Nicotiana tabacum protoplasts secretome can evidence relations among regulatory elements of exocytosis mechanisms

An alternative study involving proteome analysis of the 24 h Nicotiana tabacum protoplast culture medium was performed with the aim to confirm relations among regulatory elements of exocytotic processes. Protoplasts present many convenient features to study cellular processes during transient overexpression or suppression of specific gene's products. We performed a proteomic analysis of the culture medium fraction of protoplasts transiently expressing transgenes for 24 h to characterize the effect of various regulatory proteins dominant negative mutants. A total number of 49 spots were found reproducible in the medium. Twenty-four of these spots were identified with nano RP-HPLC-ESI-MS/MS. Only three and six spots were respectively identified as canonical and non-canonical secreted cell wall proteins. The low number of spots present in the culture medium fraction allowed us the ambitious experiment to analyze the influence of various SNAREs (SYP121, SYP122, SNAP33)and Rab (Rab11)dominant negative mutants. Missing a reasonable number of identified proteins the analyses gave rise to a similarity matrix statistically analyzed considering variation within the presence of 24 spots reproducible in presence of transient overexpression of SNAREs (SYP121 and SYP122)and Rab11 native cDNAs. The similarity confirmed the closer relation between the function of SYP122 and Rab11 as evidenced by the secRGUS based analysis. This analysis included the effect of SNAP33 DN mutant and showed that this Qb-c-SNARE influence both SYP121 and SYP122 SNARE complexes.