Plant Trans-Golgi Network/Early Endosome pH regulation requires Cation Chloride Cotransporter (CCC1)

Abstract

31The secretary and endocytic pathways intersect at the trans-Golgi network/Early Endosome 32 (TGN/EE). TGN/EE function depends on a careful balance of ions within this compartment, 33 and the electrochemical potential across its membrane. The identity of the proton pump 34 required for acidification and the transporters that catalyse cation and anion import into 35 endosome compartments are known. However, the protein needed to complete the transport 36 circuit, and that mediates cation and anion efflux from the TGN/EE has not been identified. 37Here, we characterize Cation Chloride Cotransporters (CCC1s) from Arabidopsis and rice. 38 We find that the AtCCC1 is localized to the TGN/EE, where it modulates important TGN 39 functions such as endocytosis, exocytosis, and cell wall synthesis/secretion. Loss of CCC1 40 results in severe, widespread phenotypes in both rice and Arabidopsis, including plant growth 41 and developmental perturbations, defects in root hair elongation and altered osmoregulation, 42 consistent with CCC performing a core cellular function. Complementation of the root hair 43 elongation phenotype of Atccc1 with root hair specific expression of GFP-AtCCC1 44 demonstrates CCC1 functions within the TGN/EE. Collectively, our results imply that CCC1 45 is a strong candidate for the missing component of the TGN/EE ion transportcircuit. 46 47 126 7including root hairs, and in the gynoecium, while GUS staining did not indicate expression in 127 these cells. This is likely due to the different sensitivities of the two methods. 128 129 ccc1 knockout mutants have a reduced root hair length and tip growth rate 130 As our expression analysis showed that AtCCC1 is expressed in root hairs (Fig. 1), we 131 examined whether root hair cells of the knockouts had an altered phenotype, and whether we 132 could use this model cell type to investigate the role of CCC1. Characterisation of the 133 knockouts showed that both Atccc1 and Osccc1.1 plants had a reduction in the overall length 134 of their root hairs compared to control plants (Fig. 2); and a complete lack of collet hairs in 135 Atccc1. Collet hairs are epidermal root hairs formed in some plant species in the transition 136 zone between the root and the hypocotyl (Fig. 2, Sliwinska et al., 2015). Atccc1 also had 137 branching and bulging of root hairs, although, at a low frequency, and root hairs of the 138 495 496 References 497 Balcerowicz, D., Schoenaers, S., and Vissenberg, K. (2015). Cell fate determination and 498 the switch from diffuse growth to planar polarity in Arabidopsis root epidermal cells. 499 Frontiers in Plant Science 6, 1163. 500 Bassil, E., and Blumwald, E. (2014). The ins and outs of intracellular ion homeostasis: 501 NHX-type cation/H + transporters. Current Opinion in Plant Biology 22, 1-6. 502 23 Bassil, E., Ohto, M.-a., Esumi, T., Tajima, H., Zhu, Z., Cagnac, O., Belmonte, M., Peleg, 503 Z., Yamaguchi, T., and Blumwald, E. (2011). The Arabidopsis intracellular Na + /H + 504 antiporters NHX5 and NHX6 are endosome associated ...

CLCd and CLCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack

“…Recently, the Cation Chloride Cotransporter 1 (CCC1) of Arabidopsis has been shown to be localized at the TGN/EE and to affect pH and ion homeostasis. By mediating both cation and anion efflux CCC1 complements the ion transport circuit of the TGN/EE (McKay et al, 2020). However, the circuit might not yet be complete.…”