Significance Rubisco activase (Rca) regulates the activation state of Rubisco, the carboxylating enzyme of photosynthesis. Regulation of Rca by redox status of cysteine residues in species such as Arabidopsis is well recognized, but the role of recently identified phosphorylation of Rca at threonine-78 was uncertain. We now show a regulatory role of Arabidopsis Rca phosphorylation. Surprisingly, we also observed that the conservative substitution of serine for threonine-78 results in impaired functionality of Rca in vivo that is associated with retention of phosphorylation well into the light period and with reduced plant growth. This likely reflects in part the specificity of the requisite protein kinase(s) for serine versus threonine and may explain the absence of serine at this position in terrestrial plants.

Abstract Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine-78 (Thr78) in low light and in the dark, suggesting a potential regulatory role in photosynthesis, but this has not been directly tested. To do so, we transformed an rca-knockdown mutant largely lacking redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site–directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-β wild-type control, as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II (ϕ PSII ) and reduced growth relative to control plants expressing wild-type Rca-β under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-β with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ϕ PSII relative to Rca-β controls. While expression of the wild-type Rca-β or the T78A mutant fully rescued the slow-growth phenotype of the rca-knockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-β control plants at low light (30 µmol photons m−2 s−1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

Rubisco is the CO 2 -fixing enzyme of the reductive pentose phosphate pathway and can be one of the major limitations to the rate of leaf photosynthesis (1), which is an important component of crop productivity (2, 3). The activity of Rubisco is dependent on its dedicated AAA+ helper protein, Rubisco activase (Rca), which hydrolyzes ATP to induce a conformational change at Rubisco active sites to allow release of a variety of tightly bound inhibitory sugar phosphates prior to rapid activation of Rubisco by carbamylation (4⇓–6). In Arabidopsis (Arabidopsis thaliana), Rca is encoded by 1 gene that is alternatively spliced to generate 2 protein isoforms: the full-length α-isoform and the shorter β-isoform (7). The C-terminal extension of the α-isoform contains 2 redox-active cysteine residues that form a disulfide at low light and in the dark that down-regulates Rca activity, and as a result the Rubisco activation state is reduced (8). Phosphorylation of Rca at the Thr78 site also occurs under the same conditions that result in disulfide formation (e.g., at low light and in darkness) (9, 10) and thus has the potential to contribute to the light/dark regulation of Rca activity and thereby the Rubisco activation state. A previous study (10) concluded that Rca phosphorylation is not essential for its activity, but could not rule out the possibility of a regulatory role that is redundant with that produced by redox status.

Consequently, the overall objective of the present study was to evaluate the role of Rca phosphorylation at the Thr78 site when redox regulation is strongly reduced. To do this, we transformed a strong rca-knockdown mutant with cDNAs encoding wild-type Rca-β, which lacks the redox-active cysteine residues, or Rca-β with site-directed mutations (T78A and T78S) of the phosphosite. As expected, the T78A site–directed mutation prevented phosphorylation but, unexpectedly, the T78S–directed mutant was hyperphosphorylated relative to the wild-type Rca-β. Thus, we could compare plants without phosphorylation as well as plants with greater phosphorylation relative to wild-type Rca-β, and we examined the impact on photosynthetic parameters and plant growth under different light conditions. Collectively, the results obtained provide genetic evidence to establish a negative regulatory role for Rca phosphorylation at the Thr78 site.

Discussion The results of the present study identify a regulatory role for phosphorylation of Rca and implicate the Rca Thr78 phosphosite in the evolution of terrestrial plants. We tested the regulatory role of Rca phosphorylation by expressing wild-type or mutated versions of the Rca-β isoform, which lacks the C-terminal redox regulatory domain found in the α-isoform, in an Arabidopsis rca-knockdown mutant. Relative to transgenic plants expressing wild-type Rca-β with the phosphorylatable Thr78 site, results obtained with the phospho-null T78A-directed mutant and the hyperphosphorylated T78S-directed mutant strongly suggest that phosphorylation of Rca plays a negative regulatory role in vivo that inhibits photosynthesis and growth. The phospho-null T78A mutant exhibited more rapid induction kinetics of photosynthesis and higher ϕ PSII during low- to high-light transitions (Fig. 2A), whereas the hyperphosphorylated T78S mutant exhibited slower photosynthesis induction and lower ϕ PSII (Fig. 3B) compared with the wild-type control. In addition, changes in photosynthetic parameters noted for the site-directed mutants were associated with altered growth phenotypes compared with the transgenic plants expressing wild-type Rca-β. For example, T78A plants were similar to the control plants when grown in a typical square-wave light regime, but they grew faster than controls in a fluctuating low-light/high-light regime where more of the CO 2 assimilation occurred under inductive (non-steady-state) conditions. In addition, the T78A plants had a small but significant growth advantage at low light (30 µmol photons m−2 s−1), a condition that retains phosphorylation of wild-type Rca-β (10). Thus, in the absence of the Rca phosphorylation that occurs in wild-type Rca-β, the T78A mutant plants utilized light energy more efficiently and grew slightly faster (Fig. 2B). Conversely, the hyperphosphorylated T78S plants had reduced growth in both the square-wave light and fluctuating-light regimes (Fig. 4) consistent with the retention of Rca phosphorylation observed well into the light period (Fig. 3B). Reduced growth of the T78S-directed mutant would be expected given the reduced CO 2 assimilation rate (per unit leaf area; Fig. 3B) and reduced expression of numerous growth-related genes (Table 1). Collectively, these results support the notion that phosphorylation of Rca residue-78 negatively regulates photosynthesis, with its impact more pronounced with the T78S-directed mutant compared with the wild-type Rca-β. Our finding that the T78S-directed mutant was hyperphosphorylated into the light period was based on immunoblotting with sequence- and site-specific antibodies (which recognized the phosphorylated form of Thr78 and Ser78; Fig. 3B) and on quantitative mass spectrometry–based phosphoproteomic analysis (Fig. 4B); it has important implications that are worth noting. First, the result was unexpected because substitution of serine for a threonine residue is the most conservative substitution possible; however, the change can have surprising effects because, while the 2 residues are similar, they are often not equivalent. In our system, this result suggested that the protein kinase(s) and/or protein phosphatase(s) acting on Rca discriminated slightly between serine and threonine at the phosphosite of Rca. Indeed, it is generally recognized that while many Ser/Thr kinases prefer serine as the phosphoacceptor, most protein phosphatases prefer phosphothreonine (15, 34, 35). Although the protein phosphatases acting on phospho-Rca have not been identified, cpCK2 has been established as a major protein kinase that phosphorylates Rca at the Thr78 site (10). Plastid-localized cpCK2 is a member of the nearly ubiquitous CK2 kinase family, and previous work with mammalian and yeast CK2 kinases determined that Ser is strongly preferred over Thr as the phosphoacceptor residue (34, 36). Consistent with earlier studies with nonplant CK2 kinases, our results with cpCK2 and synthetic peptides indicated a similar preference for Ser over Thr at position 78 (SI Appendix, Fig. S2B and C). A major determinant of Ser vs. Thr phosphoacceptor specificity appears to reside in a specific residue of the kinase activation segment, termed “DFG + 1” (37). The residue corresponding to DFG + 1 in cpCK2 is a leucine, which is consistent with a preference for serine and may contribute to the increased phosphorylation of the T78S-directed mutant of Rca-β in vivo. However, it is likely that the specificity of protein phosphatase(s) also plays a role in the hyperphosphorylation of the T78S mutant, but these studies await identification of the requisite phosphatases acting on phospho-Rca in vivo. Second, these results also have relevance to our understanding of the variation in residues found in Rca at the position corresponding to residue 78 in the Arabidopsis protein. Overall, the Rca protein is highly conserved in terms of sequence, but there is a surprising amount of variation among terrestrial species at position 78 (SI Appendix, Fig. S3B). However, among the 59 terrestrial species for which the National Center for Biotechnology Information Reference Sequence Database Rca sequences are available, none have a serine at the position corresponding to Thr78 of Arabidopsis Rca (SI Appendix, Fig. S3B). We speculate that the lack of serine may be explained by natural selection against this residue because of its hyperphosphorylation and negative impact on photosynthesis. While present studies have established a regulatory role for phosphorylation of Thr78 in Rca-β in vivo, a number of important questions remain for future studies. First, it will be important to examine the role of Thr78 phosphorylation of the α-isoform of Rca, which has redox regulation capability, independently as well as in plants expressing both Rca isoforms. Previous studies with a cpck2− knockout mutant concluded that phosphorylation in wild-type plants has no regulatory role (10) but, because of the potential for additional protein kinases to phosphorylate Rca at the Thr78 site, this conclusion needs further examination with directed mutants lacking the phosphosite residue. Second, and as noted earlier, it will be important to identity the protein phosphatase(s) that dephosphorylate phospho-Thr78 and determine whether there is a preference for phospho-Thr over phospho-Ser. If so, then substrate preferences of both kinase(s) and phosphatase(s) may contribute to the hyperphosphorylation of the T78S-directed mutant. Third, and perhaps most important, future efforts need to determine the mechanism by which phosphorylation of Rca affects photosynthesis (Figs. 2 and 3) and plant growth (Fig. 5). The simplest explanation is that phosphorylation of Rca-β directly inhibits Rubisco activation. To test this possibility, we performed in vitro assays of Rubisco activation using phosphorylated or nonphosphorylated Rca proteins, and found no significant inhibition by phosphorylation (SI Appendix, Fig. S6B). The phosphorylation stoichiometries of the recombinant proteins used in the Rubisco activation experiments were roughly 20% for Rca-β and 45% for T78S (SI Appendix, Fig. S6A). The phosphorylation stoichiometries of Rca-β and T78S in vivo in darkened leaves were determined by quantitative phosphoproteomic analysis (Fig. 4) to be roughly 50% for Rca-β and 75% for T78S, which were only slightly higher than the phosphorylation stoichiometries tested in vitro. Rubisco contents were not changed in T78S in light conditions (SI Appendix, Fig. S7). Thus, we tentatively conclude that phosphorylation of Rca does not directly inhibit Rubisco activation activity but rather is more complex. For example, it may not be possible to mimic the phosphorylation impact in vitro if the phospho-Thr78 Rca recruits cofactors (e.g., phosphoprotein-binding proteins) to sequester phospho-Rca or inhibit Rubisco activation in vivo. Fourth, if phosphorylation of Rca is broadly important in vivo, then understanding how phosphorylation stoichiometry is controlled warrants examination; that is, why is Rca slowly phosphorylated in the dark but rapidly dephosphorylated in the light? A role for redox in the light/dark control of Rca phosphorylation was established (10) which was consistent with increased phosphorylation in the dark; however, how the redox signal was mediated was not elaborated. Moreover, once all of the protein kinases and protein phosphatases are identified, it will be possible to determine if the changes in stromal pH and [Mg2+] that accompany light/dark changes are additional factors that regulate Rca phosphorylation. Last but not least, it will be important to unravel the full complexity of the posttranslational modifications of Rca. Next to phosphorylation and redox regulation, lysine acetylation was recently discovered on Lys438 of Rca-β2 and was found to be under the control of a plastid lysine deacetylase in low-light conditions (38). Thus, the potential for control of Rca activity by phosphorylation, lysine acetylation, and redox is apparent, and it will be important to explore these interactions further in future studies.

Materials and Methods Detailed descriptions of all materials and methods are available in SI Appendix. Briefly, the Arabidopsis rca-knockdown mutant (Salk_003204C) containing a T-DNA insertion in the Rca gene promoter was transformed with cDNAs encoding wild-type Rca-β, Rca-β (T78A), or Rca-β (T78S). Plants were grown under both nonfluctuating and fluctuating light. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting were used to confirm Rca protein expression. Peptide phosphorylation and Rubisco activation assays were conducted in vitro. Liquid chromatography–tandem mass spectrometry was used to identify phosphorylated protein fractions. Leaf photosynthesis was measured using portable gas exchange systems equipped with fluorometers. RNA was isolated for microarray expression analyses, which were confirmed using qRT-PCR.

Acknowledgments We thank Dr. Rebecca Slattery for critical reading of the manuscript. Support was provided by the Agricultural Research Service, US Department of Agriculture (National Institute of Food and Agriculture–Agriculture and Food Research Initiative grant 58-5012-025 to M.H.S. and D.R.O.) and by the Deutsche Forschungsgemeinschaft (grants FI 1655/3-1 and INST 211/744-1 FUGG to J.G., I.L., and I.F.).

Footnotes Author contributions: S.Y.K., C.M.H., J.G., I.L., A.P.C., M.H.S., I.F., D.R.O., and S.C.H. designed research; S.Y.K., C.M.H., J.G., I.L., and A.P.C. performed research; S.Y.K., C.M.H., J.G., and I.L. analyzed data; and S.Y.K., C.M.H., V.S., A.P.C., M.H.S., I.F., D.R.O., and S.C.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The microarray data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE117263).

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