Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Is Phosphorylated during Seed Development.

Claudia V. Piattoni,Ignacio Dellaferrera,Alberto Á. Iglesias,Danisa M. L. Ferrero,Abelardo Vegetti

doi:10.3389/fpls.2017.00522

Abstract

Cytosolic glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) is involved in a critical energetic step of glycolysis and also has many important functions besides its enzymatic activity. The recombinant wheat NAD-GAPDH was phosphorylated in vitro at Ser205 by a SNF1-Related protein kinase 1 (SnRK1) from wheat heterotrophic (but not from photosynthetic) tissues. The S205D mutant enzyme (mimicking the phosphorylated form) exhibited a significant decrease in activity but similar affinity toward substrates. Immunodetection and activity assays showed that NAD-GAPDH is phosphorylated in vivo, the enzyme depicting different activity, abundance and phosphorylation profiles during development of seeds that mainly accumulate starch (wheat) or lipids (castor oil seed). NAD-GAPDH activity gradually increases along wheat seed development, but protein levels and phosphorylation status exhibited slight changes. Conversely, in castor oil seed, the activity slightly increased and total protein levels do not significantly change in the first half of seed development but both abruptly decreased in the second part of development, when triacylglycerol synthesis and storage begin. Interestingly, phospho-NAD-GAPDH levels reached a maximum when the seed switch their metabolism to mainly support synthesis and accumulation of carbon reserves. After this point the castor oil seed NAD-GAPDH protein levels and activity highly decreased, and the protein stability assays showed that the protein would be degraded by the proteasome. The results presented herein suggest that phosphorylation of NAD-GAPDH during seed development would have impact on the partitioning of triose-phosphate between different metabolic pathways and cell compartments to support the specific carbon, energy and reducing equivalent demands during synthesis of storage products.

Highlights

Cytosolic glyceraldehyde-3-phosphate dehydrogenase (NADGAPDH, EC 1.2.1.12) is a homotetrameric glycolytic enzyme that catalyzes the phosphorylating-coupled oxidation of Ga3P: Ga3P + Pi + NAD+ ←→ 1,3-bisP-glycerate + NADH + H+
We describe that phosphorylation of NAD-GAPDH occurs in vivo with specific profiles of NAD-GAPDH activity, protein level and phosphorylation degree during seed development that, interestingly, were different in seeds accumulating starch or lipids
Almost no phosphorylation of the recombinant enzyme was observed (Figure 1, black columns). These results suggest that the seed protein kinase that was able to phosphorylate NAD-GAPDH was either absent or inactive in the photosynthetic tissues analyzed

Summary

Introduction

Cytosolic glyceraldehyde-3-phosphate dehydrogenase (NADGAPDH, EC 1.2.1.12) is a homotetrameric glycolytic enzyme that catalyzes the phosphorylating-coupled oxidation of Ga3P: Ga3P + Pi + NAD+ ←→ 1,3-bisP-glycerate + NADH + H+. The high energy intermediate 1,3-bisP-glycerate is metabolized by P-glycerate kinase (EC 2.7.2.3, PGK) to produce ATP and 3P-glycerate (3PGA): 1,3-bisP-glycerate + ADP ←→ 3PGA + ATP (Nelson and Cox, 2004). These two glycolytic steps are of relevance in plants. NAD-GAPDH is an abundant protein that in plants was first highly purified and kinetically characterized from pea seed (Duggleby and Dennis, 1974a,b,c). Its catalytic role in glycolysis is based on a highly reactive catalytic cysteine that is often target of oxidative modifications that blocks its enzymatic activity and in turns trigger other moonlighting non-glycolytic roles (Sirover, 2012; Zaffagnini et al, 2013)

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Conclusion