| Stomata are small pores in the epidermes of leaves that open and close in response to a variety of environmental signals including carbon dioxide (CO2). In wild type plants, high levels of CO2 cause stomata to close, as well as cause a decrease in stomatal density. However, the CO2-binding proteins that control these responses remain unknown. Through guard cell and mesophyll cell specific microarrays, two highly expressed beta-carbonic anhydrase genes, CA1 and CA4, were identified. Arabidopsis thaliana ca1ca4 double knockout mutants exhibit an insensitive stomatal closure response to 800ppm CO2 and exhibit a higher stomatal density than wild type. To determine which gene, CA1 and/or CA4, is responsible for the CO2 phenotype, the genomic DNA with native promoter of CA1 and CA4 and the cDNAs of CA1 and CA4 driven by a guard cell promoter were transformed into ca1ca4 double mutant plants. In these CA-expressing transgenic lines, stomatal density and CO2 response recovered to wild type phenotypes, suggesting that the CA1 and CA4 genes are indeed responsible for the stomatal CO2 insensitive response as well as for the high stomatal density of the ca1ca4 double mutant. To determine whether CA homologues from other species could complement the ca1ca4 phenotypes, the structurally unrelated Human CAII controlled by a guard cell promoter was also transformed into ca1ca4 plants. The HmCAII was able to restore the mutant phenotype of CO2-induced stomatal closure as well as stomatal density. These results show that CA1 and CA4 function within the CO2 signaling mechanisms that mediate CO2 control of stomatal movements and development. |
