Article Title: Pyrophosphate modulates plant stress responses via SUMOylation
Figure Lengend Snippet: Overexpression of the Arabidopsis soluble pyrophosphatase PPa5 is sufficient to complement fugu5-1 . ( A ) Comparison of 6-week-old rosettes phenotypes of Col-0, fugu5-1 and overexpression lines of Arabidopsis vacuolar pyrophosphatase AVP1 and yeast soluble pyrophosphatase IPP1. ( B ) Representative images showing the localization of UBQ:PPa5-GFP to cytosol and nucleus in shoot (upper panel) and root cells (lower panel). Scale bars: 10 µm. ( C ) Cotyledon phenotypes of 5 days old seedlings of Col-0, fugu5-1 and UBQ:PPa5-GFP in Col-0 and fugu5-1 backgrounds. ( D ) Comparison of 6-week-old short day grown rosette phenotypes of Col-0, fugu5-1 and UBQ:PPa5-GFP in Col-0 and fugu5-1 backgrounds. ( E ) Measurements of fresh weight and whole rosette area of Col-0, fugu5-1 and UBQ:PPa5-GFP in Col-0 and fugu5-1 backgrounds. Plants were grown for 6 weeks under short-day conditions. To determine rosette area Rosette Tracker plug-in of ImageJ is used. Error bars represent SD of the mean of n = 20 of 3 biological replicates. ( F ) Analysis of UBQ:PPa5-GFP protein amount in Col-0 and fugu5-1 background with anti-GFP. Soluble proteins from 6-week-old rosettes grown under short-day conditions were extracted. An internal control provided by SPL detection kit (DyeAGNOSTICS) was used for normalization. One representative image from three biological replicates is depicted. ( G ) Soluble proteins of Col-0, fugu5-1 and UBQ:PPa5-GFP in Col-0 and fugu5-1 backgrounds were used to determine K + -stimulated PP i hydrolysis. Plants were grown for 6 weeks under short-day conditions. Error bars represent SD of n = 3 biological replicates. Significant differences are indicated by different letters (Two-way ANOVA followed by Tukey’s test, p
Article Snippet: To determine the effects of PPi hydrolysis on the FRET assay, 0.8U E. coli inorganic pyrophosphatase (NEB) was used and the buffer solution that the pyrophophatase includes was added to the control wells (20 mM Tris-HCl, 100 mM NaCl, 1 mM Dithiothreitol, 0.1 mM EDTA, 50% Glycerol, pH 8.0).
Techniques: Over Expression