Limits...
Auxin Response Factor SlARF2 Is an Essential Component of the Regulatory Mechanism Controlling Fruit Ripening in Tomato.

Hao Y, Hu G, Breitel D, Liu M, Mila I, Frasse P, Fu Y, Aharoni A, Bouzayen M, Zouine M - PLoS Genet. (2015)

Bottom Line: Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin.Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits.Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening.

View Article: PubMed Central - PubMed

Affiliation: University of Toulouse, INPT, Laboratory of Genomics and Biotechnology of Fruit, Castanet-Tolosan, France.

ABSTRACT
Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.

Show MeSH

Related in: MedlinePlus

Subcellular localization and functional analysis of SlARF2A and SlARF2B by single cell system.(A) Subcellular localization of tomato SlARF2A/2B proteins. SlARF2A/2B-GFP fusion proteins were transiently expressed in BY-2 tobacco protoplasts and subcellular localization was analyzed by confocal laser scanning microscopy. The merged pictures of the green fluorescence channel (left panels) and the corresponding bright field (middle panels) are shown in the right panels. The scale bar indicates 10 μm. The top pictures correspond to control cells expressing GFP alone. The middle and bottom pictures correspond to cells expressing the SlARF2A-GFP and SlARF2B-GFP fusion proteins, respectively. (B) SlARF2A/2B protein represses the activity of DR5 in vivo. SlARF2A/2B proteins were challenged with a synthetic auxin-responsive promoter called DR5 fused to the GFP reporter gene. A transient expression assay using a single cell system was performed to measure the reporter gene activity. Tobacco protoplasts were transformed either with the reporter construct (DR5::GFP) alone or with both the reporter and effector constructs (35S::SlARF2A/2B) and incubated in the presence or absence of 50 μM 2,4-D. GFP fluorescence was measured 16 h after transfection. For each assay, three biological replicates were performed. GFP mean fluorescence is indicated in arbitrary unit (a.u.) ± standard error.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696797&req=5

pgen.1005649.g003: Subcellular localization and functional analysis of SlARF2A and SlARF2B by single cell system.(A) Subcellular localization of tomato SlARF2A/2B proteins. SlARF2A/2B-GFP fusion proteins were transiently expressed in BY-2 tobacco protoplasts and subcellular localization was analyzed by confocal laser scanning microscopy. The merged pictures of the green fluorescence channel (left panels) and the corresponding bright field (middle panels) are shown in the right panels. The scale bar indicates 10 μm. The top pictures correspond to control cells expressing GFP alone. The middle and bottom pictures correspond to cells expressing the SlARF2A-GFP and SlARF2B-GFP fusion proteins, respectively. (B) SlARF2A/2B protein represses the activity of DR5 in vivo. SlARF2A/2B proteins were challenged with a synthetic auxin-responsive promoter called DR5 fused to the GFP reporter gene. A transient expression assay using a single cell system was performed to measure the reporter gene activity. Tobacco protoplasts were transformed either with the reporter construct (DR5::GFP) alone or with both the reporter and effector constructs (35S::SlARF2A/2B) and incubated in the presence or absence of 50 μM 2,4-D. GFP fluorescence was measured 16 h after transfection. For each assay, three biological replicates were performed. GFP mean fluorescence is indicated in arbitrary unit (a.u.) ± standard error.

Mentions: The subcellular localization of SlARF2A and SlARF2B proteins was then assessed using translational fusion to the Green Fluorescent Protein (GFP) in a tobacco protoplast transient expression assay. Microscopy analysis clearly showed that SlARF2A/2B:GFP fusion proteins exclusively localized into the nucleus (Fig 3A), consistent with their putative role in transcriptional regulation activity. The ability of SlARF2A/2B proteins to regulate the activity of auxin-responsive promoters was then evaluated in a single cell system. A reporter construct, consisting of the synthetic auxin-responsive promoter DR5 fused to GFP [41], was co-transfected into tobacco protoplasts with an effector construct allowing the constitutive expression of SlARF2A or SlARF2B protein. As expected the DR5-driven GFP expression was strongly enhanced by auxin (2,4-D) treatment. However, the presence of either SlARF2A or SlARF2B proteins strongly inhibited this auxin-induced activity of DR5 promoter, clearly demonstrating that SlARF2A and SlARF2B act in vivo as strong transcriptional repressors of auxin-dependent gene transcription (Fig 3B).


Auxin Response Factor SlARF2 Is an Essential Component of the Regulatory Mechanism Controlling Fruit Ripening in Tomato.

Hao Y, Hu G, Breitel D, Liu M, Mila I, Frasse P, Fu Y, Aharoni A, Bouzayen M, Zouine M - PLoS Genet. (2015)

Subcellular localization and functional analysis of SlARF2A and SlARF2B by single cell system.(A) Subcellular localization of tomato SlARF2A/2B proteins. SlARF2A/2B-GFP fusion proteins were transiently expressed in BY-2 tobacco protoplasts and subcellular localization was analyzed by confocal laser scanning microscopy. The merged pictures of the green fluorescence channel (left panels) and the corresponding bright field (middle panels) are shown in the right panels. The scale bar indicates 10 μm. The top pictures correspond to control cells expressing GFP alone. The middle and bottom pictures correspond to cells expressing the SlARF2A-GFP and SlARF2B-GFP fusion proteins, respectively. (B) SlARF2A/2B protein represses the activity of DR5 in vivo. SlARF2A/2B proteins were challenged with a synthetic auxin-responsive promoter called DR5 fused to the GFP reporter gene. A transient expression assay using a single cell system was performed to measure the reporter gene activity. Tobacco protoplasts were transformed either with the reporter construct (DR5::GFP) alone or with both the reporter and effector constructs (35S::SlARF2A/2B) and incubated in the presence or absence of 50 μM 2,4-D. GFP fluorescence was measured 16 h after transfection. For each assay, three biological replicates were performed. GFP mean fluorescence is indicated in arbitrary unit (a.u.) ± standard error.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696797&req=5

pgen.1005649.g003: Subcellular localization and functional analysis of SlARF2A and SlARF2B by single cell system.(A) Subcellular localization of tomato SlARF2A/2B proteins. SlARF2A/2B-GFP fusion proteins were transiently expressed in BY-2 tobacco protoplasts and subcellular localization was analyzed by confocal laser scanning microscopy. The merged pictures of the green fluorescence channel (left panels) and the corresponding bright field (middle panels) are shown in the right panels. The scale bar indicates 10 μm. The top pictures correspond to control cells expressing GFP alone. The middle and bottom pictures correspond to cells expressing the SlARF2A-GFP and SlARF2B-GFP fusion proteins, respectively. (B) SlARF2A/2B protein represses the activity of DR5 in vivo. SlARF2A/2B proteins were challenged with a synthetic auxin-responsive promoter called DR5 fused to the GFP reporter gene. A transient expression assay using a single cell system was performed to measure the reporter gene activity. Tobacco protoplasts were transformed either with the reporter construct (DR5::GFP) alone or with both the reporter and effector constructs (35S::SlARF2A/2B) and incubated in the presence or absence of 50 μM 2,4-D. GFP fluorescence was measured 16 h after transfection. For each assay, three biological replicates were performed. GFP mean fluorescence is indicated in arbitrary unit (a.u.) ± standard error.
Mentions: The subcellular localization of SlARF2A and SlARF2B proteins was then assessed using translational fusion to the Green Fluorescent Protein (GFP) in a tobacco protoplast transient expression assay. Microscopy analysis clearly showed that SlARF2A/2B:GFP fusion proteins exclusively localized into the nucleus (Fig 3A), consistent with their putative role in transcriptional regulation activity. The ability of SlARF2A/2B proteins to regulate the activity of auxin-responsive promoters was then evaluated in a single cell system. A reporter construct, consisting of the synthetic auxin-responsive promoter DR5 fused to GFP [41], was co-transfected into tobacco protoplasts with an effector construct allowing the constitutive expression of SlARF2A or SlARF2B protein. As expected the DR5-driven GFP expression was strongly enhanced by auxin (2,4-D) treatment. However, the presence of either SlARF2A or SlARF2B proteins strongly inhibited this auxin-induced activity of DR5 promoter, clearly demonstrating that SlARF2A and SlARF2B act in vivo as strong transcriptional repressors of auxin-dependent gene transcription (Fig 3B).

Bottom Line: Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin.Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits.Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening.

View Article: PubMed Central - PubMed

Affiliation: University of Toulouse, INPT, Laboratory of Genomics and Biotechnology of Fruit, Castanet-Tolosan, France.

ABSTRACT
Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.

Show MeSH
Related in: MedlinePlus