Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum.

A tapetum/microspore-specific pathogenesis-related (PR) 10 gene was previously identified in lily (Lilium longiflorum Thunb.) anthers. In situ hybridization and RNA blot analysis indicated that the lily PR10 genes are expressed specifically and differentially in the tapetum of the anther wall and in microspores during anther development. The accumulation of PR10 transcripts was exogenously induced by gibberellic acid (GA) and was suppressed by ethylene. Studies using inhibitors of GA and ethylene revealed that the lily PR10 is modulated by an antagonistic interaction between GA and ethylene. The treatment of norbornadien, an ethylene inhibitor, caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole, an inhibitor of GA biosynthesis, arrested tapetal development to a status close to that of control. The expression of the lily PR10g promoter in transgenic Arabidopsis was determined using the ß-glucuronidase (GUS) reporter gene indicated that the decisive fragment required for anther specificity is located -1183 bp to -880 bp upstream of the transcription start site. The PR10gPro::barnase transgenic lines exhibited complete male sterility because of the disruption of the tapetum and the deformation of microspore/pollen. The anther specificity of lily PR10 highlights the importance of the tapetum/microspore-specific PR10g promoter for future biotechnological and agricultural applications.

Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142, D144 and E146 in Manduca sexta (tobacco hornworm) chitinase.

Chitinases (EC 3.2.1.14) are glycosyl hydrolases that catalyze the hydrolysis of beta-(1, 4)-glycosidic bonds in chitin, the major structural polysaccharide present in the cuticle and gut peritrophic matrix of insects. Two conserved regions have been identified from amino acid sequence comparisons of family 18 glycosyl hydrolases, which includes Manduca sexta (tobacco hornworm) chitinase as a member. The second of these regions in M. sexta chitinase contains three very highly conserved acidic amino acid residues, D142, D144 and E146, that are probably active site residues. In this study the functional roles of these three residues were investigated using site-directed mutagenesis for their substitutions to other amino acids. Six mutant proteins, D142E, D142N, D144E, D144N, E146D and E146Q, as well as the wild-type enzyme, were produced using a baculovirus-insect cell line expression system. The proteins were purified by anion-exchange chromatography, after which their physical, kinetic and substrate binding properties were determined. Circular dichroism spectra of the mutant proteins were similar to that of the wild-type protein, indicating that the presence of mutations did not change the overall secondary structures. E146 was required for enzymatic activity because mutants E146Q and E146D were devoid of activity. D144E retained most of the enzymatic activity, but D144N lost nearly 90%. There was a shift in the pH optimum from alkaline pH to acidic pH for mutants D142N and D144E with minimal losses of activity relative to the wild-type enzyme. The pH-activity profile for the D142E mutation resembled that of the wild-type enzyme except activity in the neutral and acidic range was lower. All of the mutant proteins bound to chitin. Therefore, none of these acidic residues was essential for substrate binding. The results indicate that E146 probably functions as an acid/base catalyst in the hydrolytic mechanism, as do homologous residues in other glycosyl hydrolases. D144 apparently functions as an electrostatic stabilizer of the positively charged transition state, whereas D142 probably influences the pKa values of D144 and E146.