Abstract: 【Objective】Comparing on differential expression proteins between stomatal closing and opening at different leaf stomata-densities, it is to be revealed how pectin metabolism regulates stomata closing and opening. The result will play an essential role in understanding how stomata functions to environment adaptation. The result will play an essential role in understanding how stomata functions to environment adaptation.【Method】Vectors, either over- or  inhibiting-expression of StEPF-2 (Solanum tuberosum EPIDERMAL PATTERNING FACTOR 2) in vivo were constructed. The fusing genes were transformed into Solanum tuberosum cultivar Kexing 1. Transgenic potato lines, either rise or lower at leaf stomatal density were generated. Gene and protein expression profiles of leaves at various stomatal densities were assayed via RNA-seq and iTRAQ. Comparing differentiation expression proteins, pectin metabolic enzymes driving stomatal movement under light and darkness were identified and confirmed by the Pulldown and LC-MS/MS. A pectin metabolism pathway regulating stomatal movement was to be proposed.【Result】At least 14 protein families, drive stomata closing and opening involved in pectin metabolism of the guard cell wall during stomatal mature. Five protein families were detected and confirmed only in the stomatal-closed leaves under darkness, including polygalacturonase inhibitor proteins (PGIP) and Rhamnose synthase (RHM) for RG side-chain biosynthesis. Four protein families, polygalacturonase (PG), pectate lyase-like (PLL), pectinmethylesterase (PME) and α-galactosidase (AGAL) were identified only in leaves at various stomatal densities under light. Additionally, five protein families were concurrently identified in both leaves of stomata closing and opening, including pectinacetylesterase (PAE) and subtilase (SBT).【Conclusion】Under light, PMEs catalyze pectin demethylesterification, afterwards, pectin was exo- and endo-hydrolyzed by PG, PLL and AGAL. Pectin losing structure was split under turgor, results in stomatal opening. Reversely, under dark, PGI inhibited pectin hydrolysis. Pectin side-chain biosynthesis was promoted by RHM. Therefore, stomata kept closing due to structurally-complete pectin with voluntary expending function.

Key words: potato, stomatal density, pectin, RNA-Seq, iTRAQ, protein