PHYTOCHROME-INTERACTING FACTOR 3 mediates light-dependent induction of tocopherol biosynthesis during tomato fruit ripening

2.1 Plant material, growth conditions, and sampling

S. lycopersicum (cv. MicroTom and Moneymaker), Nicotiana benthamiana, and Nicotiana tabacum plants were grown under standard greenhouse conditions (14-hr light at 27 ± 1°C and 10-hr dark at 22 ± 1°C). Seeds of aurea and hp2 mutants, in cv. MicroTom background, were donated by Dr Lázaro Eustáquio Pereira Peres (University of Sao Paulo, Brazil). Seeds of single and multiple phya, phyb1, and phyb2 mutants, in Moneymaker background, were provided by Dr Rameshwar Sharma (University of Hyderabad, India). In aurea, a mutation on PHYTOCHROMOBILIN SYNTHASE-encoding gene prevents the correct synthesis of the PHY chromophore PϕB, leading to a global deficiency in functional PHYs (Kendrick, Kerckhoffs, Tuinen, & Koornneef, 1997; Muramoto et al., 2005; Parks et al., 1987). The hp2 mutant is deficient for DET1 transcription factor, a negative regulator of light signal transduction (Soressi, 1975). phya, phyb1, phyb2, phyab1, phyb1b2, and phyab1b2 are loss-of-function mutants initially characterized by Kerckhoffs et al. (1996); Kerckhoffs, Schreuder, Van Tuinen, Koornneef, and Kendrick (1997); Kerckhoffs et al. (1999); Lazarova, Kerckhoffs, et al. (1998); Lazarova, Kubota, et al. (1998); and Weller, Schreuder, Smith, Koornneef, and Kendrick (2000).

For fruit ripening experiments, fruits at mature green (MG) stage were harvested about 30 days after anthesis (dpa) and were transferred to continuous white light (400 to 800 nm, approximately 50 μmol m⁻² s⁻¹) or maintained under absolute darkness until reaching distinct ripening stages in a temperature-controlled growth chamber maintained at 25 ± 2°C and air relative humidity at 80 ± 5%. Top and bottom illumination was applied to homogenize the light environment surrounding the fruits. Pericarp samples (without placenta and locule walls) were harvested at MG (displaying jelly placenta 2 days after harvesting), breaker (BR, 34 dpa displaying the first external yellow color signals), 1 day after BR (BR1), 3 days after BR (BR3), 6 days after BR (BR6), and 12 days after BR (BR12) stages.

2.2 Gene expression analysis

RNA extraction, cDNA synthesis, and quantitative polymerase chain reaction (qPCR) procedures were performed as described by Quadrana et al. (2013). The primers used for qPCR are listed in Table S1. All reactions were performed with two technical replicates and at least three biological replicates. Experiments were performed in a 7500 Real-Time PCR system (Applied Biosystem) using Power SYBR Green Master Mix (Thermo Fischer Scientific). Absolute fluorescence data were analysed with LinRegPCR software (Ruijter et al., 2009) to obtain Ct values and to calculate primer efficiency. Relative mRNA abundance was calculated and normalized with the ΔΔCt method using two reference genes as in Quadrana et al. (2013).

2.3 Tocopherol and Chl quantification

Tocopherols were extracted from approximately 25-mg dry weight as described by Lira et al. (2016). Chl extraction was carried out as described in Porra, Thompson, and Kriedemann (1989). A 1-ml aliquot of dimethylformamide was added to 200 mg of fresh weight fruit samples. After sonication for 5 min at 42 kHz and further centrifugation at 13,000 g for 5 min, the supernatant was collected. The procedure was repeated twice until total removal of tissue green colour and the supernatants were combined. Spectrophotometer measurements were performed at 664 and 647 nm. Chl a content was estimated as (12*Abs 664)-(3,11*Abs 647), whereas Chl b was calculated as (20,78*Abs 647)-(4,88*Abs 664).

2.4 Promoter analysis

Approximately 3 Kb fragments of the promoter sequences of the SlGGDR, SlVTE2, and SlVTE5 genes were retrieved from Sol Genomics Network (Fernandez-Pozo et al., 2015). To gain evidences about the eventual role of SlPIFs in the regulation of these genes, the presence of PIF-binding motifs was analysed using PlantPAN 2.0 platform (Chow et al., 2015).

2.5 Transactivation assay

Full-length cDNAs encoding SlPIF1a (Solyc09g063010), SlPIF1b (Solyc06g008030), SlPIF3 (Solyc06g008030), and SlRIN (Solyc05g012020) were amplified with the primers listed in Table S1. The fragments were cloned into pENTR/DTOPO vector using Gateway technology (Invitrogen). The entry plasmids were recombined into pK7WG2D (Karimi, Inzé, & Depicker, 2002) using LR Clonase (Invitrogen) to produce 35S::SlPIFs/SlRIN effector constructs. Fragments of 2,838 bp for SlPSY1 (Solyc03g031860), 3,123 bp for SlVTE2 (Solyc08g068570), and 2,648 bp for SlGGDR (Solyc01g067890) upstream the ATG starting codon were amplified from genomic DNA, using the primers listed in Table S1. The amplified fragments were digested with XhoI and BamHI restriction enzymes and cloned into the multicloning site of pGreenII 0800 LUC (Hellens et al., 2005) to produce the target constructs. All the constructs were sequenced and introduced into Agrobacterium tumefaciens (GV3101). For transient expression in N. tabacum leaves, A. tumefaciens cells carrying the different constructs were grown at 28°C for 48 hr in YEP medium (Sambrook, Fritsch, & Maniatis, 1989) with appropriate antibiotics. The cells were harvested, washed twice, and resuspended in infiltration buffer (50-mM MES pH 5.6, 2-mM sodium phosphate buffer pH 7, 0.5% glucose, and 200-μM acetosyringone).

Leaves of 4-week-old plants were coinfiltrated with a mix of equal volumes of effector and target cultures, both at a final OD₆₀₀ of 0.05. After 3 days, Firefly Luciferase and Renillia Luciferase activity were assayed using the Dual-Luciferase Reporter Assay System (Promega) as described by Hellens et al. (2005), with slight modifications. Two leaf discs of 2 cm were harvested and grounded in 500 μl of Passive Lysis Buffer. Ten microlitre of this crude extract were assayed in 40 μl of Luciferase Assay Reagent and the chemiluminescence was measured. Then, 40 μl of Stop and Glow™ Reagent was added and a second chemiluminescence measurement was made. Absolute relative light units (RLU) were measured by Synergy H1 (Biotek) luminometer, with a 5-s delay and 15-s measurement. Data were collected as Luciferase/Renilla activity ratio and subsequently normalized relative to the control condition (leaves infiltrated with A. tumefaciens harbouring pK7WG2D empty vector).

2.6 Subcellular localization and chromatin immunoprecipitation assay

Full-length cDNA encoding SlPIF1a, SlPIF1b, SlPIF3, and SlHY5 without the stop codon were amplified with the primers listed in Table S1. The fragments were cloned into pENTR/DTOPO using Gateway technology (Invitrogen). The entry plasmids were recombined into pK7FWG2 (Karimi et al., 2002) using LR Clonase (Invitrogen) to produce 35S::SlPIF/SlHY5-GFP fusion proteins. The constructs obtained were introduced into A. tumefaciens (GV3101) for further subcellular localization assay and for chromatin immunoprecipitation (ChIP). For subcellular localization, 35S::SlPIF/SlHY5-GFP fusion proteins were agroinfiltrated in N. benthamiana leaves and 3 days after the infiltration the fluorescence was detected using a Leica TCS SP5 confocal laser-scanning microscope. Excitation filter of 450–490 nm was used for detection of GFP fluorescence.

ChIP assay followed by qPCR was performed as described in Ricardi, González, and Iusem (2010) with some modifications. Briefly, MG fruits were agroinfiltrated with 35S::SlPIF3-GFP construct, kept for 3 days under light or dark conditions, and fixed with formaldehyde to promote the cross linking between DNA and proteins. Following nuclei enrichment with a Percoll (GE Healthcare) gradient, the chromatin was fragmented by sonication (10 s on/20 s off, amplitude 70, during 10 min using QSonica700 device) and then incubated with Dynabeads Protein-A (Invitrogen) with either anti-GFP or anti-HA antibodies (Invitrogen). Next, the immunoprecipitated DNA was purified by phenol:chloroform:isoamyl alcohol extraction and used as template for qPCR analysis. Specific primer pairs flanking the predicted transcription factor binding motifs for each promoter region and the coding region of SlACTIN4 (Fujisawa, Nakano, & Ito, 2011) as normalizer (Table S1) were used.

2.7 Fruit transient overexpression

For transient SlPIF3 overexpression assay, MG fruits were agroinfiltrated (Orzaez, Mirabel, Wieland, & Granell, 2006) with the 35S::SlPIF3 construct used for transactivation experiments, and after 3 days in the dark, the gene expression of SlGGDR and SlPIF3 was addressed by qPCR as previously described using the primers listed in Table S1.

2.8 Data analyses

Differences in parameters were analysed in Infostat software version 17/06/2015 (Di Rienzo et al., 2011). When the data set showed homoscedasticity, t test (P < 0.05) was performed to compare genotypes or treatments. In the absence of homoscedasticity, a nonparametric comparison was performed by applying Mann–Whitney test (P < 0.05). All values represent the mean of at least three biological replicates.

3.1 Tocopherol accumulation is transcriptionally regulated by light during fruit ripening

To investigate whether light regulates VTE biosynthesis during fruit ripening, tocopherol levels were quantified in fruits from wild-type (WT) plants and two light-related mutants, the light-hyposensitive PϕB deficient aurea and the light-hypersensitive DET1 deficient high-pigment 2 (hp2). Fruits at the MG stage were detached from the plants and let to ripen under constant white light or darkness (Figure 2a). As previously reported (Almeida et al., 2015), the tocopherol levels increased during ripening in WT fruits. However, the accumulation was higher when the fruits were maintained under constant white light, indicating that this stimulus promotes tocopherol biosynthesis. Interestingly, this light-associated increase in tocopherol contents was not observed in the aurea mutant (Figure 2a), strongly suggesting a PHY-mediated effect. Consistently, fruits from the light hypersensitive mutant hp2 displayed a pronounced increment of total tocopherols when ripened in the light, reaching higher absolute levels than those observed from fruits ripened in the darkness (Table S2).

The transcript levels of the genes involved in VTE biosynthesis were next profiled in WT fruits along ripening under constant light or dark conditions (Figures 2b and S1). We observed that SlGGDR, SlVTE1, SlVTE2, SlVTE4, and SlVTE5 mRNA levels were lower when the fruits were incubated in the dark. Particularly interesting were the reductions in SlGGDR, SlVTE5, and SlVTE2 mRNA levels (Figure 2b). The two formers are responsible for PDP production either de novo (SlGGDR) or from Chl degradation (SlVTE5), whereas SlVTE2 encodes the enzyme responsible for the condensation of PDP and homogentisic acid in the first committed step of tocopherol biosynthesis (Figure 1).

Together, these results revealed that tocopherol accumulation in tomato fruits is induced by light, which might be explained, at least in part, by the transcriptional regulation of tocopherol biosynthetic genes.

3.2 PHY-mediated light signal transduction enhances tocopherol accumulation

To test whether the observed positive effect of light on the regulation of fruit tocopherol content is mediated by PHYs, we analysed the tocopherol accumulation in fruits from WT and six loss-of-function PHY mutants (phya, phyb1, phyb2, phyab1, phyb1b2, and phyab1b2). Interestingly, only the double phyab1 and triple phyab1b2 mutants displayed a reduction in tocopherol content in ripe fruits (Figure 3a). These results reinforce our previous conclusion that light induces tocopherol accumulation during ripening in a PHY-mediated manner. They further reveal redundancy of individual phytochrome functions in the regulation of VTE biosynthesis. Quantification of tocopherol biosynthetic gene expression in WT and phyab1b2 mutant fruits showed reduced levels of SlGGDR, SlVTE2, and SlVTE5 (but not SlVTE6) transcripts in the PHY-defective triple mutant (Figure 3b), hence confirming that the PHY-dependent regulation of fruit tocopherol content is mediated by the transcriptional regulation of key tocopherol biosynthetic genes.

3.3 SlPIF3 represses SlGGDR transcription by physically interacting with its promoter

As PIFs are major transcription factors acting downstream of PHY-mediated light signalling, we investigated the eventual role of SlPIFs in the PHY-dependent regulation of tocopherol accumulation in tomato fruits. SlPIF loci were previously identified and transcriptionally characterized, being SlPIF1a, SlPIF1b, and SlPIF3 the most abundantly expressed in fruits (Rosado et al., 2016). Thus, we addressed whether the light treatment (Figure 2a) or the PHY deficiency (Figure 3a) affected the expression of these SlPIFs. Interestingly, these genes were found to be up-regulated in WT fruits maintained in darkness (Figure S2a), as well as in the triple mutant phyab1b2 fruits (Figure S2b), reinforcing their candidature as mediators of the PHY-dependent regulation of tocopherol accumulation. As an additional step in their functional characterization, we aimed to confirm their expected nuclear localization. In order to do so, the coding region of these genes was fused to the Green Fluorescence Protein (GFP) and transiently expressed in N. benthamiana leaves. As reported for A. thaliana PIFs (Leivar & Monte, 2014), SlPIF1a, SlPIF1b, and SlPIF3 proteins localized as nuclear speckles (Figure 4a). Such nuclear speckles, also referred to as photobodies, have been associated to the interaction of PHYs with photolabile PIFs for their phosphorylation and further degradation of these transcription factors (Al-Sady, Ni, Kircher, Schäfer, & Quail, 2006). Indeed, a GFP-tagged version of SlHY5, a transcription factor that does not directly interact with PHYs, showed a homogeneous distribution in the nucleus (Figure S3). Together, these results strongly support that, as demonstrated for SlPIF1a (Llorente et al., 2016), SlPIF1b and SlPIF3 are true PIFs, that is, PHY-interacting factors.

Being demonstrated that PHY-mediated light perception affected SlPIF expression in fruits and that SlPIFs localized in speckles, which is indicative of their light-induced PHY-mediated degradation, we investigated whether SlPIFs are responsible for the light-regulated expression of tocopherol biosynthetic genes. A de novo search for putative PIF-binding motifs in 3,000 bp fragments upstream the ATG starting codon of SlGGDR, SlVTE2, and SlVTE5 retrieved from the Heinz reference tomato genome (Sol Genomics Network, Fernandez-Pozo et al., 2015) revealed the presence of G-boxes and PBE-boxes (Chen et al., 2013; Song et al., 2014; Toledo-Ortiz et al., 2014; Zhang et al., 2013) in the promoter regions of SlGGDR and SlVTE2 (data not shown). To test the functionality of these putative PIF-binding motifs, transient transactivation assays were performed in N. tabacum (Figure 4). Promoter regions of 2,648 bp for SlGGDR and 3,123 bp for SlVTE2 were cloned from MicroTom genotype and sequenced (Figure S4). Polymorphisms were identified compared with the reference Heinz sequence, which do not alter the previously identified motifs on SlGGDR but reduce to 4 the number of PBE-boxes identified on SlVTE2 (Figure 4b). As positive control, the previously reported inductive effect of the transcription factor RIPENING INHIBITOR (SlRIN) on the SlPSY1 promoter (Martel, Vrebalov, Tafelmeyer, & Giovannoni, 2011) was also tested (Figure S5). The activity of the SlGGDR promoter was found to be down-regulated in the presence of SlPIF1a, SlPIF1b, and SlPIF3, whereas none of these SlPIFs affected the activity of the SlVTE2 promoter (Figure 4c).

The direct interaction between SlPIF3, the most highly expressed SlPIF gene in tomato fruits (Rosado et al., 2016), and the SlGGDR promoter was further addressed by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). MG fruits were infiltrated with A tumefaciens harbouring a 35S::SlPIF3-GFP construct and maintained in constant dark or light conditions for 3 days. After chromatin purification, an enrichment in SlGGDR promoter sequences harbouring PBE boxes (Figure 4b) was observed in anti-GFP immunoprecipitated samples, that is, those containing SlPIF3-GFP, in comparison with anti-HA immunoprecipitated control in those fruits maintained in the darkness. This enrichment was not observed when the fruits were kept in the light, hence indicating that SlPIF3 physically interacts with the SlGGDR promoter preferably in the darkness (Figure 4d).

3.4 Transient SlPIF3 overexpression reduces the expression of SlGGDR

The fact that SlPIF3 is the most abundantly expressed SlPIF gene in MG tomato fruits (Rosado et al., 2016), together with the results obtained by transactivation and ChIP-qPCR assays, strongly pointed this gene as the most evident candidate for down-regulating SlGGDR expression in darkness. To test this hypothesis, we transiently overexpressed SlPIF3 in MG tomato fruits by agroinfiltration and analysed the impact of increasing SlPIF3 levels on the expression level of SlGGDR. A decrease in SlGGDR mRNA levels was verified (Figure 5a) that inversely correlated with the level of SlPIF3 transcripts (Figure 5b). Altogether, the data indicate that SlPIF3 mediates the PHY-dependent regulation of VTE biosynthesis via the transcriptional inhibition of SlGGDR expression in tomato fruit.