Introduction  Plants have pores, stomata, on their leaf surfaces that allow carbon dioxide (CO2) in for photosynthesis and through which water evaporates. The specific cells that border and define these pores are guard cells (Fig 1). Guard cells literally “guard” the size of the pore by alternately swelling, which opens the pore, or shrinking, which closes the pore. Plants must respond to a variety of environmental cues and regulate their stomata accordingly so that enough CO2 gets in, but not so much water escapes that the plant dries out (Assmann and Wang, 2001). This balance between opening and closure of stomata is essential in determining a plant’s survival and how much it will grow and yield..
The swelling and shrinking of guard cells occurs through the coordinated movement of water and ions across the guard cell membrane. Ions move through regulated proteinaceous pores in the membrane called ion channels. The activity of ion channels is regulated by many signals and proteins. These include environmental signals such as ozone (O3), plant hormones such as abscisic acid (ABA), calcium ion levels (Ca2+cyt), GTP-binding proteins , kinase and phosphatase enzymes. Our laboratory studies guard cell regulation using many techniques. We can measure the amount of CO2 taken up and water lost across a leaf with the gas exchange system. We record the ion currents across the cell membrane with the patch-clamp technique. The Ca2+ ion concentration is measured in an individual cell using special dyes and confocal microscopy. The activity and identity of kinases and other proteins in guard cells is detected by biochemical assays and mass spectrometry. Molecular genetic techniques are used to clone and study the regulatory proteins and ion channels of guard cells. The main plant species that we use in our laboratory is the model plant Arabidopsis thaliana (Pandey et al., 2002). Some projects also utilize fava bean (Vicia faba).
ABA Signaling in Guard Cells Several processes are involved in controlling stomatal apertures (Fig 2). Among them, ion channel regulation is one of the key mechanisms by which ABA controls stomatal apertures. Inward potassium channels in the plasma membrane mediate K+ uptake driving stomatal opening, while slow anion channels and outward potassium channels mediate solute loss driving stomatal closure. So far, a great number of components have been revealed to be involved in ABA signaling in the guard cell. Cytosolic calcium concentration appears to be one of the central regulators. Our lab works on most of the pathways illustrated in Figure 2, especially on aspects involving G protein, AAPK, AKIP1, S1P, calcium, pH, PLC, PLD, and ROS, all indicated in red fonts.
Ozone Currently we are investigating how ozone exerts these effects. Ozone may directly oxidize the channel proteins. Using site-directed mutagenesis we will alter the K+ channel by replacing certain amino acids susceptible to oxidation with less sensitive ones. The resulting new proteins will be expressed in an insect-derived cell line and studied using the patch-clamp technique. These experiments will give us a more detailed picture of how the Kin channels work on a molecular basis and hopefully result in a channel with altered ozone-sensitivity. Another possibility is that ozone triggers a signaling cascade. Cytosolic calcium is known as an important intracellular messenger and increased levels of Ca2+cyt will inhibit the Kin channel. Therefore we will measure the cytosolic Ca2+ in guard cells in response to ozone. In these experiments the fluorescent Ca2+ indicator Fura-2 will be loaded into the cytosolic portion of guard cells (Fig 3).
Protein Kinases The ABA-activated protein kinase (AAPK) is a guard cell-specific protein kinase from fava bean (Vicia faba) (Li and Assmann, 1996). AAPK is rapidly activated by physiological levels of the plant hormone ABA. We have cloned the AAPK cDNA and demonstrated that AAPK modulates ABA-stimulated stomatal closure through regulating the activity of plasma membrane anion channels. When a dominant negative (non-functional) form of the kinase AAPK(K43A) is expressed in guard cells, ABA-induced stomatal closure is abolished (Fig 7). These results indicate that AAPK is a key component in stomatal regulation by ABA (Li et al., 2000). The Arabidopsis ortholog of AAPK, OST1 has recently been identified, and we are studying its function.
Guard Cell Nuclear Signaling
References Lee, Y.J. and Assmann, S.M. (1999) Arabidopsis thaliana 'extra-large GTP-binding protein' (AtXLG1): a new class of G- protein. Plant Mol. Biol. 40: 55-64. Li, J., Wang, X-Q, Watson, M.B. and Assmann, S.M. (2000) Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. Science 287: 300-303. Torsethaugen, G., Pell, E.J. and Assmann, S.M. (1999) Ozone inhibits guard cell K+ channels implicated in stomatal opening. Proc. Natl. Acad. Sci. USA 96:13577-13582. Wang, X-Q, Ullah, H., Jones, A.M. and Assmann, S.M. (2001) G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science 292: 2070-2072. Jacob, T., Ritchie, S, Gilroy, S. and Assmann, S.M. (1999) Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity. Proc. Natl. Acad. Sci. USA 96:12192-12197. Pandey, S, Wang, X-Q, Coursol, S.A. and Assmann, S.M (2002) Preparation and applications of Arabidopsis thaliana guard cell protoplasts. New Phytologist 153: 517-526. Assmann, S.M. and Wang, X-Q. (2001) From milliseconds to millions of years: guard cells and environmental responses. Curr. Opin. Plant Biol. 4:421-428. Li, J., Kinoshita, T, Pandey, S., Ng, C.K.-Y., Gygi, S.P, Shimazaki, K. and Assmann, S.M. (2002) Nature 418: 793-797. |
| Selected Recent Publications |
Ozone - one of the major air pollutants - causes agricultural losses of an estimated 3 billion dollars per year in the US alone. Elevated O3 levels typically occur when temperature and light intensity are high; conditions that already impose a great deal of stress on plants. Our lab has shown that ozone blocks stomatal opening by inhibiting the inwardly rectifying guard cell potassium channel (Torsethaugen et al., 1999).
Reversible protein phosphorylation is a major means of cellular regulation. Protein kinases are the enzymes responsible for phosphorylation of proteins by moving phosphate groups from ATP to proteins. The phosphate groups on the protein can be removed (dephosphorylation) by another class of enzymes called phosphatases. Therefore, protein phosphorylation and dephosphorylation work as molecular switches to regulate protein activity and thus cell functions.
We have identified an AAPK-interacting protein (AKIP1) with sequence homology to heterogeneous nuclear RNA binding proteins. Phosphorylation of AKIP1 by ABA-activated AAPK is required for AKIP1 interaction with mRNA encoding dehydrin, a protein implicated in cell protection under stress conditions (Li et al., 2002). AAPK and AKIP1 are present in the guard cell nucleus and in vivo ABA treatment enhances AKIP1 partitioning into subnuclear foci reminiscent of nuclear speckles. These results suggest that phosphorylation-regulated RNA target discrimination by hnRNP RNA-binding proteins may be a general eukaryotic phenomenon, and implicate ABA in regulation of protein dynamics during rapid subnuclear reorganization in guard cells. There are 3 AKIP1 homologs in Arabidopsis. Future work involves identifying the RNA substrates of these AKIPs, elucidating the various components of the signaling pathway regulating rapid subnuclear reorganization of AKIPs, and determining the phenotype of akip null plants.