Chitosan (CTS) is a natural, safe, and economical carbohydrate polymer produced by deacetylation of nontoxic and bio-functional chitin from shellfish such as Brachyura
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 [1
]. For example, Guan et al. reported that maize seeds priming with CTS had better germination and growth under cold stress [2
]. Pongprayoon et al. found that CTS clearly activated osmotic stress defense in rice [3
]. Bittelli et al. suggested that CTS could increase water use efficiency in pepper in response to drought stress [4
]. These findings elaborated the effects of exogenous CTS on the alterations of physiological response and gene expression under stress [1
]. 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, H2
were evaluated in response to CTS treatment [6
]. Indeed, CTS has been shown to affect drought-related genes in soybean [9
] and defense-related genes in rice [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 [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 [12
]. 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 [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?
Plants are highly sensitive to drought stress and require a relatively large amount of water for continued growth, especially in seed germination and seedling growth [32
]. Plants may respond to CTS at a series of concentrations in different manners at various stages of growth and development [10
]. In order to test this hypothesis, we applied different concentrations of CTS at seed germination and seedling stages of annual ryegrass to investigate the effects of CTS under water deficit conditions. We observed that no further positive effects on germination vigor, germination percentage, RWC, EL value, and MDA content were seen with higher CTS concentrations under osmotic stress. On the contrary, the lower CTS concentrations were shown to be more effective to induce osmotic stress defense. Moreover, 50 mg/L CTS application significantly facilitated the growth of roots and shoots at the post-germination stage, suggesting that CTS positively regulate the growth of annual ryegrass challenged with osmotic stress.
Water deficit limits photosynthesis, alters cell homeostasis, and causes an enhanced germination of ROS [34
]. To meet the challenge caused by ROS, a delicate enzymatic antioxidant system involving ascorbic acid peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutases (SOD) evolved to catalyze the reaction to change
and detoxify the H2
]. Our study supported the previous studies in demonstrating that CTS can enhance resistance to oxidative stress in plants [38
]. We found that exogenous CTS treatment resulted in significant decrease in
after three days of osmotic stress, and six days of osmotic stress showed even more reduction. However, there was no clear difference between CK and CTS treatments. This might be due to that CTS could improve the accumulation of protective enzymes (SOD, CAT, POD, and APX) in annual ryegrass seedlings under osmotic stress, indicating that CTS is able to scavenge superoxide anion as well as antioxidant during drought [40
]. In agreement with this assumption, elevated activities of SOD, APX, and POD resulted from the CTS treatment were detected in previous studies [41
]. It is worth noting that, in contrast with PEG treatment, the application of exogenous CTS under the osmotic stress remarkably increased the activity of antioxidants to reduce the damage caused by the accumulation of
. It suggests that the pre-treatment of CTS partially alleviates the formation of ROS by activating a variety of antioxidants in plants when they are exposed to unfavorable conditions. In addition to activity of the protective enzymes, chlorophyll, and three other molecules including free proline, protein, and soluble sugar functioning in osmotic regulation are considered as direct indicators to evaluate the water pressure induced damage in plants [42
]. Compared to pre-treatment of PEG alone, results demonstrated that levels of all of these indicators were elevated during the treatment course in response to the mixture of CTS and PEG pre-treatment, except that proline content was found to be declined. It suggests that the addition of CTS is capable of strengthening the osmosis regulation as indicated by the increase in protein content and soluble sugar. Furthermore, as one of the prominent osmolyte, proline has been extensively shown to be overproduced in stress condition to aid in stress tolerance by maintaining the osmotic balance [43
]. Reduction of proline content post PEG and CTS pre-treatment implies that CTS has a role in enhancing plant resistance to osmotic stress.
In general, EL levels and MDA concentrations served to indicate membrane intactness and stability [44
]. Our study demonstrated that osmotic stress would markedly increase EL and MDA content in annual ryegrass seedlings, but exogenous CTS treatments at any concentration level could mitigate these effects, suggesting that CTS could restrain damages caused by osmotic stress and maintain the membrane integrity and stability. Leaf RWC is commonly proposed as a reliable indicator of leaf water status [45
]. CTS treatment not only balances between water supply and leaf transpiration rate as supported by RWC data but also restores the chlorophyll synthesis abilities under osmotic stress [46
]. In annual ryegrass, CTS took part in processes of maintaining normal level of RWC, chlorophyll and protein content. As dehydration stress progresses, biochemical constraints may directly limit photosynthesis [47
]. CTS has been shown to enhance drought tolerance by increasing the efficiency of water use in pepper [4
]. In the current study, the exogenous CTS treatment applied to seedlings resulted in four times increase in net photosynthetic rate than untreated plants at six days of osmotic stress. Though osmotic stress adversely affects photosynthesis in this experiment, CTS treatment slightly increased the net photosynthetic rate, water use efficiency, and stomatal conductance of annual ryegrass under osmotic stress. In the present study, a multitude of genes encoding the proteins functioning in photosystem II reaction center subunit such as PsbP, Psb27, PsaN, PetF, and PetH were up-regulated in annual ryegrass seedlings with CTS pre-treatment under osmotic stress conditions. Given that the multifunction of the specific proteins in photosynthesis that are proven [48
], CTS pre-treatment might be able to promote plants to generate the proteins for the purpose of eliminating the unfavorable effects on the process of photosynthesis caused by osmotic stress. This finding elucidates the part of reason why CTS pre-treated plants can maintain to some extent higher photosynthetic rate than that of untreated plants exposed to water deficit.
Six proteins involved in carbon metabolism were identified. Among them, a transketolase encoded by the TKT
gene is a pivotal enzyme associated with both the pentose phosphate pathway and the calvin cycle of photosynthesis. As a central enzyme in glycolysis, it has been widely proved that GAPDH (EC:18.104.22.168) was involved in several abiotic stress response and improved plant tolerance against the stressful conditions [49
]. A high level of GAPDH (EC:22.214.171.124) was observed under osmotic stress, which might be a beneficial consequence of exogenous CTS treatment on seedlings of annual ryegrass. In addition, PGK (EC126.96.36.199) is one of key enzymes in the glycolytic pathway. Several studies has been focusing on the function of PGK (EC188.8.131.52) proteins in improving yield in plants under several abiotic stress such as salinity stress and drought stress [51
]. In the present study, a gene that encodes PGK was induced to enhance the plant tolerance. Ribose-5-phosphate isomerase (RPI) was identified as a key enzyme of carbon metabolism. A decreased level in RPI expression was found in photosynthesis in drought-treated rice leaves [53
]. Nevertheless, the up-regulated gene encoding RPI2 (EC:184.108.40.206) was observed in annual ryegrass, which contributes to energy consuming for resistance to osmotic stress when plants are exposed to CTS pre-treatment. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a bifunctional enzyme in charge of the Calvin cycle [54
]. Ribulose bisphosphate carboxylase small chain (rbcS) (EC:220.127.116.11) is one of the small subunits of RubisCO that widely exists in higher plants, but its function is not yet fully known [55
]. According to the results of this study, rbcS encoded by a single gene and could result in higher plant resistance to osmotic stress after exogenous CTS treatment. Shi et al. reported that PPC1 (EC:18.104.22.168) in leaves plays a pivotal role in carbon and nitrogen metabolism in Arabidopsis
]. Consistent with the previous research, the up-regulated gene encoding PPC1 (EC:22.214.171.124) that is involved in carbon metabolism was also observed in osmotic stress-treated annual ryegrass, indicating that exogenous CTS plays an important role by inducing PPC1-related genes in preventing plants from osmotic stress condition.
Based on above-reported results, a model of CTS-mediated osmotic stress response in annual ryegrass was proposed (Figure 9
). Though osmotic stress induces the ROS accumulation, osmotic pressure, and cell damage in plants, exogenous application of CTS could maintain lower MDA content and higher photosynthetic rates, change antioxidant enzyme activities, reduce proline content, and develop other unknown adaptive reactions. Meanwhile, the transcriptomic analysis revealed alterations in a variety of genes involved in photosynthesis, the carbon fixation pathway, hormone regulation, and amino acid metabolism induced by CTS pre-treatment. All these changes, in turn, relieve damage to cell integrity, ROS production, reaction to osmotic pressure, and other osmotic stress–elicited negative effects. Taken together, our study demonstrated that exogenous application of CTS could significantly improve plant performance under osmotic stress by modulating ROS accumulation, antioxidant responses, osmolyte accumulation, and expression of stress-related genes on both germination and seedling stages of annual ryegrass.