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The Russian dandelion (Taraxacum koksaghyz) is a promising source of natural rubber (NR). The synthesis of NR takes place on the surface of organelles known as rubber particles, which are found in latex – the cytoplasm of specialized cells known as laticifers. As well as the enzymes directly responsible for NR synthesis, the rubber particles also contain small rubber particle proteins (SRPPs), the most abundant of which are SRPP3, 4 and 5. These three proteins support NR synthesis by maintaining rubber particle stability. We used homology-based searches to identify the whole TkSRPP gene family and qPCR to create their spatial expression profiles. Affinity enrichment-mass spectrometry was applied to identify TkSRPP3/4/5 protein interaction partners in T. koksaghyz latex and selected interaction partners were analyzed using qPCR, confocal laser scanning microscopy and heterologous expression in yeast. We identified 17 SRPP-like sequences in the T. koksaghyz genome, including three apparent pseudogenes, 10 paralogs arranged as an inverted repeat in a cluster with TkSRPP3/4/5 , and one separate gene (TkSRPP6). Their sequence diversity and different expression profiles indicated distinct functions and the latex interactomes obtained for TkSRPP3/4/5 suggested that TkSRPP4 is a promiscuous hub protein that binds many partners from different compartments, whereas TkSRPP3 and 5 have more focused interactomes. Two interactors shared by TkSRPP3/4/5 (TkSRPP6 and TkUGT80B1) were chosen for independent validation and detailed characterization. TkUGT80B1 triterpenoid glycosylating activity provided first evidence for triterpenoid saponin synthesis in T. koksaghyz latex. Based on its identified interaction partners, TkSRPP4 appears to play a special role in the endoplasmic reticulum, interacting with lipidmodifying enzymes that may facilitate rubber particle formation. TkSRPP5 appears to be involved in GTPase-dependent signaling and TkSRPP3 may act as part of a kinase signaling cascade, with roles in stress tolerance. TkSRPP interaction with TkUGT80B1 draws a new connection between TkSRPPs and triterpenoid saponin synthesis in T. koksaghyz latex. Our data contribute to the functional differentiation between TkSRPP paralogs and demonstrate unexpected interactions that will help to further elucidate the network of proteins linking TkSRPPs, stress responses and NR biosynthesis within the cellular complexity of latex. [ABSTRACT FROM AUTHOR]

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Malate dehydrogenases (MDHs) catalyze a reversible NAD(P)‐dependent‐oxidoreductase reaction that plays an important role in central metabolism and redox homeostasis of plant cells. Recent studies suggest a moonlighting function of plastidial NAD‐dependent MDH (plNAD‐MDH; EC 1.1.1.37) in plastid biogenesis, independent of its enzyme activity. In this study, redox effects on activity and conformation of recombinant plNAD‐MDH from Arabidopsis thaliana were investigated. We show that reduced plNAD‐MDH is active while it is inhibited upon oxidation. Interestingly, the presence of its cofactors NAD+ and NADH could prevent oxidative inhibition of plNAD‐MDH. In addition, a conformational change upon oxidation could be observed via non‐reducing SDS‐PAGE. Both effects, its inhibition and conformational change, were reversible by re‐reduction. Further investigation of single cysteine substitutions and mass spectrometry revealed that oxidation of plNAD‐MDH leads to oxidation of all four cysteine residues. However, cysteine oxidation of C129 leads to inhibition of plNAD‐MDH activity and oxidation of C147 induces its conformational change. In contrast, oxidation of C190 and C333 does not affect plNAD‐MDH activity or structure. Our results demonstrate that plNAD‐MDH activity can be reversibly inhibited, but not inactivated, by cysteine oxidation and might be co‐regulated by the availability of its cofactors in vivo. [ABSTRACT FROM AUTHOR]

This article discusses the multilayered regulation of plastids and mitochondria, which are essential organelles in plant cells. The genomes of these organelles have been difficult to study, but recent developments in genome editing tools are providing new opportunities for manipulation and exploration. The article includes reviews and original articles that cover topics such as organellar genome dynamics, post-transcriptional regulation, and further multilayered regulation. The authors hope that this research will lead to a better understanding of these organelles and advancements in plant science. [Extracted from the article]

SUMMARY: Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light‐dependent post‐translational modifications (PTM) and have been studied at depth at the level of individual proteins. In contrast, a global picture of the light‐dependent PTMome dynamics is lacking, leaving the response of a large proportion of cellular function undefined. Here, we investigated the light‐dependent metabolome and proteome changes in Arabidopsis rosettes in a time resolved manner to dissect their kinetic interplay, focusing on phosphorylation, lysine acetylation, and cysteine‐based redox switches. Of over 24 000 PTM sites that were detected, more than 1700 were changed during the transition from dark to light. While the first changes, as measured 5 min after onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin–Benson cycle and the synthesis of sugars at later timepoints. Our data reveal connections between metabolism and PTM‐based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insight into the extent by which photosynthesis reprograms global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function. [ABSTRACT FROM AUTHOR]

SUMMARY: Post‐translational modifications (PTMs) of proteins play important roles in the acclimation of plants to environmental stress. Lysine acetylation is a dynamic and reversible PTM, which can be removed by histone deacetylases. Here we investigated the role of lysine acetylation in the response of Arabidopsis leaves to 1 week of salt stress. A quantitative mass spectrometry analysis revealed an increase in lysine acetylation of several proteins from cytosol and plastids, which was accompanied by altered histone deacetylase activities in the salt‐treated leaves. While activities of HDA14 and HDA15 were decreased upon salt stress, HDA5 showed a mild and HDA19 a strong increase in activity. Since HDA5 is a cytosolic‐nuclear enzyme from the class II histone deacetylase family with yet unknown protein substrates, we performed a lysine acetylome analysis on hda5 mutants and characterized its substrate proteins. Next to histone H2B, the salt stress‐responsive transcription factor GT2L and the dehydration‐related protein ERD7 were identified as HDA5 substrates. In addition, in protein–protein interaction studies, HDA18 was discovered, among other interacting proteins, to work in a complex together with HDA5. Altogether, this study revealed the substrate proteins of HDA5 and identified new lysine acetylation sites which are hyperacetylated upon salt stress. The identification of specific histone deacetylase substrate proteins, apart from histones, will be important to unravel the acclimation response of Arabidopsis to salt stress and their role in plant physiology. [ABSTRACT FROM AUTHOR]

Acetylation is one of the most common chemical modifications found on a variety of molecules ranging from metabolites to proteins. Although numerous chloroplast proteins have been shown to be acetylated, the role of acetylation in the regulation of chloroplast functions has remained mainly enigmatic. The chloroplast acetylation machinery in Arabidopsis thaliana consists of eight General control non-repressible 5 (GCN5)-related N -acetyltransferase (GNAT)–family enzymes that catalyze both N-terminal and lysine acetylation of proteins. Additionally, two plastid GNATs have also been reported to be involved in the biosynthesis of melatonin. Here, we have characterized six plastid GNATs (GNAT1, GNAT2, GNAT4, GNAT6, GNAT7 and GNAT10) using a reverse genetics approach with an emphasis on the metabolomes and photosynthesis of the knock-out plants. Our results reveal the impact of GNAT enzymes on the accumulation of chloroplast-related compounds, such as oxylipins and ascorbate, and the GNAT enzymes also affect the accumulation of amino acids and their derivatives. Specifically, the amount of acetylated arginine and proline was significantly decreased in the gnat2 and gnat7 mutants, respectively, as compared to the wild-type Col-0 plants. Additionally, our results show that the loss of the GNAT enzymes results in increased accumulation of Rubisco and Rubisco activase (RCA) at the thylakoids. Nevertheless, the reallocation of Rubisco and RCA did not have consequent effects on carbon assimilation under the studied conditions. Taken together, our results show that chloroplast GNATs affect diverse aspects of plant metabolism and pave way for future research into the role of protein acetylation. [ABSTRACT FROM AUTHOR]