Snakin-2

Potato tuber dormancy is critical for the post-harvest quality. Snakin/Gibberellic Acid Stimulated in Arabidopsis (GASA) family genes are involved in the plants' defense against pathogens and in growth and development, but the effect of Snakin-2 (SN2) on tuber dormancy and sprouting is largely unknown. In this study, a transgenic approach was applied to manipulate the expression level of SN2 in tubers, and it demonstrated that StSN2 significantly controlled tuber sprouting, and silencing StSN2 resulted in a release of dormancy and overexpressing tubers showed a longer dormant period than that of the control. Further analyses revealed that the decrease expression level accelerated skin cracking and water loss. Metabolite analyses revealed that StSN2 significantly down-regulated the accumulation of lignin precursors in the periderm, and the change of lignin content was documented, a finding which was consistent with the precursors' level. Subsequently, proteomics found that cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (COMT) and peroxidase (Prx), the key proteins for lignin synthesis, were significantly up-regulated in silencing lines, and gene expression and enzyme activity analyses also supported this effect. Interestingly, we found that StSN2 physically interacts with three peroxidases catalyzing the oxidation and polymerization of lignin. In addition, SN2 altered the hydrogen peroxide (H2O2) content and the activities of superoxide dismutase (SOD) and catalase (CAT). These results suggest that StSN2 negatively regulates lignin biosynthesis and H2O2 accumulation, and ultimately inhibits the sprouting of potato tubers.

The potato tuber is the main nutrient supply and reproductive organ; however, tuber sprouting can reduce its commercial value. Snakin-2 (StSN2) was first reported as an antimicrobial peptide that positively regulates potato disease resistance. Our recent study suggested StSN2 overexpression inhibited sprout growth, while the sprouting process was accelerated in StSN2 RNAi lines. Cytoplasmic glyceraldehyde-3- phosphate dehydrogenase 1 (StGAPC1) was identified as a candidate protein that interacts with StSN2 by coimmunoprecipitation/mass spectrometry (CoIP/MS) experiments. Here, we report that the expression levels of StSN2 and StGAPC1 decreased during sprouting compared with dormancy. Coexpression of StSN2 and StGAPC1 in bud eyes and apical buds was verified by immunofluorescence analysis of paraffin sections. In addition, interaction of StSN2 and StGAPC1 was confirmed by yeast two-hybrid, coimmunoprecipitation and split luciferase complementation assays. Overexpression of StGAPC1 depressed sprout growth, which is similar to the function of StSN2, and StSN2- and StGAPC1-overexpressing lines showed decreased glucose, fructose and galactose content. The interaction of StSN2 and StGAPC1 enhanced StGAPC1 activity and decreased its oxidative modification to inhibit sprout growth. Our results suggest that StSN2 plays a regulatory role in tuber sprout growth through interaction with StGAPC1.

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine-rich snakin-1 and -2 antimicrobial peptides by using a combination of solid-phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40-50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin-2 compared to natural snakin-2, we demonstrated that synthetic snakin-2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin-2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.