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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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Structural features and expression patterns of tomato SlARF2A and SlARF2B genes.(A) Genomic structure analysis of SlARF2A and SlARF2B genes were drawn using Fancy gene V1.4 software (http://bio.ieo.eu/fancygene/) and SlARF2A SlARF2B iTAG2.40 gene model data. The pink portion represents the promoter region; the strandlines represent intron parts; the gray boxes indicate exon parts; the yellow boxes region responsible for dimerization with Aux/IAA proteins (domain III and IV); the red boxes correspond to the DNA binding domain (DBD); ERE and AuxRE correspond to the ethylene and auxin responsive cis-elements. (B) Expression pattern of SlARF2A/2B monitored by quantitative real-time RT-PCR (qPCR) in total RNA samples extracted from root (Rt), stem (St), leaf (Le), flower (Fl), fruit (Fr), mature green fruit (MG), breaker fruit (Br) and red fruit (Re). Relative mRNA levels corresponding to SlARF2A/SlARF2B genes were normalized against actin in each RNA sample. The relative mRNA levels of SlARF2B in root and at mature green (MG) stage were used as reference (relative mRNA level 1). Error bars mean ±SD of three biological replicates.
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pgen.1005649.g001: Structural features and expression patterns of tomato SlARF2A and SlARF2B genes.(A) Genomic structure analysis of SlARF2A and SlARF2B genes were drawn using Fancy gene V1.4 software (http://bio.ieo.eu/fancygene/) and SlARF2A SlARF2B iTAG2.40 gene model data. The pink portion represents the promoter region; the strandlines represent intron parts; the gray boxes indicate exon parts; the yellow boxes region responsible for dimerization with Aux/IAA proteins (domain III and IV); the red boxes correspond to the DNA binding domain (DBD); ERE and AuxRE correspond to the ethylene and auxin responsive cis-elements. (B) Expression pattern of SlARF2A/2B monitored by quantitative real-time RT-PCR (qPCR) in total RNA samples extracted from root (Rt), stem (St), leaf (Le), flower (Fl), fruit (Fr), mature green fruit (MG), breaker fruit (Br) and red fruit (Re). Relative mRNA levels corresponding to SlARF2A/SlARF2B genes were normalized against actin in each RNA sample. The relative mRNA levels of SlARF2B in root and at mature green (MG) stage were used as reference (relative mRNA level 1). Error bars mean ±SD of three biological replicates.

Mentions: Some members of the ARF gene family were shown to play a role in regulating important aspects of tomato fruit ripening [28,32]. More recently, expression profiling of tomato ARFs revealed that some members of this gene family display a ripening-associated increase of transcript accumulation suggesting their potential involvement in regulating this process [39]. Among these, the expression pattern of ARF2 is appealing which prompted its molecular and functional characterization. In contrast to Arabidopsis where a single ARF2 gene is present, two putative orthologs are found in the tomato genome with SlARF2A (Solyc03g118290.2.1) being located in chromosome 3 and SlARF2B (Solyc12g042070.1.1) in chromosome 12 [39]. The two genomic clones share similar structural organization with, however, SlARF2A being made of 15 exons while only 14 exons are present in SlARF2B. The isolation of full-length cDNAs corresponding to SlARF2A (2541 bp) and SlARF2B (2490 bp) indicated that the deduced protein sizes are 847 and 830 amino acids, respectively (Table 1), and pairwise comparison of the two SlARF2 protein sequences revealed 83.3% amino acid identity. The search for protein domains in Expasy database (http://prosite.expasy.org/) indicated the presence of highly conserved domains typical of ARFs (Fig 1A) including the DBD (DNA Binding Domain) and the dimerization domains (protein/protein domain III and IV). Moreover, the analysis of a 2 kb promoter sequence using PLACE/signal search tool (http://www.dna.affrc.go.jp/PLACE/signalscan.html) revealed the presence of putative Ethylene Response (ERE) and Auxin Response (AuxRE) elements in both SlARF2A and SlARF2B promoters (Fig 1A).


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)

Structural features and expression patterns of tomato SlARF2A and SlARF2B genes.(A) Genomic structure analysis of SlARF2A and SlARF2B genes were drawn using Fancy gene V1.4 software (http://bio.ieo.eu/fancygene/) and SlARF2A SlARF2B iTAG2.40 gene model data. The pink portion represents the promoter region; the strandlines represent intron parts; the gray boxes indicate exon parts; the yellow boxes region responsible for dimerization with Aux/IAA proteins (domain III and IV); the red boxes correspond to the DNA binding domain (DBD); ERE and AuxRE correspond to the ethylene and auxin responsive cis-elements. (B) Expression pattern of SlARF2A/2B monitored by quantitative real-time RT-PCR (qPCR) in total RNA samples extracted from root (Rt), stem (St), leaf (Le), flower (Fl), fruit (Fr), mature green fruit (MG), breaker fruit (Br) and red fruit (Re). Relative mRNA levels corresponding to SlARF2A/SlARF2B genes were normalized against actin in each RNA sample. The relative mRNA levels of SlARF2B in root and at mature green (MG) stage were used as reference (relative mRNA level 1). Error bars mean ±SD of three biological replicates.
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pgen.1005649.g001: Structural features and expression patterns of tomato SlARF2A and SlARF2B genes.(A) Genomic structure analysis of SlARF2A and SlARF2B genes were drawn using Fancy gene V1.4 software (http://bio.ieo.eu/fancygene/) and SlARF2A SlARF2B iTAG2.40 gene model data. The pink portion represents the promoter region; the strandlines represent intron parts; the gray boxes indicate exon parts; the yellow boxes region responsible for dimerization with Aux/IAA proteins (domain III and IV); the red boxes correspond to the DNA binding domain (DBD); ERE and AuxRE correspond to the ethylene and auxin responsive cis-elements. (B) Expression pattern of SlARF2A/2B monitored by quantitative real-time RT-PCR (qPCR) in total RNA samples extracted from root (Rt), stem (St), leaf (Le), flower (Fl), fruit (Fr), mature green fruit (MG), breaker fruit (Br) and red fruit (Re). Relative mRNA levels corresponding to SlARF2A/SlARF2B genes were normalized against actin in each RNA sample. The relative mRNA levels of SlARF2B in root and at mature green (MG) stage were used as reference (relative mRNA level 1). Error bars mean ±SD of three biological replicates.
Mentions: Some members of the ARF gene family were shown to play a role in regulating important aspects of tomato fruit ripening [28,32]. More recently, expression profiling of tomato ARFs revealed that some members of this gene family display a ripening-associated increase of transcript accumulation suggesting their potential involvement in regulating this process [39]. Among these, the expression pattern of ARF2 is appealing which prompted its molecular and functional characterization. In contrast to Arabidopsis where a single ARF2 gene is present, two putative orthologs are found in the tomato genome with SlARF2A (Solyc03g118290.2.1) being located in chromosome 3 and SlARF2B (Solyc12g042070.1.1) in chromosome 12 [39]. The two genomic clones share similar structural organization with, however, SlARF2A being made of 15 exons while only 14 exons are present in SlARF2B. The isolation of full-length cDNAs corresponding to SlARF2A (2541 bp) and SlARF2B (2490 bp) indicated that the deduced protein sizes are 847 and 830 amino acids, respectively (Table 1), and pairwise comparison of the two SlARF2 protein sequences revealed 83.3% amino acid identity. The search for protein domains in Expasy database (http://prosite.expasy.org/) indicated the presence of highly conserved domains typical of ARFs (Fig 1A) including the DBD (DNA Binding Domain) and the dimerization domains (protein/protein domain III and IV). Moreover, the analysis of a 2 kb promoter sequence using PLACE/signal search tool (http://www.dna.affrc.go.jp/PLACE/signalscan.html) revealed the presence of putative Ethylene Response (ERE) and Auxin Response (AuxRE) elements in both SlARF2A and SlARF2B promoters (Fig 1A).

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