Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)



Committee Chair

W. Andy Tao

Committee Member 1

Chun-Ju Chang

Committee Member 2

Mark Hall

Committee Member 3

Jian-Kang Zhu


Protein phosphorylation is one of the important signaling mechanisms in plants which transduces environmental stimuli such as salinity, microbes, and hormones into intracellular signals and activates plant defense mechanisms. Thus, understanding the correlation between environmental stresses and alteration of plant phosphorylation requires system-wide phosphoproteomic analysis, which includes identification of kinase-substrate complexes and measurement of phosphorylation-mediated signaling changes. However, identification and quantification of plant phosphoproteome remains challenging due to the highly dynamic nature of plant proteome, interferences of cell wall, pigments, and secondary metabolites. Recently, mass spectrometry (MS) has been integrated with phosphopeptide enrichment approaches for identifying thousands of phosphorylation sites and for quantifying phosphoprotein stoichiometry. Although MS-based phosphoproteomics has revealed the global phosphorylation changes related to different physiological states of plants, many kinase-substrate networks involved in essential signaling pathways, such as the ABA-induced SNF-1-related protein kinase 2 (SnRK2) pathway and the mitogen-activated protein kinases (MAPKs) cascades, are still not completely understood. This dissertation discusses strategies for improving plant sample preparation and for identifying the direct substrates of the plant kinases. Chapter one highlights the low phosphopeptide identification rate by mass spectrometry. Chapter two details the development of a sample preparation protocol for the plant phosphoproteome analysis, and the application of the protocol for the study of tomato cold-induced phosphoproteomic changes. Chapter three shows the development of a novel approach for identification of the direct substrates of the plant kinases, whose activation regulates the signaling transductions of plant stress defense mechanisms.