# Linking Autophagy to Abiotic and Biotic Stress Responses

Santiago Signorelli, Łukasz Pawel Tarkowski, Wim van den Ende, Diane C Bassham

► To cite this version:

Santiago Signorelli, Łukasz Pawel Tarkowski, Wim van den Ende, Diane C Bassham. Linking Autophagy to Abiotic and Biotic Stress Responses. Trends in Plant Science, 2019, 24 (5), pp.413-430. 10.1016/j.tplants.2019.02.001 . hal-04651934

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# Trends in Plant Science

## Linking autophagy to abiotic and biotic stress responses

### --Manuscript Draft--

<table><tr><td>Manuscript Number:</td><td></td></tr><tr><td>Article Type:</td><td>Review</td></tr><tr><td>Corresponding Author:</td><td>Santiago Signorelli, PhD<br/>KU Leuven<br/>Leuven, Heverlee-Leuven BELGIUM</td></tr><tr><td>First Author:</td><td>Santiago Signorelli, PhD</td></tr><tr><td>Order of Authors:</td><td>Santiago Signorelli, PhD<br/>Łukasz Paweł Tarkowski, PhD Candidate<br/>Wim Van den Ende, Professor<br/>Diane C Bassham, Professor</td></tr><tr><td>Abstract:</td><td>Autophagy is a process in which damaged cellular components are delivered to lytic vacuoles to be recycled and was demonstrated to promote abiotic and biotic stress tolerance. Here, we present and discuss the latest findings showing how autophagy can contribute to stress tolerance, and its interplay with the phytohormone, MAPK/TOR/SnRK1, and reactive oxygen/nitrogen species (ROS and RNS) signaling pathways triggered by stress conditions. We also highlight the importance of osmotic adjustment for the successful performance of macro-autophagy and the avoidance of mega-autophagy. We present evidence demonstrating that ROS can affect autophagy, but also that autophagy can be considered a non-canonical antioxidant system. Finally, we highlight the importance of GABA to plant survival and its potential implications in ethylene-induced autophagy.</td></tr><tr><td>Suggested Reviewers:</td><td>Christian Meyer<br/>French National Institute for Agricultural Research<br/>cmeyer@versailles.inra.fr<br/><br/>Nicholas Smirnoff<br/>University of Exeter<br/>N.Smirnoff@exeter.ac.uk<br/><br/>Sergey Shabala<br/>University of Tasmania<br/>Sergey.Shabala@utas.edu.au<br/><br/>Elena Minina<br/>alena.minina@slu.se<br/><br/>Tamar Avin-Wittenberg<br/>tamar.avin-wittenberg@mail.huji.ac.il</td></tr><tr><td>Opposed Reviewers:</td><td></td></tr></table>
KU LEUVEN

PLANT MOLECULAR BIOLOGY
KASTEELPARK ARENBERG 31 BOX 2438
3001 LEUVEN, BELGIUM

<figure><img src="image_1.png" /></figure>

Dr Susanne C Brink,
Editor in Chief of Trends in Plant Science

OUR REFERENCE
YOUR REFERENCE
LEUVEN

2018-05-26

Dear Dr Susanne C Brink,

As previously discussed, we would like to submit our review entitled "Linking autophagy to abiotic and biotic stress responses" by Santiago Signorelli, Łukasz Paweł Tarkowski, Wim Van den Ende, and Diane C Bassham. We would like to thank again for the invitation to submit our review in Trends in Plant Science.

As expressed in the presubmission, this review focuses on the latest finding of how autophagy can contribute to stress tolerance in plants and its interplay with the signaling pathways triggered by abiotic and biotic stress conditions.

We firmly believe that this review will be of interest to the plant science community.

As reviewers we would like to suggest:

Christian Meyer (cmeyer@versailles.inra.fr) French National Institute for Agricultural Research, France.

Elena Minina (alena.minina@slu.se) Swedish University of Agricultural Sciences, Sweeden.

Sergey Shabala (Sergey.Shabala@utas.edu.au) University of Tasmania, Australia.

Tamar Avin-Wittenberg (tamar.avin-wittenberg@mail.huji.ac.il) The Hebrew University of Jerusalem, Israel.

Nicholas Smirnoff (N.Smirnoff@exeter.ac.uk) University of Exter, UK.

We hope to hear from you soon.

Kind regards,

<figure><img src="image_2.png" /></figure>

Santiago Signorelli
(on behalf of all authors)
**Highlights:**

* The energy sensors SnRK1 and TOR control autophagy not only under energy deficiency but also under diverse stress conditions.

* Autophagy enhances tolerance to many abiotic stresses and oxidative stress conditions.

* Under biotic stress, autophagy can come in advantage of the host as well as being exploited by the pathogen, depending on the pathosystem considered.

* Reactive oxygen species (ROS) contribute to the establishment of autophagy but also damage ATG proteins. Thus, tight coordination between ROS homeostasis and autophagy is required in plants.

* Osmotic adjustment under severe stress conditions contributes to sustaining intensive macro-autophagy activity and avoidance of mega-autophagy.
<figure><img src="image_3.png" /></figure>

1 **Title:**

2 Linking autophagy to abiotic and biotic stress responses

3

4 **Authors:**

5 Santiago Signorelli¹,²*, Łukasz Paweł Tarkowski¹, Wim Van den Ende¹, Diane C Bassham³

6

7 **Affiliations:**

8 ¹ Laboratory of Molecular Plant Biology, KU Leuven, Leuven, Belgium

9 ² Departamento de Biología Vegetal, Facultad de Agronomía, Universidad de la República, Montevideo 12900, Uruguay

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11 ³ Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011, United States

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14 *corresponding author: ssignorelli@fagro.edu.uy (S. Signorelli)

15 Twitter account: signorellisanti

16

17 **Keywords:**

18 ROS, TOR, GABA, SnRK1, ATG

19

20 **Abstract:**

21 Autophagy is a process in which damaged cellular components are delivered to lytic vacuoles to be recycled and was demonstrated to promote abiotic and biotic stress tolerance. Here, we present and discuss the latest findings showing how autophagy can contribute to stress tolerance, and its interplay with the phytohormone, MAPK/TOR/SnRK1, and reactive oxygen/nitrogen species (ROS and RNS) signaling pathways triggered by stress conditions. We also highlight the importance of osmotic adjustment for the successful performance of macro-autophagy and the avoidance of mega-autophagy. We present evidence demonstrating that ROS can affect autophagy, but also that autophagy can be considered a non-canonical antioxidant system. Finally, we highlight the importance of GABA to plant survival and its potential implications in ethylene-induced autophagy.

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32 **Main text:**

33 Autophagy alleviates stress-induced cell damage

34 Both abiotic and biotic stress conditions have a negative impact on plant growth, and often threaten agronomical production. In future years, an increased demand for food and a more challenging

35
environment for plant growth is expected [1]. To counteract this, a better understanding of plant
resistance to both abiotic and biotic stresses is necessary, and in particular of those processes
promoting plant survival on a cellular and organismal level. At a cellular level, abiotic and biotic
stresses lead to the overproduction of reactive oxygen and nitrogen species (ROS and RNS, see
glossary) which can damage organelles and biomolecules, affecting their functionality (see **box 1** for
further detail). Plant **macroautophagy** (here referred to as autophagy) is a process by which damaged
cellular components are encapsulated by a double membrane vesicle (**autophagosome**), which fuses
with the vacuole for recycling of its contents [2]. Recent excellent reviews were published regarding
the mechanisms and regulation of autophagosome formation (see [3–5]) and this will not be covered
in this review. This recycling process yields amino acids, fatty acids, and sugars which can be used
later by the organism [6]. In this way, autophagy can be considered as one process promoting plant
survival (**Figure 1**). In the last few years, many findings linking plant autophagy with different
signaling pathways under (a)biotic stress have emerged [7–10]. Here we discuss how autophagy can
contribute to stress tolerance and its interplay with the signaling pathways triggered upon stress
conditions. We first discuss the metabolic and redox changes that are induced by stress conditions,
and then integrate this with hormonal and sugar signaling pathways involved in autophagy activation.

**Abiotic and biotic stresses induce major metabolic changes**

As sessile organisms, plants have developed two main strategies to face adverse conditions: avoidance and tolerance [11]. The first consists of stable morphological and biochemical adaptations, whereas tolerance is the plant's potential capacity to respond and adapt to stressful conditions. Among the physiological changes observed during such processes, readjustment of primary metabolism is of critical importance [12]. Alterations in carbohydrate and amino acid fluxes were shown to be fundamental features in a plant's capacity to successfully cope with major biotic and abiotic stresses. In this regard, dynamic changes in concentration, compartmentalization and biochemical composition of soluble and insoluble sugar pools are key events. Perception and modulation of sugar status plays a prominent role in cell survival capacity under stress. The central energy-sensing SnRK1 (SNF-related kinase 1) kinase acts upstream of Target of Rapamycin (TOR) upon sugar perception to induce major changes at the cellular level, regulating plant growth and development, cell cycle progression, induction of stress responses, and autophagy [8,13–15]. Besides the signaling function of sugars, their capacity to control osmotic potential, hydrate membranes, scavenge reactive oxygen species (ROS), and their protection of the photosynthetic apparatus make them fundamental molecules in the cellular responses to chilling, freezing, heat, and drought stress [16–18]. Specific structural variants like raffinose family oligosaccharides (RFOs) and fructans were often associated with abiotic stress
tolerance [19] and increases in their contents were shown to correlate positively with drought tolerance [20].

Abiotic stress also has an impact on the amino acid pool [12]. Although a general increase in amino acid content can be a signature of protein degradation, increases in specific amino acids are associated with abiotic stress tolerance. For instance, GABA ($\gamma$-amino butyric acid) accumulates in plant tissues in response to several stresses and is thought to play a critical role in stomatal closure and water retention [21,22]. Proline is the amino acid with the greatest increase in concentration upon abiotic stress and some authors have associated this response with tolerance to drought, salt, cold and heavy metal stresses [23]. Similar to sugars, proline was suggested to have an important role as a **compatible osmolyte** and **ROS scavenger** in the protection of membranes and proteins and the maintenance of photosynthetic activity [23,24]. Nonetheless, proline is not an effective scavenger of most ROS [25,26], but is able to protect against hydroxyl radicals [27,28]. Increased levels of amino acid-derived compounds, like glycine-betaine and polyamines, may also contribute to abiotic stress tolerance in several plant species [29,30]. In our view, the **osmotic adjustment** produced by the accumulation of these compatible osmolytes in the cytosol also contributes to the equilibration of the sudden osmotic changes produced in the cell during autophagy (discussed further in **box 2**). Besides their osmoprotectant role, GABA and polyamines have been proposed as signaling molecules, mainly through the interplay with hydrogen peroxide ($H_2O_2$) and nitric oxide (NO), and by affecting $Ca^{2+}$ influx [22,31]. Below, we will also discuss their potential to affect autophagy signaling.

Likewise, the interaction of plants with pathogens has a profound impact on the plant metabolome [32]. The importance of primary metabolism modulation is linked to its role in supplying energy to cells during pathogen attack [32]. Contrary to data reported from abiotic stress studies, primary metabolites that are clearly associated with pathogen resistance are difficult to find, because such correlations are strongly dependent on the pathosystem and pathogen's lifestyle [33,34]. Furthermore, in most cases it is difficult to elucidate whether the observed metabolic changes are the result of the defence activation of the host or the manipulation of the host's physiology by the pathogen. For example, the necrotrophic pathogen *Botrytis cinerea* induced an alteration of the plant C:N balance via over-activation of the GS/GOGAT cycle together with the GABA shunt, maintaining the viability of attacked cells in tomato plants [35]. Manipulations of C reserves are documented as well, as in the case of the hemibiotroph *Zymoseptoria tritici*, able to induce an increase in the fructan pool in wheat during its switch from a biotrophic to necrotrophic lifestyle [36]. Besides microorganisms, attacks by herbivorous insects have a significant influence on primary metabolites as well. The most prominent features are a decrease in photosynthetic capacity accompanied by an increase in sucrose and decrease in starch, possibly to compensate for the decrease in carbon assimilation [37]. Regarding N
metabolism, increases in amino acid biosynthesis are usually observed, probably to accumulate
precursors (like cysteine and tryptophan) for the synthesis of nitrogen-based defense compounds such
as alkaloids [38]. The current opinion tends to accept that metabolic investment in defense
disadvantages plant growth in a trade-off process [39]. In this respect, a contribution from biotic
stress-induced autophagy (discussed below) to stimulate nutrient recycling and counteract disease
progression is a fascinating hypothesis for future investigation.

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112 Abiotic stress-induced autophagy

113 Diverse abiotic stresses were shown to induce autophagy, including osmotic and salt stress [8,40].
114 Perhaps more importantly, autophagy is relevant during oxidative stress [41], a condition associated
115 with most environmental stresses. Since **priming** is usually also associated with temporal ROS and
116 oxidative stress responses [42,43], it is possible that priming with certain molecules, such as NaCl,
117 and H₂O₂, induce autophagy too. In stress conditions, autophagy is usually considered to promote cell
118 survival [2]. The induction of autophagy upon abiotic stress is relatively fast since the expression of
119 *ATG18a* in Arabidopsis was shown to be induced within a few hours after exposure to NaCl or
120 mannitol [40]. Also in Arabidopsis, the overexpression of *ATG5* and *ATG7* increases autophagic flux
121 and results in a greater tolerance to oxidative stress [7].

122 As mentioned above, TOR protein kinase is a negative regulator of plant autophagy [9]. TOR seems
123 to be of particular importance in starvation, salt and drought-induced autophagy since the
124 overexpression of TOR is enough to block autophagy under these conditions [9]. However, under
125 oxidative and endoplasmic reticulum stress, a TOR independent mechanism was shown to be
126 involved in the control of autophagy [9]. One of the suggested candidates for this role is inositol-
127 requiring enzyme 1b (IRE1b), involved in the regulation of ER-stress related genes. Arabidopsis *ire1b*
128 mutants exhibited a decreased number of autophagosomes in response to misfolded protein
129 accumulation when compared to wild type (WT) plants [44]. The auxin 1-naphthaleneacetic acid
130 (NAA) is known to induce TOR activity, and concordantly it was shown to inhibit autophagy through
131 a reduction in the number of autophagosomes formed (Figure 2) [9]. Sugars are known to affect TOR
132 activity through the modulation of SnRK1, which can inhibit TOR activity. Trehalose-6-phosphate
133 (T6P) inhibits SnRK1 activity and was shown to inhibit autophagy in response to abiotic stress [8].
134 In Arabidopsis, the catalytic subunit of SnRK1, KIN10 (sometimes referred as AKIN10), was shown
135 to act upstream of TOR (Figure 2) [12]. KIN10 activity also strongly depends on the redox status.
136 For instance, very reducing conditions were shown to produce the highest kinase activity, whereas
137 oxidizing conditions inhibit its activity (Figure 2) [45].

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Flooding is an environmental stress that generally leads to a hypoxic state in root cells. In animals, the expression of hypoxia responsive genes, such as hypoxia induced factor-1 (HIF-1), triggers the expression of proteins belonging to the Bcl-2 family such as BNIP3 and BNP3L, both required to activate hypoxia-induced autophagy [46]. Interestingly, the presence of glucose is able to inhibit this hypoxia-induced autophagy, preventing cell death, even at 0.1% pO₂ [46], highlighting the importance of sugars in autophagy. However, when the levels of glucose are low, hypoxia can produce HIF-1-independent autophagy [46]. This hypoxia-independent autophagy seems to be controlled by TOR activity. In plants, hypoxia can not only be an acquired condition in roots due to flooding but also can be a natural condition of certain organs, such as seeds. Arabidopsis *KIN10* overexpressing lines were shown to be able to induce the tolerance to hypoxia treatments by increasing autophagy [47]. It would be interesting to investigate whether hypoxia-responsive genes are involved in hypoxia-induced autophagy. The ethylene response factors (ERFs) are known to contribute to hypoxia tolerance and this tolerance might be acquired by activating autophagy. Interestingly, it was shown that autophagy mutants are hypersensitive to submergence stress, and that submergence-induced accumulation of ERF1 is abolished in these mutants [48].

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## Biotic stress-induced autophagy

Besides its role in abiotic stress response, autophagy is a well-established component of the metazoan immune system [49]. Its activation is critical for the removal of bacterial pathogens, control of inflammatory signaling, antigen presentation, and secretion of immune mediators [49]. In plants, autophagy activation upon pathogen attack has been shown to lead to different outcomes dependent on the pathogen lifestyle [50]. The plant immune system is a complex and sophisticated machinery that relies on multiple layers of specificity to optimize defense responses [51]. One of the most well-studied plant defense mechanisms is the hypersensitive response (HR), a form of programmed cell death (PCD) which has a role in restricting pathogen invasion [52]. Importantly, necrotrophic pathogens can take advantage of the HR response, and some of them can manipulate the HR machinery to facilitate infection spreading [53]. Several studies highlighted a tight connection between autophagy and HR regulation during plant immune responses. Pioneering reports in this field provided divergent data regarding the role of autophagy in inducing or restricting HR [54,55] during *Pseudomonas* infection. Such discrepancies are probably due to the different age of the plants used in the experiments, which can be a critical factor in view of the importance of autophagy in senescence mechanisms and salicylic acid (SA) signaling [56]. In the case of necrotrophs, autophagy execution was often associated with restriction of the HR response, thus contributing to a resistance phenotype [57,58]. On the other hand, the same HR restriction has been shown to increase susceptibility to biotrophs [59]. Further research demonstrated an essential role for autophagy in the induction of HR

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[56,60], adding more layers of complexity. It was proposed that autophagy can favor HR in infection sites and restrict it in surrounding tissues [56]. This model fits well with evidence indicating an important role for autophagy in contributing to the elimination of ROS generated during the HR response [61]. Recently, overexpressing lines for ATG5 and ATG7 were shown to have a dramatically enhanced tolerance to necrotrophic pathogens [7]. Several authors have hypothesized that the role of autophagy may vary according to the specific pathogen considered [57,62]. Conversely, evidence has emerged that some pathogens have evolved strategies to modulate autophagy to their advantage [63,64]. Future research in this field is expected to shed more light on the selectivity and specificity of autophagic events during immune responses.

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## Coordination between autophagy and reactive oxygen and nitrogen species

Plants depend upon specific antioxidant systems to protect against reactive species. Usually, the antioxidant systems are divided into two types, enzymatic and non-enzymatic. The enzymatic antioxidant system includes superoxide dismutase isoforms, catalase and peroxidases, whereas the non-enzymatic system is used to refer to ROS scavengers such as glutathione, ascorbate, tocopherol and also sugars. These could be defined as the canonical antioxidant systems and they act specifically on one type of ROS and on single molecules. Since autophagy can promote the degradation of damaged organelles that can overproduce ROS and RNS when damaged, such as mitochondria, chloroplasts and peroxisomes, autophagy was suggested to also contribute to the antioxidant system [65]. In line with this, in Arabidopsis the disruption of autophagy increases H₂O₂ production, as observed in *atg5* and *atg2* mutants [66]. Moreover, silencing of the *ATG18a* gene in Arabidopsis generated hypersensitivity to oxidative stress and increased basal oxidative damage [41,67]. Hence, autophagy can be considered as a non-canonical antioxidant system acting on biomolecules or organelles instead of low molecular weight molecules. Accordingly, it was shown that entire chloroplasts are transported to the vacuole by autophagy as consequence of irradiation stress [68]. This is of particular interest since the accumulation of oxidized components can lead to cell death [65]. Additionally, as the internal redox state of cells can control cell cycle progression [69,70], it is possible that elimination of oxidized molecules is also essential to determine cell cycle fate.
Information related to reactive species and autophagy in the plant world is still limited. However, the core autophagy machinery is conserved between plants, yeast, and mammals [71]. This core of 18 ATG proteins includes the ATG9 cycling system, the phosphatidylinositol 3 kinase (PI3K) complex and the ubiquitin-like protein conjugation system [65]. Thus, in this section, we also refer to what we have learnt from other organisms. It is likely that ROS and RNS modulate autophagy either by targeting activators or repressors of this process; however direct evidence in this regard in plants is still scarce. The most detailed insights probably come from mammalian systems, in which Scherz-

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Shouval et al., (2007) showed that H₂O₂ production is induced upon starvation, regulating the activity of ATG4 by targeting a specific cysteine near the catalytic site through oxidation. Recently, the same was demonstrated for ATG3 and ATG7 [73]. These oxidations result in the inactivation of the proteins, abolishing the formation of autophagosomes (Figure 2). The redox control of ATG4 was also observed in *Chlamydomonas* [74].

The levels of ROS, site of generation and time of exposure are three important factors determining their effect on any biological process. Generally speaking, when ROS are limited, in terms of concentration, time or space, they can trigger a response, which many authors refers to the signaling role of ROS; in contrast, uncontrolled levels of ROS may have a deleterious effect on the process under study. Autophagy is not an exception. For instance, although H₂O₂ was shown to directly react with the ATG4 Cys protease, ATG3, and ATG7 inactivating their function and thus autophagy, treatment of Arabidopsis roots with exogenous H₂O₂ or methyl viologen induces autophagy [41] (Figure 2). Since methyl viologen acts on the chloroplastic electron chain, this evidence suggests that ROS produced chloroplastically are able to induce autophagy. This is in line with evidence from *Chlamydomonas*, in which a deficiency in carotenoid synthesis triggers autophagy in the light but not in the dark, where the chloroplastic electron transport is inactive [75]. Recently, the inhibition of fatty acid synthase complex in *Chlamydomonas* was shown to affect chloroplast integrity, in particular by hyperstacking of thylakoid membranes, which correlated to a higher autophagic flux in a ROS-independent way [76]. Moreover, it was recently reported that the hydrotropic response of *A. thaliana* roots requires autophagic activity, evidenced by accumulation of autophagosomes, and the presence of H₂O₂ [77]. Although H₂O₂ can directly interact with some ATG proteins, more specific mechanisms involving H₂O₂ sensors might act to mediate H₂O₂ signaling in autophagy. Cysteine-rich receptor-like kinases, glutathione peroxidase-like proteins or thiol peroxidases, are part of these H₂O₂ sensors that can mediate a more effective H₂O₂ signaling [78]. It would be interesting to see whether they participate in the regulation of autophagy mediated by H₂O₂.

In animals, superoxide is also suggested to play a role in the induction of autophagy, particularly under sugar starvation, as amino acid starvation produces both superoxide and H₂O₂, whereas upon glucose deprivation, only superoxide is induced [79]. A possible mechanism by which superoxide induces autophagy is the activation of forkhead box O3 (FOXO3) transcription factor, which upregulates ATG8 [79]. In plants, there is no FOXO3 homologue, suggesting that this superoxide-induced autophagy pathway would be absent. However, a potential alternative mechanism by which ROS can affect TOR-dependent autophagy in plants is by directly targeting TOR. In animals, the product of superoxide dismutation, H₂O₂, was shown to produce TOR oxidation and agglomeration, resulting in the loss of its functionality [80]. Hence, it is possible that in plants ROS directly inactivate
TOR, resulting in autophagy induction (Figure 2). Following this logic, a specific switch should be able to activate this ROS-induced autophagy. In plants, a potential candidate to produce the superoxide required as a signal to induce autophagy is Rboh, and this would fit perfectly with the ROS wave concept. In fact, Liu et al. (2009) showed that Rboh inhibitors were able to suppress autophagy in Arabidopsis (Figure 2). Rboh activity is induced by ABA [81], and this hormone is generally accumulated by plants under drought stress (Figure 2). Thus, Rboh would be essential to induce autophagy in the systemic response of plants to biotic and abiotic stresses, whereas the production of mitochondrial and chloroplastic ROS, through an increase in oxidized proteins, would comprise a less specific mechanism acting in particular cells subjected to unfavorable conditions. We propose that more research is needed on how chloroplastic ROS can induce autophagy. To date, the effect of mitochondrial ROS on autophagy has been better studied because this is one of the most important sources of ROS in animals. However, we have to consider that the production of ROS by mitochondria in plants is not as relevant as in animals, due to the lower respiration rate. In agreement with this, evidence for mitophagy in plants is limited [10].

Regarding the effect of RNS on plant autophagy, we consider that more research needs to be done in this field. RNS have emerged as important molecules usually overproduced under stress conditions and having regulatory roles [82]. Recently, under hypoxia ·NO was shown to target S-nitrosoglutathione reductase (GSNOR) to the autophagosome by S-nitrosylation [83]. This is of particular interest because GSNOR controls the levels of S-nitrosoglutathione (GSNO), the cellular reservoir of ·NO, having a key role in the control of crosstalk between ROS and ·NO in plants [84]. The specific targeting of GSNOR to the autophagosome by ·NO might also occur under other stresses. For example, in *Lotus japonicus* roots an induction of ·NO content was associated with a reduction of GSNOR activity under drought stress [85]. Besides targeting enzymes to autophagosomes, RNS could inhibit or enhance autophagy by posttranslational modification (protein nitration or S-nitrosylation) of the proteins involved in the autophagic process. It is not unthinkable that the exposed cysteines of ATG3, ATG4 and ATG7, which are targets of oxidation by H₂O₂, would also be targets of S-nitrosylation by ·NO. This might lead to a mechanism in which ·NO could also directly inactivate the autophagic process. Moreover, ·NO was shown to induce the expression of genes encoding hydrogen sulfide (H₂S)-synthesizing enzymes in tomato plants [86], resulting in higher levels of H₂S, which in turn is known to inhibit autophagy by an unknown mechanism [87].

## Effect of (a)biotic stress-induced metabolites and phytohormones on autophagy

In this final section we aim to connect the different phytohormones and the metabolic changes that occur under (a)biotic stress to autophagy. As discussed before, the accumulation of polyamines, proline, sugars and GABA is one of the most common responses of plants to (a)biotic stresses. A link
between polyamines and autophagy can also be suggested. The accumulation of polyamines under abiotic stress is mainly due to the induction of polyamine biosynthetic genes [88]. The enzymes polyamine oxidase and diamino oxidase, involved in the metabolism of polyamines, produce H₂O₂ as a product [89,90]. In this way, increasing concentrations of putrescine, spermidine, and spermine increase the production of H₂O₂, which in turn can induce autophagy (Figure 2). Under oxidative stress conditions, proline can yield GABA through two non-enzymatic and one enzymatic reactions [91] (Figure 2). These molecules are able to reduce ROS levels by reacting with hydroxyl radicals and in this way prevent oxidative stress-induced cell death (Figure 2). Also, GABA was shown to induce ethylene (ET) production which in turn can induce ATG8 expression and thus autophagy (Figure 2). This evidence, together with the capacity of GABA to contribute to the TCA cycle through the GABA shunt, suggests that GABA can act at different levels to promote cell survival (box 3).
ET also increases in response to extracellular pathogens through the activation of both mitogen activated protein kinase (MAPK) signaling pathways and Ca²⁺ dependent protein kinases, which increase ACS (1-aminocyclopropane-1-carboxylic acid synthase) gene expression [92] (Figure 2). Multiple reports demonstrated that intact ET signaling is required for the Rboh-dependent accumulation of ROS necessary to trigger tolerance to both biotic and abiotic stimuli [93–95], and several lines of evidence showed that ET is able to activate antioxidant systems. In particular, oxidative stress treatments were able to induce ERF1 expression, and ERF6 was shown to upregulate antioxidant enzymes under biotic and abiotic stresses [96]. ET-dependent ROS scavenging is also active during heavy metal and cold stress responses [97,98]. The role of ERFs as inducers of resistance to many abiotic stresses (salinity, cold, drought, freezing, heat, heavy metal and oxidative stress) is well documented [99]. However, the link with autophagy is still unknown, and it deserves exploration, in particular because jasmonic acid and SA are now known to regulate ERFs. On the other hand, ABA negatively regulates ERF1 and ERF6 induction; yet ERF1 induces ABA accumulation in Arabidopsis [96,100]. Given the importance of ERFs in development and stress responses, and their capacity to converge signals from different phytohormones, it would be interesting to see in future research whether ERF mutants can perform autophagy at the same rate as WT plants.

ABA is suggested to act as an endogenous messenger under abiotic and biotic stresses [101]. Although the most visible effects of ABA are in the leaves, such as the reduction in leaf expansion and stomatal closure, this phytohormone accumulates in all plant organs once the plant senses a reduction in water availability [102]. Thus, ABA is considered to be part of the plant systemic responses. In biotic stress, ABA-induced stomatal closure is proposed to be essential to avoid pathogen entrance by the stomata as a physical mechanism of defense [103]. We propose that ABA could contribute to the establishment of autophagy via the activation of Rboh (Figure 2). Autophagy
in turn, seems to be able to inhibit SA-mediated senescence and promote survival (Figure 2), since *atg2* and *atg5* mutants have increased levels of SA, as well as SA-induced senescence [66].

Over the next few years, crosstalk between sugar metabolism and autophagy will likely become clearer. In this regard, trehalose-6-phosphate phosphatase (TPP) seems to be a good candidate linking these processes. The induction and activation of TPP has been correlated with heat stress tolerance in seedlings and developmental progression in bud burst [104,105]. Since TPP activity reduces the levels of T6P, we propose that its role in promoting growth can be related to the attenuation of the T6P-mediated inhibition of autophagy (Figure 2). Additionally, the trehalose produced can have positive implications since exogenous treatments with trehalose were shown to reduce membrane damage and induce tolerance to heat stress [104] and also its catabolism via trehalase was shown to have positive implications for drought tolerance [106].

In plants, TOR has also been suggested to be activated by PI3K [107]. Although the direct mechanism for this is not described, the participation of PI3K in autophagy in plants is clear since inhibitors of PI3K activity inhibit autophagy [108]. In mammals, the PI3K complex activates autophagosome formation [109]. Since the inhibition of PI3K reduces the level of ROS, Scherz-Shouval et al. (2007) suggested that ROS production during autophagy is partially dependent on PI3K. Thus, this PI3K-dependent ROS production may be an additional way by which PI3K promotes autophagy in plants.

## Conclusions

A steadily growing body of evidence highlights autophagy as a key contributor to abiotic and biotic stress tolerance in plants. From a nutritional standpoint, sugars concentration correlates negatively with autophagy induction. However, mild abiotic stresses and many biotic stresses lead to sugar accumulation, and it is likely that new links between sugars and autophagy will arise in the coming years. In this respect, extracellular and intracellular sugar signaling pathways may differentially affect autophagy. On the other hand, the accumulation of GABA, Pro, and PA under stress conditions can indirectly promote autophagy by different pathways. These molecules also contribute to the osmotic adjustment that should be coordinated with the autophagic process to avoidance of mega-autophagy. Finally, ET, ABA, and SA are phytohormones with potential positive implications in autophagy, whereas auxins are more likely to have an inhibitory effect. Future advances in understanding autophagy dynamics under various stresses (see Outstanding Questions) may lead researchers to develop novel strategies to improve crop resistance under increasingly adverse environments.

## Acknowledgments

The authors apologize to all researchers whose work could not be cited due to space limitations. SS thanks the Scientific Research-Flanders (FWO, Belgium) for his postdoctoral fellowship. SS is an
349 associate researcher of the National System of Researchers (SNI) of Uruguay. ŁPT and WVdE are
350 supported by FWO grant no. G0A4915N. DCB is supported by grant no. 1R01GM120316-01A1 from
351 the National Institutes of Health.

352

353 References

354 1 Tilman, D. et al. (2011) Global food demand and the sustainable intensification of agriculture. *Proc. Natl. Acad. Sci.* 108, 20260–20264

356 2 Bassham, D.C. (2007) Plant autophagy - more than a starvation response. *Curr. Opin. Plant Biol.* 10, 587–593

358 3 Michaeli, S. et al. (2016) Autophagy in Plants - What's New on the Menu? *Trends Plant Sci.* 21, 134–144

360 4 Soto-Burgos, J. et al. (2018) Dynamics of Autophagosome Formation. *Plant Physiol.* 176, 219–229

362 5 Zhuang, X. et al. (2018) Autophagosome Biogenesis and the Endoplasmic Reticulum: A Plant Perspective. *Trends Plant Sci.* 23, 677–692

364 6 Mizushima, N. (2007) Autophagy: Process and function. *Genes Dev.* 21, 2861–2873

365 7 Minina, E.A. et al. (2018) Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness. *J. Exp. Bot.* 69, 1415–1432

367 8 Soto-Burgos, J. and Bassham, D.C. (2017) SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana. *PLoS One* 12, e0182591

369 9 Pu, Y. et al. (2017) TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana. *Front. Plant Sci.* 8, 1204

371 10 Avin-Wittenberg, T. (2018) Autophagy and its Role in Plant Abiotic Stress Management. *Plant. Cell Environ.* DOI: 10.1111/pce.13404

373 11 Puijalon, S. et al. (2011) Plant resistance to mechanical stress: Evidence of an avoidance-tolerance trade-off. *New Phytol.* 191, 1141–1149

375 12 Krasensky, J. and Jonak, C. (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. *J Exp Bot* 63, 1593–1608

377 13 Sablowski, R. and Carnier Dornelas, M. (2014) Interplay between cell growth and cell cycle in plants. *J. Exp. Bot.* 65, 2703–2714

379 14 Dobrenel, T. et al. (2013) Sugar metabolism and the plant target of rapamycin kinase: a sweet operaTOR? *Front. Plant Sci.* 4, 1–6

381 15 Hulsmans, S. et al. (2016) The SnRK1 Energy Sensor in Plant Biotic Interactions. *Trends Plant Sci.* 21, 648–661

383 16 Tarkowski, Ł.P. and Van den Ende, W. (2015) Cold tolerance triggered by soluble sugars: a
384 multifaceted countermeasure. *Front. Plant Sci.* 6, 203

385 17 Pommerrenig, B. *et al.* (2018) In concert: orchestrated changes in carbohydrate homeostasis are critical for plant abiotic stress tolerance. *Plant Cell Physiol* 59, 1290–1299

387 18 Sami, F. *et al.* (2016) Role of sugars under abiotic stress. *Plant Physiol. Biochem.* 109, 54–61

388 19 Van den Ende, W. (2013) Multifunctional fructans and raffinose family oligosaccharides. *Front. Plant Sci.* 4, 247

390 20 Yáñez, A. *et al.* (2017) Stem carbohydrate dynamics and expression of genes involved in fructan accumulation and remobilization during grain growth in wheat (Triticum aestivum L.) genotypes with contrasting tolerance to water stress. *PLoS One* 12, e0177667

393 21 Mekonnen, D.W. *et al.* (2016) Gamma-aminobutyric acid depletion affects stomata closure and drought tolerance of Arabidopsis thaliana. *Plant Sci.* 245, 25–34

395 22 Bown, A.W. and Shelp, B.J. (2016) Plant GABA: Not Just a Metabolite. *Trends Plant Sci.* 21, 811–813

397 23 Szabados, L. and Savouré, A. (2010) Proline: a multifunctional amino acid. *Trends Plant Sci.* 15, 89–97

399 24 Signorelli, S. (2016) The fermentation analogy: A point of view for understanding the intriguing role of proline accumulation in stressed plants. *Front. Plant Sci.* 7, 1339

400 25 Signorelli, S. *et al.* (2016) In vivo and in vitro approaches demonstrate proline is not directly involved in the protection against superoxide, nitric oxide, nitrogen dioxide and peroxynitrite. *Funct. Plant Biol.* 43, 870–879

404 26 Signorelli, S. *et al.* (2013) Proline does not quench singlet oxygen: Evidence to reconsider its protective role in plants. *Plant Physiol. Biochem.* 64, 80–83

406 27 Smirnoff, N. and Cumbes, Q.J. (1989) Hydroxyl radical scavenging activity of compatible solutes. *Phytochemistry* 28, 1057–1060

408 28 Signorelli, S. *et al.* (2014) Molecular mechanisms for the reaction between ·OH radicals and proline: insights on the role as reactive oxygen species scavenger in plant stress. *J. Phys. Chem. B* 118, 37–47

411 29 Chen, T.H.H. and Murata, N. (2011) Glycinebetaine protects plants against abiotic stress: Mechanisms and biotechnological applications. *Plant, Cell Environ.* 34, 1–20

413 30 Zarza, X. *et al.* (2017) Polyamine oxidase 5 loss-of-function mutations in Arabidopsis thaliana trigger metabolic and transcriptional reprogramming and promote salt stress tolerance. *Plant Cell Environ.* 40, 527–542

416 31 Pál, M. *et al.* (2015) Speculation: Polyamines are important in abiotic stress signaling. *Plant Sci.* 237, 16–23

418 32 Rojas, C.M. *et al.* (2014) Regulation of primary plant metabolism during plant-pathogen
419 interactions and its contribution to plant defense. *Front. Plant Sci.* 5, 1–12

420 33 Aliferis, K.A. et al. (2014) A Metabolic Profiling Strategy for the Dissection of Plant

421 Defense against Fungal Pathogens. *PLoS One* 9, e111930

422 34 Fernandez-Calvino, L. et al. (2014) Virus-Induced Alterations in Primary Metabolism

423 Modulate Susceptibility to Tobacco rattle virus in Arabidopsis. *Plant Physiol* 166, 1821–1838

425 35 Seifi, H.S. et al. (2013) Concurrent overactivation of the cytosolic glutamine synthetase and the GABA shunt in the ABA-deficient sitiens mutant of tomato leads to resistance against Botrytis cinerea. *New Phytol.* 199, 490–504

428 36 Rudd, J.J. et al. (2015) Transcriptome and Metabolite Profiling of the Infection Cycle of Zymoseptoria tritici on Wheat Reveals a Biphasic Interaction with Plant Immunity Involving Differential Pathogen Chromosomal Contributions and a Variation on the Hemibiotrophic Lifest. *Plant Physiol* 167, 1158–1185

432 37 Appel, H.M. et al. (2014) Transcriptional responses of Arabidopsis thaliana to chewing and sucking insect herbivores. *Front. Plant Sci.* 5, 565

434 38 Zhou, S. et al. (2015) Alteration of plant primary metabolism in response to insect herbivory. *Plant Physiol.* 169, 1488–1498

436 39 Eichmann, R. and Schäfer, P. Growth versus immunity-a redirection of the cell cycle?, *Curr Opin Plant Biol*, 26. (2015), 106–112

438 40 Liu, Y. et al. (2009) Autophagy is required for tolerance of drought and salt stress in plants. *Autophagy* 5, 954–963

440 41 Xiong, Y. et al. (2007) Degradation of Oxidized Proteins by Autophagy during Oxidative Stress in Arabidopsis. *Plant Physiol.* 143, 291–299

442 42 Galletti, R. et al. (2008) The AtrbohD-Mediated Oxidative Burst Elicited by Oligogalacturonides in Arabidopsis Is Dispensable for the Activation of Defense Responses Effective against Botrytis cinerea. *Plant Physiol.* 148, 1695–1706

445 43 van Aubel, G. et al. (2016) Plant immunity induced by COS-OGA elicitor is a cumulative process that involves salicylic acid. *Plant Sci.* 247, 60–70

447 44 Yang, X. et al. (2016) Activation of autophagy by unfolded proteins during endoplasmic reticulum stress. *Plant J.* 85, 83–95

449 45 Wurzinger, B. et al. (2017) Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals. *FEBS Lett.* 591, 3625–3636

451 46 Mazure, N. and Pouysségur, J. (2010) Hypoxia-induced autophagy: cell death or cell survival? *Curr. Opin. Cell Biol.* 22, 177–180

453 47 Chen, L. et al. (2017) The AMP-Activated Protein Kinase KIN10 Is Involved in the
454 Regulation of Autophagy in Arabidopsis. *Front. Plant Sci.* 8, 1201

455 48 Chen, L. *et al.* (2015) Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana. *Autophagy* 11, 2233–2246

457 49 Deretic, V. *et al.* (2013) Autophagy in infection, inflammation, and immunity. *Nat Rev Immunol.* 13, 722–737

459 50 Leary, A.Y. *et al.* (2018) Modulation of plant autophagy during pathogen attack. *J. Exp. Bot.* 69, 1325–1333

461 51 Jones, J.D. and Dangl, J.L. (2006) The plant immune system. *Nature* 444, 323–329

462 52 Coll, N.S. *et al.* (2011) Programmed cell death in the plant immune system. *Cell Death Differ.* 18, 1247–56

464 53 Lorang, J. *et al.* (2012) Tricking the guard: Exploiting plant defense for disease susceptibility. *Science* (80-.). 338, 659–662

466 54 Patel, S. and Dinesh-Kumar, S.P. (2008) Arabidopsis ATG6 is required to limit the pathogen-associated cell death response. *Autophagy* 4, 20–27

468 55 Hofius, D. *et al.* (2009) Autophagic Components Contribute to Hypersensitive Cell Death in Arabidopsis. *Cell* 137, 773–783

470 56 Coll, N.S. *et al.* (2014) The plant metacaspase AtMC1 in pathogen-triggered programmed cell death and aging: Functional linkage with autophagy. *Cell Death Differ.* 21, 1399–1408

472 57 Lenz, H.D. *et al.* (2011) ATG7 contributes to plant basal immunity towards fungal infection. *Plant Signal. Behav.* 6, 122–124

474 58 Kabbage, M. *et al.* (2013) Cell Death Control: The Interplay of Apoptosis and Autophagy in the Pathogenicity of Sclerotinia sclerotiorum. *PLoS Pathog.* 9, e1003287

476 59 Wang, Y. *et al.* (2011) The autophagy gene, ATG18a, plays a negative role in powdery mildew resistance and mildew-induced cell death in Arabidopsis. *Plant Signal. Behav.* 6, 1408–1410

479 60 Han, S. *et al.* (2015) Cytoplasmic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in *Nicotiana benthamiana*. *Plant Cell* 27, 1316–1331

482 61 Zhou, S. *et al.* (2018) Autophagy contributes to regulate the ROS levels and PCD progress in TMV-infected tomatoes. *Plant Sci.* 269, 12–19

484 62 Li, Y. *et al.* (2016) Aspartyl protease mediated cleavage of AtBAG6 is necessary for autophagy and fungal resistance in plants. *Plant Cell* 28, 233–247

486 63 Popa, C. *et al.* (2016) The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway. *Sci. Rep.* 6, 27058

488 64 Dagdas, Y.F. *et al.* (2016) An effector of the Irish potato famine pathogen antagonizes a host
489 autophagy cargo receptor. *Elife* 5, e10856
490 65 Minibayeva, F. *et al.* (2012) Oxidative stress-induced autophagy in plants: The role of mitochondria. *Plant Physiol. Biochem.* 59, 11–19
491
492 66 Yoshimoto, K. *et al.* (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, 2914–27
493
494
495 67 Xiong, Y. *et al.* (2007) Disruption of autophagy results in constitutive oxidative stress in Arabidopsis. *Autophagy* 3, 257–258
496
497 68 Izumi, M. *et al.* (2017) Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy. *Plant Cell* 29, 377–394
498
499 69 Vivancos, P.D. *et al.* (2010) Recruitment of glutathione into the nucleus during cell proliferation adjusts whole-cell redox homeostasis in Arabidopsis thaliana and lowers the oxidative defence shield. *Plant J.* 64, 825–838
500
501
502 70 Velappan, Y. *et al.* (2017) Cell cycle arrest in plants: What distinguishes quiescence, dormancy and differentiated G1? *Ann. Bot.* 120, 495–509
503
504 71 Hayward, A.P. *et al.* (2009) Autophagy and plant innate immunity: Defense through degradation. *Semin. Cell Dev. Biol.* 20, 1041–1047
505
506 72 Scherz-Shouval, R. *et al.* (2007) Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. *EMBO J.* 26, 1749–1760
507
508 73 Frudd, K. *et al.* (2018) Oxidation of Atg3 and Atg7 mediates inhibition of autophagy. *Nat. Commun.* 9, 95
509
510 74 Pérez-Pérez, M.E. *et al.* (2016) Control of Autophagy in Chlamydomonas Is Mediated through Redox-Dependent Inactivation of the ATG4 Protease. *Plant Physiol.* 172, 2219–2234
511
512
513 75 Perez-Perez, M.E. *et al.* (2012) Reactive Oxygen Species and Autophagy in Plants and Algae. *Plant Physiol* 160, 156–164
514
515 76 Heredia-Martínez, L.G. *et al.* (2018) Chloroplast damage induced by the inhibition of fatty acid synthesis triggers autophagy in Chlamydomonas. *Plant Physiol.* DOI: 10.1104/pp.18.00630
516
517
518 77 Jiménez-Nopala, G. *et al.* (2018) Autophagy mediates hydrotropic response in Arabidopsis thaliana roots. *Plant Sci.* 272, 1–13
519
520 78 Smirnoff, N. and Arnaud, D. (2018) Hydrogen peroxide metabolism and functions in plants. *New Phytol.* In press,
521
522 79 Scherz-Shouval, R. and Elazar, Z. (2011) *Regulation of autophagy by ROS: Physiology and pathology*, 36
523
524

80

Oka, S.I. et al. (2017) Thioredoxin-1 maintains mechanistic target of rapamycin (mTOR) function during oxidative stress in cardiomyocytes. *J. Biol. Chem.* 292, 18988–19000

525

81

Pel, Z.M. et al. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. *Nature* 406, 731–734

526

528

82

Del Río, L.A. (2015) ROS and RNS in plant physiology: An overview. *J. Exp. Bot.* 66, 2827–2837

529

83

Zhan, N. et al. (2018) S-Nitrosylation Targets GSNO Reductase for Selective Autophagy during Hypoxia Responses in Plants. *Mol. Cell* 71, 142–154

530

531

84

Lindermayr, C. (2017) Crosstalk between reactive oxygen species and nitric oxide in plants: Key role of S-nitrosoglutathione reductase. *Free Radic. Biol. Med.* 122, 110–115

532

534

85

Signorelli, S. et al. (2013) Water stress induces a differential and spatially distributed nitro-oxidative stress response in roots and leaves of Lotus japonicus. *Plant Sci.* 201–202, 137–146

535

536

86

da-Silva, C.J. et al. (2017) NO, hydrogen sulfide does not come first during tomato response to high salinity. *Nitric Oxide - Biol. Chem.* 76, 164–173

537

538

87

Laureano-Marín, A.M. et al. (2016) Negative regulation of autophagy by sulfide in Arabidopsis thaliana is independent of reactive oxygen species. *Plant Physiol.* 171, 1378–1391

539

540

541

88

Urano, K. et al. (2003) Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages. *Plant, Cell Environ.* 26, 1917–1926

542

543

544

89

Chen, J. et al. (2016) Hydrogen Sulfide-Mediated Polyamines and Sugar Changes Are Involved in Hydrogen Sulfide-Induced Drought Tolerance in Spinacia oleracea Seedlings. *Front. Plant Sci.* 7, 1173

545

546

547

90

Cona, A. et al. (2006) Functions of amine oxidases in plant development and defence. *Trends Plant Sci.* 11, 80–88

548

549

91

Signorelli, S. et al. (2015) Connecting proline and γ-aminobutyric acid in stressed plants through non-enzymatic reactions. *PLoS One* 10, e0115349

550

551

92

Li, S. et al. (2018) Mitogen-activated protein kinases and calcium-dependent protein kinases are involved in wounding-induced ethylene biosynthesis in Arabidopsis thaliana. *Plant. Cell Environ.* 41, 134–147

552

553

554

93

Jiang, C. et al. (2013) An Arabidopsis Soil-Salinity-Tolerance Mutation Confers Ethylene-Mediated Enhancement of Sodium/Potassium Homeostasis. *Plant Cell* 25, 3535–3552

555

556

94

Yamauchi, T. et al. (2014) Ethylene and reactive oxygen species are involved in root aerenchyma formation and adaptation of wheat seedlings to oxygen-deficient conditions. *J.*

557

558
<table>
  <tr>
    <td>559</td>
    <td>Exp. Bot. 65, 261–273</td>
  </tr>
  <tr>
    <td>560</td>
    <td>95 Mersmann, S. et al. (2010) Ethylene Signaling Regulates Accumulation of the FLS2</td>
  </tr>
  <tr>
    <td>561</td>
    <td>Receptor and Is Required for the Oxidative Burst Contributing to Plant Immunity. Plant</td>
  </tr>
  <tr>
    <td>562</td>
    <td>Physiol. 154, 391–400</td>
  </tr>
  <tr>
    <td>563</td>
    <td>96 Sewelam, N. et al. (2013) Ethylene Response Factor 6 Is a Regulator of Reactive Oxygen</td>
  </tr>
  <tr>
    <td>564</td>
    <td>Species Signaling in Arabidopsis. PLoS One 8, e70289</td>
  </tr>
  <tr>
    <td>565</td>
    <td>97 Wu, L. et al. (2008) Transcriptional Modulation of Ethylene Response Factor Protein JERF3</td>
  </tr>
  <tr>
    <td>566</td>
    <td>in the Oxidative Stress Response Enhances Tolerance of Tobacco Seedlings to Salt, Drought,</td>
  </tr>
  <tr>
    <td>567</td>
    <td>and Freezing. Plant Physiol. 148, 1953–1963</td>
  </tr>
  <tr>
    <td>568</td>
    <td>98 Schellingen, K. et al. (2015) Ethylene signalling is mediating the early cadmium-induced</td>
  </tr>
  <tr>
    <td>569</td>
    <td>oxidative challenge in Arabidopsis thaliana. Plant Sci. 239, 137–146</td>
  </tr>
  <tr>
    <td>570</td>
    <td>99 Müller, M. and Munné-Bosch, S. (2015) Ethylene Response Factors: A Key Regulatory Hub</td>
  </tr>
  <tr>
    <td>571</td>
    <td>in Hormone and Stress Signaling. Plant Physiol. 169, 32–41</td>
  </tr>
  <tr>
    <td>572</td>
    <td>100 Cheng, M.-C. et al. (2013) The Arabidopsis ETHYLENE RESPONSE FACTOR1 Regulates</td>
  </tr>
  <tr>
    <td>573</td>
    <td>Abiotic Stress-Responsive Gene Expression by Binding to Different cis-Acting Elements in</td>
  </tr>
  <tr>
    <td>574</td>
    <td>Response to Different Stress Signals. Plant Physiol 162, 1566–1582</td>
  </tr>
  <tr>
    <td>575</td>
    <td>101 Raghavendra, A.S. et al. (2010) ABA perception and signalling. Trends Plant Sci. 15, 395–</td>
  </tr>
  <tr>
    <td>576</td>
    <td>401</td>
  </tr>
  <tr>
    <td>577</td>
    <td>102 Zhang, J. and Tardieu, F. (1996) Relative contribution of apices and mature tissues to ABA</td>
  </tr>
  <tr>
    <td>578</td>
    <td>synthesis in droughted maize root systems. Plant Cell Physiol. 37, 598–605</td>
  </tr>
  <tr>
    <td>579</td>
    <td>103 Lim, C.W. et al. (2015) Function of ABA in stomatal defense against biotic and drought</td>
  </tr>
  <tr>
    <td>580</td>
    <td>stresses. Int. J. Mol. Sci. 16, 15251–15270</td>
  </tr>
  <tr>
    <td>581</td>
    <td>104 Li, Z.G. et al. (2014) Involvement of trehalose in hydrogen sulfide donor sodium</td>
  </tr>
  <tr>
    <td>582</td>
    <td>hydrosulfide-induced the acquisition of heat tolerance in maize (Zea mays L.) seedlings. Bot.</td>
  </tr>
  <tr>
    <td>583</td>
    <td>Stud. 55, 1–9</td>
  </tr>
  <tr>
    <td>584</td>
    <td>105 Signorelli, S. et al. (2018) Roles for light, energy and oxygen in the fate of quiescent axillary</td>
  </tr>
  <tr>
    <td>585</td>
    <td>buds. Plant Physiol. 176, 1171–1181</td>
  </tr>
  <tr>
    <td>586</td>
    <td>106 Van Houtte, H. et al. (2013) Overexpression of the Trehalase Gene AtTRE1 Leads to</td>
  </tr>
  <tr>
    <td>587</td>
    <td>Increased Drought Stress Tolerance in Arabidopsis and Is Involved in Abscisic Acid-Induced</td>
  </tr>
  <tr>
    <td>588</td>
    <td>Stomatal Closure. Plant Physiol 161, 1158–1171</td>
  </tr>
  <tr>
    <td>589</td>
    <td>107 Bassham, D.C. (2009) Function and regulation of macroautophagy in plants. Biochim.</td>
  </tr>
  <tr>
    <td>590</td>
    <td>Biophys. Acta - Mol. Cell Res. 1793, 1397–1403</td>
  </tr>
  <tr>
    <td>591</td>
    <td>108 Takatsuka, C. et al. (2004) 3-Methyladenine inhibits autophagy in tobacco culture cells under</td>
  </tr>
  <tr>
    <td>592</td>
    <td>sucrose starvation conditions. Plant Cell Physiol. 45, 265–274</td>
  </tr>
  <tr>
    <td>593</td>
    <td>109 Tassa, A. et al. (2003) Class III phosphoinositide 3-kinase–Beclin1 complex mediates the</td>
  </tr>
</table>
594 amino acid-dependent regulation of autophagy in C 2 C 12 myotubes. *Biochem. J* 376, 577–

595 586

596 Miller, G. *et al.* (2009) The plant NADPH oxidase RBOHD mediates rapid systemic

597 signaling in response to diverse stimuli. *Sci. Signal.* 2, ra47

598 Mittler, R. *et al.* (2011) ROS signaling: the new wave? *Trends Plant Sci.* 16, 300–9

599 Evans, M.J. *et al.* (2016) A ROS-Assisted Calcium Wave Dependent on the AtRBOHD

600 NADPH Oxidase and TPC1 Cation Channel Propagates the Systemic Response to Salt

601 Stress. *Plant Physiol.* 171, 1771–1784

602 Desikan, R. *et al.* (2000) Hydrogen peroxide-induced gene expression in Arabidopsis

603 thaliana. *Free Radic. Biol. Med.* 28, 773–778

604 Matros, A. *et al.* (2015) Sugars as hydroxyl radical scavengers: Proof-of-concept by studying

605 the fate of sucralose in Arabidopsis. *Plant J.* 82, 822–839

606 García Mata, C. and Lamattina, L. (2001) Nitric oxide induces stomatal closure and enhances

607 the adaptive plant responses against drought stress. *Plant Physiol.* 126, 1196–204

608 Lombardo, M.C. *et al.* (2006) Nitric oxide functions as a positive regulator of root hair

609 development. *Plant Signal. Behav.* 1, 28–33

610 Bethke, P.C. *et al.* (2006) Nitric oxide reduces seed dormancy in Arabidopsis. *J. Exp. Bot.*

611 57, 517–526

612 Prado, A.M. *et al.* (2004) Nitric oxide is involved in growth regulation and re-orientation of

613 pollen tubes. *Development* 131, 2707–2714

614 Rodríguez-Ruiz, M. *et al.* (2018) Arsenate disrupts ion balance, sulfur and nitric oxide

615 metabolisms in roots and leaves of pea (Pisum sativum L.) plants. *Environ. Exp. Bot.* DOI:

616 10.1016/j.envexpbot.2018.06.028

617 Signorelli, S. *et al.* (2018) Drought stress triggers the accumulation of NO and SNOs in

618 cortical cells of Lotus japonicus L. roots and the nitration of proteins with relevant metabolic

619 function. *Environ. Exp. Bot.*

620 Signorelli, S. and Considine, M.J. (2018) Nitric oxide enables germination by a four-pronged

621 attack on ABA-induced seed dormancy. *Front. Plant Sci.* 9, 296

622 Marshall, R.S. and Vierstra, R.D. (2018) Autophagy: The Master of Bulk and Selective

623 Recycling. *Annu. Rev. Plant Biol* 69, 173–208

624 Büssis, D. and Heineke, D. (1998) Acclimation of potato plants to polyethylene glycol-

625 induced water deficit. II. Contents and subcellular distribution of organic solutes. *J. Exp. Bot.*

626 49, 1361–1370

627 Miyashita, Y. and Good, A.G. (2008) Contribution of the GABA shunt to hypoxia-induced

628 alanine accumulation in roots of Arabidopsis thaliana. *Plant Cell Physiol.* 49, 92–102
629 125 Kathiresan, A. *et al*. (1997) y-Aminobutyric Acid Stimulates Ethylene Biosynthesis in Sunflower. *Plant Physiol.* 115, 129-135

631 126 Shibuya, K. *et al*. (2013) Pollination induces autophagy in petunia petals via ethylene. *J. Exp. Bot.* 64, 1111-1120

633

634
**Figure and Key Figure:**

Figure 1 is the key figure:

**Figure 1.** *Autophagy contributes to cell and plant survival under (a)biotic stress conditions.* In harsh conditions, cellular organelles can be damaged and their dysfunction increases the generation of reactive oxygen species (ROS) and oxidative damage. At the plant level this can lead to plant senescence. Damaged molecules, and even organelles such as mitochondria, chloroplasts and peroxisomes, can be recycled through autophagy. The resulting breakdown products can be used for the *de novo* synthesis of molecules and organelle biogenesis, thus promoting stress tolerance at both cellular and plant levels.

**Figure 2.** *Metabolic crosstalk in plant autophagy.* Green arrows indicate induction or promotion of a process or product. Red T-bars indicates inhibition of a process or molecule. Dashed connectors indicate that the process is suggested but not completely known in plants. ABA, abscisic acid; AUX, auxins; ATG, autophagy related genes; ET, ethylene; DAO, Diamine oxidase; GABA gamma-aminobutyric acid; H₂O₂, hydrogen peroxide; H₂S, hydrogen sulfide; MAPK, mitogen activated protein kinases; ·NO, nitric oxide; PA, polyamines; PAO, polyamine oxidase; Pro, proline; Put, putrescine; RBOH, respiratory burst homologue; ROS, reactive oxygen species; SA, salicylic acid; SnRK1, SNF-related kinase 1; Spd, spermidine; Spm, spermine; T6P, trehalose-6-phosphate; TOR, target of rapamycin; TPP, trehalose-6-phosphate phosphatase.

**Text boxes**

**Box 1:** Reactive oxygen and nitrogen species are generated under biotic and abiotic stress

Stress conditions lead to the overproduction of ROS, including superoxide (O₂⁻), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂) and hydroxyl radical (·OH). Superoxide can be produced either by electron leakage of the mitochondrial and chloroplastic electron transport chains under stress conditions, or by the respiratory burst homologue (Rboh, also known as NADPH oxidase). Stress conditions, such as cold, salinity, heat and high light, were shown to induce the expression of Rboh as a rapid systemic response [110]. Mittler et al. proposed the concept of a ROS wave [111], in which each cell senses the wave of superoxide and responds by expressing its own Rboh to propagate the wave through the apoplast. This wave is suggested to be coordinated with a calcium wave and promoted by phytohormones such as **abscisic acid** (ABA) [112]. In turn, superoxide yields H₂O₂, mainly via SOD activity. Given that H₂O₂ is considerably more stable and biological membranes are not as impermeable to it as for superoxide, it can act as a signaling molecule diffusing between different cell compartments. In fact, hundreds of genes are differentially expressed when plants are exposed to H₂O₂ [113]. It can also lead to the generation of the most reactive ROS, the hydroxyl
radical, in the presence of Fe³⁺ by the so-called Fenton reaction. This radical reacts with biomolecules in a diffusion-limited rate. The accumulated molecules under stress conditions, such as sugars and proline, react with hydroxyl radicals as a strategy to avoid damage to more essential biomolecules [28,91,114]. Singlet oxygen is mainly produced by the chloroplast when an excited chlorophyll (³Chl) reacts with molecular oxygen. High-light stress and stresses affecting the fluidity of thylakoid membranes enhance its production. In this way, damaged chloroplasts are an important source of ROS, and autophagy can contribute to the recycling of these organelles by **chlorophagy**.

Nitric oxide (·NO) is a considerably reactive molecule that can diffuse across membranes and act also as a signaling molecule, attenuating or triggering biological processes such as stomatal closure, germination and root elongation [115–118]. Under stress conditions, ·NO can be overproduced [119,120], reacting with ROS to generate more reactive species such as peroxynitrite (ONOO⁻) and nitrogen dioxide (·NO₂), collectively known as RNS. The regulatory role of ·NO and other RNS is exerted by interfering with critical components in the signaling cascade of phytohormones [121]. In this work, we discuss potential effects of ·NO on plant autophagy.

### Box 2: Coordination of autophagy with osmotic adjustment

The breakdown of more complex structures to monomers in the vacuole during the autophagic process results in increased vacuolar osmolality. Different permeases allow the transport of these recycled compounds back to the cytosol, contributing to the compensation of the osmotic gradient. However, under intense autophagic activity a transient osmotic gradient can be generated, putting the stability of the tonoplast at risk. When the tonoplast is broken, the vacuolar hydrolases are released into the cytoplasm, degrading its content. This process is known as **mega-autophagy**, and usually results in programmed cell death [122]. To avoid this, it is logical that plant cells perform *de novo* synthesis of certain metabolites (such as proline) in the cytosol to balance the increased vacuolar osmolality (**Figure I**). While for most of the recycled compounds, a higher concentration in the vacuole would ensure their transport from the vacuole to the cytosol through the permeases, some other compounds should have an opposite gradient to compensate for the osmolality. For instance, in potato plants under stress conditions proline concentrations were estimated to be 83 mM in the cytosol but 4 mM in the vacuole [123]. In conclusion, any autophagic activity contributing to increased vacuolar osmolality should be coordinated with the accumulation of cytosolic osmo-active compounds (**Figure I**).

**Figure I.** Coordination of autophagy and osmotic adjustment. When macromolecules or organelles are sent to be degraded in the vacuole (dark arrows in the left-hand side of the figure), the osmolality of the vacuole increases as soon as the degradation starts. If the cell responds by accumulating
705 compatible osmolytes in the cytosol, the osmotic pressure is compensated for (a). In the absence of
706 this response, a gradient of osmotic pressure (dark-blue arrows) will be generated between the vacuole
707 and the cytosol, producing expansion pressure on the tonoplast (sky-blue arrows, b). The water flux
708 would result in an increase in vacuolar size (c) or the rupture of the tonoplast (d). In this last scenario,
709 the vacuolar hydrolases are released into the cytosol resulting in mega-autophagy, which in turn
710 endangers the viability of the cell.

711

712 **Box 3: GABA promotes cell survival at different levels**

713 GABA is suggested to play several roles in plant responses to (a)biotic stresses, and also under
714 starvation or hypoxic conditions [124]. Under non-optimal conditions causing nutrient deficiency,
715 the GABA shunt is activated to produce reducing equivalents through the TCA cycle and feed the
716 cell (Figure I, i). Moreover, GABA has been shown to increase ET levels in sunflower cotyledons
717 [125]. Since ET was shown to promote ATG8 expression in plants [126], contributing to autophagy,
718 we hypothesize that GABA may indirectly promote autophagy (Figure I, ii). Finally, under oxidative
719 stress conditions, GABA is able to reduce ROS levels by reacting with hydroxyl radicals and in this
720 way prevent oxidative stress-induced cell death (Figure I, iii). Thus, GABA promotes cell survival
721 at three different levels, (i) feeding the cells via the GABA shunt under starvation conditions, (ii)
722 promoting plant autophagy by increasing ET signaling, and (iii) inhibiting oxidative stress-induced
723 cell death by scavenging hydroxyl radicals (Figure I).

724

725 **Figure I.** *Multiple mechanisms by which GABA promotes cell survival and stress tolerance.* This
726 scheme illustrates the proposed roles of GABA in maintaining cell viability during (a)biotic stress
727 responses. (i) GABA shunts provide energy required for cell survival. (ii) The induction of ethylene
728 (ET) by GABA can induce autophagy, which contributes to recycling damaged molecules and
729 enhances stress tolerance. (iii) GABA can directly scavenge hydroxyl radicals (·OH) and protect
730 against oxidative stress-induced cell death. Green arrows indicate promotion, whereas red T-bars
731 indicate inhibition.

732

733 **Glossary**

734 **Abscisic acid:** A plant hormone with important signaling function in development and stress
735 conditions.

736 **Autophagosome:** A double membrane compartment that delivers cytoplasmic cargo to be recycled
737 into the vacuole.

738 **Chlorophagy:** The selective degradation of damaged chloroplasts via autophagy.
739 Compatible osmolyte: Organic compounds, usually of low molecular weight, that can be
740 accumulated at high concentration in the cells without having toxic effects.

741 Ethylene: A plant hormone with important signaling function in development and stress conditions.

742 Jasmonic acid: A plant hormone with important signaling function in development and stress conditions.

744 Macroautophagy: A conserved catabolic process in which part of the cytoplasm, including
745 organelles and damaged molecules, is transferred to the vacuole for degradation.

746 Mega-autophagy: The massive degradation of the cell contents leading to programmed cell death.

747 Osmotic adjustment: The accumulation of compatible osmolytes to compensate for differing water potentials, within a cell or between the cell and the extracellular environment.

749 Priming: To induce in a plant a physiological process that prepares it for a faster and/or stronger response in case of a future stress condition.

751 Reactive oxygen species (ROS): A group of molecules derived from oxygen that are highly reactive and can oxidize other biomolecules.

753 Reactive nitrogen species (RNS): A group of small molecules containing nitrogen and oxygen which are highly reactive and can oxidize other biomolecules.

755 ROS/RNS scavengers: Molecules capable of reacting chemically with ROS/RNS, attenuating their reactivity, at relatively high frequency. This high frequency can be achieved either by high reaction rate constants or by high concentration of the molecules.

758 Salicylic acid: A plant hormone with important signaling function in development and stress conditions.

760 Systemic response: A generalized response that includes the whole plant and not only the site of stress or infection.

762

763
**Outstanding questions:**

* How can autophagy contribute to the accumulation of ERF1 under flooding conditions? In turn, do ERFs play a role during induction and establishment of autophagy?

* Are endogenously produced ROS able to oxidize TOR and promote autophagy in plants?

* Do developmental and spatio-temporal effects control the way SnRK1 is regulated by sugars? How do these differ in sink and source tissues, and does this depend on oxidizing and reducing conditions?

* What is the link between the autophagosome and H₂O₂ accumulation in the hydrotropic response of Arabidopsis roots under water stress?

* How does PI3K promote autophagy in plants?

* What are the mechanisms by which H₂S inhibits autophagy?

* Are endogenously produced RNS able to affect autophagy in plants; if so, is it due to protein nitration or nitrosylation, and which proteins are targeted by these posttranslational modifications?

* Is the autophagic response affected in plants with impaired accumulation of osmolytes? For instance, are the *p5cs1* Arabidopsis mutants (unable to accumulate proline) predisposed to activation of mega-autophagy?

* Is targeting of specific components of the autophagic machinery a diffused strategy in plant-pathogen interactions?
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(A)biotic stresses

Drought, Oxidative stress,
Pathogen attack, Wounding,
Flooding, etc.

Autophagy

Stress tolerance
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