Temperature-induced changes in wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms

Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term increase in temperature on the wheat leaf and spikelet phosphoproteome. The resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature, which will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which likely impacts protein activity.

1 0 increased temperature in wheat, we subjected both leaf and spikelet samples from the 60 min 2 2 3 time point to our phosphoproteomic workflow (Vu et al., 2016). Advances in the wheat reference sequence assembly provide a solid basis for proteome  The identified phosphosites in this study were added to our PTMViewer 2 4 5 (bioinformatics.psb.ugent.be/webtools/ptm_viewer/) (Vu et al., 2016). In addition, we found 2 4 6 1 1 several phosphosites that were differentially regulated between normal (21 or 24°C) and 2 4 7 increased ambient temperature (34°C) in wheat leaves and spikelets (Figure 2).  deregulated phosphoproteins from the statistical test were combined and analysed for in the GO terms of stress-induced processes such as response to heat, protein folding (Zhu,  Expression of HSPs is rapidly induced in the leaf by increased temperature ( Figure   2 6 8 1C), as the resulting proteins play crucial roles when plants are exposed to increased temperature (Sun et al., 2002;Kotak et al., 2007). In our leaf data set, we furthermore 2 7 0 identified several differential phosphorylation sites of HSPs at 34°C (Supplementary Table   2 7 1 1 2 S4-5), namely HSP90 (TraesCS2A01G033700.1, TaHSP90) and HSP60-3A 2 7 2 (TraesCSU01G009200.1, TaHSP60-3A) were 10.4-fold and 4.6-fold upregulated at S224 and 2 7 3 S577, respectively. However, for both proteins, another phosphosite, namely S93 of TaHSP90   2  7  4 and T420 of TaHSP60-3A, was not differentially phosphorylated after 1 h exposure to 34 °C. This suggested that HSP90 and HSP60-3A protein abundance is likely not the basis for the 2 7 6 increase in S224 and S577 phosphopeptide increase, respectively. Noticeably, our dataset indicated that the phosphoproteome of the photosynthesis 2 7 8 machinery in wheat leaves is severely affected by high temperature (Supplementary Tables   2  7  9 S4 and S5). For example, phosphorylation of T33, T37 and T39 of the subunit P of 2 8 0 photosystem I (TraesCS2A01G235000.1) was 3.2-fold downregulated after 1 h exposure to 2 8 1 Table S5). In addition, an actin-binding protein 2 8 2 (TraesCS1D01G422700.2), whose homologue in Arabidopsis (CHUP1) is important for 2 8 3

34°C (Supplementary
proper chloroplast positioning (Oikawa et al., 2008), was found to be considerably less 2 8 4 phosphorylated at S157 upon high temperature (Supplementary Table S4). Besides, a  Table S4). Both CHUP1 and KAC1 regulate the accumulation of chloroplast outer membrane (Kim et al., 2014). It is speculated that the regulatory mechanism we showed that phosphorylation of the AKR2 homologue in wheat 3 0 5 (TraesCS4A01G328600.1) at S404 is two-fold upregulated in response to higher temperature 3 0 6 (Supplementary Table S5). While protein import in chloroplasts has been shown to be indicated that this response, especially to high temperature, is highly regulated by  In conclusion, our temperature-mediated leaf phosphoproteome pinpointed 3 1 1 photosynthesis as a central target of higher temperature and identified several phosphorylated 3 1 2 residues on key components for further functional characterization. For the wheat spikelet, we identified 79 phosphosites that are only present in the 34°C  wheat spikelet dataset (phosphosites with at least 2 valid values in temperature condition;  Table   3 2 2 S7). Proteins with phosphosites uniquely identified in either condition and significantly 3 2 3 deregulated phosphoproteins from the statistical test were combined and GO analysis was folding, response to heat, response to hydrogen peroxide. Similar to the leaf GO enrichment phosphoproteins.

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In conclusion, our data suggested that an increase in ambient temperature can alter altered phosphorylation in response to increased temperature. In total, we identified 2491 identical phosphosites in both organs, which account for 48% and  Notwithstanding the considerable overlap between the phosphosites identified in both organs,  Among the common higher temperature-induced phosphosites, phosphorylation of 3 7 0 S464 of the pseudouridine synthase TraesCS2B01G177000.1 was increased 1.6-fold and 1.9- On the other hand, three different translation initiation factors are present among the 3 7 5 commonly regulated proteins with downregulated phosphosites (Supplementary Table S8).

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This is in agreement with heat stress-triggered overall pausing of translation elongation, and   signalling (Ding et al., 2015;Yu et al., 2017;Li et al., 2017;Zhao et al., 2017). Therefore, we 3 8 7 used the identified phosphosites to reveal potential phosphorylation motifs and associated 3 8 8 kinases that may act in a high-temperature responsive manner. The Motif-X algorithm was 3 8 9 applied on the set of regulated phosphosites in leaf and spikelet samples separately, using the 3 9 0 sequences of all identified phosphoproteins in either organ as reference (Figure 6). In the 3 9 1 spikelet, the common SP motif was enriched in both upregulated as well as downregulated phosphosites. This suggested that kinases (and phosphatases) targeting those sites are tightly (4.37-fold) (Figure 6). This latter trend was also found in the leaf samples (Figure 6). Despite 3 9 9 that no motif enrichment was obtained for the upregulated phosphosites in leaf, due to the 4 0 0 small size of the data set, we identified six SD motifs among these sites, which account for   Since the protein phosphosignature will determine protein behaviour (Salazar and Höfer,  (Table 1). It is thus very likely that the status of these 4 2 3 phosphosites is not affected by changes in the protein level, but rather by higher temperature-4 2 4 dependent activity of associated kinases and phosphatases. These protein phosphatases and 4 2 5 kinases might be activated by higher temperature and target the phosphosites independently to 4 2 6 generate different phosphoforms of the target protein ( Figure 7A). However, the 4 2 7 phosphorylation and dephosphorylation events might also occur in an interdependent manner 4 2 8 upon higher temperature ( Figure 7B) (Salazar and Höfer, 2009;Nishi et al., 2015). Crosstalk

3 1
A complex example is the putative protein kinase TraesCS6B01G377500.3 (Table 1) peptide (DFPIpSPSpSAR, S227 and S230) was detected 2.5-fold higher in the 21°C samples.  In addition, we also found proteins with multiple phosphosites that showed the same of these phosphosites will require additional investigation on the abundance of the proteins, 4 4 6 e.g. by analysing intact proteins or rather the different proteoforms.

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Altogether, our data indicated that multiple phosphorylation/dephosphorylation events  Interestingly, in the spikelet samples, TraesCS5B01G387800.1 (Table 1), which is a  (Supplementary Figure 4). The phosphosites are located in the WD40-  Inspecting the protein sequence, we found the two phosphorylation sites localized in a 4 6 2 sequence window of 10 amino acids of which four are either Ser or Thr ( Figure 8B). Neither homologues, we could also find high frequency of Ser and Thr residues in the same sequence 4 6 7 windows in other seed plants (Figure 8B). While the high occurrence of phosphorylatable This might provide a buffering mechanism to maintain the function of the protein by 4 7 2 differential phosphorylation of neighbouring amino acid residues depending on the 4 7 3 environmental conditions. However, we also do not rule out allosteric or orthosteric 4 7 4 regulation between the two phosphosites that might affect the activity of the protein  For the splicing factor TraesCS2D01G281200. containing only S12 (ASAETLARSPpSREPSSDPPR) is uniquely detected at 34°C, while the 4 7 8 doubly phosphorylated peptide of S10 and S12 (ASAETLARpSPpSREPSSDPPR) was 3.4-4 7 9 fold downregulated at the same temperature in the spikelets. We speculate that the context of stress responses. It is possible that temperature serves as a signalling switch for 4 9 2 such a phosphorylation toggle via regulated interaction with at least a protein kinase and/or In conclusion, we provide the scientific community with the first large scale temperature-sensitive organs. An in-depth analysis showed that the photosynthetic machinery 5 0 0 in the leaf is highly responsive to increased temperature, while epigenetic regulation in the 5 0 1 spikelets seems to be tightly regulated by higher temperature in a phosphorylation-dependent 5 0 2 manner during reproductive development. Furthermore, we observed a core set of common 5 0 3 proteins between both leaf and spikelet, suggesting some conserved mechanisms in these 5 0 4 organs when responding the higher temperature. Nevertheless, we also observed a large 5 0 5 portion of organ-specific regulation. Finally, we exposed a, so far, not reported mechanism of 5 0 6 interconversion of neighbouring phosphorylation residues, which likely plays a key role in 5 0 7 temperature signalling. Taken together, our data set increases the understanding of 5 0 8 temperature signalling in plants.         Table S10 List of proteins with multiple upregulated or multiple downregulated phosphosites We thank Michiel Van Bel for assistance in depositing the data in the PTMViewer. We thank Agronomy for Sustainable Development 37, 37.         dataset. Fold change is indicated.    phosphoform of the protein.