Figure 4.

SPY regulates the accumulation of CPN20 in guard cells and mesophyll cell chloroplasts. (A) mRNA level of CPN20 in different transgenic materials. CPN20-GFP/Col-0 or CPN20-GFP/spy-3 represents the CPN20-overexpressing line in the background of Col-0 or and spy-3. (B) Purity of chloroplasts confirmed by cytosolic marker protein HSP70 on western blot. Total proteins and chloroplast proteins were isolated from leaves of indicated materials before being subjected to SDS PAGE and western blotting using α-HSP70. Coomassie blue staining of RbcL was used as a loading control. (C) CPN20 is more abundant in chloroplasts from spy-3. GFP-tagged CPN20 was detected by α-GFP while coomassie blue staining of RbcL was used as a loading control. GFP fluorescence of CPN20-GFP in chloroplasts of stomatal guard cells in CPN20-GFP/Col-0 and CPN20-GFP/spy-3. Leaves of 40-day-old plants were observed under a confocal microscope using identical parameters. Auto, GFP, and Merge indicate chlorophyll autofluorescence, the fluorescence of CPN20-GFP, and merged image of Auto and GFP. (D) Total CPN20 abundance is largely unchanged in CPN20-GFP/spy-3 compared with CPN20-GFP/Col-0. CPN20-GFP fusion proteins in extracts from indicated genotypes were detected by western blotting using α-GFP or α-HSP70. Coomassie blue staining of RbcL was used as a loading control. (E) GFP fluorescence of CPN20-GFP in chloroplasts of stomatal guard cells in CPN20-GFP/Col-0 and CPN20-GFP/spy-3. Leaves of 40-day-old plants were observed under a confocal microscope using identical parameters. Auto, GFP, and Merge indicate chlorophyll autofluorescence, the fluorescence of CPN20-GFP, and merged image of Auto and GFP.