Several classes of receptor-like protein kinases have been isolated in plants. However, the function of these genes is not known except for the S-locus receptor protein kinase (SRK) gene of Brassica which, together with a second S-locus gene that encodes a secreted glycoprotein highly related to the extracellular domain of the receptor, is involved in pollen-stigma signaling in the self-incompatibility response. To begin to address the role of receptor protein kinases during plant development, June Nasrallah's laboratory have focused on three vegetatively expressed Arabidopsis genes, the ARK genes, which exhibit sequence similarity to the Brassica SRK gene. Transcriptional and immunological analyses have indicated that ARK genes encode two gene products, the full-length receptor and a secreted form of the extracellular domain of the receptor. This result suggests that a soluble cell-wall localized receptor is involved in the mechanism of signaling by this class of receptor kinases in vegetative cells as in pollen-stigma interactions. A role for these genes in developmental processes is suggested by analysis of ARK-overexpressing transgenic plants which are stunted, sterile, and exhibit drastically reduced cell size.
Recent in vitro evidence indicates that the chloroplast homologue of the 54 kd subunit of signal recognition particle (54CP) is required for targeting LHCP to thylakoid membranes. To investigate whether 54CP plays a fundamental role in intrachloroplast protein targeting, the Hoffman laboratory is using both antisense and dominant negative approaches in transgenic Arabidopsis. Plants transformed with either type of construct contain reduced levels of 54CP protein. These plants have a "slow to green" phenotype, i.e. the leaves are initially pale yellow but eventually green to nearly wild type levels. Targeting efficiency of chloroplast membrane proteins is currently being examined in the transgenic plants.
Kevin Pyke (University of London, UK) talked about a very interesting Arabidopsis mutant, ARC6, that is involved in proplastid and chloroplast division. The arc6 mutant of Arabidopsis was isolated from a T-DNA mutagenised seedling population by microscope based screening. Mesophyll cells from leaves of arc6 plants contain only 1 to 3 chloroplasts compared to over 80 in wild type Arabidopsis leaf cells and this low chloroplast number is maintained throughout mesophyll development and does not increase during cell expansion. The arc6 mutant shows normal Mendelian inheritance and maps to the bottom of chromosome 5. Growth and development of arc6 plants are similar to wild type, with the exception that leaves of arc6 appear twisted and curled which may result from a slight alteration in mesophyll cell shape. In compensation for the severely reduced chloroplast number in arc6 mesophyll cells, the individual chloroplasts are very large, with up to a 20 fold increase in average plan area compared to wild type chloroplasts. Consequently the total amount of chloroplast cover in arc6 and wild type mesophyll cells is similar, although arc6 chloroplasts show a 50% reduction in chloroplast thickness. Despite this radical change in chloroplast phenotype, the internal arrangement of thylakoid membranes is largely unaffected by the mutation, although the chloroplast envelope shows considerable undulations and irregularities in outline when viewed in transverse section. Ultrastructural analysis of proplastids in cells of the shoot apex and differentiating chloroplasts in leaf primordial cells show that arc6 has a major effect on plastid division at the earliest stages of cell development. Cells of the shoot apical meristem contain very few, enlarged proplastids exhibiting a complex three dimensional morphology. All arc6 apical meristem cells examined contain proplastids implying that arc6 proplastids must be capable of limited division to ensure continuity within cell lineages.
Analysis of root proplastids also shows an effect of the mutation similar to that in shoot proplastids, implying that the arc6 gene plays a role in proplastid division throughout the plant. In particular, the specialised starch-filled plastids in the columella cells, the statoliths, are also greatly increased in size and are less abundant. Another significant feature of the arc6 phenotype is an alteration in the distribution of chloroplasts in stomatal guard cells. In arc6 stomata, most guard cells contain only a single large plastid, compared to 4-5 in wild type. In 30% of stomata, one of the guard cell pair completely lacks a chloroplast. Occasionally stomata are observed in which chloroplasts are absent in both guard cells. Our characterisation of the arc6 mutant has indicated that ARC6 is the first gene in higher plants shown to play a role in the early development and division of proplastids in mitotic cells.
Research in Julian Schroeder's lab is focused on the roles of ion channels in guard cell signal transduction and in plant nutrition (1,2). Inward rectifying potassium channels allow the inward movement of potassium into cells. These ion channels are important in the uptake and movement of potassium throughout plants and in the control of cell movements, as in stomatal opening. At least three potassium channels have now been cloned from Arabidopsis. All three have structural features in common with the Shaker superfamily of potassium channels such as a predicted six membrane spanning domains and homology in the S4 (voltage sensing domain) and in the pore region. KAT1 was clone in Rick Gaber's lab, AKT1 was cloned in Herve Sentenac's lab, and ATK2 was cloned by Yongwei Cao in Nigel Crawford's and Julian Schroeder's labs. AKT2 is expressed in leaves while AKT1 is expressed in roots as shown in Northern blots. It is difficult to get a good Northern blot for KAT1 but work in Mike Sussman's and Rick Gaber's labs has demonstrated that KAT1 message is expressed in guard cells.
The pore region of potassium channels (H5 domain) contains amino acid residues which are important for blocker binding. Audrey Ichida in Julian Schroeder's lab has made point mutations at these sites to produce mutant channels with altered blocker sensitivity in an effort to study the physiological role of these guard cell potassium channels. Audrey has identified mutants that, when expressed in Xenopus oocytes, are less sensitive to block by external cesium and triethanol ammonium (TEA). These results provide valuable information concerning the orientation of KAT1 ion channels in the membrane. We are hoping that by introducing these blocker insensitive mutants into Arabidopsis we will have a dominant phenotype to study the roles of KAT1 channels in stomatal movements.
Yongwei Cao in Nigel Crawford's and Julian Schroeder's labs has made a series of chimeric ion channels using KAT1 and the Drosophila potassium channel EAG which is an outward potassium channel (3). The chimera containing the N-terminus of KAT1 through the S4 domain and the EAG pore domain encodes an inward ion channel when expressed in oocytes. These data indicate that the orientaion of KAT1 in the membrane is similar to other Shaker channels (which are outward conducting potassium channels while KAT1 is inward). Therefore differences in the gating mechanism rather than in the overall orientation in the membrane controls the direction of ion flux through these potassium channels. The mechanism of KAT1 channel gating is an important area for future molecular studies.
Stomatal closing occurs due to a decrease in guard cell turgor caused by a efflux of potassium and anions from guard cells. Analogous to the requirement for proton pumping to drive potassium influx into guard cells during stomatal opening, plasma membrane depolarization is required to drive potassium efflux during stomatal closing. Slow anion channels in the plasma membrane of guard cells are a central controller of stomatal closing. The activation of slow anion channels allows anion efflux, a requirement for stomatal closing, and also depolarizes the plasma membrane which activates outward potassium channels and drives potassium efflux.
When slow anion channels are blocked by NPPB, a benzoic acid derivative, or 9-anthacene carboxylate (9-AC), abscisic acid (ABA)-induced stomatal closing is prevented. These blockers also prevent the inhibition of stomatal opening by ABA. Work in our lab is focused on the physiological regulation of slow anion channels during ABA-induced stomatal closing. Early work by Julian Schroeder demonstrated that increases in cytoplasmic calcium activates slow anion channels and indicated that this regulation was indirect.
In patch clamp experiments Christian Schmidt in Julian Schroeder's lab found that cytoplasmic ATP was required to maintain slow anion channel activity and that ATP could not be replaced by non-hydrolyzable analogs (4). These data suggested that protein phosphorylation was involved in the regulation of slow anion channel activity. In further experiments Christian Schmidt found that K252A, a kinase inhibitor, inhibited slow anion channel activity at the whole cell and single channel level. Consistent with these results, okadeic acid, a phosphatase inhibitor, was found to maintain slow anion channel activity in the absence of cytoplasmic ATP. These results indicate that ABA either inhibits a protein phosphatase or activates a protein kinase and that slow anion channel (or associated regulatory protein) phosphorylation is required for activation of slow anion channels.
This hypothesis is supported by experiments in which the effects of kinase and phosphatase inhibitors was tested on ABA-induced stomatal closing conducted by Yuh-jen Liao and John Esser in Julian Schroeder's lab. K252A completely blocked ABA-induced stomatal closing while okadeic acid enhanced ABA-induced stomatal closing. The regulatory effects of ABA on slow anion channel activity in guard cells are currently under investigation in our lab and should further reveal the mechanism of ABA signal transduction.
2. Ward, J. M., Pei, Z.-M. and Schroeder, J. I. (1995) Roles of ion channels in initiation of signal transduction in higher plants. Plant Cell 7,833-844.
3. Cao, Y., Crawford, N. M., and Schroeder, J. I. (1995) Amino terminus and the first four membrane-spanning segments of the Arabidopsis K+ channel KAT1 confer inward-rectification property of plant-animal chimeric channels. J. Biol. Chem. (in press).
4. Schmidt, C., Schelle, I., Liao, Y.-J. and Schroeder, J. I. (1995) Activation and strong down-regulation of slow anion channels in guard cells by phosphorylation and dephosphorylation events. Proc. Natl. Acad. Sci. (in press).