Search

Petunia mutants ( Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P- ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H+P3A- ATPase, whereas PH1 is the only known eukaryoticP3B- ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5., We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms., We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A- ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events., The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P- ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes. [ABSTRACT FROM AUTHOR]

The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H+-ATPase (V-ATPase), which, in plants, act together with H+-pyrophosphatases (PPase), whereas distinct P-type H+-ATPases in the cell membrane control the pH in the cytoplasm and energize the plasma membrane. Flower colour mutants have proved useful in identifying genes controlling the pH of vacuoles where anthocyanin pigments accumulate. Here we show that PH5 of petunia encodes a P3A-ATPase proton pump that, unlike other P-type H+-ATPases, resides in the vacuolar membrane. Mutation of PH5 reduces vacuolar acidification in petals, resulting in a blue flower colour and abolishes the accumulation of proanthocyanindins (condensed tannins) in seeds. Expression of PH5 is directly activated by transcription regulators of the anthocyanin pathway, in conjunction with PH3 and PH4. Thus, flower coloration, a key-factor in plant reproduction, involves the coordinated activation of pigment synthesis and a specific pathway for vacuolar acidification. [ABSTRACT FROM AUTHOR]

In order to scan the 5′ flanking region of the chalcone synthase ( chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase ( cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5′ end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from −67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from −142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (−59 and −52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene. [ABSTRACT FROM AUTHOR]