The PRT6 N‐degron pathway restricts VERNALIZATION 2 to endogenous hypoxic niches to modulate plant development

VERNALIZATION2 (VRN2), an angiosperm‐specific subunit of the polycomb repressive complex 2 (PRC2), is an oxygen (O2) regulated target of the PCO branch of the PRT6 N‐degron pathway of ubiquitin‐mediated proteolysis. How this post‐translational regulation coordinates VRN2 activity remains to be fully established. Here we use Arabidopsis thaliana ecotypes, mutants and transgenic lines to determine how control of VRN2 stability contributes to its functions during plant development. VRN2 localises to endogenous hypoxic regions in aerial and root tissues. In the shoot apex, VRN2 differentially modulates flowering time dependent on photoperiod, whilst its presence in lateral root primordia and the root apical meristem negatively regulates root system architecture. Ectopic accumulation of VRN2 does not enhance its effects on flowering, but does potentiate its repressive effects on root growth. In late‐flowering vernalization‐dependent ecotypes, VRN2 is only active outside meristems when its proteolysis is inhibited in response to cold exposure, since its function requires concomitant cold‐triggered increases in other PRC2 subunits and co‐factors. We conclude that the O2‐sensitive N‐degron of VRN2 has a dual function, confining VRN2 to meristems and primordia, where it has specific developmental roles, whilst also permitting broad accumulation outside of meristems in response to environmental cues, leading to other functions

The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation

Chloroplast function requires the coordinated action of nuclear- and chloroplast-derived proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) proteins that regulate plastid mRNA metabolism. Despite their large number and importance, regulatory mechanisms controlling PPR expression are poorly understood. Here we show that the Arabidopsis NOT4A ubiquitin-ligase positively regulates the expression of PROTON GRADIENT REGULATION 3 (PGR3), a PPR protein required for translating several thylakoid-localised photosynthetic components and ribosome subunits within chloroplasts. Loss of NOT4A function leads to a strong depletion of cytochrome b6f and NAD(P)H dehydrogenase (NDH) complexes, as well as plastid 30 S ribosomes, which reduces mRNA translation and photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. Quantitative transcriptome and proteome analysis of the not4a mutant reveal it lacks PGR3 expression, and that its molecular defects resemble those of a pgr3 mutant. Furthermore, we show that normal plastid function is restored to not4a through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring robust photosynthetic function during development and stress-response, through promoting PGR3 production and chloroplast translation.</p

Identification and Characterization of a Novel Tyrosine Phosphatase in Plants

The study of tyrosine phosphorylation in plants has been largely neglected due to the lack of classic tyrosine kinases and underrepresentation of tyrosine phosphatases compared to humans. However, advanced phosphoproteomics studies have revealed that the abundance of phospho-tyrosine residues in plants parallels human signal transduction. This strongly suggests that in plants tyrosine phosphorylation is as important as in humans, yet we know nothing about the players responsible for these events. The Arabidopsis thaliana Rhizobiale-like phosphatase 2 (AtRLPH2) is a novel protein phosphatase not found in mammals which, according to bioinformatics analysis, clusters with the serine/threonine-specific phospho-protein phosphatase (PPP) group. In the present work, it is shown that recombinant AtRLPH2 surprisingly behaves like a tyrosine phosphatase in vitro by dephosphorylating phospho-tyrosine peptides and having essentially no activity towards phospho-serine/threonine residues. Furthermore, the endogenous AtRLPH2 from Arabidopsis thaliana was also shown to preferentially dephosphorylate tyrosine phosphorylated peptides and protein. This work shows for the first time that a member of the plant PPP family of phosphatases has the capability to dedicate its activity solely toward phospho-tyrosine. In order to understand the novel substrate specificity of AtRLPH2 its crystal structure was elucidated, providing detailed mechanistic insight into its mode of function and characteristics of its potential substrates. AtRLPH2 possesses a basic pocket next to the active site which makes it imperative for the substrate to possess a phospho-threonine/serine two amino acids upstream the phospho-tyrosine. To gain a better understanding of AtRLPH2 significance and targets, a phosphoproteomics study was undertaken to compare phospho-tyrosine peptides from wild type and atrlph2 knockout plant lines. In summary, the atypical tyrosine phosphatase AtRLPH2 was characterized by a multidisciplinary approach involving biochemistry, cell biology, phosphoproteomics and structural biology

Oxygen-dependent proteolysis regulates the stability of angiosperm polycomb repressive complex 2 subunit VERNALIZATION 2

The polycomb repressive complex 2 (PRC2) regulates epigenetic gene repression in eukaryotes. Mechanisms controlling its developmental specificity and signal-responsiveness are poorly understood. Here, we identify an oxygen-sensitive N-terminal (N-) degron in the plant PRC2 subunit VERNALIZATION(VRN) 2, a homolog of animal Su(z)12, that promotes its degradation via the N-end rule pathway. We provide evidence that this N-degron arose early during angiosperm evolution via gene duplication and N-terminal truncation, facilitating expansion of PRC2 function in flowering plants. We show that proteolysis via the N-end rule pathway prevents ectopic VRN2 accumulation, and that hypoxia and long-term cold exposure lead to increased VRN2 abundance, which we propose may be due to inhibition of VRN2 turnover via its N-degron. Furthermore, we identify an overlap in the transcriptional responses to hypoxia and prolonged cold, and show that VRN2 promotes tolerance to hypoxia. Our work reveals a mechanism for post-translational regulation of VRN2 stability that could potentially link environmental inputs to the epigenetic control of plant development