Osmo-Regulation Of Calcium-Permeable Channels In The Plasma Membrane Of Vicia Faba Guard Cells And Cloning And Characterization Of TPC1 Gene Of Arabidopsis Thaliana

Cytosolic free Ca²⁺ ([Ca²⁺]_cyt) is a ubiquitous second messenger in plant cells. A number of external and internal stimuli trigger sustained or transient elevations of [Ca²⁺]_cyt and consequntly evoke downstream stimulus-specific responses. Generation of [Ca²⁺]_cyt signals depended on Ca²⁺ influx into the cytosol from the extracellular and intracellular Ca²⁺ stores through Ca²⁺ channels. Thus, Ca²⁺ channels in the plasma membrane play an essential role in Ca²⁺-mediated signaling process. However, the direct recordings and analysis of channel-facilitated Ca²⁺ influx are very limited and upstream regulation mechanisms of the Ca²⁺-channels in plant cells remain unclear so far.The first part of this dissertation work was focused on identification and functional characterization of the Ca²⁺-permeable channels in the plasma membrane of Vicia faba guard cells by application of the patch-clamp techniques. Under the whole-cell and single-channel configurations, inward Ba²⁺-currents were observed and identified. At the single-channel level, the stretch-activated (SA) inward channels were identified and characterized. The P_o (total open probability) of the SA channels depended on the stretch across the plasma membrane patches, and the current amplitude of the SA channels was voltage-dependent. The voltage-dependent or voltage-activated (VA) and stretch-independent inward channels were also recorded and analyzed. The analysis based on the inhibitory effects of Gd³⁺ and the reversal potentials demonstrated that both types of channels were Ca²⁺-permeable channels, which was further confirmed with the experiments using Ca²⁺ instead of Ba²⁺ as current carrying ions. The further experiments demonstrated that actin polymerization and depolymerization processes regulate the activities of both types of channels. Depolymerization of the actin filaments by application of cytochalasin D activated the channels (increased P_o of the channels), and this activation was overcome when phalloidin was applied. In addition, the whole-cell Ca²⁺-currents were significantly regulated by changes in osmo-gradients across the plasma membrane. The inward Ca²⁺ currents were activated under external hypo-osmotic conditions, while the inward Ca²⁺ currents were inhibited under external hyper-osmotic conditions. More importantly, the osmo-regulation of the inward Ca²⁺-permeable channels is mediated by actin depolymerization and polymerization process. The further results support the notion that osmo-regulation of whole-cell Ca²⁺ currents were mainly contributed by the SA-type Ca²⁺-permeable channels.The second part of this work was cloning and preliminary functional characterization of AtTPC1, a putative Ca²⁺ channel in Arabidopsis thaliana (AGI code At4g03560). AtTPCl has high homology with the Two Pore Channel in rat and its structure is very similar to the half of the structure of α-subunit of voltage-activated Ca²⁺ channels in animal cells. When hetero-expressed in yeast mutant cchl (a Ca24 uptake deficient mytant), AtTPCl rescued the Ca²⁺ uptake activity of cch1. Because CCH1 encodes a homolog of the a-subunit of anima L-type Ca²⁺ channels, this result indicated that hetero-expressed AtTPCl is functional as a Ca²⁺ channel to facilitate Ca²⁺ influx.GUS staining results showed that AtTPCl is expressed in most of the organs of Arabidopsis such as roots, leaves and flowers. The result of transient expression of AtTPCl-GFP showed that AtTPCl may be located on the plasma membrane.In summary, this dissertation work investigated the Ca2+-permeable channels in the plasma membrane of Vicia faba guard cells. Two distinct types of Ca2+-permeable channels are identified and characterized. The SA-type Ca2+-permeable channels are osmo-regulated and the osmo-regulation the channels is mediated by actin polymerization/depolymerization process. A putative Ca2+-channel gene AtTPCl in Arabidopsis was cloned and function of AtTPCl for facilitating Ca2+-influx was demonstrated using yeast mutant.In addition, in collaboration with other members in the lab, regulation of the inward K+ channels in Arabidopsis pollen plasma membranes by Ca2+/CDPK signaling cascade was investigated. The results showed that the inward K+ currents were insensitive to [Ca2+]cyt in premature pollen protoplasts, whereas elevation of [Ca2+]cyt inhibited the inward K+ currents in mature pollen and pollen tube protoplasts. By analyzing the independent CDPK knockout mutants respectively, we found that the differences in [Ca2+]cyl sensitivity may result from differential expression of CDPK.