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.

Autophagy in maternal tissues contributes to Arabidopsis seed development.

Seeds are an essential food source, providing nutrients for germination and early seedling growth. Degradation events in the seed and the mother plant accompany seed development, including autophagy, which facilitates cellular component breakdown in the lytic organelle. Autophagy influences various aspects of plant physiology, specifically nutrient availability and remobilization, suggesting its involvement in source-sink interactions. During seed development, autophagy affects nutrient remobilization from mother plants and functions in the embryo. However, it is impossible to distinguish between the contribution of autophagy in the source (i.e. the mother plant) and the sink tissue (i.e. the embryo) when using autophagy knockout (atg mutant) plants. To address this, we employed an approach to differentiate between autophagy in source and sink tissues. We investigated how autophagy in the maternal tissue affects seed development by performing reciprocal crosses between wild type and atg mutant Arabidopsis (Arabidopsis thaliana) plants. Although F1 seedlings possessed a functional autophagy mechanism, etiolated F1 plants from maternal atg mutants displayed reduced growth. This was attributed to altered protein but not lipid accumulation in the seeds, suggesting autophagy differentially regulates carbon and nitrogen remobilization. Surprisingly, F1 seeds of maternal atg mutants exhibited faster germination, resulting from altered seed coat development. Our study emphasizes the importance of examining autophagy in a tissue-specific manner, revealing valuable insights into the interplay between different tissues during seed development. It also sheds light on the tissue-specific functions of autophagy, offering potential for research into the underlying mechanisms governing seed development and crop yield.

The Arabidopsis electron-transfer flavoprotein:ubiquinone oxidoreductase is required during normal seed development and germination.

The importance of the alternative donation of electrons to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex has been demonstrated. However, the functional significance of this pathway during seed development and germination remains to be elucidated. To assess the function of this pathway, we performed a detailed metabolic and transcriptomic analysis of Arabidopsis mutants to test the molecular consequences of a dysfunctional ETF/ETFQO pathway. We demonstrate that the disruption of this pathway compromises seed germination in the absence of an external carbon source and also impacts seed size and yield. Total protein and storage protein content is reduced in dry seeds, whilst sucrose levels remain invariant. Seeds of ETFQO and related mutants were also characterized by an altered fatty acid composition. During seed development, lower levels of fatty acids and proteins accumulated in the etfqo-1 mutant as well as in mutants in the alternative electron donors isovaleryl-CoA dehydrogenase (ivdh-1) and d-2-hydroxyglutarate dehydrogenase (d2hgdh1-2). Furthermore, the content of several amino acids was increased in etfqo-1 mutants during seed development, indicating that these mutants are not using such amino acids as alternative energy source for respiration. Transcriptome analysis revealed alterations in the expression levels of several genes involved in energy and hormonal metabolism. Our findings demonstrated that the alternative pathway of respiration mediated by the ETF/ETFQO complex affects seed germination and development by directly adjusting carbon storage during seed filling. These results indicate a role for the pathway in the normal plant life cycle to complement its previously defined roles in the response to abiotic stress.

The interplay between autophagy and chloroplast vesiculation pathways under dark-induced senescence.

In cellular circumstances where carbohydrates are scarce, plants can use alternative substrates for cellular energetic maintenance. In plants, the main protein reserve is present in the chloroplast, which contains most of the total leaf proteins and represents a rich source of nitrogen and amino acids. Autophagy plays a key role in chloroplast breakdown, a well-recognised symptom of both natural and stress-induced plant senescence. Remarkably, an autophagic-independent route of chloroplast degradation associated with chloroplast vesiculation (CV) gene was previously demonstrated. During extended darkness, CV is highly induced in the absence of autophagy, contributing to the early senescence phenotype of atg mutants. To further investigate the role of CV under dark-induced senescence conditions, mutants with low expression of CV (amircv) and double mutants amircv1xatg5 were characterised. Following darkness treatment, no aberrant phenotypes were observed in amircv single mutants; however, amircv1xatg5 double mutants displayed early senescence and altered dismantling of chloroplast and membrane structures under these conditions. Metabolic characterisation revealed that the functional lack of both CV and autophagy leads to higher impairment of amino acid release and differential organic acid accumulation during starvation conditions. The data obtained are discussed in the context of the role of CV and autophagy, both in terms of cellular metabolism and the regulation of chloroplast degradation.

Autophagy is required for lipid homeostasis during dark-induced senescence.

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

The significance of WRKY45 transcription factor in metabolic adjustments during dark-induced leaf senescence.

Plants are constantly exposed to environmental changes that affect their performance. Metabolic adjustments are crucial to controlling energy homoeostasis and plant survival, particularly during stress. Under carbon starvation, coordinated reprogramming is initiated to adjust metabolic processes, which culminate in premature senescence. Notwithstanding, the regulatory networks that modulate transcriptional control during low energy remain poorly understood. Here, we show that the WRKY45 transcription factor is highly induced during both developmental and dark-induced senescence. The overexpression of Arabidopsis WRKY45 resulted in an early senescence phenotype characterized by a reduction of maximum photochemical efficiency of photosystem II and chlorophyll levels in the later stages of darkness. The detailed metabolic characterization showed significant changes in amino acids coupled with the accumulation of organic acids in WRKY45 overexpression lines during dark-induced senescence. Furthermore, the markedly upregulation of alternative oxidase (AOX1a, AOX1d) and electron transfer flavoprotein/ubiquinone oxidoreductase (ETFQO) genes suggested that WRKY45 is associated with a dysregulation of mitochondrial signalling and the activation of alternative respiration rather than amino acids catabolism regulation. Collectively our results provided evidence that WRKY45 is involved in the plant metabolic reprogramming following carbon starvation and highlight the potential role of WRKY45 in the modulation of mitochondrial signalling pathways.

Exploring the Contribution of Autophagy to the Excess-Sucrose Response in Arabidopsis thaliana.

Autophagy is an essential intracellular eukaryotic recycling mechanism, functioning in, among others, carbon starvation. Surprisingly, although autophagy-deficient plants (atg mutants) are hypersensitive to carbon starvation, metabolic analysis revealed that they accumulate sugars under such conditions. In plants, sugars serve as both an energy source and as signaling molecules, affecting many developmental processes, including root and shoot formation. We thus set out to understand the interplay between autophagy and sucrose excess, comparing wild-type and atg mutant seedlings. The presented work showed that autophagy contributes to primary root elongation arrest under conditions of exogenous sucrose and glucose excess but not during fructose or mannitol treatment. Minor or no alterations in starch and primary metabolites were observed between atg mutants and wild-type plants, indicating that the sucrose response relates to its signaling and not its metabolic role. Extensive proteomic analysis of roots performed to further understand the mechanism found an accumulation of proteins essential for ROS reduction and auxin maintenance, which are necessary for root elongation, in atg plants under sucrose excess. The analysis also suggested mitochondrial and peroxisomal involvement in the autophagy-mediated sucrose response. This research increases our knowledge of the complex interplay between autophagy and sugar signaling in plants.

Autophagy and its role in plant abiotic stress management.

Being unable to move, plants are regularly exposed to changing environmental conditions, among which various types of abiotic stress, such as heat, drought, salt, and so forth. These might have deleterious effects on plant performance and yield. Plants thus need to adapt using appropriate stress responses. One of the outcomes of abiotic stress is the need to degrade and recycle damaged proteins and organelles. Autophagy is a conserved eukaryotic mechanism functioning in the degradation of proteins, protein aggregates, and whole organelles. It was previously shown to have a role in plant abiotic stress. This review will describe the current knowledge regarding the involvement of autophagy in plant abiotic stress response, mechanisms functioning in autophagy induction during stress, and possible direction for future research.

Autophagy Deficiency Compromises Alternative Pathways of Respiration following Energy Deprivation in Arabidopsis thaliana.

Under heterotrophic conditions, carbohydrate oxidation inside the mitochondrion is the primary energy source for cellular metabolism. However, during energy-limited conditions, alternative substrates are required to support respiration. Amino acid oxidation in plant cells plays a key role in this by generating electrons that can be transferred to the mitochondrial electron transport chain via the electron transfer flavoprotein/ubiquinone oxidoreductase system. Autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid pools and nutrient remobilization. Although the association between autophagy and alternative respiratory substrates has been suggested, the extent to which autophagy and primary metabolism interact to support plant respiration remains unclear. To investigate the metabolic importance of autophagy during development and under extended darkness, Arabidopsis (Arabidopsis thaliana) mutants with disruption of autophagy (atg mutants) were used. Under normal growth conditions, atg mutants showed lower growth and seed production with no impact on photosynthesis. Following extended darkness, atg mutants were characterized by signatures of early senescence, including decreased chlorophyll content and maximum photochemical efficiency of photosystem II coupled with increases in dark respiration. Transcript levels of genes involved in alternative pathways of respiration and amino acid catabolism were up-regulated in atg mutants. The metabolite profiles of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acid levels were partially compromised in atg mutants. Although an enhanced respiration in atg mutants was observed during extended darkness, autophagy deficiency compromises protein degradation and the generation of amino acids used as alternative substrates to the respiration.

An L,L-diaminopimelate aminotransferase mutation leads to metabolic shifts and growth inhibition in Arabidopsis.

Lysine (Lys) connects the mitochondrial electron transport chain to amino acid catabolism and the tricarboxylic acid cycle. However, our understanding of how a deficiency in Lys biosynthesis impacts plant metabolism and growth remains limited. Here, we used a previously characterized Arabidopsis mutant (dapat) with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase to investigate the physiological and metabolic impacts of impaired Lys biosynthesis. Despite displaying similar stomatal conductance and internal CO2 concentration, we observed reduced photosynthesis and growth in the dapat mutant. Surprisingly, whilst we did not find differences in dark respiration between genotypes, a lower storage and consumption of starch and sugars was observed in dapat plants. We found higher protein turnover but no differences in total amino acids during a diurnal cycle in dapat plants. Transcriptional and two-dimensional (isoelectric focalization/SDS-PAGE) proteome analyses revealed alterations in the abundance of several transcripts and proteins associated with photosynthesis and photorespiration coupled with a high glycine/serine ratio and increased levels of stress-responsive amino acids. Taken together, our findings demonstrate that biochemical alterations rather than stomatal limitations are responsible for the decreased photosynthesis and growth of the dapat mutant, which we hypothesize mimics stress conditions associated with impairments in the Lys biosynthesis pathway.

Autophagy-related approaches for improving nutrient use efficiency and crop yield protection.

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation.

Germination and early seedling establishment are developmental stages in which plants face limited nutrient supply as their photosynthesis mechanism is not yet active. For this reason, the plant must mobilize the nutrient reserves provided by the mother plant in order to facilitate growth. Autophagy is a catabolic process enabling the bulk degradation of cellular constituents in the vacuole. The autophagy mechanism is conserved among eukaryotes, and homologs of many autophagy-related (ATG) genes have been found in Arabidopsis thaliana. T-DNA insertion mutants (atg mutants) of these genes display higher sensitivity to various stresses, particularly nutrient starvation. However, the direct impact of autophagy on cellular metabolism has not been well studied. In this work, we used etiolated Arabidopsis seedlings as a model system for carbon starvation. atg mutant seedlings display delayed growth in response to carbon starvation compared with wild-type seedlings. High-throughput metabolomic, lipidomic, and proteomic analyses were performed, as well as extensive flux analyses, in order to decipher the underlying causes of the phenotype. Significant differences between atg mutants and wild-type plants have been demonstrated, suggesting global effects of autophagy on central metabolism during carbon starvation as well as severe energy deprivation, resulting in a morphological phenotype.

A new type of compartment, defined by plant-specific Atg8-interacting proteins, is induced upon exposure of Arabidopsis plants to carbon starvation.

Atg8 is a central protein in bulk starvation-induced autophagy, but it is also specifically associated with multiple protein targets under various physiological conditions to regulate their selective turnover by the autophagy machinery. Here, we describe two new closely related Arabidopsis thaliana Atg8-interacting proteins (ATI1 and ATI2) that are unique to plants. We show that under favorable growth conditions, ATI1 and ATI2 are partially associated with the endoplasmic reticulum (ER) membrane network, whereas upon exposure to carbon starvation, they become mainly associated with newly identified spherical compartments that dynamically move along the ER network. These compartments are morphologically distinct from previously reported spindle-shaped ER bodies and, in contrast to them, do not contain ER-lumenal markers possessing a C-terminal HDEL sequence. Organelle and autophagosome-specific markers show that the bodies containing ATI1 are distinct from Golgi, mitochondria, peroxisomes, and classical autophagosomes. The final destination of the ATI1 bodies is the central vacuole, indicating that they may operate in selective turnover of specific proteins. ATI1 and ATI2 gene expression is elevated during late seed maturation and desiccation. We further demonstrate that ATI1 overexpression or suppression of both ATI1 and ATI2, respectively, stimulate or inhibit seed germination in the presence of the germination-inhibiting hormone abscisic acid.

Deciphering energy-associated gene networks operating in the response of Arabidopsis plants to stress and nutritional cues.

Plants need to continuously adjust their transcriptome in response to various stresses that lead to inhibition of photosynthesis and the deprivation of cellular energy. This adjustment is triggered in part by a coordinated re-programming of the energy-associated transcriptome to slow down photosynthesis and activate other energy-promoting gene networks. Therefore, understanding the stress-related transcriptional networks of genes belonging to energy-associated pathways is of major importance for engineering stress tolerance. In a bioinformatics approach developed by our group, termed 'gene coordination', we previously divided genes encoding for enzymes and transcription factors in Arabidopsis thaliana into three clusters, displaying altered coordinated transcriptional behaviors in response to multiple biotic and abiotic stresses (Plant Cell, 23, 2011, 1264). Enrichment analysis indicated further that genes controlling energy-associated metabolism operate as a compound network in response to stress. In the present paper, we describe in detail the network association of genes belonging to six central energy-associated pathways in each of these three clusters described in our previous paper. Our results expose extensive stress-associated intra- and inter-pathway interactions between genes from these pathways, indicating that genes encoding proteins involved in energy-associated metabolism are expressed in a highly coordinated manner. We also provide examples showing that this approach can be further utilized to elucidate candidate genes for stress tolerance and functions of isozymes.

Metabolism and autophagy in plants-a perfect match.

Autophagy is a eukaryotic cellular transport mechanism that delivers intracellular macromolecules, proteins, and even organelles to a lytic organelle (vacuole in yeast and plants/lysosome in animals) for degradation and nutrient recycling. The process is mediated by highly conserved autophagy-related (ATG) proteins. In plants, autophagy maintains cellular homeostasis under favorable conditions, guaranteeing normal plant growth and fitness. Severe stress such as nutrient starvation and plant senescence further induce it, thus ensuring plant survival under unfavorable conditions by providing nutrients through the removal of damaged or aged proteins, or organelles. In this article, we examine the interplay between metabolism and autophagy, focusing on the different aspects of this reciprocal relationship. We show that autophagy has a strong influence on a range of metabolic processes, whereas at the same time, even single metabolites can activate autophagy. We highlight the involvement of ATG genes in metabolism, examine the role of the macronutrients carbon and nitrogen, and various micronutrients, and take a closer look at how the interaction between autophagy and metabolism impacts on plant phenotypes and yield.

Autophagy modulates the metabolism and growth of tomato fruit during development.

Although autophagy is a conserved mechanism operating across eukaryotes, its effects on crops and especially their metabolism has received relatively little attention. Indeed, whilst a few recent studies have used systems biology tools to look at the consequences of lack of autophagy in maize these focused on leaf tissues rather than the kernels. Here we utilized RNA interference (RNAi) to generate tomato plants that were deficient in the autophagy-regulating protease ATG4. Plants displayed an early senescence phenotype yet relatively mild changes in the foliar metabolome and were characterized by a reduced fruit yield phenotype. Metabolite profiling indicated that metabolites of ATG4-RNAi tomato leaves just exhibited minor alterations while that of fruit displayed bigger difference compared to the WT. In detail, many primary metabolites exhibited decreases in the ATG4-RNAi lines, such as proline, tryptophan and phenylalanine, while the representative secondary metabolites (quinic acid and 3-trans-caffeoylquinic acid) were present at substantially higher levels in ATG4-RNAi green fruits than in WT. Moreover, transcriptome analysis indicated that the most prominent differences were in the significant upregulation of organelle degradation genes involved in the proteasome or chloroplast vesiculation pathways, which was further confirmed by the reduced levels of chloroplastic proteins in the proteomics data. Furthermore, integration analysis of the metabolome, transcriptome and proteome data indicated that ATG4 significantly affected the lipid metabolism, chlorophyll binding proteins and chloroplast biosynthesis. These data collectively lead us to propose a more sophisticated model to explain the cellular co-ordination of the process of autophagy.

Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death.

The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.Abbreviations: ATG: autophagy related; CLP: caspase-like protease; HR: hypersensitive response; PCD: programmed cell death; St: Solanum tuberosum; VPE: vacuolar processing enzyme.

Anti-inflammatory effects of an inflammatory chemokine: CCL2 inhibits lymphocyte homing by modulation of CCL21-triggered integrin-mediated adhesions.

Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1- and LFA-1-mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.

The Phytotoxicity of Meta-Tyrosine Is Associated With Altered Phenylalanine Metabolism and Misincorporation of This Non-Proteinogenic Phe-Analog to the Plant's Proteome.

Plants produce a myriad of specialized (secondary) metabolites that are highly diverse chemically, and exhibit distinct biological functions. Here, we focus on meta-tyrosine (m-tyrosine), a non-proteinogenic byproduct that is often formed by a direct oxidation of phenylalanine (Phe). Some plant species (e.g., Euphorbia myrsinites and Festuca rubra) produce and accumulate high levels of m-tyrosine in their root-tips via enzymatic pathways. Upon its release to soil, the Phe-analog, m-tyrosine, affects early post-germination development (i.e., altered root development, cotyledon or leaf chlorosis, and retarded growth) of nearby plant life. However, the molecular basis of m-tyrosine-mediated (phyto)toxicity remains, to date, insufficiently understood and are still awaiting their functional characterization. It is anticipated that upon its uptake, m-tyrosine impairs key metabolic processes, or affects essential cellular activities in the plant. Here, we provide evidences that the phytotoxic effects of m-tyrosine involve two distinct molecular pathways. These include reduced steady state levels of several amino acids, and in particularly altered biosynthesis of the phenylalanine (Phe), an essential α-amino acid, which is also required for the folding and activities of proteins. In addition, proteomic studies indicate that m-tyrosine is misincorporated in place of Phe, mainly into the plant organellar proteomes. These data are supported by analyses of adt mutants, which are affected in Phe-metabolism, as well as of var2 mutants, which lack FtsH2, a major component of the chloroplast FtsH proteolytic machinery, which show higher sensitivity to m-tyrosine. Plants treated with m-tyrosine show organellar biogenesis defects, reduced respiration and photosynthetic activities and growth and developmental defect phenotypes.