Identiﬁcation and Expression Analysis of the Solanum tuberosum StATG8 Family Associated with the WRKY Transcription Factor

: Autophagy is an evolutionarily well-conserved cellular catabolic pathway in eukaryotic cells and plays an important role in cellular processes. Autophagy is regulated by autophagy-associated (ATG) proteins. Among these ATG proteins, the ubiquitin-like protein ATG8/LC3 is essential for autophagosome formation and function. In this study, the potato StATG8 family showed clade I and clade II with signiﬁcantly different sequences. Expression of the StATG8 family was also increased in senescence. Interestingly, the expression of the StATG8 and other core StATG genes decreased in potato tubers as the tubers matured. The StATG8 family also responded to a variety of stresses such as heat, wounding, salicylic acid, and salt stress. We also found that some Arabidopsis WRKY transcription factors interacted with the StATG8 protein in planta . Based on group II-a WRKY, StATG8-WRKY interaction is independent of the ATG8 interacting motif (AIM) or LC3 interacting region (LIR) motif. This study showed that the StATG8 family had diverse functions in tuber maturation and multiple stress responses in potatoes. Additionally, StATG8 may have an unrelated autophagy function in the nucleus with the WRKY transcription factor. (coIP) assays reveal that StATG8 is associated with multiple AtWRKY transcription factors. Agrobacterium -mediated transient co-expression of GFP-StATG8-2.1 or GFP control with AtWRKY-22-HF , AtWRKY-28-HF , AtWRKY-33-HF , AtWRKY-40-HF , AtWRKY-60-HF , AtWRKY-18-HF , AtWRKY-20-HF , and AtWRKY-38-HF , and AtWRKY-70-HF was performed in N. benthamiana leaves. Anti-GFP co-IPs were performed with total protein extracts and probed with anti-GFP and -FLAG antibodies. All experiments were performed for at least three times.


Introduction
Climate change has a significant impact on the productivity of various crops [1]. High temperatures, drought stress, floods, etc. have serious adverse effects on crop production. Since this adversely affects global food production, developing crops through novel molecular genetic engineering using various plant genes is required [2,3].
Selective macroautophagy/autophagy is one of the most evolutionarily conserved mechanisms and is a catabolic process induced primarily with nutrient deprivation and various stress conditions [4]. Autophagy is a basic function of eukaryotic cells and is well conserved in animals, plants, yeast, and fungi [5]. The most typical morphological feature of autophagy is the formation of bilayer autophagosomes containing selected cargo or non-selective bulk cytoplasm [4]. Autophagy is formed to remove dysfunctional organelles, abnormal cytoplasmic proteins, and pathogenic proteins; it eventually fuses with lysosomes (animals) or vacuoles (yeast and plants) to remove and recycle material [6]. Completion of the autophagosome forms a key step in autophagy that depends on a series of autophagy-related (ATG) proteins [7].
Among various ATG proteins, ATG8/LC3, a ubiquitin-like protein, performs almost all major processes of autophagosome formation [8]. Therefore, ATG8 protein is mainly used as an autophagosome marker to evaluate the induction of autophagy [9,10]. ATG8 is also required for cargo recognition through specific interactions with autophagy receptors during selective autophagy [11]. ATG8 is expressed as a cytoplasmic precursor that is processed at the C-terminal Gly residue by the ATG4 protein and is conjugated to phosphatidylethanolamine (PE) [12]. Lapidated ATG8 (ATG8-PE) protein is anchored to

The Potato StATG8 Family Contained Two Distinct Subgroups
A genome-wide analysis of the ATG family present in various crops is ongoing by many researchers [27][28][29][30][31]. Unlike other eukaryotes, the plant ATG8 family has been shown to diversify the gene family. Therefore, research on the ATG8 family present in many plants is ongoing. In the case of potatoes (Solanum tuberosum L.), only in-silco data were analyzed and there were few actual experimental results [32]. Therefore, we identified the StATG8 family (Table S1). Additionally, we analyzed the DNA and protein sequences of the potato ATG8 family. The difference was confirmed by analyzing the DNA sequence of the ATG8 family present in red pepper, tobacco, and tomato, which are representative crops of the Solanaceae family together with potato. ATG8 is divided into two main types as previously reported according to DNA sequencing [17,32]. The difference was so large that it was confirmed that there was only one clade II in each crop of Solanaceae ( Figure 1A). In addition, we found that one clade II gene was also present in pepper ( Figure 1A). The results of the comparative analysis of protein sequences were even more interesting. There is a significant difference between the two clades ( Figure 1B) protein sequences are identical, but the DNA is slightly different ( Figure 1A,B). There may be specific functional differences between the two genes due to differences in promoters.
that it was confirmed that there was only one clade II in each crop of Solanaceae ( Figure  1A). In addition, we found that one clade II gene was also present in pepper ( Figure 1A). The results of the comparative analysis of protein sequences were even more interesting. There is a significant difference between the two clades ( Figure 1B). The StATG8-2.1 and StATG8-2.2 protein sequences are identical, but the DNA is slightly different (Figures 1A  and 1B). There may be specific functional differences between the two genes due to differences in promoters. . Phylogenetic relationships of ATG8s from Solanum tuberosum with those of Nicotiana tabacum, Solanum lycopersicum, Capsicum annuum, and Arabidopsis thaliana. The tree was constructed from an analysis conducted with MEGA-X software using a maximum likelihood method. Phylogenetic tree was based on the nucleotide sequence data. Numbers along the branches (bootstrap value) show the percentage occurrence of nodes in 1000 replicates of stimulation. Sequences of ATG8 genes: Mature ATG8, processed by the ATG4 protease, is conjugated to the membrane lipid phosphatidylethanolamine (PE) and functions as a ubiquitin-like protein required for autophagosome formation [33]. The cysteine proteinase ATG4 has a dual function in . Phylogenetic relationships of ATG8s from Solanum tuberosum with those of Nicotiana tabacum, Solanum lycopersicum, Capsicum annuum, and Arabidopsis thaliana. The tree was constructed from an analysis conducted with MEGA-X software using a maximum likelihood method. Phylogenetic tree was based on the nucleotide sequence data. Numbers along the branches (bootstrap value) show the percentage occurrence of nodes in 1000 replicates of stimulation. Sequences of ATG8 genes: StATG8-1. Mature ATG8, processed by the ATG4 protease, is conjugated to the membrane lipid phosphatidylethanolamine (PE) and functions as a ubiquitin-like protein required for autophagosome formation [33]. The cysteine proteinase ATG4 has a dual function in autophagosome assembly and degradation by exposing the C-terminal glycine residue of ATG8 [34]. Interestingly, members of the clade II group of Arabidopsis AtATG8h and AtATG8i were already glycine-exposed ( Figure 1B). However, StATG8-4, NtATG8i, CaLOC107840514, and Solyc01g068060 may require ATG4 cleavage for conjugation and deconjugation in autophagosome formation ( Figure 1B). These evolutionary consequences of ATG8 clades I and II with large differences in amino acid sequence are probably characteristic of Solanaceae crops.

Promoters of StATG8s Contained Multiple Regulatory Cis-Elements
The regulatory elements of the StATG8 promoter sequence were analyzed to understand regulatory pathways and gene functions. The 1.5 kb protomer sequence of StATG8 was selected through the Spud Potato Genomics Resource DB (http://spuddb.uga.edu/) on 1 to 20 February 2022. Specific cis-acting regulatory elements of the StATG8 gene were identified using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/ on 1 to 20 February 2022) [35]. As shown in Figure 2 and Table 1, at least 15 different cis-elements were identified in the StATG8 promoter involved in development, hormone, and stress responsiveness. In the promoters of StATG8-2.1 and StATG8-2.2 having the same protein sequence, the proportion of hormone-related cis-elements in the promoters was relatively higher in  In the promoters of StATG8-2.1 and StATG8-2.2 having the same protein sequence, the proportion of hormone-related cis-elements in the promoters was relatively higher in StATG8-2.1 than in StATG8-2.2 ( Figure 2B). The promoter of StATG8-2.1 has about 35% more regulatory elements than other StATG8 members. Except for the StATG8-2.2 promoter, the abscisic acid (ABA)-responsive element ABRE was present in all StATG8 promoters [36]. Moreover, MYB cis-elements are found in all StATG8 promoters, which may be associated with drought stress [37]. Regulatory elements corresponding to methyljasmonic acid (MeJA)-responsiveness such as CGTCA/TGACG-motifs were also found [38]. Many of the StATG8 promoters exhibited wounding-response elements such as As-1, Wun, and WRE3 motifs [39,40]. It can be expected that the expression of StATG8 may also be affected by mechanical stress and immune responses [41]. WRKY transcription factor binding motif "W-box" was also found in the promoters of StATG8-1.1, StATG8-1.2, and StATG8-2.1. It can be expected that StATG8 genes can be regulated using the WRKY transcription factor [42]. From the previous reports, autophagy plays an important role in development [43,44]; the regulatory elements CCGTCC motif, RY-element, MSA-like, and GCN4-motif related to development were present in StATG8 promoters ( Figure 2 and Table 1) [45]. Thus, the diverse StATG8 genes are deeply associated with multiple stress responses and developments.

The Potato ATG8 Family Genes Are Specifically Regulated during Leaf Senescence and Tuber Maturation
It is well known that the function of autophagy is directly related to cellular senescence [46,47]. In this regard, we investigated how the gene expression of the StATG8 family changes during leaf senescence and tuber maturation, which is important for its value as a crop. In semi-quantitative RT-PCR comparing mature and senescent leaves ( Figure 3A), StATG8-2.1, StATG8-3.2, and StATG8-4 showed strong expression. On the other hand, StATG8-1.1, StATG8-1.2, StATG8-2.2, and StATG8-3.1 did not exhibit a significant increase in gene expression ( Figure 2B). Thus, the functions of StATG8 in the aging process are not all the same. We also confirmed the expression of StATG8 by sampling tubers grown in three different sizes ( Figure 3A). Interestingly, it was observed that the expression of most StATG8 genes decreased as tuber size increased ( Figure 3B). It seems different from the pattern of increasing ATG genes during the fruit ripening of tomatoes [48]. We further investigated the expression levels of other core StATG genes present in potato. It was confirmed that the expression of StATG10, StATG11, StATG13a, StATG16, and StATG101 was strongly down-regulated in leaf senescence ( Figure 3B). In addition, it was confirmed that StATG3, StATG9, StATG10, StATG11, StATG13a, StATG16, and StATG101 dramatically decreased according to tuber maturation as in the StATG8 family genes ( Figure 3B). It was first discovered that the expression of StATG8 and core StATG genes decreases in potato tubers maturation. Based on the above results, we speculated that the autophagy could be important for tuber developments. , and tubers (T1 to T3). StEF1α was used as a reference gene. The white bar represents 1 cm. Semi-quantitative RT-PCR was performed using the StATG gene-specific primers and primer information is in Supporting Table S2.

Response of StATG8 Genes Expression upon Multiple Stresses
Through previous promoter analysis, we hypothesized that the StATG8 family gene expressions would respond to wounding and heat stresses. Under heat stress, the expression of StATG8 family genes was observed in two patterns. StATG8-2.1, StATG8-2.2, StATG8-3.2, and StATG8-4 showed a strong increase in thermal stress at 3 h ( Figure 4A). On the other hand, the expression of StATG8-3.1 was significantly decreased, and StATG8-1.1 and StATG8-1.2 showed a tendency to decrease after 6 h in the heat stress ( Figure 4A). Upon wounding stress, StATG8-1.1, StATG8-2.1, and StATG8-3.2 showed an increased pattern ( Figure 4A). These results suggest that the StATG8 family will function in response to mechanical stress and wounding stress induced by insects.

Response of StATG8 Genes Expression upon Multiple Stresses
Through previous promoter analysis, we hypothesized that the StATG8 family gene expressions would respond to wounding and heat stresses. Under heat stress, the expression of StATG8 family genes was observed in two patterns. StATG8-2.1, StATG8-2.2, StATG8-3.2, and StATG8-4 showed a strong increase in thermal stress at 3 h ( Figure 4A). On the other hand, the expression of StATG8-3.1 was significantly decreased, and StATG8-1.1 and StATG8-1.2 showed a tendency to decrease after 6 h in the heat stress ( Figure 4A). Upon wounding stress, StATG8-1.1, StATG8-2.1, and StATG8-3.2 showed an increased pattern ( Figure 4A). These results suggest that the StATG8 family will function in response to mechanical stress and wounding stress induced by insects.
We also investigated whether the StATG8 family responds to the plant defense hormone salicylic acid (SA) [49]. Interestingly, only StATG8-2.1 responded to SA at 12 h ( Figure 4B). However, other StATG8 genes did not change significantly to SA treatments between 6 and 12 h. The absence of a response by SA probably means that most StATG8 genes can be regulated by other hormones such as JA and ABA. Additionally, salt stress is also one of the very important plant stresses and we tested whether StATG8 family responses to salt stress. The gene expression of StATG8-1.1, StATG8-2.1, and StATG3-2.2 showed a significantly upregulated pattern ( Figure 4B). As a result, each member of StATG8 specifically responds in a specific stress and its functions may be diversified. . For stress treatments, 4-week-old potato plants were used to stress treatment for the indicated times. StEF1α was used as a reference gene. Error bars indicate the standard deviations of three independent qRT-PCR biological replicates. Asterisks indicate significant differences from the control using the unpaired Student's t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).
We also investigated whether the StATG8 family responds to the plant defense hormone salicylic acid (SA) [49]. Interestingly, only StATG8-2.1 responded to SA at 12 h (Figure 4B). However, other StATG8 genes did not change significantly to SA treatments between 6 and 12 h. The absence of a response by SA probably means that most StATG8 genes can be regulated by other hormones such as JA and ABA. Additionally, salt stress is also one of the very important plant stresses and we tested whether StATG8 family responses to salt stress. The gene expression of StATG8-1.1, StATG8-2.1, and StATG3-2.2 showed a significantly upregulated pattern ( Figure 4B). As a result, each member of StATG8 specifically responds in a specific stress and its functions may be diversified.

Response of Core StATG Genes upon Multiple Stresses
Based on the results of investigating the gene expression of the StATG8 family for various stresses, qRT-PCR was performed to test the expression pattern of the core StATG genes. The results exhibited that StATG3, StATG9, StATG11, StATG13a, and StATG101 significantly responded to heat and wounding stresses ( Figure 5A). On the other hand, mRNA transcript levels of StATG10 and StATG16 were not significantly changed upon heat and wounding stresses ( Figure 5A). StATG11 showed a specific increase pattern in heat stress. Transcript levels of StATG9 were observed at increased patterns in both heat and wounding stresses. StATG101 exhibited a down-regulated pattern in heat, whereas increased expression was observed at 6 h for wounding stress ( Figure 5A). Like the StATG8 family, the core StATG genes are also thought to perform autophagy function in response to heat and wounding stresses. . For stress treatments, 4-week-old potato plants were used to stress treatment for the indicated times. StEF1α was used as a reference gene. Error bars indicate the standard deviations of three independent qRT-PCR biological replicates. Asterisks indicate significant differences from the control using the unpaired Student's t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Response of Core StATG Genes upon Multiple Stresses
Based on the results of investigating the gene expression of the StATG8 family for various stresses, qRT-PCR was performed to test the expression pattern of the core StATG genes. The results exhibited that StATG3, StATG9, StATG11, StATG13a, and StATG101 significantly responded to heat and wounding stresses ( Figure 5A). On the other hand, mRNA transcript levels of StATG10 and StATG16 were not significantly changed upon heat and wounding stresses ( Figure 5A). StATG11 showed a specific increase pattern in heat stress. Transcript levels of StATG9 were observed at increased patterns in both heat and wounding stresses. StATG101 exhibited a down-regulated pattern in heat, whereas increased expression was observed at 6 h for wounding stress ( Figure 5A). Like the StATG8 family, the core StATG genes are also thought to perform autophagy function in response to heat and wounding stresses.
We also investigated the expression patterns of core StATG genes during SA and NaCl treatments. The expression of StATG3, StATG9, StATG11, and StATG13a genes increased upon SA treatment ( Figure 5B). Upon salt treatment, StATG11 and StATG101 showed a significant increase pattern, though the rest of the StATG8 genes did not show a significant change of mRNA transcript levels ( Figure 5B). Thus, several StATG genes responded to SA and salt treatments. These different types of responses are expected to have features that contribute to autophagy formation and function in multiple stresses. Plants 2022, 11, x FOR PEER REVIEW 9 of 18 We also investigated the expression patterns of core StATG genes during SA and NaCl treatments. The expression of StATG3, StATG9, StATG11, and StATG13a genes increased upon SA treatment ( Figure 5B). Upon salt treatment, StATG11 and StATG101 showed a significant increase pattern, though the rest of the StATG8 genes did not show a significant change of mRNA transcript levels ( Figure 5B). Thus, several StATG genes responded to SA and salt treatments. These different types of responses are expected to have features that contribute to autophagy formation and function in multiple stresses.

StATG8 Interacts with WRKY Transcription Factor in Planta
The function of autophagy is based on what happens in the cytoplasm. However, it has recently been reported that animal LC3 and fungal Atg8, which corresponds to plant ATG8, exist in the nucleus and shuttle between the nucleus and the cytoplasm through post-translational modifications such as acetylation [23,26]. Furthermore, there are reports that cassava (Manihot esculenta) MeWRKY20 associated with MeATG8s and activated MeATG8 gene expression in the nucleus [50]. In addition, transcriptional regulator TGA9 also regulate the expression of AtATG8 genes [19]. Although the function of ATG8 in the nucleus is still largely unknown, we performed protein interaction experiments to determine whether StATG8 interacts with WRKY transcriptional regulators.
We first selected several groups of Arabidopsis WRKY ( Figure 6A). As shown in Figure 6B, we determined whether these proteins had AIM or LIR motifs known to be capable of interacting with ATG8 proteins using iLIR database (https://ilir.warwick.ac.uk) and hfAIM (http://bioinformatics.psb.ugent.be/hfAIM/). Interestingly, most of the AtWRKY . For stress treatments, 4-week-old potato plants were used for stress treatment for the indicated times. StEF1α was used as a reference gene. Error bars indicate the standard deviations of three independent qRT-PCR biological replicates. Error bars represent SD of three independent experiments. Asterisks indicate significant differences from the control using the unpaired Student's t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

StATG8 Interacts with WRKY Transcription Factor in Planta
The function of autophagy is based on what happens in the cytoplasm. However, it has recently been reported that animal LC3 and fungal Atg8, which corresponds to plant ATG8, exist in the nucleus and shuttle between the nucleus and the cytoplasm through post-translational modifications such as acetylation [23,26]. Furthermore, there are reports that cassava (Manihot esculenta) MeWRKY20 associated with MeATG8s and activated MeATG8 gene expression in the nucleus [50]. In addition, transcriptional regulator TGA9 also regulate the expression of AtATG8 genes [19]. Although the function of ATG8 in the nucleus is still largely unknown, we performed protein interaction experiments to determine whether StATG8 interacts with WRKY transcriptional regulators.
We first selected several groups of Arabidopsis WRKY ( Figure 6A). As shown in Figure 6B, we determined whether these proteins had AIM or LIR motifs known to be capable of interacting with ATG8 proteins using iLIR database (https://ilir.warwick.ac.uk on 1 to 20 February 2022) and hfAIM (http://bioinformatics.psb.ugent.be/hfAIM/ on 1 to 20 February 2022). Interestingly, most of the AtWRKY proteins contained one or two AIMs. In case of AtWRKY33, it does not have any AIM signatures and it could be used as a negative control in StATG8-WRKY interaction coIP experiments.
proteins contained one or two AIMs. In case of AtWRKY33, it does not have any AIM signatures and it could be used as a negative control in StATG8-WRKY interaction coIP experiments.

Figure 6. StATG8 associated with WRKY transcription factors in planta.
A. Phylogenetic relationships of protein sequences within the AtWRKY family. The tree was constructed from an analysis conducted with MEGA-X software using a maximum likelihood method [51]. AtWRKYs used for CoIP are shown in red boxes. B. Analysis of putative ATG8 binding motifs present in AtWRKY utilizing the iLIR database on 1 to 25 June 2022 [52]. ATG8 interacting proteins have a LC3-interacting region (LIR) and Atg8-interacting motif (AIM) responsible for interaction with ATG8 family proteins. In AtWRKYs, LIR motifs are shown in red text and AIM motifs are shown in blue text. AtWRKY18, AtWRKY40, and AtWRKY60 protein sequence analysis corresponds to group II-a. Only AtWRKY18 contains a putative LIR motif at the N-terminal end. AtWRKY33 do not contain any ATG8-binding related motifs. C. Co-immunoprecipitation (coIP) assays reveal that StATG8 is asso- We prepared co-immunoprecipitation (co-IP) samples with 35S::GFP-StATG8-2.1 and various 35S::AtWRKYs-HF combinations for Agrobacterium-mediated transient assay using Nicotiana benthamiana [53,54]. As previously reported [50], we expected that there would be enough protein interaction between WRKY and StATG8. Surprisingly, At-WRKY33, which does not contain either AIM or iLIR ( Figure 6B), can still associate with GFP-StATG8-2.1 in coIP ( Figure 6C). However, AtWRKY18 containing both AIM and iLIR did not work in coIP with StATG8-2.1 (Figures 6B and 6C). AtWRKY38 has two AIMs ( Figure 6B), but when co-expressing AtWRKY38 and StATG8-2.1, protein interactions could not be confirmed in coIP experiments ( Figure 6C). In our coIPs, StATG8-2.1 can associate with many of AtWRKY proteins. However, additional experiments are needed to determine whether the expected AIM motif can play a major role.  [52]. ATG8 interacting proteins have a LC3-interacting region (LIR) and Atg8-interacting motif (AIM) responsible for interaction with ATG8 family proteins. In AtWRKYs, LIR motifs are shown in red text and AIM motifs are shown in blue text. AtWRKY18, AtWRKY40, and AtWRKY60 protein sequence analysis corresponds to group II-a. Only AtWRKY18 contains a putative LIR motif at the N-terminal end. AtWRKY33 do not contain any ATG8-binding related motifs. (C) Co-immunoprecipitation (coIP) assays reveal that StATG8 is associated with multiple AtWRKY transcription factors. Agrobacterium-mediated transient co-expression of GFP-StATG8-2.1 or GFP control with AtWRKY-22-HF, AtWRKY-28-HF, AtWRKY-33-HF, AtWRKY-40-HF, AtWRKY-60-HF, AtWRKY-18-HF, AtWRKY-20-HF, and AtWRKY-38-HF, and AtWRKY-70-HF was performed in N. benthamiana leaves. Anti-GFP co-IPs were performed with total protein extracts and probed with anti-GFP and -FLAG antibodies. All experiments were performed for at least three times.
We prepared co-immunoprecipitation (co-IP) samples with 35S::GFP-StATG8-2.1 and various 35S::AtWRKYs-HF combinations for Agrobacterium-mediated transient assay using Nicotiana benthamiana [53,54]. As previously reported [50], we expected that there would be enough protein interaction between WRKY and StATG8. Surprisingly, AtWRKY33, which does not contain either AIM or iLIR ( Figure 6B), can still associate with GFP-StATG8-2.1 in coIP ( Figure 6C). However, AtWRKY18 containing both AIM and iLIR did not work in coIP with StATG8-2.1 ( Figure 6B,C). AtWRKY38 has two AIMs ( Figure 6B), but when co-expressing AtWRKY38 and StATG8-2.1, protein interactions could not be confirmed in coIP experiments ( Figure 6C). In our coIPs, StATG8-2.1 can associate with many of AtWRKY proteins. However, additional experiments are needed to determine whether the expected AIM motif can play a major role.

Discussion
Autophagy is well conserved in all eukaryotes and is known to play a variety of roles in many areas, including intracellular immunity, development, stress response, and waste recycling [4,55,56]. In this study, we characterized the StATG8 family among important ATG genes present in potatoes, a representative food resource under developmental and various stress conditions. Although the function of ATG8 in the nucleus is not yet clear, we found that StATG8 can interact with WRKY family proteins, one of the transcription factors, either using AIM or an unknown motif.
Plants are constantly exposed to various stresses [57]. Climate change, which has been emphasized, is creating a situation that disturbs the homeostasis of plants [58]. This climate condition is believed to be closely related to the function of autophagy, which plays an important role in maintaining intracellular homeostasis. To overcome this climate change, genetic engineering of autophagy can be one of the strategies to improve enhanced crop development. Research on core autophagy genes is ongoing [59,60]. One of them, ATG8 protein, plays an important role in the conjugation part in autophagosome formation. Plant ATG8 has several unique characteristics compared to other eukaryotes. First, plant ATG8 contains more isoforms than other eukaryotes. Animals with functions corresponding to plant ATG8 (LC3A-C, GABARAPs, and GATE-16) and fungi have 1-2 isoforms of the gene [61,62], whereas Solanaceous crops such as potatoes have about 7-8 genes ( Figure 1A). Furthermore, StATG8 members are divided into two clades ( Figure 1A). This suggests that the function of ATG8 is diversified as a plant-specific autophagy. In this study, we also confirmed that different features exist in each function in the gene expression and promoter analysis of StATG8 family. Second, ATG8 is an effector target of foreign pathogens in plant immunity. For example, StATG8 is targeted by PexRD54, an effector of the Irish potato famine pathogen Phytophthora infestans [63]. PexRD54 induces ATG8 malfunction and perturbs plant autophagy-related immunity. Barley stripe mosaic virus γb protein also utilizes a strategy to increase viral infection by disrupting the ATG7-ATG8 Interaction [64]. The pathogen probably targets ATG8 to suppress the immune function of autophagy. Interestingly, there are very large changes in the protein sequence of StATG8 members [32]. StATG8 is divided into two major clades, which are probably related to the evolution of function.
Plant leaf senescence is one of the degenerative processes of organelles or biomolecules [65]. Studies of the function of senescence-related autophagy have fairly similar results in both animals and plants [66]. Autophagy is one of the functions contributing to the maintenance of basic homeostasis in plants and is thought to play a distinct role in the breakdown of recyclable substances during leaf senescence [4]. In our experimental results, it was confirmed that the expression of StATG8-2.1, StATG8-3.2, and StATG8-4 was highly upregulated in leaf senescence ( Figure 2B). In addition, it was found that not all core ATG genes increase mRNA levels during leaf senescence. That is, each isoform of the StATG8 family may have a different part of function in senescence-associated autophagosome formation.
Potato tubers are one of the tissues used as a very important food resource [67]. To date, no information is known on the expression of autophagy-related genes in tuber formation. Through this study, we newly discovered that expression of the StATG8 family decreased during tuber maturation. This is expected to be a part that can be used for engineering by regulating the function of autophagy that regulates tuber formation. Moreover, since core ATG genes also show a decrease ( Figure 2B), autophagy activity is expected to decrease during tuber maturity. It is necessary to proceed with the study of tuber formation using Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 (CRISPR-Cas9)-based genome editing and potato StATG gene overexpression system. In general, autophagy activity increases as the fruit ripens. For example, in the fruit maturation of strawberry, the protein expression of ATG8 is increased and the ratio of ATG8-PE is increased from green fruits to red fruits [68]. Additionally, the level of NBR1 protein, a representative cargo protein, was also increased [68]. In the Solanaceae pepper, CaATG8 and CaATG5 were found to increase both protein and mRNA levels during the ripening of pepper fruit [69]. This result may be because the tuber of the potato is not a fruit, or it may be due to a difference in tissue composition. Plants are exposed to and respond to multiple stresses [25,70]. Various cis-elements in the potato StATG8 family were found to respond to MeJA, wounds, drought stress, etc. in promoter analysis (Figure 2). In fact, the StATG8 member showed various expression patterns from the wound and SA treatment results related to biotic stresses. In particular, StATG8-1.1, StATG8-2.1, and StATG8-3.2 showed significant and strong gene expression patterns in the wounding stress ( Figure 4A). On the other hand, in the SA treatment, only StATG8-2.1 exhibited significantly enhanced gene expression. Although previous studies have reported that ATG8 has multiple roles in immune processes [57], most StATG8s appear to have a specialized function in JA-related wounding stress. However, StATG8-2.1 maybe act in SA pathway in defense response. It is also worth investigating the response of StATG8 genes to additional plant hormones.
Global warming associated with climate change adversely affects plant growth at high temperatures and severely limits crop productivity [71]. Autophagy plays an important role in plant heat tolerance, in part by breaking down unusable proteins caused by heat stress [72]. We found that the StATG8 genes responded quite strongly to heat stress ( Figure 4A). All gene expression patterns, except for StATG8-1.1 and StATG8-1.2, were confirmed to be significant. This showed a similarly increased pattern for other core ATG genes. For example, there is a result that heat shock protein HsfA1a directly activates gene expression by binding to the heat-shock cis-element of Arabidopsis ATG10 and ATG18f [73]. The autophagy gene is directly regulated by transcriptional regulators such as heat shock transcription factors and is likely regulated by temperature changes; certain members of the StATG8 family are also expected to be involved.
Plant autophagy research is focused on the function in the cytoplasm where selective autophagy works [11]. However, recent studies have shown that ATG8 interacts with various proteins in the nucleus [74,75]. Pathogen proteins might affect autophagy activity through ATG8 interaction in the nucleus. The function of plant ATG8 in the nucleus is not yet well understood, but there may be interactions similar to those of almost all plants, such as the example of the protein interaction of MeATG8-MeWRKY20 [50]. In the disease resistance response, MeATG8 interacts with MeWRKY20 to increase autophagy activity and further enhances ATG8 gene expression. Using the Arabidopsis AtWRKY22 homologue of MeWRKY20, we also confirmed protein interactions with StATG8 ( Figure 6). This would be different from the general autophagy function of ATG8 in the nucleus. In addition, not all AtWRKYs interact with StATG8, which may be a versatile strategy to promote the autophagic activity of plant ATG8 with various ATG8s to respond to multiple stresses.
Recently, it has been found that UIM as well as AIM exist [16]. It can be expected that the binding motifs used by ATG8 to interact with specific targets may not yet be limited. In our experiments, AtWRKY33, a group Ia member, interacted with StATG8 despite the absence of a motif that interacts with ATG8. AtWRKY18, AtWRKY40, and AtWRKY60 formed a small group IIa with AIM but only AtWRKY18 exhibited no association with StATG8. It would be a good comparison if further studies on protein-protein interactions and StATG8 gene expression activation were performed using the potato-WRKY homologue group. Therefore, it is possible that unknown ATG8-binding motifs exist. Further studies are needed to determine whether the interaction of ATG8-WRKY is one of the regulatory actions that play a positive role in autophagy activity or autophagosome formation. If this regulatory action is common, it can be applied for crop development through dual regulation of ATG8 and WRKY.
Although functional studies of the StATG8 family are not yet complete, we presented a working model of the potato StATG8 family (Figure 7). The expression of seven members of StATG8 is regulated by specific transcription factors during leaf senescence and tuber maturation. The expression of StATG genes decreases with increasing tuber maturation. Additionally, specific StATG8 genes are up-regulated upon heat, saline, wounding, and salicylic acid treatments, ultimately contributing to autophagy formation and overcoming the stresses. The WRKY transcription regulator is expected to play a role in regulating the gene expression of StATG8. Although the exact mechanism is not yet known, StATG8 proteins can associate with WRKY proteins through specific AIM or unknown binding motifs. It is expected to enhance the expression of StATG8 and activate autophagy activity. On the other hand, a negative feedback-regulation can be expected in which StATG8 pulls WRKY out of the nucleus and degrades it. Recently, it has been found that UIM as well as AIM exist [16]. It can be expected that the binding motifs used by ATG8 to interact with specific targets may not yet be limited. In our experiments, AtWRKY33, a group Ia member, interacted with StATG8 despite the absence of a motif that interacts with ATG8. AtWRKY18, AtWRKY40, and AtWRKY60 formed a small group IIa with AIM but only AtWRKY18 exhibited no association with StATG8. It would be a good comparison if further studies on protein-protein interactions and StATG8 gene expression activation were performed using the potato-WRKY homologue group. Therefore, it is possible that unknown ATG8-binding motifs exist. Further studies are needed to determine whether the interaction of ATG8-WRKY is one of the regulatory actions that play a positive role in autophagy activity or autophagosome formation. If this regulatory action is common, it can be applied for crop development through dual regulation of ATG8 and WRKY.
Although functional studies of the StATG8 family are not yet complete, we presented a working model of the potato StATG8 family (Figure 7). The expression of seven members of StATG8 is regulated by specific transcription factors during leaf senescence and tuber maturation. The expression of StATG genes decreases with increasing tuber maturation. Additionally, specific StATG8 genes are up-regulated upon heat, saline, wounding, and salicylic acid treatments, ultimately contributing to autophagy formation and overcoming the stresses. The WRKY transcription regulator is expected to play a role in regulating the gene expression of StATG8. Although the exact mechanism is not yet known, StATG8 proteins can associate with WRKY proteins through specific AIM or unknown binding motifs. It is expected to enhance the expression of StATG8 and activate autophagy activity. On the other hand, a negative feedback-regulation can be expected in which StATG8 pulls WRKY out of the nucleus and degrades it.

Plant Materials and Stress Treatments
Arabidopsis thaliana (Col-0), tobacco (Nicotiana benthamiana), and potato (Solanum tuberosum L. cv. Sumi) plants were potted in soil and placed in a growth chamber under controlled conditions with 60% relative humidity and a 16 h:8 h, light:dark, 22 • C condition. Fourweek-old potato plants were used for salt, wounding, heat, and SA hormone treatments at the indicated times.

Phylogenetic Analysis and Protein Sequence Alignment
Phylogenetic analysis was performed for potato StATG8 with Nicotiana tabacum (Nt), Solanum lycopersicum (Sl), Capsicum annuum (Ca), and Arabidopsis thaliana (At). The tree was constructed from an analysis conducted with MEGA-X software using a maximum likelihood method [51]. Phylogenetic tree was based on the nucleotide sequence data. Numbers along the branches (bootstrap value) show the percentage occurrence of nodes in 1000 replicates of stimulation. Solanaceae crops and the identified StATG8 proteins were compared using ClustalW with basic parameters.  Table S2.

Co-Immunoprecipitation
A. tumefaciens GV3101 strains carrying the plant expression constructs were diluted in an agroinfiltration medium (10 mM MgCl 2 , 5 mM 2-[Nmorpholine]-ethanesulfonic acid [MES], pH 5.6) to a final OD 600 of 0.5. For transient co-expression assays, agro-cells were infiltrated in N. benthamiana [53]. The leaves were ground into a fine powder in liquid nitrogen with a mortar and pestle. Ground tissue was mixed with a GTEN buffer (150 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10% (w/v) glycerol; 10 mM EDTA) augmented with 10 mM dithiothreitol, 2% (w/v) PVPP, and 1% (v/v) protease inhibitor cocktail (Sigma, MO, USA). Co-IP was performed following the protocol described previously [54]. Immunoprecipitation was performed using GFP Trap A beads (Proteintech, Manchester, UK) and mixing the beads by turning end-over-end for two hours in the cool room. Immunoblot was performed with anti-GFP (Proteintech, Manchester, UK) and anti-FLAG (Sigma, MO, USA) antibodies.

Conclusions
Although the plant ATG8 family exhibits great redundancy, we found that potato StATG8 gene members are subject to specific regulatory actions at multiple stresses or developmental stages. Furthermore, the protein interaction between StATG8 and WKRY transcription factor may be one of the mechanisms regulating autophagy. Further studies