An Overview of the Molecular Mechanisms and Functions of Autophagic Pathways in Plants

ABSTRACT

Autophagy is an evolutionarily conserved pathway for the degradation of damaged or toxic components. Under normal conditions, autophagy maintains cellular homeostasis. It can be triggered by senescence and various stresses. In the process of autophagy, autophagy-related (ATG) proteins not only function as central signal regulators but also participate in the development of complex survival mechanisms when plants suffer from adverse environments. Therefore, ATGs play significant roles in metabolism, development and stress tolerance. In the past decade, both the molecular mechanisms of autophagy and a large number of components involved in the assembly of autophagic vesicles have been identified. In recent studies, an increasing number of components, mechanisms, and receptors have appeared in the autophagy pathway. In this paper, we mainly review the recent progress of research on the molecular mechanisms of plant autophagy, as well as its function under biotic stress and abiotic stress.

KEYWORDS:

• ATGs
• autophagy
• autophagosome
• abiotic stress
• arabidopsis

1. Introduction

In the process of growth and development, plants inevitably encounter various adverse conditions, such as drought, high salinity, pathogen invasion, and nutrient deficiency. Therefore, plants need a sophisticated protein degradation mechanism to cope with various stresses and maintain cell homeostasis.¹ The ubiquitin (Ub)/26S proteasome system (UPS) is a primary proteolytic pathway for abnormal and short-lived proteins in plants.² However, some large particles, such as protein complexes, protein aggregates and damaged organelles, need to be degraded by autophagy. Autophagy can be divided into three categories: chaperone-mediated autophagy, microautophagy, and macroautophagy.^3,4 Chaperone-mediated autophagy is a selective autophagy process that has been reported only in mammals.⁵ This process is independent of vesicles; it recruits individual target proteins through specific chaperone proteins and promotes their direct entry into lysosomes for degradation.⁶ In the process of microautophagy, cytoplasmic substances converge to the surface of the vacuole and are engulfed by invagination of the vacuolar membrane. Then, the tonoplast pinches off to release autophagic bodies, which are intravacuolar vesicles containing cytoplasmic components. Macroautophagy is one of the most well-known autophagic processes and is hereinafter referred to as autophagy. Once autophagy is activated, the phagophore forms and expands, eventually closing to form double membrane vesicles called autophagosomes.^7,8 Then, mature autophagosomes are delivered to lysosomes (mammals) or vacuoles (yeast and plants) and degraded by acidic hydrolases in these lytic compartments.^9,10

In the 1990s, a group of genes needed for autophagosome formation were identified in yeast (Saccharomyces cerevisiae) through forward genetics; these are called core ATG genes.^11–14 Most of the core ATG proteins are conserved in yeast, mammals and plants and can be divided into four groups: (i) the ATG1/ATG13 kinase complex; (ii) the phosphatidylinositol 3-kinase (PI3K) complex; (iii) the ATG9 cycling system; and (iv) the ATG8-PE (phosphatidylethanolamine) and ATG12-ATG5 conjugation systems.^15–17 Although ATG genes are similar in yeast and plants, some plant ATG homologs are found in the form of multigene families. In Arabidopsis (Arabidopsis thaliana), the ATG1 family contains four genes, the ATG4, ATG12 and ATG13 families each contain two genes, and the ATG18 and ATG8 families contain eight and nine genes, respectively.^18–20 Different ATG8 proteins may have different functions in plants, similar to those observed in mammals.²¹ The molecular mechanisms of autophagy are sophisticated and involve many ATG proteins; among them, the activity of the ubiquitin-like protein ATG8 is crucial for autophagy. Since ATG8 is attached to the membrane of the phagophore, autophagosome and autophagic body, it is often used as a standard marker in eukaryotic cells.^21,22

In plants, transcriptional and epigenetic regulation of autophagy has been well reviewed.^23,24 The transcriptional regulation of ATG genes is extremely important for autophagy to maintain cellular homeostasis under adverse environmental conditions. The transcription factors (TFs) including WRKY33, HY5, HsfA1a, ERF5, BZR1 are involved in this process.^25–29 Based on the yeast one-hybrid assay, it has been identified that 225 TFs from 35 families are able to bind to the promoters of 4 ATG8 genes. Meanwhile, these TFs participate in plant development and environmental stress response.³⁰ These results lay a solid foundation for us to understand the transcriptional regulation of plant autophagy. In addition, epigenetic changes such as DNA methylation, histone modification and noncoding RNAs also affect the expression of ATG genes as well as the process of autophagy.²⁴

In this review, we mainly summarize the current understanding of autophagic mechanisms in plants and supplement the latest research progress. On the other hand, the effects of autophagy on plant nutrition metabolism, growth and development and its strong relationship with various biotic and abiotic stresses are discussed.

2. Molecular Mechanisms of Autophagy in Plants

Autophagy is a conserved degradation process in plants, mammals and yeast. There are many brilliant reviews that have summarized the functions of related components and molecular mechanisms of autophagy in plants in detail.^5,6,31,32 In this section, we systematically review the plant autophagy mechanism and add some recent research results (Figure 1).

Figure 1. Schematic diagram of known or proposed steps in macroautophagy. Upon starvation, SnRK1 inhibits TOR activity, which causes ATG13 dephosphorylation and promotes the combination of ATG1, ATG13, ATG101 and ATG11 to initiate autophagy. ATG9, together with ATG18 and ATG2, facilitate the delivery of lipids to the extending phagophore. PI3K complexes synthesize PI3P and decorate the phagophore with it. Subsequently, ATG8 is modified with PE and then conjugated to the phagophore. Once formed, autophagosomes are transported to the vacuole, potentially using the microtubule cytoskeleton controlled by the ESCRT machinery. Finally, autophagosomes fuse with the tonoplast through the v-SNARE-type mechanism and release autophagic bodies into the vacuolar lumen for degradation.

2.1. Induction of Autophagy

Autophagy occurs when environmental and developmental signals affect the activity of several crucial protein kinases. In plants, the heterotrimeric complex sucrose nonfermenting 1-related protein kinase1 (SnRK1) is a major metabolic regulator in response to energy and nutrition deficiency.^33,34 The energy sensor SnRK1 consists of one catalytic α-type subunit (KIN10 and KIN11 in Arabidopsis) and two noncatalytic β-type and γ-type subunits.^35,36 In Arabidopsis, the kin10 kin11 double mutant could not survive, and overexpression of KIN10 not only delayed leaf senescence and flowering³⁷ but also increased autophagosome formation and tolerance to nutritional deficiency,³⁸ indicating that KIN10 regulates development and autophagy in plants. Sucrose nonfermenting 1 (Snf1) and AMP-activated kinase (AMPK) are orthologs of SnRK1 in yeast and animals, respectively. They are important energy and metabolic sensors that maintain homeostasis and activate the autophagy pathway during nutrient deficiency.^39,40 More importantly, they can regulate autophagy through target of rapamycin (TOR).^41,42 TOR is a significant negative regulator of autophagy, and this kinase has two effectors, namely, lethal with sec thirteen 8 (LST8) and RAPTOR. When nutrition is sufficient, TOR is in the active state, which prevents the assembly of the ATG1/ATG13 kinase complex by phosphorylating ATG13. When nutrition is deficient, TOR is in the inactive state, which causes ATG13 dephosphorylation and promotes the combination of ATG1, ATG13 and other factors, such as ATG101 and ATG11.^20,43,44 TOR deletion mutants result in embryo death,⁴⁵ while downregulation of TOR expression results in inhibition of plant growth and autophagy.^46,47 When plants are exposed to various stresses, autophagy can be activated by TOR-independent or TOR-dependent pathways, and auxin acts upstream of TOR.⁴⁸

Through the study of various mutants of the ATG1 kinase complex, researchers confirmed that there are two autophagic signaling pathways in Arabidopsis under fixed-carbon stress. Although ATG1 kinase is essential for autophagic activation during short-term fixed-carbon starvation as well as nitrogen deprivation, it is not necessary for autophagic activation induced by long-term fixed-carbon starvation. The second pathway seems to maintain strong autophagy through the direct phosphorylation of ATG6 by KIN10 in the downregulation or deficiency of the ATG1 kinase complex.⁴⁹ Little is known about the functions of Rho GTPase signaling in plant autophagy. Recently, it was found that Sec5, a subunit of the exocyst complex, could bind to activated ROP8 under stress conditions, and the ROP8-Sec5 module could further transduce upstream ROP8 signaling and promote autophagosome formation by recruiting the ATG1 and PI3K complex to the phagophore. However, at which step the Sec5 exocyst complex functions in plant autophagy is still unknown. One possibility is that Sec5 activates autophagy by recruiting ATG1/13 complexes. Another possibility is that Sec5 plays a targeting role during autophagosome progression.⁵⁰

2.2. Lipid Delivery, Vesicle Nucleation, Phagophore Expansion and Closure

After the completion of ATG1/ATG13 kinase complex assembly, the subsequent steps of autophagy are promoted. ATG9, together with ATG18 and ATG2, facilitate the delivery of essential lipids to the extending phagophore.^51,52 Unlike other ATG proteins, ATG9 is a special multispanning membrane protein and plays an important role in the formation of autophagosomes.⁵³ Recently, the 3D structure of ATG9 at 7.8 Å resolution was presented in Arabidopsis. This result has provided us with structural insights into the oligomeric mode of ATG9, identified the important interaction interfaces, and preliminarily assigned the transmembrane helices located in the membrane-embedded region.⁵⁴ Compared with atg5 and atg7 mutants, autophagy is not seriously blocked in Arabidopsis atg9 mutants.¹⁹ Moreover, upon autophagic induction in the atg9 mutant, an autophagy-related tubular structure accumulates and associates with the membrane of the endoplasmic reticulum (ER).⁵² PI3K complexes synthesize phosphatidylinositol 3-phosphate (PI3P) and decorate the phagophore with it. The PI3K complex is composed of complex I and complex II, both of which contain three core proteins: vacuolar protein sorting 34 (VPS34), VPS15 and ATG6/VPS30/Beclin1. The difference is that complex I contains ATG14, whereas complex II includes VPS38.⁵⁵ VPS38 is not necessary for autophagy in Arabidopsis.⁵⁶ Arabidopsis mutants lacking three core proteins of PI3K are sterile;^57,58 moreover, the atg14a atg14b mutants have typical autophagy-deficient phenotypes, and vps38 mutants show a severe phenotype. Compared with atg14a atg14b and vps38, the phenotype of atg14a atg14b vps38 is severely compromised but still accumulates a low level of PI3P. However, this mutant is sensitive to wortmannin, which indicates that the three core subunits of the PI3K complex are adequate to synthesize the right amount of PI3P.⁵⁵ In Arabidopsis, after treatment with wortmannin and analysis of atg double mutants, ATG11, ATG9 and PI3K were found to act upstream of ATG2. Plant ATG1 and PI3K complexes function in the initiation of autophagy, while ATG2 plays a role in the later stage of autophagic vesicle biogenesis.⁵⁹ Subsequently, starvation-induced ATG8 is modified with PE and then conjugated to the autophagosome precursor (phagophore).⁶⁰ In the Arabidopsis genome, there are two homologs of the ATG4 gene, termed ATG4a and ATG4b.¹⁹ ATG4 is a cysteine (Cys) protease that can cleave the C-terminus of an inactive precursor of ATG8 to expose the conserved glycine (Gly) residue.⁶¹ ATP-dependent ATG7 is an E1-activating enzyme that activates ATG8 and binds the Gly residue of ATG8 with its Cys residue by a thioester linkage. Then, ATG7 donates ATG8 to the Cys residue of the E2-conjugating enzyme ATG3. With the help of the E3 ligase complex, ATG8 attaches to PE and localizes to the phagophore.⁶² The other Ub-fold protein ATG12 is also activated by ATG7 and transferred to its specific E2-conjugating enzyme ATG10. Then, ATG12, through its C-terminal Gly, covalently attaches to the specific Lys of ATG5. The dimeric protein ATG16 combines with the ATG12-ATG5 conjugate to form a hexameric complex that is associated with the phagophore and facilitates ATG8 lipidation.^63–65

3D imaging analysis of growing phagophores in living Arabidopsis tissue showed that ATG5 plays a decisive role in the formation of autophagosomes. After autophagy is induced, ATG5-GFP is localized on the outer surface of the ER, and ATG8 is recruited to the initial phagophore. Then, the phagophore expands into a nearly two-dimensional cisterna, whose edge is associated with ATG5. When it expands into a cup-shaped structure, ATG5 becomes a ring-like structure to encircle the aperture of the phagophore. Finally, once the phagophore is sealed, ATG5 will leave the structure, and the autophagosome will also detach from the ER membrane.⁶⁶ SH3 domain-containing protein 2 (SH3P2) is a typical non-ATG protein in Arabidopsis. By tracing the SH3P2-GFP fusion protein, it was found that SH3P2 is mainly located in phagophore assembly site (PAS) derived from omegasome-like structures or isolated cup-shaped double-membrane structures.^67,68 When autophagy is activated, SH3P2 transfers to PAS and promotes phagophore curvature by binding to PIP, PI3K complex and ATG8. More importantly, RNAi knockdown of SH3P2 inhibits autophagosome formation.⁶⁷ After the autophagosome matures, the ATG8 proteins lining the outer membrane are released by ATG4 protease for recycling, whereas ATG8 proteins lining the inner membrane enter the vacuole and are degraded along with the cargo.^61,69

2.3. Is the Cytoskeleton Involved in the Formation of Autophagosomes?

The cytoskeletal components seem to be essential for the formation of autophagosomes in plants. It has been shown that Arabidopsis ATG8 could bind to microtubules in vitro.⁷⁰ Tobacco (Nicotiana benthamiana) protein Joka2, which is a member of a family of selective autophagy cargo receptors, can colocalize with microfilaments as well as microtubules.⁷¹ ATG6 can colocalize and interact with microtubules in tobacco, and the destruction of microtubules reduces the formation of autophagosomes.⁷² The SCAR/WAVE complex is essential for the actin-related protein 2/3 (ARP2/3) complex to control the dynamics and assembly of filamentous actin and promote actin nucleation.^73,74 All of the members of the SCAR/WAVE complex also exist in Arabidopsis.^75,76 Nck-associated protein 1 (NAP1) is a membrane-localized protein of the SCAR/WAVE complex,⁷⁷ and NAP1-GFP puncta can be triggered by constant pression. These NAP1 puncta not only associate with the cytoskeleton and ER but also colocalize with ATG8. More importantly, the nap1 mutant is sensitive to nutritional deficiency.⁷⁸ However, it is unknown whether the involvement of NAP1 in autophagy relies on its actin nucleation activity. Studies have shown that the effect of the cytoskeleton on autophagy varies across model systems. In plants, actin filaments are unexpectedly dispensable for bulk autophagy. However, actin filaments may be involved in other unknown types of autophagy or selective autophagy.⁷⁹ Arabidopsis selective autophagy cargo receptor neighbor of breast cancer 1 (NBR1) is a homolog of mammalian p62 and NBR1. It was found that YFP-AtNBR1 not only exhibited cytosolic and punctate patterns but also showed microtubule patterns especially in the cortical layer. Instead of microfilament depolymerizer Latrunculin B, treatments with microtubule depolymerizer Oryzalin disrupted the microtubule localization and influenced the vacuolar delivery of YFP-AtNBR1 upon autophagy induction. This suggests that AtNBR1 may need the assistance of microtubules to shuttle its goods to vacuoles during plant autophagy.⁸⁰ Therefore, it seems that microtubules play a more important role than microfilaments in the autophagic pathway, and the exact relationship between autophagy and cytoskeleton remains to be further studied.

2.4. The Delivery of Autophagosomes

The transport of autophagosomes to vacuoles may involve several special trafficking mechanisms. The endosomal sorting complex required for transport (ESCRT) machinery is an evolutionarily conserved protein complex that is mainly responsible for sorting ubiquitinated protein entry into the intraluminal vesicles (ILVs) of prevacuolar compartments/multivesicular bodies (PVCs/MVBs), which then fuse with lytic organelles to degrade cargos.^81,82 As a component of ESCRT, FYVE domain protein required for endosomal sorting 1 (FREE1) participates in vacuolar protein transport and the regulation of autophagic degradation^83,84 In addition, FREE1 directly interacts with SH3P2 to regulate the fusion progress between autophagosomes and vacuoles. Both autophagosomes and ATG8-PE accumulate to a certain extent in the free1 mutant.⁸³ It has been reported that ESCRT-III subunit VPS2.1 and cell death-related endosomal FYVE/SYLF protein 1 (CFS1) are also related to transport and fusion.^85,86 The deubiquitinating enzyme AMSH3 can interact with ESCRT-III, and loss of AMSH3 affects vacuolar transport and vacuole formation.⁸⁷ AMSH1 is involved in autophagy mediated degradation. More importantly, it interacts with VPS2.1, and both mutants have autophagy-related phenotypes.⁸⁵ On the other hand, the plant exocyst component EXO70B1 is also necessary for autophagosome formation and transport. The exo70B1 mutants are susceptible to nitrogen deficiency. Furthermore, the amounts of intravacuolar autophagic bodies are decreased in this mutant.⁸⁸

2.5. Fusion of Autophagosomes and Tonoplasts

Finally, autophagosomes fuse with the tonoplast through the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE)-type mechanism⁸⁹ and release autophagic bodies into the vacuolar lumen for degradation. As a member of the VTI family of v-SNAREs, VTI12 may be involved in plant autophagy. The vti12 mutant grows normally under nutrient-rich conditions, but senescence is accelerated under nutrient-poor conditions.⁸⁹ AtVPS45 and VTI12 may be functionally related because both mutants exhibit trafficking defects, and autophagy-related defects are found in VPS45-silenced lines.⁹⁰ In animals, some small Rab GTPases bind to autophagosome membranes to regulate the fusion process of lysosomes and autophagosomes by recruiting distinctive effector proteins. As one of the members, Rab7 plays an important role in the maturation of autophagosomes and endosomes and their subsequent fusion step with lysosomes (animals) or vacuoles (yeast).^91–93 RabG3b, a homologous protein of Rab7 in Arabidopsis, is located in ATG8e-labeled autophagic structures. During tracheary element (TE) differentiation, autophagy is activated, and transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) accumulate many autophagic structures. RabG3b may regulate TE differentiation as well as TE programmed cell death (PCD) through autophagy.⁹⁴ The hypersensitive response (HR) is a core component of the plant defense response to pathogens. RabG3b can facilitate HR cell death through autophagy. In Arabidopsis RabG3bCA cells, autophagosomes also increase during HR cell death.⁹⁵ In Arabidopsis, whether Rab7 can regulate the fusion process between autophagosomes and vacuoles remains to be further studied, and the function of Rab GTPase in autophagosome biogenesis is still unclear. Recently, it has been reported that a specific Rab GTPase, AtRabD2a, can specifically interact with ATG1 complex and the coat protein complex II (COPII) machinery and act as a link between autophagy and COPII.^96,97

2.6. Degradation of Autophagic Bodies in Vacuoles

Autophagic bodies can be degraded into amino acids and other small molecules for recycling by various hydrolases in the vacuole.⁹⁸ Most enzymes in vacuoles are synthesized as precursors that need to be processed into active forms. Vacuolar processing enzyme-γ (VPEγ) is a cysteine proteinase that can be induced during plant senescence and death. VPEγ degrades cargos by releasing pro-proteins into the acidic vacuole lumen to initiate processing.⁹⁹ The continuous acidification of vacuoles is extremely important for the activity of hydrolases. Vacuolar-type ATPases (V-ATPases) are ATP-dependent proton pumps that are located at the tonoplast and continuously pump H⁺ into the vacuole to maintain its acidic environment.^100,101 Concanamycin A (ConcA), a crucial inhibitor of V-ATPase, can prevent the acidification of vacuoles and inhibit the degradation of autophagic bodies, thus increasing its visibility under the microscope.^61,102 The use of this inhibitor provides a very important approach for scientific research on autophagy. Recently, it has been reported that the cytoplasmic soluble GFP was accumulated in vacuoles after treatment with ConcA.¹⁰³ Further studies showed that GFP signal colocalized with the autophagic marker mCherry-ATG8f in vacuoles, but free GFP did not display direct interaction with ATG8s. Therefore, when reporting specific cytosol components degraded by selective autophagy, colocalization analysis need to be carried out in the cytoplasm rather than in the vacuole, so as to avoid the nonselective transport of cytoplasmic components into the vacuole. In addition, other parallel experiments need to be combined to judge whether cytoplasmic proteins are degraded by selective autophagy, which will contribute to identify the cargos and receptors of selective autophagy in plants.¹⁰³

3. The Function of Autophagy in Plants

After years of research, we have a profound understanding of the functions of autophagy in plants (Figure 2 and Table 1). Under normal conditions, autophagy occurs at basal levels^104,105 and participates in a variety of biotic and abiotic stresses.^31,106,107 In Arabidopsis, most atg mutants grow normally under nonstress conditions and show a greater sensitivity to nutritional deficiency. The rosette leaves and cotyledons of the atg mutants show early senescence under short-day (SD) conditions, and the seed setting rate is lower than that of the wild type (WT).²¹ In addition, ATG genes are upregulated at the transcriptional level under carbon and nitrogen deficiency.^108,109,110

Table 1. Summary of physiological functions of autophagy related components and some regulators in plants

Functions		Genes/Proteins	References
Nutrient Recycling and Metabolism	Carbon and nitrogen recycling	AtATG2/5/7/9/10/18a, OsATG7	^Citation112,Citation113,Citation115–119,^Citation124–126,Citation131
Growth and Development	Seed development Leaf senescence TE differentiation Root meristem maintenance Pollen development	AtATG5/8f, ZmATG12/8 AtATG2/5/7/9/18a AtRabG3b AtATG2/5/7 AtATG5/6, AtVPS15/34, OsATG7, NbATG2, AtPTEN,	^Citation138–140 ^Citation19,Citation102,Citation130,Citation142,Citation173 ^Citation94,Citation144 ^Citation145 ^Citation57,Citation58,Citation115,Citation146–153
Immune Response	Pathogens Virus	NbVPS34, NbGAPCs, NbATG3/6/7, AtWRKY33 AtATG2/5/6/7/9/10/18a, AtRabG3b, AtBAG6, AtBAK1, AtExo70B2 AtGAPCs, NbATG3/5/6/7/8a/8h/8f, NbGAPCs, AtNBR1 AtATG5/7/8a,	^Citation25,Citation95,Citation159–161, ^Citation163–169,Citation174,Citation208 ^Citation181,Citation185–190
Abiotic Stress	Drought Salt Heat Oxidation Hypoxia	TdATG8,TaATG4a/4b/8a/8g/8h, SlHsfA1a, SlATG10/18f, AtKIN10, AtATG5/7/18a MtCAS31, AtCOST1, AtDSK2A/2B, AtNBR1 AtPI3K, AtATG5/7/8f/18a, AtTSPO, AtATI1/2, AtNBR1, PagNBR1, OsATG10b, TaATG8a, GmATG8c SlWRKY33, SlATG5/7, AtATG2/5/6/7/12a/12b/18a AtATI3A/3B/3C, AtNBR1, SlNBR1a/1b OsATG10b, AtATG5/7/18a AtNBR1 AtKIN10, AtATG2/5/7/10	^Citation27,Citation38,Citation106,Citation193,Citation195, ^Citation196,Citation216–218 ^Citation106,Citation193,Citation196,Citation198–201,Citation219,Citation220,Citation222,Citation226 ^Citation193,Citation203–206,Citation223 ^Citation192,Citation193,Citation200,Citation208 ^Citation38,Citation210

Figure 2. Simplified scheme of the induction, process and function of autophagy in plants. Autophagy is induced by various factors, through a series of processes such as nucleation, expansion, closure, fusion and degradation, so as to recycle various nutrients and realize diverse physiological functions.

3.1. Nutrient Recycling and Metabolism

In recent years, many studies have shown that autophagy plays a key role in nutrient cycling and reuse. As the primary carbon source of plant respiration, carbohydrates are mainly stored in chloroplasts in the form of starch for nighttime consumption.¹¹¹ Autophagy is active at night, and the content of starch in the leaves of autophagy-deficient Arabidopsis and tobacco is increased.¹¹² In Arabidopsis, the growth inhibition of atg mutants under SD will be relieved by the application of exogenous sucrose or treatment with full light conditions, and the starchless and atg double mutants have severe starvation phenotypes under SD, indicating that autophagy is involved in energy availability.¹¹³ Lipids and proteins will be used as backup carbon sources when carbohydrates are deficient in cells.¹¹⁴ For example, autophagy regulates nutritional supplements and lipid metabolism in anthers, and it is critical for anther development after meiosis in the tapetum cells of rice (Oryza sativa).¹¹⁵ In Arabidopsis, it has been found that basal autophagy facilitates the synthesis of triacylglycerol, while inducible autophagy promotes the degradation of lipid droplets. These results indicate that autophagy plays a dual role in regulating plant lipid metabolism.¹¹⁶ Autophagy has an effect on lipid homeostasis under dark-induced senescence.¹¹⁷ The statistical characterization of metabolic data indicates that autophagy deficiency has noteworthy effects on metabolite profiles in Arabidopsis leaves. Compared with the WT, 4 metabolites in Arabidopsis atg5 mutants were largely decreased, including urate, saccharopine, putrescine and N6,N6,N6-trimethyllysine, while 36 metabolites were increased. These results suggest that autophagic proteolysis is related to plant metabolic processes.¹¹⁸ Various omics analyses have shown that autophagy has a crucial effect on metabolism and leads to a morphological phenotype during starvation in Arabidopsis. Delayed growth is found in etiolated atg mutants, and the content of free amino acids also decreases.¹¹⁹ Autophagy is not only involved in amino acid cycling but also responsible for increasing the accumulation of proteogenic dipeptides during recovery from heat stress in Arabidopsis.¹²⁰ In maize (Zea mays), multi-omics analysis clearly identifies the role of autophagy in various cellular processes under nitrogen-replete and -starvation conditions, and shows that autophagic recycling has a significant impact on lipid turnover and proteome remodeling.¹²¹ Maize multi-omics also showed that autophagy plays remarkable roles in nucleotide, amino acid, and carbohydrate metabolism during fixed-carbon starvation.¹²²

In higher plants, nitrogen is the most important nutrient element and plays a decisive role in plant productivity.¹²³ Autophagy also affects nitrogen remobilization, and it has been confirmed that nitrogen use efficiency (NUE) decreases at the whole-plant level and is independent of senescence in autophagy mutants.¹²⁴ Moreover, rosette leaves show the accumulation of several proteins due to aberrant protein degradation in the Arabidopsis atg mutant.¹²⁵ Although leaf senescence of Osatg7-1 is significantly advanced, the nitrogen concentration in leaves is still high, indicating that the NUE and biomass production of Osatg7-1 are lower than those in WT.¹²⁶ In soybean (Glycine max) callus cells, the constitutive expression of GmATG8c could enhance tolerance to nitrogen deficiency, and GmATG8c overexpressing Arabidopsis could survive under long-term nutrient deficiency. In a suitable growing environment, the overexpression lines grew faster than the WT and had larger inflorescences and higher seed yields.¹²⁷ Studies have shown that overexpression of ATG8 has a beneficial effect on nitrogen remobilization at the whole-plant level without affecting plant biomass or total seed yield in Arabidopsis.¹²⁸ Consistently, compared with the WT, most Arabidopsis autophagy mutants are hypersensitive to nitrogen starvation,^{20,51,55,102,129,130} which further proves that autophagy regulates nitrogen cycling. In contrast to the decrease in free amino acids in etiolated seedlings,¹¹⁹ it was found that the content of ammonium and free amino acids in the atg mutant increased significantly at 60 days after sowing (DAS).^125,131 The output of ammonium and amino acids requires strong phloem loading, so the longevity of vein tissue is necessary.¹³² It is possible that the mutant leaves at 60 DAS affect phloem loading during senescence, resulting in the accumulation of ammonium and total amino acids.¹²⁵ The metabolome data from atg mutants showed that amino acid interconversion metabolism mainly regulates oxidative stress through various pathways and ultimately affects the concentration of amino acids.¹³¹

3.2. Growth and Development

To ensure survival, many kinds of developing seeds synthesize a large number of storage proteins, which are degraded during early germination to start photosynthesis and synthesize new plant organs.^133,134 During seed development, synthetic storage proteins are transported from the ER to protein storage vacuoles through a process similar to autophagy.^21,135,136 The expression levels of many ATG genes are upregulated during seed maturation as well as drying and approach the highest level in dry seeds in Arabidopsis.^21,137 In Arabidopsis, ATG8f is specifically expressed in seeds and siliques. Seed development is accelerated and storage protein accumulation is altered in atg5 mutants.¹³⁸ The ATG8-PE adducts are increased in starchy endosperm during seeds maturation after pollination in maize, and many ATG8-PE adducts are detected after seed germination, indicating that autophagy is beneficial to the remobilization of nutrients in endosperm during early seed development.¹³⁹ RNA-seq analysis shows that the transcription abundance of the maize ATG gene increased in the endosperm, indicating that autophagy is involved in its maturation and death process during seed development.¹⁴⁰ Autophagy is very important for grain filling in maize and Arabidopsis.^124,140 ATI1 and ATI2 are special ATG8-interacting proteins in Arabidopsis. They are partially associated with the ER membrane network under suitable conditions, and with spherical compartments under carbon starvation. In the presence of the exogenous hormone abscisic acid (ABA), the expression levels of ATI1 and ATI2 will affect the germination rate in Arabidopsis seeds.¹⁴¹

AtATG18a RNAi plants are not only sensitive to nutrient deficiency but also show premature leaf senescence. Monodansylcadaverine (MDC) staining showed that AtATG18a RNAi plants could hardly produce autophagosomes in response to nutritional starvation.¹⁴² Autophagy is beneficial to the process of proteolysis of stromal proteins, such as Rubisco activase, Rubisco large subunit and chloroplast glutamine synthetase. In Arabidopsis atg5 and atg7 mutants, the degradation of these proteins is incomplete.¹⁴³ The degradation of starch in leaves also requires the assistance of autophagy during the night. Similar to ATG6-silenced tobacco, the starch degradation ability of other ATG-silenced plants shows a decrease in varying degrees.¹¹² When E-64d was used to treat Arabidopsis root tip cells, almost no vacuolar inclusions were observed in the atg2 and atg5 mutants, but a large number of vacuolar inclusions appeared in the atg9 mutant. These results indicate that not only ATG2 but also ATG5 are essential for autophagy in Arabidopsis roots, while ATG9 is helpful but not essential. An autophagy inhibitor called 3-methyladenine could inhibit the accumulation of this vacuolar inclusion.¹⁰⁵ As an important component of autophagy, the small GTPase RabG3b may regulate TE differentiation via activating autophagy.^94,144 In Arabidopsis, autophagy could facilitate glucose-mediated root meristem maintenance by modulating reactive oxygen species (ROS) levels.¹⁴⁵ Arabidopsis PTEN, a special lipid and protein dual phosphatase homologous to animal PTENs (phosphatase and tensin homologs deleted on chromosome 10), regulates autophagy in pollen tubes by interfering with the dynamics of PI3P.¹⁴⁶ During Arabidopsis pollen development, PI3K plays a pivotal role in vacuole reorganization and nuclear division.⁵⁸ The components of PI3K complex, including AtVPS34, AtVPS15, AtATG6, participated in pollen germination.^{57,58,147–150} OsATG7 knockout mutants not only had no accumulation of starch grains and lipid bodies in pollen grains but also showed male sterility, limited anther dehiscence and decreased pollen germination activity.¹¹⁵ Autophagy is necessary for the regulation of gene expression and timely progression of tapetal PCD during rice pollen maturation.¹⁵¹ Autophagy also plays an important role in pollen development and tapetum degeneration during heat-induced tapetal PCD abortion.¹⁵² In tobacco, pollen germination is accompanied by the improvement of autophagy activity and compartmental cytoplasmic deletion is vital for male fertility.¹⁵³ The role of autophagy in plant male reproductive development has been well summarized in some reviews.^154,155

3.3. Immune Response

Unlike mammals, plants lack mobile immune cells, so they can only depend on the innate immune system to recognize pathogenic nonself molecules for defense. Plants resist the invasion of pathogenic microorganisms and maintain their normal growth and development through two immune defense systems: pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). In general, ETI can lead to cell death in host plants, referred to as HR,^156,157 a form of PCD.

The most prominent phenotype of autophagy mutants is premature senescence accompanied by accelerated cell death.¹⁰⁷ It has been reported that autophagy possesses both pro-survival and pro-death roles and may be connected with the plant defense response, especially ETI.¹⁵⁸ HR cell death may play a significant role in plant disease resistance by restricting the spread of pathogens. The most significant characteristic of HR is the accelerated death of cells in the local area around the infection. For example, tobacco protein NbATG6, an ortholog of the yeast and mammalian ATG6/VPS30/ BECLIN 1, could limit HR PCD to the infection site. In the HR PCD reaction, both infected cells and uninfected cells could induce autophagy, which is weakened in ATG6-deficient plants, indicating that autophagy plays an important role in innate immunity and negatively regulates PCD.¹⁵⁹ In AtATG6 antisense (AtATG6-AS) plants, HR PCD induced by avirulent Pst DC3000 carrying the AvrRpm1 effector protein could spread from infected sites to uninfected areas. In addition, the disease-related cell death caused by highly virulent Pst DC3000 bacterial infection is also partially misregulated in AtATG6-AS plants.¹⁶⁰ In plant innate immunity, there are several examples to prove that autophagic components contribute greatly to cell death. By detecting HR PCD in the Arabidopsis atg mutants, it was found that only a special subset of immune receptors participate in the autophagy pathway for HR execution.^161,162

Autophagy contributes to plant immunity against diverse pathogens, and the mechanisms are quite different. Even in response to the same pathogen, there are different proteins involved in autophagy by different mechanisms. In Arabidopsis, the resistance of atg5, atg10 and atg18a mutants to necrotrophic fungal pathogens is impaired, but the resistance to biotrophic bacterial invaders is enhanced. Therefore, autophagy, as a pro-death or anti-death function, largely depends on the infection strategy as well as the lifestyle of invasive microorganisms.¹⁶³ ATG7-dependent autophagy forms an anti-death plant mechanism to regulate the containment of cell death and immunity to necrotrophic fungal infection. It has been suggested that ATG7 plays an important role in plant resistance to fungal pathogens.¹⁶⁴ WRKY33 is an essential transcription factor for plant resistance to necrotrophic pathogens in Arabidopsis. ATG18a and WRKY33 could interact with each other, and both mutants are highly sensitive to the necrotrophic pathogen Botrytis cinerea, which not only induces autophagy gene expression but also promotes autophagosome formation. WRKY33- and jasmonate-mediated signaling pathways and autophagy may synergistically regulate the plant defense responses to necrotrophic pathogens.²⁵

When Arabidopsis responds to avirulent bacterial pathogens, RabG3b facilitates HR cell death by activating autophagy, and this process is partially ATG5 dependent.⁹⁵ BAG6 belongs to the Bcl-2-associated athanogene (BAG) family, whose cleavage leads to plant defense and autophagy.¹⁶⁵ In Arabidopsis, glyceraldehyde-3-phosphate dehydrogenase (GAPCs) could regulate cell death when inoculated with Pseudomonas syringae.¹⁶⁶ Similarly, ATG3 interacts with GAPCs to regulate PCD and autophagy during innate immunity in tobacco.¹⁶⁷ BAK1 is an important regulator of plant resistance that can interact with and phosphorylate ATG18a. Phosphorylation of ATG18a can inhibit the formation of autophagosomes and the subsequent transfer to vacuoles, resulting in a decrease in autophagy activity and resistance against Botrytis cinerea.¹⁶⁸ In Arabidopsis, exocyst subunit Exo70B2 interacts with ATG8 and kinase MPK3. In addition, phosphorylation of Exo70B2 by MPK3 enhances the interaction between MPK3 and ATG8. Phosphonull variants exhibit higher ETI and hypersensitivity to benzothiadiazole (BTH), which result in autophagy and secretion.¹⁶⁹

Salicylic acid (SA) is an important plant defense hormone that can effectively resist biotrophic pathogens in living plant tissues.^170–172 Studies have shown that pathogen-induced HR PCD and early senescence in atg mutants are related to SA signaling,^173,174 and SA largely accumulates in atg mutants.^163,173 In addition, pathogen infection and BTH treatment induce ATG gene expression,^25,160,173 all of which indicate that there is a negative feedback loop between SA signaling and autophagy to control each other. Furthermore, some studies have shown that autophagy can function SA-dependently and -independently.^173,174 Plant age has a significant impact on the spread of SA-dependent HR cell death in autophagy mutants. For a specific host-pathogen interaction, autophagy only has a pro-death role in a limited area during a particular period (possibly transiently and early), while autophagy plays a pro-survival role in most other periods (especially in senescent cells and tissues).^{161,173,175,176}

Autophagy is the key part of plant immunity and the crucial target of pathogens. Plant pathogens regulate host autophagy by injecting effector proteins. However, the exact molecular mechanism of autophagy manipulated by plant pathogens remains unclear. Recent studies have systematically analyzed the interaction between ATG protein of Arabidopsis and effectors of various plant pathogens and verified the interactions of a subset of effectors with biochemical and in vivo experiments. The results show that effectors of plant pathogens regulate plant autophagy through a variety of mechanisms.^177,178 PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host ATG8CL to trigger the formation of autophagosomes. PexRD54 could deplete the autophagic cargo receptor Joka2 from ATG8CL complexes and hamper the positive role of Joka2 in pathogen defense.¹⁷⁹ Additionally, the structural basis of this interaction has been analyzed.¹⁸⁰

Increasing evidence shows that autophagy also plays a significant role in the process of plant virus infection, and an excellent review has been published.¹⁸¹ During viral infection, autophagy is thought to exert both antiviral and proviral roles.^181,182 Autophagy plays an antiviral role when plants are infected by DNA viruses^{159,183–187} or RNA viruses.^188–190 Discoveries of the antiviral effect of selective autophagy have attracted much attention.^186,189,190 In turn, plant viruses can hijack, subvert, or even exploit the autophagy pathway for transmission or infection.^188,191 However, we still know little about the role of autophagy in the plant antiviral defense response, and there is no report indicating that plant viruses are completely limited by autophagy. How autophagy is inhibited in particular plant-virus interactions needs to be further studied.

3.4. Abiotic Stress

In Arabidopsis, abiotic stresses such as nutrient deficiency, drought, heat stress, oxidation and salt can induce autophagy (Figure 2) and most atg mutants are highly sensitive to these adverse conditions.^{106,142,192–194} Autophagy is an important mechanism for plant stress response. It has been reported that many ATG genes are involved in the abiotic stress response (Table 1).

Drought and high salinity are unavoidable abiotic stresses for plants. Drought can not only cause membrane damage but also decrease the activity of cytoplasmic and organelle proteins and even lead to complete denaturation. In wild emmer wheat (Triticum dicoccoides), the expression pattern of ATG8 shows that ATG8 is extremely induced under drought stress.¹⁹⁵ Drought treatment can enhance the activity of five genes, ATG4a, 4b, 8a, 8g and 8h, and prolong the effect on ATG4a and 4b in wheat (Triticum aestivum L.).¹⁹⁶ In tomato (Solanum lycopersicum), HsfA1a regulates drought tolerance by activating autophagy, and autophagy facilitates plant survival by degrading insoluble proteins produced under drought stress. HsfA1a can positively regulate drought-induced autophagy and directly bind to the ATG10 and ATG18f promoters to regulate their expression.²⁷ In Arabidopsis, the sensitivity of KIN10-OE lines to drought and hypoxia is lower than that of the WT.³⁸

High salinity can cause ion stress and osmotic stress and may lead to plant death. The main ions involved in salt stress are H⁺, Na⁺, K⁺ and Ca²⁺, and intracellular homeostasis is maintained by the interaction of these ions.¹⁹⁷ In Arabidopsis, both wortmannin-treated plants and pi3K mutants are extremely sensitive to salt.¹⁹⁸ The expression of the GFP-AtATG8f-HA fusion protein seems to make plants more sensitive to mild osmotic stress as well as salt stress.¹⁹⁹ OsATG10b mutants have a greater sensitivity to both methyl viologen (MV) and high salt treatment.²⁰⁰ Compared with the wild type, autophagy-defective RNAi-AtATG18a plants are hypersensitive to drought and salt.¹⁰⁶ Under high salt conditions, TaATG8a has the highest transcript accumulation among the three TaATG8s.¹⁹⁶ In Arabidopsis, atg mutants are extremely sensitive to osmotic and salt stress, and ATG8-OX plants show better germination than WT plants on NaCl-containing plates.²⁰¹

Heat stress is a common and important factor limiting plant productivity. It can lead to the accumulation of denatured proteins, which easily aggregate.²⁰² In tomato, heat-induced autophagy may be cooperatively regulated by ATG and WRKY33 proteins through selective degradation of protein aggregates. In addition, ATG5- and ATG7-silenced plants showed increased sensitivity to heat stress.²⁰³ Recent studies have shown that autophagy plays a specific role in resetting the memory of heat stress in plants.²⁰⁴ In Arabidopsis, ATG2 could be induced by high temperature. Moreover, the mRNA levels of ATG6, ATG5, ATG18a and ATG12a in WT plants are significantly increased after high temperature treatment.²⁰⁵ Transcriptome analysis of Arabidopsis WT and atg5 mutant plants prior to and after heat stress showed that autophagy has little effect on heat shock gene expression induced by heat, but autophagy plays an active role in the expression of numerous other stress- and defense-related genes in the early stage of plant heat stress response. These findings provide significant new insights into the extensive functions of autophagy.²⁰⁶ When the amount of unfolded or misfolded proteins within the ER increases beyond the capacity of the intracellular folding and degradation system under heat stress and other adverse conditions, it will eventually lead to ER stress.²⁰⁷

NADPH oxidase inhibitors could impede autophagy induction under salt stress and nutrient starvation, but not under osmotic stress, indicating that there are NADPH oxidase-dependent or -independent pathways for activating autophagy.¹⁰⁶ Compared with the WT, RNAi-AtATG18a transgenic plants were susceptible to MV treatment and accumulated more oxidative proteins on account of a lower degradation rate. When ConcA was applied, oxidized proteins were observed in WT root cells but not RNAi-AtATG18a root cells. These results indicate that the oxidative proteins induced by oxidative stress can be degraded by autophagy.¹⁹² In addition, overexpression of ATG5 and ATG7 can enhance the resistance of plants to oxidative stress.²⁰⁸ In mammals, both hypoxia and ROS can cause autophagy.²⁰⁹ In Arabidopsis, submergence and ethanol treatments can induce the accumulation of ROS in the atg mutants more than in the WT, the ROS signaling pathways and SA-induced defense may be involved in the regulation of autophagy-related hypoxia response.²¹⁰ Various stresses may induce ROS production, which in turn activates autophagy.¹⁷⁶ In contrast, ROS significantly accumulated in the leaves of atg2 and atg5 mutants grown in SD for 8 weeks, indicating that autophagy can partially limit ROS levels.¹⁷³ It has been reported that autophagy can control ROS homeostasis by removing oxidized peroxisomes, thus optimizing stomatal opening.²¹¹ In summary, these results indicate that autophagy components play an important role in plant tolerance to abiotic stress.

Autophagy was originally defined as a nonselective bulk degradation process. However, in recent years, accumulated evidence has shown that the recruitment of targets into autophagosomes is highly selective.^5,212 ATG8 interacts with diverse adaptor/receptor proteins to recruit specific targets for degradation by selective autophagy. These interactors usually contain an ATG8-interacting motif (AIM, or LC3-interacting region (LIR)) that contacts a hydrophobic patch on ATG8 collectively termed the LIR/AIM-docking site (LDS).²¹³ In Arabidopsis, RPN10 (regulatory particle non-ATPase 10) acts as an autophagy receptor for the ubiquitinated 26S proteasome; however, its binding to ATG8 rely on the ubiquitin-interacting motif (UIM) instead of the canonical AIM.²¹⁴ The UIM-docking site (UDS) is important for the interaction between RPN10 and ATG8.²¹⁵ UDS is located opposite to the LDS; thus, ATG8 can bind to AIM- and UIM-containing proteins simultaneously. The discovery of UIM-UDS interface greatly enlarges the range of autophagy adaptors and receptors, thereby expanding the reach of selective autophagy. There are several autophagy receptors in plants, some of which play a role in abiotic stress response (Table 1).

In Medicago truncatula, dehydrin MtCAS31 can interact with ATG8a. Under drought stress, MtCAS31 not only promotes autophagic degradation of aquaporin MtPIP2;7 but also reduces root hydraulic conductivity, thereby decreasing water loss and enhancing drought resistance.²¹⁶ COST1 is a plant-specific gene that promotes plant growth by inhibiting autophagy under suitable conditions. COST1 is degraded under drought conditions to activate autophagy and inhibit growth, thus enhancing drought tolerance.²¹⁷ DSK2 is a phosphorylation-regulated selective autophagy receptor. Due to tandem replication, there are two DSK2 paralogs genes (DSK2A and DSK2B) in Arabidopsis and both proteins are involved in BES1 degradation under drought stress conditions.²¹⁸ Arabidopsis TSPO, a tryptophan-rich sensory protein (TSPO)-related membrane protein,²¹⁹ its expression is induced by osmotic and salt stress.^219,220 The degradation of TSPO is inhibited in atg5 mutant and is sensitive to PI3K inhibitor.²²¹ Arabidopsis ATG8-interacting proteins 1/2 (ATI1/2) are AIM-motif-containing proteins identified from yeast two-hybrid screen with ATG8f as bait.¹⁴¹ ATI1 is involved in salt tolerance of Arabidopsis possibly by scavenging damaged plastid proteins.²²² Dicot-specific ATI3 proteins have a WxxL motif at the C-terminus, which is required for interaction between ATI3 and ATG8. Disruption of ATI3 significantly compromises plant heat tolerance.²²³

Perhaps NBR1 is the most well-studied autophagy receptor in plants. Under heat stress, nbr1 mutants accumulate insoluble proteins. Ubiquitinated protein aggregates derived from denatured proteins are mainly degraded by NBR1-mediated autophagy. Like autophagy-deficient atg5 and atg7, nbr1 mutants exhibit hypersensitivity to heat, salt, oxidative and drought stresses.¹⁹³ Similarly, virus-induced gene silencing (VIGS) of NBR1 compromised tomato heat tolerance.²⁰³ NBR1 also plays a key role as a receptor for selective autophagy during recovery from heat stress. It interacts with ROF1 (rotamase FKBP 1) and HSP90.1 (heat shock protein 90.1) and mediates their degradation by autophagy to regulate heat stress memory in Arabidopsis.²²⁴ In tomato, studies have shown that BRs regulate NBR1-dependent selective autophagy in response to cold stress.²²⁵ In poplar, PagNBR1 is also a significant selective autophagy receptor and confers salt tolerance by accelerating the activity of antioxidant system and autophagy.²²⁶ In addition to the important role in these abiotic stresses, plant NBR1 is also involved in the regulation of plant-virus interactions and special protein secretion. And it may play a role in stress-induced pexophagy, sulfur nutrient responses and ABA signaling.^227,228 Other selective autophagy receptors with very important functions have also been well summarized.²²⁸ ATI3s, Sec62, Reticulons (Rtn) and C53 proteins are involved in ER-phagy. Adi3 and ORM1/2 are associated with immune responses. DSK2 and GSNOR1 are closely linked to signal transduction.^228,229 RPN10 and PUXs (plant UBX domain-containing proteins) participate in proteaphagy and ERAD (ER-associated protein degradation), respectively.²²⁸ The analysis of these crucial selective autophagy receptors and related autophagy pathways have provided significant new insights into plant responses under different stress conditions.

4. Conclusions and Prospects

Autophagy can effectively degrade damaged organelles and harmful proteins in cells, and it is very important for the recycling of nutrients. Although we already have a good understanding of the mechanism of plant autophagy and the core autophagy route is highly conserved in plants, there are still some important unknown factors inherent in the sophisticated molecular mechanisms and route of autophagy, and most studies of plant autophagy are based on mammals and yeast. Therefore, we need to further study whether traditional autophagy genes have new functions and whether there are new autophagy genes and mechanisms involved. Studies have shown that the autophagy system is very complex. On the one hand, the activity of autophagy is inevitably controlled by various stresses, such as nutrient deficiency, drought, salt, heat stress, oxidation, pathogens and viruses. On the other hand, the same ATG gene can also respond to diverse abiotic stresses. We should uncover how plants integrate various environmental signals to regulate autophagy. In addition, autophagy plays an important role in plant carbon and nitrogen metabolism as well as growth and development. More importantly, the study of autophagy in crops may increase the application of autophagy in agriculture, which can improve the resistance and yield of crops. Great progress in plant autophagy research in recent decades can be used to facilitate this process.

Abbreviations

Table

AMPK	AMP-activated kinase
ATG	Autophagy-related gene
BTH	Benzothiadiazole
ConcA	Concanamycin A
COPII	Coat protein complex II
ER	Endoplasmic reticulum
ESCRT	Endosomal sorting complex required for transport
ETI	Effector-triggered immunity
GAPCs	Cytoplasmic glyceraldehyde-3-phosphate dehydrogenase
HR	Hypersensitive response
ILVs	Intraluminal vesicles
MV	Methyl viologen
MVBs	Multivesicular bodies
NBR1	Neighbour of breast cancer 1
NUE	Nitrogen use efficiency
PAS	Phagophore assembly site
PCD	Programmed cell death
PE	Phosphatidylethanolamine
PI3K	Phosphatidylinositol 3-kinase
PI3P	Phosphatidylinositol 3-phosphate
PTIPAMP	PAMP triggered immunity
PVCs	Prevacuolar compartments
ROS	Reactive oxygen species
SA	Salicylic acid
SH3P2	SH3 domain-containing protein2
SNARE	Soluble N-ethylmaleimide-sensitive factor attachment protein receptor
Snf1	Sucrose non-fermenting 1
SnRK1	Snf1-related protein kinase 1
TOR	Target of rapamycin
UPS	Ubiquitin (Ub)/26S proteasome system
V-ATPases	Vacuolar-type ATPases

Acknowledgments

This work was supported by the National Natural Science Foundation of China under Grant (31400220, 31970195); and the Fundamental Research Funds for the Central Universities under Grant (lzujbky-2021-kb05).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China under Grant [31400220, 31970195]; the Fundamental Research Funds for the Central Universities of China under Grant [lzujbky-2021-kb05].