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Autophagy and Senescence
C H A P T E R
15 Autophagy and Senescence Wei Lan, Ying Miao Center for Molecular Cell and Systems Biology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China The study of autophagy starts from the finding of a self-eating phenomenon in rat liver cells, which was termed “autophagy” by De Duve (De Duve et al., 1955; also see Novikoff et al., 1956). However, the historical landmarks of autophagy research were the discovery of the autophagy phenomenon in yeast and of the core machinery of autophagy based on the contribution of yeast as a model organism (Chiang and Schekman, 1991; Tsukada and Ohsumi, 1993; Takeshige et al., 1992; Nakatogawa et al., 2009). The core machinery of autophagy is responsible for macroautophagy, which is different from microautophagy, the other kind of autophagy. Although the core mechanism of autophagy is similar in plants and yeasts, they do have some differences. In Arabidopsis, although more than 30 homologous genes of yeasts' core autophagy-related genes (ATG) have been found and the function of most of them have been confirmed, some ATGs have not been identified in Arabidopsis, such as AtATG14, AtATG17, and AtATG31 (Hanaoka et al., 2002; Doelling et al., 2002; Xiong et al., 2005). It is still not known what other factors replace these missing genes or whether there are different processes during autophagy in plants. During the studies of ATG function, surprisingly, almost all atg mutants show early senescence and hypersensitivity to starvation conditions (Hanaoka et al., 2002; Doelling et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005; Qin et al., 2007; Phillips et al., 2008; Xiao and Chye, 2010; Chung et al., 2010; Wang et al., 2011; Suttangkakul et al., 2011). This phenomenon supports the idea that autophagy plays an important role in the senescence process. It is also known that autophagy plays a role in senescence or cell death (Minina et al., 2014). If this is so, then, how does autophagy contribute to plant senescence? In this chapter, we briefly introduce the history of mechanism of autophagy research and summarize the recent advances in our understanding of how autophagy participates in plant senescence, including the role of ATG proteins, chloroplast degradation, and senescence- associated vacuoles (SAVs).
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1 THE MECHANISM OF AUTOPHAGY 1.1 The Discovery of Autophagy and ATG Proteins De Duve et al. (1955) found a latency of acid phosphatase activity in cell fractionation of rat liver homogenate. Further analyses revealed a novel organelle, which was named “lysosome” by the researchers for its role as lytic organelles (De Duve et al., 1955). After a year, De Duve's laboratory observed an isolated, lysosome-enriched fraction by using electron microscopy (EM) for the first time, and found similar structures in various cell types (Novikoff et al., 1956). Lysosome is an organelle enwrapping various kinds of hydrolytic enzymes, which have optimal activity at acidic pH. Previously, lysosome was considered as an organelle that degraded foreign materials that entered the cell via endocytosis (De Duve and Wattiaux, 1966). However, it was reported that mitochondria, endoplasmic reticulum (ER), and ribosomes were observed in acid phosphatase-positive membrane structures by Novikoff (Novikoff et al., 1956). A year later, Clark also found that, in addition to lysosomes, irregularly shaped vacuoles contain amorphous materials and occasionally mitochondria by EM (Clark, 1957). Ashford and Porter (1962) next showed that these membrane structures could be induced by a hormone called glucagon. Based upon these observations, De Duve (1963) defined this transport process as “autophagy”, which means “self-eating”, at the Ciba Foundation Symposium on Lysosomes held in London during 12–14 February 1963. To determine the physiological function and mechanism of vacuolar proteolysis in yeast, Takeshige et al. (1992) transferred mutant yeast lacking proteinase A, B, and carboxypeptidase Y from a nutrient medium to various deficiency mediums, such as nitrogen or leucine deficiency. After incubation for several hours, more and more spherical bodies appeared in yeast cells and then moved to vacuoles. Through EM observation, these bodies were found to contain mitochondria, ribosomes, glycogen granules, and lipid granules, which were surrounded by a thinner membrane (Takeshige et al., 1992). That was the first report that cytoplasmic components can be extensively autophagic degraded under nutrient-deficient conditions in yeast cells. This phenomenon is similar to autophagy in mammalian cells (Novikoff et al., 1956; Clark, 1957; Arstila and Trump, 1968). Tsukada and Ohsumi (1993) isolated 76 apg mutants using the loss of viability as a first screening test and the autophagy defective under starvation conditions as a second screening test (Tsukada and Ohsumi, 1993). This study reveals that at least 15 APG genes were involved in autophagy in yeast. By genetic analysis, they found that there were approximately 30 ScATG genes, which encoded proteins necessary for autophagy in yeast (Saccharomyces cerevisiae) (Table 1; see also Klionsky, 2007; Cao et al., 2008). With the development of sequencing technologies, more and more organisms' genomes have been known. To date, we know that approximately 39 ATG homologs in Arabidopsis thaliana, corresponding to the 25 core ScATG genes, were identified, even though no homologs have been identified for AtATG17, AtATG31, AtATG29, AtATG23, AtATG27, AtATG41, AtATG14, and a not characterized AtATG16 (Table 1; see also Doelling et al., 2002; Hanaoka et al., 2002). In addition to A. thaliana, many ATG genes have been identified in other plants and even fungi. For instance, in rice (Oryza sativa L. (Os)), 33 OsATG homologs has been identified, which were classified into 13 ATG subfamilies, 6 of which were alternatively spliced genes (Xia et al., 2011). It also identified 37 putative ATG genes in foxtail millet (Setaria italica L.) (Li et al., 2016). In Magnaporthe oryzae, there were 22 isogenic M. oryzae mutants (Kershaw and Talbot, 2009). Those ATG genes were responsible for the autophagy process, especially the formation of autophagosome.
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TABLE 1 ATG Proteins in the Core Machinery of Autophagosome Formation and the Arabidopsis Mutants That Accelerate Senescence or Hypersensitivity to Nutrient-Limited Conditions Multifunctional Modules Atg1-Atg13 complex
Atg9 and its cycling system
ATG Proteins in Saccharomyces cerevisiae
Function of ATG Proteins
ATG Proteins in Arabidopsis thaliana
ScATG1
Is the Ser/Thr protein kinase
AtATG1a-c, AtATG1t
ScATG13
Is a regulatory subunit
AtATG13a-b
ScATG17
Is part of a scaffold
Not identified
ScATG31
Not identified
ScATG29
Not identified
Atatg13a-1/13b-2; Atatg13a-2/13b-2
ScATG11
Is a scaffold protein in selective autophagy for PAS organization
AtATG11-like
ScATG9
Is the sole multispanning membrane protein to deliver membrane lipids
AtATG9
Atatg9-1
ScATG2
Controls Atg9 movement back to the peripheral structures
AtATG2
Atatg2-2
AtATG18a-h
Atatg18a
Helps Atg9 reservoirs trafficking to the PAS
AtATG11
ScATG18 ScATG11 ScATG23
PtdIns3K complex
Atatg Mutants
Not identified
ScATG27
Not identified
ScATG41
Not identified
ScVPS34
Is the lipid kinase
AtVPS34
ScVPS15
Required for Vps34 membrane association
VPS15
ScVPS30/ ScATG6
Is a component of PtdIns3K complex I and II
AtATG6
ScATG14
Dictates the localization of the kinase complexes
Not identified
ScATG38
Is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity
Not identified
Atatg6
Continued
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TABLE 1 ATG Proteins in the Core Machinery of Autophagosome Formation and the Arabidopsis Mutants That Accelerate Senescence or Hypersensitivity to Nutrient-Limited Conditions—cont’d Multifunctional Modules Atg8 Ubl conjugation system
Atg12 Ubl conjugation system
ATG Proteins in Saccharomyces cerevisiae
Function of ATG Proteins
ATG Proteins in Arabidopsis thaliana
Atatg Mutants
ScATG8
Is a good marker of the autophagosomal membrane and a key molecule during autophagosome formation
AtATG8a-i
Atatg8
ScATG7
Activates the modified Atg8 by Atg4
AtATG7
Atatg7
ScATG3
Is the E2-like enzyme
AtATG3
Atatg3
ScATG4
Is the cysteine protease
AtATG4a-b
Atatg4a4b-1
ScATG12
The Atg12-Atg5-Atg16 complex may work as an E3 enzyme that can facilitate Atg8-PE conjugation
AtATG12a-b
Atatg12a12b
AtATG5
Atatg5-1
ScATG7
Is the E1-like enzyme
AtATG7
Atatg7-2; Atatg7-1
ScATG10
Is the E2-like enzyme
AtATG10
Atatg10-1
ScATG16 ScATG5
Not characterized
Atatg mutants: The Arabidopsis atg mutants that accelerate senescence or hypersensitivity to nutrient-limited conditions.
1.2 The Mechanism of Autophagy in Yeast and Plants There are two main types of autophagy in yeast and plants—macroautophagy and microautophagy (Bassham et al., 2006). Macroautophagy is the major intracellular route to the lysosome and vacuole in mammals and in yeast and plants, respectively. All of them can be selective or nonselective, and these processes have been best characterized in yeast (Shintani and Klionsky, 2004). In this chapter, we mainly focus on macroautophagy, which will be called autophagy for short hereafter. Thanks to the discovery of autophagy-deficient mutants and ATG genes, yeast cells became a good platform for getting insight into the autophagy mechanism. The function of ATG proteins and their regulation during autophagy initiation and the entire autophagy process have been investigated intensively in yeast. There are five multifunctional modules for the biogenesis of autophagy-related membranes: ATG1-ATG13 complex, ATG9 complex, PtdIns3K complex, and the ATG8 Ub-like and ATG12 Ub-like conjugation systems (Table 1; Feng et al., 2014; Nakatogawa et al., 2009; Farre and Subramani, 2016). The most important event in autophagy is the formation of autophagosome. Autophagosome formation relies on a series of dynamic membrane events, with the help of the five multifunctional modules (Fig. 1). The ATG1-ATG13 complex can receive stress signals by the changes in phosphorylation level. Under starvation conditions, the phosphorylation level of ATG13 is higher, which promotes the interaction of the ATG1-ATG13 and ATG17-ATG31-ATG29
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FIG. 1 The model of autophagy in plants. Autophagy consist of six steps, including initiation, nucleation, expansion, completion, docking and fusion, and degradation and recycling. ATG proteins are responsible for these processes, which have formed five multifunctional modules that orchestrate the steps of autophagy. In yeast, there are 25 proteins involved in autophagy, especially the formation of autophagosome. However, some of these proteins are missing (dotted line box) or become gene family (filled box) or not characterized (red dotted line box) in the Arabidopsis thaliana genome.
c omplexes. When the ATG1-ATG13 complex interacts with the ATG17-ATG31-ATG29 complex, they induce autophagy together, at what becomes the phagophore assembly site (PAS). With the help of the ATG9 complex, the ATG18-ATG2 complex, and the Ptdins3k complex I, a cup-shaped structure forms, which continually extends to form a double membrane-bound structure (called autophagosome) with the help of the ATG8 Ub-like and ATG12 Ub-like conjugation systems. Finally, the external membrane of autophagosome and the membrane of a vacuole fuse together. Meanwhile, the external contents of autophagosome are transported into the vacuole’s chamber to degrade, and the degradation products (such as amino acids) are transported back to the cytoplasm for recycling. Interestingly, the dynamic membrane events are conserved in almost all eukaryotes, including plants (Fig. 1). To date, it has been confirmed that the function of these five multifunctional modules is conserved in plants, especially Arabidopsis, even though some AtATG genes disappear and others appear as family genes (Li and Vierstra, 2014; Michaeli et al., 2014; Woo et al., 2014;
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FIG. 2 The responses of plants' autophagy under stress responses. External stresses can induce plant autophagy by SnRK1 and TOR, or tubulin acetylation, which then induces defense responses and plant survival. SnRK1, Snf1related protein kinase 1; TOR, target of rapamycin.
Park et al., 2014; Zhuang et al., 2017). For instance, although AtATG4 and AtATG8 contain two and nine members, respectively, AtATG4 proteins retain specificity for AtATG8 (Park et al., 2014). Furthermore, AtATG4a is the primary protease, which processes AtATG8a, AtATG8c, AtATG8d, and AtATG8i better than AtATG4b in vitro (Woo et al., 2014). In addition to the core ATG proteins, other ATG proteins, such as ATG6, or other factors, such as the A. thaliana acyl-CoA-binding protein (ACBP3), are involved in the regulating of autophagy dynamics (Qi et al., 2017; Lee et al., 2018). Interestingly, the ATG17-ATG31-ATG29 complex is missing in Arabidopsis, which acts partly as a scaffold in the initiation of autophagy (Fig. 1). This may be explained by the fact that ATG11-like proteins may act as bifunctional scaffolding proteins in both ATG11- and ATG17-related functions within the ATG1-ATG13 complex. In plants, autophagy has been suggested to be involved in seed development and germination, photomorphogenesis, chloroplast maturation, stress protection, pathogen resistance, hormonal responses, and senescence under starvation conditions (Ishida et al., 2008; Ghiglione et al., 2008; Chung et al., 2009; Izumi et al., 2015). Therefore, it is important to have insight into its mechanism in plants, especially the role of autophagy in pathogen resistance and stress protection. Recently, it has become known that many stresses can induce autophagy in plants, including abiotic stresses such as salt, starvation, drought, and ER stress; and biotic stress, which contributes to defense responses and plant survival (Fig. 2). Meanwhile, in crops, research mainly focuses on the functions of autophagy in pathogen resistance and fertile floret development in rice. Interestingly, autophagy plays a vital role in the infection-related autophagy of the rice blast fungus M. oryzae (Kershaw and Talbot, 2009; He et al., 2012; Liu et al., 2017). Here, we are interested in how autophagy promotes the senescence process and even death and contributes to defense responses and plant survival.
2 AUTOPHAGY AND SENESCENCE 2.1 Whole-Plant Senescence and Leaf-Senescence In the whole life span of plants, there are three main stages: vegetative, reproductive, and senescence. Between the vegetative and reproductive phase, there is an important event: floral
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transition. It is a major component of whole-plant senescence of monocarpic plants. During floral transition, vegetative growth ceases and flowering begins. After floral transition, the growth of fruits and seeds replaces the forming of new leaves. Fruits and seeds induce large changes in plant metabolism and nutrient redistribution, because they, as new sinks, will compete for the remaining nutrients of plants. Therefore, as a strategy, plants will mobilize and redistribute the nutrients of plants' other tissues to balance all the conflicting needs. As a tissue that captures light and absorbing carbon dioxide (CO2), leaves acquire high allocation from plants' available resources, which are good nutritional resource suppliers for fruits and seeds. Therefore, after floral transition, leaves are beginning to senesce. Leaf senescence is an active and ordered degeneration process that is not only influenced by internal factors, but also various environmental factors. The internal factors that affect leaf senescence include phytohormone and reproduction, and environmental factors include stresses such as ozone (O3), drought, high or low temperature, nutrient deficiency, and pathogen infection (Lim et al., 2007; Schippers et al., 2015). All these factors finally regulate developmental age and then initiate the onset of leaf senescence by the regulation of transcription factors, such as WRKY, NAC, AP2/EREBP, C2H2-type zinc finger, and MYB protein families (Miao et al., 2008; Schippers et al., 2015; Zentgraf et al., 2010). In addition, the cell structure and metabolism undergo orderly changes during leaf senescence. The earliest and most important change in cell structure is the chloroplast’s breakdown, while mitochondria and the nucleus collapse at the final stage of senescence. The changes in organelle structure during senescence are mainly due to protein degradation. In eukaryotes, there are two main mechanisms that are responsible for protein degradation: the lysosomal/ vacuolar degradation system and the ubiquitin/proteasome system. The ubiquitin/proteasome system is specific and degrade one after the other. However, the lysosomal/vacuolar degradation system is a degradation pathway of bulk cytosolic constituents and organelles that largely depend on autophagy that parcel the substrates for degradation through certain membrane-mediated processes. Therefore, autophagy is more efficient than the ubiquitin/ proteasome system in protein degradation, especially in cell structural breakdown.
2.2 Senescence, Autophagy, and ATG Proteins It is accompanied by decreased expression of genes (such as CAB2, RPS, and RBC) and by increased expression of senescence-associated genes (SAGs) during leaf senescence. Affymetrix GeneChip microarrays are utilized to analyze changes in global expression patterns during senescence in Arabidopsis, which has identified more than 800 SAGs (BuchananWollaston et al., 2005). Approximately 30% of SAGs involve in regulation, and 12.2% are involved in macromolecule degradation and mobilization. Amazingly, there are five autophagy genes appeared in these SAGs, including AtAPG7, AtAPG8a, AtAPG8b, AtAPG8h, and AtAPG9 (Buchanan-Wollaston et al., 2005). At the same time, most studies showed that most atg mutants exhibited premature leaf senescence after nutrient starvation treatment (Table 1, Fig. 3), especially these mutants blocking the ATG12 Ub-like conjugation system (Atatg5, Atatg10, and Atatg12a/Atatg12b double mutant), attenuating the ATG8 Ub-like conjugation system (Atatg4a/Atatg4b double), or blocking both Ub-like conjugation systems (Atatg7) (Fig. 3) (Hanaoka et al., 2002; Doelling et al., 2002; Xiong et al., 2005). All of these findings demonstrate that autophagy plays an essential role in senescence, especially the two Ub-like
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FIG. 3 Autophagy and senescence in plants. There are several kinds of autophagy-dependent bodies, including autophagosome, RuBisCO-containing bodies (RCBs), autophagy-dependent plastid bodies containing ATG8INTERACTING1 (ATI-PS) and ATI1-ATG8 spherical bodies, that degrade the cytoplasm and organelles, especially chloroplast.
conjugation systems. Therefore, any events that affect the level or the stability of ATG proteins will lead to early senescence. For instance, the A. thaliana acyl-CoA-binding protein ACBP3 regulates leaf senescence through modulating ATG8 stability (Fig. 3; see also Xiao and Chye, 2010). In yeast, the E3-like conjugating enzymes of two Ub-like conjugation systems are ATG5ATG16 for ATG12, and ATG-5-ATG16-ATG12 for ATG8. ATG8, the most commonly used maker protein for autophagic activity (Yoshimoto et al., 2010; Yoshimoto, 2012; Thompson et al., 2005),
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which involves the size of autophagosome and the cargo binding during selective autophagy (Shintani et al., 2002; Xie et al., 2008). In Arabidopsis, the atg5 and atg8 mutants show early senescence under dark or nutrient-limited conditions (Thompson et al., 2005); but can it be explained by sugar deficiency? Mukae et al. (2015) found that it has the sugar content of WT and Physcomitrella atg5 (Ppatg5) mutant are almost the same. But their amino acids content were significantly different in dark condition, which supports that the imbalance of amino acids content resulting from the deficiency of cytoplasmic degradation by autophagy may partly explain the premature phenotype in Ppatg5 mutants (Mukae et al., 2015). Therefore, autophagy may play an important role in plant senescence though maintaining the amino acid balance (Fig. 3). It still needs to confirm further whether other atg mutants promote senescence by disordering the balance of amino acid or by other pathways that can induce some stresses for plant survival.
2.3 Senescence, Autophagy, and Chloroplasts During senescence, cells increase catabolic activity to convert the intracellular accumulated materials into exportable nutrients for developing seeds, or even for survival. Ultrastructural studies showed that the earliest dismantled organelles are chloroplasts, while mitochondria and the nucleus collapse at the final stage of senescence (Butler, 1967; Dodge, 1970) In addition to internal factors, external factors can induce senescence, including ultraviolet B (UVB), nutrient limitation, heat, drought, and shading. Under nutrient limitations, plants can promote nutrient recycling and mobilization by initiating leaf senescence. When plants grow under nitrogen deficiency conditions, senescence is induced to remobilize nitrogen by chloroplast's breakdown (Masclaux et al., 2000). On the other hand, autophagy is a dynamic process that transports intracellular components, including macromolecules and even organelles, into lysosomes/vacuoles to degrade and then releases monomers such as amino acids into the cytosol for recycling. There are a good deal of evidence to support the idea that autophagy is responsible for chloroplast protein degradation during leaf senescence, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), stroma- targeted fluorescent proteins, and other proteins of chloroplasts. Chloroplasts contain up to 70% of leaf proteins, which means that they are good donators for nutrient supply (Makino and Osmond, 1991). There are a variety of enzymes in the chloroplasts, including RuBisCO, photosystems, thylakoid proteins, Calvin cycle proteins, and light harvesting complexes. Among these enzymes, RuBisCO is the most abundant protein, which is mainly synthesized during leaf expansion, and it finally accounts for 12%–30% of total leaf proteins in C3 plants (Evans, 1989). However, RuBisCO degrades rapidly during senescence. It was reported that RuBisCO and its degradation products can be transported into vacuoles via RuBisCO-containing bodies (RCBs) after darkness treatment (Chiba et al., 2003). Further, an ATG gene-dependent autophagy process has been found, which involves in the mobilization of RuBisCO and stroma-targeted fluorescent proteins without prior chloroplast destruction during naturally senescent leaves (Ishida et al., 2008). It suggests that the proteins of chloroplasts can be degraded by autophagy during natural or dark-induced senescence (Fig. 3). It remains a question what factors guide the PAS to near the chloroplast and form autophagosome-loaded buds from chloroplasts. Recently, it is known that the selective macroautophagy depends on the ATG8 protein. Through detailed real-time and three-dimensional (3D) imaging technologies, Le Bars et al. (2014) found a few ATG8 proteins gathered in PAS,
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while much ATG5 accumulates there in the initiation of autophagosome formation, accompanied by the underlying endoplasmic reticulum (Fig. 3; Le Bars et al., 2014). Therefore, the formation of RCBs may depend on this pathway with the selective of ATG8s (Fig. 3). In addition to RCBs, granum can be transported to the vacuole by autophagy-dependent plastid bodies containing ATG8-INTERACTING1 (ATI1-PS; Michaeli et al., 2014). ATI1-PS is not a typical autophagosome because it directly buds out and detaches from plastids into cytoplasm with the help of ATI1 and ATG8 proteins by a process that doesn't require active autophagy (Fig. 3; Michaeli et al., 2014). It is not clear what factors induce the budding and detaching of ATI1-PS from plastids. ATI1 also can form spherical bodies with ATG8 (ATI1ATG8 spherical bodies) under nutrient limitations, which is different from ATI1-PS. Honig et al. (2012) found that ATI1 is partially related to the ER membrane network under favorable growth conditions; however, they become mainly associated with ATI1-ATG8 spherical bodies under carbon starvation. Moreover, the overexpression or suppression of ATI1 could evoke responses from the exogenous hormone abscisic acid (ABA; Honig et al., 2012). The level of salicylic acid (SA) increases gradually with the increase of age. The higher concentration of SA allows it to interact with NPR3, which causes rapid degradation of NPR1. Then it promotes senescence and programmed cell death (PCD) through inducing ER stress by an NPR1-dependent pathway (Fig. 4; Yoshimoto et al., 2009; Breeze et al., 2011; Fu et al., 2012; Minina et al., 2014). This finding supports the fact that ATI-association autophagy may involve senescence through degrading chloroplast by ATI1-PS and the hormone signaling pathway by ATI1-ATG8 spherical bodies, which are involved in ER response.
2.4 Senescence, Autophagy, and SAVs Enzymatic activity studies showed an increase in protease activities during leaf senescence, and genomewide studies further provided a large number of protease genes that are upreg-
FIG. 4 SA signaling and ER stress in senescence. With aging, SA levels gradually increase, which causes rapid degradation of NPR1 through the action of NPR3. Then, plants get into senescence and programmed cell death. ATG8, autophagy-related protein 8; ATI-3, ATG8-INTERACTING3; NPR1/3/4, nonexpressor of pathogenesis-related (PR) genes 1/3/4; SA, salicylic acid; SnRK1, the Snf1-related protein kinase 1; TOR, target of rapamycin; UBAC2, Ubiquitin-associated protein 2.
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ulated in this development stage (Feller et al., 1977; Roberts et al., 2012). SAG12, encoding a cysteine protease, is the most famous senescence-associated protease gene. Moreover, SAG12 is the only SAG that is uniquely controlled by natural senescence, but it does not respond to environmental stresses. Therefore, it is commonly used as a marker gene for leaf senescence, which is located in small, lytic vacuolar compartments (also called SAVs) in senescent leaves (Martinez et al., 2008a,b; Otegui et al., 2005). SAVs are small (0.5–0.8 μm), and have been identified in the senescent leaves of several plants, including soybean, Arabidopsis, and tobacco, but absent from nonsenescing leaves (Martinez et al., 2008a,b). SAVs are different from central vacuole for its contents and membrane component. There are soluble chloroplast proteins (such as RuBisCO) and resident proteases (such as senescence-specific SAG12) in the acid lumen of SAVs (Pascual et al., 1994; Otegui et al., 2005; Martinez et al., 2008a,b). Meanwhile, its membrane components include vacuolar H+-pyrophosphatase but lack the aquaporin γ-TIP, a marker for the central vacuole tonoplast, and they are more acidic than central vacuoles (Jauh et al., 1999). Therefore, the SAV pathway is one of the degradation pathways of chloroplast proteins during senescence, but it is a different autophagy pathway. In oilseed rape, the SAG12 protein is important for nitrogen remobilization because it is heavily abundant in senescent leaves, especially when nitrogen remobilization is enhanced for nutrient deficiency (Desclos et al., 2009). This is coincided with the function of senescence in plant’s nutrient reuse. However, the Arabidopsis sag12 mutant showed no phenotypes (Otegui et al., 2005). Meanwhile, this finding suggests that RuBisCO degradation by SAVs is upregulated to complement the deficiency in autophagy of atg mutants, for the transcripts of SAG12 accumulate at a high level in atg2 and atg4a4b mutants and visible leaf senescence proceeds much faster than the wild type in atg2 mutants (Yoshimoto et al., 2009; Wada et al., 2009). According to these findings, the SAV pathway and the autophagy-dependent pathway are two independent and complementary pathways that contribute to degrade chloroplast proteins, which may well explain the nonphenotypes of Atsag12 mutants.
3 DISCUSSION 3.1 ATG Proteins Almost every atg mutant showed accelerating senescence under nutrient-limited conditions, but it is not clear how autophagy responds to various stresses that can induce senescence or survival in plants, such as plant age, nutrient starvation, and salt. This is an interesting question that needs to be studied. In addition to involving the core autophagy mechanisms, whether these senescence-association ATG genes can respond to other senescence-association factors or directly become senescence-association factors that activate the downstream SAG genes through a way different from autophagy? Just like the ATG18a, it can respond to WRKY33, a transcription factor, which is important for plant resistance to necrotrophic pathogens (Lai et al., 2011).
3.2 The SA Signaling in Autophagy and Senescence ER is known to be one of the important membrane material stores for autophagosome. Using 3D imaging, it has been ascertained that ER can form the membrane of autophagosomes
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and the ATI1-ATG8 spherical bodies under nutrient starvation. During these stages, it can be constantly added to autophagosomes and the ATI1-ATG8 spherical bodies, with the help of the ATG5 and ATI1 proteins, respectively (Le Bars et al., 2014; Honig et al., 2012). It is thus clear that ER plays an important role in the formation of the autophagy-association architecture. Meanwhile, it has been suggested that SA levels increase during the final phase of leaf development, which can cause rapid degradation of NPR1 through the action of NPR3. The degradation of NPR1 finally causes PCD by the NPR1-dependent, ER stress activation of autophagy (Minina et al., 2014; Yoshimoto et al., 2009; Breeze et al., 2011). In addition, the studies of autophagy induced by ER stress demonstrate that SnRK1 can respond to ER stress, which is upstream of TOR, and the ATG8-ATI3-UBAC2 complex can induce autophagy and then affect plant life (Soto-Burgos and Bassham, 2017; Zhou et al., 2018). Therefore, the senescence or PCD induced by SA through ER stress may use SnRK1 and TOR to pass on the SA signaling. The role of the ATG8-ATI3-UBAC2 complex in this pathway still needs further research (Fig. 4).
4 CONCLUSION Autophagy is a degradation pathway of bulk cytosolic constituents and organelles, which can promote plants' nutrient use efficiency and then survival from stress or reproductive death. Depending on autophagy, plants evolved several pathways to degrade chloroplast, such as RCBs and autophagy-dependent plastid bodies containing ATG8-INTERACTING1 (ATI-PS). Although SAVs pathway is different from autophagy, the deficiency of autophagy promotes RuBisCO degradation by SAVs. All these pathways apply themselves to promote plant senescence by degrading chloroplasts. However, almost every atg mutant showed early senescence under nutrient-limited conditions, which demonstrates that the role of autophagy in plant senescence are not only as a promoter but also as an inhibitor. To date, it is known that Ppatg5 mutants show an imbalance of amino acids, which partly explains the earlysenescence phenotype in Ppatg5 mutants. But more information is needed to confirm whether other atg mutants promote senescence by disordering the balance of amino acids or by other pathways that can induce certain stresses for plant survival. In addition to involving the core autophagy mechanisms, these senescence-association ATG proteins probably function as factors that respond to senescence-association factors or directly become senescence-association factors to activate the downstream SAG genes. This finding may answer the question of how autophagy receives senescence signaling and passes it on.
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Qi, H., Xia, F.N., Xie, L.J., Yu, L.J., Chen, Q.F., Zhuang, X.H., Wang, Q., Li, F., Jiang, L., Xie, Q., Xiao, S., 2017. TRAF family proteins regulate autophagy dynamics by modulating AUTOPHAGY PROTEIN6 stability in Arabidopsis. Plant Cell 29 (4), 890–911. Qin, G., Ma, Z., Zhang, L., Xing, S., Hou, X., Deng, J., Liu, J., Chen, Z., Qu, L.J., Gu, H., 2007. Arabidopsis AtBECLIN 1/ AtAtg6/AtVps30 is essential for pollen germination and plant development. Cell Res. 17 (3), 249–263. Roberts, I.N., Caputo, C., Criado, M.V., Funk, C., 2012. Senescence-associated proteases in plants. Physiol. Plant. 145 (1), 130–139. Schippers, J.H., Schmidt, R., Wagstaff, C., Jing, H.C., 2015. Living to die and dying to live: the survival strategy behind leaf senescence. Plant Physiol. 169 (2), 914–930. Shintani, T., Klionsky, D.J., 2004. Autophagy in health and disease: a double-edged sword. Science 306 (5698), 990–995. Shintani, T., Huang, W.P., Stromhaug, P.E., Klionsky, D.J., 2002. Mechanism of cargo selection in the cytoplasm to vacuole targeting pathway. Dev. Cell 3 (6), 825–837. Soto-Burgos, J., Bassham, D.C., 2017. SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana. PLoS ONE 12 (8), e0182591. Suttangkakul, A., Li, F., Chung, T., Vierstra, R.D., 2011. The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis. Plant Cell 23 (10), 3761–3779. Takeshige, K., Baba, M., Tsuboi, S., Noda, T., Ohsumi, Y., 1992. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J. Cell Biol. 119 (2), 301–311. Thompson, A.R., Doelling, J.H., Suttangkakul, A., Vierstra, R.D., 2005. Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol. 138 (4), 2097–2110. Tsukada, M., Ohsumi, Y., 1993. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333, 169–174. Wada, S., Ishida, H., Izumi, M., Yoshimoto, K., Ohsumi, Y., Mae, T., Makino, A., 2009. Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiol. 149 (2), 885–893. Wang, Y., Nishimura, M.T., Zhao, T., Tang, D., 2011. ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis. Plant J. 68 (1), 74–87. Woo, J., Park, E., Dinesh-Kumar, S.P., 2014. Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases. Proc. Natl. Acad. Sci. U. S. A. 111 (2), 863–868. Xia, K., Liu, T., Ouyang, J., Wang, R., Fan, T., Zhang, M., 2011. Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.). DNA Res. 18 (5), 363–377. Xiao, S., Chye, M.L., 2010. The Arabidopsis thaliana ACBP3 regulates leaf senescence by modulating phospholipid metabolism and ATG8 stability. Autophagy 6 (6), 802–804. Xie, Z., Nair, U., Klionsky, D.J., 2008. Atg8 controls phagophore expansion during autophagosome formation. Mol. Biol. Cell 19 (8), 3290–3298. Xiong, Y., Contento, A.L., Bassham, D.C., 2005. AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J. 42 (4), 535–546. Yoshimoto, K., 2012. Beginning to understand autophagy, an intracellular self-degradation system in plants. Plant Cell Physiol. 53 (8), 1355–1365. Yoshimoto, K., Hanaoka, H., Sato, S., Kato, T., Tabata, S., Noda, T., Ohsumi, Y., 2004. Processing of ATG8s, u biquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell 16 (11), 2967–2983. Yoshimoto, K., Jikumaru, Y., Kamiya, Y., Kusano, M., Consonni, C., Panstruga, R., Ohsumi, Y., Shirasu, K., 2009. Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell 21 (9), 2914–2927. Yoshimoto, K., Takano, Y., Sakai, Y., 2010. Autophagy in plants and phytopathogens. FEBS Lett. 584 (7), 1350–1358. Zentgraf, U., Laun, T., Miao, Y., 2010. The complex regulation of WRKY53 during leaf senescence of Arabidopsis thaliana. Eur. J. Cell Biol. 89 (2–3), 133–137. Zhou, J., Wang, Z., Wang, X., Li, X., Zhang, Z., Fan, B., Zhu, C., Chen, Z., 2018. Dicot-specific ATG8-interacting ATI3 proteins interact with conserved UBAC2 proteins and play critical roles in plant stress responses. Autophagy, 1–56. Zhuang, X., Chung, K.P., Cui, Y., Lin, W., Gao, C., Kang, B.H., Jiang, L., 2017. ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 114 (3), E426–E435.
15 Autophagy and Senescence Wei Lan, Ying Miao Center for Molecular Cell and Systems Biology, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China The study of autophagy starts from the finding of a self-eating phenomenon in rat liver cells, which was termed “autophagy” by De Duve (De Duve et al., 1955; also see Novikoff et al., 1956). However, the historical landmarks of autophagy research were the discovery of the autophagy phenomenon in yeast and of the core machinery of autophagy based on the contribution of yeast as a model organism (Chiang and Schekman, 1991; Tsukada and Ohsumi, 1993; Takeshige et al., 1992; Nakatogawa et al., 2009). The core machinery of autophagy is responsible for macroautophagy, which is different from microautophagy, the other kind of autophagy. Although the core mechanism of autophagy is similar in plants and yeasts, they do have some differences. In Arabidopsis, although more than 30 homologous genes of yeasts' core autophagy-related genes (ATG) have been found and the function of most of them have been confirmed, some ATGs have not been identified in Arabidopsis, such as AtATG14, AtATG17, and AtATG31 (Hanaoka et al., 2002; Doelling et al., 2002; Xiong et al., 2005). It is still not known what other factors replace these missing genes or whether there are different processes during autophagy in plants. During the studies of ATG function, surprisingly, almost all atg mutants show early senescence and hypersensitivity to starvation conditions (Hanaoka et al., 2002; Doelling et al., 2002; Yoshimoto et al., 2004; Thompson et al., 2005; Xiong et al., 2005; Qin et al., 2007; Phillips et al., 2008; Xiao and Chye, 2010; Chung et al., 2010; Wang et al., 2011; Suttangkakul et al., 2011). This phenomenon supports the idea that autophagy plays an important role in the senescence process. It is also known that autophagy plays a role in senescence or cell death (Minina et al., 2014). If this is so, then, how does autophagy contribute to plant senescence? In this chapter, we briefly introduce the history of mechanism of autophagy research and summarize the recent advances in our understanding of how autophagy participates in plant senescence, including the role of ATG proteins, chloroplast degradation, and senescence- associated vacuoles (SAVs).
Senescence Signalling and Control in Plants https://doi.org/10.1016/B978-0-12-813187-9.00015-9
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1 THE MECHANISM OF AUTOPHAGY 1.1 The Discovery of Autophagy and ATG Proteins De Duve et al. (1955) found a latency of acid phosphatase activity in cell fractionation of rat liver homogenate. Further analyses revealed a novel organelle, which was named “lysosome” by the researchers for its role as lytic organelles (De Duve et al., 1955). After a year, De Duve's laboratory observed an isolated, lysosome-enriched fraction by using electron microscopy (EM) for the first time, and found similar structures in various cell types (Novikoff et al., 1956). Lysosome is an organelle enwrapping various kinds of hydrolytic enzymes, which have optimal activity at acidic pH. Previously, lysosome was considered as an organelle that degraded foreign materials that entered the cell via endocytosis (De Duve and Wattiaux, 1966). However, it was reported that mitochondria, endoplasmic reticulum (ER), and ribosomes were observed in acid phosphatase-positive membrane structures by Novikoff (Novikoff et al., 1956). A year later, Clark also found that, in addition to lysosomes, irregularly shaped vacuoles contain amorphous materials and occasionally mitochondria by EM (Clark, 1957). Ashford and Porter (1962) next showed that these membrane structures could be induced by a hormone called glucagon. Based upon these observations, De Duve (1963) defined this transport process as “autophagy”, which means “self-eating”, at the Ciba Foundation Symposium on Lysosomes held in London during 12–14 February 1963. To determine the physiological function and mechanism of vacuolar proteolysis in yeast, Takeshige et al. (1992) transferred mutant yeast lacking proteinase A, B, and carboxypeptidase Y from a nutrient medium to various deficiency mediums, such as nitrogen or leucine deficiency. After incubation for several hours, more and more spherical bodies appeared in yeast cells and then moved to vacuoles. Through EM observation, these bodies were found to contain mitochondria, ribosomes, glycogen granules, and lipid granules, which were surrounded by a thinner membrane (Takeshige et al., 1992). That was the first report that cytoplasmic components can be extensively autophagic degraded under nutrient-deficient conditions in yeast cells. This phenomenon is similar to autophagy in mammalian cells (Novikoff et al., 1956; Clark, 1957; Arstila and Trump, 1968). Tsukada and Ohsumi (1993) isolated 76 apg mutants using the loss of viability as a first screening test and the autophagy defective under starvation conditions as a second screening test (Tsukada and Ohsumi, 1993). This study reveals that at least 15 APG genes were involved in autophagy in yeast. By genetic analysis, they found that there were approximately 30 ScATG genes, which encoded proteins necessary for autophagy in yeast (Saccharomyces cerevisiae) (Table 1; see also Klionsky, 2007; Cao et al., 2008). With the development of sequencing technologies, more and more organisms' genomes have been known. To date, we know that approximately 39 ATG homologs in Arabidopsis thaliana, corresponding to the 25 core ScATG genes, were identified, even though no homologs have been identified for AtATG17, AtATG31, AtATG29, AtATG23, AtATG27, AtATG41, AtATG14, and a not characterized AtATG16 (Table 1; see also Doelling et al., 2002; Hanaoka et al., 2002). In addition to A. thaliana, many ATG genes have been identified in other plants and even fungi. For instance, in rice (Oryza sativa L. (Os)), 33 OsATG homologs has been identified, which were classified into 13 ATG subfamilies, 6 of which were alternatively spliced genes (Xia et al., 2011). It also identified 37 putative ATG genes in foxtail millet (Setaria italica L.) (Li et al., 2016). In Magnaporthe oryzae, there were 22 isogenic M. oryzae mutants (Kershaw and Talbot, 2009). Those ATG genes were responsible for the autophagy process, especially the formation of autophagosome.
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TABLE 1 ATG Proteins in the Core Machinery of Autophagosome Formation and the Arabidopsis Mutants That Accelerate Senescence or Hypersensitivity to Nutrient-Limited Conditions Multifunctional Modules Atg1-Atg13 complex
Atg9 and its cycling system
ATG Proteins in Saccharomyces cerevisiae
Function of ATG Proteins
ATG Proteins in Arabidopsis thaliana
ScATG1
Is the Ser/Thr protein kinase
AtATG1a-c, AtATG1t
ScATG13
Is a regulatory subunit
AtATG13a-b
ScATG17
Is part of a scaffold
Not identified
ScATG31
Not identified
ScATG29
Not identified
Atatg13a-1/13b-2; Atatg13a-2/13b-2
ScATG11
Is a scaffold protein in selective autophagy for PAS organization
AtATG11-like
ScATG9
Is the sole multispanning membrane protein to deliver membrane lipids
AtATG9
Atatg9-1
ScATG2
Controls Atg9 movement back to the peripheral structures
AtATG2
Atatg2-2
AtATG18a-h
Atatg18a
Helps Atg9 reservoirs trafficking to the PAS
AtATG11
ScATG18 ScATG11 ScATG23
PtdIns3K complex
Atatg Mutants
Not identified
ScATG27
Not identified
ScATG41
Not identified
ScVPS34
Is the lipid kinase
AtVPS34
ScVPS15
Required for Vps34 membrane association
VPS15
ScVPS30/ ScATG6
Is a component of PtdIns3K complex I and II
AtATG6
ScATG14
Dictates the localization of the kinase complexes
Not identified
ScATG38
Is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity
Not identified
Atatg6
Continued
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TABLE 1 ATG Proteins in the Core Machinery of Autophagosome Formation and the Arabidopsis Mutants That Accelerate Senescence or Hypersensitivity to Nutrient-Limited Conditions—cont’d Multifunctional Modules Atg8 Ubl conjugation system
Atg12 Ubl conjugation system
ATG Proteins in Saccharomyces cerevisiae
Function of ATG Proteins
ATG Proteins in Arabidopsis thaliana
Atatg Mutants
ScATG8
Is a good marker of the autophagosomal membrane and a key molecule during autophagosome formation
AtATG8a-i
Atatg8
ScATG7
Activates the modified Atg8 by Atg4
AtATG7
Atatg7
ScATG3
Is the E2-like enzyme
AtATG3
Atatg3
ScATG4
Is the cysteine protease
AtATG4a-b
Atatg4a4b-1
ScATG12
The Atg12-Atg5-Atg16 complex may work as an E3 enzyme that can facilitate Atg8-PE conjugation
AtATG12a-b
Atatg12a12b
AtATG5
Atatg5-1
ScATG7
Is the E1-like enzyme
AtATG7
Atatg7-2; Atatg7-1
ScATG10
Is the E2-like enzyme
AtATG10
Atatg10-1
ScATG16 ScATG5
Not characterized
Atatg mutants: The Arabidopsis atg mutants that accelerate senescence or hypersensitivity to nutrient-limited conditions.
1.2 The Mechanism of Autophagy in Yeast and Plants There are two main types of autophagy in yeast and plants—macroautophagy and microautophagy (Bassham et al., 2006). Macroautophagy is the major intracellular route to the lysosome and vacuole in mammals and in yeast and plants, respectively. All of them can be selective or nonselective, and these processes have been best characterized in yeast (Shintani and Klionsky, 2004). In this chapter, we mainly focus on macroautophagy, which will be called autophagy for short hereafter. Thanks to the discovery of autophagy-deficient mutants and ATG genes, yeast cells became a good platform for getting insight into the autophagy mechanism. The function of ATG proteins and their regulation during autophagy initiation and the entire autophagy process have been investigated intensively in yeast. There are five multifunctional modules for the biogenesis of autophagy-related membranes: ATG1-ATG13 complex, ATG9 complex, PtdIns3K complex, and the ATG8 Ub-like and ATG12 Ub-like conjugation systems (Table 1; Feng et al., 2014; Nakatogawa et al., 2009; Farre and Subramani, 2016). The most important event in autophagy is the formation of autophagosome. Autophagosome formation relies on a series of dynamic membrane events, with the help of the five multifunctional modules (Fig. 1). The ATG1-ATG13 complex can receive stress signals by the changes in phosphorylation level. Under starvation conditions, the phosphorylation level of ATG13 is higher, which promotes the interaction of the ATG1-ATG13 and ATG17-ATG31-ATG29
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FIG. 1 The model of autophagy in plants. Autophagy consist of six steps, including initiation, nucleation, expansion, completion, docking and fusion, and degradation and recycling. ATG proteins are responsible for these processes, which have formed five multifunctional modules that orchestrate the steps of autophagy. In yeast, there are 25 proteins involved in autophagy, especially the formation of autophagosome. However, some of these proteins are missing (dotted line box) or become gene family (filled box) or not characterized (red dotted line box) in the Arabidopsis thaliana genome.
c omplexes. When the ATG1-ATG13 complex interacts with the ATG17-ATG31-ATG29 complex, they induce autophagy together, at what becomes the phagophore assembly site (PAS). With the help of the ATG9 complex, the ATG18-ATG2 complex, and the Ptdins3k complex I, a cup-shaped structure forms, which continually extends to form a double membrane-bound structure (called autophagosome) with the help of the ATG8 Ub-like and ATG12 Ub-like conjugation systems. Finally, the external membrane of autophagosome and the membrane of a vacuole fuse together. Meanwhile, the external contents of autophagosome are transported into the vacuole’s chamber to degrade, and the degradation products (such as amino acids) are transported back to the cytoplasm for recycling. Interestingly, the dynamic membrane events are conserved in almost all eukaryotes, including plants (Fig. 1). To date, it has been confirmed that the function of these five multifunctional modules is conserved in plants, especially Arabidopsis, even though some AtATG genes disappear and others appear as family genes (Li and Vierstra, 2014; Michaeli et al., 2014; Woo et al., 2014;
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FIG. 2 The responses of plants' autophagy under stress responses. External stresses can induce plant autophagy by SnRK1 and TOR, or tubulin acetylation, which then induces defense responses and plant survival. SnRK1, Snf1related protein kinase 1; TOR, target of rapamycin.
Park et al., 2014; Zhuang et al., 2017). For instance, although AtATG4 and AtATG8 contain two and nine members, respectively, AtATG4 proteins retain specificity for AtATG8 (Park et al., 2014). Furthermore, AtATG4a is the primary protease, which processes AtATG8a, AtATG8c, AtATG8d, and AtATG8i better than AtATG4b in vitro (Woo et al., 2014). In addition to the core ATG proteins, other ATG proteins, such as ATG6, or other factors, such as the A. thaliana acyl-CoA-binding protein (ACBP3), are involved in the regulating of autophagy dynamics (Qi et al., 2017; Lee et al., 2018). Interestingly, the ATG17-ATG31-ATG29 complex is missing in Arabidopsis, which acts partly as a scaffold in the initiation of autophagy (Fig. 1). This may be explained by the fact that ATG11-like proteins may act as bifunctional scaffolding proteins in both ATG11- and ATG17-related functions within the ATG1-ATG13 complex. In plants, autophagy has been suggested to be involved in seed development and germination, photomorphogenesis, chloroplast maturation, stress protection, pathogen resistance, hormonal responses, and senescence under starvation conditions (Ishida et al., 2008; Ghiglione et al., 2008; Chung et al., 2009; Izumi et al., 2015). Therefore, it is important to have insight into its mechanism in plants, especially the role of autophagy in pathogen resistance and stress protection. Recently, it has become known that many stresses can induce autophagy in plants, including abiotic stresses such as salt, starvation, drought, and ER stress; and biotic stress, which contributes to defense responses and plant survival (Fig. 2). Meanwhile, in crops, research mainly focuses on the functions of autophagy in pathogen resistance and fertile floret development in rice. Interestingly, autophagy plays a vital role in the infection-related autophagy of the rice blast fungus M. oryzae (Kershaw and Talbot, 2009; He et al., 2012; Liu et al., 2017). Here, we are interested in how autophagy promotes the senescence process and even death and contributes to defense responses and plant survival.
2 AUTOPHAGY AND SENESCENCE 2.1 Whole-Plant Senescence and Leaf-Senescence In the whole life span of plants, there are three main stages: vegetative, reproductive, and senescence. Between the vegetative and reproductive phase, there is an important event: floral
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transition. It is a major component of whole-plant senescence of monocarpic plants. During floral transition, vegetative growth ceases and flowering begins. After floral transition, the growth of fruits and seeds replaces the forming of new leaves. Fruits and seeds induce large changes in plant metabolism and nutrient redistribution, because they, as new sinks, will compete for the remaining nutrients of plants. Therefore, as a strategy, plants will mobilize and redistribute the nutrients of plants' other tissues to balance all the conflicting needs. As a tissue that captures light and absorbing carbon dioxide (CO2), leaves acquire high allocation from plants' available resources, which are good nutritional resource suppliers for fruits and seeds. Therefore, after floral transition, leaves are beginning to senesce. Leaf senescence is an active and ordered degeneration process that is not only influenced by internal factors, but also various environmental factors. The internal factors that affect leaf senescence include phytohormone and reproduction, and environmental factors include stresses such as ozone (O3), drought, high or low temperature, nutrient deficiency, and pathogen infection (Lim et al., 2007; Schippers et al., 2015). All these factors finally regulate developmental age and then initiate the onset of leaf senescence by the regulation of transcription factors, such as WRKY, NAC, AP2/EREBP, C2H2-type zinc finger, and MYB protein families (Miao et al., 2008; Schippers et al., 2015; Zentgraf et al., 2010). In addition, the cell structure and metabolism undergo orderly changes during leaf senescence. The earliest and most important change in cell structure is the chloroplast’s breakdown, while mitochondria and the nucleus collapse at the final stage of senescence. The changes in organelle structure during senescence are mainly due to protein degradation. In eukaryotes, there are two main mechanisms that are responsible for protein degradation: the lysosomal/ vacuolar degradation system and the ubiquitin/proteasome system. The ubiquitin/proteasome system is specific and degrade one after the other. However, the lysosomal/vacuolar degradation system is a degradation pathway of bulk cytosolic constituents and organelles that largely depend on autophagy that parcel the substrates for degradation through certain membrane-mediated processes. Therefore, autophagy is more efficient than the ubiquitin/ proteasome system in protein degradation, especially in cell structural breakdown.
2.2 Senescence, Autophagy, and ATG Proteins It is accompanied by decreased expression of genes (such as CAB2, RPS, and RBC) and by increased expression of senescence-associated genes (SAGs) during leaf senescence. Affymetrix GeneChip microarrays are utilized to analyze changes in global expression patterns during senescence in Arabidopsis, which has identified more than 800 SAGs (BuchananWollaston et al., 2005). Approximately 30% of SAGs involve in regulation, and 12.2% are involved in macromolecule degradation and mobilization. Amazingly, there are five autophagy genes appeared in these SAGs, including AtAPG7, AtAPG8a, AtAPG8b, AtAPG8h, and AtAPG9 (Buchanan-Wollaston et al., 2005). At the same time, most studies showed that most atg mutants exhibited premature leaf senescence after nutrient starvation treatment (Table 1, Fig. 3), especially these mutants blocking the ATG12 Ub-like conjugation system (Atatg5, Atatg10, and Atatg12a/Atatg12b double mutant), attenuating the ATG8 Ub-like conjugation system (Atatg4a/Atatg4b double), or blocking both Ub-like conjugation systems (Atatg7) (Fig. 3) (Hanaoka et al., 2002; Doelling et al., 2002; Xiong et al., 2005). All of these findings demonstrate that autophagy plays an essential role in senescence, especially the two Ub-like
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FIG. 3 Autophagy and senescence in plants. There are several kinds of autophagy-dependent bodies, including autophagosome, RuBisCO-containing bodies (RCBs), autophagy-dependent plastid bodies containing ATG8INTERACTING1 (ATI-PS) and ATI1-ATG8 spherical bodies, that degrade the cytoplasm and organelles, especially chloroplast.
conjugation systems. Therefore, any events that affect the level or the stability of ATG proteins will lead to early senescence. For instance, the A. thaliana acyl-CoA-binding protein ACBP3 regulates leaf senescence through modulating ATG8 stability (Fig. 3; see also Xiao and Chye, 2010). In yeast, the E3-like conjugating enzymes of two Ub-like conjugation systems are ATG5ATG16 for ATG12, and ATG-5-ATG16-ATG12 for ATG8. ATG8, the most commonly used maker protein for autophagic activity (Yoshimoto et al., 2010; Yoshimoto, 2012; Thompson et al., 2005),
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which involves the size of autophagosome and the cargo binding during selective autophagy (Shintani et al., 2002; Xie et al., 2008). In Arabidopsis, the atg5 and atg8 mutants show early senescence under dark or nutrient-limited conditions (Thompson et al., 2005); but can it be explained by sugar deficiency? Mukae et al. (2015) found that it has the sugar content of WT and Physcomitrella atg5 (Ppatg5) mutant are almost the same. But their amino acids content were significantly different in dark condition, which supports that the imbalance of amino acids content resulting from the deficiency of cytoplasmic degradation by autophagy may partly explain the premature phenotype in Ppatg5 mutants (Mukae et al., 2015). Therefore, autophagy may play an important role in plant senescence though maintaining the amino acid balance (Fig. 3). It still needs to confirm further whether other atg mutants promote senescence by disordering the balance of amino acid or by other pathways that can induce some stresses for plant survival.
2.3 Senescence, Autophagy, and Chloroplasts During senescence, cells increase catabolic activity to convert the intracellular accumulated materials into exportable nutrients for developing seeds, or even for survival. Ultrastructural studies showed that the earliest dismantled organelles are chloroplasts, while mitochondria and the nucleus collapse at the final stage of senescence (Butler, 1967; Dodge, 1970) In addition to internal factors, external factors can induce senescence, including ultraviolet B (UVB), nutrient limitation, heat, drought, and shading. Under nutrient limitations, plants can promote nutrient recycling and mobilization by initiating leaf senescence. When plants grow under nitrogen deficiency conditions, senescence is induced to remobilize nitrogen by chloroplast's breakdown (Masclaux et al., 2000). On the other hand, autophagy is a dynamic process that transports intracellular components, including macromolecules and even organelles, into lysosomes/vacuoles to degrade and then releases monomers such as amino acids into the cytosol for recycling. There are a good deal of evidence to support the idea that autophagy is responsible for chloroplast protein degradation during leaf senescence, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), stroma- targeted fluorescent proteins, and other proteins of chloroplasts. Chloroplasts contain up to 70% of leaf proteins, which means that they are good donators for nutrient supply (Makino and Osmond, 1991). There are a variety of enzymes in the chloroplasts, including RuBisCO, photosystems, thylakoid proteins, Calvin cycle proteins, and light harvesting complexes. Among these enzymes, RuBisCO is the most abundant protein, which is mainly synthesized during leaf expansion, and it finally accounts for 12%–30% of total leaf proteins in C3 plants (Evans, 1989). However, RuBisCO degrades rapidly during senescence. It was reported that RuBisCO and its degradation products can be transported into vacuoles via RuBisCO-containing bodies (RCBs) after darkness treatment (Chiba et al., 2003). Further, an ATG gene-dependent autophagy process has been found, which involves in the mobilization of RuBisCO and stroma-targeted fluorescent proteins without prior chloroplast destruction during naturally senescent leaves (Ishida et al., 2008). It suggests that the proteins of chloroplasts can be degraded by autophagy during natural or dark-induced senescence (Fig. 3). It remains a question what factors guide the PAS to near the chloroplast and form autophagosome-loaded buds from chloroplasts. Recently, it is known that the selective macroautophagy depends on the ATG8 protein. Through detailed real-time and three-dimensional (3D) imaging technologies, Le Bars et al. (2014) found a few ATG8 proteins gathered in PAS,
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while much ATG5 accumulates there in the initiation of autophagosome formation, accompanied by the underlying endoplasmic reticulum (Fig. 3; Le Bars et al., 2014). Therefore, the formation of RCBs may depend on this pathway with the selective of ATG8s (Fig. 3). In addition to RCBs, granum can be transported to the vacuole by autophagy-dependent plastid bodies containing ATG8-INTERACTING1 (ATI1-PS; Michaeli et al., 2014). ATI1-PS is not a typical autophagosome because it directly buds out and detaches from plastids into cytoplasm with the help of ATI1 and ATG8 proteins by a process that doesn't require active autophagy (Fig. 3; Michaeli et al., 2014). It is not clear what factors induce the budding and detaching of ATI1-PS from plastids. ATI1 also can form spherical bodies with ATG8 (ATI1ATG8 spherical bodies) under nutrient limitations, which is different from ATI1-PS. Honig et al. (2012) found that ATI1 is partially related to the ER membrane network under favorable growth conditions; however, they become mainly associated with ATI1-ATG8 spherical bodies under carbon starvation. Moreover, the overexpression or suppression of ATI1 could evoke responses from the exogenous hormone abscisic acid (ABA; Honig et al., 2012). The level of salicylic acid (SA) increases gradually with the increase of age. The higher concentration of SA allows it to interact with NPR3, which causes rapid degradation of NPR1. Then it promotes senescence and programmed cell death (PCD) through inducing ER stress by an NPR1-dependent pathway (Fig. 4; Yoshimoto et al., 2009; Breeze et al., 2011; Fu et al., 2012; Minina et al., 2014). This finding supports the fact that ATI-association autophagy may involve senescence through degrading chloroplast by ATI1-PS and the hormone signaling pathway by ATI1-ATG8 spherical bodies, which are involved in ER response.
2.4 Senescence, Autophagy, and SAVs Enzymatic activity studies showed an increase in protease activities during leaf senescence, and genomewide studies further provided a large number of protease genes that are upreg-
FIG. 4 SA signaling and ER stress in senescence. With aging, SA levels gradually increase, which causes rapid degradation of NPR1 through the action of NPR3. Then, plants get into senescence and programmed cell death. ATG8, autophagy-related protein 8; ATI-3, ATG8-INTERACTING3; NPR1/3/4, nonexpressor of pathogenesis-related (PR) genes 1/3/4; SA, salicylic acid; SnRK1, the Snf1-related protein kinase 1; TOR, target of rapamycin; UBAC2, Ubiquitin-associated protein 2.
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ulated in this development stage (Feller et al., 1977; Roberts et al., 2012). SAG12, encoding a cysteine protease, is the most famous senescence-associated protease gene. Moreover, SAG12 is the only SAG that is uniquely controlled by natural senescence, but it does not respond to environmental stresses. Therefore, it is commonly used as a marker gene for leaf senescence, which is located in small, lytic vacuolar compartments (also called SAVs) in senescent leaves (Martinez et al., 2008a,b; Otegui et al., 2005). SAVs are small (0.5–0.8 μm), and have been identified in the senescent leaves of several plants, including soybean, Arabidopsis, and tobacco, but absent from nonsenescing leaves (Martinez et al., 2008a,b). SAVs are different from central vacuole for its contents and membrane component. There are soluble chloroplast proteins (such as RuBisCO) and resident proteases (such as senescence-specific SAG12) in the acid lumen of SAVs (Pascual et al., 1994; Otegui et al., 2005; Martinez et al., 2008a,b). Meanwhile, its membrane components include vacuolar H+-pyrophosphatase but lack the aquaporin γ-TIP, a marker for the central vacuole tonoplast, and they are more acidic than central vacuoles (Jauh et al., 1999). Therefore, the SAV pathway is one of the degradation pathways of chloroplast proteins during senescence, but it is a different autophagy pathway. In oilseed rape, the SAG12 protein is important for nitrogen remobilization because it is heavily abundant in senescent leaves, especially when nitrogen remobilization is enhanced for nutrient deficiency (Desclos et al., 2009). This is coincided with the function of senescence in plant’s nutrient reuse. However, the Arabidopsis sag12 mutant showed no phenotypes (Otegui et al., 2005). Meanwhile, this finding suggests that RuBisCO degradation by SAVs is upregulated to complement the deficiency in autophagy of atg mutants, for the transcripts of SAG12 accumulate at a high level in atg2 and atg4a4b mutants and visible leaf senescence proceeds much faster than the wild type in atg2 mutants (Yoshimoto et al., 2009; Wada et al., 2009). According to these findings, the SAV pathway and the autophagy-dependent pathway are two independent and complementary pathways that contribute to degrade chloroplast proteins, which may well explain the nonphenotypes of Atsag12 mutants.
3 DISCUSSION 3.1 ATG Proteins Almost every atg mutant showed accelerating senescence under nutrient-limited conditions, but it is not clear how autophagy responds to various stresses that can induce senescence or survival in plants, such as plant age, nutrient starvation, and salt. This is an interesting question that needs to be studied. In addition to involving the core autophagy mechanisms, whether these senescence-association ATG genes can respond to other senescence-association factors or directly become senescence-association factors that activate the downstream SAG genes through a way different from autophagy? Just like the ATG18a, it can respond to WRKY33, a transcription factor, which is important for plant resistance to necrotrophic pathogens (Lai et al., 2011).
3.2 The SA Signaling in Autophagy and Senescence ER is known to be one of the important membrane material stores for autophagosome. Using 3D imaging, it has been ascertained that ER can form the membrane of autophagosomes
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and the ATI1-ATG8 spherical bodies under nutrient starvation. During these stages, it can be constantly added to autophagosomes and the ATI1-ATG8 spherical bodies, with the help of the ATG5 and ATI1 proteins, respectively (Le Bars et al., 2014; Honig et al., 2012). It is thus clear that ER plays an important role in the formation of the autophagy-association architecture. Meanwhile, it has been suggested that SA levels increase during the final phase of leaf development, which can cause rapid degradation of NPR1 through the action of NPR3. The degradation of NPR1 finally causes PCD by the NPR1-dependent, ER stress activation of autophagy (Minina et al., 2014; Yoshimoto et al., 2009; Breeze et al., 2011). In addition, the studies of autophagy induced by ER stress demonstrate that SnRK1 can respond to ER stress, which is upstream of TOR, and the ATG8-ATI3-UBAC2 complex can induce autophagy and then affect plant life (Soto-Burgos and Bassham, 2017; Zhou et al., 2018). Therefore, the senescence or PCD induced by SA through ER stress may use SnRK1 and TOR to pass on the SA signaling. The role of the ATG8-ATI3-UBAC2 complex in this pathway still needs further research (Fig. 4).
4 CONCLUSION Autophagy is a degradation pathway of bulk cytosolic constituents and organelles, which can promote plants' nutrient use efficiency and then survival from stress or reproductive death. Depending on autophagy, plants evolved several pathways to degrade chloroplast, such as RCBs and autophagy-dependent plastid bodies containing ATG8-INTERACTING1 (ATI-PS). Although SAVs pathway is different from autophagy, the deficiency of autophagy promotes RuBisCO degradation by SAVs. All these pathways apply themselves to promote plant senescence by degrading chloroplasts. However, almost every atg mutant showed early senescence under nutrient-limited conditions, which demonstrates that the role of autophagy in plant senescence are not only as a promoter but also as an inhibitor. To date, it is known that Ppatg5 mutants show an imbalance of amino acids, which partly explains the earlysenescence phenotype in Ppatg5 mutants. But more information is needed to confirm whether other atg mutants promote senescence by disordering the balance of amino acids or by other pathways that can induce certain stresses for plant survival. In addition to involving the core autophagy mechanisms, these senescence-association ATG proteins probably function as factors that respond to senescence-association factors or directly become senescence-association factors to activate the downstream SAG genes. This finding may answer the question of how autophagy receives senescence signaling and passes it on.
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