Characterization of Brachypodium QUAC1, an anion channel involved in stomatal movement

Abstract

Stomata are pores formed by guard cells in the leaf epidermis of plants. These pores are essential in responding to both abiotic and biotic stress such as drought, heat, pathogen infection, and therefore strictly regulated in plants. Uptake and release of ions from guard cells are the keys to stomatal function. For plants to close their stomata, at least two types of anion channels present in the guard cell membrane are required: the rapid (R-type) and the slow (S-type) activating anion channels. Quick anion channel 1 (QUAC1), initially named ALMT12, a member of the aluminum-activated malate transporter family, has been shown to encode an R-type anion channel in guard cells in Arabidopsis thaliana, a dicot model. Sequence alignments between dicot and monocot QUAC1 show as high as 56 % identity at the protein level. The purpose of this study was to investigate whether the monocot QUAC1s maintain the properties and functions as reported for the dicot AtQUAC1, and further characterize the monocot channel. The monocot model Brachypodium dystachyon (commonly called stiff brome) was used for this study. To examine the properties of BdQUAC1, the protein was expressed in HEK293 cells, and activities of the channel were measured using the patch clamp technique. Data collected in this study showed that BdQUAC1 is activated by malate, consistent with what had been reported for AtQUAC1. However, the malate activation occurred only in the presence of Ca2+ and was inhibited by a calmodulin (CaM) inhibitor. By computational predictions, the BdQUAC1 sequence was shown to include a CaM -binding domain (CBD). Direct interaction between the CBD and CaM was confirmed by two different methods: isothermal titration calorimetry and CaM agarose affinity pulldown. Electrophysiological analyses showed that site directed mutations at some of the basic residues in the CBD affect the activities of the channel. In addition, the hormone abscisic acid (ABA), which is known to cause an increase in cytosolic Ca2+ concentration and plays a significant role in stomatal closure, did not have any direct electrophysiological effects on BdQUAC1 protein expressed in the HEK293 system. The role of BdQUAC1 in stomatal closure was also assessed in planta. QUAC1-knockdown in Brachypodium showed an increase in the width of stomata aperture compared to wildtype. Overexpression of BdQUAC1 also showed a significant reduction of stomatal width and additionally, an effect on stripe rust infection which is known to infect via stomata. Altogether, this study has explored the regulation of BdQUAC1, suggesting an activation of BdQUAC1, that requires malate, Ca2+, and CaM. Although, in plants, the activation of anion channels is through ABA signalling pathways, ABA does not have any direct in vitro effects on BdQUAC1. This highlights the involvement of Ca2+ and CaM in the ABA signaling pathway mediating stomatal response. Altering the expression of the BdQUAC1 gene affects stomatal function and potentially pathogen infection.