Stomatal CO₂ Responses Are Mediated By Distinct Sources Of ROS

Stomata are microscopic valves on the plant epidermis essential for efficient gas exchange and plant immunity.The continuing enrichment of atmospheric CO₂(e CO₂)impacts global climate and plant physiology which have profound effects on agricultural capacity.e CO₂ reduces stomatal conductance by simultaneously promoting stomatal closure and inhibiting stomatal opening.Diverse abiotic or biotic stimuli can modulate stomatal CO₂responses,yet the molecular mechanisms remain largely unclear.In this study,based on pharmacological and genetic assays,I investigate the involvement of apoplastic ROS and plant hormones in stomatal movement and development in response to CO₂concentration changes.The major findings are as follows.1.Loss function of BIG gene can cause compromised e CO₂-induced stomatal closure,ROS accumulation and bicarbonate activation of S-type anion channel currents,as well as e CO₂ inhibited stomatal development,whereas e CO₂-inhibited stomatal opening remains fully functional in big mutants.These results indicate that the CO₂-mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two stomatal CO₂ responses.2.Both cell wall peroxidases(PRXs)(e.g.PRX33 and PRX34)and NADPH oxidases(eg.RBOHD and RBOHF)are responsible for apoplastic H₂O₂ production in e CO₂-mediated stomatal closure.3.The e CO₂-inhibited stomatal opening also requires the production of extracellular ROS.The ROS scavenger Tiron could completely inhibit the processes of e CO₂-inhibited stomatal opening and of ROS accumulation.NADPH oxidase inhibitor DPI could only partially inhibite this process,while PRXs inhibitor SHAM had no significant effect on the inhibition of stomatal opening by e CO₂.These suggest that e CO₂-inhibited stomatal opening is at least partly dependent on NADPH oxidase but completely independent on the PRXs-mediated ROS generation.4.RBOHD/F and PRX33/34 are not involved in e CO₂-inhibited stomatal opening,as both prx33-3 and prx34-2 showed normal response to this process,and there were no perceivable defects caused by the loss function of ROBHD/F in the inhibition of stomatal opening by e CO₂.5.e CO₂-induced stomatal closure and ROS accumulation are damaged in the mutants of SA biosynthesis and signaling transduction,indicating that as with ABA and JA,SA pathway is also required for e CO₂-induced stomatal closure.Interestingly,it seems that the roles of ABA,JA and SA are all exclusively limited to the stomatal closure induced by e CO₂.6.SA has an indispensable role to play in the e CO₂-modulated stomatal development.In contrast to the WT Arabidopsis which showed significant decrease of stomatal density(SD)and stomatal index(SI)when grown under e CO₂,SA deficient mutants displayed no e CO₂elicited reductions in SD or SI,whereas the SD of cim10,a mutant overaccumulating SA is two-fold greater than that of WT.And given none of rboh Drboh F,prx33-3 nor prx34-2 was responsive to e CO₂induced SD reduction,I concluded that e CO₂-inhibited stomatal development requires SA signaling pathway and the production of apoplastic ROS.In addition,we also found that NO is also required in the process of e CO₂-induced stomata closure in Arabidopsis.7.SA has an indispensable role to play in the e CO₂-modulated stomatal development.In contrast to the WT Arabidopsis which showed significant decrease of stomatal density(SD)and stomatal index(SI)when grown under e CO₂,SA deficient mutants displayed no e CO₂elicited reductions in SD or SI,whereas the SD of cim10,a mutant overaccumulating SA is two-fold greater than that of WT.And given none of rboh Drboh F,prx33-3 nor prx34-2 was responsive to e CO₂induced SD reduction,I concluded that e CO₂-inhibited stomatal development requires SA signaling pathway and the production of apoplastic ROS.In summary,we found that BIG distinguishes e CO₂-induced stomatal closure and e CO₂-inhibited stomatal opening in the molecular level.Both cell wall PRXs and NADPH oxidases are required for apoplastic H₂O₂ production during e CO₂ triggers stomatal closure.e CO₂-inhibited stomatal opening partly relies on the ROS production by NADPH oxidases whereas cell wall PRXs is dispensable in the process,suggesting additional sources for ROS production are required for CO₂ repressing stomatal opening.ABA,JA and SA pathways are all required for e CO₂-mediated stomatal closure but not for the e CO₂-inhibited stomatal opening.I have also provided evidenc that NO is required for e CO₂-induced stomata closure.Taken together,these data allow us to conclude that various plant hormones including SA condition stomatal CO₂ responses which are mediated by distinct sources of apoplastic ROS.