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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 firmness in SlARF2AB-RNAi fruits.(A) Firmness of wild-type and SlARF2AB-RNAi fruits. Fruits were harvested at breaker stage, kept at room temperate and firmness was measured day by day. A total of 15 fruits were used for each measurement and the error bars represent ±SD. AB1 = SlARF2AB-RNAi line 311; AB2 = SlARF2AB-RNAi line 223. (B) Quantitative RT-PCR relative expression of polygalacturonase gene PG2A at different ripening stages in SlARF2AB-RNAi and wild type fruits (breaker, Br; Br+2, 2 d post-breaker; Br+8, 8 d post-breaker). Relative mRNA levels in WT at the breaker (Br) stage were standardized to 1.0, referring to SlActin gene as internal control. Error bars represent ±SD of three biological replicates. Stars indicate a statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. ABL1 is SlARF2AB-RNAi line 311.
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pgen.1005649.g011: Altered firmness in SlARF2AB-RNAi fruits.(A) Firmness of wild-type and SlARF2AB-RNAi fruits. Fruits were harvested at breaker stage, kept at room temperate and firmness was measured day by day. A total of 15 fruits were used for each measurement and the error bars represent ±SD. AB1 = SlARF2AB-RNAi line 311; AB2 = SlARF2AB-RNAi line 223. (B) Quantitative RT-PCR relative expression of polygalacturonase gene PG2A at different ripening stages in SlARF2AB-RNAi and wild type fruits (breaker, Br; Br+2, 2 d post-breaker; Br+8, 8 d post-breaker). Relative mRNA levels in WT at the breaker (Br) stage were standardized to 1.0, referring to SlActin gene as internal control. Error bars represent ±SD of three biological replicates. Stars indicate a statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. ABL1 is SlARF2AB-RNAi line 311.

Mentions: The fruit color saturation assessed by Hue angle, indicative of color intensity, revealed a reduced red pigment accumulation in SlARF2AB down-regulated lines (Fig 10). Accordingly, the expression of genes involved in the carotenoid pathway was altered. PSY1, a key regulator of flux through the carotenoid pathway, was significantly down-regulated in the SlARF2AB-RNAi fruits at all ripening stages (Fig 10). Lower levels of phytoene desaturase (PDS) and phytoene synthase (ZDS) transcripts were also observed at Br+2 stage in the SlARF2AB-RNAi fruit. By contrast, transcripts corresponding to lycopene beta cyclase genes (β-LCY1, β-LCY2) displayed higher accumulation than in wild-type at all ripening stages, and those corresponding to lycopene β-cyclases (CYCB) were also up-regulated at Br and Br+2 stages in SlARF2AB-RNAi fruit (Fig 10). On the other hand, SlARF2AB-RNAi fruits maintained higher firmness than wild type throughout ripening (Fig 11). In line with this delayed softening phenotype, transcript accumulation of PG2A, a major fruit polygalacturonase gene involved in ripening-related cell wall metabolism, was significantly reduced at Br, Br+2, and Br+8 stages in SlARF2AB-RNAi fruits (Fig 11).


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 firmness in SlARF2AB-RNAi fruits.(A) Firmness of wild-type and SlARF2AB-RNAi fruits. Fruits were harvested at breaker stage, kept at room temperate and firmness was measured day by day. A total of 15 fruits were used for each measurement and the error bars represent ±SD. AB1 = SlARF2AB-RNAi line 311; AB2 = SlARF2AB-RNAi line 223. (B) Quantitative RT-PCR relative expression of polygalacturonase gene PG2A at different ripening stages in SlARF2AB-RNAi and wild type fruits (breaker, Br; Br+2, 2 d post-breaker; Br+8, 8 d post-breaker). Relative mRNA levels in WT at the breaker (Br) stage were standardized to 1.0, referring to SlActin gene as internal control. Error bars represent ±SD of three biological replicates. Stars indicate a statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. ABL1 is SlARF2AB-RNAi line 311.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005649.g011: Altered firmness in SlARF2AB-RNAi fruits.(A) Firmness of wild-type and SlARF2AB-RNAi fruits. Fruits were harvested at breaker stage, kept at room temperate and firmness was measured day by day. A total of 15 fruits were used for each measurement and the error bars represent ±SD. AB1 = SlARF2AB-RNAi line 311; AB2 = SlARF2AB-RNAi line 223. (B) Quantitative RT-PCR relative expression of polygalacturonase gene PG2A at different ripening stages in SlARF2AB-RNAi and wild type fruits (breaker, Br; Br+2, 2 d post-breaker; Br+8, 8 d post-breaker). Relative mRNA levels in WT at the breaker (Br) stage were standardized to 1.0, referring to SlActin gene as internal control. Error bars represent ±SD of three biological replicates. Stars indicate a statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. ABL1 is SlARF2AB-RNAi line 311.
Mentions: The fruit color saturation assessed by Hue angle, indicative of color intensity, revealed a reduced red pigment accumulation in SlARF2AB down-regulated lines (Fig 10). Accordingly, the expression of genes involved in the carotenoid pathway was altered. PSY1, a key regulator of flux through the carotenoid pathway, was significantly down-regulated in the SlARF2AB-RNAi fruits at all ripening stages (Fig 10). Lower levels of phytoene desaturase (PDS) and phytoene synthase (ZDS) transcripts were also observed at Br+2 stage in the SlARF2AB-RNAi fruit. By contrast, transcripts corresponding to lycopene beta cyclase genes (β-LCY1, β-LCY2) displayed higher accumulation than in wild-type at all ripening stages, and those corresponding to lycopene β-cyclases (CYCB) were also up-regulated at Br and Br+2 stages in SlARF2AB-RNAi fruit (Fig 10). On the other hand, SlARF2AB-RNAi fruits maintained higher firmness than wild type throughout ripening (Fig 11). In line with this delayed softening phenotype, transcript accumulation of PG2A, a major fruit polygalacturonase gene involved in ripening-related cell wall metabolism, was significantly reduced at Br, Br+2, and Br+8 stages in SlARF2AB-RNAi fruits (Fig 11).

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