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Overexpression of Banana ATG8f Modulates Drought Stress Resistance in Arabidopsis

Autophagy is essential for plant growth, development, and stress resistance. However, the involvement of banana autophagy-related genes in drought stress response and the underlying mechanism remain elusive. In this study, we found that the transcripts of 10 banana ATG8s responded to drought stress in different ways, and MaATG8f with the highest transcript in response to drought stress among them was chosen for functional analysis. Overexpression of MaATG8f improved drought stress resistance in Arabidopsis, with lower malonaldehyde level and higher level of assimilation rate. On the one hand, overexpression of MaATG8f activated the activities of superoxide dismutase, catalase, and peroxidase under drought stress conditions, so as to regulate reactive oxygen species accumulation. On the other hand, MaATG8f-overexpressing lines exhibited higher endogenous abscisic acid (ABA) level and more sensitivity to abscisic acid. Notably, the autophagosomes as visualized by CaMV35S::GFP–MaATG8f was activated after ABA treatment. Taken together, overexpression of MaATG8f positively regulated plant drought stress resistance through modulating reactive oxygen species metabolism, abscisic acid biosynthesis, and autophagic activity.


Introduction
Drought stress is one of the most serious environmental threats, resulting in large yield loss in agriculture [1]. With the change of global warming, drought stress is becoming more and more serious through influencing the dynamic regulation between crops and soil [2]. In order to survive under adverse environmental stresses such as drought stress, plants have developed complex mechanisms [3].
So far, abscisic acid (ABA) is widely known for its involvement in plant drought stress responses. ABA was firstly found in wheat under drought stress conditions [4]. Thereafter, the correlation between ABA and drought stress has been studied and proved in sugarcane [5], sorghum [6], maize [6], rice [7], barley [8], soybean [9], sunflower [10], and cowpea [11]. Endogenous ABA is largely and quickly induced in leaves under drought stress in plants [11]. Many genes, such as Arabidopsis ABA insensitive 1 (AtABI1), modulate drought stress resistance in the ABA-signaling pathway [12,13]. In addition, the increase of ABA level enhances plant tolerance to cold, waterlogging, and salt stresses [14]. In recent decades, more and more studies have shown the relationship between autophagy and ABA in plants, especially under drought stress conditions [15][16][17][18].
As a conserved procession, autophagy participates in the protein degradation pathway in lysosome and vacuole [19,20]. In this process, substances to be degraded such as misfolded proteins in the cell are surrounded by autophagosome possessing bilaminar membrane, and then digested by

Plant Transformation and Screening of Transgenic Plants
The recombinant plasmid pEGAD-MaATG8f (PromoterCaMV35S::GFP-MaATG8f ) has been described previously [44,46]. Briefly, the sequence of MaATG8f was cloned into pEGAD vector in the C-terminal in frame with green fluorescent protein (GFP) to form the constructs of PromoterCaMV35S::GFP-MaATG8s. In this study, PromoterCaMV35S::GFP-MaATG8f was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into the wild-type (WT, Col-0) Arabidopsis, according to the reported method [47]. The MaATG8f -overexpressing lines were selected by Basta resistance and confirmed by PCR and quantitative real-time PCR (qRT-PCR) analysis. The primers used are listed in Table S1.

Plant Materials and Treatments
Banana seedlings and Arabidopsis seedlings (WT and MaATG8f -overexpressing lines) were cultivated in the mixed soil (vermiculite/nutrition soil = 2:1, V:V) in green house at 25 ± 1 • C, with 16 h light/8 h dark cycles. For drought stress treatment, about 1-month-old banana and Arabidopsis seedlings in the soil were withheld water for designed days, while the control seedlings were watered every 4 days. At designed time-points of treatments, banana or Arabidopsis leaves were collected for the assays of physiological parameters. Total RNA extraction, purification, and first-strand cDNA synthesis were carried out by using the  RNAprep Pure Plant Plus Kit (TIANGEN, DP441, Beijing, China), RNase-freeDNase (NEB, M0303S,  Ipswich, MA, USA), and the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, K1622, Waltham, MA, USA), respectively. All procedures were conducted according to the manufacturer's instructions. The mixture of cDNA, primers, and TransStart Tip Green qPCR SuperMix (TransGen Biotech, AQ141, Beijing, China) were reacted in the LightCycler ® 96 Real-Time PCR System (Roche, Basel, Switzerland) for quantitative real-time PCR. All the transcript levels were analyzed using the comparative ∆∆Ct method in comparison to the reference gene Musa acuminata Ribosomal Protein S (MaRPS) [48]. The primers used are listed in Table S1.

Measurement of Assimilation Rate
The assimilation rate in the 5th to 8th leaves were determined using the Portable Photosynthesis System (Hansatech, CIRAS-3, Norfolk, UK) with 1.75 cm 2 area chamber. The internal light PAR was set at 1200 µmol m −2 s −1 , and the carbon dioxide concentration was 380-400 ppm. Then, the assimilation rate was directly calculated by CIRAS-3.

Analysis of ABA Sensitivity
ABA level was detected by ABA ELISA Kit (Haling, HL97003, Shanghai, China). Five-day-old seedlings were transferred on MS with 0, 1, 5, and 10 µmol/L ABA for 8 days to measure primary root length. Simultaneously, surface-sterilized seeds from each MaATG8f -overexpressing line and the control were cultured on MS with or without 0, 0.5, 1, and 2 µmol/L ABA for 6 days to record the ratio of green cotyledons.

Observation of Autophagosome
Five-day-old seedlings of WT and MaATG8f -overexpressing lines growing on MS medium were transferred into MS medium with or without 20 µmol/L ABA for 1 day. Then, the fluorescence at 900 ms through GFP channel in elongation zone of roots was examined by fluorescence microscope (Leica, DM5000, Wetzlar, Germany).

Statistical Analysis
There were at least three biological replicates for every experiment. All data in this study were analyzed using ANOVA and Student's t-test, and expressed as means ± standard deviation (SD). The asterisk (*) was used to indicate the difference at p < 0.05 in comparison to WT.

The Transcript Levels of MaATG8s in Response to Drought Stress
First of all, the transcript levels of MaATG8s were analyzed by qRT-PCR under drought stress treatment for 0-25 days ( Figure 1). Generally, the transcript levels of all MaATGs were regulated under drought stress conditions by different ways. Among them, the transcript level of MaATG8f showed a constant rising trend within 25 days, and it was much higher than the transcript levels of other MaATG8s at 25 days of drought stress treatment ( Figure 1). Due to the highest expression in response to drought stress treatment, MaATG8f was chosen for functional analysis.

The Transcript Levels of MaATG8s in Response to Drought Stress
First of all, the transcript levels of MaATG8s were analyzed by qRT-PCR under drought stress treatment for 0-25 days ( Figure 1). Generally, the transcript levels of all MaATGs were regulated under drought stress conditions by different ways. Among them, the transcript level of MaATG8f showed a constant rising trend within 25 days, and it was much higher than the transcript levels of other MaATG8s at 25 days of drought stress treatment ( Figure 1). Due to the highest expression in response to drought stress treatment, MaATG8f was chosen for functional analysis.

Overexpression of MaATG8f Increases Drought Stress Resistance in Arabidopsis
To investigate the function of MaATG8f, MaATG8f was overexpressed in Arabidopsis. In total, 18 MaATG8f-overexpressing lines were obtained and validated by PCR (Figure 2A), as well as qRT-PCR ( Figure 2B). Based on the transcript levels of MaATG8f in the transgenic lines, four independent transgenic lines (OE1, OE4, OE6, and OE7) were selected for the analysis of phenotype. Under normal condition, there was no difference of phenotype among these four transgenic lines. Under drought stress conditions, the leaves of MaATG8f-overexpressing lines leaves were less shriveling and greener than that of WT ( Figure 2C). Consistently, OE1, OE4, OE6, and OE7 showed significantly lower MDA level than WT during drought stress treatment (7, 14, and 21 days), especially at 21 days of drought stress treatment ( Figure 2D). This result shows that MaATG8f-overexpressing lines exhibited much better performance than WT in response to drought stress, indicating the protective role of MaATG8f in drought stress response.

Overexpression of MaATG8f Increases Drought Stress Resistance in Arabidopsis
To investigate the function of MaATG8f, MaATG8f was overexpressed in Arabidopsis. In total, 18 MaATG8f -overexpressing lines were obtained and validated by PCR (Figure 2A), as well as qRT-PCR ( Figure 2B). Based on the transcript levels of MaATG8f in the transgenic lines, four independent transgenic lines (OE1, OE4, OE6, and OE7) were selected for the analysis of phenotype. Under normal condition, there was no difference of phenotype among these four transgenic lines. Under drought stress conditions, the leaves of MaATG8f -overexpressing lines leaves were less shriveling and greener than that of WT ( Figure 2C). Consistently, OE1, OE4, OE6, and OE7 showed significantly lower MDA level than WT during drought stress treatment (7, 14, and 21 days), especially at 21 days of drought stress treatment ( Figure 2D). This result shows that MaATG8f -overexpressing lines exhibited much better performance than WT in response to drought stress, indicating the protective role of MaATG8f in drought stress response.

Determination of ROS and the Activity of Antioxidant Enzymes
The endogenous H 2 O 2 and O 2 − levels that were visualized by DAB staining and NBT staining, respectively, can directly reflect the degree of oxidative damage in plants. Under control conditions, there were no significant differences in both brown color by DAB staining and blue zone by NBT staining between WT and MaATG8f -overexpressing leaves. Under drought stress conditions, MaATG8f -overexpressing leaves displayed lighter brown color by DAB staining and less blue zone by NBT staining than WT leaves ( Figure 3A).

Determination of ROS and the Activity of Antioxidant Enzymes
The endogenous H2O2 and O2 − levels that were visualized by DAB staining and NBT staining, respectively, can directly reflect the degree of oxidative damage in plants. Under control conditions, there were no significant differences in both brown color by DAB staining and blue zone by NBT staining between WT and MaATG8f-overexpressing leaves. Under drought stress conditions, MaATG8f-overexpressing leaves displayed lighter brown color by DAB staining and less blue zone by NBT staining than WT leaves ( Figure 3A). Consistently, quantification of H2O2 ( Figure 3B) and O2 − ( Figure 3C) demonstrated that MaATG8f-overexpressing lines accumulated much lower levels of H2O2 and O2 − than WT in the leaves during drought stress.
To further reveal the role of MaATG8f on the activity of antioxidant enzymes, three key enzymes (POD, SOD, and CAT) were chosen for analysis. SOD serves as the key antioxidant enzyme to catalyze O2 − into H2O2 and O2 − , while POD and CAT can directly break down H2O2 to H2O [52]. Transgenic lines displayed significantly higher POD and SOD activities than WT upon drought stress ( Figure 3D,E). In addition, CAT activity in MaATG8f-overexpressing lines was higher than that in WT ( Figure 3F). Therefore, higher activity of these antioxidant enzymes might contribute to lower ROS accumulation in MaATG8f-overexpressing leaves in response to drought stress.  In these assays, the 5th to 8th leaves in the rosette were taken for the study. n = 3, *p < 0.05.

Overexpression of MaATG8f Promotes Photosynthesis Efficiency
To explore the relationship between autophagy and photosynthesis, assimilation rate was detected during drought stress treatment. Under drought stress conditions for 21 days, the photosynthesis rate in four overexpressing lines was significantly higher than WT leaves (Figure 4). This result indicates that overexpression of MaATG8f could maintain effective photosynthesis rate in response to drought stress. activity. (F) Catalase (CAT) activity. In these assays, the 5th to 8th leaves in the rosette were taken for the study. n = 3, * p < 0.05.
To further reveal the role of MaATG8f on the activity of antioxidant enzymes, three key enzymes (POD, SOD, and CAT) were chosen for analysis. SOD Figure 3D,E). In addition, CAT activity in MaATG8f -overexpressing lines was higher than that in WT ( Figure 3F). Therefore, higher activity of these antioxidant enzymes might contribute to lower ROS accumulation in MaATG8f -overexpressing leaves in response to drought stress.

Overexpression of MaATG8f Promotes Photosynthesis Efficiency
To explore the relationship between autophagy and photosynthesis, assimilation rate was detected during drought stress treatment. Under drought stress conditions for 21 days, the photosynthesis rate in four overexpressing lines was significantly higher than WT leaves (Figure 4). This result indicates that overexpression of MaATG8f could maintain effective photosynthesis rate in response to drought stress. In these assays, the 5th to 8th leaves in the rosette were taken for the study. n = 3, *p < 0.05.

Overexpression of MaATG8f Promotes Photosynthesis Efficiency
To explore the relationship between autophagy and photosynthesis, assimilation rate was detected during drought stress treatment. Under drought stress conditions for 21 days, the photosynthesis rate in four overexpressing lines was significantly higher than WT leaves (Figure 4). This result indicates that overexpression of MaATG8f could maintain effective photosynthesis rate in response to drought stress.

Overexpression of MaATG8f Increases the Sensitivity to ABA in Arabidopsis
Under control condition, the endogenous ABA level in MaATG8f -overexpressing leaves was significantly higher than that in WT leaves ( Figure 5). When they grew on MS plate with different concentrations of ABA, MaATG8f -overexpressing lines were more sensitive to ABA, with lower green cotyledon rate and shorter primary root length ( Figure 6A-D). Generally, MaATG8f -overexpressing lines regulated both endogenous ABA level and plant sensitivity to exogenous ABA.

Overexpression of MaATG8f Increases the Sensitivity to ABA in Arabidopsis
Under control condition, the endogenous ABA level in MaATG8f-overexpressing leaves was significantly higher than that in WT leaves ( Figure 5). When they grew on MS plate with different concentrations of ABA, MaATG8f-overexpressing lines were more sensitive to ABA, with lower green cotyledon rate and shorter primary root length ( Figure 6A-D). Generally, MaATG8f-overexpressing lines regulated both endogenous ABA level and plant sensitivity to exogenous ABA.

Overexpression of MaATG8f Promotes the Generation of Autophagosomes byABA
Under normal condition, the signal of GFP-MaATG8f was detected in the membrane and nucleus. When ABA was applied, the fluorescence of GFP-MaATG8f was visualized in both the membrane and cell cytoplasm. Notably, many fluorescent spots were induced by ABA in the elongation zone of MaATG8f -overexpressing roots (CaMV35S::GFP-MaATG8f ) (Figure 7), indicating the generation of autophagosomes by ABA treatment.

Overexpression of MaATG8f Promotes the Generation of Autophagosomes byABA
Under normal condition, the signal of GFP-MaATG8f was detected in the membrane and nucleus. When ABA was applied, the fluorescence of GFP-MaATG8f was visualized in both the membrane and cell cytoplasm. Notably, many fluorescent spots were induced by ABA in the elongation zone of MaATG8f-overexpressing roots (CaMV35S::GFP-MaATG8f) (Figure 7), indicating the generation of autophagosomes by ABA treatment.

Discussion
Autophagy that is mediated by ATGs is vital for plant metabolism in plant stress resistance and defense responses [53]. To date, plant ATGs are widely involved in plant stress responses. High salt and osmotic stresses induce the transcript level of AtATG18a, and deficiency of ATG18a decreases the resistance to salt and drought [25]. Moreover, the transcript levels of nine ATGs in Capsicum annuum L. are upregulated under salt stress, and silence of CaATG8c decreases plant resistance to osmotic and salt stress [43]. On the contrary, overexpression of ATGs can enhance stress resistance in multiple plant species [16]. In our previous study, 32 ATGs were identified in banana. Among them, MaATG8s with 10 members is the largest subfamily and the transcripts of them were commonly regulated by Fusarium oxysporum f. sp. cubense [44]. Herein, the transcript level of most MaATG8s was upregulated by drought stress. Consistent with ATGs in other plant species, overexpression of MaATG8f in Arabidopsis enhanced the adaptation to drought stress and alleviated the damage of lipid peroxidation

Discussion
Autophagy that is mediated by ATGs is vital for plant metabolism in plant stress resistance and defense responses [53]. To date, plant ATGs are widely involved in plant stress responses. High salt and osmotic stresses induce the transcript level of AtATG18a, and deficiency of ATG18a decreases the resistance to salt and drought [25]. Moreover, the transcript levels of nine ATGs in Capsicum annuum L. are upregulated under salt stress, and silence of CaATG8c decreases plant resistance to osmotic and salt stress [43]. On the contrary, overexpression of ATGs can enhance stress resistance in multiple plant species [16]. In our previous study, 32 ATGs were identified in banana. Among them, MaATG8s with 10 members is the largest subfamily and the transcripts of them were commonly regulated by Fusarium oxysporum f. sp. cubense [44]. Herein, the transcript level of most MaATG8s was upregulated by drought stress. Consistent with ATGs in other plant species, overexpression of MaATG8f in Arabidopsis enhanced the adaptation to drought stress and alleviated the damage of lipid peroxidation in plants, confirming that MaATG8f is a positive regulator of drought stress resistance. Therefore, the function of plant ATG8s may be conserved in multiple plants, and they may be used as ideal genes for stress related molecular breeding.
ROS, including H 2 O 2 , O 2 − , and free radicals, can oxidize lipids, nucleic acids, and amino acid precursors, which could be considered as oxidative stress [54,55]. Oxidative stress leads to mitochondrial dysfunction and cell injury [56]. In this study, overexpression of MaATG8f alleviated the oxidative damage through modulation of ROS accumulation under drought stress conditions. The relationship between autophagy and ROS has been revealed. On the one hand, autophagy might be involved in degradation of ROS [31]. On the other hand, ROS could also initiate autophagosome formation and autophagic degradation, acting as cellular signaling molecules [57]. Meanwhile, autophagy can assist to scavenge oxidative damage and decrease ROS level through promoting the degradation of misfolded proteins and injured organelles [58]. Briefly, ROS plays an important role in the process of autophagy and is modulated by autophagy. Therefore, the dual roles of MaATG8f in scavenging the burst of ROS under drought stress conditions and triggering autophasomes might be directly related, and the complex effect resulted in improved drought stress resistance. Photosynthesis is restrained by drought stress in plants [59,60]. Herein, overexpression of MaATG8f in plants could keep photosynthesis with higher assimilation rate under drought stress conditions, which might contribute to improved drought stress resistance. This result is consistent with the higher assimilation rate in MdATG18a-overexpressing plants [16]. Therefore, overexpression of ATG keeps the efficiency of photosynthetic carbon assimilation, which may facilitate nutrient accumulation and promote carbon transportation under stress conditions. Chloroplasts are vital for solar light utilization and photosynthesis. Autophagy has a critical role in chloroplast degradation and might constitute a mechanism of dynamic control in chloroplasts [61]. Based on the vital role of autophagy in photosynthesis, the higher assimilation rate in MaATG8f -overexpressing lines might serve for better photosynthesis under drought stress conditions. In chloroplasts, ROS induced by abiotic stress break the balance of the redox system. In addition, the enhancement of assimilation in MaATG8f -overexpressing lines might also be a result of the improvement of the antioxidant system under drought stress. However, the underlying mechanism of autophagy in modulating the photosynthetic implementation or assisting in repairing important proteins needs to be investigated.
As an isoprenoid phytohormone, ABA regulates many plant stress responses, especially drought stress response [62]. Meanwhile, abiotic stress can activate ABA biosynthesis and the ABA signaling transduction pathway as a kind of systemic acquired acclimation [63]. Not only does ABA modulate stomatal closing to keep plant leaf water status and decrease water loss, but also the ABA signaling transduction pathway, including ABA receptor, the related kinases, transcription factors, and downstream genes, is involved in plant drought stress response [64][65][66][67][68][69][70]. Therefore, endogenous ABA level is directly related to plant leaf water status, and the activation of the key components in the ABA signaling transduction pathway is also related to plant drought stress resistance [71,72]. In this study, MaATG8f -overexpressing lines showed higher endogenous ABA level and more sensitivity to ABA than WT. Because of the essential role of ABA level and ABA sensitivity in plant drought stress response [64][65][66][67][68], the modulation of MaATG8f on ABA biosynthesis and ABA sensitivity might be directly related to its role in drought stress response. Although MaATG8f positively regulates drought stress resistance in the ABA-dependent pathway, the role of MaATG8f in the activation of the key components in the ABA signaling transduction pathway remains to be further investigated.
Consistent with the induction of the transcripts of plant ATGs by multiple stresses and ABA [73], the formation of autophagosomes can also be significantly induced by them [37], but inhibited by the silencing of ATGs [74]. Autophagy can regulate the protein level of TSPO (tryptophan-rich sensory protein) in ABA signaling, especially under drought stress conditions [75]. Herein, we found that autophagosomes labeled by GFP-MaATG8f were activated by ABA treatment, indicating the involvement of MaATG8f and autophagy in ABA-mediated plant stress responses. We have to note that MaATG8f may have multiple targets, which may be regulated at the protein level. The identification of direct targets of MaATG8f may provide novel insight into the correlation between MaATG8f -mediated autophagy, ABA biosynthesis and signaling, as well as ROS metabolism. Although there may be some differences in the function of MaATG8s between Arabidopsis and banana, this study, together with other studies about plant MaATG8s [45], indicates that MaATG8f might be used as an ideal candidate for crop genetic breeding.

Conclusions
In conclusion, this study has shown that overexpression of MaATG8f results in improved drought stress resistance through activating the activities of antioxidant enzymes, regulating ROS accumulation, and activating endogenous ABA level and autophagic activity.