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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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The expression of a number of ripening-related genes is altered in SlARF2AB-RNAi plants.Quantitative RT-PCR relative expression of ripening regulator genes in wild-type (WT) and SlARF2AB-RNAi lines during fruit ripening. Total RNA was extracted from the indicated developmental stages of fruit (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at the breaker (Br) stage were standardized to 1.0, referring to the SlActin gene as internal control. Error bar means ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. AP2a, APETALA2/ERF gene; CNR, colorless non-ripening; HB-1, HD-Zip homeobox; NOR, non-ripening; RIN, ripening inhibitor; TAGL1, tomato AGAMOUS-LIKE 1. FUL1, FUL2 MADS domain transcription factors; E4, E8 ethylene-responsive and ripening-regulated genes.
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pgen.1005649.g012: The expression of a number of ripening-related genes is altered in SlARF2AB-RNAi plants.Quantitative RT-PCR relative expression of ripening regulator genes in wild-type (WT) and SlARF2AB-RNAi lines during fruit ripening. Total RNA was extracted from the indicated developmental stages of fruit (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at the breaker (Br) stage were standardized to 1.0, referring to the SlActin gene as internal control. Error bar means ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. AP2a, APETALA2/ERF gene; CNR, colorless non-ripening; HB-1, HD-Zip homeobox; NOR, non-ripening; RIN, ripening inhibitor; TAGL1, tomato AGAMOUS-LIKE 1. FUL1, FUL2 MADS domain transcription factors; E4, E8 ethylene-responsive and ripening-regulated genes.

Mentions: The expression of key ripening regulators assessed at the transcript level was strongly reduced throughout ripening in the SlARF2 RNAi line. Compared to wild type fruit, transcript levels corresponding to RIN and CNR genes were significantly lower at Br, Br+2 and Br+8 stages (Fig 12). Likewise, the NOR gene displayed reduced expression at Br and Br+8 stages, TAGL1 showed the same tendency at Br and Br+2 stages, FUL1 at Br and Br+2 stages, and FUL2 at Br+2 and Br+8 stages. The altered expression of these genes is consistent with the dramatically altered ripening of SlARF2AB-RNAi fruits. Likewise, the low expression level of E8 and E4, two ethylene-responsive and ripening- associated genes, is consistent with the altered expression of ethylene biosynthesis and signaling genes. By contrast, mRNA levels of LeHB-1, another ripening regulator gene, did not display significant change in SlARF2AB-RNAi fruits compared to wild type (Fig 12).


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)

The expression of a number of ripening-related genes is altered in SlARF2AB-RNAi plants.Quantitative RT-PCR relative expression of ripening regulator genes in wild-type (WT) and SlARF2AB-RNAi lines during fruit ripening. Total RNA was extracted from the indicated developmental stages of fruit (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at the breaker (Br) stage were standardized to 1.0, referring to the SlActin gene as internal control. Error bar means ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. AP2a, APETALA2/ERF gene; CNR, colorless non-ripening; HB-1, HD-Zip homeobox; NOR, non-ripening; RIN, ripening inhibitor; TAGL1, tomato AGAMOUS-LIKE 1. FUL1, FUL2 MADS domain transcription factors; E4, E8 ethylene-responsive and ripening-regulated genes.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005649.g012: The expression of a number of ripening-related genes is altered in SlARF2AB-RNAi plants.Quantitative RT-PCR relative expression of ripening regulator genes in wild-type (WT) and SlARF2AB-RNAi lines during fruit ripening. Total RNA was extracted from the indicated developmental stages of fruit (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at the breaker (Br) stage were standardized to 1.0, referring to the SlActin gene as internal control. Error bar means ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. AP2a, APETALA2/ERF gene; CNR, colorless non-ripening; HB-1, HD-Zip homeobox; NOR, non-ripening; RIN, ripening inhibitor; TAGL1, tomato AGAMOUS-LIKE 1. FUL1, FUL2 MADS domain transcription factors; E4, E8 ethylene-responsive and ripening-regulated genes.
Mentions: The expression of key ripening regulators assessed at the transcript level was strongly reduced throughout ripening in the SlARF2 RNAi line. Compared to wild type fruit, transcript levels corresponding to RIN and CNR genes were significantly lower at Br, Br+2 and Br+8 stages (Fig 12). Likewise, the NOR gene displayed reduced expression at Br and Br+8 stages, TAGL1 showed the same tendency at Br and Br+2 stages, FUL1 at Br and Br+2 stages, and FUL2 at Br+2 and Br+8 stages. The altered expression of these genes is consistent with the dramatically altered ripening of SlARF2AB-RNAi fruits. Likewise, the low expression level of E8 and E4, two ethylene-responsive and ripening- associated genes, is consistent with the altered expression of ethylene biosynthesis and signaling genes. By contrast, mRNA levels of LeHB-1, another ripening regulator gene, did not display significant change in SlARF2AB-RNAi fruits compared to wild type (Fig 12).

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