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.