Limits...
A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening.

Lin Z, Hong Y, Yin M, Li C, Zhang K, Grierson D - Plant J. (2008)

Bottom Line: Inhibition of ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development.Inhibition of LeHB-1 mRNA accumulation in tomato fruit, using virus-induced gene silencing, greatly reduced LeACO1 mRNA levels, and inhibited ripening.Our findings suggest that LeHB-1 is not only involved in the control of ripening but also plays a critical role in floral organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK.

ABSTRACT
Ethylene is required for climacteric fruit ripening. Inhibition of ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development. LeHB-1, a previously uncharacterized tomato homeobox protein, was shown by gel retardation assay to interact with the promoter of LeACO1, an ACC oxidase gene expressed during ripening. Inhibition of LeHB-1 mRNA accumulation in tomato fruit, using virus-induced gene silencing, greatly reduced LeACO1 mRNA levels, and inhibited ripening. Conversely, ectopic overexpression of LeHB-1 by viral delivery to developing flowers elsewhere on injected plants triggered altered floral organ morphology, including production of multiple flowers within one sepal whorl, fusion of sepals and petals, and conversion of sepals into carpel-like structures that grew into fruits and ripened. Our findings suggest that LeHB-1 is not only involved in the control of ripening but also plays a critical role in floral organogenesis.

Show MeSH

Related in: MedlinePlus

Effects of silencing LeHB-1 on ripening and LeACO1 expression.(a) The PVX/GFP vector (Wezel et al., 2002) is shown together with the wild-type LeHB-1 and the mutated LeHB-1 (asterisked; Table 1) genes used to create PVX/LeHB1::GFP and PVX/mLeHB1::GFP, respectively. The PVX 166K-RDRP, movement proteins (25, 12 and 8K) and coat protein (CP) are indicated. The triangles show the positions of primers (Table 1) for detecting the transgene.(b) Fruit injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (panels 1–4) and fruit injected with PVX/GFP (panels 5, Ctl). Photographs were taken 4 weeks post-injection.(c) Silencing endogenous LeHB-1 downregulated LeACO1 in virus-induced gene silencing (VIGS) fruits. Total RNA (10 μg) from delayed-ripening fruits injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (from panels 1, 2 and 3) (lanes 1–3) and PVX/GFP control fruit (lane ctl) were used for northern analysis using the 5′-UTR of LeHB-1 (HB1) and the first exon of LeACO1 (ACO1) as probes. The quantification of LeHB-1 and LeACO1 mRNA by 32P radioactivity emission is given as a percentage. Viral transient LeHB-1 (HB1-trans) in the PVX genome and subgenomes was detected in PVX/LeHB1::GFP- or PVX/mLeHB1::GFP-injected fruits (lanes 1–3), but not in control fruits (lane Ctl). Ethidium bromide stained rRNA (rRNA) indicates RNA loading.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2607530&req=5

fig04: Effects of silencing LeHB-1 on ripening and LeACO1 expression.(a) The PVX/GFP vector (Wezel et al., 2002) is shown together with the wild-type LeHB-1 and the mutated LeHB-1 (asterisked; Table 1) genes used to create PVX/LeHB1::GFP and PVX/mLeHB1::GFP, respectively. The PVX 166K-RDRP, movement proteins (25, 12 and 8K) and coat protein (CP) are indicated. The triangles show the positions of primers (Table 1) for detecting the transgene.(b) Fruit injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (panels 1–4) and fruit injected with PVX/GFP (panels 5, Ctl). Photographs were taken 4 weeks post-injection.(c) Silencing endogenous LeHB-1 downregulated LeACO1 in virus-induced gene silencing (VIGS) fruits. Total RNA (10 μg) from delayed-ripening fruits injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (from panels 1, 2 and 3) (lanes 1–3) and PVX/GFP control fruit (lane ctl) were used for northern analysis using the 5′-UTR of LeHB-1 (HB1) and the first exon of LeACO1 (ACO1) as probes. The quantification of LeHB-1 and LeACO1 mRNA by 32P radioactivity emission is given as a percentage. Viral transient LeHB-1 (HB1-trans) in the PVX genome and subgenomes was detected in PVX/LeHB1::GFP- or PVX/mLeHB1::GFP-injected fruits (lanes 1–3), but not in control fruits (lane Ctl). Ethidium bromide stained rRNA (rRNA) indicates RNA loading.

Mentions: Three LeACO1 promoter regions (F1-1, F3-1 and F4-1), containing predicted homeobox cis-elements similar to the AtHB-1 binding sequence (CAATA/TATTG) (Figure 3c), were selected for the GST::HD-Zip/DNA binding analysis. The 141-bp F1-1 promoter fragment contains a 10-bp sequence AATA(AA)TATT with dyad symmetry, F3-1 (119 bp) has a 9-bp sequence CAAT(A)ATGG, and F4-1 (128 bp) has a 9-bp sequence AATA(A)TATT with dyad symmetry (Figure 3c). These three fragments were PCR amplified and sequenced. Incubation of GST::HD-Zip with either F1-1 or F4-1 resulted in a DNA–protein complex that showed an electrophoretic mobility shift compared with free DNA fragments (Figure 3d). The formation of the DNA–protein complex was specific, and was out-competed by a 200-fold molar excess of unlabelled starting DNA of each respective promoter region. However, the GST::HD-Zip fusion did not produce a similar complex with the fragment F3-1 (data not shown). No DNA–protein complex was formed between any of the promoter fragments and free GST (Figure 4d). These findings demonstrate that the LeHB-1 HD-Zip is capable of binding to the LeACO1 promoter, probably by recognizing the 9 or 10-bp DNA sequences with dyad symmetry, indicating that LeHB-1 might be involved in transcriptional regulation of LeACO1.


A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening.

Lin Z, Hong Y, Yin M, Li C, Zhang K, Grierson D - Plant J. (2008)

Effects of silencing LeHB-1 on ripening and LeACO1 expression.(a) The PVX/GFP vector (Wezel et al., 2002) is shown together with the wild-type LeHB-1 and the mutated LeHB-1 (asterisked; Table 1) genes used to create PVX/LeHB1::GFP and PVX/mLeHB1::GFP, respectively. The PVX 166K-RDRP, movement proteins (25, 12 and 8K) and coat protein (CP) are indicated. The triangles show the positions of primers (Table 1) for detecting the transgene.(b) Fruit injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (panels 1–4) and fruit injected with PVX/GFP (panels 5, Ctl). Photographs were taken 4 weeks post-injection.(c) Silencing endogenous LeHB-1 downregulated LeACO1 in virus-induced gene silencing (VIGS) fruits. Total RNA (10 μg) from delayed-ripening fruits injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (from panels 1, 2 and 3) (lanes 1–3) and PVX/GFP control fruit (lane ctl) were used for northern analysis using the 5′-UTR of LeHB-1 (HB1) and the first exon of LeACO1 (ACO1) as probes. The quantification of LeHB-1 and LeACO1 mRNA by 32P radioactivity emission is given as a percentage. Viral transient LeHB-1 (HB1-trans) in the PVX genome and subgenomes was detected in PVX/LeHB1::GFP- or PVX/mLeHB1::GFP-injected fruits (lanes 1–3), but not in control fruits (lane Ctl). Ethidium bromide stained rRNA (rRNA) indicates RNA loading.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2607530&req=5

fig04: Effects of silencing LeHB-1 on ripening and LeACO1 expression.(a) The PVX/GFP vector (Wezel et al., 2002) is shown together with the wild-type LeHB-1 and the mutated LeHB-1 (asterisked; Table 1) genes used to create PVX/LeHB1::GFP and PVX/mLeHB1::GFP, respectively. The PVX 166K-RDRP, movement proteins (25, 12 and 8K) and coat protein (CP) are indicated. The triangles show the positions of primers (Table 1) for detecting the transgene.(b) Fruit injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (panels 1–4) and fruit injected with PVX/GFP (panels 5, Ctl). Photographs were taken 4 weeks post-injection.(c) Silencing endogenous LeHB-1 downregulated LeACO1 in virus-induced gene silencing (VIGS) fruits. Total RNA (10 μg) from delayed-ripening fruits injected with PVX/LeHB1::GFP or PVX/mLeHB1::GFP (from panels 1, 2 and 3) (lanes 1–3) and PVX/GFP control fruit (lane ctl) were used for northern analysis using the 5′-UTR of LeHB-1 (HB1) and the first exon of LeACO1 (ACO1) as probes. The quantification of LeHB-1 and LeACO1 mRNA by 32P radioactivity emission is given as a percentage. Viral transient LeHB-1 (HB1-trans) in the PVX genome and subgenomes was detected in PVX/LeHB1::GFP- or PVX/mLeHB1::GFP-injected fruits (lanes 1–3), but not in control fruits (lane Ctl). Ethidium bromide stained rRNA (rRNA) indicates RNA loading.
Mentions: Three LeACO1 promoter regions (F1-1, F3-1 and F4-1), containing predicted homeobox cis-elements similar to the AtHB-1 binding sequence (CAATA/TATTG) (Figure 3c), were selected for the GST::HD-Zip/DNA binding analysis. The 141-bp F1-1 promoter fragment contains a 10-bp sequence AATA(AA)TATT with dyad symmetry, F3-1 (119 bp) has a 9-bp sequence CAAT(A)ATGG, and F4-1 (128 bp) has a 9-bp sequence AATA(A)TATT with dyad symmetry (Figure 3c). These three fragments were PCR amplified and sequenced. Incubation of GST::HD-Zip with either F1-1 or F4-1 resulted in a DNA–protein complex that showed an electrophoretic mobility shift compared with free DNA fragments (Figure 3d). The formation of the DNA–protein complex was specific, and was out-competed by a 200-fold molar excess of unlabelled starting DNA of each respective promoter region. However, the GST::HD-Zip fusion did not produce a similar complex with the fragment F3-1 (data not shown). No DNA–protein complex was formed between any of the promoter fragments and free GST (Figure 4d). These findings demonstrate that the LeHB-1 HD-Zip is capable of binding to the LeACO1 promoter, probably by recognizing the 9 or 10-bp DNA sequences with dyad symmetry, indicating that LeHB-1 might be involved in transcriptional regulation of LeACO1.

Bottom Line: Inhibition of ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development.Inhibition of LeHB-1 mRNA accumulation in tomato fruit, using virus-induced gene silencing, greatly reduced LeACO1 mRNA levels, and inhibited ripening.Our findings suggest that LeHB-1 is not only involved in the control of ripening but also plays a critical role in floral organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK.

ABSTRACT
Ethylene is required for climacteric fruit ripening. Inhibition of ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development. LeHB-1, a previously uncharacterized tomato homeobox protein, was shown by gel retardation assay to interact with the promoter of LeACO1, an ACC oxidase gene expressed during ripening. Inhibition of LeHB-1 mRNA accumulation in tomato fruit, using virus-induced gene silencing, greatly reduced LeACO1 mRNA levels, and inhibited ripening. Conversely, ectopic overexpression of LeHB-1 by viral delivery to developing flowers elsewhere on injected plants triggered altered floral organ morphology, including production of multiple flowers within one sepal whorl, fusion of sepals and petals, and conversion of sepals into carpel-like structures that grew into fruits and ripened. Our findings suggest that LeHB-1 is not only involved in the control of ripening but also plays a critical role in floral organogenesis.

Show MeSH
Related in: MedlinePlus