Pleiotropic and Novel Phenotypes in The \u3cem\u3eDrosophila\u3c/em\u3e Gut Caused by Mutation of \u3cem\u3eDrop-Dead\u3c/em\u3e

Normal gut function is vital for animal survival, and deviations from such function can contribute to malnutrition, inflammation, increased susceptibility to pathogens, diabetes, neurodegenerative diseases, and cancer. In the fruit fly Drosophila melanogaster, mutation of the gene drop-dead (drd) results in defective gut function, as measured by enlargement of the crop and reduced food movement through the gut, and drd mutation also causes the unrelated phenotypes of neurodegeneration, early adult lethality and female sterility. In the current work, adult drd mutant flies are also shown to lack the peritrophic matrix (PM), an extracellular barrier that lines the lumen of the midgut and is found in many insects including flies, mosquitos and termites. The use of a drd-gal4 construct to drive a GFP reporter in late pupae and adults revealed drd expression in the anterior cardia, which is the site of PM synthesis in Drosophila. Moreover, the ability of drd knockdown or rescue with several gal4 drivers to recapitulate or rescue the gut phenotypes (lack of a PM, reduced defecation, and reduced adult survival 10–40 days post-eclosion) was correlated to the level of expression of each driver in the anterior cardia. Surprisingly, however, knocking down drd expression only in adult flies, which has previously been shown not to affect survival, eliminated the PM without reducing defecation rate. These results demonstrate that drd mutant flies have a novel phenotype, the absence of a PM, which is functionally separable from the previously described gut dysfunction observed in these flies. As the first mutant Drosophila strain reported to lack a PM, drd mutants will be a useful tool for studying the synthesis of this structure

Laccase-dependent lignification of secondary cell walls of protoxylem tracheary elements in Arabidopsis thaliana

Lignin is a phenolic polymer that plays important roles in the structural integrity of plants. Both peroxidases and laccases have been implicated in the polymerization of lignin, and mutant analyses have conclusively demonstrated a role for laccases in lignification of Arabidopsis thaliana stems. However, the oxidative enzymes that polymerize lignin in protoxylem tracheary elements (TEs) have not been defined. Induction of the master transcription factor VASCULAR RELATED NAC-DOMAIN 7 (VND7) causes systemic transdifferentiation into protoxylem TEs, providing an inducible-experimental model system to study protoxylem TE differentiation. The transcriptome of these lines has been well characterized, and two laccases, LAC4 and LAC17, are strongly expressed following induction of protoxylem TE development. To test if LAC4 and LAC17 are necessary for the lignification of protoxylem TEs, the inducible VND7 construct was transformed into the lac4-2/lac17 double mutant background and fluorescently labeled monolignols were exogenously applied to differentiating protoxylem TEs. Labeled polymerized lignin was only detected in the wild-type protoxylem TEs, but not in lac4-2/lac17 protoxylem TEs. To test if laccases alone are sufficient to promote lignification, the constitutive 35S promoter was used to drive either LAC4 or LAC17 in wild-type plants, resulting in strong ectopic lignification of primary cell walls upon application of fluorescently labeled monolignols. Fluorescently tagged laccases were transformed into the inducible protoxylem TEs system, where they specifically localize to the secondary, but not primary, cell walls of protoxylem tracheary elements. This research shows that LAC4 and LAC17 are necessary and sufficient for the lignification of secondary cell wall domains of protoxylem TEs and that they are specifically localized to these domains.Science, Faculty ofBotany, Department ofGraduat