Participation of autophagy in the response of plants to the action of abiotic stressors

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Participation of autophagy in the response of plants to the action of abiotic stressors

Zh.Z. Guralchuk,

D.S. Gurianov



Annotation

The data on the process of plant autophagy, its importance for the functioning of plants under optimal and stressful conditions are reviewed. Autophagy, a highly intricate and conserved process, remains at a basal level under optimal conditions, and is induced when plants fall under stress. This process stands as one of the central mechanisms enabling plants to adapt to adverse environmental factors.

The activation of autophagy in response to various abiotic stressors, encompassing extreme temperatures, salinity, drought, nutrient deprivation, and herbicides is discussed. The regulatory mechanisms of autophagy are thoroughly considered, encompassing post-translational protein modifications crucial for its activation and progression, transcriptional regulation, and signaling through phytohormones.

The pivotal role of autophagy activation is underscored in eliminating damaged organelles, and providing vital nutrients required for plant functionality and survival when exposed to abiotic stressors. Furthermore, the potential involvement of autophagy in herbicide-induced pathogenesis is discussed, with a dual perspective: its potential role in regulating programmed cell death (PCD), and its role in mitigating herbicide effects through the plant's homeostatic resistance mechanisms.

Key words: autophagy, abiotic factors, herbicides.



Анотація

Ж.З. Гуральчук, Д.С. Гур'янов. Участь аутофагії у реакції рослин на дію абіотичних стресорів

Розглядаються процеси, пов'язані з аутофагією рослин, її значення для функціонування рослин за оптимальних і стресових умов. Аутофагія є складним висококонсервативним процесом, активність якого знаходиться на базовому рівні за оптимальних умов та індукується за стресових. Вона є одним із ключових процесів адаптації рослин до дії несприятливих чинників середовища.

Описується активація аутофагії у рослин у відповідь на дію різних абіотичних чинників (високих і низьких температур, засолення, посухи, нестачі поживних речовин, гербіцидів). Обговорюються механізми регуляції аутофагії, пов'язані з посттрансляційними модифікаціями білків, необхідних для активації та прогресування аутофагії, регуляцією транскрипції та фітогормональною сигналізацією. Підкреслюється значення активації аутофагії для видалення пошкоджених органел і реутилізації поживних речовин, необхідних для функціонування і виживання рослин за дії абіотичних чинників.

Припускається можлива участь аутофагії в індукованому гербіцидами патогенезі. Зазначається, що роль аутофагії в індукованому гербіцидами патогенезі, з одного боку, може бути пов'язана з її участю у регуляції програмованої загибелі клітин (ПЗК), з іншого - з послабленням їх дії, зумовленим гомеостатичною резистентністю рослин.

Ключові слова: аутофагія, абіотичні чинники, гербіциди.



Introduction

Autophagy is a highly conserved process among eukaryotes. Under optimal conditions, it functions at a low basal level, and is essential for maintaining metabolic homeostasis. However, its activation is frequently observed in response to changes in environmental conditions. The triggers for such activation can encompass a range of stressful factors [1]. Autophagy ensures the degradation and recycling of various types of cellular components [1]. This process plays a crucial role in the survival of plants exposed to abiotic stressors. Autophagy is significantly important in plant metabolism and energy balance, and closely linked to maintaining stable plant productivity, especially in suboptimal conditions [2].

The term «autophagy» was initially introduced in 1963 by Christian de Duve to describe the process of degrading cytoplasmic components within the lysosome/vacuole [3]. A pivotal moment in autophagy research was when

Yoshinori Ohsumi identified the first 15 genes related to autophagy, denoted as ATG (autophagy-related genes), through genetic screening using yeast [3-5]. Currently, more than 40 ATG genes have been identified in yeast, with the majority of them having close homologues in plants, from algae to vascular plants [6, 7]. Notably, Arabidopsis thaliana has approximately 40 ATGs [8] (some sources mention 47 [9]), rice (Oryza sativa L.) - 33 [10], and tea (Camellia sinensis (L.) O. Kuntze) - near 80 [11].

It's worth mentioning that, unlike yeast, many ATG genes in plants are organized into small families. It is assumed that genes within these families are induced differently in response to stressful conditions and developmental stages, allowing for finer regulation of autophagy activity [12]. In contrast to the ubiquitin-proteasome system, which involves nearly 1300 genes, constituting about 5 % of the Arabidopsis proteome [13], autophagy can target and degrade nearly all substrates or aggregates within cells, including proteins and their aggregates, redundant biomolecules, damaged organelles, and certain invasive microorganisms [14, 15].

There are three types of autophagy in plants: microautophagy, macroautophagy, and megaautophagy [16]. In microautophagy, cytoplasmic material is gathered on the surface of the vacuole, and enters it through the invagination of the tonoplast. Eventually, the tonoplast breaks down to release autophagic bodies, which are intravacuolar vesicles containing cytoplasmic material. In macroautophagy, the predominant and most studied form of autophagy in plants [17], biomaterial is insulated by cytoplasmic vesicles formed de novo. These vesicles originate from the expansion of the cup-shaped phagophore (or isolation membrane) surrounding the cytoplasm, which ultimately condenses to form an autophagosome surrounded by a double membrane [2]. The outer membrane of the autophagosome then merges with the tonoplast to release the inner vesicle as an autophagic body into the vacuole's lumen. In both microand macroautophagy, disruption of the autophagic cell membrane exposes its content to vacuolar hydrolases, which cleave this content into its constituent parts, subsequently transported back to the cytoplasm for reuse [18].

In megaautophagy, the tonoplast ruptures or becomes permeable, leading to the release of vacuolar hydrolases directly into the cytoplasm, where they degrade the cytoplasmic material in situ [16]. Megaautophagy often represents the final stage of programmed cell death during development (e.g., during xylogenesis) or in response to pathogen invasion (e.g., during the hypersensitivity reaction) [2].

Autophagy, hereafter referred to as macroautophagy, is a complex, stepwise, and dynamic process involving highly conserved ATG genes responsible for the initiation and formation of autophagosomes [19, 20]. ATG proteins can be categorized into four functional groups [20]:

1) The ATG1/ATG13 kinase complex, which triggers the formation of a phagophore in response to the phosphorylation status of ATG13;

2) The phosphatidylinositol-3-kinase complex (PI3K), responsible for incorporating phospholipids into the expanding phagophore;

3) The ATG2- ATG9-ATG18 complex, which plays a role in expanding the phagophore membrane and advancing the autophagosome;

4) The ATG8 and ATG12 conjugation systems, which catalyze the covalent binding of the ATG8 protein to phosphatidylethanolamine (PE) and are crucial for the expansion and maturation of the phagophore [21-27].

ATG8 plays a central role in plant autophagy. In Arabidopsis thaliana, nine homologues of ATG8 (ATG8a-ATG8i) have been described, each with different expression patterns and functions [19, 28]. In maise, there are five ATG8 homologues [29], while rice has six [10], wheat has 14, and rapeseed (Brassica napus L.) has 22 [30].

ATG8-PE conjugates, found on both the outer and inner membranes of the autophagosome, contribute to phagophore expansion and vesicle closure during autophagosome formation. They also play a key role in recognizing autophagic materials, facilitated by adapter proteins and receptors interacting with ATG8 [6, 17, 31].

While autophagy was traditionally viewed as a process for the extensive degradation of cytoplasmic material [32], recent studies have revealed its capacity for selectivity, enabling quality control over specific organelles. These include chlorophagy (chloroplasts) [33], mitophagy (mitochondria) [22, 34], reticulophagy (endoplasmic reticulum) [35-38], pexophagy (peroxisomes) [39-41], ribophagy (ribosomes) [42-44], proteaphagy (proteasomes) [45, 46], as well as the clearance of protein aggregates [47-49]. Autophagy can be triggered both in response to various abiotic stress factors and for nutrient remobilization [1, 2, 50-52], as well as for defense against pathogens [53-56].

The selectivity of autophagy is adjusted by the interaction between ATG8 and specific autophagic receptors that contain ATG8-interacting motifs (AIM) or ubiquitin-interacting motifs (UIM) [17, 45, 57, 58]. These autophagic receptors mediate the selective autophagy of cell organelles, protein aggregates, and even certain pathogens [2].



The influence of abiotic factors on plant autophagy

Autophagy is induced by specific environmental factors to maintain a balance between plant growth and stress resistance [59]. It plays a crucial role in plant responses to various abiotic factors such as high and low temperatures, nutrient deficiency, salinity, drought, ultraviolet radiation, and more.

For instance, autophagy can be activated in response to elevated temperatures. Numerous studies have demonstrated the accumulation of autophagosomes in Arabidopsis and tomato plants subjected to heat stress [47, 48, 60]. Notably, Arabidopsis mutants lacking key autophagy-related genes, atg5 and atg7, displayed increased sensitivity to heat stress, manifested by heightened wilting, elevated electrolyte leakage, and reduced photosynthesis, in comparison to wild-type plants. Furthermore, atg7 mutant plants exhibited the accumulation of insoluble protein aggregates labeled with ubiquitin [47]. Given that heat stress leads to protein misfolding and denaturation, it is hypothesized that it induces autophagy by triggering endoplasmic reticulum (ER) stress, which results in the accumulation of unfolded proteins within the ER, forming protein aggregates [36, 60]. These aggregates can be degraded by autophagy [48, 49].

It's worth noting that, under heat stress conditions in pepper plants (Capsicum annuum L.), the number of regulated CaATG genes, and the increase in autophagosomal punctate were more pronounced in the heat-resistant line R9 compared to the heat-sensitive B6. This observation suggests a strong link between autophagy and tolerance to high temperatures [61].

Following heat stress, plants undergo a thermal recovery phase, during which various processes enable them to return to normal growth and development. It has been observed, that autophagy is induced by thermopriming (moderate heat stress), and remains elevated long after the stressor is removed [62]. Throughout the thermal recovery phase, in the absence of persistent heat stress, the numbers of autophagosomes and autophagic bodies progressively increase in Arabidopsis thaliana. This process has been demonstrated to aid in the degradation of HSP during the late stages of the thermal recovery phase, contributing to the resetting of cellular memory regarding heat stress [62].

Recent findings [63] indicate an unexpected role for the ATG8 protein in restoring the vacuolated Golgi apparatus after short-term acute heat stress. ATG8 has been shown to translocate to the extended Golgi apparatus membrane and participate in the recruitment of clathrin light chain 2 (CLC2) to the cisternal membrane, which, in turn, facilitates the reassembly of the Golgi apparatus.

Low temperatures have been shown to increase the expression of CaATGId and induce the formation of autophagosomes in pepper plants [61]. Similarly, alfalfa (Medicago sativa L.) demonstrates an elevation in MsATGI3 expression in response to cold stress [64]. In tobacco plants overexpressing MsATGI3, exposure to low temperatures resulted in a larger number of autophagosomes compared to wild-type plants [64]. This increase in autophagy is attributed to the activation of other ATGs necessary for autophagosome formation. Additionally, in transgenic tobacco plants with excessive expression of MsATGI3, the levels of proline content and the activity of antioxidant enzymes increased in response to cold stress, while the levels of reactive oxygen species (ROS) decreased.

As previously mentioned, gene homologues within families can exhibit varying expression patterns under the influence of different stress factors. For example, in rice, three ATG6 homologues display distinct responses to high, low temperatures, and drought. All three stress factors increase the expression of OsATG6c, while heat stress decreases the expression of OsATG6a, and cold stress reduces the expression of OsATG6b [65]. In contrast, drought leads to increased expression levels of all three homologues (OsATG6a-OsATG6c) in rice. Similarly, in Tatar buckwheat (Fagopyrum tataricum), all FtATG8 homologues are inducibly expressed during drought, low temperature, and salt stress, exhibiting different expression patterns [9]. Among them, FtATG8a expression increases under drought conditions, while FtATG8e significantly decreases.

Changes in the expression levels of CaATG genes in response to various abiotic factors (high and low temperatures, drought, salt stress) have also been demonstrated in pepper plants [61]. This diversity of expression patterns may suggest multiple functions of homologous ATG genes, which could be linked to the variety of motifs present in ATG proteins [28].

Autophagy plays a pivotal role in promoting plant survival by maintaining cellular homeostasis under drought conditions [66]. Evidence demonstrates that Arabidopsis atg5 and atg7 mutants, as well as RNAiATG18a plants where autophagy is blocked, exhibit heightened sensitivity to drought [47, 67]. Reduced expression of ATG8d and ATG18h in tomato (Solanum lycopersicum), and ATG6 in wheat (Triticum aestivum), also increases their vulnerability to drought [68, 69]. Conversely, the overexpression of ATG18a in apple (Malus domestica) leads to increased autophagy, and enhanced drought resistance [70].

In apple plants, overexpressing MdATG8i under drought conditions, several positive changes were observed. These include a reduction in ROS levels compared to wild-type plants, an increase in the activity of antioxidant enzymes, a decrease in the accumulation of insoluble or oxidized proteins, and higher amounts of amino acids and flavonoids. Additionally, root water absorption improved, resulting in higher water use efficiency, contributing to increased drought resistance [71, 72]. Drought also triggers the activation of autophagy in tomatoes and wheat, leading to increased expression of ATG genes and enhanced formation of autophagosomes [68, 69, 73].

In peach (Prunus persicae L.), of the 23 autophagy-related genes identified, 20 responded to drought stress. Under the influence of drought, peach leaves exhibited suppressed photosynthetic processes and increased antioxidant enzyme activity. Autophagy and ATG expression were notably enhanced, particularly PpATG8 and some PpATG18 genes. Interestingly, the activity of antioxidant enzymes displayed an inverse correlation with the expression levels of PpATG [74].

Medicago truncatula possesses 39 autophagy-related genes (ATG), with most of them being strongly induced during seed development and in response to drought, highlighting the essential role of autophagy in both seed development and the plant's response to drought stress [75].

Autophagy is involved in the degradation of aquaporins belonging to the PIP subfamily (plasma membrane intrinsic protein) during drought conditions, contributing to a reduction in water loss [66]. Using Arabidopsis plants as an example, it has been demonstrated that PIP 2;7 is degraded through autophagy, and this process is mediated by the selective autophagy receptor - tryptophan-rich sensory protein (TSPO). When autophagy is blocked using inhibitors or through the use of the atg5 mutant, both TSPO and PIP 2;7 stabilization occurs [76].

In Medicago truncatula, the autophagic degradation of aquaporin PIP 2;7 follows a slightly different pathway. It involves the participation of dehydrin CAS31 (cold acclimation-specific 31), acting as a receptor for the selective autophagic degradation of PIP 2;7 during drought [77].

Recently, a plant-unique protein known as COST 1 (constitutively stressed 1) has been identified. This protein is essential for plant growth but can exert a negative regulatory influence on their resistance to drought through its direct interaction with the autophagy receptor protein ATG8e [78]. In Arabidopsis thaliana plants with overexpressed COST 1, autophagy is suppressed, rendering them hypersensitive to drought. Conversely, the cost 1 mutant displays reduced growth but a heightened level of drought resistance [79].

It has been demonstrated that both MG132, an inhibitor of the 26S proteasome pathway, and concanamycin A, an inhibitor of the autophagymediated vacuolar degradation pathway, can hinder the degradation of COST 1 during dehydration [79]. This observation suggests the involvement of both pathways in the efficient removal of COST 1, which eliminates its inhibition of autophagy and, in turn, enhances drought tolerance [78].

Under conditions of salt stress, the expression of MdATG10 in apple plants, especially in the roots, is induced [80]. Transgenic apple plants overexpressing MdATG10 demonstrated increased salt tolerance, characterized by less impaired photosynthesis, improved growth, reduced Na+ accumulation, and a lower Na+/K+ ratio compared to wild-type plants. Transgenic apple plants with overexpression of MdATG8i also exhibited an amelioration of negative effects caused by salt stress [81]. This improvement was accompanied by increased autophagic activity and the accumulation of proline, arginine, and polyamines, which play a crucial role in enhancing plant resistance to salt stress.

In Arabidopsis, salinity induces autophagy, with its peak occurring within 30 minutes of salt stress initiation [82]. Autophagy-defective mutants do not display such induction. During a 3-hour salt treatment, the accumulation of oxidized proteins decreases in the wild type; however, this reduction is not observed in autophagy-defective atg2 or atg7 mutants. Moreover, it has been demonstrated that autophagy is essential for sequestering Na+ in the central vacuole of root cortex cells under salinity conditions [82].

Autophagy is enhanced under conditions of limited nutrient supply, as it serves to remobilize nutrients in plants and provide cellular material. Depletion of nitrogen, inorganic phosphate (Pi), and limited sugar reserves are known triggers for autophagy in cultured cells, as demonstrated in the case of transformed BY-2 tobacco cells [83-86]. This indicates that such a response occurs at the cellular level [84]. In nutrient-deficient conditions, including Pi deficiency, the induction of autophagy in cultured BY-2 tobacco cells takes approximately 12 hours or even less, whereas intact plants require a significantly longer duration to initiate such a response [85]. Autophagy plays an important role in nitrogen transport and its assimilation. Overexpression of ATG6 promoted autophagy and biomass accumulation in tomato plants under low nitrogen content in the nutrient medium [52]. At the same time, the nitrogen content in atg6 mutant plants, the activity of nitrate reductase and nitrite reductase decreased, while their increase was observed in lines with overexpression of ATG6 under low nitrogen supply.

Experiments conducted using autophagy-defective mutants have provided evidence of autophagy's involvement in nutrient remobilization, particularly in conditions where nutrient supply is limited [19, 51, 87]. In Arabidopsis plants, during nitrogen starvation, the expression of most ATG8 genes is induced, except for ATG8E and ATG8F [88]. Notably, remobilization of ¹⁵N in several Arabidopsis atg mutants significantly decreased compared to wild-type plants under varying nitrogen availability [87]. Furthermore, the atg mutants accumulated higher levels of ammonium, amino acids, and proteins in rosette leaves compared to wild-type plants, while displaying lower sugar content [51]. This observation suggests that autophagy plays a pivotal role in controlling the C/N status and protein content in Arabidopsis leaves.

In the atg5 mutant of Arabidopsis, it has been demonstrated that not only nitrogen but also sulfur remobilization from rosette leaves to seeds is impaired when compared to control lines [89]. Additionally, the efficiency of iron (Fe) translocation from vegetative organs to seeds is significantly reduced in the atg5-1 mutant, even when Arabidopsis plants are adequately supplied with this element during seed formation [90].

In the atg12 mutant of maize, under conditions of insufficient nitrogen nutrition, increased leaf senescence, impaired nitrogen remobilization, and reduced productivity were observed [91].

Recent studies have revealed that autophagy induced by Pi starvation is regulated by the ER stress response [92]. Additionally, evidence suggests that in Arabidopsis thaliana, ER-phagy facilitates Pi recycling during the initial stages of Pi starvation (2-3 days), while chlorophagy replenishes Pi reserves when phosphorus starvation is combined with a substantial (5-fold) nitrate supply to plants and a reduced C/N ratio [93-95].

It has been demonstrated that the expression of Arabidopsis ATG8 genes (AtATG8f and AtATG8h) is significantly induced in the roots when exposed to low Pi supply. This regulation is closely associated with their promoter activity and can be suppressed in the phosphate mutant response 1 (phr1) [96]. The researchers propose that induction of AtATG8f and AtATG8h by Pi starvation may not directly facilitate Pi recycling but rather relies on a subsequent wave of transcriptional activation triggered by PHR1. This second phase fine-tunes cell type-specific autophagic activity. Further research is necessary to elucidate the mechanisms of autophagy activation and its role in plant responses to phosphorus starvation.

The lack of zinc also induces autophagy in plants, as demonstrated on the example of Arabidopsis [97]. Among all the members of the ATG8 gene family, ATG8d responds most strongly to this factor. Researchers have shown that in the mutants atg5-4 and atg10-1, a more significant increase in O₂"^- and H₂O₂ levels is observed under zinc deficiency compared to wild-type plants. In other words, autophagy suppresses the accumulation of ROS that form under conditions of limited zinc supply to plants.

Literature evidence suggests that intracellular self-degradation through autophagy in Arabidopsis plants replenishes the pool of zinc ions from proteins and organelles to increase the amount of usable zinc when it is scarce [98]. This replenishment of free zinc ions helps mitigate photosynthesis related chlorosis caused by a Fenton-like reaction during zinc starvation in Arabidopsis. Overall, the increased expression of ATG genes and the formation of autophagic structures in response to nutrient deficiencies, including nitrogen, carbon, phosphorus, zinc, and others, suggest the involvement of autophagy in plant adaptation to mineral nutrient deficiencies [99].

There is compelling evidence in the literature that autophagy is triggered in photosynthesizing organisms in response to stress, often associated with increased formation of reactive oxygen species, regardless of the source and location of ROS generation within the cell [100, 101]. Elevated ROS production can result from a different abiotic factor, including nutrient deficiencies, drought, salinity, high and low temperatures, intense lighting, and exposure to herbicides. ROS can lead to the accumulation of damaged or toxic materials in cells, and the degradation of these materials occurs through the process of autophagy [102].

Significant oxidative stress and induction of autophagy were observed in Arabidopsis plants upon exposure to H₂O₂ [100], as well as in Chlamydomonas [103]. When treated with methyl viologen (the herbicide paraquat), which is known to generate ROS, Arabidopsis seedlings with impaired autophagy (RNAi-ATG18a) exhibited stunted growth and pronounced discoloration compared to wild-type plants [100]. In the RNAiATG18a plants, unlike in the wild type, there was no accumulation of autophagosomes under the influence of methyl viologen, and the level of oxidized proteins in these plants was significantly higher, suggesting the role of autophagy in degrading oxidized proteins during oxidative stress. Similar results were observed when rice mutant atg10b was treated with methyl viologen [104]. It is worth noting that Arabidopsis mutants with over expression of ATG5 and ATG7 showed increased resistance to methyl viologen treatment compared to wild-type plants [105]. Under conditions inducing ROS with methyl viologen (10 pM), Nicotiana benthamiana plants exhibited a 3.25-fold increase in basal autophagy activity, as determined by the number of structures labeled with CFP-ATG8f [106]. In carotenoid deficient mutants of Chlamydomonas reinhardtii, an increase in ROS levels in chloroplasts was accompanied by a significant increase in autophagy [101]. Treatment of Chlamydomonas reinhardtii cells with the herbicide norflurazon, which inhibits phytoene desaturases and suppresses carotenoid synthesis, led to autophagy activation only in the presence of light. This suggests that autophagy is associated with photooxidative damage caused by ROS production in the presence of light [102].

Since microtubules are believed to play a role in autophagy, herbicides that cause their depolymerization by binding to tubulins may impact autophagy-related processes in plants. For example, the herbicides amiprofosmethyl and oryzalin significantly reduced the formation of autophagosomes and decreased the level of autophagy by approximately half [106].

It is known, that autophagy can be induced through the interaction of ROS and nitric oxide (NO) [107]. Therefore, data on the effect of the NO donor on the phytotoxic effects of herbicides, such as the acetyl-CoA-carboxylase inhibitor fenoxaprop [108], the protoporphyrinogen oxidase (PPO) inhibitor carfentrazone, and the synthetic auxin 2,4-D [109], provide indirect evidence of the possible involvement of programmed cell death, and specifically, autophagy, in the pathogenesis induced by these herbicides.

In the current literature, three types of programmed cell death (PCD) are considered: apoptosis (type 1), autophagy (type 2), and necrosis (type 3) [110]. While there is literature data on the role of programmed cell death in herbicide-induced pathogenesis [111], it remains unclear whether programmed cell death occurs via necrosis or autophagy. Simultaneously, evidence of the involvement of programmed cell death in herbicide-induced pathogenesis [112] has been obtained for both acetyl-CoA-carboxylase (ACC) inhibitor herbicides, whose action is mediated by the formation of ROS [113, 114], and for ALS inhibitor herbicides, whose action is independent of ROS.

Direct evidence of the role of autophagy in determining the plants sensitivity to the action of ALS inhibitor herbicides has been obtained. Since ALS is a key enzyme in the biosynthesis pathway of branched-chain amino acids valine, leucine, and isoleucine, the activation of autophagy under the action of ALS inhibitor herbicides may be attributed to a deficiency of these amino acids. For instance, treatment of rapeseed plants with the ALS inhibitor herbicide tribenuron-methyl has been shown to deplete branched-chain amino acids in anthers, ultimately leading to autophagic cell death [115]. Activation of GCN2, a protein kinase that regulates mRNA translation, was observed under the influence of tribenuron-methyl in Arabidopsis plants [116, 117]. The activation of autophagy and GCN2 by tribenuron-methyl, as well as their reversal by exogenous valine, leucine, and isoleucine, suggest that the starvation of branchedchain amino acids induced by tribenuron-methyl is responsible for the activation of autophagy and GCN2 [117]. Genetic and biochemical analyses conducted by these researchers revealed a lower proportion of free branched-chain amino acids and more sensitive phenotypes in the single mutants atg5, atg7, and gcn2 than in wild-type seedlings after herbicide treatment. Additionally, the lowest proportion of such amino acids and the most sensitive phenotypes were found in the double mutants atg5 gcn2 and atg7 gcn2. It is likely that tribenuron-methyl-induced starvation for branched-chain amino acids activates autophagy, possibly through the inactivation of the target of rapamycin (TOR) [117].

Activation of autophagy in plants treated with herbicides containing ALS inhibitors can lead to an increase in the content of branched-chain amino acids, thereby weakening the phytotoxic effects of the herbicides. This contributes to the development of herbicide resistance, known as homeostatic tolerance [117], in addition to the previously recognized target and metabolic tolerance. Homeostatic tolerance provides cellular protection when exposed to non-lethal stress. However, it should be noted that the long-term effects of herbicides may eventually lead to autophagic cell death.

Regulation of autophagy under the action of abiotic stresses

Autophagy is a pivotal process for the adaptation of plants to adverse conditions. Its activation under stressful circumstances is essential for eliminating damaged organelles and recycling essential nutrients to ensure plant survival. This complex process involves a significant number of proteins, receptors, and signaling pathways. To safeguard plant cells, autophagy must be carefully and strictly regulated. The regulatory pathways controlling autophagy activation are still being unraveled. These pathways encompass post-translational protein modifications necessary for initiating and advancing autophagy, control over the protein stability of the autophagy machinery, and transcriptional regulation leading to changes in the expression of autophagy-related genes [20].

Post-translational modifications of key proteins involved in the plant autophagy mechanism include phosphorylation, ubiquitination, lipidization, S-sulfhydration, S-nitrosylation, and acetylation [118]. During these modifications, chemical groups such as phosphate, ubiquitin, persulfide,

acetate, methyl, and others covalently alter specific amino acids in target proteins, enabling changes in their functions, dynamics, and stability [20]. Serine and threonine residues are the most frequently phosphorylated, while tyrosine is less common [119]. In some instances, histidine and arginine residues can also undergo these modifications [120, 121].

A key role in the activation of autophagy is played by kinases, one of which is the TOR, and the other is SNF-related kinase 1 (sucrose nonfermenting 1-related protein kinase 1 (SnRK1)) [122]. TOR functions as a serine/threonine protein kinase and as a nutrient sensor that integrates multiple output signals [123, 124]. The antibiotic rapamycin suppresses its activity. Both of these kinase complexes are evolutionarily conserved among eukaryotes and coordinate many signaling pathways in response to energy deprivation and various stress conditions, including those that activate autophagy [125]. SnRK1 is activated by energy deficiency, often associated with stress, to restore homeostasis, while TOR is activated under nutrient-rich conditions to promote growth [125]. TOR controls the processes of plant growth and development under favorable conditions, while SnRK1 inhibits TOR with low supply of energy and resources, or in stressful conditions [126]. SnRK1 is a master regulator of responses to nutrient and energy deprivation, in particular, as evidenced by the fact that a double mutant in the two catalytic subunits of SnRK1 (KIN10 and KIN11) in Arabidopsis is lethal [127]. Transgenic Arabidopsis lines overexpressing KIN10 (KIN10-OE) demonstrated induction of autophagy and autophagosome formation by affecting ATG1 phosphorylation, resulting in delayed leaf senescence and increased tolerance to nutrient starvation. In addition, KIN10-OE lines were less sensitive to drought and hypoxia compared to the wild type [128]. At the same time, the kin10 mutant had a basal level of autophagy under normal conditions similar to wild-type plants, but the activation of autophagy by many abiotic stresses was blocked, indicating that SnRK1 is required for the induction of autophagy by a diversity of stress conditions [129].

SnRK1 is a positive regulator of autophagy. It activates this process by directly phosphorylating the ATG1 protein or by inhibiting TOR [128, 129]. Conversely, TOR, which responds to nutrient deficiency, acts as a negative regulator of autophagy in plants [124]. For instance, overexpression of TOR suppresses the induction of autophagy in nutrient-deprived conditions, as well as in the presence of salt and osmotic stress. However, it does not affect autophagy induction in response to oxidative stress or ER stress (which occurs when a large number of unfolded proteins accumulate under unfavorable conditions) [130, 131].

In addition to the TOR and SnRK1 kinases described above, other kinases, particularly which involved in the transmission of signals from brassinosteroids [132-135] or ABA [136-137], can also participate in the phosphorylation of substrates related to autophagy. Furthermore, protein phosphatases may play a role in these processes [138]. Recently, considerable research has been dedicated to per sulfidation, a post-translational modification process in which cysteine thiol groups of proteins are converted to persulfide groups [139]. This modification extends to proteins involved in autophagy [140]. According to [141], 5214 proteins that have undergone persulfide modification were identified in the roots of Arabidopsis. In Arabidopsis, the deficiency of DES1 (cytosolic L-cysteine desulfhydrase), responsible for cysteine degradation and hydrogen sulfide (H₂S) generation, leads to autophagy induction [142].

In Arabidopsis, an autophagy-related target protein that undergoes persulfidation is the Cys-protease ATG4, which cleaves the C-terminal extension of ATG8, is crucial for autophagosome formation [143]. As the intracellular ABA level increases, the persulfidation of ATG4 temporarily decreases, contributing to ATG8 processing and autophagy progression [143]. The persulfidation of ATG18a at C103 in Arabidopsis regulates autophagy under endoplasmic reticulum stress, and the disruption of this process affects both the number and size of autophagosomes [144]. The literature reports data on the negative regulation of autophagy by sulfide [142, 145-147]. However, the role of H₂S as a negative or positive autophagy regulator may depend on specific abiotic factors and the potential involvement of other regulators in the autophagy signaling pathway [20, 144, 148]. activation autophagy damaged organelle nutrient survival plant

Under the influence of stress factors, post-translational protein modifications ensure the rapid activation of autophagy. To support autophagic activity, plants increase the expression of ATG genes [20]. The correct expression of ATG genes is mediated by transcription factors. Correlation analysis between the expression of FtATG8 and related transcription factor genes in buckwheat plants under drought and salt stress indicates that FiATG8a, FtATG8c. and FtATG8g exhibit a strong correlation with numerous stress-related transcription factors, suggesting that FtATG8 may be regulated by transcription [9]. There is a high degree of correlation between the expression levels of FtATG8 and most of the stress-related transcription factors, including FiMYBK), FiMYB21, Itblll.HA, FNAC2, and FtNAC6, further indicating the potential involvement of ltATG8 in salt stress and drought in Tatar buckwheat [9].

Spatio-temporal specificity of ltATG8 response to abiotic factors (cold, drought, salinity) is evident. Some autophagy genes exhibit early responses, while others respond at a later stage. For instance, the maximum expression of FtATG8a and FtATG8e was observed after 3 hours, whereas other F1ATG8 genes, especially FtATG8f, showed their peak expression after 12 hours [9]. It is important to note that autophagy is a long-term regulatory process. In grape plants, for example, certain genes related to autophagy are induced after 20 days of drought [149].

In tomato (Solanum lycopersicum), silencing WRKY33, a representative of one of the largest families of transcription factors in plants [150], results in reduced expression of ATG5, ATG7, and NBR1. This disruption hampers the formation of autophagosomes, leading to an accumulation of insoluble protein aggregates and decreased plant resistance to elevated temperatures [48]. It is hypothesized that under the influence of elevated temperatures, WRKY33 and ATG proteins collaboratively regulate autophagy. This process is involved in the selective removal of heat-induced protein aggregates, contributing to the plant's resistance to this abiotic factor. However, the precise interaction between ATG proteins and WRKY transcription factors necessitates further investigation and clarification [151].

WRKY33 can promote the selective degradation and recycling of protein aggregates by enhancing the expression of NBR1 under elevated temperatures. NBR1 (neighbour of BRCA1 gene), the first identified receptor for selective autophagy, interacts with both ATG8 and ubiquitin, facilitating the encapsulation of ubiquitinated protein aggregates in autophagosomes [48, 152].

Another transcription factor, TGA9, serves as a positive regulator of autophagy, upregulating the expression of various ATG genes to enhance autophagy in Arabidopsis plants under osmotic stress [88]. Overexpression of TGA9 activates autophagy in both control and stress conditions and amplifies the transcriptional expression of ATG8B, ATG8E, and other ATG genes by binding to their promoters.

BZR1 (brassinazole-resistant1) is a transcription factor activated by brassinosteroids. It acts as a positive regulator of the brassinosteroid signaling pathway and plays a role in the activation of autophagy [152]. This transcription factor can induce autophagy and the accumulation of NBR1, a receptor facilitating the selective autophagy of protein aggregates, in tomato plants under cold conditions [153]. BZR1 enhances the expression of ATG2 genes and ATG6 under the influence of cold and nitrogen starvation, as well as genes NBR1 (NBRla and NBRlb) under cold stress by directly binding to their promoters, thereby promoting autophagy [152, 153]. Consequently, the accumulation of insoluble protein aggregates decreases, resulting in increased resistance of tomato plants to cold and nitrogen starvation [152, 153]. However, it's worth noting that the activation and role of BZR1 in autophagy regulation may depend on the specific abiotic factor and the plant species [20]. Therefore, further research is necessary to draw more comprehensive conclusions.

In experiments with tomato plants, it was demonstrated that under drought conditions, autophagy is activated through the induction of the ethylene response transcription factor 5 (ERF5) [68]. Autophagy, facilitated in part by the binding of ERF5 (ethylene response factor 5) to the promoters of ATG8d and ATG18h, contributes to the ethylene-mediated drought tolerance of tomatoes [68].

The heat shock transcription factor, HsfA1a, plays a significant role in tomato drought tolerance, particularly by its involvement in the induction of autophagy [73]. HsfA1a-mediated autophagy, enabled by the binding of this factor to the promoters of ATG10 and ATG18f, reduces the accumulation of insoluble ubiquitinated protein aggregates and enhances resistance to drought.

Numerous recent studies have highlighted the significance of hormonal signaling in the regulation of autophagy and plant responses to stress factors. However, it is essential to note that the molecular mechanisms governing such regulation still require further investigation [122].

In summary, autophagy is a complex and highly conserved process that plays a crucial role in the functioning of eukaryotes, both under optimal and stressful conditions. Autophagy is activated in response to various abiotic factors, and it participates in the processing and recycling of damaged biomolecules, organelles, aggregated proteins, and more. This, in turn, enables plants to survive and thrive under adverse environmental conditions.

Literature data indicate that the activation of autophagy occurs not only in response to abiotic factors such as high and low temperatures, drought, salinity, and nutrient deficiency but also in plants treated with herbicides. The action of herbicides, in particular, is associated with the production of reactive oxygen species, disorganization of photosynthesis, and inhibition of specific enzymes. The specific mechanisms by which herbicides influence the activation of autophagy and its role in programmed cell death still require further elucidation.



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