The Mechanisms Underlying Chlorella-induced Stomatal Closure And Its Anti-transpiration Effect In Plants

Stomata play a prominent role in controlling gas exchanges between plants and environments. The size of stomatal pores can be tuned by multiple environmental cues. However, transpiration and photosynthesis do not respond to the changing stomatal aperture identically. Historically, 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 and microbe-associated molecular patterns (MAMPs) can actively induce stomatal closure. Mechanisms underlying stomata-based defense in crops have caused intensive studies in crops in decades. Furthermore, nonpathogenic bacteria and MAMPs have been proved to meliorate the water use efficiency and yield of crops notably through mediating stomatal closure. Previous studies have suggested that Chlorella and its extracts have good biological and medical effects on human and animals. However, it is unknown whether Chlorella can induce stomatal closure and improve water use efficiency (WUE) in plants. Based on the differences of effects of stomatal pores on transpiration and photosynthesis of plants and the mechanisms underlying stomtal closure induced by bacteria microbes, we hypothesize that Chlorella may trigger stomatal closure via activating a series of guard cell signaling pathway differed from bacterica-induced and influence transpiration, photosynthesis, WUE and yields in plants. Certain monocotyledon (e.g. Oryza sativa L), dicotyledon (e.g. Vicia faba and Arabidopsis thaliana) and microalgae (e.g. Chlorella vulgaris) provide ideal material models for the research. The dynamics and mechanisms of stomatal closure induced by Chlorella and its effects on anti-transpiration and yield of plants will be investigated through microscopic dynamic observation, pharmacological analysis, reactive oxygen species (ROS) fluorescence assay, nitric oxide (NO) fluorescence assay, gas-exchange measurements and chlorophyll fluorescence imaging. The principal results of our studies are as follows:(1) Chlorella-triggered stomatal closure improved leaf instantaneous water use efficiency (WUEj) in Vicia faba. The chlorophyll fluorescence and content analysis further demonstrated that short-term foliar use of Chlorella did not influence the activities of plant photosynthetic reactions center.(2) The effects of foliar application of Chlorella suspension on WUEi in rice were different at different growing stages. Foliar application of Chlorella suspension largely increased plant height, grain yield, thousand-grain weight and grain:straw, significantly reduced straw weight, while not affected the content of nitrogen (N), phosphorus (P) and the ratios of nitrogen to phosphorus of leaf and stalk of rice grown under well-watered (100% soil water content) and moderate drought stress (60% soil water content) conditions.(3) ROS production, NO production, Ca2+, Ca2+channel and water channel were involved in Chlorella-induced stomatal closure in Vicia faba.(4) Glucose and its isomer mannose could induce stomatal closure in epidermal strips of broad bean. Pharmacological analysis further demonstrated that ROS production, Ca2+, Ca2+ channel and water channel were involved in glucose- and mannose-triggered stomatal closure in Vicia faba.(5) Chlorella and glucose could induce stomatal closure in Arabidopsis. Subsequent studies with Arabidopsis mutant lines indicated that Chlorella-and glucose-induced stomatal closure were dependent on ABA receptor protein PYR/RCAR, protein kinase OST1, type 2 protein phosphatase ABI, slow anion channel SLAC1, calcium-dependent protein kinase CPK6, nitrate reductase NR and hexokinase HXK1, while not dependent on flagellin receptor kinase FLS2.In conclusion, these results suggest that (1) Just as bacterial microbes, Chlorella can induce stomatal closure and improve anti-transpiration and yields of plants when applied as foliar spray; (2) Glucose may be a key elicitor responsible for Chlorella-triggered stomatal closure; (3) Chlorella-triggered stomatal closure is dependent on ABA and glucose signaling in Arabidopsis. These main results deepen our knowledge of the mechanism underlying interactions of plant-microbe and provide scientific evidence for the development of new carbon-negative bio-antitranspirant and advancement of crop water-saving and product-increasing technology.