Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans

Abstract

The terminal process of xylogenesis, autolysis, is essential for the formulation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H2O2 challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.

Flow cytometric analysis of tracheary element differentiation in Zinnia elegans cells

“…Protease synthesis and proteosome activity have been associated with secondary cell wall development (16)(17)(18), and there is evidence for budding or vesiculation of the nucleus and the cytoplasm in Zinnia and pine TEs (19,20); vesicular removal of cell contents is a prime feature of mammalian cell apoptosis. Although there is no information on specific chromatin changes in the process (2), some elements of DNA repair also occur (1).…”

Delayed expression of SAGs correlates with longevity in CMS wheat plants compared to its fertile plants

“…the reaction was stopped immediately without further incubation and without substrate. One unit of proteolytic activity in 1 cm cuvette was defined as an increment of 0.01 in A340 in 1 h. For inhibitor studies, the reaction mixture containing 50 μl of the crude extract was preincubated for 10 min with one of the following inhibitors −3.8 μl of 2 mM PMSF, 18.8 μl of 10 mM EDTA, 9.4 μl of 25 μM DL-norleucine and 3.8 μl of 10 mM iodoacetamide for serine-, metallo-, aspartate-, and cysteine proteases, respectively (Ye and Varner 1996). The remaining procedure was the same as for the endopeptidase activity.…”

Programmed cell death during vascular system formation

“…In addition, a serine protease of 145 kD was detected specifically in differentiating TEs on substrate-impregnated gels (Beers and Freeman, 1997). An approximately 60 kD serine protease was also reported to appear preferentially in TE-inducing culture (Ye and Varner, 1996;Beers and Freeman, 1997). The activity of the 60 kD protease is higher above pH 7, which is unlike the pH optimum of the autolysis-specific cysteine protease, suggesting that at least two compartments with different pHs are involved in the autolysis.…”

“…Organellar degeneration is coupled with a high activity of proteases (Minami and Fukuda, 1995;Ye and Varner, 1996;Beers and Freeman, 1997). Single-cell protease assay has indicated that differentiating TEs themselves have high proteolytic activities, which suggests cellautonomous protein degradation in TEs (Beers and Freeman, 1997).…”