Characterization of MdMADS2, a member of the SQUAMOSA subfamily of genes, in apple

Abstract

A MADS-box gene, MdMADS2, was isolated from the apple (Malus x domestica Borkh.) var Fuji and its developmental expression pattern was studied during flower development. MdMADS2 shares a high degree of amino acid sequence identity with the SQUAMOSA subfamily of genes. RNA blot analysis showed that MdMADS2 is transcribed through all stages of flower development, and its transcription was seen in the four floral organs. RNA in situ hybridization revealed that the MdMADS2 mRNA is expressed both in the inflorescence meristem and in the floral meristem. The MdMADS2 transcript was detected at all stages of flower development. Protein localization analysis showed that MdMADS2 protein was excluded from the stamen and carpel primordia, in which a considerable MdMADS2 mRNA signal was detected. This indicates that posttanscriptional regulation may be involved in the MdMADS2-mediated control of flower development. Transgenic tobacco expressing the MdMADS2 gene from the cauliflower mosaic virus 35S promoter showed early flowering and shorter bolts, but did not show any homeotic changes in the floral organs. These results suggest that MdMADS2 plays an important role during early stages of flower development.

Figure 1

Comparison of the amino acid sequence of the MdMADS2 protein with other MADS proteins in the SQUA subfamily. A, Sequence alignment of the MADS domain. Shown here are the MADS-box sequences of MdMADS2 (accession no. U78948), silver birch BpMADS5 (accession no. X99655), tomato TM4 (accession no. X60757), potato PTOM1-1 (accession no. U23757), white campion SLM5 (accession no. X80492), Arabidopsis AGL8 (accession no. U33473), white mustard SaMADSB (accession no. U25695), snapdragon SQUA (accession no. X63701), Arabidopsis AP1 (accession no. S35631), and broccoli Bio2AP1 (accession no. U67452). The asterisks represent amino acid residues identical to the corresponding residues in MdMADS2. The numbers at the left represent the positions of the first amino acid residues shown for each sequence. B, Alignment of the K domains. The asterisks and the numbers at the left are as represented in A. C, Alignment of the C-terminal regions. The last 20 amino acid residues are shown. The left numbers are the positions of the first amino acid residues shown for each sequence. The numbers at the right represent the percentage of identical amino acid residues with MdMADS2.

Figure 2

A, RNA blot analysis of the MdMADS2 transcript in apple flower bud. Lane 1, Mature leaves; lane 2, stage 1 bud (length = 4– 5 mm); lane 3, stage 2 bud (length = 7–8 mm); lane 4, stage 3 bud (petals begin to emerge from sepals); lane 5, mature flowers; lane 6, post-anthesis flowers; lane 7, sepals; lane 8, petals; lane 9, stamens; and lane 10, carpels of mature flower. B, rRNA stained with ethidium bromide showing the equivalence of RNA loading between the lines.

Figure 3

In situ hybridization patterns of MdMADS2 mRNA in a developing flower bud. Each section was photographed using a blue filter under bright-field optics. The transcript signal is blue. Bar = 300 μm. A, Stage 1 flower bud with the inflorescence meristem. The arrowhead indicates the early floral meristem emerging from the inflorescence meristem. B, Stage 2 flower bud with young flower primordia. C and D, Flower buds at early and late stage 3, respectively. Floral organs arise and differentiate at these stages. E and F, Stage 4 flower bud with fused carpels and mature flower, respectively. G through I, Hybridization with a sense probe of MdMADS2 in flower bud at stage 1 (G), stage 3 (H), and stage 4 (I). a, Anther; b, bract; c, carpel; f, filament; fp, flower primordium; ft, floral tube; g, gynoecium; im, inflorescence meristem; l, leaf appendage; o, ovule; p, petal; pc, procambium; s, style; se, sepal; st, stamen.

Figure 4

MdMADS2 protein expressed in E. coli and protein gel blots of MdMADS2 and other MADS proteins with affinity-purified anti-MdMADS2 antibodies. A, Coomassie Blue-stained protein gel showing truncated MdMADS2 protein expressed in E. coli. Lane M, M_r marker; lane −, before IPTG induction; lane +, after IPTG induction; lane P, purified MdMADS2 protein. B, Protein gel blots of truncated MADS proteins with affinity-purified anti-MdMADS2 antibodies. Each protein was loaded in two adjacent lanes. Md2, MdMADS2; Md3, MdMADS3; Md4, MdMADS4; Os1, OsMADS1.

Figure 5

In situ immunolocalization of the MdMADS2 protein in a developing flower bud. Sections were hybridized with affinity-purified anti-MdMADS2 antibodies. Sections were photographed using a blue filter under bright-field optics. The protein signal is purple. Bar = 100 μm. A, Flower bud with the inflorescence meristem (stage 1). B, Preimmune serum control for A. C, Vegetative bud with the stem meristem. D, Preimmune serum control for C. E, Flower bud at stage 2. F, Preimmune serum control for E. G, Flower bud at stage 3. H, Preimmune serum control for G. b, Bract; c, carpel primordium; im, inflorescence meristem; fp, flower primordium; l, leaf appendage; p, petal primordium; pc, procambium; rm, rib meristem; se, sepal primordium; sm, stem meristem; st, stamen primordium.

Figure 6

RNA blot analysis of primary transgenic tobacco plants. The numbers indicate independent transgenic tobacco lines of two different cultivars, SR1 (A) and Xanthi (B). Twenty micrograms of total RNA was isolated from mature leaves and hybridized with the ³²P-labeled MdMADS2 probe. As a control, a probe prepared from the TSC29 cDNA (Gao et al., 1994) was hybridized to the same membrane after washing off the MdMADS2 probe.

Figure 7

Ectopic phenotypes of transgenic tobacco plant. A, Homozygous transgenic line S4 (left two plants) and wild type (right two plants) of the tobacco cv SR1. B, Homozygous transgenic line X4 (left two plants) and wild type (right two plants) of the tobacco cv Xanthi.