Figure 1.
Alignment and phylogenetic analysis of SUP-like genes.
(A) Alignment of CsSUP and SUP-like genes. The amino acid sequences of CsSUP, Csa001112 and Cas010435 in cucumber, AtSUP and RBE in Arabidopsis, NtSUP in tobacco, PhSUP in petunia, SlSUP in white campion and Os05g0286100 in rice were aligned using ClustalW in the MEGA5 software package. The black and gray areas indicate identical and similar amino acid, respectively. Zinc-finger and leucine zipper (LZ)-like domains were indicated in black lines. The DLELRL domain [15] was showed in red box. (B) An unrooted phylogenetic tree constructed using the amino acid sequences of CsSUP, Csa001112, Cas010435, AtSUP, RBE, NtSUP, PhSUP, SlSUP and Os05g0286100 based on the neighbor joining method. Branch length is proportional to evolutionary distance.
Figure 2.
Quantitative RT-PCR (qRT-PCR) analysis of CsSUP in different organs of cucumber (A) or different parts of cucumber fruit (B–C).
Three biological replicates were used for each sample, and 18S rRNA was used as internal control. Bars represent the standard error. (A) CsSUP is predominately expressed in the female flower bud and developing fruit. lf: leaves, te: tendrils, mb: male flower buds, fb: female flower buds, mf: male flowers, ff: female flowers, fr-4: fruit of 4 days before flower opening, fr: fruit on flower opening, fr+3: fruit of 3 days after flower opening. (B) Transverse sections of commercially mature cucumber fruit. ep: epicarp, me: mesocarp, en:endocarp, ve:ventricle. (C) CsSUP is highly expressed in the ventricle of cucumber fruit where the ovules are located.
Figure 3.
In situ hybridization of CsSUP transcripts in developing flowers and fruits of cucumber.
Longitudinal sections of the shoot apex (A), stage 2 flower (B), stage 4 flower (C) and stage 8 female flower (D) reveal that CsSUP is expressed throughout in the IM, FM and young floral primordia (stage 1–2), and then limited to the boundary between petal and stamen (arrow in C), and the developing ovary (arrow in D). Transverse sections reveal that CsSUP is specifically expressed in the boundary of developing ovary (arrow in E), and the developing ovules (arrow in F). CsSUP is undetectable in the male flower primordia (G). Control hybridizations with CsSUP sense probe in male (H) or female flower primordia (I) show no signal. Bar = 200 µm except for (I), in which bar = 100 µm.
Figure 4.
Ectopic CsSUP expression can partially rescue the phenotype of sup-5 mutant Arabidopsis.
Flowers of sup-5 (A, D), pAtSUP::CsSUP;sup-5 (B, E) and 35S::CsSUP;sup-5 (C, F) shows the complement of excess stamen upon ectopic CsSUP expression. (G) Representative siliques of sup-5 (top), pAtSUP::CsSUP;sup-5 (top middle), 35S::CsSUP;sup-5 (bottom middle) and Ler (bottom) indicate the partial rescue of the sup-5 silique development by ectopic expression of CsSUP in Arabidopsis. (H) Opened siliques of sup-5 (top) and pAtSUP::CsSUP;sup-5 (bottom) at similar developmental stages. (I) Expression of CsSUP in transgenic Arabidopsis. Lane 1–2: sup-5 plants, lane 3–4: 35S::CsSUP;sup-5 lines, lane 5–6: pAtSUP::CsSUP;sup-5 lines. Actin2 was used as internal control to normalize the expression data. Bars = 1 mm.
Table 1.
Quantification of silique phenotype.
Table 2.
Partial complement of sup-5 mutant upon ectopic expression of CsSUP.
Figure 5.
Phenotypes of overexpression of CsSUP in wild type Arabidopsis.
(A–B) Flowers of wild type (A) and 35S::CsSUP (B) show the disturbed petal organization. (C–D) Stamens of wild type (C) and 35S::CsSUP (D) indicate the suppressed stamen development in the transgenic lines. (E–F) Siliques (E) and opened siliques (F) of wild type (top) and 35S::CsSUP (bottom) show the reduced silique length and decreased seed numbers. Bars = 1 mm.
Table 3.
Reduced stamen numbers in 35S::CsSUP transgenic plants.
Table 4.
Reduced silique length in 35S::CsSUP transgenic plants.
Table 5.
Reduced seed numbers in 35S::CsSUP transgenic plants.