The Artificial Promoter rMdAG2I Confers Flower-specific Activity in Malus

Abstract

Genetic modifications of floral organs are important in the breeding of Malus species. Flower-specific promoters can be used to improve floral organs specifically, without affecting vegetative organs, and therefore developing such promoters is highly desirable. Here, we characterized two paralogs of the Arabidopsis thaliana gene AGAMOUS (AG) from Malus domestica (apple): MdAG1 and MdAG2. We then isolated the second-intron sequences for both genes, and created four artificial promoters by fusing each intron sequence to a minimal 35S promoter sequence in both the forward and reverse directions. When transferred into tobacco (Nicotiana benthamiana) by Agrobacterium tumefaciens-mediated stable transformation, one promoter, rMdAG2I, exhibited activity specifically in flowers, whereas the other three also showed detectable activity in vegetative organs. A test of the four promoters' activities in the ornamental species Malus micromalus by Agrobacterium-mediated transient transformation showed that, as in tobacco, only rMdAG2I exhibited a flower-specific expression pattern. Through particle bombardment transformation, we demonstrated that rMdAG2I also had flower-specific activity in the apple cultivar 'Golden Delicious'. The flower-specific promoter rMdAG2I, derived from M. domestica, thus has great potential for use in improving the floral characteristics of ornamental plants, especially the Malus species.

Keywords: AGAMOUS; Malus; flower-specific promoter; intron.

Figure 1

Phylogenetic analysis of AG protein sequences. The phylogenetic tree was constructed using the neighbor-joining method of MEGA 6.0 software based on the protein sequences of Phaseolus vulgaris AG (Phvul.006G169600), Medicago truncatula AG (XP_013462626.1), Arachis hypogaea AG (XP_025640343.1), Malus domestica AG2 (MDP0000250080), Malus domestica AG1 (MDP0000324166), Vitis vinifera AG (NP_001268097.1), Primula vulgaris AG (AZZ09252.1), Sesamum indicum AG (AIS82595.1), Nicotiana tabacum AG (NP_001312829.1), Populus trichocarpa AG1 (AAC06237.1), Theobroma cacao AG (XP_007025252.1), Citrus clementina AG (XP_006449577.1), Citrus sinensis AG (XP_024950555.1) and Arabidopsis thaliana AG (NP_001328877.1). Values at nodes indicate bootstrap percentages.

Figure 2

Relative expression levels of MdAG1 and MdAG2 in various tissues of Malus domestica ‘Golden Delicious’. Expression levels of MdActin were used as an internal reference. Data represent means of three biological replicates ± SE. Bars with different letters are significant different at p < 0.05 based on one-way analysis of variance (ANOVA).

Figure 3

Distributions of ten types of predicted cis-regulatory elements in the second introns of MdAG1 and MdAG2. The different distribution modes may cause different activities when these are used as promoters.

Figure 4

Gene constructs used for tobacco and Malus transformation. 35S, cauliflower mosaic virus 35S gene promoter sequence; GUS, coding sequence of the β-glucuronidase gene; tNos, nopaline synthase terminator; fMdAG1I, forward orientation of the second intron of Malus AGAMOUS1 (MdAG1); rMdAG1I, reverse orientation of the second intron of MdAG1; fMdAG2I, forward orientation of the second intron of MdAG2; rMdAG2I, reverse orientation of the second intron of MdAG2; Min35S, 60 bases of the 35S gene promoter sequence.

Figure 5

GUS staining in tobacco transformed with the four artificial promoters and 35S::GUS control. (A–C) GUS expression using 35S::GUS as a positive control. All organs are stained. (D) GUS expression in non-transformed flowers. None of the plant was stained. (E–G) GUS expression of tobacco tissues transformed with fMdAG1I::GUS (E), rMdAG1I::GUS (F), and fMdAG2I::GUS (G), respectively. Flowers, leaves and stems all show different degrees of blue color. (H–I) Two lines of rMdAG2I::GUS transgenic tobacco. Only flowers are stained.

Figure 6

GUS staining of Malus micromalus with Agrobacterium-mediated transient transformation. (A) 35S::GUS-transformed flowers (positive control). Stem and flower show blue staining. (B) Non-transformed flowers (negative control) have no blue staining. (C–F) fMdAG1I::GUS- (C) rMdAG1I::GUS- (D) fMdAG2I::GUS- (E) and rMdAG2I::GUS- (F) transformed flowers show blue staining.

Figure 7

GUS staining of ‘Golden Delicious’ transformed with rMdAG2I::GUS through particle bombardment. (A) Flower organs with blue staining. RE, receptacle; SE, sepal; PE, petal; ST, stamen; PI, pistil. (B) Stem without blue staining.