Functional redox links between lumen thiol oxidoreductase1 and serine/threonine-protein kinase STN7

Abstract

In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

Figure 1

Co-expression analysis based on the RNAseq database of LTO1. Co-expression data with RNAseq of LTO1 were obtained from the latest version (10.1) of the ATTED-II website (http://atted.jp/). STN7, LHCII kinase. STN8, PSII core protein kinase. TAP38, LHCII phosphatase. PBCP, PSII core phosphatase. CcdA and HCF164 operate in the trans-thylakoid thiol-reducing pathway. PsbO1/2, subunits of PSII complex. The PCC value of RNAseq database (Cor-r) is indicated at the top of each panel. LTO1 with itself has an obvious linear correlation (Cor-r = 1.00).

Figure 2

LTO1 directly interacts with STN7 in vitro and in vivo. A, Pull-down assay with LTO1 and STN7. The lumen domain of STN7 fused to MBP-His was incubated with immobilized empty GST and GST-tagged LTO1 lumenal-domain. After washing three times, bound proteins were eluted and fractionated by Tricine–PAGE for staining (CBB) or SDS–PAGE for immunoblot analysis with His antibody. B, Split ubiquitin-based yeast two-hybrid analysis. The full-length LTO1 was fused to the N and C termini of Cub-LexA-VP16 fragment in the pNCW and pCCW vectors as the prey, respectively. The same strategy was used for STN7 as the bait, fused with the NubG fragment of the pDSLNx and pDL2xN plasmids at the N and C termini. NubG, negative control; NubI, positive control. Plasmid construction and yeast two-hybrid analysis were performed as described in “Materials and methods” section. Left, yeast colonies on synthetic dropout medium without Trp-Leu-His (SD-Trp-Leu-His); Right, staining with X-Gal of the left plate. C, Protein overlay analysis of LTO1 protein interactions. Overlay of the reconstructed LTO1-LD with His tag and STN7-HA. Blotting of stn7 mutant or STN7-HAtransgenic Arabidopsis thylakoid proteins (20 μg per lane) was followed by immunodetection with anti-His and anti-HA antibody. Input, thylakoid proteins of stn7 mutant and STN7-HA transgenic Arabidopsis; Control, blotting membrane incubated with empty buffer. LTO1-His, blotting membrane incubated with LTO1-His-fused proteins. D, Co-immunoprecipitation analysis. Thylakoid proteins of STN7-HA transgenic Arabidopsis were immunoprecipitated with nonimmune serum (control) or antibodies against LTO1, then analyzed by immunoblotting. Input indicates that 5 mg (50%) and 10 mg (100%) protein was loaded on the gel. lto1, negative control for LTO1 antibody; cross-linked-IP, immunoprecipitation with the cross-linker DSP; Wash, third washing buffer; Elution, the elution buffer with bound proteins.

Figure 3

The thioredoxin-like domain of LTO1 catalyzes disulfide bond formation in the lumen domain of STN7. Recombinant proteins (lumen domain of STN7 and LTO1) were treated with AMS, separated by nonreducing SDS–PAGE and stained with CBB. The different states of LTO1 and STN7 are indicated on the right of the figure. AMS is an alkylating reagent and AMS treatment of exposed thiols in reduced LTO1 and STN7 results in an increased molecular mass of the alkylated molecules. The conserved cysteines of STN7 and LTO1 in the lumen domains can form dimers and/or oligomers under oxidizing conditions. A, DTT-dependent reduction of the disulfides in LTO1. Lane 1, recombinant LTO1-lumen domain in reducing buffer as the control. Lane 2, marker. Lanes 3–7, oxygen-oxidized LTO1 was reduced with increasing concentrations of DTT for 90 min then loaded with nonreducing buffer. LTO1 monomer is present in several shifted bands corresponding to fully oxidized (ox1), partially oxidized (ox2), partially reduced (red1), fully reduced (red2) forms under different incubation conditions. The dimer and oligomer can also be detected. B, DTT-dependent reduction of the disulfide in STN7. Lane 1, recombinant STN7-lumen domain with MBP tag in reducing buffer as the control. Lanes 2 and 8, marker. Lanes 3–7, O₂-oxidized STN7 was reduced with increasing concentrations of DTT for 90 min, then loaded with nonreducing buffer. The shifted and unshifted forms of the STN7 monomer cannot be resolved because the molecular weight of AMS (0.54 kDa) is very small compared to the fused protein (50 kDa). The dimer and oligomer of the fusion proteins can be detected under oxidizing conditions and gradually decrease with increasing concentration of DTT. C, LTO1-dependent oxidation of STN7 sulfhydryls. Lanes 1 and 10, the control in reducing buffer. Lanes 2 and 9, markers. Lanes 3 and 8, oxygen-oxidized LTO1 and STN7, respectively. Lanes 4 and 7, DTT-reduced LTO1 and STN7, respectively. Lanes 5 and 6, co-incubation of oxidized LTO1 and reduced STN7. The concentration (mg/mL) of loaded samples is shown above the figures.

Figure 4

Phosphorylation patterns in lto1 and stn7 mutants during STs. A, Thylakoid membrane proteins extracted from WT, lto1 and stn7 mutants in state 1 (ST1, far-red light) and state 2 (ST2, white-light) were separated by SDS–PAGE and immunoblotted with anti-phosphothreonine antiserum (top) and LHCII antibodies (medium). Equal protein loading was verified with CBB staining. B, Relative level of phosphorylated proteins in figure A. The amount of protein of the relevant bands was estimated by Image J software and analyzed with three biological repeats. The relative levels of the phosphorylated bands and LHCII bands in every lane were normalized with the amount of WT under ST2 condition. Bars indicate standard deviations. Asterisks indicate significant differences (**P <0.01 by Student’s t test) compared to WT.

Figure 5

Cysteines in the lumen domain of LTO1 affect STs. The four cysteines in the lumen domain of LTO1 were changed to alanine. These Cys variants which include double sites (named as C12 and C34) and four sites (named as C1234) mutants were introduced into the lto1 mutant. The phenotype of WT, stn7, lto1, LTO1 complemented line (LTO1/lto1) and Cys variants were examined by fluorescence imaging. A, B, STs measured with Method-2 in WT (A) and stn7 (B). D, dark-adaptation. B, blue-light. B+FR, blue and far-red light. Fo, minimum fluorescence after dark-adaptation; Fm, maximum fluorescence after dark-adaptation; Fm1, maximum fluorescence in state 1. Fm2, maximum fluorescence in state 2. a.u., arbitrary units. C, False color of ST fluorescence using method-2. D, State transition (qST) was estimated by (Fm1-Fm2)/Fm1. This value is positive in WT and close to zero or negative in stn7. The mean values obtained from three independent measurements of qST are shown. Bars indicate standard deviations. ns, not significant. Asterisks indicate significant differences (**P <0.01 by Student’s t test) compared to WT. E, Brightfield image. F, Fv/Fm was obtained from (Fm-Fo)/Fm. Three independent measurements were performed. Bars indicate standard deviations. ns, not significant. Asterisks indicate significant differences (**P <0.01 by Student’s t test) compared to WT.