Recently, Takahashi et al. (2022) identified a protein kinase, HIGH LEAF TEMPERATURE 1 (HT1), which plays a central role in CO2-mediated stomatal movement (Horak et al. 2016). A previous report by Matrasova et al. (2015) revealed that HT1 protein is a negative regulator of CO2 induced stomatal closure. However, recently Takahashi et al. (2022) have suggested that HT1 is a Raf-like protein kinase which regulates the opening and closing of stomata by inducing protein phosphorylation of other Raf-like protein kinases such as CONVERGENCE OF BLUE LIGHT (CBC1 and CBC2). In addition, mitogen-activated protein kinases (MPK4 and MPK12) were also identified as key regulators of stomatal movements (Toldsepp et al. 2018). MPK4/12 was reported to be an essential upstream signaling component of CO2 sensing mechiansm. However, this MAPK4/12 was not directly modulated by CO2 and hence it was speculated that MPK4/12 is not a direct CO2/bicarbonate sensor.
Experimental evidence presented by Takahashi et al. (2022) also reached the same conclusion and authors suggested that MPK4/12 is an essential positive component of the CO2 sensing mechanism as its mutation causes plants to become insensitive towards high CO2 concentration. The authors further revealed that the CO2 sensor in plants is composed of MPK4/12 and HT1 protein. High CO2 concentration causes interaction of MPK4/12 with HT1 protein which in turn inhibits HT1 kinase activity and thus CBC1 remains un-phosphorylated and inactivated.
Through a phosphorylation assay using radioactive 32P-ATP, His-HT1, and glutathione-S-transferase-CBC1, performed in the recent experiment of Takahashi et al. (2022), authors showed that the kinase activity of CBC1 is dependent on its phosphorylation induced by HT1 protein. CBC1/2 protein possesses HT1-mediated phosphorylation sites at Thr256 and Ser280 and the substitution of these two amino acids by alanine resulted in reduced stomatal conductance in Arabidopsis. Further, CBC1 phosphorylation was observed in presence of sodium bicarbonate (NaHCO3) whereas the addition of sodium chloride (NaCl) failed to induce CBC1 phosphorylation, suggesting CBC1 phosphorylation is dependent on bicarbonate and not on sodium ions. In vitro experiments with different buffer solutions indicated the occurrence of CBC1 phosphorylation at pH is 7.5. This shows that bicarbonate ion is the main compound causing CBC1 kinase regulation whereas CO2 is secondary in impact, as it is present abundantly at low pH. Besides, the addition of MPK4/12 also failed to induce CBC1 phosphorylation.
Arabidopsis plants exhibiting mutations in the dominant allele of HT1 genes [ht1-G89R, ht1-173Q, and A-109V(ht1-8D)] were found to be insensitive toward elevated CO2 in the plants. Whole plant gas exchange analysis showed that ht1-G89R and ht1-R173Q plants exhibit impaired stomatal conductance. In particular, ht1-G89R plants showed low stomatal conductance whereas ht1-R173Q showed partly impaired stomatal conductance in comparison to the wild-type plants. On the other hand, recombinant HT1-G89R and HT1-R173Q plants displayed activated CBC1 protein. Further in vitro phosphorylation assays revealed that the HT1-G89R isoform did not show NaHCO3-mediated inhibition of CBC1 whereas HT1-R173Q showed a partial reduction in NaHCO3-mediated inhibition of CBC1 protein. Thus, it was clear that both the dominant mutants, ht1-G89R, and recombinant HT1-G89R isoforms showed low stomatal conductance. Other dominant ht1 mutants (ht1-R102K and ht1-A109V) exhibited a higher CO2 insensitivity. Besides, these point mutations did not cause enhancement in the kinase activity of HT1.
On having phosphorylation assay of other more dominant mutants, ht1-A109V and ht1-R102K using MPK4/12 and HT1, it was found that NaHCO3-mediated inhibition of CBC1 kinase activity was disrupted and stomata remain open. Moreover, the importance of CBC1 kinase was pronounced in triple mutants of cbc1/cbc2, ht1-A109V which exhibited closed stomata. On recalling the above results, it was found that HT1 provides an important regulator that senses the CO2 concentration.
The addition of NaHCO3 to CBC1, MPK4/12, or HT1 did not affect their kinase activity. However, the addition of NaHCO3 to MPK4/12 and HT1 inhibited the kinase activity of HT1, suggesting that MPK4/12 and HT1 together could function as a CO2/bicarbonate sensor. Additionally, an in vitro pull-down assay showed that HCO3- has a positive impact on the binding interaction of MPK4/12 and HT1 whereas the removal of HCO3- showed a negative impact. This clarifies the reversible impact of HCO3- towards MPK4/12 and HT1 interaction.
Bimolecular fluorescence complementation analysis (BiFC) showed an improved interaction of MPK4/12 with HT1 in the presence of increased CO2. Additionally, co-immunoprecipitation assays further confirmed this observation as HCO3- expressing Arabidopsis mesophyll cells exhibited a better interaction of MPK4/12 and HT1. These results clearly demonstrate that MPK4/12 and HT1 are the probable CO2/bicarbonate sensor. This sensor inhibits the CBC1 phosphorylation by HT1 protein which subsequently leads to the closing of stomata.
In essence, these results highlight that the reversible interaction of MPK4/12 and HT1 is responsible for sensing CO2, rather than MAP kinase-mediated phosphorylation of HT1 protein. A rise in atmospheric CO2 concentrations triggers interaction between MPK4/12 and HT1 protein which inhibit the HT1-mediated phosphorylation of CBC1, causing the closing of stomata (Figure 1). Future research work regarding stomatal proteins could further enhance our understanding of CO2 sensing mechanisms in plants. Moreover, the list of identified proteins involved in the CO2 sensing mechanism will provide the possibility to manipulate them for maximizing plants' fitness in the continuously increasing environmental CO2 concentrations to ensure food security for the global population.