qRT-PCR. The transcript level ofCRCT in the leaf sheath was low at the beginning of the light period but then increased and peaked at the end of the light period. Throughout the day, the transcript level of CRCT in the leaf blade was higher than that in the leaf sheath. The protein level of CRCT, however, was barely detectable in the leaf blade (Figure 3b). In the leaf sheath, the CRCT protein was clearly detected and varied in a pattern similar to the transcript level. Thus, in contrast to the transcript level, the protein level of CRCT in the leaf sheath was always higher than that in the leaf blade.