Mechanisms for calcium sensing receptor-regulated stomatal closure in response to the extracellular calcium signal

Abstract

In Arabidopsis, extracellular calcium (Ca²⁺_o) promotes intracellular calcium (Ca²⁺_i) transients and stomatal closure, which has been found to be regulated by the calcium sensing receptor (CAS). However, the detailed pathways for transducting the Ca²⁺_o signal by CAS are still unclear. We found that nitric oxide (NO) and the hydrogen peroxide (H₂O₂) accumulated in the guard cell chloroplast were the two elements that act downstream of the CAS signaling and trigger the stomatal closure by prolonging Ca²⁺_i transients.¹ Here we provide more commentary on CAS-regulated H₂O₂ generation from chloroplast and Ca²⁺_i transients in response to Ca²⁺_o, as well as other potential mechanisms that may be involved in the CAS signaling pathway.

Calcium sensing receptor CAS, a protein localized in chloroplast thylakoid membranes in Arabidopsis,² was suggested to be activated by Ca²⁺_o, which can result in Ca²⁺_i transients and stomatal closure.³ Recently, our study revealed the effects of Ca²⁺_o on H₂O₂ generation from the chloroplast in the wild type of Arabidopsis and demonstrated that Ca²⁺_o-driven H₂O₂ generation in guard cell was impaired in cas antisense line (CASas), suggesting the involvement of H₂O₂ in CAS signaling.¹

Previous studies have shown that Ca²⁺_o could initiate prolonged Ca²⁺_i transients in the guard cell of the wild type.^4,5 However, CASas or cas mutant exhibited a first limited Ca²⁺_i increase without the consequent Ca²⁺_i transients in response to Ca²⁺_o simulation, resulting the failure of stomatal closure.^2,3 In addition, according to Siegel et al.,⁶ Ca²⁺_o causes a first Ca²⁺_i increase in both control and Ca²⁺ chelator BAPTA-AM treated guard cells. However, the second step of the prolonged Ca²⁺_i transients were inhibited in BAPTA-AM treated guard cells. Interestingly, Ca²⁺_o was able to close the stomata of cas mutant by imposing Ca²⁺_i transients upon the incubation of stomata in depolarizing buffer.⁷ These results suggested that CAS could regulate the prolonged Ca²⁺_i transients, which functioned to result in stomatal closure when exposed to the Ca²⁺_o signal. Our results also proposed the possibility that H₂O₂ generation regulated by CAS mainly drove the prolonged Ca²⁺_i transients because stomatal closure was observed in H₂O₂-treated guard cells in CASas and H₂O₂ was found to activate the Ca²⁺ channel and induce several Ca²⁺_i transients.^5,8 The first step in Ca²⁺_i elevation is likely to activate the thylakoid-localized CAS and make subsequent H₂O₂ generation, leading to prolonged Ca²⁺_i transients and stomatal closure.

Usually, Ca²⁺_i level is maintained at a low concentration (~0.1 μM) and considered to elevate up to 10 μM when cells are stimulated.⁹ Comparing to our experiment, Ca²⁺-driven H₂O₂ generation from the isolated chloroplast was detected at the Ca²⁺ concentration over 0.6 mM.¹ Physiological levels of Ca²⁺ (1–10 μM) seem to be not effective in causing H₂O₂ generation according to our results. However, we argue that 10 μM was an average Ca²⁺_i concentration of the whole guard cells while the concentration from some sections of the cell may exceed this level. According to the guard cell images shown by Allen et al.,^4,5 only a few regions in the guard cell showed strong Ca²⁺_i elevation in response to Ca²⁺_o stimulation. The concentration of Ca²⁺_i from these parts should be much higher than the average at 10 μM. It is likely that Ca²⁺_i burst around these parts in response to Ca²⁺_o signal promoted CAS-induced H₂O₂ generation in chloroplasts. In addition, the Ca²⁺ treatments of isolated chloroplasts were performed in vitro in our study. This result could only reflect the relationship between Ca²⁺ and the chloroplast H₂O₂ generation. The exact correlation between the concentration of Ca²⁺_i and the chloroplast H₂O₂ generation in vivo needs further studies.

In our present study, H₂O₂ and NO were found to be involved in the CAS signaling.¹ However, other biomolecules that play roles in abiotic or biotic stimulation are also the potential candidates functioning in Ca²⁺_o and CAS signaling as well as in stomatal closure. Inositol 1,4,5-triphosphate (Ins(1,4,5)P₃)-mediated Ca²⁺_i transients have been demonstrated to be involved in abiotic stresses, including salt,^10,11 drought^12,13 and cold.^14,15 Reduction of Ins(1,4,5)P₃ or phospholipase C (PLC) activity attenuate plant responses to stress, ABA-induced Ca²⁺_i transients and stomatal closure.^16-18 On the contrary, release of Ins(1,4,5)P₃ caused Ca²⁺_i elevation and stomatal closure,¹⁹ suggesting the involvement of Ins(1,4,5)P₃ pathway in ABA signaling. In this putative Ins(1,4,5)P₃ pathway, CAS was also found to regulate Ins(1,4,5)P₃ level, leading to Ca²⁺_i transients and stomatal closure.²⁰ Interestingly, guard cells of transgenic Arabidopsis with reduced soluble inositol phosphates were less responsive to stomatal closure in the presence of high Ca²⁺_o,²¹ indicating a relationship between Ca²⁺_o signaling and the phosphoinositide pathway. It should also be noted that U73122, an inhibitor of PLC, can block NO-induced stomatal closure,²² indicating that PLC-Ins(1,4,5)P₃ signaling may act downstream of the NO signaling cascade during stomatal closure.

Phosphatidic acid (PA), which is emerging as a second messenger in plants, was found to accumulate in response to ABA treatment, drought or salt stress.²³ PA can be generated via phospholipase D (PLD) and PLC in concert with diacylglycerol kinase (DGK) pathway, which are activated by H₂O₂ and NO^22,24 and could inhibit H⁺-ATPase and inward K⁺ channels, leading to K⁺ efflux and stomatal closure.^25-27

We are far from fully understanding the mechanisms of CAS-mediated H₂O₂ generation in guard cells. However, cytosolic alkalinization, a common and early messenger preceding the production of reactive oxygen species and NO during stomatal closure by variable signals, including ABA and methyl jasmonate,^28,29 are likely to occur during Ca²⁺_o-induced stomatal closure. These instances prompt us to wonder whether the complex mechanisms underlying the exact correlation between CAS, pH, H₂O₂, NO, PLC-Ins(1,4,5)P₃ pathway as well as PA in Ca²⁺_o signaling.

Wang W-H, Yi X-Q, Han A-D, Liu TW, Chen J, Wu FH, et al. Calcium-sensing receptor regulates stomatal closure through hydrogen peroxide and nitric oxide in response to extracellular calcium in Arabidopsis. J Exp Bot. 2012;63:177–90. doi: 10.1093/jxb/err259.