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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 ethylene synthesis and ethylene perception genes is altered in SlARF2AB-RNAi plants.(A) Expression of ethylene synthesis pathway genes in SlARF2AB-RNAi lines assed by Quantitative RT-PCR. ACO1, ACO2, ACO3, ACO4 aminocyclopropane-1-carboxylic acid oxidase; ACS1, ACS2, ACS3, ACS4, ACS6 aminocyclopropane-1-carboxylic acid synthases. (B) Expression of ethylene perception genes in SlARF2AB-RNAi assessed by Quantitative RT-PCR. EIN2 ethylene signaling protein; EIL2 and EIL3 are EIN3-like proteins; ETR1, ETR2, ETR3 (NR, never-ripe), ETR4, ETR5, ETR6 ethylene receptors; CTR1 ethylene-responsive protein kinase. ABL1 refers to SlARF2AB-RNAi line 311. Total RNA was extracted from different fruit developmental stages (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 bars mean ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01.
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pgen.1005649.g008: The expression of ethylene synthesis and ethylene perception genes is altered in SlARF2AB-RNAi plants.(A) Expression of ethylene synthesis pathway genes in SlARF2AB-RNAi lines assed by Quantitative RT-PCR. ACO1, ACO2, ACO3, ACO4 aminocyclopropane-1-carboxylic acid oxidase; ACS1, ACS2, ACS3, ACS4, ACS6 aminocyclopropane-1-carboxylic acid synthases. (B) Expression of ethylene perception genes in SlARF2AB-RNAi assessed by Quantitative RT-PCR. EIN2 ethylene signaling protein; EIL2 and EIL3 are EIN3-like proteins; ETR1, ETR2, ETR3 (NR, never-ripe), ETR4, ETR5, ETR6 ethylene receptors; CTR1 ethylene-responsive protein kinase. ABL1 refers to SlARF2AB-RNAi line 311. Total RNA was extracted from different fruit developmental stages (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 bars mean ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01.

Mentions: The ripening defect phenotype prompted us to monitor the climacteric ethylene production in the SlARF2AB-RNAi line. Ethylene production, assessed either on fruits kept on the plant or detached (Fig 7), is significantly low throughout ripening and reaches its peak with 3 days delay as compared to wild type (Fig 7). Assessing the expression of ethylene biosynthesis genes by qPCR (Fig 8A) revealed reduced levels of ACO1, ACS2, ACS3 and ACS4 transcripts in the SlARF2AB RNAi line at all ripening stages (Breaker, Breaker+2 and Breaker+8). However, the reduced ethylene production cannot account for the ripening defects because exogenous ethylene treatment failed to reverse the ripening phenotype (Fig 6D). We therefore examined the expression of ethylene receptor genes (Fig 8B). transcript levels corresponding to ETR3 (NR) and ETR4 are dramatically low in the transgenic lines compared to wild type at all stages of fruit ripening (Br, Br+2, and Br+8) and the expression of other receptor genes (ETR1, ETR2, and ETR5) is also down-regulated at the breaker+8 stage. The disturbed expression of ethylene receptor genes is likely to result in altered ethylene perception in the transgenic lines. In addition, the expression of EIN2 and two EIN3-like genes (EIL2 and EIL3), which encode major components of ethylene transduction pathways, was also down-regulated during ripening of SlARF2A/B RNAi fruit (Fig 8B). More striking, the expression of a high number of ERF genes (Fig 9), known to mediate ethylene responses, was also altered with SlERF.A1, SlERF.A2, SlERF.A3, SlERF.C1, SlERF.C3, SlERF.C6, SlERF.D1, SlERF.D2, SlERF.D4, SlERF.E1, SlERF.E3 and SlERF.E4 being down-regulated while SlERF.B1, SlERF.B2, SlERF.B3, SlERF.D3, SlERF.F2 are up-regulated. Altogether, these data strongly suggest that ethylene responses are highly impaired in the transgenic lines.


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 ethylene synthesis and ethylene perception genes is altered in SlARF2AB-RNAi plants.(A) Expression of ethylene synthesis pathway genes in SlARF2AB-RNAi lines assed by Quantitative RT-PCR. ACO1, ACO2, ACO3, ACO4 aminocyclopropane-1-carboxylic acid oxidase; ACS1, ACS2, ACS3, ACS4, ACS6 aminocyclopropane-1-carboxylic acid synthases. (B) Expression of ethylene perception genes in SlARF2AB-RNAi assessed by Quantitative RT-PCR. EIN2 ethylene signaling protein; EIL2 and EIL3 are EIN3-like proteins; ETR1, ETR2, ETR3 (NR, never-ripe), ETR4, ETR5, ETR6 ethylene receptors; CTR1 ethylene-responsive protein kinase. ABL1 refers to SlARF2AB-RNAi line 311. Total RNA was extracted from different fruit developmental stages (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 bars mean ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696797&req=5

pgen.1005649.g008: The expression of ethylene synthesis and ethylene perception genes is altered in SlARF2AB-RNAi plants.(A) Expression of ethylene synthesis pathway genes in SlARF2AB-RNAi lines assed by Quantitative RT-PCR. ACO1, ACO2, ACO3, ACO4 aminocyclopropane-1-carboxylic acid oxidase; ACS1, ACS2, ACS3, ACS4, ACS6 aminocyclopropane-1-carboxylic acid synthases. (B) Expression of ethylene perception genes in SlARF2AB-RNAi assessed by Quantitative RT-PCR. EIN2 ethylene signaling protein; EIL2 and EIL3 are EIN3-like proteins; ETR1, ETR2, ETR3 (NR, never-ripe), ETR4, ETR5, ETR6 ethylene receptors; CTR1 ethylene-responsive protein kinase. ABL1 refers to SlARF2AB-RNAi line 311. Total RNA was extracted from different fruit developmental stages (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 bars mean ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01.
Mentions: The ripening defect phenotype prompted us to monitor the climacteric ethylene production in the SlARF2AB-RNAi line. Ethylene production, assessed either on fruits kept on the plant or detached (Fig 7), is significantly low throughout ripening and reaches its peak with 3 days delay as compared to wild type (Fig 7). Assessing the expression of ethylene biosynthesis genes by qPCR (Fig 8A) revealed reduced levels of ACO1, ACS2, ACS3 and ACS4 transcripts in the SlARF2AB RNAi line at all ripening stages (Breaker, Breaker+2 and Breaker+8). However, the reduced ethylene production cannot account for the ripening defects because exogenous ethylene treatment failed to reverse the ripening phenotype (Fig 6D). We therefore examined the expression of ethylene receptor genes (Fig 8B). transcript levels corresponding to ETR3 (NR) and ETR4 are dramatically low in the transgenic lines compared to wild type at all stages of fruit ripening (Br, Br+2, and Br+8) and the expression of other receptor genes (ETR1, ETR2, and ETR5) is also down-regulated at the breaker+8 stage. The disturbed expression of ethylene receptor genes is likely to result in altered ethylene perception in the transgenic lines. In addition, the expression of EIN2 and two EIN3-like genes (EIL2 and EIL3), which encode major components of ethylene transduction pathways, was also down-regulated during ripening of SlARF2A/B RNAi fruit (Fig 8B). More striking, the expression of a high number of ERF genes (Fig 9), known to mediate ethylene responses, was also altered with SlERF.A1, SlERF.A2, SlERF.A3, SlERF.C1, SlERF.C3, SlERF.C6, SlERF.D1, SlERF.D2, SlERF.D4, SlERF.E1, SlERF.E3 and SlERF.E4 being down-regulated while SlERF.B1, SlERF.B2, SlERF.B3, SlERF.D3, SlERF.F2 are up-regulated. Altogether, these data strongly suggest that ethylene responses are highly impaired in the transgenic lines.

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