MdATG8i functions positively in apple salt tolerance by maintaining photosynthetic ability and increasing the accumulation of arginine and polyamines

Environmental and Experimental Botany 172 (2020) 103989

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MdATG8i functions positively in apple salt tolerance by maintaining photosynthetic ability and increasing the accumulation of arginine and polyamines

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Liuqing Huoa,1, Zijian Guoa,1, Ping Wanga, Zhijun Zhanga, Xin Jiaa, Yiming Suna, Xun Sunb, Xiaoqing Gonga,*, Fengwang Maa,* a State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, 712100, China b Center of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Autophagy Salt stress MdATG8i Stoma Arginine Polyamines

Autophagy has been widely reported to play critical roles in plant adaption to various abiotic and biotic stressors. Although many studies have shown that autophagy-deficient mutants exhibit sensitivity to salt stress, our understanding of the detailed function of autophagy in response to salt stress remains limited. In this study, we found that the expression of MdATG8i was induced by salt stress. We treated transgenic apple plants overexpressing MdATG8i with salt stress, and explored its biological role in response to salt treatment. The results showed that the growth limitation, reactive oxygen species generation and Na+ accumulation caused by salt stress were alleviated in transgenic plants. The shrinkage of stomata and the damage to photosynthetic ability caused by salt stress were diff ;erentially mitigated. These changes were accompanied by the elevated autophagic activity and the accumulation of proline, arginine and its downstream polyamine products in two transgenic lines, which play important roles in plant tolerance to salt stress. In summary, these results revealed that MdATG8i-mediated autophagy enhanced salt tolerance in apple, primarily by alleviating the decrease in carbon assimilation and the accumulation of compatible osmolytes.

1. Introduction Salinity stress is becoming a significant environmental challenge due to its adverse effects on plant growth and development. It is estimated that soil salinity affects more than 800 million hectares of arable land, accounting for one-third of worldwide food production (Yang and Guo, 2018). Among all types of salt, the most soluble and widely used salt is NaCl (Ismail et al., 2014). The harmful effects of salinity can change almost every aspect of the plant, including photosynthesis, respiration, chlorophyll content and fluorescence, ion compartmentation and exclusion, water absorption, antioxidant defences and membrane stability, all of which apparently result in yield reductions (Muscolo et al., 2015; Zhang et al., 2017a). High salt levels generally induce osmotic stress, ionic stress and secondary stress. First, excess soluble salts reduce water availability to the plant, leading to a water deficit, which is the osmotic effect of salinity (Munns, 2005). Second, ionic

toxicity associated with high concentrations of Na+ in the cytoplasm leads to deficiencies in other ions, which has adverse effects on many metabolic pathways (Acosta-Motos et al., 2017). Third, long-term salt stress can cause secondary stress in plants due to the disruption of the nutrient balance (Yang and Guo, 2018). In addition, excessive uptake of salt can induce the accumulation of reactive oxygen species (ROS) in plant cells (Mittova et al., 2004), which would affect the photosynthetic ability of plants. All of these responses to salinity cause detrimental effects on plants, leading to limitations in plant growth and productivity. Numerous studies have been conducted to unravel the mechanisms regulating plant adaptation and tolerance to salt stress. Salt-induced ROS generation causes serious damage to plants; therefore, the ROS concentration in plants, which is regulated by enzymatic and non-enzymatic systems, has been widely researched in plants under salt stress (Miller et al., 2010). The salt overly sensitive (SOS) signalling cascade

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Corresponding authors. E-mail addresses: [email protected] (X. Gong), [email protected], [email protected] (F. Ma). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.envexpbot.2020.103989 Received 23 September 2019; Received in revised form 13 January 2020; Accepted 15 January 2020 Available online 17 January 2020 0098-8472/ © 2020 Elsevier B.V. All rights reserved.

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2. Materials and methods

has been discovered, with which plants maintain ion homeostasis in tissues and cells to avoid an excess of toxic substances (Ji et al., 2013; Zhao et al., 2011). For example, the plasma membrane Na+/H+ exchanger SOS1 controls net root Na+ uptake and long-distance Na+ transport to shoots, thereby playing a major role in salt tolerance of rice (El Mahi et al., 2019). In addition, the ability of plants to maintain appropriate photosynthetic performance could be an important factor in plant salt tolerance (Acosta-Motos et al., 2017). The biosynthesis and accumulation of compatible osmolytes, such as sugar alcohols (Eggert et al., 2016), polyamines (PAs) (Mansour and Ali, 2017) and proline (Pro) (Zapata et al., 2017), is a primary adaptive strategy under salt stress, which reduces the osmotic potential of the cell. Large changes in amino acids and carbohydrates have been observed under salt stress (Fougere et al., 1991; Zhang et al., 2017b). For example, as a protective osmolyte, Pro has been reported to have antioxidant properties, and thus Pro metabolism is conjointly related to salt, osmotic and dehydration stress tolerance (Dai et al., 2018; EstradaMelo et al., 2015). Additionally, arginine (Arg), one of the most metabolically versatile amino acids, participates in various physiological processes (Shi et al., 2013). Arg is an important amino acid with a high N/C ratio in plants, serving as a medium for the nitrogen reserve in apple (Gao et al., 2009). As a substrate for PAs and nitric oxide, both endogenous and exogenous Arg have been reported to participate in various physiological processes (Hassan and Mohamed, 2019; Patel et al., 2017). Recently, changes in the PAs, spermidine (Spd) and spermine (Spm), together with their diamine precursor putrescine (Put), have been suggested to be an important response of plants to salt stress (Liu et al., 2006; Yin et al., 2019). All of these studies demonstrate that the biosynthesis and accumulation of compatible osmolytes is a very important adaptive strategy for the stress response. Autophagy is a highly conserved and regulated catabolic process that allows the recycling of cell components into primary molecules. It is a ubiquitous recycling system that is involved in the degradation of cytoplasmic constituents, including proteins and organelles, allowing the maintenance of amino acid pools and nutrient remobilization (AvinWittenberg et al., 2018; Li and Vierstra, 2012). In plants, autophagy has been shown to be induced during abiotic stress, including a high salt condition (Liu et al., 2009; Luo et al., 2017). Several lines of evidence suggest that NaCl treatment induces the transcription of several autophagy-related (ATG) genes. In Arabidopsis, RNAi-AtATG18a plants are more sensitive to salt conditions than wild-type (WT) plants (Liu et al., 2009). The autophagy-deficient mutants atg5 and atg7 exhibit sensitivity towards drought and salt treatments (Luo et al., 2017), and plants overexpressing ATG8 perform better than the WT in germination assays under salt treatment (Zhou et al., 2013). These findings suggest that salt stress rapidly triggers autophagy and facilitates bulk protein turnover; thus, providing the molecules and energy required for plant survival. Additionally, autophagy has been demonstrated to be an important component of amino acid metabolism. For example, autophagy provides a supply of energy, such as amino acids, instead of sugars in plants exposed to constant darkness (Izumi et al., 2013). In our previous study, we characterized the ATG8s gene, MdATG8i, in apple, and demonstrated that it possesses a conserved function in the apple autophagy process (Wang et al., 2016). To better understand the involvement of autophagy in the apple stress response, we sought to functionally analyse the reaction of MdATG8i-overexpressing apple plants under salt stress. We found that transgenic plants overexpressing MdATG8i exhibited enhanced salt tolerance, as evidenced by the mitigation of growth limitation, ROS generation, Na+ accumulation and damage to the photosynthetic ability caused by salt stress. In addition, these responses were accompanied by the accumulation of Pro, Arg and PAs in transgenic plants, which was also closely associated with MdATG8i-mediated salt tolerance. This study increases our understanding of the metabolic mechanisms underlying the autophagy-related amelioration of salinity stress in plants.

2.1. Plant materials and salt treatments Tissue-cultured GL-3 plants were cultured as described previously (Sun et al., 2018b). After 30 d on rooting media, transgenic and nontransgenic apple (Malus domestica) plantlets were transferred to small plastic pots (8 × 8 cm) containing a mixture of loam/perlite (1:1, v:v). After 3 weeks of adaptation to the growth chamber, the plants were moved to medium plastic pots (10 × 10 cm) and grown in a glasshouse. They were watered regularly and supplied with half-strength Hoagland’s nutrient solution (pH 6.0) once a week. Transgenic and WT apple plants of similar size were transferred to a hydroponics system after 40 d of growth and cultured for the hydroponics salt tolerance assay as described previously (Hu et al., 2018). After a 2-week preincubation, plants of uniform size were selected for treatment in half-strength Hoagland’s nutrient solution supplemented with 75 mM NaCl. Plants treated without NaCl were used as the control. At 0, 2, 4, 8, 12, and 24 h after applying the salt, the third and fourth leaves from the apex of the stem were sampled from six GL-3 plants for the MdATG8i expression analysis. After 10 and 20 d of this experiment, the third through sixth leaves from the apex of the stem (fully mature leaves) were sampled from six plants per strain for physiological analyses. The samples were stored at −80 °C after being frozen quickly in liquid nitrogen. 2.2. Construction of plasmids and generation of transgenic apple plants To construct the vector for the overexpressing lines, the MdATG8i coding region was introduced into the pCambia2300 vector, which was driven by the CaMV 35S promoter and carried the kanamycin (Kan) selectable marker in plants. The sequencing-confirmed plasmid was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. The primers used for constructing the vector are listed in Supplemental Table S3. Transformation of apple was generated from leaf fragments using GL-3 as the genetic background, as described previously (Dai et al., 2013). Regenerated Kan-resistant buds were sub-cultured every 3 weeks on MS medium containing 25 mg L−1 Kan as a selectable marker. Lines showing normal growth were evaluated by polymerase chain reaction (PCR) analysis of the extracted DNA. Overexpression of MdATG8i was confirmed by quantitative real-time PCR (qRT-PCR). Untransformed GL-3 plants were cultured in the same way without selection pressure and served as the control. 2.3. RNA extraction, DNA isolation and qRT-PCR analysis Total RNA was extracted using a Wolact plant RNA isolation kit (Wolact, Hong Kong, China), and first-strand cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) with 1 μg total RNA. Genomic DNA was isolated with a Wolact Plant Genomic DNA purification kit (Wolact, Hong Kong, China). The qRT-PCR analysis was carried out as described previously after 20 d of salt stress (Gong et al., 2017). The primers used are listed in Supplemental Table S3. 2.4. Physiological analysis and histochemical staining Relative electrolyte leakage (REL), relative water content (RWC) and chlorophyll concentrations in the leaves were determined according to a method described previously (Sun et al., 2018b; Wang et al., 2011). Chlorophyll concentrations were represented by SPAD values, as tested with the SPAD-502 Plus device (Konika-Minolta, Tokyo, Japan). The activities of superoxide dismutase (SOD) and catalase (CAT), and the levels of H2O2, malondialdehyde (MDA), and superoxide radical (O2−) were determined using detection kits (Suzhou 2

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Comin Biotechnology Co., Ltd., Suzhou, China) following the manufacturer’s instructions. In situ accumulation of H2O2 and O2− was examined by histochemical staining with DAB (3,3’- diaminobenzidine) and NBT (nitro blue tetrazolium), respectively (Sun et al., 2018b). Na+ and K+ concentrations were determined by flame spectrometry (M410; Sherwood Scientific, Cambridge, UK) as described previously (Liang et al., 2018). 2.5. Growth measurements To evaluate the difference in seedling growth of WT and transgenic apples after the salt treatment, nine plants per strain were collected after the hydroponic salt treatment. Plant heights were measured from the stem base to the terminal bud of the main stem. Whole plants were divided into root, stem and leaf portions. The fresh and dry weights were measured as described previously (Liang et al., 2018). Briefly, after the fresh weight of each sample plant was recorded, the dry weight was obtained after the plants were fixed at 105 °C for 15 min and oven-dried at 75 °C for at least 72 h to a constant weight. Relative growth was calculated as the weight of the salt-treated plant divided by the weight of the control plant.

Fig. 1. Changes in the expression of MdATG8i after treatment with salt in GL-3 apple plants. Data are shown as the means of three replicates with SE.

% perchloric acid buffer and incubated on ice for 30 min. After centrifugation, the supernatant was filtered through a 0.22-μm filter for analysis with the same system and column as applied for the AAs. The solvent system consisted of water containing 0.1 % (v/v) formic acid (A) and acetonitrile containing 0.1 % (v/v) formic acid (B). ABA was extracted as described previously (Guo et al., 2019) and measured using the LC–MS system. Briefly, 200 mg of frozen leaf sample was extracted in 2 ml solvent (methanol: isopropanol, 20: 80 (v/ v) with 1 % glacial acetic acid). After centrifugation, the supernatant was filtered through a 0.22-μm filter for analysis using the same system described above but equipped with the InertSustain AQ-C18 column (4.6 × 150 mm, 5 μm) at a flow rate of 0.5 ml/min. The solvent system consisted of water containing 0.1 % (v/v) formic acid (A) and methanol (B).

2.6. Evaluation of photosynthetic characteristics and chlorophyll fluorescence On days 5, 10, 15 and 20 of the hydroponics experiment, the net photosynthesis rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) were monitored between 9:00 and 11:00 a.m. using a CIRAS-3 portable photosynthesis system (CIRAS, Amesbury, MA, USA). All measurements were recorded at 1000 μmol photons m−2 s-1 and a constant airflow rate of 500 μmol s-1. The concentration of CO2 was set to 400 ± 5 cm3 m-3. Data were collected from fully expanded, fully light-exposed leaves from eight plants. Chlorophyll fluorescence transients were measured on leaves at the same position from selected plants after 20 min of dark adaptation using the Open FluorCam FC 800-O as described previously (PerezBueno et al., 2015), and Fv/Fm ratios were calculated with Fluorcam7 software (PSI, Brno, Czech Republic).

3. Results 3.1. Overexpression of MdATG8i leads to enhanced salt tolerance in apple In this study, the expression of MdATG8i was induced by the NaCl treatment in GL-3 plants (Fig. 1). To investigate the possible functions of MdATG8i under salt stress in apple, we generated two overexpressing (OE) lines in which MdATG8i expression levels were noticeably elevated compared with the WT. The transcripts were constitutively increased by 5.4- and 10.1-fold in lines OE-1 and OE-6, respectively (Fig. 2a, b). To determine the function of MdATG8i in the defence mechanism of apple in response to salt stress, we used a hydroponics system to investigate the performance of plants after 20 d of treatment with 75 mM NaCl. Under normal hydroponic conditions, the phenotypes did not differ apparently between the OE lines and WT (Fig. 2c). Treatment with NaCl for 20 d resulted in decreased apple plant height, but the transgenic lines were affected to a less serious extent than the WT (Fig. 2d). The fresh and dry weights of the transgenic plants were less affected by NaCl than that of the WT plants compared to culturing under normal conditions (Fig. 2e, f). The growth limitation of each tissue caused by salt stress was also lessened in the OE lines than in WT (Table S1, S2). Additionally, the REL and MDA concentrations increased significantly due to the injury caused by salt stress, but they were still much lower in the transgenic lines than in the WT plants (Fig. 2g, h). Total chlorophyll concentrations decreased after the salt treatment, but the reduction was much smaller in OE plants compared with the WT (Fig. 2i). All of these results show that overexpressing MdATG8i enhanced salt tolerance in apple.

2.7. Observations of leaf stomata and autophagosomes On the last day of the experiment, mature leaves at the same position were excised from selected plants and immediately cut into small pieces in glutaraldehyde solution. The leaf stomata were observed with a JSM-6360LV scanning electron microscope as described previously (Liang et al., 2018). The autophagosomes were observed under a JEOL1230 transmission electron microscope (TEM, Hitachi, Tokyo, Japan) as described previously (Sun et al., 2018b). 2.8. Measurements of abscisic acid (ABA), amino acids (AAs) and free PAs AAs were extracted and measured as described previously (Jin et al., 2019), with a minor modification. Briefly, 200 mg of frozen leaf sample was extracted in 2 mL 50 % ethanol (include 0.1 M HCl) and centrifuged at 13,000 g for 10 min. The supernatant was added with methanol to a final volume of 10 mL. Samples were filtered through a 0.22μm filter to analyse the metabolites with a liquid chromatography mass spectrometry (LC–MS) system (SCIEX, QTRAP5500) equipped with an Inertsil ODS-4 C18 column (4.6 × 250 mm, 5 μm) at a flow rate of 0.3 mL/min. The solvent system consisted of water containing 0.1 % (v/v) formic acid (A) and acetonitrile (B). Data were quantified by comparing the peak surface areas with those obtained using standard AAs (Sigma, St. Louis, MO, USA). Free PAs were extracted and measured as described previously (Gong et al., 2017; Wang et al., 2017), with a slight modification. Briefly, 200 mg of frozen leaf sample was homogenized with 2 ml of 5

3.2. Apple lines overexpressing MdATG8i accumulate less ROS under salt stress The salt-induced stress response includes the accumulation of ROS, which can cause oxidative damage by disrupting cytomembranes and 3

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Fig. 2. Overexpression of MdATG8i confers enhanced salt tolerance to apple. (a) PCR confirmation of transgenic apple plants. Lanes: M, molecular marker DL2000; V, positive vector containing pCambia2300-MdATG8i plasmid; WT, non-transformed wild-type; OE-1 and -6, MdATG8i-transgenic apple lines. (b) qRT-PCR analysis of MdATG8i transcripts in Lines OE-1 and OE-6. (c) Phenotypes of WT and transgenic apple plants under normal hydroponic conditions and after 20 d of treatment with 75 mM NaCl. Bars: 5 cm. (d) Plant height, (e) total fresh weight, (f) total dry weight, (g) electrolyte leakage, (h) malondialdehyde (MDA) and (i) chlorophyll concentration in WT and transgenic plants treated with or without salt. Data are shown as the means of six replicates with SEs. Different letters indicate significant differences between treatments, according to one-way ANOVA followed by Tukey’s multiple range test (P < 0.05).

3.3. Overexpression of MdATG8i reduces the accumulation of Na+ in apple under salt stress

macromolecules, such as enzymes, DNA and lipids (Miller et al., 2010; Mittova et al., 2004). We used histochemical staining to examine the in situ accumulation of H2O2 and O2–. When staining separately with DAB for H2O2 and NBT for O2–, we found more intense brown coloration or blue patches on WT leaves compared to the transgenic plants in the presence of NaCl, suggesting that the OE lines accumulated less ROS than the WT (Fig. 3a). Moreover, all lines were similarly and lightly stained in the absence of salt stress. These results were further confirmed by quantitative measurements (Fig. 3b, c). In addition, the activity of SOD (O2−-scavenging enzyme) and CAT (H2O2-scavenging enzyme) increased significantly after the salt treatment, and a greater increase was detected in the transgenic lines (Fig. 3d, e). These data suggest that the transgenic lines had stronger toxic ROS-scavenging ability under salt stress than the WT.

As salinity is known to disturb Na+ and K+ homeostasis, we measured their concentrations in the roots, stems and leaves of seedlings. No distinct changes in Na+ or K+ concentrations were observed among the genotypes under normal growth conditions (Fig. 4a–f). Na+ increased dramatically during the response to salt stress, but all tissues of WT seedlings contained higher Na+ than the same tissues of the transgenic plants (Fig. 4a–c). K+ concentrations in the roots and stems decreased significantly in response to the salt stress, and there was no significant difference among the genotypes (Fig. 4d–f). As a result, the Na+/ K+ ratios in the roots and leaves were markedly lower in the transgenic lines compared with the WT (Fig. 4g–i). These findings indicate that the tolerance of transgenic apple plants to salt stress was correlated with the reduced Na+ accumulation. 4

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Fig. 3. Overexpression of MdATG8i in apple leads to enhanced ROS-scavenging ability under salt stress. (a) In situ accumulation of H2O2 and superoxide radical (O2−) in leaves treated with (right panels) and without (left panels) salt treatment revealed by DAB and NBT staining, respectively. Quantitative measurement of (b) H2O2 and (c) O2− concentrations in apple leaves with and without salt treatment. Activities of (d) superoxide dismutase (SOD) and (e) catalase (CAT) with and without salt treatment. Data are shown as the means of three replicates with SEs. Different letters indicate significant differences between treatments, according to one-way ANOVA followed by Tukey’s multiple range test (P < 0.05).

of salt treatment (Fig. 5c). In addition, we measured the maximum photochemical efficiency of photosystem II (PSII) photochemistry (Fv/ Fm), which was lower in WT plants than in the transgenic lines after the salt treatment, indicating reduced salt damage in PSII of the OE lines (Fig. 5d, e). These data suggest that the photosynthetic ability of plants overexpressing MdATG8i was less damaged during the salt treatment.

3.4. Apple lines overexpressing MdATG8i maintain a higher photosynthetic capacity under salt stress As there was a significant difference in biomass production between the OE lines and WT after salt treatment, we thought that the photosynthetic ability might be different among the genotypes. We applied continuous measurements of the gas exchange parameters for all lines during the salt treatment. Under normal growing conditions, Pn, Gs and Ci were consistent, and no conspicuous difference was detected between the WT and transgenic lines (Fig. 5a–c). These three parameters all decreased in response to the salt treatment, but the reduction was much less in the transgenic plants. In particular, Gs declined drastically after 10 d of salt treatment in the WT, but it was not apparently reduced in the OE lines until 15 d of salt stress (Fig. 5b). We further observed an increase in Ci that was not due to a change in Gs in WT plants after 20 d

3.5. Overexpression of MdATG8i in apple alleviates the shrinkage of stomata under salt stress Decreases in the stomatal aperture due to salinity have been found in several different plant species, limiting CO2 uptake by leaves and affecting photosynthesis (Banon et al., 2012; Gomez-Bellot et al., 2013). In our study, no difference in stomatal morphology was observed among the genotypes under normal conditions. We detected a reduced 5

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Fig. 4. Overexpression of MdATG8i in apple reduces the accumulation of Na+ under salt stress. Changes in concentrations of Na+ (a–c), K+ (d–f) and Na+: K+ ratios (g–i) in the roots, stems, and leaves of WT and transgenic apple plants. Data are shown as the means of three replicates with SEs. Different letters indicate significant differences between treatments, according to one-way ANOVA followed by Tukey’s multiple range test (P < 0.05).

salt stress, we analysed the concentrations of 15 AAs (Fig. 7). The analysis revealed that Ala, Arg, Glu, His, Ile, Pro and Ser increased significantly in all genotypes after 20 d of salt treatment. Intriguingly, some metabolites exhibited distinct changes with respect to the WT after saline stress, such as Ala, Arg, Glu, His and Pro, which increased greater in transgenic plants. For example, the level of Pro increased almost eight times compared with the control in all genotypes in response to salt stress, but it was nearly 1.4 times that of WT in the OE lines. In addition to Pro, we focused on the change in Arg concentrations. After salt treatment, the Arg contents in OE-1 and OE-6 were respectively 2.4 and 3 times that of WT. These data suggest that MdATG8i overexpression in apple increased the accumulation of certain AAs under salt treatment, which contributed to plant resistance to salt stress.

stomatal aperture caused by saline stress, particularly in WT plants, but no significant difference was found in stomatal density among the genotypes after the salt treatment (Fig. 6a–c). In addition, the shrinkage of the stomatal aperture caused by salt stress was less pronounced in the OE lines compared with the WT (Fig. 6d). As stomatal closure in saltstressed plants can be induced by the accumulation of ABA (Fricke et al., 2004; Mulholland et al., 2003), we measured ABA content in seedlings. Consistent with the stomatal behaviour, saline stress increased the level of ABA in apple seedlings. No difference in ABA concentration was observed between the OE and WT apples under favourable growth conditions, but ABA levels increased more in the WT than in the transgenic plants after the salt treatment, which might explain the difference in stoma morphology among the genotypes (Fig. 6e). Furthermore, the expression levels of MdNCED1 and MdNCED3, two ABA biosynthetic genes in apple, increased slightly in transgenic plants than in the WT after salt stress (Fig. 6f, g). These data suggest that endogenous ABA and stomatal closure were less sensitive in plants overexpressing MdATG8i under salt stress and therefore likely account for the MdATG8i-mediated higher photosynthetic capacity in the presence of salt.

3.7. MdATG8i overexpression enhances polyamine accumulation and increases the expression of polyamine-related genes during salt stress As a significant difference in Arg contents was observed between the OE lines and WT plants after the salt treatment, we measured the levels of PAs that used Arg as a substrate among the genotypes. PAs have been reported to activate antioxidant systems, thereby protecting plants from salt injury (Liu et al., 2006; Yin et al., 2019). Coincident with the change in Arg, we also detected more Put, Spm and Spd accumulation in transgenic plants compared to the WT plants after the salt treatment. After the saline stress, the Put and Spd levels in the OE lines were almost 1.4-fold that of the WT, while Spm content was more than twofold that of WT (Fig. 8a–c). To determine whether the change was

3.6. Amino acid metabolism is involved in MdATG8i-mediated salt tolerance When exposed to saline stress, a range of nitrogenous compounds, including AAs accumulate in plants, a phenomenon that is usually correlated with plant salinity tolerance (Mansour, 2000; Zhang et al., 2017b). To examine the relationship between AAs and MdATG8i upon 6

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Fig. 5. Overexpression of MdATG8i in apple leads to higher photosynthetic capacity under salt stress. Changes in the (a) net photosynthesis rate (Pn), (b) stomatal conductance (Gs) and (c) intercellular CO2 concentration (Ci) were determined every five days during the salt treatment. (d) Chlorophyll fluorescence images and (e) Fv/Fm ratios of WT and transgenic plants treated with and without salt. Data are shown as the means of three replicates with SEs. Different letters indicate significant differences between treatments, according to one-way ANOVA followed by Tukey’s multiple range test (P < 0.05).

3.8. Overexpression of MdATG8i in apple upregulates the expression of other MdATGs and elevates autophagic activity under salt stress

consistent during the entire salt treatment, we measured the Arg, Put, Spd and Spm concentrations in the seedlings after 10 d of salt treatment and observed a similar pattern (Fig. S1). As the apple genes involved in PA synthesis have been identified previously (Gong et al., 2017), we used qPCR analysis to examine their expression. As shown in Fig. 6d, ADC2, SAMDC1 and SAMDC3 were induced by salt stress, and the expression level of ADC2 was significantly higher in the transgenic lines than in the WT (Fig. 8d, h, i). In addition, while SPDS1 only slightly changed and SPDS5 was downregulated by salt stress in WT plants, they were both upregulated in transgenic plants (Fig. 8f, g). The expression level of ODC2 was consistently lower in transgenic plants than in WT with or without saline (Fig. 8e). These results indicate that the synthesis of PAs under salt stress was enhanced by MdATG8i overexpression in apple.

To analyse whether autophagic activity changed among the genotypes under salt stress, we first examined the expression of nine important MdATGs. As shown in Fig. 9a, the transcript levels of all detected MdATG genes were induced by salt stress. In addition, except for the expression of MdATG7a and MdATG7b, which were slightly lower in OE plants than in WT after salt stress, the other MdATGs were all expressed at higher levels in the OE lines. To further confirm these results, we observed autophagosome formation by transmission electron microscopy to monitor autophagic activity in response to salt stress (Fig. 9b). Under normal hydroponic conditions, we observed low numbers of autophagosome structures and autophagic bodies in WT 7

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Fig. 6. Stomatal aperture and ABA content in WT and MdATG8i-OE plants after salt stress. (a) Scanning electron microscopy (SEM) images of stomata from plants treated with and without salt stress. Changes in (b) stomatal length, (c) stomatal density, (d) stomatal aperture and (e) ABA content under salt stress. Changes in the expression of (f) MdNCED1 and (g) MdNCED3 under salt stress. Total RNA was isolated from leaf samples collected after 20 d of salt treatment. Data are shown as the means of six replicates with SEs. Different letters indicate significant differences between treatments, according to one-way ANOVA followed by Tukey’s multiple range test (P < 0.05).

4. Discussion

and OE plants. After salt treatment, the numbers of autophagic bodies increased in WT and OE plants, but the increase in the OE lines was almost 1.5-fold that in WT (Fig. 9c). Taken together, these results demonstrate that overexpression of MdATG8i in apple promotes the occurrence of autophagy under salt stress.

A growing body of evidence has indicated the important roles of autophagy in stress adaptation, with upregulated autophagic activity in plants in response to various abiotic and biotic stressors, such as drought, high salinity, high temperature, and pathogenic infection (Luo 8

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Fig. 7. Amino acids levels in the leaves of WT and MdATG8i-OE plants after salt stress, as measured by LC–MS.

mutants exhibit sensitivity to salt treatment (Liu et al., 2009; Luo et al., 2017; Zhou et al., 2013). Here, we discovered that apple plants overexpressing MdATG8i, which showed higher autophagic activity under salt stress, presented enhanced salt tolerance (Figs. 2,9). In addition to the reduced extent of damage in OE plants caused by salt stress, we also observed lower ROS accumulation in transgenic plants (Fig. 3). A previous study showed that MdATG18a enhances apple drought tolerance partly by reducing the accumulation of harmful ROS, and we thought that this pathway might also exist in MdATG8i-mediated salt tolerance. Ionic toxicity is one of the main threats imposed by salt stress in plants, which is associated with excessive Na+ uptake leading to K+ deficiency and other nutrient imbalances (Zhu, 2002). Herein, we also found that enhanced salt tolerance of the OE lines was consistent with reduced accumulation of Na+ and lower Na+: K+ ratios (Fig. 4). Continuous measurements during salt treatment revealed the

et al., 2017; Signorelli et al., 2019; Sun et al., 2018a). Notably, although several lines of evidence suggest that autophagy is positively involved in plant salt stress adaptation, knowledge is still limited concerning the function and mode of action of autophagy in non-model perennial woody plants. In our previous study, MdATG8i was responsive to leaf senescence, nitrogen depletion, and oxidative stress, and overexpression of MdATG8i in Arabidopsis and apple calli enhanced their tolerance to nitrogen and carbon starvation, which demonstrated that the MdATG8i protein functioned in a conserved way as a positive regulator in autophagy. In the present study, expression of MdATG8i was induced by NaCl treatment (Fig. 1). Next, we generated MdATG8iOE apple plants and used a hydroponic salt treatment to analyse the specific role of autophagy in the resistance mechanisms of apple in response to salt stress. Several studies have reported that autophagy-deficient Arabidopsis 9

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Fig. 8. MdATG8i overexpression induces the accumulation of free polyamines and increases the expression of PA-related genes upon saline stress. Changes in the concentrations of (a) putrescine (Put), (b) spermidine (Spd) and (c) spermine (Spm) under salt stress. Changes in the expression of (d) ADC2, (e) ODC2, (f) SPDS1, (g) SPDS5, (h) SAMDC1 and (i) SAMDC3 under salt stress. Total RNA was isolated from leaf samples collected after 20 d of salt treatment. Data are shown as the means of three replicates with SEs. Different letters indicate significant differences between treatments, according to one-way ANOVA followed by Tukey’s multiple range test (P < 0.05).

to salt stress (Dai et al., 2018). Here, we also observed a large accumulation of Pro in response to salt stress (Fig. 7). The increase in Pro was higher in transgenic plants than that in WT plants, which might contribute to the antioxidant system in the OE lines and, thereby, be partly responsible for the higher salt tolerance. More attention has been paid to the function of Pro under salt stress, with less concern focused on the other AAs. However, in this study, the concentration of Arg, which is a vital AA for the transport and storage of nitrogen (Brauc et al., 2012; Flores et al., 2008), was also significantly induced by salt stress (Fig. 7). During plant Arg metabolism, Arg can be used as a precursor for the synthesis of PAs and nitric oxide (Gao et al., 2009), which are involved in the regulation of plant growth and development, as well as in the effects on components of the photosynthetic machinery (Ma et al., 2013). Reduced Arg levels in mammals and plants are often correlated to higher arginase activity (Flores et al., 2008; Shi et al., 2013). The mutational elimination of AtARGAHs in Arabidopsis has been shown to improve salt tolerance as an important route for Arg metabolism, while overexpression leads to reduced stress resistance (Shi et al., 2013). Although the interactions between AA metabolism and the autophagic process have long been established in plants under nutrient-starved conditions (Avin-Wittenberg et al., 2015; Barros et al., 2017), this correlation with plant tolerance to salt stress remains largely unknown. As Arg content is relatively high in apple leaves, and it increased in OE plants more than twice the levels in WT after salt treatment, Arg accumulation appears to contribute, at least in part if not entirely, to MdATG8i-mediated salt tolerance. Because the same pattern of reduced levels of most AAs have been previously observed in autophagy-deficient atg mutants following

consistently superior photosynthetic ability of the OE lines than the WT under salt stress, which might be part of the reason for the large difference in biomass production among the genotypes (Fig. 5). Salt stress has been reported to induce decreases in the stomatal aperture, limiting CO2 uptake of leaves (Yang and Guo, 2018). In the present study, shrinkage of stomata caused by saline was less pronounced in the OE lines, suggesting that the decrease in carbon assimilation caused by stomatal limitations under salt stress could be alleviated by MdATG8i overexpression. A recent study showed that autophagy allows stomatal opening by controlling ROS homeostasis in guard cells (Yamauchi et al., 2019). This finding is consistent with our results, demonstrating the close relationship between autophagy and stomatal opening, which plays an important role in plant resistance. In addition, salt treatment increases ABA concentrations in plant cells (Duan et al., 2013; Yang and Guo, 2018), which promote stomatal closure in guard cells and regulate the expression of many genes that may function in plant stress tolerance (Geilfus et al., 2015). In this study, consistent with the stomatal behaviour, we also observed a greater increase in ABA accumulation in WT plants after salt treatment (Fig. 6), but the definite relationship between autophagy and ABA accumulation requires further exploration. Salt stress also restricts plant development by causing detrimental hyperosmotic stress, so that any protective mechanisms that serve to counteract these detrimental effects are highly correlated with the protection of plant survival under salt stress (Acosta-Motos et al., 2017; Zhang et al., 2017b). Many studies have reported that AA metabolism in plants changes to accommodate various abiotic stressors (Zhang et al., 2017b; Zorb et al., 2004). In particular, it has been widely suggested that Pro metabolism plays an indispensable role in the response 10

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Fig. 9. Expression of other MdATGs and formation of autophagosomes in apple leaves under salt stress. (a) Changes in the expression of other MdATGs in WT and MdATG8i OE plants following salt stress. (b) Representative TEM images of autophagic structures in mesophyll cells from WT and MdATG8i OE plants. Autophagic bodies are indicated by arrows. Bars: 1 μm. (c) Relative autophagic activity normalized to the activity of WT or MdATG8i OE plants shown in (b). More than 10 cells were used to quantify structures. Data are shown as the means of six replicates with SEs. Different letters indicate significant differences between treatments, according to one-way ANOVA followed by Tukey’s multiple range test (P < 0.05).

plant cells (Liu et al., 2006), including Put, Spd and Spm. Put is synthesized via the arginine decarboxylase (ADC) or ornithine decarboxylase pathways either from Arg or ornithine. The synthesis of Spd and Spm involves spermidine synthase (SPDS) and S-adenosylmethionine decarboxylase (SAMDC) (Liu et al., 2015). In the present study, in accordance with the change in Arg, we also detected more Put, Spm and Spd accumulation in transgenic plants. In addition, the expression level of ADC was induced to a higher extent in the transgenic plants than in WT following salt stress (Fig. 8), further indicating a link between the MdATG8i-mediated Arg increase and greater accumulation of PAs in OE plants. Many studies have demonstrated that PAs play important roles in plant tolerance to different abiotic stressors, including salt stress. Altering the level of endogenous PAs by transgenic methods can also affect plant salt tolerance. For example, overexpression of the

carbon starvation (Avin-Wittenberg et al., 2015; Barros et al., 2017), our data indicate that functional autophagy is probably required to increase salt resistance by altering AA metabolism. Although our results provide circumstantial evidence, the direct link between autophagy and the provision of AAs, particularly Arg, remains unclear. A recent study found that release of the Arg-degrading enzyme arginase I is prevented in the circulation of Atg7-deficient hosts, leading to a decline in Arg contents in vivo, which demonstrated the relationship between Arg metabolism and the autophagic process in animals (Poillet-Perez et al., 2018). However, whether this specific regulatory mechanism is conserved in plants requires further exploration. In addition, Arg also serves as an important precursor for the synthesis of PAs (Brauc et al., 2012; Flores et al., 2008). PAs are a class of low-molecular-weight organic cations that are widely distributed in 11

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authors are grateful to Dr. Zhihong Zhang for providing tissue-cultured GL-3 apple plants. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.envexpbot.2020. 103989. References Acosta-Motos, J.R., Ortuno, M.F., Bernal-Vicente, A., Diaz-Vivancos, P., Sanchez-Blanco, M.J., Hernandez, J.A., 2017. Plant responses to salt stress: adaptive mechanisms. Agronomy-Basel 7, 38. Avin-Wittenberg, T., Bajdzienko, K., Wittenberg, G., Alseekh, S., Tohge, T., Bock, R., Giavalisco, P., Fernie, A.R., 2015. Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation. Plant Cell 27, 306–322. Avin-Wittenberg, T., Baluska, F., Bozhkov, P.V., Elander, P.H., Fernie, A.R., Galili, G., Hassan, A., Hofius, D., Isono, E., Le Bars, R., Masclaux-Daubresse, C., Minina, E.A., Peled-Zehavi, H., Coll, N.S., Sandalio, L.M., Satiat-Jeunemaitre, B., Sirko, A., Testillano, P.S., Batoko, H., 2018. Autophagy-related approaches for improving nutrient use efficiency and crop yield protection. J. Exp. Bot. 69, 1335–1353. Banon, S., Miralles, J., Ochoa, J., Sanchez-Blanco, M.J., 2012. The effect of salinity and high boron on growth, photosynthetic activity and mineral contents of two ornamental shrubs. Hortic. Sci. 39, 188–194. Barros, J.A.S., Cavalcanti, J.H.F., Medeiros, D.B., Nunes-Nesi, A., Avin-Wittenberg, T., Fernie, A.R., Araujo, W.L., 2017. Autophagy deficiency compromises alternative pathways of respiration following energy deprivation in Arabidopsis thaliana. Plant Physiol. 175, 62–76. Brauc, S., De Vooght, E., Claeys, M., Geuns, J.M.C., Hofte, M., Angenon, G., 2012. Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea. Plant Biol. 14, 39–45. Dai, H.Y., Li, W.R., Han, G.F., Yang, Y., Ma, Y., Li, H., Zhang, Z.H., 2013. Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple. Sci. Hortic. 164, 202–208. Dai, W.S., Wang, M., Gong, X.Q., Liu, J.H., 2018. The transcription factor FcWRKY40 of Fortunella crassifolia functions positively in salt tolerance through modulation of ion homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs. New Phytol. 219, 972–989. Duan, L.N., Dietrich, D., Ng, C.H., Chan, P.M.Y., Bhalerao, R., Bennett, M.J., Dinneny, J.R., 2013. Endodermal ABA signaling promotes lateral root quiescence during salt stress in Arabidopsis seedlings. Plant Cell 25, 324–341. Eggert, E., Obata, T., Gerstenberger, A., Gier, K., Brandt, T., Fernie, A.R., Schulze, W., Kuhn, C., 2016. 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Fig. 10. A proposed model of action for explaining regulator function of MdATG8i in response to salt in apple.

LcSAMDC1 gene from Leymus chinensis in Arabidopsis increases the levels of different forms of PAs and enhances salt tolerance (Liu et al., 2017). The impact of PAs on salt tolerance is influenced by a number of factors, such as their types and forms (Pottosin and Shabala, 2014). Increasing evidence has demonstrated that PAs might act as an important factor regulating the antioxidant defence system in plants (Liu et al., 2015). In addition to functioning directly as antioxidants, PAs might activate antioxidant enzymes (Pottosin and Shabala, 2014). Thereby, MdATG8i-mediated salt tolerance is also associated with increased PA concentrations. In conclusion, we have provided comprehensive analyses of apple MdATG8i-mediated autophagy in plant salt tolerance (Fig. 10). We demonstrated that MdATG8i-mediated salt tolerance was related to the maintenance of photosynthetic ability and AA metabolism under salt stress. The less pronounced shrinkage of stomata in transgenic plants partly mitigated the decrease in carbon assimilation caused by salt stress. MdATG8i-mediated Arg and its downstream accumulation of PA product were important components of apple salt tolerance. The metabolic response is an important part of resisting experimental stress during plant development, and the significant contribution of autophagy to metabolic processes affecting plant developmental fitness has been acknowledged. The current study provides sufficient evidence for autophagy-mediated salt tolerance, as well as insight into the metabolic importance of autophagy in the response of plants to salt stress during development. Author contributions FM, XG, and LH designed the experiments; LH, ZG, PW and ZZ performed the experiments and analyzed the data, assisted by XJ, YS and XS; LH, XG and FM wrote the paper with contributions from all authors. Declaration of Competing Interest The authors declare no competing interests. Acknowledgements This work was supported by National Key Research and Development Program of China (2018YFD1000303) and by the Earmarked Fund for China Agriculture Research System (CARS-27). The 12

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