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. 2015 Dec 16:2:15059.
doi: 10.1038/hortres.2015.59. eCollection 2015.

A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia

Jing Yin  1 , Xiaoxiao Chang  2 , Takao Kasuga  3 , Mai Bui  3 , Michael S Reid  4 , Cai-Zhong Jiang  3
Affiliations

A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia

Jing Yin et al. Hortic Res. .

Abstract

The basic helix-loop-helix (bHLH) transcription factors (TFs) play important roles in regulating multiple biological processes in plants. However, there are few reports about the function of bHLHs in flower senescence. In this study, a bHLH TF, PhFBH4, was found to be dramatically upregulated during flower senescence. Transcription of PhFBH4 is induced by plant hormones and abiotic stress treatments. Silencing of PhFBH4 using virus-induced gene silencing or an antisense approach extended flower longevity, while transgenic petunia flowers with an overexpression construct showed a reduction in flower lifespan. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was significantly changed in petunia PhFBH4 transgenic flowers. Furthermore, silencing or overexpression of PhFBH4 reduced or increased, respectively, transcript abundances of important ethylene biosynthesis-related genes, ACS1 and ACO1, thereby influencing ethylene production. An electrophoretic mobility shift assay showed that the PhFBH4 protein physically interacted with the G-box cis-element in the promoter of ACS1, suggesting that ACS1 was a direct target of the PhFBH4 protein. In addition, ectopic expression of this gene altered plant development including plant height, internode length, and size of leaves and flowers, accompanied by alteration of transcript abundance of the gibberellin biosynthesis-related gene GA2OX3. Our results indicate that PhFBH4 plays an important role in regulating plant growth and development through modulating the ethylene biosynthesis pathway.

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Figures

Figure 1
Figure 1
PhFBH4 transcripts in different flower stages. (A) Different stages of petunia flower after anthesis. D0: the day of anthesis, D2, D4, D6, D7: 2, 4, 6, and 7 days after anthesis, respectively. (B) Expression level of PhFBH4 in different flower stages by qRT-PCR. Error bars show SD of the means of three biological replicates.
Figure 2
Figure 2
Expression pattern of PhFBH4 under hormone and abiotic treatment by qRT-PCR. (A) Expression of PhFBH4 under hormone treatment. Petunia flowers harvested at anthesis were placed in tubes with water (mock), 0.1 mM ABA, 50 μM GA3, treated with ethylene (3 µl/l), or with 1-MCP for 4 h before ethylene treatment. (B) Expression of PhFBH4 under abiotic stress treatment. Petunia flowers were placed in tubes with water at 29 °C and 4 °C, without water (drought), or with 100 mM NaCl at room temperature (salt). Error bars show SD of the means of three biological replicates.
Figure 3
Figure 3
Silencing PhFBH4 using the VIGS system extended flower longevity. (A) Phenotype of plants using different constructs at 5 weeks after inoculation. (B) Flower longevity of attached flowers in different plants. CHS/TRV (W), white flowers in CHS/TRV plants; PhFBH4/CHS/TRV (W), white flowers in PhFBH4/CHS/TRV plants. Error bars indicate SD (n ≥ 10). Different letters denote significant differences at p > 0.05 analyzed by Tukey's test (C) Abundance of related gene expression in flowers on D7 in different plants.
Figure 4
Figure 4
Ectopic expression of PhFBH4 affected flower longevity. (A) Expression of PhFBH4 in WT, PhFBH4 overexpression and antisense silencing transgenic plants by qRT-PCR. PhFBH4-OX-2, PhFBH4-OX-7, different lines of PhFBH4 overexpression. PhFBH4-AS-1, PhFBH4-AS-3, different lines of PhFBH4 antisense silencing. Error bars show SE of the means of three biological replicates. (B) Different flower stages in WT petunia and PhFBH4 transgenic petunia. D0: the day of anthesis; D5, D7: 5 and 7 days after anthesis, respectively. (C) Flower longevity in WT and PhFBH4-OX and AS transgenic plants. Error bars indicate SD (n ≥ 10). Different letters denote significant differences at p ≤ 0.05 analyzed by Tukey's test. (D) Senescence marker genes, SAG12 and SAG29, expression in the flower of wide type petunia and transgenic petunia on D5.
Figure 5
Figure 5
Expression of ethylene biosynthesis-related genes in D5 petunia flowers. Abundance of transcripts of genes associated with ethylene biosynthesis was determined at D5 in WT and PhFBH4 transgenic plants. Error bars show SD of the means of three biological replicates. Different letters denote significant differences at p ≤ 0.05 analyzed by Tukey's test.
Figure 6
Figure 6
Effect of ectopic expression of PhFBH4 on ethylene production of petunia flowers in WT and transgenic plants. Ethylene production was measured at D5. Error bars show SD of the means of five biological replicates. Different letters denote significant differences at p ≤ 0.05 analyzed by Tukey's test.
Figure 7
Figure 7
Electrophoretic mobility shift assay of the PhFBH4 protein. The biotin-labeled oligonucleotide of proACS1 was mixed with GST-tagged protein control (lane 1) or PhFBH4 proteins prepared from cells transfected with pGST::PhFBH4 plasmid (lanes 2–7). PhFBH4 protein physically binds a cis-element (G-box) in the PhACS1 promoter (lane 2). Binding was gradually abolished by the addition of an unlabeled oligonucleotide competitor in 10-fold (lane 3), 100-fold (lane 4), and 1000-fold (lane 5) molar excess, whereas specific binding was maintained when the same amount of the excess mutant oligonucleotide competitor was added (lanes 6 and 7). PhFBH4 protein failed to bind the E-boxes in the promoter of ACO1 (lane 8).
Figure 8
Figure 8
Phenotypic traits of the PhFBH4 overexpression (OX) and antisense silencing (AS) transgenic petunia plants. (A) Whole plants of different lines. (B) Leaf size and flower size of different plants. Flowers were harvested on D4. Leaves were collected at the sixth from the top.
Figure 9
Figure 9
Expression analysis of GA-related genes in the flower of WT and transgenic petunia on D4 by qRT-PCR. Abundance of transcripts of genes associated with GA was determined at D4 in WT and PhFBH4 transgenic plants. Error bars show SD of the means of three biological replicates. Different letters denote significant differences at p ≤ 0.05 analyzed by Tukey's test.
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