Fig 2: Phenotypic changes in transgenic grapevine plants expressing the repressor NAC33-EAR and the expression level of VviNAC33 targets. (a) Whole-plant phenotype caused by the stable expression of VviNAC33-EAR in the three EARNAC33 lines compared to the vector control. (b) Fully expanded leaf phenotype caused by the stable expression of VviNAC33-EAR in the three EARNAC33 lines compared to the vector control. Total Chl and carotenoid (‘Car’) levels are indicated by the bars next to the pictures. Data are mean ± SD (n = 4). Asterisks indicate significant differences (*, P < 0.05; t-test) between the EARNAC33 lines and vector control. (c) Expression levels of SGR1, ATG8f, sucrase, PIN1, RopGEF1 and ATPC1 in the leaves of transgenic plants overexpressing VviNAC33 (OXNAC33) or expressing VviNAC33-EAR (EARNAC33), determined by quantitative polymerase chain reaction (qPCR). Each value corresponds to the mean ± SD of three technical replicates relative to the VviUBIQUITIN1 (VIT_16s0098g01190) control. Asterisks indicate significant differences (*, P < 0.05; t-test) between the OXNAC33 or EARNAC33 lines and the vector control. MNE, mean expression level.

Fig 3: Photosynthetic parameters and organization of thylakoid membranes. (a) Dependence of photosystem II operating efficiency (FPSII), (b) 1-qL (estimates the fraction of PSII centers with reduced first quinone acceptor (QA)), (c) electron transport rate (ETR) and (d) total proton motive force (ECS) on actinic light intensity for the OX2 line compared to the vector control. (e) Immunotitration of thylakoid proteins using specific antibodies against PSAA, LHCII and PSAA corrected for Chl content, and (f) ATPC1 corrected for total protein content. (g) Sucrose density gradient fractionation. The composition of the green bands is shown on the left, and the relative abundance of the band (normalized to total green content on the gradient) is shown on the right. All data are expressed as mean ± SD (n = 4). Asterisks (*) indicate significant differences (*, P < 0.05; t-test) between the OX2 line and the vector control.

Fig 4: Chloroplast RNA editing profile in the atpc1 mutant. (A) Sequencing chromatogram analysis shows that the RNA editing level in chloroplasts is affected in the atpc1 mutant (The four sites having =20% editing level changes are listed). The peak for C is in blue, the peak for T is in red, the peak for A is in green, and the peak for G is in black. The edited sites are highlighted by dark blocks, and the calculated C to T (equal C to U in RNA) editing levels are labeled. (B) The comparison of editing levels of 36 RNA editing sites in Arabidopsis chloroplasts between wild-type and atpc1 mutant plants. The x axis indicates the different RNA editing sites. The y axis represents the editing level of each site. Data are mean ± SEM from three biological replicates. Asterisks represent the significance level (* p < 0.05, ** p < 0.01, *** p < 0.001) compared with the wild type using a two-tailed Student’s t test.

Fig 5: ATPC1 interacts with MORF8, MORF9, ORRM1, and OZ1. The detailed legends are the same as those noted in Figure 5. (A) Y2H assays show the interaction of ATPC1 with MORF8, MORF9, ORRM1, and OZ1 in yeast. (B) ATPC1 interacts with MORF8, MORF9, ORRM1, and OZ1 in Arabidopsis mesophyll chloroplasts, as shown by BiFC assays (Scale bar, 5 µm). (C) LCI assays indicate the interaction of ATPC1 with MORF8, MORF9, ORRM1, and OZ1 in tobacco epidermal cells.