The lowdown on breakdown: Open questions in plant proteolysis.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and post-harvest regulation), plant responses to environmental signals (endoplasmic-reticulum associated degradation, chloroplast-associated degradation, drought tolerance, the growth-defense tradeoff)), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor.

Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAalavia conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

FTSH PROTEASE 3 facilitates Complex I degradation through a direct interaction with the Complex I subunit PSST.

Complex I (CI) (NADH dehydrogenase), the largest complex involved in mitochondrial oxidative phosphorylation, is composed of nuclear- and mitochondrial-encoded subunits. CI assembly occurs via the sequential addition of subdomains and modules. As CI is prone to oxidative damage, its subunits continually undergo proteolysis and turnover. We describe the mechanism by which CI abundance is regulated in a CI-deficient Arabidopsis thaliana mutant. Using a forward genetic approach, we determined that the CI Q-module domain subunit PSST interacts with FTSH PROTEASE 3 (FTSH3) to mediate the disassembly of the matrix arm domain for proteolysis and turnover as a means of protein quality control. We demonstrated the direct interaction of FTSH3 with PSST and identified the amino acid residues required for this interaction. The ATPase function of FTSH3, rather than its proteolytic activity, is required for this interaction, as its mutation was compensated for by a proteolytically inactive form of FTSH3. This study reveals the mechanistic process by which FTSH3 recognizes CI for degradation at amino acid resolution.

The biogenesis and regulation of the plant oxidative phosphorylation system.

Mitochondria are central organelles for respiration in plants. At the heart of this process is oxidative phosphorylation (OXPHOS) system, which generates ATP required for cellular energetic needs. OXPHOS complexes comprise of multiple subunits that originated from both mitochondrial and nuclear genome, which requires careful orchestration of expression, translation, import, and assembly. Constant exposure to reactive oxygen species due to redox activity also renders OXPHOS subunits to be more prone to oxidative damage, which requires coordination of disassembly and degradation. In this review, we highlight the composition, assembly, and activity of OXPHOS complexes in plants based on recent biochemical and structural studies. We also discuss how plants regulate the biogenesis and turnover of OXPHOS subunits and the importance of OXPHOS in overall plant respiration. Further studies in determining the regulation of biogenesis and activity of OXPHOS will advances the field, especially in understanding plant respiration and its role to plant growth and development.

Presequence translocase-associated motor subunits of the mitochondrial protein import apparatus are dual-targeted to mitochondria and plastids.

The import and assembly of most of the mitochondrial proteome is regulated by protein translocases located within the mitochondrial membranes. The Presequence Translocase-Associated Motor (PAM) complex powers the translocation of proteins across the inner membrane and consists of Hsp70, the J-domain containing co-chaperones, Pam16 and Pam18, and their associated proteins Tim15 and Mge1. In Arabidopsis, multiple orthologues of Pam16, Pam18, Tim15 and Mge1 have been identified and a mitochondrial localization has been confirmed for most. As the localization of Pam18-1 has yet to be determined and a plastid localization has been observed for homologues of Tim15 and Mge1, we carried out a comprehensive targeting analysis of all PAM complex orthologues using multiple in vitro and in vivo methods. We found that, Pam16 was exclusively targeted to the mitochondria, but Pam18 orthologues could be targeted to both the mitochondria and plastids, as observed for the PAM complex interacting partner proteins Tim15 and Mge1.

An MCIA-like complex is required for mitochondrial complex I assembly and seed development in maize.

During adaptive radiation, mitochondria have co-evolved with their hosts, leading to gain or loss of subunits and assembly factors of respiratory complexes. Plant mitochondrial complex I harbors ∼40 nuclear- and 9 mitochondrial-encoded subunits, and is formed by stepwise assembly during which different intermediates are integrated via various assembly factors. In mammals, the mitochondrial complex I intermediate assembly (MCIA) complex is required for building the membrane arm module. However, plants have lost almost all of the MCIA complex components, giving rise to the hypothesis that plants follow an ancestral pathway to assemble the membrane arm subunits. Here, we characterize a maize crumpled seed mutant, crk1, and reveal by map-based cloning that CRK1 encodes an ortholog of human complex I assembly factor 1, zNDUFAF1, the only evolutionarily conserved MCIA subunit in plants. zNDUFAF1 is localized in the mitochondria and accumulates in two intermediate complexes that contain complex I membrane arm subunits. Disruption of zNDUFAF1 results in severe defects in complex I assembly and activity, a cellular bioenergetic shift to aerobic glycolysis, and mitochondrial vacuolation. Moreover, we found that zNDUFAF1, the putative mitochondrial import inner membrane translocase ZmTIM17-1, and the isovaleryl-coenzyme A dehydrogenase ZmIVD1 interact each other, and could be co-precipitated from the mitochondria and co-migrate in the same assembly intermediates. Knockout of either ZmTIM17-1 or ZmIVD1 could lead to the significantly reduced complex I stability and activity as well as defective seeds. These results suggest that zNDUFAF1, ZmTIM17-1 and ZmIVD1 probably form an MCIA-like complex that is essential for the biogenesis of mitochondrial complex I and seed development in maize. Our findings also imply that plants and mammals recruit MCIA subunits independently for mitochondrial complex I assembly, highlighting the importance of parallel evolution in mitochondria adaptation to their hosts.

Late Embryogenesis Abundant (LEA)5 Regulates Translation in Mitochondria and Chloroplasts to Enhance Growth and Stress Tolerance.

The late embryogenesis abundant (LEA)5 protein is predominantly expressed in Arabidopsis leaves in the dark, the levels of LEA5 transcripts decreasing rapidly upon illumination. LEA5 is important in plant responses to environmental stresses but the mechanisms involved have not been elucidated. We therefore explored LEA5 functions in Arabidopsis mutants (lea5) and transgenic Arabidopsis plants constitutively expressing LEA5 (OEX 2-5), as well as in transgenic barley lines expressing the Arabidopsis LEA5 gene. The OEX 2-5 plants grew better than controls and lea5 mutants in the presence of the prooxidants methyl viologen and menadione. Confocal microscopy of Arabidopsis mesophyll protoplasts expressing a LEA5-YFP fusion protein demonstrated that LEA5 could be localized to chloroplasts as well as mitochondria in Arabidopsis protoplasts. Tandem affinity purification (TAP) analysis revealed LEA5 interacts with the chloroplast DEAD-box ATP-dependent RNA helicase 22 (RH22) in Arabidopsis cells. Split YFP analysis confirmed the interaction between RH22 and LEA5 in chloroplasts. The abundance of translated protein products in chloroplasts was decreased in transgenic Arabidopsis plants and increased in lea5 knockout mutants. Conversely, the abundance of translated mitochondrial protein products was increased in OEX 2-5 plants and decreased in lea5 mutants. Mitochondrial electron transport rates were higher in the OEX 2-5 plants than the wild type. The transformed barley lines expressing the Arabidopsis LEA5 had increased seed yields, but they showed a greater drought-induced inhibition of photosynthesis than controls. Taken together, these data demonstrate that LEA5 regulates organellar translation, in order to enhance respiration relative to photosynthesis in response to stress.

Mitochondrial activity and biogenesis during resurrection of Haberlea rhodopensis.

Haberlea rhodopensis is a resurrection plant that can tolerate extreme and prolonged periods of desiccation with a rapid restoration of physiological function upon rehydration. Specialized mechanisms are required to minimize cellular damage during desiccation and to maintain integrity for rapid recovery following rehydration. In this study we used respiratory activity measurements, electron microscopy, transcript, protein and blue native-PAGE analysis to investigate mitochondrial activity and biogenesis in fresh, desiccated and rehydrated detached H. rhodopensis leaves. We demonstrate that unlike photosynthesis, mitochondrial respiration was almost immediately activated to levels of fresh tissue upon rehydration. The abundance of transcripts and proteins involved in mitochondrial respiration and biogenesis were at comparable levels in fresh, desiccated and rehydrated tissues. Blue native-PAGE analysis revealed fully assembled and equally abundant OXPHOS complexes in mitochondria isolated from fresh, desiccated and rehydrated detached leaves. We observed a high abundance of alternative respiratory components which correlates with the observed high uncoupled respiration capacity in desiccated tissue. Our study reveals that during desiccation of vascular H. rhodopensis tissue, mitochondrial composition is conserved and maintained at a functional state allowing for an almost immediate activation to full capacity upon rehydration. Mitochondria-specific mechanisms were activated during desiccation which probably play a role in maintaining tolerance.

Proteolytic regulation of mitochondrial oxidative phosphorylation components in plants.

Mitochondrial function relies on the homeostasis and quality control of their proteome, including components of the oxidative phosphorylation (OXPHOS) pathway that generates energy in form of ATP. OXPHOS subunits are under constant exposure to reactive oxygen species due to their oxidation-reduction activities, which consequently make them prone to oxidative damage, misfolding, and aggregation. As a result, quality control mechanisms through turnover and degradation are required for maintaining mitochondrial activity. Degradation of OXPHOS subunits can be achieved through proteomic turnover or modular degradation. In this review, we present multiple protein degradation pathways in plant mitochondria. Specifically, we focus on the intricate turnover of OXPHOS subunits, prior to protein import via cytosolic proteasomal degradation and post import and assembly via intra-mitochondrial proteolysis involving multiple AAA+ proteases. Together, these proteolytic pathways maintain the activity and homeostasis of OXPHOS components.

Inhibition of chloroplast translation as a new target for herbicides.

The rise in herbicide resistance over recent decades threatens global agriculture and food security and so discovery of new modes of action is increasingly important. Here we reveal linezolid, an oxazolidinone antibiotic that inhibits microbial translation, is also herbicidal. To validate the herbicidal mode of action of linezolid we confirmed its micromolar inhibition is specific to chloroplast translation and did not affect photosynthesis directly. To assess the herbicide potential of linezolid, testing against a range of weed and crop species found it effective pre- and post-emergence. Using structure-activity analysis we identified the critical elements for herbicidal activity, but importantly also show, using antimicrobial susceptibility assays, that separation of antibacterial and herbicidal activities was possible. Overall these results validate chloroplast translation as a viable herbicidal target.

The mitochondrial LYR protein SDHAF1 is required for succinate dehydrogenase activity in Arabidopsis.

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.

The dual-targeted prolyl aminopeptidase PAP1 is involved in proline accumulation in response to stress and during pollen development.

Plant endosymbiotic organelles such as mitochondria and chloroplasts harbour a wide array of biochemical reactions. As a part of protein homeostasis to maintain organellar activity and stability, unwanted proteins and peptides need to be completely degraded in a stepwise mechanism termed the processing pathway, where at the last stage single amino acids are released by aminopeptidases. Here, we determined the molecular and physiological functions of a prolyl aminopeptidase homologue PAP1 (At2g14260) that is able to release N-terminal proline. Transcript analyses demonstrate that an alternative transcription start site gives rise to two alternative transcripts, generating two in-frame proteins PAP1.1 and PAP1.2. Subcellular localization studies revealed that the longer isoform PAP1.1, which contains a 51 residue N-terminal extension, is exclusively targeted to chloroplasts, while the truncated isoform PAP1.2 is located in the cytosol. Distinct expression patterns in different tissues and developmental stages were observed. Investigations into the physiological role of PAP1 using loss-of-function mutants revealed that PAP1 activity may be involved in proline homeostasis and accumulation, required for pollen development and tolerance to osmotic stress. Enzymatic activity, subcellular location, and expression patterns of PAP1 suggest a role in the chloroplastic peptide processing pathway and proline homeostasis.

The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly.

ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidizing NADH and transferring electrons to the ubiquinone pool. Typically, Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP-dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named as rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterized Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

A mitochondrial prolyl aminopeptidase PAP2 releases N-terminal proline and regulates proline homeostasis during stress response.

Most mitochondrial proteins are synthesised in the cytosol and targeted into the organelle via N-terminal targeting peptides that are cleaved upon import. The free targeting peptide is subsequently processed in a stepwise manner, with single amino acids released as final products. Here, we have characterised a proline-cleaving aminopeptidase in Arabidopsis thaliana, prolyl aminopeptidase-2 (PAP2, At3g61540). Activity assays show that PAP2 has a preferred activity to hydrolyse N-terminal proline. Protein localisation studies revealed that PAP2 is exclusively targeted to mitochondria. Characterisation of pap2 mutants show defective pollen, enhanced dark-induced senescence and increased susceptibility to abiotic stresses, which are likely attributed to a reduced level of accumulated free proline. Taken together, these results demonstrate the role of PAP2 in proline cleavage from mitochondrial peptides and proline homeostasis, which is required for the development of male gametophyte, tolerance to abiotic stresses, and leaf senescence.

Mitochondrial CLPP2 Assists Coordination and Homeostasis of Respiratory Complexes.

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.

The peptidases involved in plant mitochondrial protein import.

The endosymbiotic origin of the mitochondrion and the subsequent transfer of its genome to the host nucleus has resulted in intricate mechanisms of regulating mitochondrial biogenesis and protein content. The majority of mitochondrial proteins are nuclear encoded and synthesized in the cytosol, thus requiring specialized and dedicated machinery for the correct targeting import and sorting of its proteome. Most proteins targeted to the mitochondria utilize N-terminal targeting signals called presequences that are cleaved upon import. This cleavage is carried out by a variety of peptidases, generating free peptides that can be detrimental to organellar and cellular activity. Research over the last few decades has elucidated a range of mitochondrial peptidases that are involved in the initial removal of the targeting signal and its sequential degradation, allowing for the recovery of single amino acids. The significance of these processing pathways goes beyond presequence degradation after protein import, whereby the deletion of processing peptidases induces plant stress responses, compromises mitochondrial respiratory capability, and alters overall plant growth and development. Here, we review the multitude of plant mitochondrial peptidases that are known to be involved in protein import and processing of targeting signals to detail how their activities can affect organellar protein homeostasis and overall plant growth.

A Mitochondrial LYR Protein Is Required for Complex I Assembly.

Complex I biogenesis requires the expression of both nuclear and mitochondrial genes, the import of proteins, cofactor biosynthesis, and the assembly of at least 49 individual subunits. Assembly factors interact with subunits of Complex I but are not part of the final holocomplex. We show that in Arabidopsis (Arabidopsis thaliana), a mitochondrial matrix protein (EMB1793, At1g76060), which we term COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), contains a LYR domain and is required for Complex I assembly. T-DNA insertion mutants of CIAF1 lack Complex I and the Supercomplex I+III. Biochemical characterization shows that the assembly of Complex I is stalled at 650 and 800 kD intermediates in mitochondria isolated from ciaf1 mutant lines.I. Yeast-two-hybrid interaction and complementation assays indicate that CIAF1 specifically interacts with the 23-kD TYKY-1 matrix domain subunit of Complex I and likely plays a role in Fe-S insertion into this subunit. These data show that CIAF1 plays an essential role in assembling the peripheral matrix arm Complex I subunits into the Complex I holoenzyme.

An interstitial peptide is readily processed from within seed proteins.

The importance of de novo protein evolution is apparent, but most examples are de novo coding transcripts evolving from silent or non-coding DNA. The peptide macrocycle SunFlower Trypsin Inhibitor 1 (SFTI-1) evolved over 45 million years from genetic expansion within the N-terminal 'discarded' region of an ancestral seed albumin precursor. SFTI-1 and its adjacent albumin are both processed into separate, mature forms by asparaginyl endopeptidase (AEP). Here to determine whether the evolution of SFTI-1 in a latent region of its precursor was critical, we used a transgene approach in A. thaliana analysed by peptide mass spectrometry and RT-qPCR. SFTI could emerge from alternative locations within preproalbumin as well as emerge with precision from unrelated seed proteins via AEP-processing. SFTI production was possible with the adjacent albumin, but peptide levels dropped greatly without the albumin. The ability for SFTI to be processed from multiple sequence contexts and different proteins suggests that to make peptide, it was not crucial for the genetic expansion that gave rise to SFTI and its family to be within a latent protein region. Interstitial peptides, evolving like SFTI within existing proteins, might be more widespread and as a mechanism, SFTI exemplifies a stable, new, functional peptide that did not need a new gene to evolve de novo.

Accumulation of endogenous peptides triggers a pathogen stress response in Arabidopsis thaliana.

The stepwise degradation of peptides to amino acids in plant mitochondria and chloroplasts is catalyzed by a network of oligopeptidases (presequence protease PreP, organellar oligopeptidase OOP) and aminopeptidases. In the present report, we show that the lack of oligopeptidase activity in Arabidopsis thaliana results in the accumulation of endogenous free peptides, mostly of chloroplastic origin (targeting peptides and degradation products). Using mRNA sequencing and deep coverage proteomics, allowing for the identification of 17 000 transcripts and 11 000 proteins, respectively, we uncover a peptide-stress response occurring in plants lacking PreP and OOP oligopeptidase activity. The peptide-stress response results in the activation of the classical plant defense pathways in the absence of pathogenic challenge. The constitutive activation of the pathogen-defense pathways imposes a strong growth penalty and a reduction of the plants reproductive fitness. Our results indicate that the absence of organellar oligopeptidases PreP1/2 and OOP results in the accumulation of peptides that are perceived as pathogenic effectors and activate the signaling pathways of plant-defense response.

Plant mitochondrial protein import: the ins and outs.

The majority of the mitochondrial proteome, required to fulfil its diverse range of functions, is cytosolically synthesised and translocated via specialised machinery. The dedicated translocases, receptors, and associated proteins have been characterised in great detail in yeast over the last several decades, yet many of the mechanisms that regulate these processes in higher eukaryotes are still unknown. In this review, we highlight the current knowledge of mitochondrial protein import in plants. Despite the fact that the mechanisms of mitochondrial protein import have remained conserved across species, many unique features have arisen in plants to encompass the developmental, tissue-specific, and stress-responsive regulation in planta. An understanding of unique features and mechanisms in plants provides us with a unique insight into the regulation of mitochondrial biogenesis in higher eukaryotes.