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

Developing plants tolerant of low phosphate (Pi) availability is essential to reduce reliance on fertilizers and achieve 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 Arabidopsis thaliana CORNICHON HOMOLOG 5 (AtCNIH5) encodes a Pi deficiency-induced ER cargo receptor that regulates Pi homeostasis. To find potential membrane cargoes of AtCNIH5, 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 proteins in cnih5. Besides PHOSPHATE TRANSPORTER 1 proteins (PHT1s), downregulation of the biosynthetic or modifying enzymes for cell wall polysaccharides, very long-chain fatty acids, and their derivative extracellular aliphatic compounds is over-represented. Using the yeast split-ubiquitin and the in-planta tripartite split-GFP assays, we verified the interaction of AtCNIH5 with various downregulated transporters in cnih5, including AtPHT1s, AtOCT1, AtURGT6, AtDTX21, and AtDTX35. In addition, we demonstrated that the C-terminal acidic residue of AtCNIH5 is required for interaction with AtOCT1 but not with AtPHT1;1 or AtDTX21, indicating distinct cargo selection mechanisms. More importantly, enhancing in-situ AtCNIH5 expression/activity enhances plant growth. By analogy with transcriptional factors that govern gene expression, we propose that AtCNIH5 acts as a low Pi-responsive hub to facilitate ER export of specific membrane cargoes, providing a potential engineering strategy to improve plant fitness under suboptimal Pi supply.