Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular and Cellular Biology

Date of Defense

12-16-2013

Graduate Advisor

Xuemin Wang, PhD

Committee

Dr. Xuemin Wang

Dr. Bethany Zolman

Dr. Mark Running

Dr. Wendy Olivas

Abstract

The Arabidopsis genome has 12 phospholipase D (PLD) genes that are classified into six types, PLDα, PLDβ, PLDγ, PLDδ, PLDε, and PLDζ, based on sequence similarities, domain structures, and biochemical properties. Phosphatidic acids (PA) produced by PLDs have been identified as important lipid signaling molecules in cell growth, development, and stress responses. This study was undertaken to determine the role of PLD and PA in plant response to abscisic acid (ABA) and reactive oxygen species (ROS). The lipid mediator PA was found to interact with sphingosine kinases (SPHKs) in Arabidopsis. Two unique SPHK cDNAs were cloned and expressed. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs). PA binds to and stimulates both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. SPHK-deficient and PLDα1-deficient mutants were employed to determine the cellular and physiological functions of the PA-SPHK interaction in plants. Compared to wild-type (WT) plants, sphk and pldα1 mutants all displayed decreased sensitivity to ABA-promoted stomatal closure. The data indicate that SPHK and PLDα1 act together in ABA response and that SPHK and phyto-S1P act upstream of PLDα1 and PA in mediating the ABA response. On the other hand, PA is involved in the activation of SPHK, forming a positive feedback loop in signaling plant response to ABA. In addition, another PLD, PLDδ, was found to be involved in the ROS and ABA signaling pathways. PLDδ functions downstream of PLDα1 and H2O2 to mediate the ABA-induced stomatal closure. The study has identified that cytosolic glyceraldehydes-3-phosphate dehydrogenase (GAPC) acts as a molecular link between H2O2 and PLDδ activation. H2O2 inhibited GAPC activity but promoted GAPC-PLDδ interaction and the PLDδ activity. The loss of both GAPCs decreased plant insensitivity to ABA- and H2O2-induced stomatal closure, like PLDδ. In addition, GPAC-deficient plants produced less PA than wild-type in response to ABA and H2O2. These results indicate that GAPCs mediate H2O2-activation of PLDδ in Arabidopsis response to ABA. The interaction of a cytosolic metabolic enzyme GAPCs and a membrane-associated PLDδ transduces ROS signals in plant response to ABA and oxidative stress.

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