key: cord-0323695-9m29tuek authors: Gałgańska, Hanna; Gałgański, Łukasz title: Mitogen-activated protein kinases are carbon dioxide receptors in plants date: 2020-05-10 journal: bioRxiv DOI: 10.1101/2020.05.09.086116 sha: 3e4e56f2153ade83991bac222ea764ce85ef2e4d doc_id: 323695 cord_uid: 9m29tuek The amount of CO2 in the atmosphere is increasing continuously in the industrial era, posing a threat to the ecological balance on Earth. There are two ways to reduce elevated CO2 concentrations ([CO2]high): reducing human emissions or increasing their absorption by oceans and plants. However, in response to [CO2]high, plants diminish gas exchange and CO2 uptake by closing stomata. Surprisingly, we do not know how plants sense CO2 in their environment, and the basic mechanisms of the plant response to [CO2]high are very poorly understood. Here, we show that mitogen-activated protein kinases (MAPKs) are plant CO2 receptors. We demonstrate that MPK4, a prominent MAPK that is known to be involved in the stomatal response to [CO2]high1–3, is capable of binding CO2 and is directly activated by a very low increase in [CO2] in vivo and in vitro. Unlike MPK4 activation by infections4, stress and hormones within known MAPK signalling cascades, [CO2]high-induced MPK4 activation is independent of the upstream regulators MKK1 and MKK2. Moreover, once activated, MPK4 is prone to inactivation by bicarbonate. The identification of stress-responsive MPK4 as a CO2 receptor sheds new light on the integration of various environmental signals in guard cells, setting up MPK4 as the main hub regulating CO2 availability for photosynthesis. This result could help to find new ways to increase CO2 uptake by plants. 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 ). Similar to 665 dissolved CO 2 , KHCO 3 regulates MPK4 activity. c, MPK4 activation in response to [CO 2 ] high 666 occurs in just a few seconds. d, Very high CO 2 /HCO 3 -concentrations can positively or 667 negatively regulate MPK4 activity. The lack of MPK4 activation in response to millimolar 668 CO 2 /HCO 3 -concentration is consistent with a previous report 2 In b, d, MPK4 activity was determined by MBP in vitro thiophosphorylation and 671 detected by immunoblotting with anti-TE. MPK4 was stained with CBB, and MBP was 672 stained with Ponceau S. Experiments were carried out using MPK4 purified from bacteria and 673 dephosphorylated by FastAP phosphatase Representative results from three independent experiments are presented Elevation of [HCO 3 -] at constant [CO 2 ] triggers MPK4 inactivation. In vitro 680 thiophosphorylation reactions with different [CO 2 /HCO 3 -] in the pH series were allowed 681 exchange with ambient air for 30 min ATPγS were added, and in vitro 683 thiophosphorylation reactions were carried out for only 2 min. High pH and concomitant high 684 Lowering the pH to 6.6 (increase in free [CO 2 ] and dissolved CO 2 . MBP was used as a substrate of tag-free MPK4, and MPK4 690 activity was detected by immunoblotting with anti-phospho-MBP. MBP loading was 691 visualized by Ponceau S, and MPK4 loading was visualized by CBB Scheme of gel 698 filtration-based CO 2 binding assay. b, Graphs of CO 2 binding at 10-30 M CO 2 /HCO 3 -in 699 individual pH series from the graph shown in Fig. 3b; data normalization was based on values 700 of no-protein controls from each experiment; mean ±SD, n=3 experiments. c, MPK4 activity 701 under the conditions applied for the CO 2 binding assay shown in b. Dephosphorylated MBP 702 was used as a substrate of dephosphorylated tag-free MPK4; FastAP-fast alkaline 703 phosphatase. The intensity of MBP phosphorylation was detected by immunoblotting with 704 anti-phospho-MBP. The amount of MBP was determined by Ponceau S, and the amount of 705 MPK4 was determined by CBB. Mean ± SD CO 2 ] high -induced TEY phosphorylation is not inhibited by HCO 3 -at pH ≥7, unlike the 711 decrease in MPK4 activity, defined as substrate phosphorylation intensity. TEY and MBP 712 phosphorylation is shown by immunoblotting with anti-phospho-TEY and anti Both analyses were carried out from one set of in vitro phosphorylation 714 reactions. The amounts of dephosphorylated MBP and dephosphorylated MPK4 on the 715 nitrocellulose membrane were specified by Ponceau S staining MPK4 influences stomatal development. a, Stomata of WT Arabidopsis. b-d, Enlarged and 725 elongated stomata in mpk4-2 leaves. Scale bars 20 m. As reported for stomata of an N tabacum line with silenced NtMPK4 1 , mpk4-2 stomata display a much wider range of length Anti-phospho-TEY antibody and MBP in vitro phosphorylation 733 experiments failed to detect the activity of guard cell One-STrEP-Tag-MPK4 in contrast to 734 We 735 used a powerful method for specific purification of One-STrEP-tagged plant proteins under 736 native conditions within several minutes 35,36 . In contrast to high-yield One-STrEP-Tag-MPK4 737 purification from epidermal peels, we were not able to detect One-STrEP-Tag-MPK4 activity 738 by in vitro MBP phosphorylation we hypothesize that MPK4 activatable by [CO 2 ] high is 740 connected to the cell membrane. In addition, the use of phenol-SDS extraction (Fig Supplementary Fig. 1), which increases membrane protein solubilization and decreases 742 protein interactions, underlies the successful detection of MPK4 activity Alignment (using ClustalX 2.1) of amino acid sequences of Arabidopsis MPK4 and its two 747 barley homologues. The response of barley stomata to darkness is the quickest among the 748 studied species 34 . It may be speculated that this results from the presence of two specialized 749 MPK4 homologues in barley guard cells. The protein encoded by the BAJ95789 locus shares 750 lower identity (82%) 752 frame); therefore, the barley MPK encoded in the BAJ97968 locus was included in the 753 comparative analysis of [CO 2 ] high -induced MPK activity in Fig. 4a, and the expression in 754 barley protoplasts is presented in b. b, Barley MPK4-YFP in barley mesophyll protoplasts is 755 localized in the proximity of the cell membrane no-protein control (NPC). The experimental design is illustrated in Supplementary Fig. 5a.