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EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae). Knockout of ENOD93 in Arabidopsis (Arabidopsis thaliana) causes a short root phenotype and early flowering. ENOD93 is associated with a protein complex the size of CIV in mitochondria, but neither CIV abundance nor its activity changed in ruptured organelles of enod93. However, a progressive loss of ADP-dependent respiration rate was observed in intact enod93 mitochondria, which could be recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 in a CIV-dependent manner, but ATP synthesis and ADP depletion rates progressively decreased. The respiration rate of whole enod93 seedlings was elevated, and root ADP content was nearly double that in wild type without a change in ATP content. We propose that ENOD93 and HYPOXIA-INDUCED GENE DOMAIN 2 (HIGD2) are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded by 2 distinct genes., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Loss of Lon1 led to stunted plant growth and accumulation of nuclear-encoded mitochondrial proteins including Lon1 substrates. However, an in-depth label-free proteomics quantification of mitochondrial proteins in lon1 revealed that the majority of mitochondrial-encoded proteins decreased in abundance. Additionally, we found that lon1 mutants contained protein aggregates in the mitochondrial that were enriched in metabolic enzymes, ribosomal subunits and PPR-containing proteins of the translation apparatus. These mutants exhibited reduced general mitochondrial translation as well as deficiencies in RNA splicing and editing. These findings support the role of Lon1 in maintaining a functional translational apparatus for mitochondrial-encoded gene translation. Transcriptome analysis of lon1 revealed a mitochondrial unfolded protein response reminiscent of the mitochondrial retrograde signalling dependent on the transcription factor ANAC017. Notably, lon1 mutants exhibited transiently elevated ethylene production, and the shortened hypocotyl observed in lon1 mutants during skotomorphogenesis was partially alleviated by ethylene inhibitors. Furthermore, the short root phenotype was partially ameliorated by introducing a mutation in the ethylene receptor ETR1. Interestingly, the upregulation of only a select few target genes was linked to ETR1-mediated ethylene signalling. Together this provides multiple steps in the link between loss of Lon1 and signalling responses to restore mitochondrial protein homoeostasis in plants., (© 2024 John Wiley & Sons Ltd.)

Spontaneous mutations are rare in mitochondria and the lack of mitochondrial transformation methods has hindered genetic analyses. We show that a custom-designed RNA-binding pentatricopeptide repeat (PPR) protein binds and specifically induces cleavage of ATP synthase subunit1 (atp1) mRNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis (Arabidopsis thaliana). The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits and lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates, or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP synthase-depleted lines showed higher respiratory rates and elevated steady-state levels of numerous amino acids, most notably of the serine family. The results show the value of using custom-designed PPR proteins to influence the expression of specific mitochondrial transcripts to carry out reverse genetic studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy., Competing Interests: HL, JC, AG, KC, JB, SS, AM, RV, KE, MO No competing interests declared, (© 2023, Lee et al.)

Identification of autophagic protein cargo in plants in autophagy-related genes (ATG) mutants is complicated by changes in protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis (Arabidopsis thaliana) atg5 and atg11 mutants. 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 glycolytic enzymes and of other cytosol, chloroplast, and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts. Protein localization in transformed protoplasts and degradation assays in the presence of inhibitors confirm a role for autophagy in degrading glycolytic enzymes. Autophagy induction by phosphate (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 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., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light–induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of the photosystem II (PSII) D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate-citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilization in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Metabolism, auxin signaling and reactive oxygen species (ROS) all contribute to plant growth, and each is linked to plant mitochondria and the process of respiration. Knockdown of mitochondrial succinate dehydrogenase assembly factor 2 (SDHAF2) in Arabidopsis thaliana lowered succinate dehydrogenase activity and led to pH-inducible root inhibition when the growth medium pH was poised at different points between 7.0 and 5.0, but this phenomenon was not observed in wildtype (WT). Roots of sdhaf2 mutants showed high accumulation of succinate, depletion of citrate and malate and up-regulation of ROS-related and stress-inducible genes at pH 5.5. A change of oxidative status in sdhaf2 roots at low pH was also evidenced by low ROS staining in root tips and altered root sensitivity to H2O2. sdhaf2 had low auxin activity in root tips via DR5-GUS staining but displayed increased indole-3-acetic acid (IAA, auxin) abundance and IAA hypersensitivity, which is most likely caused by the change in ROS levels. On this basis, we conclude that knockdown of SDHAF2 induces pH-related root elongation and auxin hyperaccumulation and hypersensitivity, mediated by altered ROS homeostasis. This observation extends the existing evidence of associations between mitochondrial function and auxin by establishing a cascade of cellular events that link them through ROS formation, metabolism and root growth at different pH values., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation conditions 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 12 h post-illumination. We observed that while gene expression for photosynthetic machinery during de-etiolation was largely unaffected in atg mutants, several genes involved in photosystem assembly were transcriptionally downregulated. We also 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 chloroplast development. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected de-etiolation. Moreover, we showed that lower levels of carbon resources exacerbated the slow greening phenotype whereas higher levels of carbon resources had an opposite effect. This work suggests a lack of autophagy machinery limits chloroplast development during de-etiolation, and this is exacerbated by limited lipid turnover (lipophagy) that physically or energetically restrains chloroplast development., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate 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 putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana 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 was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases 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., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Recent studies in Arabidopsis (Arabidopsis thaliana) have reported conflicting roles for NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), a transcription factor regulating mitochondria-to-nuclear signaling, and its closest paralog NAC DOMAIN CONTAINING PROTEIN 16 (ANAC016), in leaf senescence. By synchronizing senescence in individually darkened leaves of knockout and overexpressing mutants from these contrasting studies, we demonstrate that elevated ANAC017 expression consistently causes accelerated senescence and cell death. A time-resolved transcriptome analysis revealed that senescence-associated pathways such as autophagy are not constitutively activated in ANAC017 overexpression lines, but require a senescence-stimulus to trigger accelerated induction. ANAC017 transcript and ANAC017-target genes are constitutively upregulated in ANAC017 overexpression lines, but surprisingly show a transient "super-induction" 1 d after senescence induction. This induction of ANAC017 and its target genes is observed during the later stages of age-related and dark-induced senescence, indicating the ANAC017 pathway is also activated in natural senescence. In contrast, knockout mutants of ANAC017 showed lowered senescence-induced induction of ANAC017 target genes during the late stages of dark-induced senescence. Finally, promoter binding analyses show that the ANAC016 promoter sequence is directly bound by ANAC017, so ANAC016 likely acts downstream of ANAC017 and is directly transcriptionally controlled by ANAC017 in a feed-forward loop during late senescence., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized 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 amino acid sequences and correlation with independent studies of protein labelling with 15 N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples 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 post-translational modifications in plants., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Multiple studies have shown ribulose-1,5-bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39; Rubisco) to be subject to Lys-acetylation at various residues; however, opposing reports exist about the biological significance of these post-translational modifications. One aspect of the Lys-acetylation that has not been addressed in plants generally, or with Rubisco specifically, is the stoichiometry at which these Lys-acetylation events occur. As a method to ascertain which Lys-acetylation sites on Arabidopsis Rubisco might be of regulatory importance to its catalytic function in the Calvin-Benson cycle, we purified Rubisco from leaves in both the day and night-time and performed independent mass spectrometry based methods to determine the stoichiometry of Rubisco Lys-acetylation events. The results indicate that Rubisco is acetylated at most Lys residues, but each acetylation event occurs at very low stoichiometry. Furthermore, in vitro treatments that increased the extent of Lys-acetylation on purified Rubisco had no effect on Rubisco maximal activity. Therefore, we are unable to confirm that Lys-acetylation at low stoichiometries can be a regulatory mechanism controlling Rubisco maximal activity. The results highlight the need for further use of stoichiometry measurements when determining the biological significance of reversible PTMs like acetylation., (© 2020 The Author(s).)

Protein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear- and mitochondrial-encoded subunits for complex assembly in mitochondria await discovery. We generated knockout lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis ( Arabidopsis thaliana ). Mutants showed a higher abundance of transcripts from mitochondrial genes encoding oxidative phosphorylation protein complexes, whereas nuclear genes encoding other subunits of the same complexes showed no change in transcript abundance. By contrast, the protein abundance of specific nuclear-encoded subunits in oxidative phosphorylation complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Complexes with subunits mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import and function of complex I revealed that while function was retained, protein homeostasis was disrupted, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and homeostasis of protein complexes encoded across mitochondrial and nuclear genomes., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O 2 consumption rate (R N ) in mature leaves of Arabidopsis ( Arabidopsis thaliana ). Multi-hour R N measurements following leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids) identified phospho enol pyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf R N Using metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling R N Many amino acids, as well as Glc analogs, were found to potently inhibit the R N stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro- and Ala-stimulated R N were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway. Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes., (© 2020 American Society of Plant Biologists. All rights reserved.)

Pyruvate dehydrogenase is the first enzyme (E1) of the PDH complex (PDC). This multienzyme complex contains E1, E2, and E3 components and controls the entry of carbon into the mitochondrial tricarboxylic acid cycle to enable cellular energy production. The E1 component of the PDC is composed of an E1α catalytic subunit and an E1β regulatory subunit. In Arabidopsis ( Arabidopsis thaliana ), there are two mitochondrial E1α homologs encoded by IAA-CONJUGATE-RESISTANT 4 ( IAR4 ) and IAR4-LIKE ( IAR4L ), and one mitochondrial E1β homolog. Although IAR4 was reported to be involved in auxin conjugate sensitivity and auxin homeostasis in root development, its precise role remains unknown. Here, we provide experimental evidence that mitochondrial PDC E1 contributes to polar auxin transport during organ development. We performed genetic screens for factors involved in cotyledon development and identified an uncharacterized mutant, macchi-bou 1 ( mab1 ). MAB1 encodes a mitochondrial PDC E1β subunit that can form both a homodimer and a heterodimer with IAR4. The mab1 mutation impaired MAB1 homodimerization, reduced the abundance of IAR4 and IAR4L, weakened PDC enzymatic activity, and diminished mitochondrial respiration. A metabolomics analysis showed significant changes in metabolites including amino acids in mab1 and, in particular, identified an accumulation of Ala. These results suggest that MAB1 is a component of the Arabidopsis mitochondrial PDC E1. Furthermore, in mab1 mutants and seedlings where the TCA cycle was pharmacologically blocked, we found reduced abundance of the PIN-FORMED (PIN) auxin efflux carriers, possibly due to impaired PIN recycling and enhanced PIN degradation in vacuoles. Therefore, we suggest that mab1 induces defective polar auxin transport via metabolic abnormalities., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

The combined action of the electron transport chain (ETC) and ATP synthase is essential in determining energy efficiency in plants, and so is important for cellular biosynthesis, growth and development. Owing to the sessile nature of plants, mitochondria must operate over a wide temperature range in the environment, necessitating a broad temperature tolerance of their biochemical reactions. We investigated the temperature response of mitochondrial respiratory processes in isolated mitochondria and intact plants of Arabidopsis thaliana and considered the effect of instantaneous responses to temperature and acclimation responses to low temperatures. We show that at 4°C the plant mitochondrial ATP synthase is differentially inhibited compared with other elements of the respiratory pathway, leading to decreased ADP : oxygen ratios and a limitation to the rate of ATP synthesis. This effect persists in vivo and cannot be overcome by cold-temperature acclimation of plants. This mechanism adds a new element to the respiratory acclimation model and provides a direct means of temperature perception by plant mitochondria. This also provides an alternative explanation for non-phosphorylating ETC bypass mechanisms, like the alternative oxidase to maintain respiratory rates, albeit at lower ATP synthesis efficiency, in response to the sensitivity of ATP synthase to the prevailing temperature., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Succinate dehydrogenase (Complex II; SDH) plays an important role in mitochondrial respiratory metabolism. The SDH complex consists of four core subunits and multiple cofactors, which must be assembled correctly to ensure enzyme function. To date, only an assembly factor (SDHAF2) required for FAD insertion into subunit SDH1 has been identified in plants. Here, we report the identification of Arabidopsis ( Arabidopsis thaliana ) At5g67490 as a second SDH assembly factor. Knockout of At5g67490 ( sdhaf4 ) did not cause any phenotypic variation in seedlings but resulted in a decrease in both SDH activity and the succinate-dependent respiration rate as well as increased accumulation of succinate. Mass spectrometry analyses revealed stable levels of FAD-SDH1 in sdhaf4 , together with increased levels of the FAD-SDH1 assembly factor, SDHAF2, and reduced levels of SDH2 compared with the wild type. Loss of SDHAF4 in sdhaf4 inhibited the formation of the SDH1/SDH2 intermediate, leading to the accumulation of soluble SDH1 in the mitochondrial matrix and reduced levels of SDH1 in the membrane. The increased levels of SDHAF2 suggest that the stabilization of soluble FAD-SDH1 depends on SDHAF2 availability. We conclude that SDHAF4 acts on FAD-SDH1 and promotes its assembly with SDH2, thereby stabilizing SDH2 and enabling its full assembly with SDH3/SDH4 to form the SDH complex., (© 2018 American Society of Plant Biologists. All rights reserved.)

Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live)., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Lon is a highly conserved protein family in eukaryotes and eubacteria and its members all contain both a chaperone and a proteolytic domain that are important for Lon function. Loss of mitochondrial Lon1 leads to deleterious phenotypes in yeast and plants, and causes developmental disorders and aging-related diseases in humans. In Arabidopsis, we have recently reported the multiple roles of Lon1 in mitochondrial protein homeostasis through an evaluation of changes in protein degradation rates in the absence of Lon1. 1 In this addendum, we extend our discussion to the roles of Lon1 in mitochondrial post-transcriptional regulation by considering the effects of its loss on ribosome proteins required for protein synthesis and mitochondrial PPR proteins required for RNA regulation.

Mitochondrial Lon1 loss impairs oxidative phosphorylation complexes and TCA enzymes and causes accumulation of specific mitochondrial proteins. Analysis of over 400 mitochondrial protein degradation rates using 15 N labelling showed that 205 were significantly different between wild type (WT) and lon1-1. Those proteins included ribosomal proteins, electron transport chain subunits and TCA enzymes. For respiratory complexes I and V, decreased protein abundance correlated with higher degradation rate of subunits in total mitochondrial extracts. After blue native separation, however, the assembled complexes had slow degradation, while smaller subcomplexes displayed rapid degradation in lon1-1. In insoluble fractions, a number of TCA enzymes were more abundant but the proteins degraded slowly in lon1-1. In soluble protein fractions, TCA enzymes were less abundant but degraded more rapidly. These observations are consistent with the reported roles of Lon1 as a chaperone aiding the proper folding of newly synthesized/imported proteins to stabilise them and as a protease to degrade mitochondrial protein aggregates. HSP70, prohibitin and enzymes of photorespiration accumulated in lon1-1 and degraded slowly in all fractions, indicating an important role of Lon1 in their clearance from the proteome., (© 2016 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

We applied 15 N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15 N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome., (© 2017 American Society of Plant Biologists. All rights reserved.)

Background: Papain-like Cys Proteases (PLCPs) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly expressed proteases during leaf senescence in Arabidopsis. Using activity-based protein profiling (ABPP), a method that enables detection of active enzymes within a complex sample using chemical probes, the activities of PLCPs and VPEs were investigated in individually darkened leaves of Arabidopsis, and their role in senescence was tested in null mutants., Results: ABPP and mass spectrometry revealed an increased activity of several PLCPs, particularly RD21A and AALP. By contrast, despite increased VPE transcript levels, active VPE decreased in individually darkened leaves. Eight protease knock-out lines and two protease over expressing lines were subjected to senescence phenotype analysis to determine the importance of individual protease activities to senescence. Unexpectedly, despite the absence of dominating PLCP activities in these plants, the rubisco and chlorophyll decline in individually darkened leaves and the onset of whole plant senescence were unaltered. However, a significant delay in progression of whole plant senescence was observed in aalp-1 and rd21A-1/aalp-1 mutants, visible in the reduced number of senescent leaves., Conclusions: Major Cys protease activities are not essential for dark-induced and developmental senescence and only a knock out line lacking AALP shows a slight but significant delay in plant senescence.

The SUBcellular location database for Arabidopsis proteins (SUBA4, http://suba.live) is a comprehensive collection of manually curated published data sets of large-scale subcellular proteomics, fluorescent protein visualization, protein-protein interaction (PPI) as well as subcellular targeting calls from 22 prediction programs. SUBA4 contains an additional 35 568 localizations totalling more than 60 000 experimental protein location claims as well as 37 new suborganellar localization categories. The experimental PPI data has been expanded to 26 327 PPI pairs including 856 PPI localizations from experimental fluorescent visualizations. The new SUBA4 user interface enables users to choose quickly from the filter categories: 'subcellular location', 'protein properties', 'protein-protein interaction' and 'affiliations' to build complex queries. This allows substantial expansion of search parameters into 80 annotation types comprising 1 150 204 new annotations to study metadata associated with subcellular localization. The 'BLAST' tab contains a sequence alignment tool to enable a sequence fragment from any species to find the closest match in Arabidopsis and retrieve data on subcellular location. Using the location consensus SUBAcon, the SUBA4 toolbox delivers three novel data services allowing interactive analysis of user data to provide relative compartmental protein abundances and proximity relationship analysis of PPI and coexpression partners from a submitted list of Arabidopsis gene identifiers., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondria are the sites of a diverse set of essential biochemical processes in plants. In order to facilitate the analysis of these functions, this chapter presents protocols for the isolation of intact mitochondria from a range of plant tissues as well two workflows for fractionation into their four subcompartments; the inner and outer membranes and the two aqueous compartments, the inter membrane space and matrix. Protocols for the assessment of mitochondrial integrity and purity through enzymatic function and suggestions of commercially available compartment marker antibodies are provided.

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Mitochondria are crucial for plant viability and are able to communicate information on their functional status to the cellular nucleus via retrograde signalling, thereby affecting gene expression. It is currently unclear if retrograde signalling in response to constitutive mitochondrial biogenesis defects is mediated by the same pathways as those triggered during acute mitochondrial dysfunction. Furthermore, it is unknown if retrograde signalling can effectively improve plant performance when mitochondrial function is constitutively impaired. Here we show that retrograde signalling in mutants defective in mitochondrial proteins RNA polymerase rpotmp or prohibitin atphb3 can be suppressed by knocking out the transcription factor ANAC017. Genome-wide RNA-seq expression analysis revealed that ANAC017 is almost solely responsible for the most dramatic transcriptional changes common to rpotmp and atphb3 mutants, regulating classical marker genes such as alternative oxidase 1a (AOX1a) and also previously-uncharacterised DUF295 genes that appear to be new retrograde markers. In contrast, ANAC017 does not regulate intra-mitochondrial gene expression or transcriptional changes unique to either rpotmp or atphb3 genotype, suggesting the existence of currently unknown signalling cascades. The data show that ANAC017 function extends beyond common retrograde transcriptional responses and affects downstream protein abundance and enzyme activity of alternative oxidase, as well as steady-state energy metabolism in atphb3 plants. Furthermore, detailed growth analysis revealed that ANAC017-dependent retrograde signalling provides benefits for growth and productivity in plants with mitochondrial defects. In conclusion, ANAC017 plays a key role in both biogenic and operational mitochondrial retrograde signalling, and improves plant performance when mitochondrial function is constitutively impaired., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1:At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell., (Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.)

Glutaredoxins (Grxs) are small proteins that function as oxidoreductases with roles in deglutathionylation of proteins, reduction of antioxidants, and assembly of iron-sulfur (Fe-S) cluster-containing enzymes. Which of the 33 Grxs in Arabidopsis (Arabidopsis thaliana) perform roles in Fe-S assembly in mitochondria is unknown. We have examined in detail the function of the monothiol GrxS15 in plants. Our results show its exclusive mitochondrial localization, and we are concluding it is the major or only Grx in this subcellular location. Recombinant GrxS15 has a very low deglutathionylation and dehydroascorbate reductase activity, but it binds a Fe-S cluster. Partially removing GrxS15 from mitochondria slowed whole plant growth and respiration. Native GrxS15 is shown to be especially important for lipoic acid-dependent enzymes in mitochondria, highlighting a putative role in the transfer of Fe-S clusters in this process. The enhanced effect of the toxin arsenic on the growth of GrxS15 knockdown plants compared to wild type highlights the role of mitochondrial glutaredoxin Fe-S-binding in whole plant growth and toxin tolerance., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Precursor proteins containing mitochondrial peptide signals are cleaved after import by a mitochondrial processing peptidase. In yeast (Saccharomyces cerevisiae) and human (Homo sapiens), intermediate cleavage peptidase55 (ICP55) plays a role in stabilizing mitochondrial proteins by the removal of single amino acids from mitochondrial processing peptidase-processed proteins. We have investigated the role of a metallopeptidase (At1g09300) from Arabidopsis (Arabidopsis thaliana) that has sequence similarity to yeast ICP55. We identified this protein in mitochondria by mass spectrometry and have studied its function in a transfer DNA insertion line (icp55). Monitoring of amino-terminal peptides showed that Arabidopsis ICP55 was responsible for the removal of single amino acids, and its action explained the -3 arginine processing motif of a number of mitochondrial proteins. ICP55 also removed single amino acids from mitochondrial proteins known to be cleaved at nonconserved arginine sites, a subset of mitochondrial proteins specific to plants. Faster mitochondrial protein degradation rates not only for ICP55 cleaved protein but also for some non-ICP55 cleaved proteins were observed in Arabidopsis mitochondrial samples isolated from icp55 than from the wild type, indicating that a complicated protease degradation network has been affected. The lower protein stability of isolated mitochondria and the lack of processing of target proteins in icp55 were complemented by transformation with the full-length ICP55. Analysis of in vitro degradation rates and protein turnover rates in vivo of specific proteins indicated that serine hydroxymethyltransferase was affected in icp55. The maturation of serine hydroxymethyltransferase by ICP55 is unusual, as it involves breaking an amino-terminal diserine that is not known as an ICP55 substrate in other organisms and that is typically considered a sequence that stabilizes rather than destabilizes a protein., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Motivation: Knowing the subcellular location of proteins is critical for understanding their function and developing accurate networks representing eukaryotic biological processes. Many computational tools have been developed to predict proteome-wide subcellular location, and abundant experimental data from green fluorescent protein (GFP) tagging or mass spectrometry (MS) are available in the model plant, Arabidopsis. None of these approaches is error-free, and thus, results are often contradictory., Results: To help unify these multiple data sources, we have developed the SUBcellular Arabidopsis consensus (SUBAcon) algorithm, a naive Bayes classifier that integrates 22 computational prediction algorithms, experimental GFP and MS localizations, protein-protein interaction and co-expression data to derive a consensus call and probability. SUBAcon classifies protein location in Arabidopsis more accurately than single predictors., Availability: SUBAcon is a useful tool for recovering proteome-wide subcellular locations of Arabidopsis proteins and is displayed in the SUBA3 database (http://suba.plantenergy.uwa.edu.au). The source code and input data is available through the SUBA3 server (http://suba.plantenergy.uwa.edu.au//SUBAcon.html) and the Arabidopsis SUbproteome REference (ASURE) training set can be accessed using the ASURE web portal (http://suba.plantenergy.uwa.edu.au/ASURE)., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild-type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady-state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild-type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis., (© 2013 John Wiley & Sons Ltd.)

In reverse genetic knockout (KO) studies that aim to assign function to specific genes, confirming the reduction in abundance of the encoded protein will often aid the link between genotype and phenotype. However, measuring specific protein abundance is particularly difficult in plant research, where only a limited number of antibodies are available. This problem is enhanced when studying gene families or different proteins derived from the same gene (isoforms), as many antibodies cross react with more than one protein. We show that utilizing selected reaction monitoring (SRM) mass spectrometry allows researchers to confirm protein abundance in mutant lines, even when discrimination between very similar proteins is needed. Selecting the best peptides for SRM analysis to ensure that protein- or gene-specific information can be obtained requires a series of steps, aids, and interpretation. To enable this process in Arabidopsis (Arabidopsis thaliana), we have built a Web-based tool, the Arabidopsis Proteotypic Predictor, to select candidate SRM transitions when no previous mass spectrometry evidence exists. We also provide an in-depth analysis of the theoretical Arabidopsis proteome and its use in selecting candidate SRM peptides to establish assays for use in determining protein abundance. To test the effectiveness of SRM mass spectrometry in determining protein abundance in mutant lines, we selected two enzymes with multiple isoforms, aconitase and malate dehydrogenase. Selected peptides were quantified to estimate the abundance of each of the two mitochondrial isoforms in wild-type, KO, double KO, and complemented plant lines. We show that SRM protein analysis is a sensitive and rapid approach to quantify protein abundance differences in Arabidopsis for specific and highly related enzyme isoforms.

The turnover of the proteomes of organelles in plant cells are known to be governed by both whole cell and organelle-specific processes. However, the rate and specificity of this protein turnover has not been explored in depth to understand how it affects different organellar processes. Here we have used progressive ¹⁵N labeling of Arabidopsis cells, and focused on the turnover rate of proteins in mitochondria. We provide estimates of degradation rate (K(d)) for 224 mitochondrial proteins, showing a range of over 50-fold in K(d). Protein complexes, most notably the respiratory chain complexes, had K(d) values that were generally coordinated and we have interpreted these measurements to outline how protein K(d) differs within protein complexes and between functional categories. The fastest turnover rates were reported for DNA/RNA metabolism enzymes, chaperones, and proteases.

An individually darkened leaf model was used to study protein changes in the Arabidopsis mutant stay-green1 (sgr1) to partially mimic the process of leaf covering senescence that occurs naturally in the shaded rosettes of Arabidopsis plants. Utilizing this controlled and predictable induced senescence model has allowed the direct comparison of sgr1 with Col-0 during the developmental period preceding the retention of chlorophyll and light harvesting complex II (LHCII) in sgr1 and the induction of senescence in Col-0. Quantitative proteomic analysis of soluble leaf proteins from sgr1 and Col-0 before the initiation of senescence has revealed a range of differences in plastid soluble protein abundance in sgr1 when compared to Col-0. Changes were also observed in membrane located machinery for photosystem II (PSII), in Calvin cycle components, proteins involved in redox control of the stromal compartment and ammonia assimilation that differentiated sgr1 during the early stages of the senescence process. The changes in PSII abundance were accompanied with a lower capacity of photosynthetic CO(2) assimilation in sgr1 than Col-0 after return of plants to lighted conditions following 3 and 5 days of darkness. A light-harvesting chlorophyll-a/b binding protein (LHCB2) was retained during the later stages of senescence in sgr1 but this was accompanied by an enhanced loss of oxygen evolving complex (OEC) subunits from PSII, which was confirmed by Western blotting, and an enhanced stability of PSII repair proteins in sgr1, compared to Col-0. Together these data provide insights into the significant differences in the steady-state proteome in sgr1 and its response to senescence, showing this cosmetic stay-green mutant is in fact significantly different to wild-type plants both before and during leaf senescence.

Lon1 is an ATP-dependent protease and chaperone located in the mitochondrial matrix in plants. Knockout in Arabidopsis (Arabidopsis thaliana) leads to a significant growth rate deficit in both roots and shoots and lowered activity of specific mitochondrial enzymes associated with respiratory metabolism. Analysis of the mitochondrial proteomes of two lon1 mutant alleles (lon1-1 and lon1-2) with different severities of phenotypes shows a common accumulation of several stress marker chaperones and lowered abundance of Complexes I, IV, and V of OXPHOS. Certain enzymes of the tricarboxylic acid (TCA) cycle are modified or accumulated, and TCA cycle bypasses were repressed rather than induced. While whole tissue respiratory rates were unaltered in roots and shoots, TCA cycle intermediate organic acids were depleted in leaf extracts in the day in lon1-1 and in both lon mutants at night. No significant evidence of broad steady-state oxidative damage to isolated mitochondrial samples could be found, but peptides from several specific proteins were more oxidized and selected functions were more debilitated in lon1-1. Collectively, the evidence suggests that loss of Lon1 significantly modifies respiratory function and plant performance by small but broad alterations in the mitochondrial proteome gained by subtly changing steady-state protein assembly, stability, and damage of a range of components that debilitate an anaplerotic role for mitochondria in cellular carbon metabolism.

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.

Specialization of the mitochondrial proteome in Arabidopsis has the potential to underlie the roles of these organelles at different developmental time points and in specific organs; however, most research to date has been limited to studies of mitochondrial composition from a few vegetative tissue types. To provide further insight into the extent of mitochondrial heterogeneity in Arabidopsis, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, bolt stem, and silique, were analyzed. Of the 286 protein spots on a 2-D gel of the mitochondrial proteome, the abundance of 237 spots was significantly varied between different samples. Identification of these spots revealed a nonredundant set of 83 proteins which were differentially expressed between organ/cell types, and the protein identification information can be analyzed in an integrated manner in an interactive fashion online. A number of mitochondrial protein spots were identified as being derived from the same genes in Arabidopsis but differed in their pI, indicating organ-specific variation in the post-translational modifications, or in their MW, suggesting differences in truncated mitochondrial products accumulating in different tissues. Comparisons of the proteomic data for the major isoforms with microarray analysis showed a positive correlation between mRNA and mitochondrial protein abundance and 60-90% concordance between changes in protein and transcript abundance. These analyses demonstrate that, while mitochondrial proteins are controlled transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the state or regulation of proteins in key biochemical pathways in plant mitochondria for specific functions. The integration of protein abundance and protein modification data with respiratory measurements, enzyme assays, and transcript data sets has allowed the identification of organ-enhanced differences in central carbon and amino acid metabolism pathways and provides ranked lists of mitochondrial proteins that are strongly transcriptionally regulated vs those whose abundance or activity is strongly influenced by a variety of post-transcriptional processes.

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.

Mitochondria undertake the process of oxidative phosphorylation yielding ATP for plant cell maintenance and growth. The principles of isolation and fractionation of plant mitochondrial proteins have been improved over decades, and surveys of the mitochondrial proteome in a number of plants species have been performed. Over time, many quantitative analyses of changes in the plant mitochondrial proteome have been performed by 2-D gel analyses revealing the induction, degradation and modification of mitochondrial proteins in responses to mutation, stress and development. Here, we present a saturating MS analysis of 2-D gel separable protein spots from a typical purification of Arabidopsis mitochondria identifying 264 proteins, alongside an LC-MS/MS survey by non-gel methods identifying 220 proteins. This allowed us to characterise the major mitochondrial proteins that are not observed on 2-D gels, the common contaminants and the abundance of the protein machinery of key mitochondrial biochemical pathways, and consider the impact of N-terminal pre-sequence cleavage and phosphorylation as explanations of multiple protein spots and the co-ordinates of proteins on 2-D gels., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The plant cell cytosol is a dynamic and complex intracellular matrix that, by definition, contains no compartmentalization. Nonetheless, it maintains a wide variety of biochemical networks and often links metabolic pathways across multiple organelles. There have been numerous detailed proteomic studies of organelles in the model plant Arabidopsis thaliana, although no such analysis has been undertaken on the cytosol. The cytosolic protein fraction from cell suspensions of Arabidopsis thaliana was isolated and analyzed using offline strong cation exchange liquid chromatography and LC-MS/MS. This generated a robust set of 1071 cytosolic proteins. Functional annotation of this set revealed major activities in protein synthesis and degradation, RNA metabolism and basic sugar metabolism. This included an array of important cytosol-related functions, specifically the ribosome, the set of tRNA catabolic enzymes, the ubiquitin-proteasome pathway, glycolysis and associated sugar metabolism pathways, phenylpropanoid biosynthesis, vitamin metabolism, nucleotide metabolism, an array of signaling and stress-responsive molecules, and NDP-sugar biosynthesis. This set of cytosolic proteins provides for the first time an extensive analysis of enzymes responsible for the myriad of reactions in the Arabidopsis cytosol and defines an experimental set of plant protein sequences that are not targeted to subcellular locations following translation and folding in the cytosol.

Malate dehydrogenase (MDH) catalyzes a reversible NAD(+)-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO(2) assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO(2) assimilation/intercellular CO(2) curves at low CO(2), and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.

Respiratory oxidative phosphorylation is a cornerstone of cellular metabolism in aerobic multicellular organisms. The efficiency of this process is generally assumed to be maximized, but the presence of dynamically regulated nonphosphorylating bypasses implies that plants can alter phosphorylation efficiency and can benefit from lowered energy generation during respiration under certain conditions. We characterized an Arabidopsis (Arabidopsis thaliana) mutant, ndufs4 (for NADH dehydrogenase [ubiquinone] fragment S subunit 4), lacking complex I of the respiratory chain, which has constitutively lowered phosphorylation efficiency. Through analysis of the changes to mitochondrial function as well as whole cell transcripts and metabolites, we provide insights into how cellular metabolism flexibly adapts to reduced phosphorylation efficiency and why this state may benefit the plant by providing moderate stress tolerance. We show that removal of the single protein subunit NDUFS4 prevents assembly of complex I and removes its function from mitochondria without pleiotropic effects on other respiratory components. However, the lack of complex I promotes broad changes in the nuclear transcriptome governing growth and photosynthetic function. We observed increases in organic acid and amino acid pools in the mutant, especially at night, concomitant with alteration of the adenylate content. While germination is delayed, this can be rescued by application of gibberellic acid, and root growth assays of seedlings show enhanced tolerance to cold, mild salt, and osmotic stress. We discuss these observations in the light of recent data on the knockout of nonphosphorylating respiratory bypass enzymes that show opposite changes in metabolites and stress sensitivity. Our data suggest that the absence of complex I alters the adenylate control of cellular metabolism.

Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2-DE separations of Arabidopsis mitochondrial proteins and affinity-enriched phosphoproteins using the Pro-Q Diamond phospho-specific in-gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.

Transcription of mitochondrial genes in animals, fungi, and plants relies on the activity of T3/T7 phage-type RNA polymerases. Two such enzymes, RPOTm and RPOTmp, are present in the mitochondria of eudicotyledonous plants; RPOTmp is additionally found in plastids. We have characterized the transcriptional role of the dual-targeted RNA polymerase in mitochondria of Arabidopsis thaliana. Examination of mitochondrial transcripts in rpoTmp mutants revealed major differences in transcript abundances between wild-type and rpoTmp plants. Decreased levels of specific transcripts were correlated with reduced abundances of the respiratory chain complexes I and IV. Altered transcript levels in rpoTmp were found to result from gene-specific transcriptional changes, establishing that RPOTmp functions in distinct transcriptional processes within mitochondria. Decreased transcription of specific genes in rpoTmp was not associated with changes in promoter utilization; therefore, RPOTmp function is not promoter specific but gene specific. This implies that additional gene-specific elements direct the transcription of a subset of mitochondrial genes by RPOTmp.

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.

Summary The Arabidopsis thaliana chloroplast contains 20 group-II introns in its genome, and seven known splicing factors are required for the splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group-II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group-I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein, but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2, and also influences the splicing of several other group-IIa introns. Loss of OTP51 has far-reaching consequences for photosystem-I and photosystem-II assembly, and for the photosynthetic fluorescence characteristics of mutant plants.

Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical gene-specific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene- or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co- and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These site-specific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis.