Pik1-H4 andPik2-H4 co-express in planta
The expression patterns of BDGs can be classified as co-regulated,
anti-regulated or with one direction exclusively regulated(Trinkleinet al. , 2004). Given the cooperative function ofPik1-H4 and Pik2-H4 , it is
logical to infer that the Pik-H4 gene pair is co-regulated. To
validate this hypothesis,
PPik1-H4 ::GUS /Nip,
PPik2-H4 ::GUS /Nip andRFP ::PPik-H4 ::GFP /Nip plants were
generated to examine tissue specificity and promoter activity ofPik-H4 . As shown in Figure 3A and Figure S3A,
PPik-H4 displayed activity in both directions in
roots, stems, leaf sheaths, leaves, spikelets, and seeds in both GUS
staining assay and fluorescent plants, exhibiting constitutive
expression patterns. Detecting the fluorescence intensity of RFP and GFP
and relative expression levels of Pik-H4 in Pik-H4Lijiangheituanxingu (LTH) near-isogenic lines (NILs, hereafter,Pik-H4 NILs) showed similar expression trends of Pik-H4(Figure 3B, 3D). In accordance with the tobacco filtration assay, the
activity of PPik1-H4 was stronger than
PPik2-H4 . Employing the Pearson’s correlation
test, the fluorescence intensity, representing the transcription levels
of Pik1-H4 and Pik2-H4 ,
demonstrated a significantly positive co-regulation in planta(Figure 3C) and across all tissues (Figure S3B-G). In addition, RFP and
GFP signals were particularly robust within vascular bundles in roots,
stems, leaf sheaths, and leaves, revealing high
PPik-H4 activity in vascular bundles compared to
mesophyll (Figure 3E). Zooming into the cellular level (Figure 3F), thePik-H4 expression patterns were also measured. Since the leaf
sheaths from rice seedlings are thin enough for laser confocal
microscopy, we used leaf sheaths for further research. The transcription
activity of PPik-H4 in both directions exhibited
a linear correlation in both mesophyll cells (Figure 3G) and vascular
cells (Figure 3H) in leaf sheaths. Importantly, the fluorescence
intensity of PPik-H4 was higher in vascular cells
(Figure 3I), aligning with the observed trends at the tissue level.