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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.

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Altered ripening phenotypes of SlARF2 down-regulated lines.(A) Ripening phenotypes of SlARF2A-RNAi; SlARF2B-RNAi and SlARF2AB-RNAi fruits at mature green (upper panel) and ripe (lower panel) stages. The SlARF2A/SlARF2B-RNAi fruits show spiky phenotype at mature green stage and ripe stage fruits, SlARF2AB-RNAi mutant displays inhibited ripening. (B) Time (number of days) from anthesis to breaker in wild type and two independent SlARF2AB-RNAi lines. (C) Ripening phenotypes of wild-type (WT) and SlARF2AB-RNAi fruits. Transgenic fruits never reach a full red color. Br = breaker stage; Br+3 = 3 days post-breaker stage; Br+5 = 5 days post-breaker stage; Br+7 = 7 days post-breaker stage. (D) Effect of ethylene treatment on wild type (WT) and SlARF2AB-RNAi fruit. Mature green fruits from WT and SlARF2AB-RNAi lines were treated 2 hours and 3 times per day with 10 ppm ethylene or with air for 3 days. After 7 days, both ethylene treated and untreated wild type fruit reached full red while SlARF2AB-RNAi fruits treated or untreated displayed orange sectors on the fruit surface and never get red.
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pgen.1005649.g006: Altered ripening phenotypes of SlARF2 down-regulated lines.(A) Ripening phenotypes of SlARF2A-RNAi; SlARF2B-RNAi and SlARF2AB-RNAi fruits at mature green (upper panel) and ripe (lower panel) stages. The SlARF2A/SlARF2B-RNAi fruits show spiky phenotype at mature green stage and ripe stage fruits, SlARF2AB-RNAi mutant displays inhibited ripening. (B) Time (number of days) from anthesis to breaker in wild type and two independent SlARF2AB-RNAi lines. (C) Ripening phenotypes of wild-type (WT) and SlARF2AB-RNAi fruits. Transgenic fruits never reach a full red color. Br = breaker stage; Br+3 = 3 days post-breaker stage; Br+5 = 5 days post-breaker stage; Br+7 = 7 days post-breaker stage. (D) Effect of ethylene treatment on wild type (WT) and SlARF2AB-RNAi fruit. Mature green fruits from WT and SlARF2AB-RNAi lines were treated 2 hours and 3 times per day with 10 ppm ethylene or with air for 3 days. After 7 days, both ethylene treated and untreated wild type fruit reached full red while SlARF2AB-RNAi fruits treated or untreated displayed orange sectors on the fruit surface and never get red.

Mentions: Considering the ripening-associated pattern of both SlARF2A and SlARF2B, we sought to analyze the fruit phenotypes of SlARF2A and SlARF2B single and double knockdown tomato lines. In both SlARF2A and SlARF2B-RNAi single knockdown lines, the fruit exhibited dark green spots at immature and mature green stages, and then displayed a mottled pattern of ripening with yellow/orange spots on the skin remaining till the full mature stage (Fig 6). The double silenced lines exhibited more severe ripening defects with yellow and orange patches never reaching the typical red color of wild type or out-segregating lines, again suggesting that SlARF2A and SlARF2B may have redundant function in fruit ripening (Fig 6A). Assessing the time period from anthesis to breaker stage revealed a slight but statistically significant delay (2 to 3 days delay) in the onset of ripening between wild type and double knockdown lines (Fig 6B). The fruit color in SlARF2AB-RNAi lines never get fully red (Fig 6C) and full ripening cannot be recovered upon exogenous ethylene treatment of the SlARF2A/B RNAi double knockdown fruits which suggests a possible alteration in ethylene perception or response (Fig 6D).


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)

Altered ripening phenotypes of SlARF2 down-regulated lines.(A) Ripening phenotypes of SlARF2A-RNAi; SlARF2B-RNAi and SlARF2AB-RNAi fruits at mature green (upper panel) and ripe (lower panel) stages. The SlARF2A/SlARF2B-RNAi fruits show spiky phenotype at mature green stage and ripe stage fruits, SlARF2AB-RNAi mutant displays inhibited ripening. (B) Time (number of days) from anthesis to breaker in wild type and two independent SlARF2AB-RNAi lines. (C) Ripening phenotypes of wild-type (WT) and SlARF2AB-RNAi fruits. Transgenic fruits never reach a full red color. Br = breaker stage; Br+3 = 3 days post-breaker stage; Br+5 = 5 days post-breaker stage; Br+7 = 7 days post-breaker stage. (D) Effect of ethylene treatment on wild type (WT) and SlARF2AB-RNAi fruit. Mature green fruits from WT and SlARF2AB-RNAi lines were treated 2 hours and 3 times per day with 10 ppm ethylene or with air for 3 days. After 7 days, both ethylene treated and untreated wild type fruit reached full red while SlARF2AB-RNAi fruits treated or untreated displayed orange sectors on the fruit surface and never get red.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696797&req=5

pgen.1005649.g006: Altered ripening phenotypes of SlARF2 down-regulated lines.(A) Ripening phenotypes of SlARF2A-RNAi; SlARF2B-RNAi and SlARF2AB-RNAi fruits at mature green (upper panel) and ripe (lower panel) stages. The SlARF2A/SlARF2B-RNAi fruits show spiky phenotype at mature green stage and ripe stage fruits, SlARF2AB-RNAi mutant displays inhibited ripening. (B) Time (number of days) from anthesis to breaker in wild type and two independent SlARF2AB-RNAi lines. (C) Ripening phenotypes of wild-type (WT) and SlARF2AB-RNAi fruits. Transgenic fruits never reach a full red color. Br = breaker stage; Br+3 = 3 days post-breaker stage; Br+5 = 5 days post-breaker stage; Br+7 = 7 days post-breaker stage. (D) Effect of ethylene treatment on wild type (WT) and SlARF2AB-RNAi fruit. Mature green fruits from WT and SlARF2AB-RNAi lines were treated 2 hours and 3 times per day with 10 ppm ethylene or with air for 3 days. After 7 days, both ethylene treated and untreated wild type fruit reached full red while SlARF2AB-RNAi fruits treated or untreated displayed orange sectors on the fruit surface and never get red.
Mentions: Considering the ripening-associated pattern of both SlARF2A and SlARF2B, we sought to analyze the fruit phenotypes of SlARF2A and SlARF2B single and double knockdown tomato lines. In both SlARF2A and SlARF2B-RNAi single knockdown lines, the fruit exhibited dark green spots at immature and mature green stages, and then displayed a mottled pattern of ripening with yellow/orange spots on the skin remaining till the full mature stage (Fig 6). The double silenced lines exhibited more severe ripening defects with yellow and orange patches never reaching the typical red color of wild type or out-segregating lines, again suggesting that SlARF2A and SlARF2B may have redundant function in fruit ripening (Fig 6A). Assessing the time period from anthesis to breaker stage revealed a slight but statistically significant delay (2 to 3 days delay) in the onset of ripening between wild type and double knockdown lines (Fig 6B). The fruit color in SlARF2AB-RNAi lines never get fully red (Fig 6C) and full ripening cannot be recovered upon exogenous ethylene treatment of the SlARF2A/B RNAi double knockdown fruits which suggests a possible alteration in ethylene perception or response (Fig 6D).

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