UC San Diego

As atmospheric carbon dioxide levels continue to rise, agricultural yields will be impacted worldwide. Our laboratory obtained evidence that specific MAPKs inhibit the activity of HT1, a negative regulator of CO2-induced stomatal closure. The findings showed that HT1 can phosphorylate CBC1/2 but not whether HT1 regulates CBC1/CBC2’s activity. This study analyzed stomatal conductance responses to changing CO2 levels with a cbc1/cbc2 double knockout line, a dominant ht1(A109V) line with a permanently active HT1 protein, and a cbc1/cbc2/ht1(A109V) triple mutant line with knocked out CBC1 and CBC2 genes and a constantly active HT1 protein. The cbc1/cbc2/ht1(A109V) mutant showed stomatal conductance values similar to cbc1/cbc2 samples, not ht1(A109V). The results suggest that the CBC1/CBC2 are epistatic to and function downstream of HT1 in the signaling pathway. Next, mutant lines of pGC1:MPK12(K70R)-GFP/mpk12, with a K70R mutation in MPK12, and pGC1:MPK12-GFP/mpk12, a rescue line, both showed stomatal conductance responses similar to Col-0. Meanwhile, mpk12 samples showed responses to changes in CO2 but higher stomatal conductance levels compared to Col-0. The data suggests that the kinase activity of MPK12 is not required to inhibit HT1. The final experiments used a line with two mutations in mapped HT1-mediated CBC1 phosphorylation sites, pGC1:CBC1(T256A/S280A)-GFP/cbc1cbc2, showed stomatal conductance similar to the cbc1/cbc2 samples while the rescue line pGC1:CBC1-GFP/cbc1cbc2 with a wildtype CBC1 gene, showed stomatal responses similar to Col-0. This suggests that the two mutated sites could be important for HT1 phosphorylation. The information gathered here can contribute to developing agricultural lines that can withstand rising levels of CO2.