Evolutionary conservation and multilevel post-translational control of S-adenosyl-homocysteine-Hydrolase in land plants

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH) activity, and in doing so essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and multilevel post-translational control of SAHH in land plants. We find that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants, and provide evidence that the predominant enzyme is a tetramer. By analyzing light-stress-induced adjustments occurring on SAHH in Arabidopsis thaliana and Physcomitrella patens, we demonstrate that both angiosperms and bryophytes undergo regulatory adjustments in the levels of protein complex formation and post-translational modification of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.

Here we report on the evolutionary conservation and biochemical characteristics of SAHH in 91 land plants. We find that the predominant oligomeric SAHH complex 4 can be detected in 92 phylogenetically divergent land plants, and provide evidence suggesting that the protein 93 complex is a tetrameric form of the enzyme. By analyzing regulatory adjustments occurring on 94 SAHH in high-light-exposed Arabidopsis and Physcomitrella patens (hereafter 95 Physcomitrella), we demonstrate that both angiosperms and bryophytes respond to light-96 induced stress by regulatory adjustments in this metabolically central enzyme.       Table). Perturbations in which at least 60% (24 out of 39) of the genes for the 182 AMC enzymes and selected MTs were differentially expressed with a p-value <0.05 were 183 selected to build the cluster heatmap. the AMC enzymes (S1 Table). *This gene represents both AT5G17920 and AT3G03780 as 202 they were indistinguishable because they share the same probe in Affymetrix Arabidopsis 203 ATH1 microarray chip. that SAHH localized to multiple sub-cellular compartments (Fig 2). However, SAHH1 was not 210 uniformly localized within the cells, but rather highly organized to various cellular structures.      does not co-localize with SAHH (Fig 2A) and is therefore highly unlikely to interact with 271 SAHH. Based on these findings and the apparent 200 kDa MW of the complex, it can be 272 deduced that the SAHH complex 4 is composed by a tetramer of the enzyme. Shattered of 273 complexed 1 and 2 with 0.25% treatment generate the increase in abundance of SAHH 274 complex 4 containing spot in the 0.25% SDS treatment (Fig 3 A and B).   14 293 oleracea and P. patens (Fig 4). Arabidopsis and B. oleracea are closely related species and 294 showed 99% SAHH amino acid sequence similarity (Fig 4 A and B, Table 1). Even between 295 the more distantly related species, pair-wise amino acid comparison between L. luteus and 296 Physcomitrella SAHH indicated 90% similarity (Fig 4 A and B PTMs sites were conserved in the plant species studied (Fig 4A), suggesting that post-300 translational regulation of SAHH could be a conserved feature among land plants (Fig 4A).

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To assess stress-induced adjustments in SAHH, we next exposed the two well-established, 330 phylogenetically different model plants, Arabidopsis  high-light-induced decrease in SAHH complex 2 abundance, and a corresponding increase in 333 the abundance of SAHH complexes 5 and 6 in Arabidopsis (Fig 5A). Physcomitrella, in 334 contrast, showed a clear increase in the abundance of a SAHH complex, which corresponds to 335 SAHH complex 2 in Arabidopsis (Fig 5A). The total abundance of SAHH did not differ 336 between the treatments (Fig 5C). The high light treatment slightly decreased SAHH complex 337 4 abundance in Arabidopsis, whereas Physcomitrella accumulated slightly more SAHH 338 complex 4 (Fig 5A). Hence, both model species responded to high light treatment at the level 339 of SAHH complex formation, but exhibited opposite outcomes. Analysis of SAHH phosphorylation by Phos-tag gel electrophoresis suggested also light-357 dependent phosphoregulation in both species (Fig 5B). In Arabidopsis, a slow-migrating form 358 of SAHH was detected in leaf extracts isolated from growth light conditions, while high-light-359 exposed leaves did not contain such phosphorylated form of SAHH (Fig 5B). In

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Physcomitrella, the intensity of one phosphorylated SAHH species increased, while another 361 SAHH species disappeared upon high light illumination (Fig 5B). Hence, both Arabidopsis and

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Physcomitrella responded to light-induced stress by regulatory adjustments in SAHH, but the 363 responses differed between the angiosperm and bryophyte models. membrane, but not chloroplasts or mitochondria (Fig 2). While SAHH1 has not been localized into the chloroplast (Fig 2) [18], we detected SAHH1 as 393 a ring in the immediate vicinity around the photosynthetic organelles (Fig 2) migration of potential complex-forming proteins when assessed by 2D SDS-PAGE (Fig 3A).

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The physiological significance of this abundant SAHH complex present in various land plants, 419 including the moss Physcomitrella (Fig 4C) [15], and phosphorylation at these sites could therefore affect the activation state of the enzyme.

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The phosphorylated residues S20 and T44 of SAHH1 in turn reside on the surface of the Excess light also triggers chloroplast-to-nucleus signaling, whereby stress-exposed 454 chloroplasts induce coordinated adjustments in the expression of nuclear genes [46,47]. Light- interacts with hormonal signaling to drive stomatal closure, is operational in angiosperms, 459 mosses and ferns [48]. Our studies provide evidence that high-light-induced signals, likely 460 originating from chloroplasts, are reflected by regulatory adjustments in SAHH at the level of 461 complex formation and phosphorylation in both Physcomitrella and Arabidopsis (Fig 5A).