Genes encoding the vacuolar Na+/H+ exchanger and flower coloration.

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.

Identification of new chalcone synthase genes for flower pigmentation in the Japanese and common morning glories.

New cDNA sequences for chalcone synthase (CHS), a key enzyme in the flavonoid biosynthesis, were obtained from the Japanese and common morning glories; they are more closely related to other CHS sequences than the six previously described CHS genes from the same plants. The newly isolated CHS-D gene is abundantly expressed in the pigmented flower buds, while its expression is drastically reduced in the white flower buds. Thus CHS-D appears to produce major CHS transcripts for flower pigmentation.

Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence.

Using amplified restriction fragment length polymorphism (AFLP) technology, we have developed a new protocol for the fingerprinting of mRNA that allows systematic comparison of the differential expression of genes between mRNA samples. The major advantage of our protocol is the use of only a single restriction enzyme that recognizes a 4-bp sequence but allows screening of large numbers of different cDNAs. Using this new protocol, we compared mRNA samples obtained from the flower buds of two lines of the common morning glory (Ipomoea purpurea) with red and white flowers, respectively. Approximately 50 bands were observed in each lane of a denaturing polyacrylamide gel and the results were highly reproducible, as indicated by the results of analysis of two sets of independent mRNA samples. Two cDNA fragments, which were differentially amplified in the samples from the two lines, were shown to have been derived from a single gene that was actively expressed in the buds of red flowers but not in those of white flowers. A full-length cDNA of this gene was cloned from a bud cDNA library. Sequence analysis showed that this cDNA carries a sequence highly homologous to the chalcone synthase (CHS) genes, the key enzyme in the flavonoid biosynthetic pathway.