Abstract
BackgroundThe increase in atmospheric CO2 is causing a number of changes in plant growth such as increases in leaf area and number, branching, plant size and biomass, and growth rate. Despite the importance of stomatal responses to CO2, little is known about the genetic and molecular mechanisms that mediate stomatal development and movement in response to CO2 levels. Deciphering the mechanisms that sense changes in CO2 and/or HCO3− concentration is critical for unraveling the role of CO2 in stomatal development movement. In Arabidopsis, CO2-induced stomatal closure is strongly Ca2+-dependent. To further dissect this signaling pathway and identify new components in the CO2 response pathway, we recorded [Ca2+]cyt changes in mutagenized Arabidopsis leaves and screened for mutants with abnormal guard cell behavior in response to CO2/HCO3−.ResultsWe observed that 1 mM HCO3− induces [Ca2+]cys transient changes in guard cells and stomatal closure both in light and darkness. The changes in [Ca2+]cys induced by HCO3− could be detected by an aequorin-based calcium imaging system. Using this system, we identified a number of Arabidopsis mutants defective in both [Ca2+]cyt changes and the stomatal response to CO2/HCO3−.ConclusionsWe provide a sensitive method for isolating stomatal CO2/HCO3− response genes that function early in stomatal closure and that have a role in regulating [Ca2+]cyt. This method will be helpful in elucidating the Ca2+-dependent regulation of guard cell behavior in response to CO2/HCO3−.
Highlights
The increase in atmospheric CO2 is causing a number of changes in plant growth such as increases in leaf area and number, branching, plant size and biomass, and growth rate
The results showed that HCO3−-induced stomatal closure whether in light or darkness; by using the aequorin-based system, it will be possible to indentify abnormal-response mutants for both stomatal movement and [Ca2+]cys transient change at 1 mM K HCO3
We first monitored the stomatal movement of mci1 and mcs1 in response to HCO3−/CO2.The results clearly showed that 1 mM HCO3− could induce stomatal closure within 30 min in mcs1 but not in wild type (Fig. 4a)
Summary
The stomata, which are formed by pairs of guard cells, can be considered the gas-exchange valves of plants. Through isolation and analysis of genetic mutants, a number of proteins have been identified that function in CO2-controlled stomatal movement, including the SLAC1 anion channel [7, 8], the PATROL1 Munc 13-like protein [9], the AtALMT12/QUAC1 R-type anion channel [10], and the RHC1 MATE transporter [11] The characterization of these proteins has contributed to our understanding of the mechanisms of C O2-regulated guard cell behavior. We used the C a2+ reporter aequorin (AEQ) to record [Ca2+]cyt changes in Arabidopsis leaves in real time in order to visualize locally induced [Ca2+]cyt elevations in response to CO2 or HCO3− stimulus This screening method had already been used for analyzing the responses of Arabidopsis to different stimuli such as salt stress, ABA, sorbitol, and cold [15], it had not been tried for screening mutants with altered stomatal responses to CO2 or/and HCO3−.
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