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theses

Stomatal guard cells (GC) mainly exist in the epidermis of plant leaves and stems. They mediate the gas exchange between plants and the environment, thus are important for plant growth and development. In Arabidopsis, stomatal development and patterning are regulated by a linear signal pathway, mainly comprised of a set of peptide ligands, a suite of receptor-like protein/kinases, a mitogen-activated protein kinase (MAPK) cascade, and a series of transcriptional factors. Plant hormones and environmental factors regulate stomatal development through crosstalk with multiple components in this pathway. The MAPK cascade receives upstream signals from the cell surface to transduce to downstream intracellular factors, thus plays a central role in the regulation of stomatal development. The major goal of my Ph.D. thesis is to understand the signaling events occurring upstream and downstream of the MAPK cascade and how these signaling events are integrated to regulate stomatal development and patterning in Arabidopsis.
In the first chapter of my thesis, I identify the protein phosphatase 2A (PP2A) as new regulators in stomatal development and determine that they function antagonistically with MAPK3/6 and other kinases on the regulation of a master transcription factor in Arabidopsis. The PP2A phosphatase is a heterotrimeric complex, which is composed of three subunits (A, B, and C). In my studies, loss-of-function mutations in the genes encoding the subunits A and C both lead to suppressed stomatal production, suggesting the positive role of PP2A. The genetic analysis places PP2A function downstream of the MAPK cascade but upstream of the key transcription factor SPEECHLESS (SPCH). Pharmacological treatment supports the positive role of PP2A on SPCH protein stability. I further show that SPCH directly binds to PP2A-A subunits in vitro. Consistent with the hypothesis that PP2A dephosphorylates and stabilizes SPCH, in plants, the protein abundance of phospho-deficient SPCH appears less sensitive to the PP2A activities. Previously, multiple kinases, including MAPK3/6, BIN2, and CDKA;1, were identified to regulate SPCH protein phosphorylation that is related to protein stability and function, but it remained unknown which phosphatase combats these processes. Thus, my research identifies PP2A as the missing phosphatase that functions antagonistically with the known kinases to balance the phosphorylation status of the master transcription factor SPCH in Arabidopsis stomatal development. This work is currently under view at PNAS, on which I am first-authored.
In the second chapter of my thesis, the research investigates the D6 protein kinase (D6PK) for their functional connection with cell polarity in the regulation of stomatal development and patterning. This work was initiated from the collaboration with Dr. Claus Schwechheimer (TUM, Germany) and conducted by myself and the collaborative effort of Dr. Kezhen Yang (visiting scholar in our lab). We provide genetic evidence that D6PK regulates stomatal development and patterning by participating in the BASL-YDA polarity complex in Arabidopsis stomatal lineage cells. BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) is a plant-specific protein, which has a unique polarized localization at the cell cortex. The loss-of-function mutant of BASL shows clustered stomata and abnormal cell division pattern. A recent study indicated phosphorylated BASL recruits the MAPKKK YODA (YDA) and MPK3/6 to the cell cortex to active the MAPK cassette to inhibit stomatal cell fate. However, what other components in the polarity complex and how the BASL-YDA module is regulated by other genetic components remain largely unknown. In this study, we report the D6PK family as a new regulator in stomatal development and patterning. I show that the D6PK family loss-of-function mutants and the overexpression of D6PK both generate stomatal defects in Arabidopsis. In the stomatal lineage cells, we found that D6PK associates with BASL-YDA polarity complex at the cell cortex, and D6PK polarity is dependent on the presence of BASL. Further, our genetic analysis suggests D6PK functions upstream of the BASL-YDA complex, and D6PK possibly regulates BASL protein stability. Thus, our data suggest the D6PK protein family regulates stomatal development and patterning by participating and regulating the BASL-YDA complex.
The last chapter of my thesis is achieved by my collaborative effort with Dr. Xueyi Xue (Post-doctoral fellow in the lab). Here, we identify and characterize a new family of MAPK substrates for their function in Arabidopsis stomatal development. The MAPK cascades play critical roles in many aspects in plant growth, development and stress responses. Stomatal development and patterning in Arabidopsis are tightly regulated by a canonical MAPK signaling cascade composed of the MAPKK kinase YODA, MAPK Kinase 4 and 5 (MKK4/5) and MAPK 3 and 6 (MPK3/6). Three MAPK SUBSTRATES IN THE STOMATAL LINEAGE (MASS) proteins were isolated from a large-scale screen of substrate peptides modified by Arabidopsis MPK3 and MPK6. The loss-of-function and overexpression phenotypes suggest MASS functions as a positive regulator of stomatal production in Arabidopsis. Furthermore, we provide experimental evidence supporting that MAPK-mediated phosphorylation is important for the subcellular localization and function of MASS, in turn, the MASS proteins interact with YDA, likely to suppress YDA’s function at the plasma membrane. Thus, the functional connection between the MASS family and the YDA MAPK cascade represent a negative feedback loop, providing a mechanism to diverge the MAPK functional output in the regulation of stomatal development. The manuscript was submitted to PLOS Genetics, on which I am a co-first author.

ETD_10296

Ph.D.

Includes bibliographical references

doi:10.7282/t3-m1ft-r605

ETD doctoral

The author owns the copyright to this work.