Generation of Yellow Flowers of the Japanese Morning Glory by Engineering Its Flavonoid Biosynthetic Pathway toward Aurones.

Wild-type plants of the Japanese morning glory (Ipomoea nil) produce blue flowers that accumulate anthocyanin pigments, whereas its mutant cultivars show wide range flower color such as red, magenta and white. However, I. nil lacks yellow color varieties even though yellow flowers were curiously described in words and woodblocks printed in the 19th century. Such yellow flowers have been regarded as 'phantom morning glories', and their production has not been achieved despite efforts by breeders of I. nil. The chalcone isomerase (CHI) mutants (including line 54Y) bloom very pale yellow or cream-colored flowers conferred by the accumulation of 2', 4', 6', 4-tetrahydoroxychalcone (THC) 2'-O-glucoside. To produce yellow phantom morning glories, we introduced two snapdragon (Antirrhinum majus) genes to the 54Y line by encoding aureusidin synthase (AmAS1) and chalcone 4'-O-glucosyltransferase (Am4'CGT), which are necessary for the accumulation of aureusidin 6-O-glucoside and yellow coloration in A. majus. The transgenic plants expressing both genes exhibit yellow flowers, a character sought for many years. The flower petals of the transgenic plants contained aureusidin 6-O-glucoside, as well as a reduced amount of THC 2'-O-glucoside. In addition, we identified a novel aurone compound, aureusidin 6-O-(6″-O-malonyl)-glucoside, in the yellow petals. A combination of the coexpression of AmAS1 and Am4'CGT and suppression of CHI is an effective strategy for generating yellow varieties in horticultural plants.

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny.

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.

Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice.

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive-negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding beta-glucuronidase fused with the endogenous promoter of MET1a, one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T(0)) transgenic knock-in plants obtained were found to carry only one copy of GUS, with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter-fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive-negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.

Characterization of a novel Na+/H+ antiporter gene InNHX2 and comparison of InNHX2 with InNHX1, which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory.

The reddish-purple buds of the wild-type Japanese morning glory (Ipomoea nil) change into blue open flowers, and the shift in the flower coloration correlates with an increase in the vacuolar pH of the flower epidermal cell. In the mutant deficient in the InNHX1 gene for the vacuolar Na(+)/H(+) antiporter, the vacuolar alkalization occurs only partially, and reddish-purple buds become purple open flowers. While most of the plant NHX genes characterized are generally expressed in leaves, stems and roots and induced by NaCl treatment, the InNHX1 gene is expressed predominantly in the flower limbs at around 12 h before flower opening. It is expressed very sparsly in leaves, stems and roots, and no induction occurs in response to NaCl treatment. Here, we identified a novel vacuolar Na(+)/H(+) antiporter gene InNHX2, which is expressed in leaves, stems and roots and is induced in response to NaCl treatment. In addition, relatively higher expression of InNHX2 was observed in the flower limbs shortly before flower opening. We also discovered that both the InNHX1 and InNHX2 proteins can catalyze both Na(+) and K(+) transport into vacuoles. These results suggest that InNHX2 performs dual functions: to confer salt tolerance on the plant and to promote partial vacuolar alkalization in the petals. The implication is that the InNHX2 protein is probably one of the components responsible for converting reddish-purple buds into purple open flowers by partially increasing the vacuolar pH in the absence of major InNHX1 activity.