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.