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A MADS-box gene NtSVP regulates pedicel elongation by directly suppressing a KNAT1-like KNOX gene NtBPL in tobacco (Nicotiana tabacum L.).

Wang D, Chen X, Zhang Z, Liu D, Song G, Kong X, Geng S, Yang J, Wang B, Wu L, Li A, Mao L - J. Exp. Bot. (2015)

Bottom Line: Moreover, an Arabidopsis BREVIPEDECELLUS/KNAT1 homologue NtBP-Like (NtBPL) was significantly up-regulated in NtSVP-RNAi plants.Disruption of NtBPL decreased pedicel lengths and shortened cortex cells.Microarray analysis showed that down-regulation of NtBPL resulted in differential expression of genes associated with a number of hormone biogenesis and signalling genes such as those for auxin and gibberellin.

View Article: PubMed Central - PubMed

Affiliation: National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), MOA Key Laboratory of Crop Germplasm and Biotechnology, Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China.

No MeSH data available.


Related in: MedlinePlus

NtSVP as a direct transcription repressor of NtBPL. (A) The design of the wild-type (WT) probe containing the CArG-box (proNtBPL-wt) and the mutated probe (proNtBPL-m) in the NtBPL promoter. Mutated bases are highlighted in red. Numbers indicate sequence locations. (B) Yeast one-hybrid assays showing interactions between the NtSVP protein and the NtBPL promoter. pGADT7 (AD) was used as control. (C) EMSA showing MBP–NtSVP fusion protein binding to the probes of proNtBPL as described in (A). m represents proNtBPL-m as a control. + indicates the presence and – the absence of corresponding components as indicated. (D) The design of reporter constructs for the dual-luciferase assay. The WT and two mutant version of proNtBPL (M1, M2) are indicated. (E) LUC activities in N. benthamiana leaves infiltrated with the Agrobacterium strain harbouring the indicated reporter in the presence (+) or absence (–) of the co-transfected Agrobacterium strain harbouring the plasmid expressing the NtSVP protein. (F) Dual-luciferase assay of relative reporter activity of samples shown in (E). The relative LUC activities were normalized to REN activity and presented as relative expression units (REUs, n=5). **P<0.01.
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Figure 4: NtSVP as a direct transcription repressor of NtBPL. (A) The design of the wild-type (WT) probe containing the CArG-box (proNtBPL-wt) and the mutated probe (proNtBPL-m) in the NtBPL promoter. Mutated bases are highlighted in red. Numbers indicate sequence locations. (B) Yeast one-hybrid assays showing interactions between the NtSVP protein and the NtBPL promoter. pGADT7 (AD) was used as control. (C) EMSA showing MBP–NtSVP fusion protein binding to the probes of proNtBPL as described in (A). m represents proNtBPL-m as a control. + indicates the presence and – the absence of corresponding components as indicated. (D) The design of reporter constructs for the dual-luciferase assay. The WT and two mutant version of proNtBPL (M1, M2) are indicated. (E) LUC activities in N. benthamiana leaves infiltrated with the Agrobacterium strain harbouring the indicated reporter in the presence (+) or absence (–) of the co-transfected Agrobacterium strain harbouring the plasmid expressing the NtSVP protein. (F) Dual-luciferase assay of relative reporter activity of samples shown in (E). The relative LUC activities were normalized to REN activity and presented as relative expression units (REUs, n=5). **P<0.01.

Mentions: Since genetic analysis indicated that NtBPL acted downstream of NtSVP, experiments were carried out to determine whether NtBPL was a direct target of NtSVP regulation. To test this hypothesis, ~1kb of the promoter sequence of NtBPL was cloned and cis-element prediction was performed using the PLACE program. A CArG-box (5’CAATATTAAG3’), a consensus MADS-box transcription factor binding site, was found on the NtBPL promoter at –866bp to –856bp from the initiation codon (Fig. 4A; Supplementary Fig. S6 at JXB online), providing a condition for direct binding of NtSVP to the promoter of NtBPL. Yeast one-hybrid assay showed that NtSVP could indeed activate the expression of a reporter gene driven by the NtBPL promoter (Fig. 4B). This was further confirmed by EMSA using the MBP–NtSVP fusion protein. As shown in Fig. 4C, the MBP–NtSVP fusion protein was able to bind to DNA probes containing the wild-type CArG-box, but failed to bind to the mutated version. Furthermore, increasing the concentration of unlabelled wild-type probes in the binding reactions resulted in much weaker retarded bands. Meanwhile, the formation of proNtBPL and the MBP–NtSVP complex was not inhibited by an excess amount of mutated probes, indicating that NtSVP may specifically bind to the NtBPL promoter under these conditions.


A MADS-box gene NtSVP regulates pedicel elongation by directly suppressing a KNAT1-like KNOX gene NtBPL in tobacco (Nicotiana tabacum L.).

Wang D, Chen X, Zhang Z, Liu D, Song G, Kong X, Geng S, Yang J, Wang B, Wu L, Li A, Mao L - J. Exp. Bot. (2015)

NtSVP as a direct transcription repressor of NtBPL. (A) The design of the wild-type (WT) probe containing the CArG-box (proNtBPL-wt) and the mutated probe (proNtBPL-m) in the NtBPL promoter. Mutated bases are highlighted in red. Numbers indicate sequence locations. (B) Yeast one-hybrid assays showing interactions between the NtSVP protein and the NtBPL promoter. pGADT7 (AD) was used as control. (C) EMSA showing MBP–NtSVP fusion protein binding to the probes of proNtBPL as described in (A). m represents proNtBPL-m as a control. + indicates the presence and – the absence of corresponding components as indicated. (D) The design of reporter constructs for the dual-luciferase assay. The WT and two mutant version of proNtBPL (M1, M2) are indicated. (E) LUC activities in N. benthamiana leaves infiltrated with the Agrobacterium strain harbouring the indicated reporter in the presence (+) or absence (–) of the co-transfected Agrobacterium strain harbouring the plasmid expressing the NtSVP protein. (F) Dual-luciferase assay of relative reporter activity of samples shown in (E). The relative LUC activities were normalized to REN activity and presented as relative expression units (REUs, n=5). **P<0.01.
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Figure 4: NtSVP as a direct transcription repressor of NtBPL. (A) The design of the wild-type (WT) probe containing the CArG-box (proNtBPL-wt) and the mutated probe (proNtBPL-m) in the NtBPL promoter. Mutated bases are highlighted in red. Numbers indicate sequence locations. (B) Yeast one-hybrid assays showing interactions between the NtSVP protein and the NtBPL promoter. pGADT7 (AD) was used as control. (C) EMSA showing MBP–NtSVP fusion protein binding to the probes of proNtBPL as described in (A). m represents proNtBPL-m as a control. + indicates the presence and – the absence of corresponding components as indicated. (D) The design of reporter constructs for the dual-luciferase assay. The WT and two mutant version of proNtBPL (M1, M2) are indicated. (E) LUC activities in N. benthamiana leaves infiltrated with the Agrobacterium strain harbouring the indicated reporter in the presence (+) or absence (–) of the co-transfected Agrobacterium strain harbouring the plasmid expressing the NtSVP protein. (F) Dual-luciferase assay of relative reporter activity of samples shown in (E). The relative LUC activities were normalized to REN activity and presented as relative expression units (REUs, n=5). **P<0.01.
Mentions: Since genetic analysis indicated that NtBPL acted downstream of NtSVP, experiments were carried out to determine whether NtBPL was a direct target of NtSVP regulation. To test this hypothesis, ~1kb of the promoter sequence of NtBPL was cloned and cis-element prediction was performed using the PLACE program. A CArG-box (5’CAATATTAAG3’), a consensus MADS-box transcription factor binding site, was found on the NtBPL promoter at –866bp to –856bp from the initiation codon (Fig. 4A; Supplementary Fig. S6 at JXB online), providing a condition for direct binding of NtSVP to the promoter of NtBPL. Yeast one-hybrid assay showed that NtSVP could indeed activate the expression of a reporter gene driven by the NtBPL promoter (Fig. 4B). This was further confirmed by EMSA using the MBP–NtSVP fusion protein. As shown in Fig. 4C, the MBP–NtSVP fusion protein was able to bind to DNA probes containing the wild-type CArG-box, but failed to bind to the mutated version. Furthermore, increasing the concentration of unlabelled wild-type probes in the binding reactions resulted in much weaker retarded bands. Meanwhile, the formation of proNtBPL and the MBP–NtSVP complex was not inhibited by an excess amount of mutated probes, indicating that NtSVP may specifically bind to the NtBPL promoter under these conditions.

Bottom Line: Moreover, an Arabidopsis BREVIPEDECELLUS/KNAT1 homologue NtBP-Like (NtBPL) was significantly up-regulated in NtSVP-RNAi plants.Disruption of NtBPL decreased pedicel lengths and shortened cortex cells.Microarray analysis showed that down-regulation of NtBPL resulted in differential expression of genes associated with a number of hormone biogenesis and signalling genes such as those for auxin and gibberellin.

View Article: PubMed Central - PubMed

Affiliation: National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), MOA Key Laboratory of Crop Germplasm and Biotechnology, Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China.

No MeSH data available.


Related in: MedlinePlus