Edited by Mike Shipston In plants, strict regulation of stomatal pores is critical for modulation of CO 2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca 2؉) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminumactivated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate-and voltage-dependent. However, this was shown to be true only in the presence of Ca 2؉. Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca 2؉dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca 2؉ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca 2؉ /CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

The mucosal-to-serosal flux of 14C 3- O-methyl-d-glucose was compared against the electrogenic transport of d-glucose across ex vivo intestinal segments of Nile tilapia, rainbow trout, and pig in Ussing chambers. The difference in affinities ( Km “fingerprints”) between pig flux and electrogenic transport of glucose, and the absence of this difference in tilapia and trout, suggest two absorptive pathways in the pig and one in the fish species examined. More specifically, the total mucosal-to-serosal flux revealed a super high-affinity, high-capacity (sHa/Hc) total glucose transport system in tilapia; a super high-affinity, low-capacity (sHa/Lc) total glucose transport system in trout and a low-affinity, low-capacity (La/Lc) total glucose transport system in pig. Comparatively, electrogenic glucose absorption revealed similar Km in both fish species, with a super high-affinity, high capacity (sHa/Hc) system in tilapia; a super high-affinity/super low-capacity (sHa/sLc) system in trout; but a different Km fingerprint in the pig, with a high-affinity, low-capacity (Ha/Lc) system. This was supported by different responses to inhibitors of sodium-dependent glucose transporters (SGLTs) and glucose transporter type 2 (GLUT2) administered on the apical side between species. More specifically, tilapia flux was inhibited by SGLT inhibitors, but not the GLUT2 inhibitor, whereas trout lacked response to inhibitors. In contrast, the pig responded to inhibition by both SGLT and GLUT2 inhibitors with a higher expression of GLUT2. Altogether, it would appear that two pathways are working together in the pig, allowing it to have continued absorption at high glucose concentrations, whereas this is not present in both tilapia and trout.