CORNICHON HOMOLOG 5-dependent ER export of membrane cargoes in phosphate-starved Arabidopsis root as revealed by membrane proteomic analysis

Developing plants with tolerance to low phosphate (Pi) is of interest to reducing the reliance on fertilizers and thus achieving agricultural sustainability. One strategy is to enhance the endoplasmic reticulum (ER) export of cargoes associated with Pi starvation and their trafficking to final destinations. However, the mechanisms underlying this process are underexplored. We recently discovered that the Arabidopsis thaliana CORNICHON HOMOLOG 5 ( AtCNIH5 ) encodes a Pi deficiency-induced ER cargo receptor regulating Pi homeostasis. To find potential membrane cargoes of At CNIH5, we applied the UV-cleavable 4-hexylphenylazosulfonate (Azo)-solubilized microsomal protein extraction for iTRAQ-based proteomic analysis. We identified 4,317 proteins in Pi-limited Arabidopsis roots, with 372 upregulated and 106 downregulated in cnih5 . Besides PHOSPHATE TRANSPORTER 1 proteins (PHT1s), downregulation of enzymes catalyzing the biosynthesis of very long-chain fatty acids and their derivative extracellular aliphatic compounds and nucleotide sugar for cell walls is over-represented. Using the yeast split-ubiquitin and the in-planta tripartite split-GFP assays, we verified the interaction of At CNIH5 with the downregulated transporters in cnih5 , including At PHT1s, At OCT1, At URGT6, At DTX21, and At DTX35. Furthermore, At CNIH5 increases in the root of the Pi overaccumulator pho2 , and its C-terminal acidic residue is not required for the interaction with At PHT1;1, implying that At CNIH5 employs a distinct selection mechanism for At PHT1;1. Importantly, increasing in-situ At CNIH5 expression/activity boosts plant growth under Pi repletion and limitation. We propose At CNIH5 as a low Pi-responsive hub that controls the ER export of specific membrane cargoes, thus providing a potential engineering strategy to improve plant fitness under suboptimal Pi supply.