Abstract Water deficit adversely affects the growth and productivity of annual ryegrass (Lolium multiflorum Lam.). The exogenous application of chitosan (CTS) has gained extensive interests due to its effect on improving drought resistance. This research aimed to determine the role of exogenous CTS on annual ryegrass in response to water stress. Here, we investigated the impact of exogenous CTS on the physiological responses and transcriptome changes of annual ryegrass variety “Tetragold” under osmotic stress induced by exposing them to 20% polyethylene glycol (PEG)-6000. Our experimental results demonstrated that 50 mg/L exogenous CTS had the optimal effect on promoting seed germination under osmotic stress. Pre-treatment of annual ryegrass seedlings with 500 mg/L CTS solution reduced the level of electrolyte leakage (EL) as well as the contents of malondialdehyde (MDA) and proline and enhanced the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbic acid peroxidase (APX) under osmotic stress. In addition, CTS increased soluble sugars and chlorophyll (Chl) content, net photosynthetic rate (A), stomatal conductance (gs), water use efficiency (WUE), and transpiration rate (E) in annual ryegrass seedlings in response to three and six days of osmotic stress. Transcriptome analysis further provided a comprehensive understanding of underlying molecular mechanisms of CTS impact. To be more specific, in contrast of non-treated seedlings, the distinct changes of gene expressions of CTS-treated seedlings were shown to be tightly related to carbon metabolism, photosynthesis, and plant hormone. Altogether, exogenous CTS could elicit drought-related genes in annual ryegrass, leading to resistance to osmotic stress via producing antioxidant enzymes and maintaining intact cell membranes and photosynthetic rates. This robust evidence supports the potential of the application of exogenous CTS, which will be helpful for determining the suitability and productivity of agricultural crops. Keywords: Lolium multiflorum Lam., antioxidant enzymes, osmotic stress, transcriptome, exogenous chitosan, physiological and photosynthetic characterizes 1. Introduction Chitosan (CTS) is a natural, safe, and economical carbohydrate polymer produced by deacetylation of nontoxic and bio-functional chitin from shellfish such as Brachyura, Caridea and Procambarus clarkia. During the past several decades, CTS has been proven to improve plant production and induce abiotic stress tolerance as a plant growth regulator [[34]1]. For example, Guan et al. reported that maize seeds priming with CTS had better germination and growth under cold stress [[35]2]. Pongprayoon et al. found that CTS clearly activated osmotic stress defense in rice [[36]3]. Bittelli et al. suggested that CTS could increase water use efficiency in pepper in response to drought stress [[37]4]. These findings elaborated the effects of exogenous CTS on the alterations of physiological response and gene expression under stress [[38]1,[39]5]. In plants under stress conditions, changes in antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) activities, electrolyte leakage (EL), relative water content (RWC), malondialdehyde (MDA) concentration, H[2]O[2], and [MATH: O2−· :MATH] were evaluated in response to CTS treatment [[40]6,[41]7,[42]8]. Indeed, CTS has been shown to affect drought-related genes in soybean [[43]9] and defense-related genes in rice [[44]10] on transcriptional level. However, few studies have taken into consideration of the critical parameters involved in CTS application, such as CTS concentration, the duration of treatment, and the developmental stages of the plants. Annual ryegrass (Lolium multiflorum Lam.), an economically and agriculturally important cool season grass species, is broadly grown throughout the world for its high productivity and forage value [[45]11]. Unfortunately, just like other crops, annual ryegrass has been suffering from prolonged water deficit mainly caused by global climate change, which thus severely reduced its potential yields in the natural pastures of semi-arid condition [[46]12,[47]13]. Hence, engineering annual ryegrass with improved resistance to water stress is in urgent need of full implementation. Recently, the exogenous application of CTS has gained extensive interests due to its effect on enhancing drought tolerance. If exogenous CTS also acts as a water stress enhancer for annual ryegrass, the underlying molecular mechanism yet remains unknown. The aim of this study was to unravel the yet-unknown role of exogenous CTS on annual ryegrass in response to water deficit. Polyethylene glycol (PEG) 6000–induced osmotic stress can result in water deficit without causing any direct physiological damage in plants [[48]14]. The alterations of physiological traits, photosynthetic traits, and transcriptome of CTS pre-treated and mock-treated ryegrass seedlings were examined under the PEG 6000–induced osmotic stress to address three main questions as following: (1) which levels of exogenous CTS are optimal to improve resistance to osmotic stress in annual ryegrass, (2) how does the exogenous CTS prevent annual ryegrass seedlings from osmotic stress, and (3) which are the CTS-induced biological processes and corresponding osmotic stress-responsive genes functioning to improve the resistance of annual ryegrass against osmotic stress? 2. Materials and Methods 2.1. Plant Materials and Chitosan Treatments The annual ryegrass cultivar “Tetragold” was provided by Barenbrug Co. (Beijing, China). The seeds were sterilized in 20% NaClO for 5 min, and rinsed five times with distilled water, and then 2.5 g of seeds was sown in trays (30 cm × 16 cm × 10 cm) with 1/2 Hoagland’s solution in growth room. The environmental conditions were as follows: 25 °C (day) and 20 °C (night), and photosynthetically active radiation 300 μmol m^−2s^−1. For the CTS treatment, when the plants had 3–4 leaves, roots of annual ryegrass were pre-treated in 1/2 Hoagland’s solution mixed with CTS for three days. To optimize the concentration of CTS, increasing concentrations of CTS (0, 50, 100, 250, 500, 1000, 2000 mg/L (W/V)) were chosen to determine the changes of leaf relative water content (RWC), leaf electrolyte leakage (EL), and malondialdehyde (MDA) content after four days of osmotic stress. As commonly used in previous studies to induce the osmotic stress in annual ryegrass, 20% polyethylene glycol (PEG)-6000 was selected for treatment in this study [[49]15,[50]16]. Mock- (0 mg/L CTS) or CTS-treated plants were then exposed to PEG or 1/2 Hoagland’s solution resulting in the following four different combinatory treatment groups: (1) CK, mock-treated plants exposed to 1/2 Hoagland’s solution; (2) CTS, CTS pre-treatment plants exposed to 1/2 Hoagland’s solution; (3) PEG, mock-treated plants exposed to 1/2 Hoagland’s solution containing 20% PEG-6000; and (4) PEG + CTS, CTS pre-treatment plants exposed to 1/2 Hoagland’s solution containing PEG-6000. Leaf samples were collected after 0, 3, and 6 days of osmotic stress, respectively. For each treatment group, the physiological parameters were measured, and qRT-PCR analyses were further performed in three independent replicates. Our preliminary experimental results showed that the germination of annual ryegrass seeds was inhibited under 400 mg/L (W/V) CTS. To characterize the seed germination, the sterilized seeds were soaked in lower concentration [0, 10, 20, 30, 40, 50, 80, 100, and 200 mg/L (W/V)] CTS for 24 h at 25 °C, respectively. Each treatment had six independent replicates (50 seeds per replicate). The soaked seeds experienced germination in 9 cm diameter petri dishes cushioned with two pieces of filter paper treated with 3 ml of 20% (W/V) PEG-6000. The petri dishes were then placed in a growth chamber where the day/night temperature was set to 25 °C and 20 °C, photosynthetically active radiation 300 μmol m^−2s^−1 and 75% relative humidity. Seeds were sampled at four and seven days after germination for germination vigor and germination percentage measurements, respectively. Another batch was sampled at seven days after germination for determinations of shoot length, root length, and fresh weight. 2.2. Seedling Physiological Analysis The RWC in leaf was measured following the protocol of Barrs and Weatherley [[51]17]. Upon the removal of leaves from the plant, the fresh weight (FW) of leaves was determined immediately. After 12 h submersion of the leaves in deionized water at 4 °C, turgid weight (TW) was measured once the turgid leaves were blotted dry. The dry weight (DW) of leaves was then obtained following incubation at 80 °C for no less than 72 h. The formula RWC (%) = [(FW − DW)/(TW − DW)] × 100% was applied to determine the Leaf RWC. Leaf chlorophyll (Chl) concentration was determined by assessing the absorbance at 645 nm and 663 nm. Following the 72 h immersion of leaves (0.1 g) in 95% ethanol (10 mL) without the light, a spectrophotometer was used to assay the Chl content in the leaf extract. Based on the protocol by Bates et al. [[52]18], free proline was quantified. Upon immersing the leaves (0.1 g) in 3% sulfosalicylic acid (10 mL), the leaf tissues were then boiled in water for 20 min. Once the temperature drops to room temperature, the solution mixed with leaf extract (1 mL), glacial acetic acid (1 mL) and 2.5% ninhydrin test reaction reagent (1.5 mL) was heated in boiling water for 40 min. The ice bath incubation terminated the reaction which was then added in 2.5 mL toluene for the analytical separation. After 1 min vigorously mixing the reaction, the phases were separated, and the proline content was determined by measuring the absorbance of chromophore-containing toluene at 520 nm. Leaf electrolyte leakage (EL) was gauged according to the method of Blum and Ebercon [[53]19]. After washing the leaves (0.1 g) with deionized water three times, the leaves were immersed and shaken in deionized water (30 mL) for 24 h. The initial conductivity (Ci) was determined by the conductivity meter and applied to express the solution conductance. Leaves were subjected to autoclave at 120 °C for 20 min and cooled down to room temperature. The maximum conductance (Cmax) was then assayed for the tissue conductivity. The percentage of Ci/Cmax was used to express EL. By applying the sulfanilamide method [[54]20], the formation rate of [MATH: O2−· :MATH] was determined. The leaf extracts were made by mixing 0.1 g grinding leaves with 1.5 mL 65 mM PBS (pH 7.8). Upon centrifugation at 10,000 rpm for 30 min at 4 °C, the supernatant was separated for further use in the reaction. The supernatant (0.5 mL) was incubated at 25 °C water bath in the mixture with 0.5 mL PBS and 0.1 mL 10 mM hydrochloride for 20 min. Then, the reaction mixture was incubated for additional 20 min at 25 °C upon the addition of 1 mL 58 mM sulfanilamide plus 1 mL 7 mM a-naphthylamine. Finally, the absorbance of reaction being extracted via 2 mL chloroform was determined at 530 nm. H[2]O[2] was assayed using the potassium iodide method [[55]21]. Also, 0.1 g of leaf tissue homogenate in 5 mL 0.1% TCA was centrifuged for 20 min at 12,000 rpm. The reaction was conducted by mixing 0.5 mL leaf supernatant with 0.5 mL 10 mM potassium phosphate and 1 mL 1 M KI. The results were recorded by measuring the absorbance at 390 nm. MDA content was examined following the protocol by Heath and Packer [[56]22]. The leaves weighing 0.2 g were homogenized in solution of 2 mL of 20 % (W/V) trichloroacetic acid on ice. The supernatant was collected following centrifugation of tissue homogenates at 12,000 rpm for 10 min. Upon the addition of 0.5 mL supernatant along with 1 mL reaction solution (20% trichloroacetic acid and 0.5% thiobarbituric acid) into the pellet, the reaction was heated for 10 min at 95 °C and then placed in cold water to cool it down. Following the centrifugation of the reaction at 8000 rpm for 10 min, the absorbance of the reaction was assayed at 532 and 600 nm. To determine the antioxidant activities, 0.3 g of fresh leaves were collected per tray on each sampling day in a random manner, subjected to “snap freezing” in liquid nitrogen, and later stored at −80 °C for future analyses. For extraction, the sample was ground on ice with 4 ml of 50 mM phosphate buffer (pH 7.8). The homogenate was centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was used for assays of antioxidant enzyme activity. The ascorbic acid peroxidase (APX) activities were assayed based on the protocol of Nakano and Asada [[57]23]. Here, 0.05 mL of supernatant was mixed with 1.5 mL reaction reagent containing 10 mM ascorbic acid, 0.003 mM EDTA, 5 mM H[2]O[2], and 100 mM PBS (pH 5.8). The absorbance of reaction was determined at 290 nm every 10 s for 1 min. The peroxidase (POD) and catalase (CAT) activities were measured folloing the method of Maehly and Chance [[58]24]. Here, 0.05 mL enzyme extract was mixed with 1.5 ml reaction reagent either composed of 0.05 mL of 0.75% H[2]O[2], 0.5 mL of 0.25% guaiacol solution, and 0.995 mL of 100 mM PBS, pH 5.0 for POD assay or 0.5 mL of 45 mM H[2]O[2] and 1 mL of 50 mM PBS, pH 7.0 for CAT assay. Results of POD or CAT assays were recorded as the absorbance measured every 10 s for 1 min at 470 or 240 nm, respectively. The SOD activities were examined using a total superoxide dismutase (SOD) Assay Kit (S0102; Haimen Beyotime, Haimen, China). All these relative enzyme activities were expressed as fold change. Protein concentration (mg·g^−1 DW (dry weight)) was analyzed using bovine serum albumin (BSA) as a standard according to Bradford [[59]25]. 2.3. Measurement of Photosynthetic Characteristics Six seedling leaves from three trays were measured for the photosynthetic parameters by using a narrow leaf chamber connected to a CIRAS-3 (PP Systems, Amesbury, MA, US) [[60]26]. In this study, 400 µmol mol^−1 of carbon dioxide was constantly retained, and 800 µmol m^−2s^−1 of light intensity was maintained with the light-emitting diode light sources placed in the leaf chamber. 2.4. RNA Extraction and Transcriptome Sequencing Analysis Three independent replicates of the mock-treated and 3 d of CTS pre-treatment seedlings were collected for RNA-seq analysis. Total RNA was isolated from plant leaf with Plant Total RNA Kit (Qiagen, Germantown, MD, USA). The RNA samples were qualified and quantified by running 1% agarose gels and Qubit^® 2.0 Flurometer (Life Technologies, Foster, CA, USA). RNA-seq libraries were constructed by using NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (NEB, Ipswich, MA, USA) according to the manufacture’s protocol. Followed by the manufacture’s protocol, a cBot Cluster Generation System was used to perform the clustering of the index-coded samples. An Illumina (San Diego, CA, USA) Hi-seq platform was used to sequence the library, and the paired-end reads were produced. The software Trinity was used to assemble the annual ryegrass transcriptome based on left.fq and right.fq [[61]27], and all the parameters used the default settings except that the parameter “min kmer cov” was set to 2. Seven public databases—cluster of orthologous groups of proteins database (COG), kyoto encyclopaedia of genes and genomes database (KEGG), gene ontology database (GO), non-redundant protein database (NR), non-redundant nucleotide database (NT), Swiss-Prot protein database (Swiss-Prot), and protein family database (Pfam)—were used as references to annotate the gene functions.