Targeted knock-out of a gene encoding bryoporin in the moss Physcomitrella patens affects leafyshoot tolerance to dehydration
Yang Song, Li Li, Li Wang, Xinxing Dang, Yikun He, Suxia Cui* 

College of Life Sciences, Capital Normal University, Beijing 100048, China.
This work was supported by grants from National Natural Science Foundation of China (30870195) and Earmarked Research Grant (2011ZX001-003) of Ministry of Agriculture of China.
^*Corresponding author. E-mail: sxcui@cnu.edu.cn

Abstract
Physcomitrella patens is an extremely dehydration-tolerant moss. Using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE) combined with MALDI TOF/TOF MS, a small group of bryoporins (PpBPs) which showed remarkably high level of induced expression in dehydrated and rehydrated leafyshoots were identified. Bryoporins are homologue of pore-forming cytotoxin protein studied extensively in sea anemone. In this study, the moss PpBP2 was deleted via targeted gene disruption. The targeted knockout mutant of PpBP2 are viable on phototrophic media and displayed similar growth phenotype compared with the wild type. However, physiological assays for the content of water and chlorophyll demonstrated that the leafyshoot of the null mutant (Δbp2) could not recover from around 75% of water deficiency. The result indicates that PpBP2 knockout plants were less resistant to dehydration treatment than wild type. To understand the molecular details of dehydration response in the null mutant, we performed a proteomics study. Analyses of the Δbp2 mutant and wild type under dehydration condition identified more than a dozen proteins, which might associtate with the dehydration tolerance in the absence of PpBP2. They included arabinogalactan protein, glucose-6-phosphate dehydrogenase, phosphatidylethanolamine-binding protein and dehydrin. The study provides novel insight into the molecular network that links PpBP2 to specific cellular and physiological responses to dehydration tolerance.

Keywords: Physcomitrella patens, bryoporin，gene knockout, dehydration tolerance，proteomics