UNDERSTANDING AND EXPLOITING THE ROLES OF AUTOPHAGY IN PLANTS THROUGH MULTI-OMICS APPROACHES

Abstract

Autophagy is a highly conserved pathway in eukaryotes that promotes nutrient recycling and cellular homeostasis through the degradation of excess or damaged cytoplasmic constituents. In plants, autophagy is increasingly recognized as a key contributor to development, reproduction, metabolism, leaf senescence, endosperm and grain development, pathogen defense, and tolerance to abiotic and biotic stresses. Characterizing the functional transcriptomic, proteomic, and metabolomic networks relating to autophagy in plants subjected to various extra- and intra-cellular stimuli may help to identify components associated with the pathway. As such, the integration of multi-omics approaches (i.e., transcriptomics, proteomics and metabolomics), along with cellular, genetic and functional analyses, could provide a global perspective regarding the effects of autophagy on plant metabolism, development and stress responses. In this mini-review, recent research progress in plant autophagy is discussed, highlighting the importance of high-throughput omics approaches for defining the underpinning molecular mechanisms of autophagy and understanding its associated regulatory network.

1. Introduction¹

Macroautophagy (hereafter termed autophagy) is a highly conserved and regulated degradation pathway in eukaryotes, through which cytoplasmic constituents including long-lived proteins, dysfunctional complexes and organelles, and invading pathogens are degraded in the vacuole (in plants and yeast) or lysosome (in animals) [1, 2]. During autophagy, a set of AUTOPHAGY-RELATED (ATG) proteins are recruited to a pre-autophagosomal structure, where they orchestrate the formation of a cup-shaped double membrane structure called the phagophore [2, 3]. As the phagophore elongates and ultimately closes, cytoplasmic cargo becomes encapsulated within a double membrane-bound vesicle termed the autophagosome. Upon fusion of its outer membrane with the tonoplast, the internal vesicle of the autophagosome is delivered to the vacuolar lumen as an autophagic body. Subsequently, the autophagic body is degraded by vacuolar hydrolases, and the resulting products are recycled back into the cytosol for re-use [2].

The molecular mechanisms of autophagy were initially defined by a genetic screen in budding yeast (Saccharomyces cerevisiae), in which the first ATG gene that encodes the central autophagy machinery was identified [3, 4]. Thus far, ~40 conserved ATG genes required for this sophisticated process have been identified through numerous studies in yeast, animals and, later, plants [3, 5] They have been classified based on cellular, proteomic and genetic analyses into four protein complexes [6]: (i) the ATG1/13 kinase complex; (ii) the ATG9 membrane delivery complex; (iii) the class III phosphatidylinositol-3-kinase (PI3K) complex; (iv) the ATG8/12 conjugation pathways. The autophagy process is initiated by activation of the ATG1/13 kinase complex, which functions as a key regulator by integrating upstream nutrient and energy signals, and recruiting downstream ATG proteins to the pre-autophagosomal structure to initiate phagophore expansion [7]. The upstream nutrient and energy sensors/regulators of the autophagy pathway include the TARGET OF RAPAMYCIN (TOR) kinase and the AMP-ACTIVATED PROTEIN KINASE (AMPK, known as SnRK1 in plants), while the downstream ATG protein complexes stimulated upon ATG1/13 activation include the PI3K and ATG9 complexes [5, 6, 8, 9].

The PI3K complex is assembled with VACUOLAR PROTEIN SORTING 34 (VPS34), which provides the kinase activity, together with three accessory subunits: ATG6/VPS30 (Beclin-1 in mammals), ATG14 and VPS15 [10]. This complex plays an important role in vesicle nucleation, presumably by decorating the phagophore with its product phosphatidylinositol-3-phosphate (PI3P), which in turn serves as a signal to recruit further downstream ATG proteins. The ATG9 complex participates in the delivery of lipids required for phagophore expansion [2, 11]. ATG9 is the sole transmembrane protein required for autophagy, and its trafficking to and from the PAS requires a sophisticated targeting and retrieval system involving ATG2 and ATG18 [12, 13]. Subsequently, elongation and closure of the phagophore to form the sealed autophagosome is mediated by two ubiquitin-like proteins, ATG12 and ATG8. During autophagy initiation, ATG12 is conjugated to ATG5 and forms a complex with ATG16. The ATG12-ATG5-ATG16 complex then provides E3 ligase activity to conjugate ATG8 to a phosphatidylethanolamine (PE) moiety. The ATG8-PE adduct subsequently coats the expanding phagophore, promoting its closure by recruiting members of the SH3-BAR family that mediate membrane curvature [14]. ATG8-PE decorating the inner membrane can also serve as a docking platform for autophagy receptors that recruit specific cargo during selective autophagy (see below) [15]. The autophagy machinery is highly conserved in eukaryotes, with most ATG proteins discovered in yeast having also been identified in animals and plants. However, unlike in yeast, many ATG components in plants are present as multiple isoforms; for example, while ATG8 is encoded by a single gene in yeast, nine and five isoforms are found in Arabidopsis thaliana and maize (Zea mays), respectively [16–19].

Numerous studies with yeast, mammalian cells, and plants have shown that autophagy plays a central role in nutrient recycling and cell homeostasis [20, 21]. As such, Arabidopsis mutants defective in the autophagy system are hypersensitive to carbon and nitrogen starvation, and display early senescence even under nutrient-rich conditions [7, 18, 22, 23]. More recently, direct evidence implicating autophagy as a key process in nutrient recycling has come from ¹⁵N labeling experiments. Three studies in Arabidopsis, rice (Oryza sativa) and maize have demonstrated that the efficiency of nitrogen re-mobilization from leaves and other senescing tissues into seeds is significantly decreased in autophagy-defective mutants [24–26]. In addition, the autophagy machinery was shown to be up-regulated upon various nutrient starvations and in senescing tissues. For example, 34 out of 42 maize ATG genes are transcriptionally up-regulated in senescing leaves [24]. Even under optimal growth conditions, autophagic activity increases during the night, with autophagy seeming to play a role in starch breakdown to regulate night-time energy availability, as Arabidopsis and tobacco (Nicotiana benthamiana) autophagy mutants accumulate starch in their leaves [27].

Accumulating evidence indicates that autophagy also plays important roles in stress adaptation, with plant autophagic activity being up-regulated in response to various abiotic and biotic stresses, such as drought, high salinity, high temperature, and during pathogen infection [28]. For example, two Arabidopsis autophagy-defective mutants, atg5 and atg7, plus ATG18a-RNAi plants, displayed enhanced sensitivity to drought, high salinity and methyl viologen treatment compared with wild-type plants [29–31]. Additionally, a recent study in Arabidopsis showed that several autophagy-defective mutants were hypersensitive to hypoxia stress, indicating that autophagy enhances tolerance to low oxygen conditions [32]. Autophagy-mediated turnover therefore seems to function in maintaining cellular homeostasis under adverse growth conditions, presumably by removing abnormal/dysfunctional organelles or protein aggregates.

Autophagy was originally identified as a response to nutrient starvation, and was long considered exclusively a bulk degradative process. However, a multitude of recent studies in yeast and animals have highlighted autophagy as an ordered and highly selective process, with targets including protein aggregates, organelles and invading pathogens. In plants, increasing evidence has suggested that selective autophagic routes play critical roles in clearance of damaged or excess chloroplasts (chlorophagy), mitochondria (mitophagy), peroxisomes (pexophagy), proteasomes (proteaphagy), ribosomes (ribophagy), endoplasmic reticulum components (reticulophagy), ubiquitylated protein aggregates (aggrephagy), invading pathogens (xenophagy) and free phototoxic porphyrins (recently reviewed in [5]). Recent studies have also revealed that autophagy can also selectively degrade specific proteins. Two such cases are ARGONAUTE 1 (AGO1) [33], an important component of the RNA-silencing complex, and BRI1-EMS SUPRESSOR 1 (BES1) [34], a master regulator of brassinosteroid (BR) signaling. Degradation of BES1 is mediated by the ubiquitin receptor protein DOMINANT SUPPRESSOR OF KAR 2 (DSK2); binding of DSK2 to BES1 is mediated by ubiquitylation via the SINAT2 E3 ligase, while binding of DSK2 to ATG8 is regulated by the BIN2 kinase [34].

Our knowledge about autophagy in plants has been delivered by over a decade of dedicated research using traditional genetic, biochemical, and cellular approaches, and these methods still continue to provide unexpected insights into the complexities associated with plant autophagy. However, as autophagy influences such a wide variety of important processes in plants, ranging from development to stress adaption to pathogen resistance, forward genetics approaches have limitations and are not able to provide full appreciation of the impact of autophagy. It is therefore naturally of interest to dissect autophagy from a broader perspective, using more global techniques. Lately, omics approaches have begun to be successfully employed to dissect the molecular mechanisms of plant autophagy. In this current review, findings from transcriptomic, proteomic and metabolic studies of plant autophagy are presented (see Table 1 for summary).

Table 1.

A summary of omics studies on plant autophagy

Study	Species/tissue	Experimental condition	Platform	Key points of interest
Transcriptomics
Contento et al., (2004) [35]	A.thaliana suspension culture cells	Sucrose starvation	Microarray	An ATG8 gene was up-regulated under sucrose starvation conditions.
Caldana et al., (2011) [36]	A.thaliana leaf	Carbon starvation (Dark treatment)	Microarray	Several ATG genes were up-regulated under dark conditions
Buchanan-Wollaston et al., (2005) [37]	A.thaliana leaf	Natural senescence	Microarray	Five ATG genes were identified as up-regulated during leaf senescence.
van der Graaff et al., (2006) [38]	A.thaliana leaf	Natural senescence, DIS and DET	Microarray	19 ATG genes were found to be transcriptionally activated in all three senescence conditions.
Breeze et al., (2011) [39]	A.thaliana leaf	Natural senescence	Microarray	15 ATG genes were transcriptionally up-regulated during leaf senescence with nine of them increasing their expression levels in still-expanding leaves.
Álvarez et al., (2012) [47]	A.thaliana leaf	Growth with exogenous sulfide	Microarray	Transcriptome studies confirm the role of sulfide as a repressor of autophagy.
Caldana et al., (2013) [48]	A.thaliana leaf	Normal growth conditions	Microarray	Transcriptome studies confirm the role of TOR as a negative regulator of autophagy.
Masclaux-Daubresse et al., (2014) [50]	A.thaliana leaf	Short day, low or high nitrogen	Microarray	Genes involved in flavonoid biosynthesis are down-regulated, and pathways for glutathione, methionine, raffinose and galacturonate are altered, in atg mutants.
Garapati et al., (2015) [49]	A.thaliana leaf	Normal growth conditions	Microarray	Transcription factor ATAF1 is identified by gene-expression profiling as a potential repressor of autophagy under carbon starvation conditions.
Li et al., (2015) [24]	Z. mays leaf, endosperm	Normal growth conditions	RNA-seq	A large set of ATG genes are transcriptionally up-regulated during leaf senescence and in developing endosperm.
Pérez-Martín et al., (2015) [46]	C. reinhardtii	Heavy metal toxicity	RNA-seq	Autophagy activity is up-regulated upon heavy metal treatment, and such up-regulation is independent of CRR1.
Williams et al., (2015) [44]	T. loliiformis leaf	Desiccation	RNA-seq	13 ATG genes are up-regulated throughout dehydration and desiccation.
Zhu et al., (2015) [45]	B. hygrometrica leaf	Rapid dehydration, slow dehydration, and rapid dehydration after acclimation	RNA-seq	Autophagy activity is up-regulated by slow dehydration and rapid dehydration after acclimation but not in rapid dehydration.
Minina et al., (2018) [51]	A.thaliana leaf	Natural senescence	Microarray	Transcriptional changes found in plants over-expressing ATG genes are the opposite of those seen in autophagy-defective mutants.
Proteomics
Avin-Wittenberg et al., (2015) [57]	A.thaliana seedling	Carbon starvation (etiolated seedlings grown without sugar)	SDS-PAGE MS	Several proteins accumulate in atg mutants.
Wang et al., (2018) [58]	A.thaliana leaf	V. dahliae infection	iTRAQ	780 proteins were identified through iTRAQ to be differentially abundant in wild-type versus atg mutant plants; most are involved in defense responses, oxidative stress responses, phenylpropanoid and lignin metabolism, and mitochondrial function.
Havé et al., (2018) [59]	A. thaliana leaf	Short day, low or high nitrogen	LC-MS/MS	Chloroplast protease abundance was reduced and levels of proteasome subunits and some cysteine proteases were increased in atg mutants.
Metabolomics
Izumi et al., (2013) [65]	A.thaliana leaf	Carbon starvation (short day-grown starchless mutants harvested at the end of the night)	CE-TOFMS	The release of branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) through protein degradation are partially impaired in starchless atg double mutants.
Kurusu et al., (2014) [67]	O. sativa mature anther	Normal growth conditions	LC-MS	PC editing and lipid desaturation during pollen maturation are compromised in an atg mutant.
Masclaux-Daubresse et al., (2014) [50]	A.thaliana leaf	Short day, low or high nitrogen	GC-MS; LC-MS; starch assay kit	Reduced levels of starch, hexoses and anthocyanins, but higher levels of several amino acids, were detected in atg mutants.
Avin-Wittenberg et al., (2015) [57]	A.thaliana seedling	Carbon starvation (etiolated seedlings grown without sugar)	GC-MS; LC-MS	Reduced levels of free amino acids and altered lipid composition are observed in carbon-starved atg mutants.
Barros et al., (2017) [66]	A.thaliana leaf	Carbon starvation (extended darkness)	GC-MS	The release of many amino acids during protein degradation is partially impaired in dark-treated atg mutants.

Abbreviations are DIS: darkening-induced senescence of individual leaves attached to the plant; DET: senescence in dark-incubated detached leaves; iTRAQ: isobaric tags for relative and absolute quantification; GC-MS: gas chromatography mass spectrometry; CE-TOF MS: capillary electrophoresis time-of-flight mass spectrometry; MS: Mass Spectrometry; LC-MS: liquid chromatography-mass spectrometry; PC: Phosphatidylcholine.

2. Genome-Wide Transcriptome Profiling Helps Unravel the Regulatory Mechanism(s) of Plant Autophagy

The transcriptome is the complete set of RNA molecules present in a specific type of cell or tissue. Transcriptome studies (transcriptomics) using DNA microarrays, particularly in Arabidopsis, have greatly advanced our understanding of how plant autophagy responds to various unfavorable stresses, and have revealed transcriptional regulatory mechanism(s) underpinning plant autophagy.

As a vital pathway for nutrient recycling, the expression of many plant ATG genes is intensely up-regulated under nutrient starvation conditions, and in senescing leaves. The first attempt to use a transcriptomics approach to investigate the regulation of plant ATG genes was conducted in Arabidopsis suspension culture cells [35]. An ATG8 gene was found to display elevated transcript levels upon sucrose starvation, which suggested autophagic activity was increased in response to the lack of carbon. Such energy deprivation-induced autophagy was also observed in dark-treated Arabidopsis seedlings, as multiple autophagy genes including ATG8g were shown to be up-regulated after several hours in darkness [36]. Furthermore, two studies using DNA microarrays investigated how autophagy pathways are activated during senescence in Arabidopsis. In the first study, gene-expression profiles were assessed in senescent leaves from plants at the mid flowering stage, and five autophagy genes, including ATG7, ATG8a, ATG8b, ATG8h and ATG9, were identified among 827 up-regulated genes [37]. A similar but more detailed comparative analysis was conducted with Arabidopsis naturally senescing leaves, individually shaded leaves still attached to the plant, and detached darkincubated leaves [38]. Under all three senescence conditions, 19 ATG genes were found to be transcriptionally activated, suggesting that up-regulation of autophagy activity is a part of the leaf senescence program, perhaps playing a role in nutrient re-mobilization during this process. Recently, a high-resolution, multiple time-course microarray analysis also detected that 15 ATG genes were transcriptionally up-regulated during leaf senescence [39]. Interestingly, expression of nine of these 15 genes began to increase in leaves that were not yet fully expanded, indicating that there may be a role for autophagy other than in nutrient recycling. Further analysis of the overall expression patterns revealed that expression of four ATG genes (ATG7, ATG8a, ATG8b and ATG8h) was highly correlated and rapidly increased at the onset of leaf senescence. Given that ATG7 is required for ATG8 lipidation, the authors proposed that activation of ATG8 by ATG7 may represent the limiting step of autophagy in senescing leaf cells [39].

Genome-wide transcriptome studies to investigate the expression pattern of ATG genes during plant growth and development have subsequently expanded to cover a greater diversity of plants in addition to Arabidopsis [24, 40–42]. For example, an RNA-seq analysis was recently conducted to study the transcript profiles of maize ATG genes in 80 different tissues [24]. This study identified 30 ATG genes that were up-regulated in older leaves, with 27 of these genes up-regulated in the leaf tip, which represents the oldest and most mature portion of a maize leaf. Furthermore, this study also revealed that the transcript abundance of 29 ATG genes increased in the endosperm, but not in the embryo, during seed development. While the role of autophagy in seed development and maturation remains to be fully elucidated, a recent study confirmed that the expression of ATG genes is strongly induced during silique development in Arabidopsis, and that atg mutant plants show increased rates of seed abortion and altered deposition of seed storage proteins in the seeds that remain viable [43].

Transcriptome studies using microarrays or RNA-seq have also indicated a role for autophagy in response to multiple environmental stresses, including desiccation and heavy metal toxicity. Global transcriptome analysis of Tripogon loliiformis, a largely uncharacterized resurrection grass, found that the transcripts of 13 ATG genes were more abundant during periods of dehydration and desiccation [44]. Combined with other cellular and biochemical evidence, the authors proposed that autophagy functions as an important pro-survival mechanism to prevent senescence during desiccation. A recent comparative transcriptome analysis was also conducted in the small perennial resurrection plant Boea hygrometrica, where plants were subjected to either rapid dehydration (desiccation sensitive), slow dehydration (desiccation tolerant), or rapid dehydration following acclimation (desiccation tolerant) [45]. This study highlighted that autophagy activity was up-regulated in plants stressed by slow dehydration or rapid dehydration after acclimation, but not in plants rapidly dehydrated without acclimation, suggesting that autophagy, along with other processes such as protein quality control and energy supply, plays a crucial role in desiccation tolerance. Additionally, transcriptomic analysis revealed an increase in expression of ATG genes in a unicellular green alga (Chlamydomonas reinhardtii) treated with nickel ions [46]. This study also highlighted that the up-regulation of autophagy is independent of COPPER RESPONSIVE REGULATOR 1 (CRR1), a global regulator of copper signaling in Chlamydomonas [46].

The profiling of gene expression has provided valuable insights into the regulatory mechanism(s) of plant autophagy. In Arabidopsis, for example, autophagic degradation is negatively regulated by hydrogen sulfide (H₂S), an observation re-inforced by transcriptomic studies of a mutant defective in the L-CYSTEINE DESULFHYDRASE 1 (DES1) enzyme [47]. These des1 mutant plants have impaired H₂S generation in the cytosol and display elevated autophagy, as indicated by the accumulation of lipidated ATG8 protein. A comparison of the transcriptional profiles of des1 mutants grown with or without exogenous sodium sulfide (Na₂S) led to the conclusion that sulfide acts as a repressor of autophagy. The idea that plant TOR kinase functions as a negative regulator of autophagy was also strengthened by global transcriptome analysis in Arabidopsis. Gene profiling revealed a substantial enrichment for degradative processes such as autophagy and senescence in inducible amiR-tor plants, where the TOR gene is down-regulated [48]. Transcriptome analysis of ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR 1 (ATAF1) also provided clues about how expression levels of ATG genes are regulated in response to carbon starvation [49]. ATAF1 is a NAC transcription factor involved in leaf senescence, and its over-expression results in a transcriptome profile similar to that observed upon carbon starvation, including enhanced expression of ATG genes. Likewise, loss of ATAF1 results in decreased autophagic activity, suggesting that ATAF1 may act as a key regulator that integrates energy status with the expression of ATG genes [49].

In an effort to assess the impacts of autophagy on plant growth and development, the transcriptomes of Arabidopsis autophagy mutants were profiled using microarrays, revealing that genes involved in salicylic acid and ethylene biosynthesis were up-regulated in atg mutants compared with wild type, which is consistent with the increased levels of these phytohormones and the early senescence phenotype found in atg mutants [50]. Furthermore, the transcript levels of many transcription factors involved in stress responses or senescence, such as those from the WRKY and NAC families, were significantly increased. Notably, several master genes involved in flavonoid biosynthesis were found to be down-regulated, which is in agreement with the lower flavonoid content of Arabidopsis atg mutants. The above findings were further confirmed by transcriptomic analyses with transgenic lines over-expressing various ATG genes [51]; many genes involved in salicylic acid signaling, proteolysis, and lipid degradation were down-regulated, while transcripts involved in flavonoid biosynthesis and anthocyanin production, as well as the oxidative stress response, were up-regulated in these plants.

The above studies clearly demonstrate that numerous ATG genes are transcriptionally regulated in plants, and have potentially implicated various groups of stress-responsive transcription factors in this process. However, the underlying molecular mechanisms remain to be elucidated. Numerous factors regulating the transcription of ATG genes have been identified in other model systems, including the transcriptional activators Gat1, Gcn4 and Gln3, and the transcriptional repressors Ume6 and Pho23, in yeast, and their counterparts TFEB and ZKSCAN3, respectively, in mammals [52, 53]. Identification of the responsible transcriptional activators and repressors in plants is an important next step toward the full appreciation of autophagy regulation. Despite this, the current large-scale transcriptomic studies of ATG genes via DNA microarray or RNA-seq have certainly advanced our understanding of the expression patterns and regulatory mechanism(s) of plant autophagy.

3. Proteomics Approaches for Analyzing Autophagy-related Protein Dynamics

As described above, transcriptome analyses have established a simplistic but powerful approach that has greatly advanced our knowledge about the expression patterns and regulatory mechanisms of ATG genes in plants. However, given that proteins represent important players in cellular life, it is naturally of interest to study autophagy using proteomic approaches. Thus far, mass spectrometry (MS)-based proteomics has proven to be a powerful tool for dissecting the autophagy pathway in yeast and mammalian systems (reviewed in [54, 55]). For example, whole cell proteomic approaches in both yeast and mammals have identified changes in protein abundance under certain growth or disease states that have provided clues to novel functions or regulatory mechanisms of autophagy. On the other hand, sub-cellular fractionation studies have provided invaluable insights into the localization, protein-protein interactions and post-translational modifications of ATG proteins by combining proteomic approaches with other classic biochemical techniques, including affinity enrichment/purification of protein complexes [54–56].

In an effort to assess the contribution of autophagy to seedling establishment in Arabidopsis, the protein contents of etiolated wild-type and atg mutant seedlings were comparatively analyzed using a gel-based approach [57]. Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands displaying differential intensities were excised from the gel and analyzed via liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS). Seven proteins, including seed storage proteins and proteins involved in β-oxidation of fatty acids and seedling establishment, showed increased abundance in the atg mutants, indicating that their degradation was likely impaired. This work, although limited owing to the methods used, provided an initial protein list contributing to our understanding of the role of autophagy in protein degradation during early seedling establishment.

In more recent work, proteomic changes in Arabidopsis during Verticillium dahlia infection were investigated, to help appreciate the contribution of autophagy to pathogen resistance [58]. Using the isobaric tags for relative and absolute quantification (iTRAQ) technique, the authors identified 780 differentially abundant proteins between wild-type and atg mutant plants. Most of these proteins are involved in defense responses, oxidative stress responses, phenylpropanoid and lignin metabolism, and mitochondrial function. Notably, further cell biology studies revealed that mitophagy appears in infected roots, suggesting that selective mitochondria degradation may have a role in pathogen defense.

Additionally, a shotgun proteomics study focusing on protease activity revealed reduced levels of the chloroplast FILAMENTATION TEMPERATURE SENSITIVE H (FTSH) and DEGRADATION OF PERIPLASMIC PROTEINS (DEG) proteases in Arabidopsis atg mutants, likely reflecting the impaired plastid maintenance of these plants [59]. Levels of several extracellular serine proteases, including subtilases and serine carboxypeptidase-like proteases, were also reduced. By contrast, proteasome-related proteins and cytosolic or vacuolar cysteine proteases were more abundant in the atg mutant plants, which were likely due to either reduced autophagic degradation of proteasomes and/or an attempt to up-regulate alternative proteolytic pathways to compensate for the lack of autophagy. Thus, while the application of proteomics in plant autophagy is still in its infancy, such approaches seem to have great potential for uncovering new information about the global impacts of this process. As such, learning lessons from yeast and mammalian proteomic studies of autophagy will provide additional opportunities to uncover the fundamental role(s) of autophagy in plants.

4. Metabolomic Analyses for Assessing the Effects of Autophagy on Plant Development

Metabolomics involves the systemic analysis of changes in metabolite profiles within cells, tissues or biological systems. Measuring the abundance of metabolites permits accurate portrayal of the functional and physiological states of a plant, and thus may reveal the underlying causes of the phenotypic effects of stress on plants [60, 61]. Currently, metabolomic profiling is usually conducted using two powerful and complementary techniques, namely nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). As a pivotal cellular catabolic mechanism, autophagy is responsible for breaking down a huge variety of both unwanted or dysfunctional organelles and major macromolecular complexes into simple building blocks such as amino acids, hence dysregulation of autophagic processes often leads to metabolic disorders. Furthermore, as a pathway tightly regulated by cellular metabolic status, autophagy is often influenced by the availability of metabolites such as glucose, amino acids and lipids. It is therefore of great interest to study autophagic processes using metabolomic approaches, which can not only reflect the impact of autophagy on the metabolism of certain cells, tissues or organs, but may also provide insight into regulatory mechanisms of autophagy by metabolic factors.

Under carbon-starvation conditions, plants break down proteins and lipids into simple monomers and use them as alternative carbon sources [62–64]. To address the contribution of plant autophagy in responses to carbon starvation, the metabolite contents of wild-type Arabidopsis plants were compared to those of starchless mutants, atg mutants, or starchless atg double mutants grown under a short day photoperiod for 40 days and harvested at the end of the night [65]. Compared to wild-type plants, starchless mutants accumulated significantly higher levels of free amino acids, particularly branched chain amino acids, aromatic amino acids and lysine, which function both as alternative energy sources and as electron donors to the mitochondrial electron transport chain under carbon-starvation conditions [62, 63]. Further analysis revealed that although total amino acid content was largely unchanged, the levels of branched chain amino acids, aromatic amino acids and lysine were much lower in starchless atg double mutants compared with starchless single mutants, suggesting that autophagy is essential for the supply of amino acids during carbon starvation. Later, two similar observations were reported in 6 day-old etiolated Arabidopsis seedlings grown without sugar, and in 4 week-old plants subjected to extended darkness [57, 66]. Metabolic profiling showed that, compared with wild-type plants, etiolated atg mutant seedlings accumulated lower levels of most free amino acids and some tricarboxylic acid cycle intermediates such as malate, fumarate and dehydroascorbate, together with higher levels of some carbon metabolites, such as fructose, glucose and sucrose [57]. Subsequently, another study examining atg mutant plants subjected to nine days of darkness showed that atg mutants accumulated reduced amounts of branched chain amino acids (leucine, isoleucine and valine), aromatic amino acids (tyrosine) and lysine than wild-type plants [66]. Interestingly, they also found that atg mutants accumulated higher levels of 2-oxoglutarate, malate, fumarate and dehydroascorbate, but had lower levels of glucose and fructose. The contradictory results in relation to carbon metabolites reported in the above two studies may stem from the age of the studied plants.

The role of plant autophagy in response to nitrogen starvation has also been investigated using a metabolomic approach in rosette leaves from 60 day-old wild-type or atg mutant Arabidopsis plants grown under low or high nitrogen conditions [50]. The presented data showed that the atg mutants accumulated a large set of amino acids, including branched chain amino acids and the aromatic amino acid phenylalanine, as well as related compounds such as glutamate, methionine, glutathione and shikimate, regardless of the nitrogen conditions employed for growth. In contrast, the atg mutants accumulated lower levels of hexoses, quercetins and anthocyanins. Interestingly, the corresponding sugar alcohol and aldonic acids of these sugars, such as mannitol, sorbitol, gluconate and malonate, accumulated to higher levels in the atg mutants, particularly when grown under nitrogen-limiting conditions. The authors proposed that the depletion of hexoses and the concomitant increases in sugar alcohols and acids indicated dysregulation of the redox management of sugar components in the atg mutants [50]. In addition to amino acids and carbohydrates, this study and others have also reported distorted lipid profiles in Arabidopsis and rice atg mutants by metabolite profiling, suggesting an important role for autophagy in maintaining cellular lipid homeostasis [50, 67]. However, further specialized lipidomic approaches will likely be needed to fully appreciate this aspect of plant autophagy.

5. Conclusions and Future Perspectives

In this review, we have discussed the recent advances in the study of plant autophagy from a multi-omics perspective. Global analyses, such as transcriptome studies, have been performed to elucidate the expression profiles of ATG genes and uncover novel transcriptional regulatory mechanisms of autophagy. In addition, proteomic and metabolomic profiling have increasingly been used to assess the effects of autophagy on plant growth and development. Nonetheless, many questions remain open, and integrative analysis of multiple layers of omics information is essential to acquire a precise picture of plant autophagy, and to better dissect its regulatory mechanism at a transcriptional, translational and post-translational level. However, there are currently few integrative studies on plant autophagy. Using etiolated Arabidopsis seedlings as a model system, the importance of autophagy in early seedling establishment was clearly demonstrated by integrating metabolomic data with proteomic data [57]. Additionally, transcriptomic data were integrated with metabolic data of Arabidopsis rosette leaves to reveal links between autophagy, transcription and metabolism in plants, and to decipher the diverse effects caused by defective autophagy [50]. However, integrated multi-omics analyses will be required to fully appreciate the importance of autophagy to whole plant physiology.

An additional avenue for future research involves the exploration of autophagosome or organellar proteomics during selective autophagy, which may facilitate the identification of autophagy receptors and reveal the nature of the cargo of various selective autophagy pathways in plants. Such studies have been undertaken previously in mammalian systems [68, 69], but they remain untried in plants. In addition, it is necessary to extend our omics studies to crop plants. With knowledge acquired through such studies, it should be possible to re-engineer crops that more efficiently use nitrogen and other nutrients, that display improved tolerance to various unfavorable growth conditions, and that ultimately provide better grain yields. In summary, future integrative multi-omics approaches are well-suited for deciphering the complex regulatory mechanisms involved in autophagy. We therefore envisage that these approaches will play a key role in enhancing our knowledge of the molecular mechanisms underlying plant autophagy.

Highlights.

• The regulatory mechanism(s) of autophagy was uncovered by transcriptomic studies.

• Proteomics approaches were used to analyze the dynamics of ATG Proteins.

• The effects of autophagy on plant development were assessed by metabolomic analyses.

• Integrative analysis of multi-omics data could provide a precise picture of plant autophagy.

Glossary

ATG

autophagy-related

MS

mass spectrometry