Current
Research activities of the Laboratory focus on studies of the molecular basis of regulatory processes in plants. "Regulation of growth and development in plants: the role of reversible cytokinin conjugation" represents the major current research programme of the Laboratory. The programme consists of the following projects:
1. Structure - function relationships of the cytokinin-glucoside-specific b-glucosidase Zm- p60.1.
Knowledge of the structure-function relationships in b-glucosidase Zm-p60.1 is necessary for understanding its specificity and catalytic activity, and for rational engineering of its catalytic properties. Our previous experiments indicated an important role for cysteine residues in catalytic activity, and structural stability of Zm-p60.1. A combination of molecular and biochemical techniques is employed to assign function to individual cysteine residues in Zm-p60.1. Site-directed mutagenesis is used to replace cysteine residues by specific amino acids. Analysis of changed catalytic and structural properties of the enzyme will allow us to draw
conclusions about the role of individual cysteine residues in activity and architecture of the enzyme. In mutants which lose activity we will search, by random mutagenesis, for second-site mutations restoring the enzyme activity. Thereby we will gain novel information on plasticity of the enzyme structure.
2. A cytokinin-glucoside-specific b-glucosidase Zm-p60.1 as a tool to investigate the subcellular compartmentation of cytokinin conjugation.
The subcellular compartmentation of cytokinin conjugation and release can play an important role in control mechanisms regulating reversible conversion between free and conjugated cytokinins. To get an insight into the control mechanisms, we are combining
(i) classical techniques of subcellular fractionation followed by cytokinin metabolite analysis using HPLC-MS,
(ii) redirection of b-glucosidase Zm-p60.1, which is specific for cytokinin-glucosides, into individual subcellular compartments followed by analysis of changes in cytokinin metabolites in individual subcellular compartment as well as in whole organs. We expect to gain both new knowledge of the role of subcellular compartmentation in regulation of reversible cytokinin glucosylation, and a unique tool to manipulate the subcellular distribution of free and conjugated cytokinins.
3. Analysis of cell autonomous pollen sterility in pollen accumulating Zm-p60.1, a
b- glucosidase specific for cytokinin glucosides
Knowledge of the molecular basis of pollen differentiation from sporophytic tissue and the role of plant hormones in this process is rather limited. Insight into pollen development may be gained by analysis of pollen mutants. Recently we have identified a transgenic tobacco plant exhibiting cell autonomous pollen sterility (CAPS) associated with accumulation of
Zm-p60.1, a maize b-glucosidase specific for cytokinin glucosides, in pollen. Characterisation of the CAPS using combined biochemical and genetic engineering approaches will allow a detailed dissection of the pollen defect, and provide insights into the role of cytokinins in this process. Knowledge gained from this project may form the basis for engineering a novel regulatable male sterility in important crops.
We use functional analysis of Arabidopsis thaliana genome to identify genes that represent key players in metabolic and regulatory processes of plant growth. The long-term goal of this work is to contribute towards our knowledge of these processes in the areas of both plant physiology and plant genetics. Our results will be used to evaluate the potential of using ectopic gene expression in plants to modify the properties and growth of plants in useful and well-defined ways.
Research is carried out by staff members, PhD. students, postdoctoral fellows, and partially also by diploma students.
Part of the personnel (research assistants) and instrumentation form the basis of a newly formed Central Laboratory of Advanced Methods of Molecular Biology at the Masaryk University.
