Combinatory actions of CP29 phosphorylation by STN7 and stability regulate leaf age-dependent disassembly of photosynthetic complexes

Abstract

A predominant physiological change that occurs during leaf senescence is a decrease in photosynthetic efficiency. An optimal organization of photosynthesis complexes in plant leaves is critical for efficient photosynthesis. However, molecular mechanisms for regulating photosynthesis complexes during leaf senescence remain largely unknown. Here we tracked photosynthesis complexes alterations during leaf senescence in Arabidopsis thaliana. Grana stack is significantly thickened and photosynthesis complexes were disassembled in senescing leaves. Defects in STN7 and CP29 led to an altered chloroplast ultrastructure and a malformation of photosynthesis complex organization in stroma lamella. Both CP29 phosphorylation by STN7 and CP29 fragmentation are highly associated with the photosynthesis complex disassembly. In turn, CP29 functions as a molecular glue to facilitate protein complex formation leading phosphorylation cascade and to maintain photosynthetic efficiency during leaf senescence. These data suggest a novel molecular mechanism to modulate leaf senescence via CP29 phosphorylation and fragmentation, serving as an efficient strategy to control photosynthesis complexes.

Figure 1

Grana stack thickness gradually increases with leaf age. (A) Chloroplast ultrastructure in aging Arabidopsis leaves. Representative Arabidopsis leaves were taken at each leaf age stage. Chloroplast images in red-dashed box and thylakoid membrane in yellow-dashed box were enlarged. White bars indicate 2 μm. (B) Quantitative measurement of the grana thickness from several hundreds of grana stacks. Leaves from DAE34 could not be determined owing to most of the chloroplast disruption. Data are mean ± SE from 3 biological replicates, and p-value was displayed after ordinary one-way ANOVA test. (C) Representative images of thylakoid membrane ultrastructure from WT, stn7, stn8, pph1-3, and cp29 mutants at DAE18 and DAE30. Scale bars indicate 500 nm. (D) Measurement of grana thickness in WT, stn7, stn8, pph1-3, and cp29 mutants at DAE18 and DAE30. Data are mean ± SE from 3 biological replicates, and p-value was displayed after two-way ANOVA test. Asterisks indicate *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

Defects in STN7 and CP29 showed accelerated leaf senescence phenotype. (A) Visual leaf senescence phenotypes and Fv’/Fm’ images of WT, stn7, stn8, pph1-3, and cp29. (B) Leaf age-dependent changes of chlorophyll a/b ratio. Data are mean ± SD from 8 leaves. (C) Measurement of the PSII maximum efficiency during leaf aging. (D) Nonphotochemical quenching during leaf aging. (C and D) Data are mean ± SE from 4 to 5 biological trials. (E) Age-associated gene expression during leaf senescence. Data are mean ± SE from 6 to7 biological trials. Asterisk indicates p-value after two-way ANOVA test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 3

STN7 dependent CP29 phosphorylation and CP29 degradation are highly associated with leaf-age disassembly of photosynthetic protein complex in stroma lamella. (A) Age-dependent degradation of photosynthetic protein complexes in the stroma lamella. This blue native gel is a full length gel image. (B) Photosynthesis-related protein abundance along with leaf age. (C) Quantification of their abundance from 3 biological trials. Data are mean ± SE, and p-value was displayed after two-way ANOVA test. Asterisks indicate **p < 0.01 and ****p < 0.0001. (D) Leaf age-dependent decrease of phosphorylation of Ser/Thr residues in key photosynthetic proteins. Blot images of (D) and (E) were from a full-gel image each. (E) Changes in CP29 protein phosphorylation and fragmentation in senescing leaves. Phosphorylated CP29 (CP29-P) bands are indicated with red-arrows.

Figure 4

CP29.1 and CP29.2 are sufficient to reconstitute photosynthetic protein complex. (A) Protein structure of three CP29 isoforms. Yellow boxes represent a low-complexity region and red lines indicate known phosphorylation sites. (B) CP29 dosage-dependent complementation of photosynthetic protein complexes. Thylakoid membrane which expresses strong (S), medium (M), and weak (W) protein levels of CP29 isoforms were loaded. These samples were run in three different gels with WT and cp29 controls. C) Working model for the molecular mechanism of STN7 and CP29 regulating photosynthetic protein complex organization during leaf senescence.