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Succinate dehydrogenase (SDH, complex II), which plays an essential role in mitochondrial respiration and tricarboxylic acid metabolism, requires the assembly of eight nuclear-encoded subunits and the insertion of various cofactors. Here, we report on the characterization of an Arabidopsis thaliana leucine-tyrosine-arginine (LYR) protein family member SDHAF1, (At2g39725) is a factor required for SDH activity. SDHAF1 is located in mitochondria and can fully complement the yeast SDHAF1 deletion strain. Knockdown of SDHAF1 using RNA interference resulted in a decrease in seedling hypocotyl elongation and reduced SDH activity. Proteomic analyses revealed a decreased abundance of various SDH subunits and assembly factors. Protein interaction assays revealed that SDHAF1 can interact exclusively with the Fe-S cluster-containing subunit SDH2 and HSCB, a cochaperone involved in Fe-S cluster complex recruitment. Therefore, we propose that in Arabidopsis, SDHAF1 plays a role in the biogenesis of SDH2 to form the functional complex II, which is essential for mitochondrial respiration and metabolism.

A link between Pro catabolism and mitochondrial reactive oxygen species production has been established across eukaryotes and in plants increases in leaf respiration rates have been reported following Pro exposure. Here we investigated how alternative oxidases (AOXs) of the mitochondrial electron transport chain accommodate the large, atypical flux resulting from Pro catabolism and limit oxidative stress during Pro breakdown in mature Arabidopsis leaves. Following Pro treatment, AOX1a and AOX1d accumulate at transcript and protein levels, with AOX1d approaching the level of the typically dominant AOX1a isoform. We therefore sought to determine the function of both AOX isoforms under Pro respiring conditions. Oxygen consumption rate measurements in aox1a and aox1d leaves suggested these AOXs can functionally compensate for each other to establish enhanced AOX catalytic capacity in response to Pro. Generation of aox1a.aox1d lines showed complete loss of AOX proteins and activity upon Pro treatment, yet full respiratory induction in response to Pro remained possible via the cytochrome pathway. However, aox1a.aox1d leaves suffered increased levels of oxidative stress and damage during Pro metabolism compared to WT or the single mutants. During recovery from salt stress, when high rates of Pro catabolism occur naturally, photosynthetic rates in aox1a.aox1d recovered slower than WT or the single aox lines, showing that both AOX1a and AOX1d are beneficial for cellular metabolism during Pro drawdown following osmotic stress. This work provides physiological evidence of a beneficial role for AOX1a but also the less studied AOX1d isoform in allowing safe catabolism of alternative respiratory substrates like Pro.One sentence summaryThe alternative oxidase of plant mitochondria contributes to Pro catabolism by preventing oxidative stress in the electron transport chain and this aids recovery of leaf metabolic rates following salinity stress.

SummaryThe impact of salinity on wheat plants is often studied by analysis of shoot responses, even though the main mechanism of tolerance is shoot Na+ exclusion. There is a need to understand the molecular responses of root tissues that directly experience rising NaCl concentrations. We have combined analysis of root growth, ion content and respiration with proteome responses in wheat root tip and mature root tissues under saline conditions. We find significant changes in translation and protein synthesis, energy metabolism and amino acid metabolism in a root tissue specific manner. Translation and protein synthesis related proteins showed significant decreases in abundance only in root tips, as did most of the glycolytic enzymes and selected TCA cycle enzymes and ATP synthase subunits. This selective root tip proteome response indicates protein synthesis capacity and energy production were impaired under salt stress, correlating with the anatomical response of roots and reduced root tip respiration rate. Wheat roots respond directly to soil salinity, therefore shoot responses such as reduction in shoot growth and photosynthetic capacity need to be considered in light of these effects.

ePlant was introduced in 2017 for exploring large Arabidopsis thaliana data sets from the kilometre to nanometre scales. In the past four years we have used the ePlant framework to develop ePlants for 15 agronomically-important species: maize, poplar, tomato, Camelina sativa, soybean, potato, barley, Medicago truncatula, eucalyptus, rice, willow, sunflower, Cannabis sativa, wheat and sugarcane. We also updated the interface to improve performance and accessibility, and added two new views to the Arabidopsis ePlant – the Navigator and Pathways viewers. The former shows phylogenetic relationships between homologs in other species and their expression pattern similarities, with links to view data for those genes in the respective ePlants. The latter shows Plant Reactome metabolic reactions. We also describe new Arabidopsis data sets including single cell RNA-seq data from roots, and how to embed ePlant eFP expression pictographs into any web page.

Identification of autophagic protein cargo in plants by their abundance in autophagy related genes (ATG) mutants is complicated by changes in both protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis atg5 and atg11 mutant plants. These data show that less than a quarter of proteins changing in abundance are probable cargo and revealed roles of ATG11 and ATG5 in degradation of specific cytosol, chloroplast and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts. Our data support a role for autophagy in degrading glycolytic enzymes and the chaperonin containing T-complex polypeptide-1 complex. Autophagy induction by Pi limitation changed metabolic profiles and the protein synthesis and degradation rates of atg5 and atg11 plants. A general decrease in the abundance of amino acids and increase in several secondary metabolites in autophagy mutants was consistent with altered catabolism and changes in energy conversion caused by reduced degradation rate of specific proteins. Combining measures of changes in protein abundance and degradation rates, we also identify ATG11 and ATG5 associated protein cargo of low Pi induced autophagy in chloroplasts and ER-resident proteins involved in secondary metabolism.Single Sentence SummaryProtein cargo of autophagy in plants can be discovered by identifying proteins that increase in abundance and decrease in degradation rate in mutants deficient in autophagy machinery

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation condition and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12h post illumination. We investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in photomorphogenesis. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected photomorphogenesis. Moreover, we showed that lower levels of carbon resources exacerbated the delay in photomorphogenesis whereas higher levels of carbon resources had an opposite effect. This work provides evidence that autophagic process operate during de-etiolation in a manner that contributes to photomorphogenesis through increasing lipid turnover to physically or energetically sustain photomorphogenesis.

Determining which proteins are actively synthesised at a given point in time and extracting them for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables BONCAT (Bio-Orthogonal Non-Canonical Amino acid Tagging) of proteins being synthesised in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met-sites throughout protein AA sequences and correlation with independent studies of protein labelling with 15N verified the data. We provide evidence that HPG-based BONCAT tags nascent plant proteins more efficiently than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that AHA’s induction of Met metabolism and greater inhibition of cell growth rate than HPG likely limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for determining which plant proteins are being synthesised at a given time point and enriches new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of PTMs in plants.

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate (OAA) and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration.13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis were largely maintained except for alanine and glutamate, indicating that transamination contributes to restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases (AlaAT) when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

The cytosolic 80S ribosome is composed of protein and RNA molecules and its function in protein synthesis is modulated through interaction with other cytosolic components. Defining the role of each of the proteins associated with ribosomes in plants is a major challenge which is hampered by difficulties in attribution of different proteins to roles in ribosome biogenesis, the mature cytosolic ribosome (r-proteins) or to the broader translatome associated with functioning ribosomes. Here we refined the core r-protein composition in plants by determining the abundance of proteins in low, partially and highly purified ribosomal samples from Arabidopsis thaliana cell cultures. To characterise this list of proteins further we determined their degradation (KD) and synthesis (KS) rate by progressive labelling with 15N combined with peptide mass spectrometry analysis. The turnover rates of 55 r-proteins, including 26 r-proteins from the 40S subunit and 29 r-proteins from the 60S subunit could be determined. Overall, ribosome proteins showed very similar KD and KS rates suggesting that half of the ribosome population is replaced every 3-4 days. Three proteins showed significantly shorter half-lives; ribosomal protein P0D (RPP0D) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The ribosomal RPP0D protein is a homolog of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role as a 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins in Arabidopsis. Of this set, 26 were enriched more than 10-fold during ribosome purification. A number have known roles in translation or ribosome-association while others are newly proposed ribosome-associated factors in plants. This analysis provides a more robust understanding of Arabidopsis ribosome content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and reveals how protein turnover can identify r-proteins involved in ribosome biogenesis or regulation in plants. Data are available via ProteomeXchange with identifier PXD012839.