Determination of the Activity Signature of Key Carbohydrate Metabolism Enzymes in Phenolic-rich Grapevine Tissues.

Elizabeth Dunn Covington,Marina Dermastia,Thomas Roitsch

doi:10.17344/acsi.2016.2484

Abstract

Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf. As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteria 'Candidatus Phytoplasma solani', known to alter carbohydrate metabolism in grapevine. The described adaptations may be useful for determination of metabolic fingerprints for physiological phenotyping of other plant species with inherently high levels of phenolic compounds.

Highlights

Carbohydrates are the main energy and carbon source for all organisms, and in plants are important signaling factors.[1]
Significant changes in carbohydrate metabolism are associated with development of disease symptoms on grapevine infected with phytoplasmas,[2,3,4] the causal agents of important crop and fruit tree diseases, including phytoplasmal grapevine diseases.[5]
The changes of carbohydrate metabolism in grapevine infected with these phytopathogenic bacteria have been detected mainly as increased expression of genes encoding the key enzymes associated with carbohydrate production

Summary

Introduction

Carbohydrates are the main energy and carbon source for all organisms, and in plants are important signaling factors.[1]. The workflow for measuring the enzyme activities of AGPase, SuSy, cytINV, cwINV and vacINV is depicted in the assays’ scheme (Figure 1). The method we established to determine an activity signature for key enzymes of carbohydrate metabolism performed well with various monocot and dicot model and crop plants[11] and proved to be useful for physiological phenotyping,[8] we encountered a number of difficulties in applying the “universal” extraction protocol to grapevine, probably due to interfering phenolic and anthocyanin compounds in the extracts.[12,13]

Results

Conclusion