A carnation anthocyanin mutant is complemented by the glutathione S-transferases encoded by maize Bz2 and petunia An9.

Particle bombardment was used to elucidate the function of Flavonoid3, a late-acting anthocyanin gene of the ornamental plant, carnation ( Dianthus caryophyllus L.). The fl3 mutation conditions dilute anthocyanin coloration that closely resembles phenotypes produced by the anthocyanin mutants bz2 of maize and an9 of petunia. Bz2 and An9 encode glutathione S-transferases (GSTs) involved in vacuolar sequestration of anthocyanins. Constructs containing either of these or another late-function maize gene, Bronze1 (UDPglucose:flavonol 3- O-glucosyltransferase), were introduced via microprojectile bombardment into fl3 petals. Complementation resulted only from Bz2 and An9, indicating that Fl3 encodes a GST involved in the transport of anthocyanins to the vacuole. The observed result in carnation, an angiosperm phylogenetically distant from maize and petunia, indicates that GST activity might be a universal step in the anthocyanin pathway. Microprojectile bombardment was used to identify late-pathway anthocyanin mutations, which may be responsible for the pale anthocyanin coloration of important cultivars in many species but which can be difficult to characterize by other means.

Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1.

A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2.

Glutathione S-transferases (GSTs) are enzymes that detoxify heterocyclic compounds (xenobiotics) by covalently linking glutathione to the substrate, forming a glutathione S-conjugate. A glutathione pump in the vacuolar membrane of barley actively sequesters herbicide-glutathione S-conjugates; glutathionation allows recognition and entry of the conjugates into vacuoles. The protein encoded by the Bronze-2 gene in maize performs the last genetically defined step in anthocyanin biosynthesis, resulting in the deposition of red and purple pigments in the vacuoles of maize tissues. We show here that Bz2 encodes a GST with activity in maize, transformed Arabidopsis thaliana plants and Escherichia coli. We demonstrate that anthocyanins extracted from maize protoplasts expressing BZ2 are conjugated with glutathione, and that vanadate, a known inhibitor of the glutathione pump in plant vacuolar membranes, inhibits the accumulation of anthocyanins in the vacuole. These results provide a biochemical function for BZ2, and suggest a common mechanism for the ability of plants to sequester structurally similar but functionally diverse molecules in the vacuole.

Molecular characterization of a maize B chromosome centric sequence.

Supernumerary chromosomes are widespread in the plant kingdom but little is known of their molecular nature or mechanism of origin. We report here the initial cloning of sequences from the maize B chromosome. Our analysis suggests that many sequences are highly repetitive and shared with the normal A chromosomes. However, all clones selected for B-specificity contain at least one copy of a particular repeat. Cytological mapping using B chromosome derivatives and in situ hybridization show that the B specific repeats are derived from the centric region of the chromosome. Sequence analysis of this repeat shows homology to motifs mapped to various plant and animal centromeres and to the maize neocentromere. A precise localization of these sequences among breakpoints within the B centromere and an homology to a facultative centromere, suggest a role for this sequence in centromere function.

Contractile proteins in Drosophila development.

In summary, we have used a multidisciplinary approach to the analysis of actomyosin-based motility during Drosophila embryogenesis. We have documented the movements of early embryogenesis with modern, video methods. We have characterized the cytoplasmic myosin polypeptide, made specific polyclonal antisera to the molecule, studied its distribution during early embryogenesis, cloned and partially characterized the gene that encodes it, and have recently completed the nucleotide sequence of a nearly full length cDNA that encodes the entire protein-coding region. We have initiated studies on myosin function in living embryos both by direct microinjection of antibodies and through classical genetics. To better understand how myosin function is regulated, we have begun analysis of its light chains. Finally, to investigate the molecular mechanism by which its function is integrated into a labile cytoskeleton, whose architecture is constantly changing, we have also investigated Drosophila spectrins. Together, these studies are designed to shed light on the dynamics of biologic form at the cellular level, with current focus on such complex processes as cytokinesis and morphogenesis.