Acclimation to cold involves changes in the proteome of both Col-0 and fum2.1
In a previous study of dynamic acclimation of photosynthetic capacity, in response to increased light, we saw that acclimation entails an increase in enzyme concentrations involved in multiple metabolic processes (Milleret al. , 2017). In Col-0, cold exposure for 7 days resulted in a significant increase in leaf protein content (Figure 4a), with an approx. 30% increase in protein content per unit fresh weight of leaf. In fum2.1 , protein content did not change significantly. Nevertheless, analysis of the proteome shows that there were changes occurring in both Col-0 and fum2.1 , albeit to a much smaller extent in the latter. We were able to estimate the relative abundance of 2427 polypeptides, based on a minimum of 3 unique peptides per protein. Principal Component Analysis of proteomic data indicates that the proteomes of Col-0 and fum2.1 differ already under 20oC conditions, however there is a clear separation of cold acclimated plants from their corresponding 20oC controls in both genotypes (Figure 4b). Cluster analysis was performed using data from the 2015 proteins which showed significantly altered expression in one of more conditions. As expected, given the total increase in protein, the most common response to cold is for proteins to increase in Col-0 but less so or not at all infum2.1 (Figure 4c Clusters 1, 4). A far smaller cluster of proteins increased in both genotypes following acclimation (Cluster 2) whilst a few proteins decreased in response to cold in fum2.1(Cluster 3).
Examination of the relative concentration of proteins involved in the photosynthetic electron transport chain demonstrated that only subtle changes were occurring (Table S1). There were increases in the relative abundance of various peripheral PSII proteins, including isoforms of PSBS, and in PSB29, which has been implicated in PSII assembly (Kerenet al. , 2005), however components of the PSII core did not change significantly in response to cold. Amongst the proteins of the photosynthetic electron transport chain, 2 of the 4 detected Cyt b6f complex subunits increased significantly in Col-0, while measurements for the other subunit were too variable to allow a confident assessment of changes in abundance. Overall, this suggests a tendency to increase cytochrome b6f abundance in Col-0, whilst infum2.1 there is no evidence for a change in the abundance of this complex. While plastocyanin showed no change in abundance in either genotype, the only detected ferredoxin isoform increased in both genotypes, as did one of the four detected FNR isoforms. 4 of the detected 8 ATP synthase subunits were upregulated in Col-0, whilst 2 showed a significant change in fum2.1 . Taking these data overall, we conclude that there were no changes in photosystem stoichiometry in response to cold in either genotype and that changes in electron transport proteins in Col-0 were either reduced or absent infum2.1 . There were however consistent and significant differences between the genotypes both in warm and cold conditions, with a greater abundance of subunits of all complexes being seen in Col-0.
In contrast to the components of the photosynthetic electron transport chain, changes in the enzymes associated with the Benson Calvin cycle gave a clearer and more consistent pattern of response (Figure 5). The CO2 fixing enzyme, Rubisco, is by far the most abundant protein in the leaf. We were able to quantify the chloroplast-encoded large subunit (RBCL) and 2 isoforms of the nuclear-encoded small subunit, RBCS. All increased significantly in Col-0 in response to cold, with a mean 1.8-fold increase in relative abundance. In fum2.1there was no significant change in RBCL abundance. One isoform of RBCS increased significantly, whilst the other decreased to a similar extent. Combining data from both isoforms, there was no significant change in RBCS abundance. For other reactions associated with the Benson Calvin cycle, we were able to quantify a total of 20 distinct proteins, including isoforms of specific enzymes. In Col-0, 14 of these significantly increase, whilst 5 decreased. In fum2.1 , although 6 enzymes involved in the Benson Calvin cycle came out as significantly increased, the overall extent of this change was lower.
Across other major metabolic pathways – including starch and sucrose synthesis, glycolysis and the tricarboxylic acid cycle, similar patterns of acclimation were observed, with most proteins increasing in the cold in Col-0 and fum2.1 but to a lesser extent in the latter case (Table S1). In the sucrose synthesis pathway, most enzymes increased their concentration in response to cold in both genotypes, but with the relative abundance of these tending to be lower in fum2.1 (Figure S2). A notable exception to this was sucrose phosphate phosphatase, which did not increase in fum2.1 .
To summarise, acclimation of photosynthesis to low temperature in Col-0 involves an increase in the abundance of some electron transfer proteins, though not reaction centres, and substantial changes in the amount of a broad range of Benson Calvin cycle enzymes. These changes are largely or completely absent in fum2.1 . Changes in the proteome across metabolism show a similar tendency but a different extent in other metabolic pathways. These data indicate that fumarate accumulation or FUM2 protein or activity plays a central role in high-level processes regulating acclimation of metabolism.