Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf‐like kinase HT1 as a required step for stomatal movements caused by changes in CO2concentration. However, whether MPK12 kinase activity is required for regulation of CO2‐induced stomatal responses warrants in‐depth investigation. We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO2signaling that relies on allosteric inhibition of HT1. We show that CO2/HCO3−‐enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase‐dead and constitutively active versions of MPK12 in a plant line where These data add to the model that MPK12 kinase‐activity‐independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO2concentration.
- Award ID(s):
- 1900567
- NSF-PAR ID:
- 10408465
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 8
- Issue:
- 49
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary MPK12 is deleted, we confirmed that CO2‐dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2/HCO3−and present structural modeling that explains the MPK12:HT1 interaction interface. -
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Stomatal pore apertures are narrowing globally due to the continuing rise in atmospheric [CO2]. CO2elevation and the plant hormone abscisic acid (ABA) both induce rapid stomatal closure. However, the underlying signal transduction mechanisms for CO2/ABA interaction remain unclear. Two models have been considered: (
i ) CO2elevation enhances ABA concentrations and/or early ABA signaling in guard cells to induce stomatal closure and (ii ) CO2signaling merges with ABA at OST1/SnRK2.6 protein kinase activation. Here we use genetics, ABA-reporter imaging, stomatal conductance, patch clamp, and biochemical analyses to investigate these models. The strong ABA biosynthesis mutantsnced3/nced5 andaba2-1 remain responsive to CO2elevation. Rapid CO2-triggered stomatal closure in PYR/RCAR ABA receptor quadruple and hextuple mutants is not disrupted but delayed. Time-resolved ABA concentration monitoring in guard cells using a FRET-based ABA-reporter, ABAleon2.15, and ABA reporter gene assays suggest that CO2elevation does not trigger [ABA] increases in guard cells, in contrast to control ABA exposures. Moreover, CO2activates guard cell S-type anion channels innced3/nced5 and ABA receptor hextuple mutants. Unexpectedly, in-gel protein kinase assays show that unlike ABA, elevated CO2does not activate OST1/SnRK2 kinases in guard cells. The present study points to a model in which rapid CO2signal transduction leading to stomatal closure occurs via an ABA-independent pathway downstream of OST1/SnRK2.6. Basal ABA signaling and OST1/SnRK2 activity are required to facilitate the stomatal response to elevated CO2. These findings provide insights into the interaction between CO2/ABA signal transduction in light of the continuing rise in atmospheric [CO2]. -
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Abstract Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP‐sensitive potassium channel (KATP) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATPlocally produces the ATP that inhibits KATPactivity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside‐out patches inhibited KATP. To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATPactivity using mouse beta cells and INS‐1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATPactivity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well‐known action of protons to inhibit KATP. In cell‐attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATPchannel closure by glucose, further suggesting that PK is not an important regulator of KATP. In contrast, addition of mitochondrial inhibitors robustly increased KATPactivity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATPin pancreatic beta cells.
image Key points Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATPactivity in excised patches. PEP solutions only inhibit KATPactivity if the pH is unbalanced.
Modulating pyruvate kinase has minimal effects on KATPactivity.
Mitochondrial inhibition, in contrast, robustly potentiates KATPactivity in cell‐attached patches.
Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP.
Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATPactivity of pancreatic beta cells.
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/sciadv.abq6161
