Suppression of reactive oxygen species by glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classical glycolytic enzyme, is involved in cellular energy production and has important housekeeping functions. In this report, we show that a GAPDH from Arabidopsis, GAPDHa, has a novel function involved in H(2)O(2)-mediated cell death in yeast and Arabidopsis protoplasts. GAPDHa was cloned along with other plant genes that suppress Bax-induced cell death in yeast. Flow cytometry analyses with dihydrorhodamine 123 indicated that H(2)O(2) production mediated by Bax expression in yeast cells was greatly reduced when Bax was coexpressed with GAPDHa. In plants, GAPDHa transcript levels were greatly increased by H(2)O(2) treatment. Furthermore, transformation of GAPDHa into Arabidopsis protoplasts strongly suppressed heat shock-induced H(2)O(2) production and cell death. Together, our results indicate that GAPDH controls generation of H(2)O(2) by Bax and heat shock, which in turn suppresses cell death in yeast and plant cells.

NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants.

NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H(2)O(2)-mediated mitogen-activated protein kinase signaling in plants. H(2)O(2) stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking AtNDPK2 had higher levels of ROS than wild type. H(2)O(2) treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H(2)O(2)-activated A. thaliana mitogen-activated protein kinases. In the absence of H(2)O(2) treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H(2)O(2)-mediated MAPK signaling in plants.

Soybean ascorbate peroxidase suppresses Bax-induced apoptosis in yeast by inhibiting oxygen radical generation.

Bax, a mammalian proapoptotic member of the Bcl-2 family, can induce cell death when expressed in yeast or plant cells. To identify plant Bax inhibitors, we cotransformed a soybean cDNA library and the Bax gene into yeast cells and screened for expressed genes that prevented Bax-induced apoptosis. From the Bax-inhibiting genes isolated, ascorbate peroxidase (sAPX) was selected for characterization. The transcription of sAPX in plants was specifically induced by oxidative stress. Moreover, overexpression of sAPX partially suppressed the H(2)O(2)-sensitive phenotype of yeast cytosolic catalase T (Deltactt)- and thermosensitive phenotype of cytochrome c peroxidase (Deltaccp)-deleted mutant cells. Examination of reactive oxygen species (ROS) production using the fluorescence method of dihydrorhodamine 123 oxidation revealed that expression of Bax in yeast cells generated ROS, which was greatly reduced by coexpression with sAPX. Our results collectively suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast.