The tomato floral homeotic protein FBP1-like gene, SlGLO1, plays key roles in petal and stamen development

Abstract

MADS-box transcription factors play important role in plant growth and development, especially floral organ identities. In our study, a MADS-box gene SlGLO1- tomato floral homeotic protein FBP1-like gene was isolated. Its tissue-specific expression profile analysis showed that SlGLO1 was highly expressed in petals and stamens. RNAi (RNA interference) repression of SlGLO1 resulted in floral organ abnormal phenotypes, including green petals with shorter size, and aberrant carpelloid stamens. SlGLO1-silenced lines are male sterile. Total chlorophyll content was increased and chlorophyll biosynthetic genes were significantly up-regulated in SlGLO1-silenced petals and stamens. Furthermore, B-class genes expression analysis indicated that the repressed function of SlGLO1 led to the enhanced expression of TAP3 and the down-regulation of TPI in the petals and stamens, while the expression of TM6 was reduced in petals and increased in stamens and carpels of SlGLO1-RNAi plants. Additionally, pollen grains of transgenic lines were aberrant and failed to germinate and tomato pollen-specific genes were down-regulated by more than 90% in SlGLO1-silenced lines. These results suggest that SlGLO1 plays important role in regulating plant floral organ and pollen development in tomato.

Figure 1. Sequence and expression analysis of SlGLO1 in WT.

(A) Phylogenetic analysis of SlGLO1 and other MADS-Box proteins was performed by the neighbor-joining method, bootstrap analysis of 1,000 replicates. SlGLO1 is marked with asterisk. Accession numbers for other proteins are listed as follows: PhGLO1 (Q03488.1), NbGLO1 (HQ005417), AmGLO (Q03378.1), PhGLO2 (CAA49568.1), NbGLO2 (HQ005418), TPI (DQ674531), TM6 (X60759), NbTM6 (AY577817), PhTM6 (AAS46017.1), AmDEF (CAA44629.1), NbDEF (DQ437635), PhDEF (Q07472), TAP3 (DQ674532). (B) Multiple sequence alignment of SlGLO1 and other MADS-Box proteins. SlGLO1 is marked with asterisk. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. (C) The relative expression patterns of SlGLO1 in WT. RT, root; ST, stem; YL, young leaf; ML, muture leaf; SL, senescent leaf; F, flower; SE, sepal of flower in anthesis; IMG, immature green fruit; MG, mature green fruit; B, breaker fruit; B4, 4 days after breaker fruit; B7, 7 days after breaker fruit. The relative expression of SlGLO1 in the four-whorl floral organs (D) of WT. Se, sepal; Pe, petal; St, stamen; Ca, carpel. Each value represents the mean ± SE of three replicates.

Figure 2. Expression profiles of SlGLO1 between WT and SlGLO1-RNAi lines.

The expression data of WT plants were normalized to 1. Each value represents the mean ± SE of three replicates.

Figure 3. Phenotypes of petals in SlGLO1-RNAi lines.

Wild type, (A–C); SlGLO1-RNAi, (D–F). Bars = 300 μm in (B,E), 60 μm in (C,F). (G) is digital photograph, and (B,C,E,F), are scanning electron micrographs. (A) Open wild-type flower. (B,C) Wild-type adaxial petal surface. (D) Flower from SlGLO1-RNAi line. (E,F) SlGLO1-RNAi line adaxial petal surface. (G) Silencing of SlGLO1 results in smaller size petal. (H) Petal length of WT and transgenic lines. Error bars represent the standard error of the mean (n = 10). Asterisks indicate a significant difference (P < 0.05) between WT and transgenic lines.

Figure 4. Phenotypes of stamens in SlGLO1-RNAi lines.

Wild type, (A–D); SlGLO1-RNAi, (E–I). Bars = 60 μm in (C,D,H,I). (A,B,E–G) are digital photographs, and (C,D,H,I), are scanning electron micrographs. (A) Wild-type stamen. (B) Split wild-type stamen. (C) Wild-type adaxial stamen proximal end. (D) Wild-type adaxial stamen distal end. (E) Stamen from SlGLO1-RNAi line. (F) Stamens of SlGLO1-RNAi lines are unclosed. (G) Silencing of SlGLO1 results in carpelloid stamens. (H) SlGLO1-RNAi line adaxial stamen proximal end. (I) SlGLO1-RNAi line adaxial stamen distal end.

Figure 5. Total chlorophyll content of petals and chlorophyll biosynthetic genes expression of wild-type and silenced-SlGLO1 lines.

(A) Total chlorophyll content of petals from wild-type and silenced-SlGLO1 lines. (B–D) respectively represents expression analysis of SlDCL, SlGLK1, SlGLK2 in petals of wild-type and transgenic lines. Each value represents the mean ± SE of three replicates. Asterisks indicate a significant difference (P < 0.05) between WT and transgenic lines.

Figure 6. Total chlorophyll content of stamens and chlorophyll biosynthetic genes expression of wild-type and silenced-SlGLO1 lines.

(A) Total chlorophyll content of stamens from wild-type and silenced-SlGLO1 lines. (B–D) respectively represents expression analysis of SlDCL, SlGLK1, SlGLK2 in stamens of wild-type and transgenic lines. Each value represents the mean ± SE of three replicates. Asterisks indicate a significant difference (P < 0.05) between WT and transgenic lines.

Figure 7. Expression Patterns of other floral organ identity genes in wild-type and RNAi Lines.

Se, sepal; Pe, petal; St, stamen; Ca, carpel. (A,B) respectively represents expression analysis of TAP3, TPI, TM6 (B-class genes) in wild-type and transgenic lines. (D–G) respectively represents expression analysis of MC (A-class gene), TAG1(C-class gene), TM5 and TAGL2 (E-class genes) in wild-type and transgenic lines. Each value represents the mean ± SE of three replicates. Asterisks indicate a significant difference (P < 0.05) between WT and transgenic lines.

Figure 8. Fruit phenotype, pollen germination and cross between WT and SlGLO1-RNAi lines.

(A) The SlGLO1-silenced lines are male sterile. (B) Pollen germination of WT and SlGLO1-RNAi lines. Pollen grains are germinated following 5 h culture at 25 °C in vitro containing liquid culture medium with 120 g/L sucrose, 120 mg/L boric acid, 4 mg/L gibberellin and 0.5 mg/L thiamine. Bars = 20 μm. (C) Fruit of the transgenic line produces normally seeds through manual crossing assay.

Figure 9. Expression analysis of tomato pollen-specific genes in the pollen of wild-type and transgenic plants.

(A–E) respectively represents expression of pollen-specific genes SlCRK1, SlPMEI, LePRK3, SlPRALF and LAT52 in pollen of wild-type and transgenic lines. Each value represents the mean ± SE of three replicates. Asterisks indicate a significant difference (P < 0.05) between WT and transgenic lines.