Phosphorylation, rather than acetylation, of SR-rich splicing factors is involved in the upregulation of alternative splicing (AS) genes in response to high temperatures in grape leaves

Alternative splicing involves the formation of two or more different transcripts from the same pre-mRNA molecule. The identity of the splice sites is largely determined by RNA-binding proteins, most notably SR proteins and heterogeneous nuclear ribonucleoproteins, which promote and inhibit binding of the spliceosome, respectively31. The precise formation of mRNA splice variants is determined by splicing factors that define the exon–intron boundaries between. SR proteins modulate alternative splicing via concentration-dependent and phosphorylation-dependent splice site selection, while the activity and localization of SR proteins are regulated by phosphorylation32. In plants, notable changes in alternative splicing are triggered by a wide variety of abiotic stresses33,34,35. A subset of human SR proteins comprise nucleocytoplasmic shuttling proteins with diverse roles in aspects of postsplicing, such as mRNA export, stability, and mRNA translation36. In our previous study, we observed a rapid change in pre-mRNA splicing in grape leaves at 35 °C, as 206 differential splicing events were detected, whereas 1075 and 748 were detected at 40 °C and 45 °C, respectively6. Moreover, we found that the abundance of SR45, SR30, and SR34 in grape leaves increased gradually as ambient temperatures increased6. These results are consistent with additional alternative splicing events that occur in response to high temperatures in grape leaves. We also found that the phosphorylation levels of 11 SR-rich splicing factors were mainly upregulated at 35 °C, 40 °C or 45 °C, which included RS2Z32, SR34A, SCL30, SCL30A, SC35, RSZ22A, RSZ22, RS31, and RS41. However, the downregulated phosphosites at each high temperature were associated only with SCL28. These results indicated that phosphorylation upregulation of SR-rich splicing factors is important for promoting alternative splicing during the response to high temperatures in grape leaves (Fig. 5; Supplementary Table S2). Some studies have also demonstrated that SCL30A, SR34, RS2Z33, and RS41 are involved in plant responses to salinity or high/low temperatures37,38,39. The increase in the phosphorylation level of these SR-rich splicing factors would strengthen their own activities and promote alternative splicing, which further explains why increased numbers of alternative splicing events occurred in grape leaves in response to high temperature. However, SR proteins did not appear to be acetylated during the grape leaf response to high temperature. Therefore, we conclude that phosphorylation, rather than acetylation, of SR-rich splicing factors is involved in the upregulation of alternative splicing during the grape leaf response to high temperatures.

Fig. 5: Phosphorylation level change of SR-rich splicing factors at 35 °C, 40 °C and 45 °C in grape leaves. Each block represents a phosphosite, and the different colors of each block represent up- or downregulation of the phosphorylation level Full size image

Acetylation modulates more photosynthesis proteins and is more sensitive to high temperatures than is phosphorylation during responses to high temperatures in grape leaves

Photosynthesis involves highly heat-sensitive enzymes, and previous studies have shown that phosphorylation of photosynthesis proteins plays an important role in regulating photosynthesis40,41. The photosynthetic light reactions are catalyzed by thylakoid-embedded pigment–protein complexes (PSII, Cytb6f, and PSI), while the photooxidation of water during the light reactions of photosynthesis involves proteins of the oxygen-evolving complex (OEC), which are termed PsbO, PsbP, and PsbQ. In addition, PsbR has an auxiliary role in PsbP binding to PSII. In this study, we found that the phosphorylation levels of PsbR (at phosphosites Y52, S67, and Y77) at 35 °C, 40 °C, or 45 °C were upregulated (Fig. 6a; Table S3a). Furthermore, the acetylation levels of all oxygen-evolving complex components (PsbQ2, PsbP1, and PsbO2) were upregulated at 35 °C, 40 °C, or 45 °C, while their phosphorylation levels did not change (Fig. 6c). These results indicated that the oxygen-evolving complex may utilize acetylation and not phosphorylation to coordinate the response to high temperatures in grape leaves.

Fig. 6: Schematic representation of phosphoprotein or acetylproteins involved in photosynthesis. Changes in phosphorylation levels (a) and acetylation levels (c) of proteins related to the light reactions of photosynthesis at 35 °C, 40 °C, and 45 °C in grape leaves. Changes in phosphorylation levels (b) and acetylation levels (d) of proteins related to the Calvin cycle of photosynthesis at 35 °C, 40 °C, and 45 °C Full size image

The photosystem II (PSII) core proteins D1 and D2, as well as the inner antenna protein CP43 and the minor PSII subunit PsbH, are phosphorylated, mostly by the kinase STN8, at Thr residues42,43,44. In this study, we found that the phosphorylation levels of PsbH (phosphosites T3 and T5) in grape leaves at 35 °C, 40 °C, or 45 °C were downregulated (Fig. 6a), and that the acetylation levels of PsbD2 at 35 °C were downregulated (Fig. 6c). The phosphorylation levels of PsaF and the acetylation levels of PsaH2 at the three high temperatures were upregulated (Fig. 6a, c). These results constitute the first report indicating that the acetylation of PSI proteins represents a response to high temperature in plants.

The light-harvesting proteins LHCB1, LHCB2, and LHCB4 are phosphorylated by the STN7 kinase42,45. In our study, we found that the phosphorylation levels of LHCB1 and LHCB6 were upregulated, but the phosphorylation levels of LHCB4 were downregulated in grape leaves in response to high temperatures (Fig. 6a). In addition to phosphorylation, LHC proteins undergo other PTMs such as Lys acetylation46,47. Acetylation can have a range of biochemical and biological effects, including the determination of enzyme activity, alteration of protein stability and modification of protein–protein interactions48,49,50. As an example, acetylation of LHCB1 and LHCB2 appears to influence LHC attachment to PSII complexes: the peripheral LHC antenna that is more loosely bound to PSII shows more extensive Lys acetylation than do PSII–LHCII supercomplexes47. In our study, the acetylation level of LHCB6 at high temperatures was downregulated or unchanged in grape leaves (Fig. 6c; Supplementary Table S3b). In addition, the phosphorylation level of LHCA3 and the acetylation level of the LHCA1 protein at high temperatures were downregulated in grape leaves (Fig. 6a, c; Supplementary Table S3a, b). These results indicated that the light-harvesting proteins of PSII and PSI responded to the phosphorylation or acetylation levels in the grape leaves in response to high temperatures.

RuBisCO is the first enzyme in carbon fixation, and it exists as a holocomplex of eight small subunits (RBCS) and eight large subunits (RBCL) that contain multiple phosphosites51,52 and are reversibly phosphorylated in many plant species52,53. RCA is an ancillary photosynthesis protein essential for RuBisCO activity. It has been reported that phosphorylation of RuBisCO results in a substantial increase in its activity, while desphosphorylation results in a major decrease in its activity54. However, the phosphorylation of RCA may result in decreased activity of RuBisCO40,41. In our study, the phosphorylation levels and phosphosites of RBCL and RBCS1A increased at high temperatures (Fig. 6b; Supplementary Table S3a), and the RCA showed the same trend as did RuBisCO. Based on these data, we concluded that the phosphorylation of RuBisCO may compensate for its decreased activity due to the phosphorylation of RCA. In addition, RuBisCO subunits undergo Lys acetylation in response to different light conditions55. The RuBisCO holocomplex has multiple Lys acetyl sites, which are located either in the RuBisCO catalytic center46,56, at the interface between the two RBCL subunits46,57, or at a location that is important in defining the RuBisCO tertiary structure57. Lys acetylation is thought to affect RuBisCO activity, as well as interactions between subunits and other molecules, and recent studies have shown that Lys acetylation suppresses RuBisCO activity46,55. In this study, almost all of the identified acetyl sites of RuBisCO (RBCL and RBCSA1) and RCA were upregulated at different high temperatures compared with the control (Fig. 6d; Supplementary Table S3b). Thus, acetylation and phosphorylation of RuBisCO and RCA may jointly help coordinate the light reactions and carbon assimilation in response to the carbon status of the cell under different high temperatures. Moreover, acetylation may have a larger effect on function rather than phosphorylation with respect to RuBisCO modification. In the green alga Chlamydomonas reinhardtii, RCA is phosphorylated at S53 by a thylakoid-localized kinase58. RCA is mainly localized in the stroma, but a small portion of the enzyme is associated with the thylakoid membrane59. It has been proposed that phosphorylation of RCA promotes its attachment to the membrane, thereby protecting Stt7 from proteolysis58,60. The relocation may also reduce the activity of RuBisCO under specific environmental conditions58. In Arabidopsis, RCA is phosphorylated at two sites, T78 and S17241, and in the dark, the proportion of phosphorylated T78 sites increases61,62. In this study, the phosphorylation levels of six sites of three RCA proteins (F6HNV2, D7SKB2, and D7THJ7) and the acetylation levels of three sites of one RCA protein (D7SKB2) were upregulated compared with the control in grape leaves under high temperature (Fig. 6b, d; Supplementary Table S3).

Other enzymes of the Calvin cycle have bene identified as being phosphorylated. Phosphoglycerate kinase (PGK) is known to be phosphorylated in Arabidopsis, rice, and maize;53,61,63,64 while the second two species have the same phosphosite, Arabidopsis PGK is phosphorylated in a domain near the N-terminus. GAPDH has several phosphorylation sites, but they differ considerably between different organisms; thus, it has been suggested that phosphorylation may not be a key factor in regulating GAPDH activity in chloroplasts64. It has been noted that PGK and GAPDH are also targets of Lys acetylation46,65, and the activities of both enzymes increase upon deacetylation. Thus, Calvin cycle enzymes are subject to complex regulation by PTMs, but the enzymes involved in such modifications have yet to be identified64,66. In our study, in addition to those of RuBisCO and RCA, the phosphorylation levels of FBA and GAPB increased at high temperatures (Fig. 6b; Supplementary Table S3a). Moreover, the acetylation levels of PGK, FBA, GAPB, RPE, and TIM increased under the heat treatments (Fig. 6d). However, the acetylation level of three sites of transketolase (TKT) decreased (Fig. 6d; Supplementary Table S3b). Therefore, both phosphorylation and acetylation are associated with the modulation of key enzymes of carbon assimilation in response to high temperatures (Fig. 6b, d). Moreover, it appears that the Calvin cycle may be affected more by acetylation than by phosphorylation. In addition, for all the investigated photosynthesis proteins in grape leaves, acetylation modification occurred largely at 35 °C, and phosphorylation modification was not prevalent (Fig. 6), which suggests that acetylation of grape leaf photosynthesis proteins is more influenced than phosphorylation is by high temperatures. We conclude that photosynthesis is fundamentally affected by high temperatures, and can be regulated by both phosphorylation and acetylation. Moreover, the acetylation of photosynthesis proteins was more sensitive to high temperatures than was phosphorylation.

HSPs exhibit more extensive phosphorylation than acetylation in response to heat exposure in grape leaves

Many studies have characterized large-scale responses to heat-shock responses in a variety of cells and organisms by the use of approaches, such as transcriptional profiling, differential displays, and proteomic analyses. HSPs act as molecular chaperones, reducing the aggregation and resolubilization of denatured proteins, promoting the folding of nascent polypeptides, and facilitating the refolding of denatured proteins67,68,69. In plants, HSPs can be classified into five groups based on molecular mass: small HSP (sHsp) proteins, chaperonins (GroEL and Hsp60), Hsp70 (DnaK) proteins, Hsp90 proteins, and members of the Hsp100 (Clp) family. Many studies have reported that high temperatures lead to increases in HSP levels in plants70. However, there are few studies on the acetylation and phosphorylation of HSPs in plants under heat or other stresses71. Characterized the phosphoproteome of different tissues of wheat cultivars that exhibit different degrees of drought tolerance following simulated drought and recovery. Notably, the phosphorylation levels of HSP60 and HSP90 were found to be upregulated in response to drought in the drought-tolerant cultivar. In another study72, reported the effects of a short-term and moderate increase in temperature on the wheat leaf and spikelet phosphoproteome, which involved a substantial increase in the abundance of S224 of HSP90 and S577 of HSP60-3A. The acetylation of HSP70 has been described as a regulatory mechanism that temporally balances protein refolding/degradation in response to stress73,74, while HSP20 phosphorylation in mammalian systems has been implicated in a variety of pathophysiological processes but most prominently in cardiovascular disease75. In this study, we found that the phosphorylation or acetylation levels of a number of HSPs were upregulated in grape leaves in response to heat treatment. Phosphorylation levels increased substantially at two sites (S10 and S154) of HSP17.6C (F6HNM7), one site (S12) at HSP23.6 (D7TN47), and two sites (S27 and S56) of HSP17.6 (A5AQ47) at three high temperatures (Fig. 7). The phosphorylation levels of other HSPs, including HSP17.4A, HSP17.6 (F6H3Q4), HSP17.6 (A5AQ47), putative HSP40, HSP40.3 (A5ALT5), HSP40.3 (D7T3A9), HSP81.1 (F6H6C3), and HSP81.2 (F6HCU9), were regulated at high temperatures. However, phosphorylation levels declined at one site of HSP20 (D7UDS4), one site of HSP20 (D7T1L1), and two sites of HSP40.10 (Fig. 7; Supplementary Table S4). The acetylation level increased at one site (K191) in HSP70.1 (A5C0Z3) and declined at another site (K613) in HSP70.6 (F6GTP0) (Fig. 7; Supplementary Table S4). In our previous study, 11 HSPs were coupregulated at both the transcriptional and translational levels in grape leaves upon exposure to the 40 °C and 45 °C treatments compared with the control, but not by low-temperature treatments; these proteins included HSP17.4, HSP17.6, HSP23.6, HSP40, HSP25.3, HSP70.1, and HSP81.16. The results from this study indicate that these HSPs not only function in response to high temperatures by upregulating transcript and protein levels but also are involved in the heat tolerance of grape leaves via phosphorylation more so than acetylation. This study therefore underlines the importance of HSPs in grape thermotolerance.

Fig. 7 Phosphorylation and acetylation changes in heat shock protein (HSPs) at 35 °C, 40 °C, and 45 °C in grape leaves Full size image

Crosstalk between protein phosphorylation and acetylation in response to high temperatures in grape leaves

Some proteins can undergo multiple modifications, and PTM crosstalk has recently been reported76,77,78,79,80. Xiong et al. found that an acetyl site (K324) on an enolase protein from rice was located next to a phosphosite (S325)81. In addition, the acetyl sites and phosphosites in two spliced isoforms of a gamma-interferon-inducible lysosomal thiol reductase precursor from rice were found to be in close proximity. Ahn et al. provided evidence for acetylation and phosphorylation crosstalk between two neighboring histone residues (S10 and K11) in vivo and in vitro in H2B (HIB4) in yeast82. Protein phosphorylation and acetylation have been associated with many cellular functions, as recently reported in Mycoplasma pneumonia83. In this study, 19 proteins exhibiting possible crosstalk between phosphorylation and acetylation were associated with the photosynthetic light reactions and the Calvin cycle, glycolysis, the TCA cycle, DNA and protein synthesis, cell division, and the cell cycle (Table S1). These results indicated that key proteins of some biological processes, especially photosynthesis, are regulated by concurrent phosphorylation and acetylation in grape leaves in response to high temperature.

Emerging evidence suggests that protein PTMs that are adjacent or in close proximity within a single protein often lead to regulation of protein function or are important in signaling84. Carlomagno et al. demonstrated that acetylation of Lys321 (contained within a KCGS motif) inhibited phosphorylation of Ser-324 in tau proteins85. Cook et al. also noted a similar competitive relationship between phosphorylation and acetylation of KIGS motifs86. Previous studies have shown that mutagenesis of the S113 and K230 sites of isocitrate dehydrogenase (Idh) results in reduced enzyme activity and substrate affinity, underlining the significance of both phosphorylation and acetylation in regulating Idh in E. coli87,88. Direct evidence of crosstalk between acetylation and phosphorylation was recently demonstrated in M. pneumonia83; however, there are no reports of phosphorylation and acetylation of the same protein in plants under stress. In this study, acetylation levels in a number of sites of RBCL increased in the grape leaves in response to 35 °C, 40 °C, and 45 °C, while the phosphorylation levels of a few sites increased only at 45 °C (Table S1). Acetylation levels at five sites and phosphorylation levels at two sites of RBCS1A increased in response to heat treatment (Table S1). These results indicated that RBCL may undergo different PTMs than does RBCS1A under high temperature in grape leaves and that their coordination modulates RuBisCO activity. Recent studies have indicated negative regulation of RuBisCO activity by Lys acetylation46,55, and we previously reported that RuBisCO activity significantly decreased in grape leaves under 43 °C high temperature24. In this study, RCA appeared to exhibit more acetylation than phosphorylation (Table S1). Other studies have indicated that the activity of RCA decreases in many plant species under high temperature89. Therefore, upregulation of acetylation levels may be a factor in the decrease in RuBisCO and RCA activities in the grape leaf response to high temperatures. In addition, at different high temperatures, the phosphorylation and acetylation levels of proteins differed. For example, the LHCB6 phosphorylation (T180) and acetylation (K219 and K327) levels were upregulated at 35 °C, but did not change or were downregulated at 40 °C and 45 °C. Grape protein modification by acetylation appears to be as common as phosphorylation, and acetylation may provide a balance to phosphorylation in the regulation of protein activity. Congruent with previous studies in humans and microbes83,87,88, an upregulated acetyl site (K183) and an upregulated phosphosite (S180) are near one another in RBCS1A, and an upregulated acetyl site (K151) and a downregulated phosphosite (S154) are very close in a SAP domain-containing protein (E0CRG0). The regulatory mechanism of protein activity under high temperature is clearly very complex.

In summary, phosphoproteomic and acetylproteomic analyses were conducted on leaves of grape plants subjected to four different temperature regimes (25 °C, 35 °C, 40 °C, and 45 °C). The results revealed significant changes in phosphosites and acetyl sites, mainly following exposure to 40 °C and 45 °C. Phosphorylation, rather than acetylation, of SR-rich splicing factors was involved in the increase in AS events. Moreover, compared with phosphorylation modification, acetylation modification modulated more photosynthesis-related proteins and was more sensitive to high temperatures. Conversely, we conclude that modifications of HSPs during heat tolerance responses in grape leaves involve phosphorylation more than acetylation. We identified 19 proteins with significantly changed phosphorylation and acetylation levels, which is indicative of crosstalk between these PTMs. Acetylation may balance phosphorylation with regard to protein activity. Additional studies are needed to determine how key phosphorylation and acetylation proteins influence grape heat tolerance.