Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii.

The NiFe hydrogenase from Azotobacter vinelandii is a membrane-bound alpha beta heterodimer that can oxidize H2 to protons and electrons and thereby provide energy. Genes encoding the alpha and beta subunits, hoxG and hoxK respectively, followed by thirteen contiguous accessory genes potentially involved in H2 oxidation, have been previously sequenced. Mutations in some of these accessory genes give rise to inactive enzyme containing an alpha subunit with decreased electrophoretic mobility. Mass spectral analysis of the subunits demonstrated that the alpha subunit had a molecular weight 1,663 Da less than that predicted from hoxG. Since the N-terminal sequence of the purified alpha subunit matches the sequence predicted from hoxG we suggest this difference is due to removal of the C-terminus of the alpha subunit which may be an important step linked to metal insertion, localization, and formation of active hydrogenase.

The control of root, vegetative shoot and flower morphogenesis in tobacco thin cell-layer explants (TCLs).

Thin cell-layer explants (TCLs) have been proposed as favorable tissues for the study of root, vegetative shoot and flower formation. We tested the effects of pH, light quality, light quantity, and IBA and kinetin concentrations on the morphogenesis of TCLs cultured individually on a liquid medium. Alterations of the amounts of exogenously supplied IBA and kinetin were sufficient to induce the formation of roots, vegetative shoots and flowers on TCLs cultured on otherwise identical media. The type and number of organs formed were sensitive to the intensity of light (55, 75, 100 and 120 muEinsteins m-2 sec-1) under which TCLs were grown. Evidence was obtained that the effects of light on TCL morphogenesis were associated with photochemical degradation of IBA in the medium. Evaluation of the organogenesis that occurred in TCLs cultured on a medium containing a range of IBA and kinetin concentrations showed that the number and type of organs formed, and overall growth, were dependent upon the initial concentrations of auxin and cytokinin. We have developed the TCL culture system into a sensitive and reproducible bioassay for the study of morphogenesis. The advantages of using the TCL morphogenesis bioassay for the identification and study of molecules (e.g. cell wall oligosaccharides) that may regulate morphogenesis are discussed.

Structure and function of plant cell wall polysaccharides.

Studies of the primary structures of polysaccharides of growing plant cell walls have shown that these structures are far more complex than was anticipated just a few years ago. This complexity can best be appreciated by considering xyloglucan, a hemicellulose present in the cell wall of both monocots and dicots, and rhamnogalacturonan II (RG-II) and rhamnogalacturonan I (RG-I), two structurally unrelated pectic polysaccharides. This realization led us to postulate that cell wall polysaccharides have functions beyond determining the size, shape and strength of plants. Some years ago we demonstrated that oligosaccharide fragments of a branched beta-linked glucan of fungal cell walls can elicit the production of phytoalexins (antibiotics) in plants by inducing the formation of the enzymes responsible for synthesis of the phytoalexins. It has now been ascertained and confirmed by synthesis that the elicitor activity resides in a very specific hepta-beta-D-glucoside. The heptaglucoside has been shown to elicit phytoalexins by activating the expression of specific genes, that is, by causing the synthesis of the mRNAs that encode the enzymes that synthesize phytoalexins. In other words, complex carbohydrates can be regulatory molecules. Further experiments established that oligosaccharide fragments of polysaccharides, produced by acid or base hydrolysis or by enzymolysis of primary cell walls of plants, also evoked defence responses in plants. Subsequently, we learned that defined fragments of polysaccharides, released from covalent attachment within plant cell walls, can function as regulators of various physiological processes such as morphogenesis, rate of cell growth and time of flowering and rooting, in addition to activating mechanisms for resisting potential pathogens. Examples of plant oligosaccharides with regulatory properties (called oligosaccharins) will be described.