Beyond Glycolysis: GAPDHs Are Multi-functional Enzymes Involved in Regulation of ROS, Autophagy, and Plant Immune Responses

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in energy metabolism with diverse cellular regulatory roles in vertebrates, but few reports have investigated the importance of plant GAPDH isoforms outside of their role in glycolysis. While animals possess one GAPDH isoform, plants possess multiple isoforms. In this study, cell biological and genetic approaches were used to investigate the role of GAPDHs during plant immune responses. Individual Arabidopsis GAPDH knockouts (KO lines) exhibited enhanced disease resistance phenotypes upon inoculation with the bacterial plant pathogen Pseudomonas syringae pv. tomato. KO lines exhibited accelerated programmed cell death and increased electrolyte leakage in response to effector triggered immunity. Furthermore, KO lines displayed increased basal ROS accumulation as visualized using the fluorescent probe H2DCFDA. The gapa1-2 and gapc1 KOs exhibited constitutive autophagy phenotypes in the absence of nutrient starvation. Due to the high sequence conservation between vertebrate and plant cytosolic GAPDH, our experiments focused on cytosolic GAPC1 cellular dynamics using a complemented GAPC1-GFP line. Confocal imaging coupled with an endocytic membrane marker (FM4-64) and endosomal trafficking inhibitors (BFA, Wortmannin) demonstrated cytosolic GAPC1 is localized to the plasma membrane and the endomembrane system, in addition to the cytosol and nucleus. After perception of bacterial flagellin, GAPC1 dynamically responded with a significant increase in size of fluorescent puncta and enhanced nuclear accumulation. Taken together, these results indicate that plant GAPDHs can affect multiple aspects of plant immunity in diverse sub-cellular compartments.

Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types during Infection

“…Infiltration with Pst DC3000 (EV) did not induce an HR in any lines. To quantify macroscopic HR in the CSP lines, we measured ion leakage, a proxy for cell death (Henry et al, 2015). Consistent with the limited expression of RIN4 in different cell types, we found the ion leakage in the CSP lines was reduced with respect to the positive controls Col-0 and r2 after infiltration with Pst DC3000 (avrB) ( Figure 1E).…”

The Coiled-Coil and Nucleotide Binding Domains of BROWN PLANTHOPPER RESISTANCE14 Function in Signaling and Resistance against Planthopper in Rice

“…Images used for quantification (as shown in Figure 4A) were obtained using a Leica (TCS SP8) epifluorescent microscope with a GFP cube. Fluorescence was quantified using ImageJ from a minimum of 25 protoplasts across 10 or more images per genotype (Henry et al, 2015).…”

Hydrogen Sulfide Regulates the Cytosolic/Nuclear Partitioning of Glyceraldehyde-3-Phosphate Dehydrogenase by Enhancing its Nuclear Localization

“…Numerous publications investigating the S-sulfhydration of GAPDH and its regulation have been highlighted in recent years due to the importance of this protein in diverse biological functions, in addition to its classical glycolytic role. These include its participation in mRNA regulation, DNA replication, endocytosis or regulation of apoptosis (Sirover 2011, Henry et al 2015, White and Garcin 2016. Mammalian GAPDH was first described as an S-sulfhydrated protein at Cys150 and this modification results in an increase in enzymatic activity in mice (Mustafa et al 2009).…”