FIGURE 9.

HbNCED5 localization in chloroplasts is altered by EqCSEP01276 expression in Nicotiana benthamiana. (A). Co-expression of EqCSEP01276^ΔSP-GFP and HbNCED5-RFP in leaves of N. benthamiana were examined by a fluorescence microscope. The other sets were used as controls. Each image of GFP, RFP, or chlorophyll channel was photographed by using the Z-stack tool of confocal fluorescence microscopy to scan one leaf area containing epidermal and mesophyll cells. White arrows indicate the chloroplast-localized EqCSEP01276^ΔSP. Bar = 10 μm. (B) HbNCED5-RFP puncta per 0.5 mm² area were analyzed with ImageJ software. Three independent replicates with three areas per replicate were examined. Individual values (n = 9) are indicated by dots. Mean values are indicated by “×.” Median values are indicated by the middle line. Asterisks indicate significant differences (P < 0.01). (C) Western blot analysis of total and chloroplast proteins extracted from N. benthamiana leaves expressing proteins of interest. The expression of EqCSEP01276^ΔSP-GFP reduced HbNCED5-RFP abundance in chloroplasts. Asterisk indicates total protein used as positive control in analysis with anti-β-actin antibody.