Arabidopsis guard cell CO2/HCO3− response mutant screening by an aequorin-based calcium imaging system
Plant Methods volume 16, Article number: 59 (2020)
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. 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−.
We 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−.
We 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−.
The stomata, which are formed by pairs of guard cells, can be considered the gas-exchange valves of plants. Stomatal aperture is regulated by several factors including phytohormone levels, carbon dioxide (CO2) concentration, humidity, light, and pathogens.
A higher ambient CO2 concentration increases leaf intercellular CO2 concentration and mediates stomatal closure in plants, whereas a lower CO2 concentration triggers stomatal opening. CO2 influences not only the stomatal response, but also the number of stomata per unit leaf. This number is decreasing due to the long-term effect of continuing CO2 concentration increases .
Despite the importance of stomatal responses to CO2, little is known about the genetic and molecular mechanisms mediating stomatal development and movement in response to elevation in CO2. CO2 levels have been increasing steadily, and it is estimated that atmospheric CO2 will reach 550 ppm in 2050 compared with 400 ppm presently , so it is increasingly urgent to discover the underlying mechanisms of guard cell regulation in response to CO2 levels.
CO2 sensing in animals is mainly linked to α-carbonic anhydrases (α-CAs) , which are also important for CO2 perception in fungi [3, 4]. Carbonic anhydrases (CAs) can accelerate the conversion of CO2 into HCO3− and H+, which in turn induce related responses. In plants, CO2 also can be converted into HCO3− and H+ by anhydrases . The key question in understanding stomatal movement in response to CO2 is the mechanism for perception of changes in CO2 and/or HCO3− concentration. Despite the importance of anhydrase enzymes in CO2 perception in mammalian and fungal systems [3, 4], no orthologous α-CAs has been identified in plants. There are six β-CAs in Arabidopsis thaliana. CA1- and CA4-related stomatal movements were controlled by CO2 in guard cells, whereas a ca1 ca4 double mutant exhibited insensitive stomatal movement response to CO2 . Expression of a mammalian α-CA in the ca1 ca4 double mutant restored the stomatal response to CO2, implying that CA-mediated CO2 catalysis to HCO3− and H+ in guard cells is the key step for transmission of the CO2 signal .
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 , the AtALMT12/QUAC1 R-type anion channel , and the RHC1 MATE transporter . The characterization of these proteins has contributed to our understanding of the mechanisms of CO2-regulated guard cell behavior. For example, transporter protein RHC1 acts as a bicarbonate sensor, and the high-CO2-induced stomatal closure mediated by RHC1 is controlled by inhibition of HT1 (HIGH LEAF TEMPERATURE1) activity [11, 12].
HT1 is regarded as a negative regulator in the CO2 signaling pathway: it functions by promoting phosphorylation of OST1 and thus inhibiting its kinase activity . Furthermore, OST1 protein kinase has been proved essential for high-CO2-induced stomatal closure [13, 14]. But still, many points remain controversial, such as the mechanism underlying CO2 sensing; the identities of the CAs involved in this pathway; the function of CAs under low-CO2 conditions; and the interaction of CO2 with light, temperature, humidity, and phytohormones in influencing stomatal movement.
The primary requirement for solving these questions is the isolation of mutants. Screening dependent on thermal imaging is quite common for isolating Arabidopsis mutants with abnormal guard cell behavior. Almost all of the mutants obtained until now, including ht1, rhc1, and patrol1, were obtained using this method. Although this method has been effective in unraveling the regulation network in CO2-mediated stomatal movement, it is still not clear of this regulation network; thus, it is urgent to develop new screening methods.
Calcium ion (Ca2+) has been shown to act as a key cellular second messenger in numerous plant processes. In Arabidopsis thaliana, abscisic acid (ABA), hydrogen peroxide, cold, and CO2 all can stimulate cytosolic Ca2+ ([Ca2+]cyt) oscillation, which causes stomatal closure . CO2-induced stomatal closure is strongly Ca2+-dependent in Arabidopsis, consistent with previous findings in Commelina guard cells [16,17,18]. Cytosolic Ca2+ regulates stomatal closure by two mechanisms: short-term Ca2+-reactive closure and long-term Ca2+-programmed closure .
Extracellular CO2 induces changes of the [Ca2+]cyt in Arabidopsis guard cells. To further dissect this signaling pathway, new components in the CO2 response pathway that are related to the [Ca2+]cyt changes need to be identified. Here, we used a novel approach for screening genetic mutants to identify proteins involved in CO2 response. In this study, we used the Ca2+ 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. Although this screening method had already been used for analyzing the responses of Arabidopsis to different stimuli such as salt stress, ABA, sorbitol, and cold , it had not been tried for screening mutants with altered stomatal responses to CO2 or/and HCO3−. By using this system, we obtained several mci (mutant of HCO3−/CO2insensitive) and mcs (mutant of HCO3−/CO2sensitive) mutants. Further study with these mutants will be helpful for uncovering the mechanism for calcium-dependent CO2-regulated guard cell movement.
[Ca2+]cys changes induced by HCO3− can be detected by an aequorin-based calcium imaging system
In our first experiment, we tested whether AEQ-transgenic Arabidopsis plants could be used to detect [Ca2+]cys changes induced by HCO3−. As it is already known that the pH of incubation buffer (50 mM KCl, 0.1 mM CaCl2, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES) and 10 μM coelenterazine) cannot be stabilized at 7.0 when the concentration of KHCO3 is above 5 mM, a lower concentration (1 mM) that has previously been used for analyzing guard cell behavior  was selected to avoid the putative influence of pH.
The AEQ-transgenic Arabidopsis leaves were treated with 1 mM KHCO3, and after 5 min, dramatic increases in [Ca2+]cys were detected in the leaves by analyzing the AEQ luminescence image (Fig. 1a, left). The average luminescence values increased from about 200 to 2300 RLU (Relative luminescence units, which represents the electrical signal values generated by stimulated photons) within 2 s of KHCO3 addition (Fig. 1a, right). We also added incubation buffer and same concentration of KCl as controls, and found that these only caused small changes of calcium (Fig. 1a, right). These suggested that 1 mM KHCO3 is effective for checking the cytosolic calcium changes with the AEQ system. Furthermore, when we analyzed individual guard cells within leaf epidermal strips under 1 mM KHCO3 treatment, a visible increase in luminescence of the guard cell was found after 1 min; when luminescence values were collected continuously for about 10 min, the quantified data of luminescence images of the guard cells confirmed the visually increase in [Ca2+]cys to KHCO3 (Fig. 1b).
To further validate the AEQ-based screening method, we adopted another Ca2+ indicator, yellow Cameleon 3.6 (YC3.6), for measuring CO2/HCO3−-induced [Ca2+]cyt increases in guard cells. The YC3.6 transgenic plants showed a marked increase in [Ca2+]cyt when treated with 1 mM KHCO3. This was consistent with the results of AEQ, suggesting that the aequorin-based system is a reliable method of [Ca2+]cyt measurement (Fig. 1c).
1 mM HCO3− induces closure of Arabidopsis thaliana stomata whether in light or darkness
Since the aequorin-based system requires that samples first be incubated in incubation buffer in the dark for several hours, we measured the stomatal responses in both light and darkness. For this experiment, detached leaves of 3-week-old plants were incubated in a glass chamber with quicklime to remove CO2 from the chamber.
After 2 h of incubation in the chamber under light conditions, almost all stomata had opened very well. The opened stomata were closing after 20 min of 1 mM KHCO3 treatment, and 60 min later, the stomatal apertures (width/length) were 0.71 ± 0.01 and 0.56 ± 0.01 for control and treated leaves, respectively (Fig. 2a).
Compared with the responses in light, the stomatal apertures were less after incubation in darkness without CO2. However, the stomata closed 5 min after 1 mM KHCO3 was added, and stomatal aperture decreased to 0.20 ± 0.01 for treated leaves at 60 min; while stomatal aperture was 0.42 ± 0.01 for the untreated at this time (Fig. 2b). The results showed that HCO3−-induced stomatal closure whether in light or darkness; thus, 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 KHCO3.
High-throughput screening for CO2/HCO3− response mutants
For high-throughput genetic screening with the aequorin-based system, we used the protocol shown schematically in Fig. 3. About 5000 AEQ-expressing Arabidopsis seeds were treated with 0.3% (w/v) ethyl methane sulfonate (EMS) and sown on soil. M2 seeds were collected individually and screened as described in Fig. 3. The leaves of 3-week-old M2 plants were placed in a 96-well plate and 100 µL of freshly prepared incubation buffer was added to each well for 4-6 h. AEQ luminescences of leave treatment with 1 mM KHCO3 were then identified by using a luminescence reader (LB960, Berthold) (Fig. 3). So far, approximately 35,000 M2 plants have been screened, and about 120 sensitive and 80 insensitive putative mutants have been identified.
The selected plants were examined further for their stomatal response to KHCO3 to narrow down the target mutants. HCO3−/CO2-induced stomatal closure of the putative mutants was assayed in M2 and again in M3, 6 out of 80 putative mutants with lower luminescence showed an insensitive stomatal response to HCO3−/CO2, and 4 out of 120 putative mutants with higher luminescence displayed a hypersensitive response.
Characterization of mutants obtained by the aequorin-based screening method
By using the aequorin-based screening procedure, we identified HCO3−/CO2 response mutants that appeared abnormal in both [Ca2+]cys and stomatal movement. To examin the stability of the mutants obtained by this method, two were selected for further analysis and named mci1 (insensitive response) and mcs1 (hypersensitive response).
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). For mci1, even 3 mM HCO3− could not induce stomatal closure after 1 h (Fig. 4b).
Consistent with the results of the screen, by comparing with wild type, AEQ luminescence intensities increased dramatically in mcs1, while no significant change was observed in mci1 in response to HCO3− treatment (Fig. 4c). These results further suggest that the products of MCS1 and MCI1 participate in HCO3− signal transduction pathways regulating both [Ca2+]cys and stomatal movement.
It is necessary to make sure that only a single gene locus functions in controlling a phenotype of interest before conducting gene mapping. After crossing each of the mutants with wild type, we analyzed the segregation of the F2 progeny. Phenotypes of F2 plants showed 3:1 (wild-type:mci1 or mcs1) segregation, suggesting that mci1 or mcs1 was resulted from a recessive mutation. These two mutants are appropriate for subsequent gene mapping work.
Together, these data demonstrated that the high-throughput methods developed in this study are valuable for identifying new calcium-related components in the HCO3−/CO2-mediated stomatal closure signaling network pathway.
CO2 influences both stomatal movement and stomatal development; however, the mechanisms of guard cell perception and transduction are still not fully clear, and the sensors that mediate CO2-controlled stomatal movement remain enigmatic. Previous studies have suggested that intracellular bicarbonate acts as a second messenger in guard cells involved in mediating CO2 signal transduction [19,20,21]. To date, a number of proteins with critical roles in this signaling pathway have been identified, such as CA1 and CA4, HT1, SLAC1, RHC1, and others.
Because Ca2+ is a key cellular second messenger, transient change in [Ca2+]cyt reflects most physiology processes including CO2-regulated guard cell behavior. GROWTH CONTROLLED BY ABSCISIC ACID 2 (GCA2) has been proved to function downstream of both CO2 signaling and ABA signaling by regulating [Ca2+]cyt. gca2 mutant plants display decreased sensitivity of stomata to elevated CO2 and show an abnormal [Ca2+]cyt pattern in guard cells . This altered pattern of [Ca2+]cyt in CO2/HCO3−-treated guard cells prompted us to design a screening method to identify genes implicated in [Ca2+]cyt regulation during stomatal response to CO2.
AEQ photoprotein has been extensively used in the Ca2+ signaling field for almost 40 years. Because it is convenient, fast, sensitive, easy to use, and applicable to real-time measurement of [Ca2+]cyt changes, we chose an AEQ-based system for our genetic screen. According to our data showing that the CO2/HCO3−-induced increase of [Ca2+]cyt happened no more than 1 s after CO2/HCO3− application, almost climbed to the highest value, then dropped almost back to the baseline; the whole process only lasted for about 3 s (Fig. 1a, right), suggesting that the variation of [Ca2+]cyt happened both early and rapidly in this physiological process. To identify components underlying this response, Arabidopsis mutants were usually isolated by analyzing their leaf temperature through thermal imaging. This traditional method was convenient and common, however thermal imaging takes hours to reach a steady state before detection, which may miss some important components that function earlier in the response to CO2.
According to a previous report about detecting stomatal responses to bicarbonate, 1 mM KHCO3 has been used for screening . As shown in Figs. 1, 2, both significantly increased bioluminescence and remarkable stomatal closure can be detected at this concentration. Before the screen, a period of dark treatment for AEQ incubation is necessary, so we conducted another preliminary test because dark can influence guard cell status. We found that even in dark treatment, 1 mM KHCO3 still can cause closure of the stomata (Fig. 2b), further suggesting the suitability of this screening method.
By using this AEQ-based method and treatment with 1 mM KHCO3, we obtained mci and mcs mutants from about 35,000 M2 seeds. We will continue to analyze these mutants and characterize the function of these genes. This series of experiments will shed light on the mechanism of calcium-mediated CO2/HCO3− response in the guard cell, which appears to occur early during CO2/HCO3−-induced stomatal closure.
We have developed a sensitive method for isolating stomatal CO2/HCO3− response genes that function early in the response and play a role in regulating [Ca2+]cyt transient changes. This method will be helpful in elucidating the Ca2+-dependent regulation of stomatal response.
Plant material and growth conditions
Lines of Arabidopsis thaliana ecotype Col-0 constitutively expressing the intracellular Ca2+ indicator AEQ (pMAQ2; a gift from Marc R. Knight) or Cameleon (YC3.6; a gift from Simon Gilroy) were used. Plants homozygous for the AEQ-transgenic Arabidopsis plant were selected from the second generation after transformation (T1 plants). One such plant, expressing a high level of AEQ, was selected for subsequent experiments.
Plants were grown in soil or in medium containing Murashige and Skoog salts (MS; PhytoTechnology Laboratories), 3% (w/v) sucrose (Sigma), and 0.6% agar (Solarbio) in controlled environmental rooms at 20 ± 2 °C. The fluency rate of white light was ~ 80–100 μmol m−2 s−1. The photoperiod was 16 h light/8 h dark. Seeds were sown on MS medium, placed at 4 °C for 3 days in the dark, and then transferred to growth rooms.
AEQ bioluminescence-based Ca2+ imaging
[Ca2+]cys was measured using Arabidopsis plants expressing AEQ. Leaves (1 per well) were treated evenly with 150 μL of 10 μM coelenterazine (Sigma, C2230) in 96-well white culture plates 4 to 6 h before imaging and placed in the dark and in the glass chamber to remove CO2. AEQ bioluminescence imaging was performed using a Berthold LB985 system equipped with a light-tight box and a cryogenically cooled, back-illuminated CCD camera. The recording of luminescence was started 30 s prior to treatment and lasted for 5 min. All the treatments were carried out in the dark, and the experiments were carried out at room temperature (22–24 °C).
Similarly, guard cells were used for AEQ bioluminescence imaging. Rosette leaf epidermal peels from 3- to 4-week-old plants were placed in a microwell chamber in incubation buffer for 4–6 h in the dark. AEQ bioluminescence imaging of guard cells was performed using a bioluminescence microscope (Sclis; Biocover) equipped with a light-tight box and a cryogenically cooled, back-illuminated CCD camera. The recording of luminescence was started 60 s prior to treatment and lasted for 5 min. Bright-field images were taken after AEQ imaging. All treatments were carried out in the dark, and the experiments were carried out at room temperature (22–24 °C).
Arabidopsis seeds expressing AEQ were mutagenized with EMS as described previously . Briefly, about 5000–10,000 seeds were imbibed overnight and then shaken in 0.3% EMS (v/v) for 15 h. The M1 seeds were rinsed thoroughly with tap water, sterilized with 10% bleach for 30 min, and washed with sterilized water 5–8 times. M2 seeds were harvested separately from individual M1 plants. For screening, M2 seeds were individually planted in soil and grown for 3 weeks. Leaves from M2 plants were placed in a 96-well plate and 100 µL of freshly prepared incubation buffer was added to each well. Kinetic luminescence measurements were performed with an automated microplate luminescence reader (LB960; Berthold) every 0.2 s. After 3 s of luminescence counts, 100 µL of 2 mM KHCO3 solution was automatically injected into each well to obtain a final concentration of 1 mM. Bioluminescence was recorded for 30 s per well.
Stomatal aperture bioassay
Leaves of 3- to 4-week-old seedlings were used in the stomatal aperture assays . Leaves were detached before the light period started. For monitoring stomatal response to KHCO3 in light or dark, whole leaves were incubated in stomatal buffer and then exposed to light (100 μmol m−2 s−1) or dark for 2 h at 25 °C in the glass chamber.
The stomatal buffer contained 50 mM KCl, 0.1 mM CaCl2, and 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), adjusted to pH 7.0 with Tris (hydroxymethyl) aminomethane (Tris) [25, 26]. Two hours later, the effects of 1 mM KHCO3 on stomatal closure were tested. For characterization of stomatal response mutants, 3 mM KHCO3 was also used. Prior to measuring the stomatal aperture, the adaxial epidermis and mesophyll layers were gently separated, and the epidermal strips were placed on microslides containing a drop of stomatal buffer with the desired concentration of KHCO3 and covered with coverslips. Pictures of stomata were acquired using an inverted microscope (IX73; Olympus) at 40× magnification. Approximately 100 stomatal apertures from different leaves of each plant type were measured using Image J software (Broken Symmetry Software), and three independent experiments were performed.
Cameleon-based [Ca2+]cys imaging in guard cells
The wild-type plants constitutively expressing GFP fluorescence resonance energy transfer (FRET)-based Ca2+ sensor YC3.6, and 10 homozygous lines were generated. Rosette leaf epidermal peels from 3-week-old plants were placed in a microwell chamber in the stomatal buffer for 2 h under light (100 μmol m−2 s−1). Epidermal peels were treated with 1 mM KHCO3 and ratiometric Ca2+ imaging was performed using a confocal microscope (LSM710; Zeiss) as described previously . The YC3.6 Ca2+ sensor was excited with the 458 nm line of the argon laser. The cyan fluorescent protein (CFP; 473–505 nm) and FRET-dependent Venus (562–536 nm) emission were collected using a 458 nm primary dichroic mirror and the Meta detector of the microscope. Emission images (562–536 nm and 473–505 nm) of epidermal peels were taken, and ratiometric images before and 10 s after addition of 1 mM KHCO3.
Identification of MCI1 and MCS1 by MutMap analysis
We backcrossed mci1 or mcs1 to AEQ-expressing Col-0 and produced F2 individuals. Plants with the mci1 or mcs1 phenotype were then subjected to MutMap analysis to find the mutated gene . DNA of 30 F2 progeny showing the mutant phenotype was isolated and then bulked using an equal amount of DNA from each plant. This bulked DNA was then subjected to MutMap analysis.
Availability of data and materials
The raw data from all experiments as well as the material used in this manuscript can be obtained from the corresponding authors upon reasonable request.
SLOW ANION CHANNEL-ASSOCIATED 1
A MUNC 13 ortholog in Arabidopsis controls the tethering of an H+-ATPase
ALUMINUM-ACTIVATED MALATE TRANSPORTER 12
RESISTANT TO HIGH CO2
OPEN STOMATA 1
HIGH LEAF TEMPERATURE 1
- [Ca2+]cyt :
Free calcium ion concentration in cytosol
- Mci :
Mutant of HCO3−/CO2insensitive
- Mcs :
Mutant of HCO3−/CO2sensitive
Ethyl methane sulfonate
GROWTH CONTROLLED BY ABSCISIC ACID 2
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We thank Marc R. Knight of Durham University (UK) for the kind gift of pMAQ2 and Simon Gilroy of the University of Wisconsin, Madison for the generous gift of the YC3.6 vector.
This work was supported by the National Natural Science Foundation of China (31900239 and 31570287) and the Henan Education Department Key Foundation (19A180012).
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Tang, M., Zhao, X., Hu, Y. et al. Arabidopsis guard cell CO2/HCO3− response mutant screening by an aequorin-based calcium imaging system. Plant Methods 16, 59 (2020). https://doi.org/10.1186/s13007-020-00600-w