The Enhancement of Drought Tolerance in Arabidopsis Plants Induced by Pretreatment with Sulfur Dioxide

Sulfur dioxide (SO 2 ) is a common air pollutant that has multiple effects on plants. Here, the effect of prior exposure to SO 2 on the improvements of drought tolerance and possible regulation mechanisms were investigated in Arabidopsis plants. The experimental results showed that pre-exposure to 30 mg/m 3 SO 2 for 72 h could reduce leaf water loss, and enhance the drought tolerance of Arabidopsis plants. SO 2 pre-exposure decreased leaf stomatal conductance (Gs) and transpiration rate (Tr) but increased net photosynthetic rate (Pn), water use eciency (iWUE) and photosynthetic pigment contents under drought conditions. Importantly, the activities of superoxide dismutase (SOD) and peroxidase (POD) were signicantly increased, while the contents of hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) were decreased in SO 2 -pretreated Arabidopsis plants under drought stress. Additionally, the activity of o-acetylserine(thio)lyase (OASTL) and the content of cysteine (Cys), the rate-limiting enzyme and the rst organic product of sulfur assimilation, were increased signicantly in drought-stressed plants after SO 2 pretreatment, along with the increases of other thiol-containing compounds glutathione (GSH) and non-protein thiol (NPT). Meanwhile, SO 2 pre-exposure induced a higher level of proline accumulation, accompanied by the increased activity of proline synthase P5CS, the decreased activity of proline dehydrogenase ProDH and the corresponding alteration of gene transcription. Collectively, the enhanced drought tolerance afforded by SO 2 might be related to the improvement of plant photosynthesis, antioxidant defense, sulfur assimilation and osmotic adjustment. These ndings provide new insights in understanding the role of SO 2 in plant adaptation to environmental stress. tolerance in Arabidopsis grown in water shortage conditions by increasing the photosynthesis performance, antioxidant defense, sulfur assimilation, and osmoprotectant accumulation. The present study reveals the molecular mechanism of cross-adaptation of plants under SO 2 and drought stress, and provides a new perspective for future researches on stress-resistance mechanisms. However, more detailed analyses might be still needed to better understand the role of SO 2 in plant adaptation to drought stress.


Introduction
Sulfur dioxide (SO 2 ) is one of the important gaseous pollutants that have an irreparable impact on plants, animals and ecosystem. Airborne SO 2 mainly enter plants through stomata and rapidly hydrolyzed into sul te and bisul te (Hänsch and Mendel 2005). Sul te can be oxidized to sulfate accompanied by reactive oxygen species (ROS) production, or be fed into sulfur assimilation pathway to form cysteine (Cys) (Zhao and Yi 2014). The phytotoxicity of SO 2 is strongly dependent on its concentration and duration. High concentrations of SO 2 could cause leaf necrosis, growth inhibition and even plant death  provide a speci c plant physiological status contributing to drought tolerance. There were several studies reporting the enhanced drought tolerance in SO 2 -exposed Arabidopsis and foxtail millet plants (Wang and Yi 2017;Han et al. 2019). However, the exact molecular mechanisms of SO 2 -induced drought tolerance still remain largely unknown. Therefore, the objective of this study was to explore the effects of SO 2 pretreatment on the physiology and biochemistry of drought resistance in Arabidopsis plants to reveal the possible regulatory mechanisms.

Plant cultivation and treatments
The seeds of Arabidopsis thaliana wild type Columbia-0 (Col-0) were vernalized at 4°C for 24 h, and then sown on a culture medium of high-nutrient soil (KlasmannDeilmann, Germany) and vermiculite at a ratio of 3:1 in square pots. The cultures were maintained in a controlled growth room under a 60-70% relative humidity, temperature 23 ± 2°C, light/dark regime of 16/8 h and light intensity of 140 µmol m − 2 s − 1 . These plants were watered regularly and watered with a nutrient solution after three weeks.
Four-week-old Arabidopsis plants were randomly divided into four groups: control, SO 2 , drought and SO 2 + drought. (1) control: the plants with neither SO 2 nor drought treatment; (2) SO 2 : the plants exposed with SO 2 only; (3) drought: the plants were exposed with ltered pollutant-free air and subsequently treated by drought stress. (4) SO 2 + drought: the plants were fumigated with SO 2 and subjected to drought stress later. For the control and SO 2 treatment, the plants were exposed with pollutant-free air or 30 mg/m 3 SO 2 for 72 h in a chamber and subsequently watered regularly. Drought stress were performed by withholding irrigation.

Measurement of relative water content (RWC)
The detection of leaf RWC was performed according to the method of Nawaz et al. (2015). The RWC was calculated as follows: RWC (%) = (FW-DW) / (TW-DW) ×100%. FW, DW and TW represent fresh weight, dry weight and turgid weight, respectively.
Determination of leaf gas exchange and photosynthetic pigment contents The photosynthetic gas exchange parameters were measured with a portable photosynthetic apparatus (SY-1020, Shiyakeji, Shijiazhuang), as described previously (Li and Yi 2020). The contents of leaf chlorophylls and carotenoids were determined by measuring the absorbances at 440 nm, 663 nm, 645 nm and 652 nm in 80% anhydrous acetone extracts according to Lichtenthaler (1987).
Measurement of relative electrical conductivity, malondialdehyde (MDA) and H 2 O 2 contents The relative electrical conductivity of Arabidopsis leaves was measured using DDS-307 Conductivity Meter (LEICI Company, China). Simply, twenty round leaf sections were cut from different individual plants and immediately immersed in 5 mL deionized water, followed by vacuum in ltration. The initial electrical conductivity (A 1 ) of the samples was detected. Then the samples were boiled for 30 min to release all electrolytes. The ultimate electrical conductivity (A 2 ) was measured again when the samples had cooled to 25°C. The relative electrical conductivity = A 1 /A 2 ×100%.

Assay of antioxidant enzyme activities
The activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were measured using the method reported by Li and Yi (2012b). SOD activity was assayed by recording the decrease in optical density of nitroblue tetrazolium (NBT) at 560 nm. POD activity was measured by using guaiacol as a hydrogen donor and monitoring the increase in absorbance at 470 nm.
CAT activity was determined by the rate of H 2 O 2 decomposition at 240 nm.
The glutathione peroxidase (GPX) activity was determined by the rate of oxidation of reduced glutathione (GSH) by H 2 O 2 oxidation following previously described method of Wendel (1981). The glutathione reductase (GR) activity was determined by the rate of reduction of oxidized glutathione (GSSG) by NADPH oxidation according to the method of Foyer and Halliwell (1976).
Determination of o-acetylserine(thio)lyase (OASTL) activity and the contents of cysteine (Cys), glutathione (GSH) and non-protein thiol (NPT) The OASTL activity was assayed by quanti cation of the reaction product cysteine according to the method of Riemenschneider et al. (2005) with minor modi cation. Brie y, the plant shoots were homogenized with 20 mM Tris-HCl (pH 8.0) and centrifugated for the assay of enzyme activity. The reaction mixture, containing 20 mM Tris-HCl (pH 8.0), 50 mM sodium sul de (Na 2 S), 50 mM dithiothreitol (DTT), 50 mM o-acetylserine (OAS) and the supernatant. One unit (U) of OASTL activity was de ned as the amount of enzyme required to catalyze the formation of 1 µmol of Cys per min at 25°C.
The contents of Cys, GSH and NPT were determined according to previously described methods (Li and Yi 2020). The assay of Cys level was performed by acid ninhydrin method and the absorbance of reaction mixture was measured at 560 nm. The contents of GSH and NPT were detected by the reaction of the supernatant and 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), and the absorbance of reaction mixture was measured at 412 nm. The supernatants of GSH and NPT were extracted by 3% (w/v) trichloroacetic acid and 5% sulfosalicylic acid followed by centrifugation, respectively.

Measurement of the contents of proline and soluble sugar, and the activities of P5CS and ProDH
The determination of proline content was performed by using the ninhydrin method described by Bates et al. (1973). The Arabidopsis shoots were ground with 3% (w/v) sulfosalicylic acid and then the supernatant collected by centrifugation was mixed with acid-ninhydrin reagent for the analysis of proline content. The soluble sugar was extracted with distilled water and then incubated at 50°C for 15 min, followed by centrifugation. The supernatant was reacted with anthrone reagent and the absorbance of reaction mixture at 630 nm was measured for the assay of soluble sucrose content.
The P5CS activity was measured following the previously described method (Lei et al. 2007). The crude enzyme was extracted with 50 mM phosphate buffer (pH 7.0) containing 1% (w/v) PVP, 1 mM EDTA, 0.6 M KCl and 5 mM MgCl 2 , followed by centrifugation. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 50 mM glutamic acid, 10 mM ATP, 2 mM MgCl 2 , 1 mM NADH and the supernatant. One unit (U) of P5CS activity was de ned as a change in absorbance of 0.01 at 440 nm per min.
The ProDH activity was determined from the change of NAD reduction at 340 nm according to previously described methods (Ren et al. 2018). The samples were extracted with 50 mM Tris-HCl buffer (pH 7.5) containing 5% (w/v) PVP, 7 mM MgCl 2 , 3 mM EDTA, 1 mM DTT and 0.6 M KCl. The supernatant collected by centrifugation was used to measure the ProDH activity.

RNA isolation and quantitative RT-PCR analysis
RNAs were isolated from Arabidopsis shoots using Trizol reagent (TransGen Biotech, China) and were used as templates to synthesize cDNAs with a PrimeScript RT reagent kit (TaKaRa, Japan) according to the manufacturer's protocol. The transcription levels of genes were detected by quantitative RT-PCR with speci c primers ( Table 1). The changes of gene expression under different treatments were analyzed by using the 2 −ΔΔCT method (Livak and Schmittgen 2001). The Arabidopsis Actin2 was used as a standard control.

Statistical analysis
Each experiment was repeated with at least three times and the values have been subjected to statistical analysis. One-way analysis of variance (ANOVA) and Duncan's multiple range test were performed to compare the means of different data sets. For all the analyses, the signi cant difference was considered at P < 0.05.

Results
SO 2 pre-exposure enhanced the tolerance of Arabidopsis plants to drought stress The morphology of Arabidopsis plants did not show obvious differences between SO 2 and control groups after 72 h of SO 2 exposure except a few transparent small spots appeared on the leaves of SO 2fumigated plants. Under drought condition, leaf curling and wilting were occurred and gradually aggravated with prolonged drought stress. However, after SO 2 pre-exposure, the progress of leaf wilting and curling was slowed down under drought condition (Fig. 1A), and obvious differences were occurred at 8th days of drought treatment. The leaf RWCs constantly decreased with prolonged drought treatment, while the leaf RWCs in SO 2 + drought group were signi cantly higher than in drought group. The results of leaf RWCs were consistent with the change of plant morphology under drought stress, indicating that SO 2 pre-exposure improved the leaf RWCs and enhanced the adaptability of Arabidopsis plants to drought stress. Therefore, the Arabidopsis plants at 8th day of drought stress was selected as the materials to further explore the mechanisms underlying improvements in drought adaptability afforded by SO 2 . SO 2 pre-exposure enhanced photosynthetic rate and photosynthetic pigment contents in Arabidopsis plants under drought stress Photosynthesis, the basic process of green plants, is very sensitive to environmental stresses. As shown in Fig. 2, drought stress decreased leaf gas exchange in Arabidopsis plants. Compared with the drought group, Gs and Tr decreased markedly by 28.6% and 20.0%, respectively ( Fig. 2B and C), meanwhile Pn and iWUE increased signi cantly in SO 2 + drought group ( Fig. 2A and E). No signi cant difference was found in Ci between drought and SO 2 + drought groups (Fig. 2D). These results indicated that preexposure to 30 mg/m 3 SO 2 affected photosynthetic performance of Arabidopsis plants under drought stress.
Photosynthetic pigments are the important molecules responsible for photosynthesis. As shown in Fig. 2, the contents of photosynthetic pigments, including chlorophyll a, chlorophyll b and carotenoid, were signi cantly decreased under drought stress. Compared with drought treatment alone, the contents of chlorophyll a, chlorophyll b and carotenoid were increased by 14.03%, 8.15% and 12.40%, respectively, in SO 2 + drought group (Fig. 2). These results indicated that SO 2 pre-exposure could e ciently inhibit the decline of photosynthetic pigments in Arabidopsis plants to support effective photosynthetic performance under drought stress. SO 2 pre-exposure induced antioxidant defense responses to alleviate drought stress Drought stress could induce ROS accumulation and disrupt the redox balance in plants. As shown in Fig.  3, the activities of SOD and POD were increased with the increases of H 2 O 2 and MDA levels in droughtstressed Arabidopsis plants. Compared to drought group, the activities of SOD and POD in SO 2 + drought group were signi cantly increased, meanwhile the contents of H 2 O 2 and MDA were markedly decreased, although no signi cant effect on CAT activity was found. Similarly, drought treatment caused the increase of the relative electrical conductivity, while SO 2 pre-exposure decreased the increment of relative electrical conductivity in drought-treated Arabidopsis plants. These results indicated that SO 2 pretreatment could induce antioxidant defense responses and effectively alleviate oxidative stress caused by drought stress. SO 2 pre-exposure promoted sulfur assimilation and the biosynthesis of thiol-containing compounds in Arabidopsis plants under drought stress Drought tolerance is associated with enhanced sulfur metabolism. As shown in Fig. 4, the activity of OASTL, the rate-limiting enzyme in sulfur assimilation pathway, was increased signi cantly in Arabidopsis plants under drought stress. Compared to drought stress alone, SO 2 pretreatment further enhanced the activity of OASTL in drought-treated Arabidopsis plants. Cys, the rst organic product of sulfur assimilation, increased signi cantly under drought conditions, along with the increases of GSH and NPT contents. The contents of Cys, GSH and NPT were increased markedly in SO 2 + drought group as compared with drought stress alone (Fig. 4). The activities of GPX and GR ( Fig. 4E and F) increased signi cantly under drought stress and showed a noticeably higher increment in SO 2 + drought group, which could accelerate turnover of the GSH redox cycle to reduce ROS accumulation under drought stress. Collectively, these results indicated that SO 2 pretreatment promoted sulfur assimilation and the biosynthesis of thiol-containing compounds, which could enhance the ROS-scavenging capacity to help maintain the cellular redox status under drought stress. SO 2 pre-exposure enhanced osmotic adjustment of Arabidopsis plants under drought stress Osmotic adjustment is a prime adaptive response to drought stress (Blum 2017). As shown in Fig. 5, the contents of proline and soluble sugar, the important osmoregulatory substances, were signi cantly increased under drought stress. And more, the increment was higher in SO 2 + drought group than in drought stress alone (Fig. 5A and B). These results demonstrated that SO 2 pretreatment could enhance the osmotic adjustment capacity to cope with drought stress by accumulation of osmoregulatory substances.
To further explore the responses of free proline accumulation, the activities of P5CS and ProDH, which are key enzymes functioning at proline synthesis and decomposition pathways, respectively, were measured. As shown in Fig. 5, the activities of P5CS and ProDH were increased markedly in droughtstressed Arabidopsis plants, while P5CS activity was signi cantly increased by 43.0% and ProDH activity was decreased by 27.2% in SO 2 + drought group as compared with drought treatment alone. Our ndings indicated that SO 2 pretreatment promoted the proline accumulation for osmotic adjustment by increasing proline biosynthesis and decreasing proline decomposition in Arabidopsis plants.

SO 2 pre-exposure in uenced the transcription of droughtresponsive genes in Arabidopsis plants under drought stress
Transcriptional regulation is a key step in plant responses to environmental stress. The results showed that the transcription levels of genes encoding antioxidant enzymes (SOD, POD and CAT) were signi cantly up-regulated in Arabidopsis under drought condition. Moreover, the transcription levels of these genes were higher in SO 2 + drought group than in drought group. Similarly, the transcriptional levels of P5CS1, P5CS2 (coding for Δ1-pyrroline-5-carboxylate synthetase) and P5CR (coding for pyrroline-5carboxylate reductase) were up-regulated signi cantly in drought group and with much high increase in SO 2 + drought group, accompanied with the down-regulation of ProDH (coding for proline decomposing enzyme). The changes in gene expression were consistent with the alterations of their corresponding enzymes activities. Therefore, SO 2 pretreatment could in uence gene expression regulating the physiological and cellular processes to cope with drought-induced oxidative stress, water de cit and other related changes in plant cells.

Discussion
Plants are often challenged with various environmental stresses simultaneously or consecutively. Crossadaptation is the phenomenon that the adaptive changes caused by one stressor may make the organism more t to resist the adverse effects of another type of stressor, which plays important roles in plant adapting to complex and changeable environments ( In the present study, we report that SO 2 pre-exposure resulted in an enhanced tolerance of Arabidopsis plants to drought stress. The enhanced drought tolerance is mainly attributed to the improving photosynthetic performance, the increasing biosynthesis of osmotic adjustment substances, and the enhancing antioxidant capability. Photosynthesis of green plants is easily affected by drought stress (Chaves et al. 2009). Under water de cit conditions, the reduction of leaf RWC, the alteration of leaf gas exchange and the decrease of photosynthetic pigment contents could cause inhibition of photosynthesis (Degl'Innocenti et al. 2009; Misson et al. 2010;Hejnák et al. 2015). Here, SO 2 pretreatment effectively decreased the Gs in droughtstressed plant leaves, which may be bene cial for the reduction of Tr and the improvement of RWC under drought stress (Fig. 2B and C). However, the decreases of Gs and Tr in SO 2 + drought group did not directly lead to a lower Pn, which may be due to the enhanced iWUE in SO 2 pre-exposed Arabidopsis plants (Fig. 2E). The higher leaf RWCs and the improvement of iWUE induced by SO 2 pretreatment were of great relevance for drought tolerance of Arabidopsis plants under drought stress. Additionally, the increases of chlorophylls and carotenoid contents in SO 2 pretreatment group (Fig. 2) The sulfur assimilation and downstream metabolic pathways play important roles in plant response to drought stress (Chan et al. 2013;Stanislav et al. 2019). In the present study, SO 2 pre-exposure promoted the sulfur assimilation in the leaves of drought-stressed Arabidopsis plants, as evidenced by the enhanced OASTL activity and the increased Cys content. The increased Cys content is bene cial for the biosynthesis of other thiol-containing compounds, including GSH and NPT (Fig. 4). High GSH level together with increased GPX and GR activities could enhance the ROS scavenging capacity of GSH-GSSG cycle in drought-treated Arabidopsis plants, and reduce drought-caused damage. Taken together, SO 2 pretreatment enhanced the sulfur assimilation and further promoted the synthesis of GSH and NPT to complete the detoxi cation under drought stress.
The accumulation of osmoprotectants (such as soluble sugar, proline, and betaine) is a signi cant strategy for plants to maintain water potential and membrane stability to avoid drought-induced damage (Fang and Xiong 2015). In this study, drought stress resulted in a signi cant increase of the contents of proline and soluble sugar in Arabidopsis plants. Moreover, SO 2 pretreatment induced a higher level of proline accumulation in drought-stressed Arabidopsis plants, consistent with the increased P5CS activity and decreased ProDH activity. In accordance with this result, the alteration of the transcription level of key genes involved in proline biosynthesis pathway (P5CS1, P5CS2 and P5CR) and degradation pathway (ProDH) showed the similar tendencies, indicating the involvement of gene expression and cellular metabolism in SO 2 pretreatment-induced drought tolerance. In addition to its accepted role as an osmolyte, proline can act as a potent nonenzymatic antioxidant induced by ROS, contributing to overall antioxidant activity in plant response to limited water supply (Ben Rejeb et al. 2014). Therefore, proline accumulation induced by SO 2 pretreatment might protect plant cells by osmotic adjustment, and also promote redox balance by scavenging ROS under drought stress, which contribute to the improvement of drought tolerance in Arabidopsis plants.
In conclusion, this study provides a systematic description of key events leading to enhanced drought tolerance in SO 2 -pretreated Arabidopsis plants (Fig. 7). SO 2 pretreatment signi cantly reduced leaf water loss and maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS activity under water de cit conditions. Importantly, SO 2 -induced antioxidant enzymes, together with high levels of nonenzymatic antioxidants increased the ROS scavenging capacity to recover drought-induced cellular redox imbalance. Taken together, SO 2 exposure could enhance drought tolerance in Arabidopsis grown in water shortage conditions by increasing the photosynthesis performance, antioxidant defense, sulfur assimilation, and osmoprotectant accumulation. The present study reveals the molecular mechanism of cross-adaptation of plants under SO 2 and drought stress, and provides a new perspective for future researches on stress-resistance mechanisms. However, more detailed analyses might be still needed to better understand the role of SO 2 in plant adaptation to drought stress.

Declarations
Author Contribution Lijuan Li measured photosynthetic parameters, analysed antioxidant indexes and gene expression, and wrote the manuscript. Huilan Yi conceived and guided the project, and reviewed and edited manuscript. All authors read and approved the nal manuscript. Effects of SO2 pretreatment on morphology (A) and leaf relative water content (B) of four-week-old Arabidopsis plants under drought stress conditions. The plants were exposed to 30 mg/m3 SO2 for 72 h and then subjected to drought stress by withholding watering. Columns labelled with different letters (a, b, c, d, e) indicate signi cant differences at P<0.05.

Figure 2
Effects of SO2 pretreatment on leaf gas exchange and photosynthetic pigments of four-week-old Arabidopsis plants under drought stress. The plants were exposed to 30 mg/m3 SO2 for 72 h and then subjected to drought stress for 8 days. A, Net photosynthetic rate (Pn); B, Stomatal conductance (Gs); C, Transpiration rate (Tr); D, Intercellular CO2 concentration (Ci); E, Intrinsic water use e ciency (iWUE); F,  Effects of SO2 pretreatment on the expression of genes in four-week-old Arabidopsis plants under drought stress. The plants were pre-exposed to 30 mg/m3 SO2 for 72 h and then were subjected to drought treatment for 8 days. Different letters (a, b, c, d) indicate signi cant differences at P<0.05. A schematic model for a systematic description of key events leading to enhanced drought tolerance in SO2-pretreated Arabidopsis plants.