Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels.
Citation: Li Y, Xu S-S, Gao J, Pan S, Wang G-X (2014) Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba. PLoS ONE 9(3): e93290. https://doi.org/10.1371/journal.pone.0093290
Editor: Ji-Hong Liu, Key Laboratory of Horticultural Plant Biology (MOE), China
Received: October 20, 2013; Accepted: March 4, 2014; Published: March 31, 2014
Copyright: © 2014 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the National High-Tech R & D Program (863 Program) for the 12th Five-Year Plan (2011AA100503), Natural Science Fund of China (31330010), and Zhejiang Provincial Natural Science Foundation of China (LZ13C030002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Stomata are natural microscopic pores located in the epidermis of the aerial parts of plants that play a pivotal role in regulating influx of CO2 for photosynthesis and water loss through transpiration . However, transpiration and photosynthesis do not respond to the changing stomatal aperture identically , . As the stomatal aperture decreases above the threshold, leaf transpiration rates always diminish prior to the CO2 assimilation rates, favoring a higher instantaneous water use efficiency (WUEi) –. It provides a basis for the suggestions that partial closure of the stomata might conduce to the improvement of WUEi in plants , . It is well known that moderate stomatal closure can be triggered by numerous abiotic cues including high light intensity, high CO2 concentrations, low air humidity, and especially drought stress, see the review . Moreover, previous studies have also shown that stomatal closure can be induced by multiple biotic cues such as pathogenic microbes and purified MAMPs –. However, some pathogenic microbes can reopen closed stomata via various virulence factors such as coronatine, fusicoccin and oxalic acid , , , . Noticeably, a kind of nonpathogenic yeast Saccharomyces cerevisiae is observed to induce stomatal closure via ROS production mediated mainly by salicylhydroxamic acid-sensitive peroxidase in a recent study . Besides yeast, whether other nonpathogenic microbes can trigger stomatal closure remains less known. Just as nonpathogenic as Saccharomyces cerevisiae, Chlorella vulgaris is a unicellular photosynthetic microorganism that can employ light energy and CO2, with higher photosynthetic activity than plants . Chlorella vulgaris can synthesize many bioactive substances such as carbohydrates, proteins, function lipids, amino acids and vitamins, with positive effects both on humans and animals, as well as the improvement of seed germination capacities, root growth and nutrient uptake of cash crops –. However, the effects of Chlorella on stomatal movement and WUEi of plants have not been studied. We therefore explore whether Chlorella can be sensed by guard cells to induce partial stomatal closure and improve WUEi of plants when applied as foliar spray.
In plant cells, ROS act as key second messengers in mediating stomatal closure triggered by abiotic and biotic signals –, , . ROS production induced by various stimuli in guard cells is modulated by specific enzymes, including NADPH oxidase, peroxidase, xanthine oxidase, oxalate oxidase and amine oxidase. The various enzymes have different functions in distinct signaling pathways –. For instance, ROS production mediated by diphenylene iodonium chloride (DPI)-sensitive plasma membrane NADPH oxidase is involved in abscisic acid (ABA)-, methyl jasmonate (MeJA)-, ozone-, darkness, ethylene-, allyl isothiocyanate (AITC)-, low dose of ultraviolet B (UV-B)-, bacterial flagellum (flg22)-, bacterial elongation factor Tu (elf18)- and bacterial lipopolysaccharides (LPS)-induced stomatal closure , –, while ROS production modulated by salicylhydroxamic acid (SHAM)-sensitive cell wall peroxidase is implicated in salicylic acid (SA)-, high dose of UV-B-, chitosan-, yeast elicitor (YEL)-, methylglyoxal- and yeast-triggered stomatal closure , , –. However, it is unknown whether Chlorella induces ROS production and what is the enzyme source of ROS production in guard cells.
Certain dicotyledon (e.g. Vicia faba L.) and microalgae (e.g. Chlorella vulgaris) provide ideal material models for exploring the effects of Chlorella on stomatal movement and WUEi in V. faba. Using epidermal strip bioassay, H2O2 fluorescence assay, gas-exchange measurements, chlorophyll fluorescence and content analysis, we were to determine 1) whether Chlorella can be perceived by guard cell to trigger partial stomatal closure in epidermal strips of broad bean; 2) whether this action requires ROS production, and if so, which enzyme mediates ROS production; 3) whether this action improves WUEi in V. faba.
Materials and Methods
Seeds of broad bean (V. faba L. cv. Da qing pi) were selected and sterilized in 70% ethanol for 30 min, then cleaned with distilled water. Sterilized Seeds were soaked in water until they were germinated, then transplanted into pots (5 cm×10 cm) containing a mixture of growing medium: vermiculite (3∶1, v/v). Plants were grown in a controlled growth chamber with a temperature of 20°C–25°C, a relative humidity of 70%, photosynthetic active radiation (PAR) of 300 µmol m−2 s−1 and a photoperiod of 14 h light/10 h dark, and watered daily. When plants were 4 weeks old, the even-aged fully expanded leaves were used as experimental material.
Molecular probe 2′, 7′-dichlorofluorescin diacetate (H2DCF-DA, Sigma-Aldrich, St Louis, MO, USA) was dissolved in dimethyl sulfoxide to produce a stock solution, which was aliquoted. Salicylhydroxamic acid (SHAM), diphenyleneiodonium chloride (DPI), catalase (CAT, bovine liver) and ethanesulfonic acid (MES) were obtained from Sigma-Aldrich. Besides these chemicals, the remaining chemicals were purchased from Chinese companies. All the chemicals used were of the highest analytical grade.
Culture of Chlorella cells
The Chlorella (Chlorella vulgaris) sample used in this study was obtained from FACHB (Wuhan, People's Republic of China). Chlorella cells were cultured in Erlenmeyer flasks (500 mL) containing 250 mL BG11 medium (PH 6.8) and shaken at 150 rpm in a rotary shaker under controlled conditions at a temperature of day 25°C/night 22°C, photosynthetic active radiation (PAR) of 50 µmol m−2 s−1 and a photoperiod of 16 h light/8 h dark. After 7 days of culture, the Chlorella cells were collected by centrifugation, and then rinsed twice with sterile water. Finally the cells were resuspended in sterile double distilled water to yield the different concentrations of Chlorella suspension (1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1) as determined by optical density and serial dilutions with plate counts.
Stomatal bioassay experiments were performed as described , ,  with slight modifications. Briefly, the epidermis was first peeled carefully from the abaxial surface of the youngest, fully expanded leaves of 4-week-old plants, and cut into strips, then incubated in opening buffer (10 mM MES, 50 mM KCl, pH 6.15) for 2 h under light condition (photon flux density of 300 µmol m−2 s−1) at 22°C–25°C to promote stomatal opening. Once the stomata were fully open, the epidermal strips were transferred to opening buffer containing different concentrations of Chlorella suspension (0, 1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1) for another 2 h. When inhibitors or scavenger (SHAM, DPI, or CAT) were used, they were added 30 min prior to the Chlorella treatments. Finally, stomata were digitized using a Canon PowerShot G10 camera coupled to a DSZ5000X microscope (UOP, Chongqing, China). The width and length of stomatal pore were measured on digital images with the Image-Pro plus6.0 software (Media Cybernetics, Silver Springs, MD). The stomatal aperture was calculated as pore width/length. Fifty stomata originated from three separate plants were randomly chosen for each treatment and the experiments were repeated three times , . These data represented 150 measurements ± SE. To obtain time response course, the stomatal aperture was examined at 20 min intervals. Twenty stomata were randomly selected for three independent experiments. These data represented 60 measurements ± SE.
Measurement of ROS in guard cells
Reactive oxygen species (ROS) in guard cells were examined by loading epidermal preparations with 2′, 7′-dichlorofluorescin diacetate (H2DCF-DA). After the treatment described in the above experiment, the epidermal strips were transferred to opening buffer containing 50 µM H2DCF-DA for 15–20 min in the dark at room temperature. The excess dye was washed out with opening buffer and fluorescence photographs of guard cells were taken with a Canon PowerShot G10 camera coupled to a DSZ5000X microscope (UOP, Chongqing, China) after 15–20 min. The acquired fluorescence images were processed using Image-Pro plus 6.0 software (Media Cybernetics, Silver Springs, MD). Average fluorescence intensities of treated cells were normalized to the control value taken as 100%. In each treatment, three epidermal strips from different plants were measured and the experiment was repeated three times. These data represented 100 measurements ± SE.
Gas-exchange measurements and leaf stomatal bioassay
When plants were 4 weeks old, the different concentrations of Chlorella suspension (1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1) and water were sprayed onto all fully expanded leaves present with a hand sprayer until the both sides of leaves were uniformly wet. After treatment for 48 h, the net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci) and transpiration rate (E) of the fourteen random fully expanded leaves were measured on seven individual plants per treatment under a photosynthetic active radiation (PAR) of 1000 µmol m−2 s−1 with a portable photosynthetic open-system between 10:00 a.m. and noon (CI-340, 4845NW Camas Meadows Drive, Camas, WA, 98607, USA) . Before measurements, the plants were allowed to acclimate to a sufficient light irradiance for more than 1 h. In operation, air temperature in the assimilation chamber was kept at 25°C, air flow was set to 500 mL min−1 and ambient CO2 concentration was maintained at 380 ppm. Data were recorded after 3–4 min, when the steady-state photosynthesis was achieved. WUEi was calculated as Pn/E .
To further test the effects of Chlorella on stomatal aperture in leaves level, the epidermal strips of expanded leaves of broad beans sprayed with different concentrations of Chlorella suspension and water for 48 h were peeled off and immediately observed under a microscope. Finally, stomata were digitized using a Canon PowerShot G10 camera coupled to a DSZ5000X microscope (UOP, Chongqing, China). The width and length of stomatal pore were measured on digital images with the Image-Pro plus6.0 software (Media Cybernetics, Silver Springs, MD). 120 stomata were randomly selected for each treatment from different epidermal strips of seven individual plants. These data represented 120 measurements ± SE.
Chlorophyll fluorescence measurements
Maximal photochemical efficiency (Fv/Fm), the effective quantum yield of PSII (ΦPSII), non-photochemical quenching (NPQ), the coefficient for photochemical quenching (qP) and electron transportation rate (ETR) were measured on the same leaves used for gas exchange analysis with the MAXI version of the IMAGING-PAM M-Series chlorophyll fluorescence system (Heinz-Walz GmbH, Effeltrich, Germany). The experiment procedures and nomenclature used for fluorescence measurements were based on the descriptions in previous studies . Saturating light pulses were given every 20 s , .
The same leaves selected for gas-exchange and chlorophyll fluorescence analysis were used for immediate chlorophyll determination with a spectrophotometer (SP752, Shanghai, China). 0.1 g leaf tissue was first homogenized in 10 mL acetone: ethanol (1∶1, v/v) for 24 h, then the chlorophyll content of the supernatant was measured and supernatant absorption spectrum was set at 645 and 663 nm. The chlorophyll a and b content were calculated as [(12.71×A663)−(2.69×A645)]/[100×(fresh weight of leaves)] and [(22.88×A645)−(4.67×A633)]/[100×(fresh weight of leaves)], respectively. The unit was reported as mg Chl. per g FW, as described in , .
Chlorella-induced stomatal closure in broad bean
To determine whether Chlorella has any effect on stomatal movement, the abaxial epidermal peels of broad bean were separately treated with 1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1 of Chlorella suspension. After 2 h, the stomatal apertures were reduced by 7.5% (P<0.001), 22.0% (P<0.001), 33.8% (P<0.001), 39.7% (P<0.001), and 39.4% (P<0.001), respectively, and showed a dosage effect (Figure 1A). Furthermore, as noted in Figure 1B, 1.0×109 ind mL−1 of Chlorella suspension induced stomatal closure in a time-dependent manner, reaching the maximum effect at 2 h after treatment, under which conditions the stomatal apertures were reduced by 40.8% (P<0.001).
(A) The dosage effect of Chlorella-induced stomatal closure. Epidermal peels of broad beans preincubated for 2 h in opening buffer under light were treated with various concentrations of Chlorella suspension, and stomatal apertures were measured after 2 h. Each bar represents the mean ± SE of three biological replicates (n = 150). (B) Time response course of stomatal closure triggered by Chlorella (1.0×109 ind mL−1) and stomatal apertures were quantified every 20 min. Data are the mean of 60 measurements ± SE from three biological repeats. Solid square: Control, solid circle: Chlorella. Means with different letters are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
The effects of CAT, DPI and SHAM on Chlorella-induced stomatal closure
ROS play a vital role in regulating stomatal movement , , . To confirm the involvement of ROS in Chlorella-induced stomatal closure and elucidate the enzymatic sources of ROS production, the abaxial epidermal strips of broad bean were incubated with 1.0×109 ind mL−1 of Chlorella suspension with or without CAT, DPI or SHAM, all of which remove or decrease ROS level , , . We observed that Chlorella-induced stomatal closure was almost entirely restored by a H2O2 scavenger, CAT, at 100 UmL−1 (P = 0.03), strongly inhibited by an NADPH oxidase inhibitor, DPI, at 20 µM (P<0.001), while slightly suppressed by a peroxidase inhibitor, SHAM, at 2 mM (P<0.001) (Figure 2). Additionally, there was no statistical change in stomatal aperture when epidermal strips of broad bean were treated with DPI, SHAM or CAT alone (Figure S1).
Epidermal peels of broad beans preincubated for 2 µM DPI, 2 mM SHAM and 100 UmL−1 CAT for 30 min, then, floated on 1.0×109 ind mL−1 Chlorella suspension and stomatal apertures were measured after 2 h. These data are the mean ± SE of the representative results from three biological repeats (n = 150 per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Chlorella-induced ROS production in guard cells of broad bean
ROS act as important second messengers in mediating stomatal closure , , , . ROS production via NADPH oxidase has been demonstrated to participate in Chlorella-triggered stomatal closure in the stomatal bioassay experiments. Therefore, we further monitored Chlorella-induced ROS production in guard cells using 2′, 7′-dichlorofluorescin diacetate (H2DCF-DA). As shown in Figure 3, the ROS level in guard cells was significantly improved when 1.0×109 ind mL−1 of Chlorella suspension was applied (P<0.001). Moreover, the Chlorella-induced ROS production was completely inhibited by 100 UmL−1 CAT (P<0.001), largely suppressed by 20 µM DPI (P<0.001) while not affected by 2 mM SHAM (P = 0.949) (Figure 3).
Epidermal peels of broad beans without (control) (A) or with 120 min of treatment with Chlorella alone (B), with Chlorella and DPI (C), with Chlorella and SHAM (D) or with Chlorella and CAT (E) were loaded with 50 µM of H2DCF-DA for 15–20 min in the dark. After a brief wash with opening buffer, photographs were taken for representative pairs of guard cells from each treatment using fluorescence microscopy (A–E). The scale bar in E is 100 µm, and applies to all photographs. (F)The average fluorescence intensity of guard cells in images (A–E). The vertical scale represents percentage response relative to fluorescence value taken as 100% in control treatments. These data are the mean ± SE of the representative results from three biological repeats (100 total guard cells per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
The effects of Chlorella on gas-exchange
To survey the effects of Chlorella on plant photosynthetic gas-exchange, 1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1 of Chlorella suspension were evenly sprayed onto leaves of broad beans. After 48 h, the stomatal apertures were reduced by 0.4% (P = 0.999), 1.1% (P = 0.863), 15.2% (P<0.001), 21.8% (P<0.001), and 27.2% (P<0.001), respectively (Figure 4), the gs was severally reduced by 1.6‰ (P = 0.919), 8.9‰ (P = 0.563), 6.3% (P<0.001), 13.9% (P<0.001) and 19.3% (P<0.001), and the E was reduced by 0.2‰ (P = 0.992), 3‰ (P = 0.872), 7.3% (P<0.001), 14.7% (P<0.001) and 20.4% (P<0.001), respectively (Figure 5). Furthermore, the Pn and Ci were hardly affected when the concentrations of Chlorella suspension were less than or equal to 1.0×109 ind mL−1 (P>0.05), while were significantly inhibited at a higher concentration (P<0.001) (Figure 5). As a result, the WUEi were separately improved by 1.5% (P = 0.082), 4‰ (P = 0.600), 8.6% (P<0.001), 19.2% (P<0.001) and 10.2% (P<0.001) (Figure 5).
After spraying uniformly different concentrations of Chlorella suspension (1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1) and water onto the leaves of broad bean for 48 h, the epidermal strips were peeled off and immediately observed under a microscope. Photographs were taken for representative pairs of guard cells from each treatment using microscopy (A–F). The scale bar in F is 40 µm, and applies to all photographs. (G) The average stomatal aperture shown in images (A–F). These data are the mean ± SE of the representative results (n = 120 per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Alterations in (A) net photosynthetic rate (Pn), (B) stomatal conductance (gs), (C) intercellular CO2 concentration (Ci), (D) transpiration rate (E), and (E) instantaneous intrinsic water use efficiency (WUEi), of broad bean leaves at 48 h after treatment with different concentrations of Chlorella suspension (1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1). These data are the mean ± SE (n = 14 per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Chlorophyll fluorescence and content analysis
To investigate whether plant photosynthetic activities are influenced by Chlorella, leaf chlorophyll fluorescence was tested on the same leaves used for gas exchange analysis. No statistical alterations in the values of minimal fluorescence (F0) and maximal fluorescence (Fm) were observed after different treatments (Table S1). As a result, there was no pronounced change in maximal photochemical efficiency, Fv/Fm (P>0.05) (Table 1). Furthermore, other chlorophyll fluorescence parameters such as effective quantum yield of PSII (ΦPSII), non-photochemical quenching (NPQ), the coefficient for photochemical quenching (qP) and electron transportation rate (ETR) were not significantly affected by different concentrations of Chlorella suspension (P>0.05) (Table 1). Moreover, as noted in Table 1, the contents of chlorophyll a, chlorophyll b and total chlorophyll and the ratio of chlorophyll a/b were not different among treatments (P>0.05).
The effects of Chlorella on stomatal movement in broad bean
Stomatal openings have long been recognized as passive access to inner leaf tissues for many plant bacteria. However, recent studies have revealed that potential microbes can induce stomatal closure by activating a series of signaling transduction pathways , , , , , . In the present study, we found that Chlorella as an available autotrophic microorganism induced stomatal closure in epidermal strips of broad beans in a dose-and time-dependent manner, arriving at the maximum effect at 1.0×109 ind mL−1 of Chlorella suspension and 2 h after treatment (Figure 1). These results further validate that microbes can actively trigger stomatal closure. Mounting evidence suggests that microbe-triggered stomatal closure relies on the perception of MAMPs by pattern recognition receptors (PRRs) –. MAMPs are conserved molecules on the surface of microorganisms, such as flg22, elf18, LPS, fungal cell wall derived chitin, ergosterol, glucans and chitosan oligosaccharides, yeast elicitor, plant cell wall derived oligogalacturonic acid, as well as various glycopeptides and glycoproteins, most of which are capable of inducing stomatal closure –. It is known that Chlorella cell wall and extracellular organic matter (EOM) attached at the Chlorella cell surface are mainly composed of polysaccharides, glycoproteins, lipids, glucosamine, chitin-like glycans and other matrix , . Consequently, we speculate that some components of Chlorella cell wall and EOM are involved in Chlorella-triggered stomatal closure. Further work will be done to unveil which component induces stomatal closure.
The effects of Chlorella on ROS production in broad bean
In plant cells, ROS functions as an integral intermediate in MAMPs- and ABA-dependent stomatal closure , , , , . Our work primarily indicated that Chlorella-induced stomatal closure was accompanied by ROS production (Figure 3), which is similar to live pathogenic microbes, yeast and MAMPs-induced stomatal closure , , , , . ROS production in guard cells is mediated by various enzymes in response to different stimuli, including NADPH oxidase, peroxidase, xanthine oxidase, oxalate oxidase and amine oxidase –, , , . To clarify which enzyme catalyzes ROS production mediating Chlorella-induced stomatal closure, we further assessed the effects of Chlorella on stomatal movement and ROS production with pharmacological inhibitors or scavenger such as DPI, SHAM and CAT , . We observed that Chlorella-induced stomatal closure was almost completely reversed by an H2O2 scavenger, CAT, strongly inhibited by an NADPH oxidase inhibitor, DPI, while slightly suppressed by a peroxidase inhibitor, SHAM (Figure 2). In addition, it was shown that the Chlorella-induced ROS production was completely abolished by CAT, largely restrained by DPI, whereas hardly affected by SHAM (Figure 3). Taken together, these results suggest that Chlorella-induced stomatal closure is mediated by ROS production mainly via DPI-sensitive plasma membrane NADPH oxidase but not SHAM-sensitive peroxidase. It is analogous to ABA-, MeJA-, ozone-, darkness-, ethylene-, AITC-, low dose of UV-B-, flg22-, LPS- and elf18-induced stomatal closure modulated by DPI-sensitive NADPH oxidase , –, while different from SA-, high dose of UV-B-, chitosan-, YEL-, methylglyoxal- and yeast-triggered stomatal closure via SHAM-sensitive peroxidase , , –. These results will provide insights for the prediction of elicitors from Chlorella. In plants, the NADPH oxidase respiratory burst oxidase homologs (RBOHs) are encoded by 10 different RBOHs genes, four of which have been characterized, namely RBOHB, RBOHC, RBOHD and RBOHF . Among four RBOHs, RBOHD is the primary NADPH oxidase responsible for the rapid ROS production in response to MAMPs, RBOHF on the other hand, is the main ROS-producing enzymes mediating ABA-dependent ROS production . Although our work showed that Chlorella-induced ROS production was mainly dependent on NADPH oxidase, which RBOH catalyzes the generation of ROS remains unsloved. Mechanical studies have revealed that stomatal closure triggered by biotic and abiotic stresses is related to the ABA-mediated complex network of signalling events, including activation of G-proteins and the guard cell specific OST1 kinase, generation of ROS and nitric oxide (NO), elevation of cytosolic Ca2+ levels and Ca2+ oscillations, protein phosphorylation/dephosphorylation, reorganization of the cytoskeleton, elevation of cytosolic pH, and activation of cation and anion channels . Besides ROS, whether other components of ABA signaling pathways are involved in Chlorella-induced stomatal closure need to be explored in the future. The stomatal system of Arabidopsis mutant will provide an attractive tool for dissecting novel aspects of signaling events.
Involvement of Chlorella-induced stomatal closure in the improvement of WUEi in broad bean
Stomata play a prominent role in controlling gas exchanges between plants and environments. Numerous studies have demonstrated that moderate stomatal closure is an extraordinary adaptation to various environmental stresses in plants , –. When the plant encounters mild stress, the leaf stomatal conductance usually decreases earlier than photosynthesis, leading to the increase of WUEi in plants , , , . Therefore, Chlorella-induced stomatal closure can be expected to improve WUEi of plants by preventing more water loss through transpiration. Our studies showed that Chlorella caused reductions in leaf stomatal aperture, gs and E until the concentration reached 1.0×108 ind mL−1 in intact leaves (Figure 4 and Figure 5). The images in Figure 4 provided further visible proof that reductions in transpiration rate and stomatal conductance in whole leaves when the concentration reached 1.0×108–1.0×1010 ind mL−1 were only due to the Chlorella-induced stomatal closure rather than mechanical obstruction of stomata caused by stacked Chlorella cells. Comparing the results in Figure 1, Figure 4 and Figure 5, it could been seen that there was a linear response in stomatal closure when floating epidermal peels on increasing Chlorella concentration from 1.0×106 up to 1.0×109 ind mL−1 (Figure 1), however, stomatal aperture, transpiration rate and stomatal conductance (Figure 4 and Figure 5) remained unchanged up to 1.0×107 ind mL−1 in intact leaves. These differences between stomata in intact leaves and epidermal peels responding to various treatments may be attributed to the stomatal sensitivity to different stimuli because of the structure discrepancies. It agreed with a previous study that stomata in epidermal peels of Commelina communis L. were more sensitive to ABA than that in intact leaves . The study revealed that the cuticle and mesophyll of the whole leaf might limit the stomatal sensitivity to applied stimulus . Furthermore, the Pn and Ci were hardly affected after treatment with less than or equal to 1.0×109 ind mL−1 of Chlorella suspension, whereas were significantly repressed at a higher concentration (Figure 5). As a consequence, the WUEi of broad beans was improved greatly when proper concentrations of Chlorella suspension were applied, while less affected at higher concentrations due to the inhibition of leaf Pn (Figure 5). Previous studies have indicated that photosynthesis is not only restricted by stomatal limitations of the CO2 influx but also by non-stomatal limitations that influence activities of photosynthetic reactions center such as photosynthetic apparatus, ATP synthesis, and electron transfer –. The comprehensive information about the photosynthetic activity can be obtained from chlorophyll fluorescence and content analysis . Our data demonstrated that no pronounced alterations in the chlorophyll content and fluorescence parameters, including Fv/Fm, ΦPSII, NPQ, qP, and ETR, were detected 48 h after application of various concentrations of Chlorella suspension (Table 1). These phenomena suggest that short-term Chlorella treatment neither affect the photosynthetic transport of electrons nor lead to photoinhibition at the PSII complexes , . The data together with the changes of Pn and Ci reveal that inhibition of photosynthesis at a higher concentration of Chlorella suspension is only attributed to a decrease in CO2 transfer from atmosphere. To sum up, these results initially present that optimal concentrations of Chlorella suspension improve the WUEi in plants after exogenous application, mainly ascribing to a reduction in stomatal conductance above the threshold, under which transpiration rates decrease greatly whereas photosynthetic rates are barely affected.
In conclusion, Chlorella could be sensed by guard cell to trigger partial stomatal closure via NADPH oxidase-dependent ROS production and improve WUEi in V. faba when applied as foliar spray. In addition, heated-killed Chlorella cells could trigger stomatal closure and improve WUEi, just as live Chlorella cells did (Figure S2 and Figure S3). Further work will be needed to comprehensively understand the effects of Chlorella on stomatal movement and WUEi of a wide of agricultural crops. It will provide scientific evidence for the development of new bio-antitranspirant and advancement of water-saving technology for solving fresh water scarcity.
Effects of 20 µM DPI, 2 mM SHAM and 100 UmL−1 CAT on stomatal aperture in V. faba. Epidermal peels of broad beans preincubated for 2 h in opening buffer under light were treated with 20 µM DPI, 2 mM SHAM and 100 UmL−1 CAT and stomatal apertures were measured after 2 h. These data are the mean ± SE of the representative results from three biological repeats (n = 150 per bar). Same letters above the bars indicate mean values that are not significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Heated-killed Chlorella-triggered stomatal closure in V. faba. (A) The dosage effect of heated-killed Chlorella-induced stomatal closure in epidermal peel experiments. Epidermal peels of broad beans preincubated for 2 h in opening buffer under light were treated with different concentrations of heated-killed Chlorella suspension, and stomatal apertures were measured after 2 h. These data are the mean ± SE of three biological replicates (n = 150 per bar). (B) The dosage effect of heated-killed Chlorella-triggered stomatal closure in intact leaves. After evenly spraying various concentrations of heated-killed Chlorella suspension and water onto the leaves of broad bean for 48 h, the epidermal strips were peeled off and immediately observed under a microscope. Each bar represents the mean ± SE of the representative results (n = 120). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
The effects of Heated-killed Chlorella on instantaneous water use efficiency (WUEi) in V. faba. The changes in instantaneous intrinsic water use efficiency (WUEi) of broad bean leaves 48 h after treatment with different concentrations of heated-killed Chlorella suspension (1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1). These data are the mean ± SE (n = 14 per bar). Different letters above the bars demonstrate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
The minimal fluorescence (F0) and maximal fluorescence (Fm) of broad bean leaves after treatment with different concentrations of Chlorella suspension raging from 0 to 1.0×1010 ind mL−1 for 48 h. Each value represents the mean ± SE (n = 14). Means estimates with same letters are not significantly different among treatments as determined by ANOVA (LSD test, P<0.05).
Conceived and designed the experiments: YL GXW. Performed the experiments: YL SSX JG SP GXW. Analyzed the data: YL GXW. Contributed reagents/materials/analysis tools: JG GXW. Wrote the paper: YL GXW.
- 1. Jiang K, Sorefan K, Deeks MJ, Bevan MW, Hussey PJ, et al. (2012) The ARP2/3 complex mediates guard cell actin reorganization and stomatal movement in Arabidopsis. Plant Cell 24: 2031–2040.
- 2. Hetherington AM, Woodward FI (2003) The role of stomata in sensing and driving environmental change. Nature 424: 901–908.
- 3. Chaves M, Oliveira M (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J Exp Bot 55: 2365–2384.
- 4. Condon A, Richards R, Rebetzke G, Farquhar G (2002) Improving intrinsic water-use efficiency and crop yield. Crop Sci 42: 122–131.
- 5. Grimmer MK, Foulkes MJ, Paveley ND (2012) Foliar pathogenesis and plant water relations: a review. J Exp Bot 63: 4321–4331.
- 6. Wang GX, Zhang J, Liao JX, Wang JL (2001) Hydropassive evidence and effective factors in stomatal oscillations of Glycyrrhiza inflata under desert conditions. Plant Sci 160: 1007–1013.
- 7. Acharya BR, Assmann SM (2009) Hormone interactions in stomatal function. Plant Mol Biol 69: 451–462.
- 8. Lee S, Choi H, Suh S, Doo I-S, Oh K-Y, et al. (1999) Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiol 121: 147–152.
- 9. Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate immunity against bacterial invasion. Cell 126: 969–980.
- 10. Schulze-Lefert P, Robatzek S (2006) Plant pathogens trick guard cells into opening the gates. Cell 126: 831–834.
- 11. Bari R, Jones JD (2009) Role of plant hormones in plant defence responses. Plant Mol Biol 69: 473–488.
- 12. Koers S, Guzel-Deger A, Marten I, Roelfsema MRG (2011) Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard-cell S-type anion channels. Plant J 68: 670–680.
- 13. Melotto M, Underwood W, He SY (2008) Role of stomata in plant innate immunity and foliar bacterial diseases. Ann Rev Phytopathol 46: 101–122.
- 14. Zeng W, Melotto M, He SY (2010) Plant stomata: a checkpoint of host immunity and pathogen virulence. Curr Opin Biotech 21: 599–603.
- 15. Gao J, Wang N, Wang G-X (2013) Saccharomyces cerevisiae-induced stomatal closure mainly mediated by salicylhydroxamic acid-sensitive peroxidases in Vicia faba. Plant Physiol Bioch 65: 27–31.
- 16. Keffer J, Kleinheinz G (2002) Use of Chlorella vulgaris for CO2 mitigation in a photobioreactor. J Ind Microbiol Biot 29: 275–280.
- 17. Guzman S, Gato A, Calleja J (2001) Antiinflammatory, analgesic and free radical scavenging activities of the marine microalgae Chlorella stigmatophora and Phaeodactylum tricornutum. Phytother Res 15: 224–230.
- 18. Miranda M, Sato S, Mancini-Filho J (2001) Antioxidant activity of the microalga Chlorella vulgaris cultered on special conditions. Boll Chim Farm 140: 165–168.
- 19. Jong-Yuh C, Mei-Fen S (2005) Potential hypoglycemic effects of Chlorella in streptozotocin-induced diabetic mice. Life Sci 77: 980–990.
- 20. Park JY, Cho HY, Kim JK, Noh KH, Yang JR, et al. (2005) Chlorella dichloromethane extract ameliorates NO production and iNOS expression through the down-regulation of NFκB activity mediated by suppressed oxidative stress in RAW 264.7 macrophages. Clin Chim Acta 351: 185–196.
- 21. Lee HS, Kim MK (2009) Effect of Chlorella vulgaris on glucose metabolism in Wistar rats fed high fat diet. J Med Food 12: 1029–1037.
- 22. Li X, Jiang ML, He LQ, Li LL, Ji DX, et al. (2010) The effects of extracts of Chlorella vulgaris on seed germination of cruciferous vegetables. Jiangsu Agr Sci 4: 160–162 (In Chinese).
- 23. Yun HJ, Kim I, Kwon SH, Kang JS, Om AS (2011) Protective effect of Chlorella vulgaris against lead-induced oxidative stress in rat brains. J Health Sci 57: 245–254.
- 24. Khokon MAR, Hossain MA, Munemasa S, Uraji M, Nakamura Y, et al. (2010a) Yeast elicitor-induced stomatal closure and peroxidase-mediated ROS production in Arabidopsis. Plant Cell Physiol 51: 1915–1921.
- 25. Luis A, Corpas FJ, Sandalio LM, Palma JM, Gómez M, et al. (2002) Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes. J Exp Bot 53: 1255–1272.
- 26. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7: 405–410.
- 27. Vranova E, Inzé D, Van Breusegem F (2002) Signal transduction during oxidative stress. J Exp Bot 53: 1227–1236.
- 28. Cona A, Rea G, Angelini R, Federico R, Tavladoraki P (2006) Functions of amine oxidases in plant development and defence. Trends Plant Sci 11: 80–88.
- 29. Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, et al. (2003) NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO J 22: 2623–2633.
- 30. Desikan R, Cheung MK, Clarke A, Golding S, Sagi M, et al. (2004) Hydrogen peroxide is a common signal for darkness-and ABA-induced stomatal closure in Pisum sativum. Funct Plant Biol 31: 913–920.
- 31. Suhita D, Raghavendra AS, Kwak JM, Vavasseur A (2004) Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate-and abscisic acid-induced stomatal closure. Plant Physiol 134: 1536–1545.
- 32. Joo JH, Wang S, Chen J, Jones A, Fedoroff NV (2005) Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone. Plant Cell 17: 957–970.
- 33. Desikan R, Last K, Harrett-Williams R, Tagliavia C, Harter K, et al. (2006) Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. Plant J 47: 907–916.
- 34. Munemasa S, Oda K, Watanabe-Sugimoto M, Nakamura Y, Shimoishi Y, et al. (2007) The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production. Plant Physiol 143: 1398–1407.
- 35. Khokon M, Jahan MS, Rahman T, Hossain MA, Muroyama D, et al. (2011) Allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis. Plant Cell Environ 34: 1900–1906.
- 36. He J-M, Ma X-G, Zhang Y, Sun T-F, Xu F-F, et al. (2013) Role and interrelationship of Gα protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiol 161: 1570–1583.
- 37. Mori IC, Pinontoan R, Kawano T, Muto S (2001) Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba. Plant Cell Physiol 42: 1383–1388.
- 38. Khokon MAR, Uraji M, Munemasa S, Okuma E, Nakamura Y, et al. (2010b) Chitosan-induced stomatal closure accompanied by peroxidase-mediated reactive oxygen species production in Arabidopsis. Biosci Biotech Bioch 74: 2313–2315.
- 39. He J, Yue X, Wang R, Zhang Y (2011) Ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent hydrogen peroxide synthesis in Vicia faba L. J Exp Bot 62: 2657–2666.
- 40. Khokon MAR, Okuma E, Hossain MA, Munemasa S, Uraji M, et al. (2011) Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis. Plant Cell Environ 34: 434–443.
- 41. Hoque TS, Uraji M, Ye W, Hossain MA, Nakamura Y, et al. (2012) Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis. J Plant Physiol 979–986.
- 42. Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, et al. (2001) Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol 126: 1438–1448.
- 43. Yang HM, Zhang XY, Wang GX, Li Y, Wei XP (2003) Cytosolic calcium oscillation may induce stomatal oscillation in Vicia faba. Plant Sci 165: 1117–1122.
- 44. Iriti M, Picchi V, Rossoni M, Gomarasca S, Ludwig N, et al. (2009) Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure. Environ Exp Bot 66: 493–500.
- 45. Condon A, Richards R, Rebetzke G, Farquhar G (2004) Breeding for high water-use efficiency. J Exp Bot 55: 2447–2460.
- 46. Krupenina NA, Bulychev AA, Roelfsema MRG, Schreiber U (2008) Action potential in Chara cells intensifies spatial patterns of photosynthetic electron flow and non-photochemical quenching in parallel with inhibition of pH banding. Photoch Photobio Sci 7: 681–688.
- 47. Guidi L, Degl'Innocenti E, Genovesi S, Soldatini GF (2005) Photosynthetic process and activities of enzymes involved in the phenylpropanoid pathway in resistant and sensitive genotypes of Lycopersicon esculentum L. exposed to ozone. Plant Sci 168: 153–160.
- 48. Kościelniak J, Biesaga-Kościelniak J (2006) Photosynthesis and non-photochemical excitation quenching components of chlorophyll excitation in maize and field bean during chilling at different photon flux density. Photosynthetica 44: 174–180.
- 49. Zhang H, Xie X, Kim MS, Kornyeyev DA, Holaday S, et al. (2008) Soil bacteria augment Arabidopsis photosynthesis by decreasing glucose sensing and abscisic acid levels in planta. Plant J 56: 264–273.
- 50. Silva Júnior JMd, Rodrigues M, Castro EMd, Bertolucci SKV, Pasqual M (2012) Changes in anatomy and chlorophyll synthesis in orchids propagated in vitro in the presence of urea-. Acta Sci Agron 35: 65–72.
- 51. Pei ZM, Murata Y, Benning G, Thomine S, Klüsener B, et al. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406: 731–734.
- 52. Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002) Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14: 3089–3099.
- 53. Bright J, Desikan R, Hancock JT, Weir IS, Neill SJ (2006) ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J 45: 113–122.
- 54. Gómez-Gómez L, Boller T (2000) FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5: 1003–1011.
- 55. Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, et al. (2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125: 749–760.
- 56. Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, et al. (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. PNAS 104: 19613–19618.
- 57. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JD, et al. (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428: 764–767.
- 58. Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60: 379–406.
- 59. Sawinski K, Mersmann S, Robatzek S, Böhmer M (2013) Guarding the green: pathways to stomatal immunity. Mol Plant Microbe In 26: 626–632.
- 60. Henderson RK, Baker A, Parsons SA, Jefferson B (2008) Characterisation of algogenic organic matter extracted from cyanobacteria, green algae and diatoms. Water Res 42: 3435–3445.
- 61. Gerken HG, Donohoe B, Knoshaug EP (2013) Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta 237: 239–253.
- 62. Cuevas JC, Sánchez DH, Marina M, Ruiz OA (2004) Do polyamines modulate the Lotus glaber NADPH oxidation activity induced by the herbicide methyl viologen? Funct Plant Biol 31: 921–928.
- 63. Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signaling and abiotic stress. Physiol Plantarum 133: 481–489.
- 64. Marino D, Dunand C, Puppo A, Pauly N (2012) A burst of plant NADPH oxidases. Trends Plant Sci 17: 9–15.
- 65. Macho AP, Boutrot F, Rathjen JP, Zipfel C (2012) Aspartate oxidase plays an important role in Arabidopsis stomatal immunity. Plant Physiol 159: 1845–1856.
- 66. Underwood W, Melotto M, He SY (2007) Role of plant stomata in bacterial invasion. Cell Microbiol 9: 1621–1629.
- 67. Chaves MM, Santos TP, Souza CR, Ortuno M, Rodrigues M, et al. (2007) Deficit irrigation in grapevine improves water-use efficiency while controlling vigour and production quality. Ann Appl Biol 150: 237–252.
- 68. Neill S, Barros R, Bright J, Desikan R, Hancock J, et al. (2008) Nitric oxide, stomatal closure, and abiotic stress. J Exp Bot 59: 165–176.
- 69. Wan J, Griffiths R, Ying J, McCourt P, Huang Y (2009) Development of drought-tolerant Canola (Brassica napus L.) through genetic modulation of ABA-mediated stomatal responses. Crop Sci 49: 1539–1554.
- 70. Vahisalu T, Puzõrjova I, Brosché M, Valk E, Lepiku M, et al. (2010) Ozone-triggered rapid stomatal response involves the production of reactive oxygen species, and is controlled by SLAC1 and OST1. Plant J 62: 442–453.
- 71. De Souza CR, Maroco JP, Dos Santos TP, Rodrigues ML, Lopes CM, et al. (2005) Impact of deficit irrigation on water use efficiency and carbon isotope composition (δ13C) of field-grown grapevines under Mediterranean climate. J Exp Bot 56: 2163–2172.
- 72. Trejo CL, Davies WJ, Ruiz L (1993) Sensitivity of stomata to abscisic acid (an effect of the mesophyll). Plant Physiol 102: 497–502.
- 73. Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51: 659–668.
- 74. Lawlor D, Cornic G (2002) Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants. Plant Cell Environ 25: 275–294.
- 75. Rivero RM, Shulaev V, Blumwald E (2009) Cytokinin-dependent photorespiration and the protection of photosynthesis during water deficit. Plant Physiol 150: 1530–1540.
- 76. Pereira EG, Oliva MA, Rosado-Souza L, Mendes GC, Colares DS, et al. (2012) Iron excess affects rice photosynthesis through stomatal and non-stomatal limitations. Plant Sci 201–202: 81–92.