Fig 2: PetL overaccumulates in comparison to the cyt b6f core subunit PetB during acclimation to low temperature. Upper: Immunoblot analyses of PetL, PetB, and AtpB accumulation at the indicated time points after cold shift. One microgram of thylakoid proteins isolated from mature leaves of acclimating (8 or 14 days after shift to 12°C) and control plants (grown at 24°C to the same developmental stage) were used for immunoblot analyses. In addition, a dilution series is shown for the control plants on the left (dilutions as indicated on top of the blots). Lower: quantification of immunoblot signal intensities. Each blot was normalized to the average signal of all samples for all analyzed proteins (to eliminate technical deviations causing different signal intensities between independent protein membranes and antibodies). Note that this analysis is intended to enable the comparison of signal intensities for a given protein between different conditions but does not allow any comparative assessments for the accumulation of different proteins. Normalized signal intensities were averaged for results obtained from three thylakoid isolations derived from mature leaves of individual plants used as independent biological replicates (left ordinate, green and blue bars for 24°C and 12°C, respectively). The ratios of signal intensities between 12°C and 24°C were calculated for each developmental stage and protein (right ordinate, gray bars). The diagrams for plants that were cold-shifted for 8 and 14 days have identical y-axes scales. Error bars denote the standard deviation. Asterisks indicate statistically significant differences (P < 0.05, two-sided Student’s t test, Supplemental Data Set 1). Norm.: normalized; a.u.: arbitrary units.

Fig 3: Changes in the amount of large (RbcL) and small (RbcS) subunit of Rubisco, Rubisco activase (RCA), chalcone synthase (CHS) and phytochrome interacting factor 5 (PIF5) in leaves of Solanum lycopersicum L. cv. Malinowy Ozarowski grown under different LED illumination. ATPB is the loading control. A Coomassie staining (RbcL and RbcS) and immunoblotting (RCA, CHS, PIF5 and ATPB) were performed using extracts containing leaf proteins. To visualize RbcL, RbcS, RCA and ATPB, the aliquots containing 5 µg of total protein were used, whilst for PIF5 and CHS, the aliquots containing 60 µg of total protein were loaded to each lane in the gel. The quantification of the Coomassie-stained protein bands of B RbcL and D RbcS, as well as DAB-stained F RCA, C CHS and E PIF5, was carried out using ImageJ densitometric analysis. The relative amount of protein of interest was assessed based on the assumption that the densitometric value recorder for C plants is unity (100%). The values are means of four replicates ± SD. Different letters (a–d) indicate significant differences between treatments at p = 0.05 with a Tukey’s HSD test. AU arbitrary unit, MW molecular weight

Fig 4: Analyses of the NAD(P)H dehydrogenase (NDH) complex in otp970. (A) Analysis of the NDH complex and the major photosynthetic complexes. Thylakoid membrane proteins of wild-type (Col-0) and otp970 mutant plants were immunodetected with antisera raised against thylakoid protein. The antibodies against photosynthetic proteins were purchased from Agrisera. Total thylakoid protein samples containing equal amounts of chlorophyll were loaded in each lane. NdhB, a subunit of NDH; Cytf, a subunit of the Cytb6f complex; PsaB, a subunit of PSI; PsbO, a subunit of PSII; ATPB, a subunit of the ATP synthase complex. (B) BN-PAGE analysis of thylakoid proteins in Col-0, otp970, and crr2-2. Thylakoid membrane proteins in 10 µg of chlorophyll were loaded in each lane and were stained with Coomassie brilliant blue (CBB). NDH-PSI super complex (band I) is indicated with an arrow. PSI-NDH supercomplex-less mutant (crr2-2) was used for contrast. (C) NDH activity analysis by chlorophyll fluorescence measurements. Leaves were exposed to actinic light (AL) (50 µmol photons m-2 s-1) for 5 min, after which AL was turned off, and changes in the chlorophyll fluorescence levels were detected. Platquinone reduction based on NDH activity determined the transient increase in chlorophyll fluorescence. Insets are magnified traces from the boxed area. Fluorescence levels were normalized relative to the maximum fluorescence (Fm) at closed PSII centers in the dark. ML, measuring light; SP, a saturating pulse of white light.

Fig 5: Western blot analysis of selected candidate proteins (AtpB, 2Fe-2S, GLN2, Psb R, PRK, ALD) with their corresponding molecular weight in rice leaves during As stress under various S regimes (A); RuBisCo large subunit a band in coomassie blue staining (CBB) SDS gel served as a loading control (B), which used for normalization of detected proteins in densitometry studies (C) fold change indicated with respect of control samples.