In Vivo Evidence for a Regulatory Role of Phosphorylation of Arabidopsis Rubisco Activase at the Thr78 Site

In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site

Sang Yeol Kima,b, Christopher M. Harveya,b, Jonas Giesec, Ines Lassowskatc, Vijayata Singhb,d, Amanda P. Cavanaghb,e, Martin H. Spaldingf, Iris Finkemeierc, Donald R. Ortb,e,1, and Steven C. Hubera,b

aAgricultural Research Service, US Department of Agriculture, Urbana, IL 61801; bDepartment of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; cPlant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster 48143, Muenster, Germany; dDepartment of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523; eCarl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and fDepartment of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011

Edited by Bob B. Buchanan, University of California, Berkeley, CA, and approved August 1, 2019 (received for review July 27, 2018) Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine- Consequently, the overall objective of the present study was to 78 (Thr78) in low light and in the dark, suggesting a potential reg- evaluate the role of Rca phosphorylation at the Thr78 site when ulatory role in photosynthesis, but this has not been directly tested. redox regulation is strongly reduced. To do this, we transformed To do so, we transformed an rca-knockdown mutant largely lacking a strong rca-knockdown mutant with cDNAs encoding wild-type redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala Rca-β, which lacks the redox-active cysteine residues, or Rca-β (T78A) or Thr78-to-Ser (T78S) site–directed mutations. Interestingly, with site-directed mutations (T78A and T78S) of the phospho- the T78S mutant was hyperphosphorylated at the Ser78 site relative site. As expected, the T78A site–directed mutation prevented to Thr78 of the Rca-β wild-type control, as evidenced by immuno- phosphorylation but, unexpectedly, the T78S–directed mutant blotting with custom antibodies and quantitative mass spectrome- was hyperphosphorylated relative to the wild-type Rca-β. Thus, try. Moreover, plants expressing the T78S mutation had reduced we could compare plants without phosphorylation as well as photosynthesis and quantum efficiency of photosystem II (ϕPSII) plants with greater phosphorylation relative to wild-type Rca-β,

and reduced growth relative to control plants expressing wild- and we examined the impact on photosynthetic parameters and PLANT BIOLOGY type Rca-β under all conditions tested. Gene expression was also plant growth under different light conditions. Collectively, the altered in a manner consistent with reduced growth. In contrast, results obtained provide genetic evidence to establish a negative plants expressing Rca-β with the phospho-null T78A mutation had regulatory role for Rca phosphorylation at the Thr78 site. faster photosynthetic induction kinetics and increased ϕPSII relative to Rca-β controls. While expression of the wild-type Rca-β or the Results T78A mutant fully rescued the slow-growth phenotype of the rca- Transgenic Expression of Rca-β Rescued the Slow-Growth Phenotype of knockdown mutant grown in a square-wave light regime, the T78A an rca-Knockdown Mutant. We obtained a Salk line (SALK_003204c) mutants grew faster than the Rca-β control plants at low light with a T-DNA insertion in the promoter of the Rca gene (At2g39730) (30 μmol photons m−2 s−1) and in a fluctuating low-light/high-light that produces a strong knockdown of both the α-andβ-isoforms of environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory Significance role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species. Rubisco activase (Rca) regulates the activation state of Rubisco, the carboxylating enzyme of photosynthesis. Regulation of Rca Rubisco activase | photosynthesis | phosphorylation by redox status of cysteine residues in species such as Arabi- dopsis is well recognized, but the role of recently identified ubisco is the CO2-fixing enzyme of the reductive pentose phosphorylation of Rca at threonine-78 was uncertain. We now Rphosphate pathway and can be one of the major limitations show a regulatory role of Arabidopsis Rca phosphorylation. to the rate of leaf photosynthesis (1), which is an important Surprisingly, we also observed that the conservative substitution component of crop productivity (2, 3). The activity of Rubisco is of serine for threonine-78 results in impaired functionality of Rca + dependent on its dedicated AAA helper protein, Rubisco activase in vivo that is associated with retention of phosphorylation well (Rca), which hydrolyzes ATP to induce a conformational change into the light period and with reduced plant growth. This likely at Rubisco active sites to allow release of a variety of tightly bound reflects in part the specificity of the requisite protein kinase(s) inhibitory sugar phosphates prior to rapid activation of Rubisco by for serine versus threonine and may explain the absence of carbamylation (4–6). In Arabidopsis (Arabidopsis thaliana), Rca is serine at this position in terrestrial plants. encoded by 1 gene that is alternatively spliced to generate 2 pro- 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. de- tein isoforms: the full-length α-isoform and the shorter β-isoform signed research; S.Y.K., C.M.H., J.G., I.L., and A.P.C. performed research; S.Y.K., C.M.H., (7). The C-terminal extension of the α-isoform contains 2 redox- 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. active cysteine residues that form a disulfide at low light and in the wrote the paper. dark that down-regulates Rca activity, and as a result the Rubisco The authors declare no conflict of interest. activation state is reduced (8). Phosphorylation of Rca at the This article is a PNAS Direct Submission. Thr78 site also occurs under the same conditions that result in Published under the PNAS license. disulfide formation (e.g., at low light and in darkness) (9, 10) and 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 thus has the potential to contribute to the light/dark regulation of no. GSE117263). Rca activity and thereby the Rubisco activation state. A previous 1To whom correspondence may be addressed. Email: [email protected]. study (10) concluded that Rca phosphorylation is not essential for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. its activity, but could not rule out the possibility of a regulatory role 1073/pnas.1812916116/-/DCSupplemental. that is redundant with that produced by redox status. Published online August 26, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1812916116 PNAS | September 10, 2019 | vol. 116 | no. 37 | 18723–18731 Downloaded by guest on October 2, 2021 Rca, which severely reduces plant growth (Fig. 1A). However, the Phosphorylation of Rca at the Thr78 site occurs in the dark and at plants survive and grow slowly at ambient [CO2], unlike the full low light (10), and thus the results in Fig. 2A suggest that the − rca knockout that requires high [CO2] (11). We transformed the phosphorylation of Rca that occurred in the transgenic plants Salk rca-knockdown lines with a cDNA encoding Rca-β that re- expressing wild-type Rca-β was sufficient to negatively impact pho- stored normal growth in T1 plants (Fig. 1B). Likewise, trans- tosynthetic induction profiles. In steady-state light, Rca is required formation of the rca-knockdown line with the phospho-null T78A- to sustain Rubisco activation, but wild-type Rca activity is not lim- directed mutant also restored growth and, as expected, only the iting, which is consistent with dephosphorylation of the protein after residual wild-type Rca-β protein in the knockdown mutant was 15–30 min of exposure to high light. phosphorylated at the Thr78 site when plants were darkened. Because phosphorylation of Rca was retained at low light, we Dark-harvested Col-0 and rca-knockdown plants were used as speculated that the transgenic plants expressing wild-type Rca-β controls (Fig. 1C). Rca-β and T78A homozygous plants were or the phospho-null T78A mutation would differ in growth at low established. These transgenic plants provided a convenient ex- photosynthetic photon flux density (PPFD). As expected, plant perimental system to determine whether phosphorylation at the growth (monitored as shoot fresh weight) decreased progressively − − Thr78 site has a regulatory role in vivo when redox regulation of as light decreased from 150 to 30 μmol photons m 2 s 1 in both Rca activity is strongly reduced. Several independent lines of each genotypes, but a different shoot mass between genotypes was transformation event were selected (See SI Appendix,Fig.S1;2of observed only for plants grown at the lowest light tested, with each are shown) and used for further characterization in the lower mass in the wild-type Rca-β plants compared with the present study. phospho-null T78A plants (significance: 1-tailed, unpaired t test, P value < 0.001; Fig. 2B). The difference in growth between the − − Prevention of Phosphorylation of Rca-β Enhanced Photosynthetic 2 genotypes at 30 μmol photons m 2 s 1 was also documented by Induction and Growth at Low Light. Rca is one of the major de- continuous monitoring of the increase in total rosette leaf area as terminants of the rate of photosynthetic induction during low- to a function of time (Fig. 2C). high-light transition (12, 13). Accordingly, we monitored induction kinetics in the transgenic plants with and without the Substitution of Ser for Thr78 in Rca-β Resulted in Hyperphosphorylation Thr78 phosphosite. As shown in Fig. 2A, plants expressing the in the Light and Reduced Photosynthesis. While the functional sig- phospho-null Rca (T78A) mutation had higher rates of net CO2 nificance of a phosphosite is usually tested by generating a assimilation following transition from low to high light compared phospho-null (e.g., the T78A mutant) or by substituting an acidic with plants expressing the wild-type Rca-β. Photosystem II op- residue that sometimes acts as a phosphomimetic, another ap- erating efficiency (ϕPSII) was correspondingly higher in the T78A proach is to substitute a serine for a threonine (or vice versa). The plants following the transition, which is consistent with higher Thr-to-Ser approach is a more subtle modification that has the rates of CO2 assimilation. In fully light-adapted plants, the potential to impact phosphosite function because the 2 residues maximum rate of carboxylation, Vcmax, and the maximum rate of are similar but often not equivalent at the molecular level (14–16). electron transport, Jmax, were not significantly different in the Accordingly, we generated transgenic plants expressing the T78S transgenic lines (Fig. 2A, Inset). This suggests that all lines site–directed mutant in the rca-knockdown mutant background reached similar final levels of Rubisco activation and is consistent (SI Appendix,Fig.S1). Fortunately, the custom phospho-specific with the Rca function limiting Rubisco activation to a greater ex- antibodies that recognize phospho-Thr78 also recognized the tent during inductive, compared with steady-state, photosynthesis. cpCK2-mediated phosphorylated form of the T78S site–directed

A SALK_003204C B TAA

ATG Col-0 rca_Salk

IB: an- Rca-α Rca ab Rca-β

C Col-0 rca_Salk Col-0 Rca-β-5 T78A-3 IB: an- Rca-α IB: an- Rca-α Rca ab Rca-β Rca ab Rca-β

CBB RbcL * IB: an- Rca-α pT78 ab Rca-β

Fig. 1. Wild-type Rca-β and the T78A site–directed mutant rescue the slow-growth phenotype of the strong rca-knockdown mutant (rca_SALK). (A, Top) − Gene map showing the position of the T-DNA insertion in the promoter of the Rca gene (At2g39730). (Middle) Comparison of the rca mutant and Col-0 plants grown in a 16-h photoperiod for 24 d. (Bottom) Immunoblot showing the large reduction in Rca isoforms in the knockdown mutant and the Coomassie Brilliant Blue (CBB)–stained blot showing the Rubisco large subunit as a loading control. (B, Top) T1 plants of Rca-β or T78A rescue the rca- knockdown dwarf phenotype. Plants were photographed 21 d after germination and were grown in a long-day photoperiod. (Bottom) Immunoblot showing increased levels of Rca-β in T1 transgenic plants. (C) Transgenic homozygous plants expressing Rca-β or the T78A site–directed mutation were harvested in the dark; phosphorylation of Rca-β at the Thr78 site, but not the phospho-null T78A mutant, was confirmed using sequence- and phosphorylation-specific (anti- pT78) antibodies. Dark-harvested Col-0 and rca_SALK were used as controls. The asterisk identifies an off-target protein that is recognized by anti- pT78 antibodies (10).

18724 | www.pnas.org/cgi/doi/10.1073/pnas.1812916116 Kim et al. Downloaded by guest on October 2, 2021 A 20 μmol 400 μmol m-2 s-1 20 μmol 400 μmol m-2 s-1

Rca-β T78A

Vcmax 28.8 ± 3.5 25.9 ± 0.8

Jmax 61.1 ± 7.7 66.5 ± 2.7

Rd 1.5 ± 0.3 1.3 ± 0.2

PAR: 30 (μmol m-2 s-1) BCPAR: 150 (μmol m-2 s-1) PAR: 80 (μmol m-2 s-1) PAR: 30 (μmol m-2 s-1) Rcaβ-5 & -6 T78A-3 & -4

2.5 30 ) 2 2 25 20 1.5 15 1 10 PLANT BIOLOGY Leaf area (cm Fresh weight (g) Fresh weight 0.5 5 0 0 PAR 150 PAR 80 PAR30 0 4 8 121620242832 Days RCAβ-5 & -6 T78A-3 & -4

Fig. 2. Plants expressing T78A have faster low- to high-light induction kinetics of photosynthesis and increased growth at low light compared with plants

expressing wild-type Rca-β.(A)CO2 assimilation (Left) and PSII quantum efficiency (Right) during photosynthetic induction (n ≥ 11; error bars represent ±SE). Measurements were performed at 22 °C on leaves from mature rosettes prior to bolting. Leaves were dark-adapted for at least 30 min prior to the start of each experiment, which consisted of 20 min at low light followed by 30 min at high light. Horizontal bars above the chart indicate experimental light levels.

Plotted points are the average of at least 11 biological replicates, ± SE. (Inset) Parameters estimated from CO2 response curves (n = 3, ±1 SE) conducted during steady-state photosynthesis. (B) Growth of Rca-β and T78A plants as a function of light intensity. The difference between Rca-β and T78A plants at PAR 30 was statistically significant based on a 1-tailed, unpaired t test (P value < 0.001). All plants were 4 wk old at harvest for shoot fresh-weight determination. (C) Leaf area accumulation of plants growing at 30 μmol photons m−2 s−1. Ten plants were used to generate values of average and SE for B and C.

mutant (SI Appendix,Fig.S2A). Indeed, recombinant cpCK2 peptide containing residue 78 and the corresponding phosphopep- phosphorylated a synthetic peptide containing the T78S substitu- tide, we could estimate site-specific phosphorylation stoichiometries. tion more rapidly than the corresponding T78 peptide (SI Ap- In plants expressing wild-type Rca-β, phosphorylation stoichiometry pendix, Fig. S2 B and C). was ∼51% in the dark and reduced to near zero levels after 1 h of It is interesting that, while phospho-Thr78 in transgenic plants light (Fig. 4A). The transgenic plants expressing the T78S-directed expressing wild-type Rca-β was completely dephosphorylated 1 h mutant also contained some residual wild-type Rca protein isoforms after the transfer of plants from dark to light (125 μmol photons (α, β1, and β2), and thus phosphorylation at Thr78 could also be − − m 2 s 1 in these experiments), phosphorylation at the Ser78 site monitored in these plants (as noted previously in relation to im- in the T78S-directed mutant was removed much more slowly and munoblot analysis in Fig. 3). A similar phosphorylation stoichiom- persisted for several hours into the light period (Fig. 3A). In the etry at the Thr78 site in the rca-knockdown background of 50% was dark samples, phosphorylation of Thr78 in Rca-α, which is pre- observed, but again the phosphosite was completely dephosphory- sent at low levels in the rca-knockdown mutant, was also readily lated after exposure to 1 h of light (Fig. 4A). In the T78S mutant, apparent in the immunoblots, and that signal was completely phosphorylation at the engineered Ser78 site was confirmed with removed within 1 h of illumination (Fig. 3A), as expected (10). high confidence by mass spectrometric analysis (Fig. 4 C–E); the Thus, phospho-Thr78 was rapidly dephosphorylated in marked phosphorylation stoichiometry was high in the dark (∼76%) and contrast to phospho-Ser78 in the transgenic plants expressing the reduced in the light but only to ∼52% after 1 h (Fig. 4B). T78S mutant protein. We then monitored photosynthetic induction kinetics follow- These results were confirmed and extended by quantitative ing transition of whole rosettes from low to high light. As shown phosphoproteomic analysis of transgenic plants expressing Rca-β in Fig. 3B, plants expressing T78S had substantially slower or the T78S mutation, specifically targeting Rca-β peptides photosynthetic rates relative to plants expressing wild-type Rca-β (identified in Dataset S1) and, in particular, the phosphosite at in the rca-knockdown background. As expected, there was a residue 78. Leaves of plants were harvested after 8 h in the dark corresponding reduction in ϕPSII in plants expressing T78S that −2 −1 or after subsequent exposure to light (125 μmol photons m s ) was consistent with differences in CO2 assimilation (Fig. 3 B, for 1 h. By quantifying the amount of unphosphorylated tryptic Right). ACi curves performed on fully light-adapted T78S plants

Kim et al. PNAS | September 10, 2019 | vol. 116 | no. 37 | 18725 Downloaded by guest on October 2, 2021 Rca-β-6 T78S-7 A D8 L1 L2 L4 (Hour) D8 L1 L2 L3 L4 L6 L8 (Hour)

IB: an- Rca-α IB: an- Rca-α Rca ab Rca-β RCA ab Rca-β

Oﬀ target Oﬀ target IB: an- IB: an- Rca-α Rca-α pT78 ab pT78 ab Rca-β Rca-β

80 60

60 40 40 20

20 (pT78/ Rca) (pT78/ Rca) Densitometry Densitometry 0 0 D8 L1 L2 L4 D8 L1 L2 L3 L4 L6 L8

B 20 PAR 400 PAR 20 PAR 400 PAR

T78S Vcmax 24.6 ± 1.4 Jmax 61.6 ± 2.4 Rd 0.9 ± 0.1

Fig. 3. Plants expressing T78S retain phosphorylation into the light period and have impaired photosynthesis relative to plants expressing wild-type Rca-β. (A) Two-week-old seedlings were harvested after 8 h of darkness (D8) or after transfer to light for 1–8 h (L1–L8), as indicated, prior to harvest for extraction of protein and immunoblot assay. Anti-pT78 antibodies were used to detect phosphorylation of both Rca-β-T78 (Left) and T78S (Right); the histogram shows

densitometry of the immunoblots where the pT78 (or pS78) phospho-signal associated with Rca-β was normalized to the Rca-β protein signal. (B)CO2 as- − − similation (Left) and PSII quantum efficiency (Right) during photosynthetic induction (n ≥ 5, ±SE). Plants were grown in Cone-tainers at 140 μmol photons m 2 s 1 for ∼3 wk. Induction curves were obtained with a whole-rosette chamber (6400-40 Leaf Chamber Fluorometer, LI-COR) and the RGB light source. Fluorescence

was measured on a separate day with the Technologica Fluorimager to assess changes in quantum efficiency of ϕPSII. Horizontal bars above the chart indicate experimental light levels. (Inset) parameters estimated from CO2 response curves (n = 6, ±SE) conducted during steady-state photosynthesis.

−2 −1 yielded Vcmax = 24.6 ± 1.4 μmol m s and Jmax = 61.6 ± 2.4 μmol with the fact that maximum Rca activity is not limiting in vivo − − m 2 s 1, neither of which was significantly different from the values during steady-state photosynthesis, suggests that the reduced for the wild-type Rca-β.TheACi measurements were performed on growth and reduced photosynthetic activity of T78S plants cannot different days than the low- to high-light induction profiles and were be attributed to the intrinsic activity of the fully unphosphorylated also performed from 3 to 8 h into the photoperiod, at which time T78S mutant protein. It is also worth noting that, while there is phosphorylation of T78S at the engineered S78 phosphosite was considerable variation among terrestrial plants in the amino acid likely to have been substantially reduced (Fig. 3B). As was the case found at position 78 in Rca, Ser is not known in nature; the most for T78A, this was consistent with the notion that Rca functions common residue is Thr or a hydrophobic residue (Ile, Met, Val, or primarily during inductive photosynthesis but is also essential for Phe; See SI Appendix,Fig.S3B and Dataset S2). maintaining Rubisco activation under steady-state conditions. One explanation for the reduced growth and photosynthetic Rca-β Phosphorylation Reduced Plant Growth in a Rapidly Fluctuating performance of transgenic plants expressing the T78S-directed Light Environment. If phosphorylation of Rca reduces the rate mutant could be that the conservative substitution of Ser for Thr at which photosynthesis increases following a low- to high-light directly affects the ability of the protein variant to activate Rubisco transition, then growing plants with wild-type Rca in a fluctuating even in the unphosphorylated state. To test this possibility, we light regime should negatively impact plant growth. To test this prepared wild-type Rca-β and the T78A- and T78S-directed mutants notion, we monitored the increase in rosette leaf area as a function β as His6-tagged recombinant proteins for use in Rubisco activation of time for transgenic plants differing in phosphorylation of Rca- assays in vitro. While both the T78A- and T78S-directed mutants at the Thr78 site and growing in fluctuating-light or a square-wave were less active in vitro compared with wild-type Rca-β, there was light regime. In the square-wave regime, transgenic plants not a statistically significant difference between the T78A and expressing wild-type Rca-β and the phospho-null T78A-directed T78S proteins (SI Appendix,Fig.S3A). This observation, coupled mutant grew similarly and had higher total leaf areas compared

18726 | www.pnas.org/cgi/doi/10.1073/pnas.1812916116 Kim et al. Downloaded by guest on October 2, 2021 PLANT BIOLOGY

Fig. 4. Quantitative phosphopeptide analysis of the phosphosite at position 78 in the wild-type Rca-β and the T78S-directed mutant. (A) The wild-type form of Rca-β was completely dephosphorylated at position T78 in Rca-β–complemented and T78S-complemented knockdown plants. Significance: ***P < 0.001. (B)

The engineered T78S was not significantly dephosphorylated at position 78 in the light. Average log2 intensities of phosphopeptide and unmodified peptide were calculated (n = 3, Rca-β2_dark n = 2). A Student’s t test was applied for light–dark comparison. Phosphosite occupancy on the Rca protein is stated above the corresponding peptide and sample. (C and D) Fragmentation spectra for GLAYD(p)TSDDQQDITP and GLAYD(p)STSDDQQDITP, respectively. The b (in blue) and y (in red) represent ion series of the fragmented peptides (E) Scores of phosphorylated Rca-β2 peptides.

with the hyperphosphorylated T78S mutant (Fig. 5A). Inflores- terns as leaf area, indicating that the differing growth was not due cence induction occurred at a similar rosette size in all lines, de- to differences in specific leaf area (Fig. 5C). Under square-wave spite being slightly delayed in T78S. In contrast, when plants were light, the dry weights of T78A and wild-type Rca-β were identical, grown in the fluctuating-light regime, the transgenic plants express- while T78A plants were ∼30% heavier than the wild-type Rca-β ing the phospho-null T78A-directed mutant had the highest total plants under fluctuating light. This difference was statistically sig- leaf area, followed by wild-type Rca-β and the T78S mutant (Fig. nificant at a 10% confidence interval based on a 1-tailed, unpaired 5B). Rosette dry weights at terminal harvest showed similar pat- t test (P value = 0.099).

Kim et al. PNAS | September 10, 2019 | vol. 116 | no. 37 | 18727 Downloaded by guest on October 2, 2021 AB

Fig. 5. Growth curves of wild-type Rca-β, T78A, and T78S plants under square-wave light or fluctuating-light regimes. (A) Accumulation of leaf area in the − − square-wave light regime (n ≥ 16, ±SE). Light was constant at 125 μmol photons m 2 s 1 for the entire 16-h light period. Arrows indicate the average plant age at inflorescence initiation. (B) Accumulation of leaf area in the fluctuating light regime consisting of low light (25 μmol m−2 s−1 PPFD) and high light (125 μmol m−2 s−1 PPFD; 10 min each) for the entire 16-h photoperiod (n ≥ 11, ±SE). Data in A and B were normalized (Rca-β mean = 1 at 38 d after planting) to account for variation in average growth rate between experiments. (C) Rosette dry weight comparisons 40 d after sowing (n ≥ 4, ±SE). Significant differences (t test, P ≤ 0.1) in fluctuating light are indicated by different uppercase letters.

Altered Gene Expression in the Rca-β T78S-Directed Mutant Was branched chain acyltransferase (BCE2): 1.71-fold], DIN4 (AT3G13450; Consistent with Reduced Photosynthetic Activity and Slow Growth. branched chain α-keto acid dehydrogenase E1 β: 1.59-fold), DIN9 In order to explore the underlying causes of reduced growth in [AT67070; phosphomannose isomerase 2 (PMA2): 1.89-fold], and plants expressing the T78S-directed mutation, we performed DIN10 [AT5G20250; raffinose synthase 6 (RS6): 1.40-fold]. While microarray analysis comparing transgenic seedlings expressing wild- DIN gene transcripts accumulate in darkness, their levels in type Rca-β or the T78S-directed mutation (all in the rca-knockdown darkened leaves can be repressed by sucrose feeding, suggesting a mutant background). After the results were sorted to include only role for cellular sugar levels. Many of the DIN genes are also in- samples with expression values greater than 6.5 and significant P duced in senescing leaves (19, 20), as are several other genes that values, false discovery rate (FDR) correction was applied, which were up-regulated in the T78S plants, including a senescence- yielded 752 genes up-regulated in T78S compared with wild-type associated gene (SAG13) (21), a nitrilase gene (NIT4) (22), and Rca-β, with 80 of those genes up-regulated more than 2.5-fold. A an alternative oxidase 3 gene (AOX1D) (23) (Table 1). It is im- total of 460 genes were down-regulated, and 43 of those were down- portant to note, however, that the T78S plants did not show signs regulated with fold changes less than 0.5. To confirm the changes in of senescence and, in fact, did not flower or mature earlier than gene expression identified by microarray analysis, we quantified the the control plants expressing wild-type Rca-β (SI Appendix, Fig. expression level of several up- and down-regulated genes using S1). Because the DIN genes are considered to be low-energy status qRT-PCR (SI Appendix,Fig.S4A). Although we only examined a markers, their up-regulation in T78S plants is consistent with re- total of 8 transcripts from the list of differentially expressed genes, duced photosynthetic activity in these plants (Fig. 5A). there was a positive correlation (R2 = 0.71) for the fold changes As noted previously, a number of genes were down-regulated obtained by the 2 methods and the linear regression line passed in the T78S plants, and many of those genes are associated with through the origin (SI Appendix,Fig.S4B). These results demon- growth. For example, cell growth involves several different cell strate the overall reliability of the microarray results. wall–loosening proteins that facilitate movement of cell wall A selected list of genes that were more than 2-fold up- or polymers to accommodate water uptake and cell expansion, in- down-regulated is presented in Table 1. Interestingly, a number cluding glycosyl hydrolases, α-expansins, and various small cell of the up-regulated genes are DARK-INDUCED (DIN) genes wall–binding proteins (24, 25). Thus, it is noteworthy that 2 glycosyl (17, 18) and include DIN2 (β-glucosidase; AT3G60140), DIN6 hydrolases, GUN6 and GUN11, were down-regulated in the T78S (asparagine synthetase ASN1; AT3G47340), and DIN11 (2-oxoacid– plants relative to the Rca-β control plants (Table 1). Likewise, the dependent dioxygenase; AT3G49620). Other DIN genes were sig- α-expansin AtEXP8 (26) was down-regulated, as were several nificantly up-regulated but with fold changes below the cutoff used arabinogalactan proteins (AGP7, AGP21, and AGP41), which play for Table 1, including DIN1 [AT4G35770; senescence-associated multiple roles in plant development, including promotion of gene 1 (SEN1): 1.87-fold], DIN3 [AT3G06850; dihydrolipoamide growth (27). Pectins are major components of the cell wall and

18728 | www.pnas.org/cgi/doi/10.1073/pnas.1812916116 Kim et al. Downloaded by guest on October 2, 2021 Table 1. Differentially regulated genes in transgenic plants expressing the T78S-directed mutation relative to wild-type Rca-β in the − rca knockdown mutant background Fold Change

The Arabidopsis Information Resource Symbol Gene Product (T78S/RCA-β)

Up-regulated genes Genes induced by low carbon, dark, or senescence AT3G49620 DIN11 Encodes 2-oxoacid–dependent dioxygenase. 8.38 AT3G60140 DIN2 Beta-glucosidase 30 (SEN2) 7.44 AT2G29350 SAG13 Senescence-associated gene SAG13 3.67 AT5G22300 AtNIT4 Encodes a nitrilase isomer 3.12 AT1G32350 AOX1D Alternative oxidase 3: up-regulated in senescence 2.95 AT1G35140 EXL1 Required for growth at low carbon availability 2.88 AT3G47340 DIN6 Glutamine-dependent asparagine synthetase 2.66 Down-regulated genes Hydrolases and expansins AT2G32990 AtGH9B8 Cellulase; GUN11 −3.22 AT1G64390 AtGH9C2 Glycosyl hydrolase; GUN6 −2.03 AT3G53190 PLL17 Pectate lyase–like −2.01 AT1G04680 PLL26 Pectate lyase–like −2.50 AT2G40610 ATEXP8 Alpha-expansin gene family −1.99 Lignin-related AT5G62360 NA Pectin methylesterase inhibitor −2.32 AT5G62350 NA Pectin methylesterase inhibitor −2.07 AT4G21960 PRXR1 Cell wall peroxidase, possibly involved in lignin −2.04 Development AT3G25717 DVL6 ROTUNDIFOLIA-like 16 −2.15 PLANT BIOLOGY Cell wall–binding proteins AT5G24105 AGP41 Arabinogalactan protein (AGP41) −2.24 AT5G65390 AGP7 Arabinogalactan protein 7: involved in development −2.04 AT1G55330 AGP21 Arabinogalactan protein −1.99 AT4G18280 NA Glycine-rich cell wall–like protein −2.16 AT3G16670 NA Extensin-like protein −2.14 AT1G12090 ELP Extensin-like protein (ELP) −1.98 Hormone-related AT1G52400 ATBG1 Glycosyl hydrolase family 1 protein generates active form of ABA −3.58 AT3G22231 PCC1 Circadian clock–regulated gene; flowering and ABA responses −2.57 AT3G22240 NA PCC1-like protein −1.99 AT1G19050 ARR7 Arabidopsis response regulator (ARR) protein, cytokinin −2.30 signaling, and meristem stem cell maintenance AT5G18010 SAUR19 Positive effector of cell expansion −2.13 AT5G18030 SAUR21 Positive effector of cell expansion −2.10 AT5G18060 SAUR23 Positive effector of cell expansion −2.18 AT5G18050 SAUR22 Positive effector of cell expansion −2.16

often involve complex structures that can be modified or degraded this subfamily of SAUR genes would be expected to contribute to to alter cell wall properties. Accordingly, pectin-related enzymes reduced growth of the T78S transgenic plants. are important in cell expansion, and 2 pectate lyase–like genes Functional interactions within the differentially expressed and 2 genes encoding pectin methylesterase inhibitors were genes were explored using the STRING database (31). Analyses down-regulated in the present study. Pectate lyases are associated were carried out using a medium confidence threshold and all with depolymerization of pectins and cell separation, whereas lines of evidence except neighborhood and gene fusion. Within pectin lyase–like (PLL) proteins are thought to be involved in the up-regulated genes, there was a large network of interactors numerous aspects of growth potentially independent of cell sep- containing 56 nodes and 136 edges (SI Appendix,Fig.S5A). aration (28); 2 PLL genes were down-regulated in T78S plants. AT1G26380 and CYP71A12 were the most central nodes, based on Methyl-esterified pectin is secreted in cell walls, and removal of weighted closeness centrality (32). These genes were implicated methyl-esters from pectin by pectin methylesterases (PMEs) can recently in the production of cyanogens during pathogen response generate negative charges that coordinate calcium ions to form (33). Up-regulation of this network may therefore suggest activation cross-links that affect cell wall extensibility. Because PME inhibi- of stress response pathways in T78S. Among the down-regulated tors block this action, down-regulation of PME inhibitors could genes, no large interaction networks were found (SI Appendix,Fig. result in cell walls with reduced capacity for expansion (29). In- S5B), only a small cluster of the aforementioned SAUR genes. terestingly, genes encoding 2 PME inhibitors were down-regulated in T78S plants. Additional growth-related genes include the Discussion SMALL AUXIN UP RNA (SAUR) genes that are auxin-responsive The results of the present study identify a regulatory role for and often correlated with auxin-mediated cell expansion. In par- phosphorylation of Rca and implicate the Rca Thr78 phosphosite ticular, genetic evidence implicates the SAUR19–24 subfamily as in the evolution of terrestrial plants. We tested the regulatory role positive effectors of cell expansion (30); the down-regulation of of Rca phosphorylation by expressing wild-type or mutated versions

Kim et al. PNAS | September 10, 2019 | vol. 116 | no. 37 | 18729 Downloaded by guest on October 2, 2021 of the Rca-β isoform, which lacks the C-terminal redox regulatory role in the hyperphosphorylation of the T78S mutant, but these domain found in the α-isoform, in an Arabidopsis rca-knockdown studies await identification of the requisite phosphatases acting on mutant. Relative to transgenic plants expressing wild-type Rca-β phospho-Rca in vivo. Second, these results also have relevance to with the phosphorylatable Thr78 site, results obtained with the our understanding of the variation in residues found in Rca at the phospho-null T78A-directed mutant and the hyperphosphorylated position corresponding to residue 78 in the Arabidopsis protein. T78S-directed mutant strongly suggest that phosphorylation of Overall, the Rca protein is highly conserved in terms of sequence, Rca plays a negative regulatory role in vivo that inhibits photo- but there is a surprising amount of variation among terrestrial synthesis and growth. The phospho-null T78A mutant exhibited species at position 78 (SI Appendix,Fig.S3B). However, among more rapid induction kinetics of photosynthesis and higher the 59 terrestrial species for which the National Center for Bio- ϕPSII during low- to high-light transitions (Fig. 2A), whereas the technology Information Reference Sequence Database Rca se- hyperphosphorylated T78S mutant exhibited slower photosyn- quences are available, none have a serine at the position thesis induction and lower ϕPSII (Fig. 3B)comparedwiththe corresponding to Thr78 of Arabidopsis Rca (SI Appendix, Fig. wild-type control. In addition, changes in photosynthetic param- S3B). We speculate that the lack of serine may be explained by eters noted for the site-directed mutants were associated with al- natural selection against this residue because of its hyper- tered growth phenotypes compared with the transgenic plants phosphorylation and negative impact on photosynthesis. expressing wild-type Rca-β. For example, T78A plants were sim- While present studies have established a regulatory role for ilar to the control plants when grown in a typical square-wave light phosphorylation of Thr78 in Rca-β in vivo, a number of impor- regime, but they grew faster than controls in a fluctuating low- tant questions remain for future studies. First, it will be impor- light/high-light regime where more of the CO2 assimilation oc- tant to examine the role of Thr78 phosphorylation of the curred under inductive (non-steady-state) conditions. In addition, α-isoform of Rca, which has redox regulation capability, in- the T78A plants had a small but significant growth advantage at dependently as well as in plants expressing both Rca isoforms. − − − low light (30 μmol photons m 2 s 1), a condition that retains Previous studies with a cpck2 knockout mutant concluded that phosphorylation of wild-type Rca-β (10). Thus, in the absence of phosphorylation in wild-type plants has no regulatory role (10) the Rca phosphorylation that occurs in wild-type Rca-β,theT78A but, because of the potential for additional protein kinases to mutant plants utilized light energy more efficiently and grew phosphorylate Rca at the Thr78 site, this conclusion needs fur- slightly faster (Fig. 2B). Conversely, the hyperphosphorylated ther examination with directed mutants lacking the phosphosite T78S plants had reduced growth in both the square-wave light and residue. Second, and as noted earlier, it will be important to fluctuating-light regimes (Fig. 4) consistent with the retention of identity the protein phosphatase(s) that dephosphorylate Rca phosphorylation observed well into the light period (Fig. 3B). phospho-Thr78 and determine whether there is a preference for Reduced growth of the T78S-directed mutant would be expected phospho-Thr over phospho-Ser. If so, then substrate preferences given the reduced CO2 assimilation rate (per unit leaf area; Fig. 3B) of both kinase(s) and phosphatase(s) may contribute to the and reduced expression of numerous growth-related genes (Table hyperphosphorylation of the T78S-directed mutant. Third, and 1). Collectively, these results support the notion that phosphory- perhaps most important, future efforts need to determine the lation of Rca residue-78 negatively regulates photosynthesis, with mechanism by which phosphorylation of Rca affects photosyn- its impact more pronounced with the T78S-directed mutant com- thesis (Figs. 2 and 3) and plant growth (Fig. 5). The simplest pared with the wild-type Rca-β. explanation is that phosphorylation of Rca-β directly inhibits Our finding that the T78S-directed mutant was hyper- Rubisco activation. To test this possibility, we performed in vitro phosphorylated into the light period was based on immunoblotting assays of Rubisco activation using phosphorylated or non- with sequence- and site-specific antibodies (which recognized the phosphorylated Rca proteins, and found no significant inhibition phosphorylated form of Thr78 and Ser78; Fig. 3B) and on quan- by phosphorylation (SI Appendix, Fig. S6B). The phosphorylation titative mass spectrometry–based phosphoproteomic analysis (Fig. stoichiometries of the recombinant proteins used in the Rubisco 4B); it has important implications that are worth noting. First, the activation experiments were roughly 20% for Rca-β and 45% for result was unexpected because substitution of serine for a threo- T78S (SI Appendix, Fig. S6A). The phosphorylation stoichiom- nine residue is the most conservative substitution possible; how- etries of Rca-β and T78S in vivo in darkened leaves were de- ever, the change can have surprising effects because, while the termined by quantitative phosphoproteomic analysis (Fig. 4) to 2 residues are similar, they are often not equivalent. In our system, be roughly 50% for Rca-β and 75% for T78S, which were only this result suggested that the protein kinase(s) and/or protein slightly higher than the phosphorylation stoichiometries tested in phosphatase(s) acting on Rca discriminated slightly between ser- vitro. Rubisco contents were not changed in T78S in light con- ine and threonine at the phosphosite of Rca. Indeed, it is gener- ditions (SI Appendix, Fig. S7). Thus, we tentatively conclude that ally recognized that while many Ser/Thr kinases prefer serine phosphorylation of Rca does not directly inhibit Rubisco acti- as the phosphoacceptor, most protein phosphatases prefer vation activity but rather is more complex. For example, it may phosphothreonine (15, 34, 35). Although the protein phosphatases not be possible to mimic the phosphorylation impact in vitro if the acting on phospho-Rca have not been identified, cpCK2 has been phospho-Thr78 Rca recruits cofactors (e.g., phosphoprotein- established as a major protein kinase that phosphorylates Rca binding proteins) to sequester phospho-Rca or inhibit Rubisco at the Thr78 site (10). Plastid-localized cpCK2 is a member of activation in vivo. Fourth, if phosphorylation of Rca is broadly the nearly ubiquitous CK2 kinase family, and previous work with important in vivo, then understanding how phosphorylation stoi- mammalian and yeast CK2 kinases determined that Ser is strongly chiometry is controlled warrants examination; that is, why is Rca preferred over Thr as the phosphoacceptor residue (34, 36). Con- slowly phosphorylated in the dark but rapidly dephosphorylated in sistent with earlier studies with nonplant CK2 kinases, our results the light? A role for redox in the light/dark control of Rca phos- with cpCK2 and synthetic peptides indicated a similar preference phorylation was established (10) which was consistent with in- for Ser over Thr at position 78 (SI Appendix,Fig.S2B and C). A creased phosphorylation in the dark; however, how the redox major determinant of Ser vs. Thr phosphoacceptor specificity signal was mediated was not elaborated. Moreover, once all of the appears to reside in a specific residue of the kinase activation protein kinases and protein phosphatases are identified, it will be + segment, termed “DFG + 1” (37). The residue corresponding to possible to determine if the changes in stromal pH and [Mg2 ] that DFG + 1 in cpCK2 is a leucine, which is consistent with a pref- accompany light/dark changes are additional factors that regulate erence for serine and may contribute to the increased phosphor- Rca phosphorylation. Last but not least, it will be important to ylation of the T78S-directed mutant of Rca-β in vivo. However, it unravel the full complexity of the posttranslational modifications is likely that the specificity of protein phosphatase(s) also plays a of Rca. Next to phosphorylation and redox regulation, lysine

18730 | www.pnas.org/cgi/doi/10.1073/pnas.1812916116 Kim et al. Downloaded by guest on October 2, 2021 acetylation was recently discovered on Lys438 of Rca-β2andwas sulfate polyacrylamide gel electrophoresis and immunoblotting were used found to be under the control of a plastid lysine deacetylase in low- to confirm Rca protein expression. Peptide phosphorylation and Rubisco activa- light conditions (38). Thus, the potential for control of Rca activity tion assays were conducted in vitro. Liquid chromatography–tandem mass by phosphorylation, lysine acetylation, and redox is apparent, and spectrometry was used to identify phosphorylated protein fractions. Leaf it will be important to explore these interactions further in photosynthesis was measured using portable gas exchange systems equipped future studies. with fluorometers. RNA was isolated for microarray expression analyses, which were confirmed using qRT-PCR. Materials and Methods ACKNOWLEDGMENTS. We thank Dr. Rebecca Slattery for critical reading of Detailed descriptions of all materials and methods are available in SI Ap- the manuscript. Support was provided by the Agricultural Research Service, pendix. Briefly, the Arabidopsis rca-knockdown mutant (Salk_003204C) US Department of Agriculture (National Institute of Food and Agriculture– containing a T-DNA insertion in the Rca gene promoter was transformed Agriculture and Food Research Initiative grant 58-5012-025 to M.H.S. and with cDNAs encoding wild-type Rca-β, Rca-β (T78A), or Rca-β (T78S). Plants D.R.O.) and by the Deutsche Forschungsgemeinschaft (grants FI 1655/3-1 and were grown under both nonfluctuating and fluctuating light. Sodium dodecyl INST 211/744-1 FUGG to J.G., I.L., and I.F.).

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