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Tomato R2R3-MYB Proteins SlANT1 and SlAN2: Same Protein Activity, Different Roles.

Kiferle C, Fantini E, Bassolino L, Povero G, Spelt C, Buti S, Giuliano G, Quattrocchio F, Koes R, Perata P, Gonzali S - PLoS ONE (2015)

Bottom Line: Despite the fact that cultivated tomato varieties do not accumulate anthocyanins in the fruit, the biosynthetic pathway can be activated in the vegetative organs by several environmental stimuli.Little is known about the molecular mechanisms regulating anthocyanin synthesis in tomato.Here, we carried out a molecular and functional characterization of two genes, SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors.

View Article: PubMed Central - PubMed

Affiliation: PlantLab, Institute of Life Sciences, Scuola Superiore Sant'Anna, Pisa, Italy.

ABSTRACT
Anthocyanins are water-soluble polyphenolic compounds with a high nutraceutical value. Despite the fact that cultivated tomato varieties do not accumulate anthocyanins in the fruit, the biosynthetic pathway can be activated in the vegetative organs by several environmental stimuli. Little is known about the molecular mechanisms regulating anthocyanin synthesis in tomato. Here, we carried out a molecular and functional characterization of two genes, SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors. We show that both can induce ectopic anthocyanin synthesis in transgenic tomato lines, including the fruit. However, only SlAN2 acts as a positive regulator of anthocyanin synthesis in vegetative tissues under high light or low temperature conditions.

No MeSH data available.


Related in: MedlinePlus

Virus Induced Gene Silencing of SlAN2 and SlANT1 in vegetative tissues of tomato plants.Phenotype of SlAN2 and SlANT1 silenced leaves and stems (TRV/SlAN2, TRV/SlANT1, respectively) compared to non-silenced controls (TRV) (A). Quantitative analysis of transcript levels for SlAN2, SlAN1, SlAN11, SlDFR, SlANT1 and SlJAF13 in SlAN2 silenced tomato plants (TRV/SlAN2) (B) and in SlANT1 silenced tomato plants (TRV/SlANT1) (C) compared to non-silenced controls (TRV). Expression levels are shown as relative units, with the value of one of the biological replicates of control TRV samples set to one. Data are means of three biological replicates ± SD.
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pone.0136365.g005: Virus Induced Gene Silencing of SlAN2 and SlANT1 in vegetative tissues of tomato plants.Phenotype of SlAN2 and SlANT1 silenced leaves and stems (TRV/SlAN2, TRV/SlANT1, respectively) compared to non-silenced controls (TRV) (A). Quantitative analysis of transcript levels for SlAN2, SlAN1, SlAN11, SlDFR, SlANT1 and SlJAF13 in SlAN2 silenced tomato plants (TRV/SlAN2) (B) and in SlANT1 silenced tomato plants (TRV/SlANT1) (C) compared to non-silenced controls (TRV). Expression levels are shown as relative units, with the value of one of the biological replicates of control TRV samples set to one. Data are means of three biological replicates ± SD.

Mentions: To further elucidate the respective contribution of each of the two MYB genes in the activation of the anthocyanin pathway, tomato seedlings were grown under stress conditions (low temperature plus limiting soil) to promote strong anthocyanin accumulation and Virus Induced Gene Silencing (VIGS) of SlAN2 or SlANT1 was carried out. As shown in Fig 5A, the silencing of SlAN2 caused a strong reduction of anthocyanin accumulation both in leaves and in the stem, as compared to control plants. qPCR analysis showed a strong down-regulation of SlAN2 mRNA in the silenced tissues, confirming the effectiveness of the VIGS, as well as a strong repression of the expression of SlDFR (Fig 5B). Furthermore, silenced SlAN2 tissues showed significant reduction of the bHLHs SlAN1 and SlJAF13 transcripts providing evidence that SlAN2 is involved in the transcriptional regulation of these genes. Interestingly, the expression in petals of the petunia bHLH factor PhAN1 is similarly down-regulated in mutants for the MYB protein PhAN4 [6]. On the contrary, the expression of SlAN11 was not altered in silenced SlAN2 tissues, further confirming that SlAN11 is expressed independently from SlAN2. SlANT1 was not significantly affected by the silencing of SlAN2 (Fig 5B), suggesting that this gene did not play an important role in the anthocyanin accumulation observed in the not silenced plants. This was further confirmed by the silencing of SlANT1 itself. In tomato seedlings growing in the same stressing conditions and accumulating elevated quantities of anthocyanins, the reduced expression of SlANT1 obtained by VIGS and confirmed by qPCR did not result in loss of pigmentation (Fig 5A) or in changes in the expression of the genes analyzed (Fig 5C). All these results, together with those shown in Fig 4, suggest that SlAN2, together with SlAN1 and probably SlJAF13, is involved in the regulation of anthocyanin biosynthesis in vegetative tissues of tomato plants upon light and cold induction and that SlDFR is a target gene of these regulators. Furthermore, the role of SlANT1 in the regulation of pigment accumulation seems to be at most marginal.


Tomato R2R3-MYB Proteins SlANT1 and SlAN2: Same Protein Activity, Different Roles.

Kiferle C, Fantini E, Bassolino L, Povero G, Spelt C, Buti S, Giuliano G, Quattrocchio F, Koes R, Perata P, Gonzali S - PLoS ONE (2015)

Virus Induced Gene Silencing of SlAN2 and SlANT1 in vegetative tissues of tomato plants.Phenotype of SlAN2 and SlANT1 silenced leaves and stems (TRV/SlAN2, TRV/SlANT1, respectively) compared to non-silenced controls (TRV) (A). Quantitative analysis of transcript levels for SlAN2, SlAN1, SlAN11, SlDFR, SlANT1 and SlJAF13 in SlAN2 silenced tomato plants (TRV/SlAN2) (B) and in SlANT1 silenced tomato plants (TRV/SlANT1) (C) compared to non-silenced controls (TRV). Expression levels are shown as relative units, with the value of one of the biological replicates of control TRV samples set to one. Data are means of three biological replicates ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556288&req=5

pone.0136365.g005: Virus Induced Gene Silencing of SlAN2 and SlANT1 in vegetative tissues of tomato plants.Phenotype of SlAN2 and SlANT1 silenced leaves and stems (TRV/SlAN2, TRV/SlANT1, respectively) compared to non-silenced controls (TRV) (A). Quantitative analysis of transcript levels for SlAN2, SlAN1, SlAN11, SlDFR, SlANT1 and SlJAF13 in SlAN2 silenced tomato plants (TRV/SlAN2) (B) and in SlANT1 silenced tomato plants (TRV/SlANT1) (C) compared to non-silenced controls (TRV). Expression levels are shown as relative units, with the value of one of the biological replicates of control TRV samples set to one. Data are means of three biological replicates ± SD.
Mentions: To further elucidate the respective contribution of each of the two MYB genes in the activation of the anthocyanin pathway, tomato seedlings were grown under stress conditions (low temperature plus limiting soil) to promote strong anthocyanin accumulation and Virus Induced Gene Silencing (VIGS) of SlAN2 or SlANT1 was carried out. As shown in Fig 5A, the silencing of SlAN2 caused a strong reduction of anthocyanin accumulation both in leaves and in the stem, as compared to control plants. qPCR analysis showed a strong down-regulation of SlAN2 mRNA in the silenced tissues, confirming the effectiveness of the VIGS, as well as a strong repression of the expression of SlDFR (Fig 5B). Furthermore, silenced SlAN2 tissues showed significant reduction of the bHLHs SlAN1 and SlJAF13 transcripts providing evidence that SlAN2 is involved in the transcriptional regulation of these genes. Interestingly, the expression in petals of the petunia bHLH factor PhAN1 is similarly down-regulated in mutants for the MYB protein PhAN4 [6]. On the contrary, the expression of SlAN11 was not altered in silenced SlAN2 tissues, further confirming that SlAN11 is expressed independently from SlAN2. SlANT1 was not significantly affected by the silencing of SlAN2 (Fig 5B), suggesting that this gene did not play an important role in the anthocyanin accumulation observed in the not silenced plants. This was further confirmed by the silencing of SlANT1 itself. In tomato seedlings growing in the same stressing conditions and accumulating elevated quantities of anthocyanins, the reduced expression of SlANT1 obtained by VIGS and confirmed by qPCR did not result in loss of pigmentation (Fig 5A) or in changes in the expression of the genes analyzed (Fig 5C). All these results, together with those shown in Fig 4, suggest that SlAN2, together with SlAN1 and probably SlJAF13, is involved in the regulation of anthocyanin biosynthesis in vegetative tissues of tomato plants upon light and cold induction and that SlDFR is a target gene of these regulators. Furthermore, the role of SlANT1 in the regulation of pigment accumulation seems to be at most marginal.

Bottom Line: Despite the fact that cultivated tomato varieties do not accumulate anthocyanins in the fruit, the biosynthetic pathway can be activated in the vegetative organs by several environmental stimuli.Little is known about the molecular mechanisms regulating anthocyanin synthesis in tomato.Here, we carried out a molecular and functional characterization of two genes, SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors.

View Article: PubMed Central - PubMed

Affiliation: PlantLab, Institute of Life Sciences, Scuola Superiore Sant'Anna, Pisa, Italy.

ABSTRACT
Anthocyanins are water-soluble polyphenolic compounds with a high nutraceutical value. Despite the fact that cultivated tomato varieties do not accumulate anthocyanins in the fruit, the biosynthetic pathway can be activated in the vegetative organs by several environmental stimuli. Little is known about the molecular mechanisms regulating anthocyanin synthesis in tomato. Here, we carried out a molecular and functional characterization of two genes, SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors. We show that both can induce ectopic anthocyanin synthesis in transgenic tomato lines, including the fruit. However, only SlAN2 acts as a positive regulator of anthocyanin synthesis in vegetative tissues under high light or low temperature conditions.

No MeSH data available.


Related in: MedlinePlus