Abscisic acid (ABA) is the best studied regulator of stomatal closure, and for many 30 years, ABA-induced signalling events were thought to direct stomatal closure triggered by 31 [CO 2 ] high . Recent studies, however, have proposed otherwise, suggesting that both pathways 32 48 MPK4 is activated by CO 2 in vivo 49 No kinase involved in plant CO 2 signalling has been shown to be activated by 50 [CO 2 ] high in vivo to date; therefore, we decided to study MPK4 activation in response to 51 [CO 2 ] high in epidermal peels. In line with the reported high activity of the MPK4 promoter in 52 guard cells 4 , we detected very high MPK4 expression in Arabidopsis epidermal peels using 53 immunoblotting (Fig. 1a) . 54 The activity of MPK4 was extremely low compared to that of the highly active MPK3 55 and MPK6. The lack of MPK4 activation in the control samples indicates the maintenance of 56 stress-free conditions in the experimental system used. We assumed that the method used to 57 study [CO 2 ] high -induced MPK activation should utilize direct analysis of the protein extract 58 without lengthy sample preparation steps at indoor [CO 2 ] under native conditions. Therefore, 59 we rejected the classic in-gel kinase assay following kinase immunoprecipitation. 
To obtain further insight into the opposing effects of HCO 3 and CO 2 on MPK activity,
we measured the [CO 2 ] high -induced activation of several MPKs at pH 7.0 ( Fig. 4a- acts as an additional activity enhancer of inactive or incompletely activated MPK4.
All the mutants tested were negatively regulated by HCO 3 at pH 7.0 (~85% HCO 3 -167 and ~15% CO 2 ) (Fig. 4c ). Lowering the pH to 6.6 (~62% HCO 3 and ~38% CO 2 ) eliminated suggested recently to be a protective factor against the development of COVID-19 symptoms.
Importantly, CO 2 is a natural and safe gas in the lungs, and short-term CO 2 inhalation is 224 beneficial for the respiratory, nervous 28-30 and circulatory 31,32 systems. increasing temperature and because of ice production from high-pH water in our laboratory.
All solutions were prepared using acidified (pH 4.8-5.2) CO 2 -free water in rooms with fresh 234 air. Solutions were stored frozen, or the pH was adjusted immediately before use. MPK 235 purification or modification (e.g., dephosphorylation or protease digestion) was followed by Tris-glycine SDS-PAGE was carried out in a discontinuous buffer system with a 5% stacking 296 gel (pH 6.8) and 9% resolving gel (pH 8.8). A total of 30-50 g of total protein was loaded with the following setting: a single pulse at 150 V with an 8-ms pulse duration. Immediately, 360 1 ml of ice-cold 0.615 M mannitol was added, and protoplasts were transferred to 2-ml tubes.
The protoplasts were allowed to sediment for 20 min at room temperature before resuspension Fig. 2) S staining and immunoblotting with anti-MPK3, anti-MPK4 and anti-MPK6 antibodies.
Representative results from three independent experiments are presented. Error bars represent 576 the standard deviation (SD). *, ** and *** indicate significant differences in MPK4 activity 577 (p<0.05, p<0.01 and p<0.001, respectively) compared to the control. The above data were 578 obtained on proteins isolated by phenol-SDS extraction for immediate separation of ATP 33 579 from MPKs to prevent their extracellular activation. In contrast, we were unable to detect the 580 activity of guard cell MPK4 purified under native conditions ( Supplementary Fig. 9 ). 