A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Abstract

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal-specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal-specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β-glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal-specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA-like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal-specific promoter in molecular breeding of floricultural crops.

Keywords: Ipomoea nil; MYB; floricultural crops; molecular breeding; petal-specific promoter.

Figure 1

Histochemical assay for GUS activity in InMYB1_3kb:: GUS and InMYB1_1kb:: GUS transgenic Arabidopsis. (a) histochemical assay for GUS activity in whole plants of the InMYB1_1kb:: GUS transgenic Arabidopsis. (b) histochemical assay for GUS activity in flowers, leaves and stems of InMYB1_1kb:: GUS and InMYB1_3kb:: GUS transgenic Arabidopsis and wild type. Bars = 1 cm in (a), 500 μm in (b).

Figure 2

Histochemical assay for GUS activity in InMYB1_1kb:: GUS Arabidopsis grown in stress conditions. Control: seedlings grown on MS medium containing 1% sucrose in 16‐h photoperiod. 5% sucrose: seedlings grown on MS medium containing 5% sucrose in 16‐h photoperiod. Continuous light: seedlings grown on MS medium containing 1% sucrose in continuous light condition. Bars = 500 μm.

Figure 3

Histochemical assay for GUS activity in InMYB1_1kb:: GUS lines in flower buds and petals. (a) flower developmental stages of Arabidopsis. Stages are defined as follows: stage 1, ratio of transverse diameter to median diameter of flower bud is 1; stage 2, ratio of transverse diameter to median diameter of flower bud is 2; stage 3, petal length and sepal length are identical; stage 4, petal length is slightly greater than sepal length; stage 5, petal length is 1.5 times greater than sepal length; stage 6, carpel length is greater than sepal length. (b) hand‐opened flower at stage 1. Bars = 250 μm.

Figure 4

Histochemical assay for GUS activity in flower, stem, mature leaf and young leaf of InMYB1_1kb:: GUS transgenic Eustoma. Bars = 2 mm.

Figure 5

Transient assay for GUS activity of InMYB1_1kb:: GUS in petals of various floricultural crops by particle bombardment. CaMV35S:: GUS was used as positive control. ND: not determined. Bars = 1 mm.

Figure 6

Angiosperm Phylogeny Group III (APG III) system of flowering plant and InMYB1 promoter activity in various plant species. APG III is modified from Angiosperm Phylogeny Group (2009). Strong, low or no GUS staining of InMYB1_1kb:: GUS in transient assay is shown by (++), (+) or (−), respectively. Origin of InMYB1 promoter, Japanese morning glory, is shown by red letters. The plants showed InMYB1 promoter activity by stable transformation. Arabidopsis and Eustoma are shown in blue lettering.

Figure 7

Phenotypes of InMYB1pro::AtMYB106‐SRDX or InMYB1pro::AtMYB106‐VP16 transgenic Arabidopsis. (a) flowers, petals and SEM image of petal epidermal cells of WT and InMYB1pro::AtMYB106‐SRDX Arabidopsis. (b) flowers and SEM image of petal epidermal cells of InMYB1pro::AtMYB106‐VP16. Nanoridge structures are shown with white arrowheads. Bars = 1 mm in pictures of flowers and petals, 10 μm in SEM images in (a), 5 μm in SEM images in (b).