Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight
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Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab.
The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.
KeywordsChalcone 3-hydroxylase (CH3H) Dihydrochalcones Erwinia amylovora 3-Hydroxyphloretin 3-Hydroxyphloridzin Malus × domestica Venturia inaequalis
Materials and methods
[14C ]-Phloretin was synthesized as described previously (Halbwirth et al. 2006).
3-Hydroxyphloretin was purchased from Apin Chemicals (http://www.apinchemicals.com). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were obtained from Sigma-Aldrich (www.sigmaaldrich.com/austria.html).
Production and in vitro testing of recombinant CsCH3H
Heterologous expression of CsCH3H and F3′H in yeast and testing of substrate acceptance was performed as described previously (Schlangen et al. 2010b).
Vector construction for plant transformation
The binary plasmid vector p9u10-35S::CH3H was provided by DNA Cloning Service e.K. (http://dna-cloning.com) using a cDNA clone of C. sulphureus (NCBI accession FJ216429) and the p9u10-35S plasmid that contains the nptII selectable marker gene driven by the ubiquitin promotor from A. thaliana. The cDNA clone was ligated between the SpeI and MluI restriction sites using the primers SpeCH3H and CH3HMlu (Suppl. Table S1).
Proliferating axillary shoot cultures of PinS (JKI, Dresden-Pillnitz, Germany), a seedling of the apple (Malus × domestica BORKH.) cv. ‘Pinova’ were used for plant transformation. Transformation was performed using the Agrobacterium tumefaciens strain EHA105 (Hood et al. 1993) containing the binary plasmid vector p9u10-35S::CH3H. Regeneration, selection of transgenic plants, rooting and acclimatization were carried out as previously described (Li et al. 2007). For the studies on gene expression, enzyme activities and polyphenol contents, the first (L1), second (L2) and third (L3) leaves of the shoots were collected separately, shock-frozen in liquid nitrogen and stored at −80 °C until use. Three independent samples were harvested.
RT-PCR and qRT-PCR analysis
Young leaves of greenhouse-grown shoots (transgenic and non-transgenic) were collected and frozen in liquid nitrogen. The total RNA from leaf tissue was extracted using the InviTrap Spin Plant RNA Mini Kit (Stratec Biomedical AG, Berlin, Germany). First strand cDNA synthesis was performed as described by Flachowsky et al. (2007). Transcripts of nptII and CH3H were detected by reverse transcriptase (RT)-PCR using the primers nptII_F/R for nptII and SpeCH3H/CH3HMlu for CH3H.
The relative mRNA expression level of genes of the flavonoid genes encoding ANS, ANR and FGT was determined by quantitative Real-Time PCR SYBR® Green PCR Master Mix according to the supplier’s instruction on a StepOnePlus system (Applied Biosystems, Darmstadt, Germany) with first-strand cDNA as template using the primers listed in Suppl. Table S1.
Southern hybridization experiments were performed using 10 µg of DNA digested with 100 U SalI (Life Technologies, Darmstadt, Germany) at 37 °C overnight. The restricted DNA was separated on a 0.8 % agarose gel in 1× Trisacetate-EDTA (TAE) buffer and blotted onto a positively charged nylon membrane (Roche Deutschland Holding GmbH, Mannheim, Germany) by capillary transfer (Southern 1975) with 20× SSC (0.15 M NaCl, 0.015 M tri-sodium citrate dihydrate, pH 7.0) as transfer buffer. The membrane was hybridized with PCR-amplified, digoxygenin-labeled probes of nptII and CH3H, respectively. Probes were amplified using the PCR DIG Probe Synthesis Kit (Roche). Amplification was performed using the primers nptII_F/R for nptII and SpeCH3H/CH3HMlu (Suppl. Table S1) for CH3H, respectively. Detection was performed using Anti-DIG-AP (Roche) and ECF™ substrate (Amersham Biosciences Europe, Freiburg, Germany) on a ChemiDoc™ XRS + System (Bio-Rad Laboratories, Munich, Germany).
Analysis of phenolic compounds
Determination and identification of phenolic compounds were performed according to Roemmelt et al. (2003) with small modifications. Freeze-dried leaves were ground in a ball mill. The extraction was performed by adding 500 µL of methanol containing 3-methoxyflavone as internal standard to 100 mg of powder for a period of 30 min in a cooled ultrasound water bath (7 °C). After centrifugation (10,000g, 10 min, 4 °C), the clear supernatant was transferred to an Eppendorf tube. A 10 µL sample of the extract was injected for HPLC analysis. The phenolic compounds were separated on a Nucleosil column (250 × 4 mm, Macherey–Nagel) and eluted with a mixture of H2O containing 5 % HCO2H (solvent A) and MeOH (solvent B). The following gradient was applied using a flow rate of 0.5 mL/min: 0–5 min, 5 % B; 5–10 min, 5–10 % B; 10–15 min, 10 % B; 15–35 min, 10–15 % B; 35–55 min, 15 % B; 55–70 min, 15–20 % B; 70–80 min, 20 % B; 80–95 min, 20–25 % B; 95–125 min, 25–30 % B; 125–145 min, 30–40 % B; 145–160 min, 40–50 % B; 160–175 min, 50–90 % B; 175–195 min, 90 % B (Treutter et al. 1994). For the quantification of phloridzin the extract was diluted 200-fold with methanol and analyzed using a short column (12.5 × 4 mm I.D.) prepacked with LiChrospher 100 RP18, 5 µm particle size, and a gradient range from 40 to 90 % aqueous methanol. Dihydrochalcones, hydroxycinnamic acids, and flavonols were detected at 280, 320 and 350 nm. The monomeric flavan-3-ols and the procyanidins were estimated using a chemical reaction detection (HPLC-CRD) at 640 nm after postcolumn derivatization with 4-dimethylaminocinnamicaldehyde (DMACA) (Treutter 1989). Quantification was performed as follows: phloridzin and phloretin were available as standard; 3-hydroxyphloridzin was calculated as phloridzin; hydroxycinnamic acids as chlorogenic acid; flavonols as rutin; catechin, epicatechin and procyanidin B2 were available as standards; procyanidin B5 and EB-5 were calculated as procyanidin B2. Peak identification is published elsewhere (Mayr et al. 1995; Roemmelt et al. 2003).
Identification of 3-hydroxyphloridzin
The putative 3-hydroxyphloridzin peak showed an UV spectrum with λ max at 284 nm which is similar with those of phloridzin in front of each is eluted at t R = 97 min. The same elution behavior was reported by Leu et al. (2006). The compound was isolated by several analytical HPLC runs and respective fractions were combined. The hydrolysis with glycosidase was performed as described by Regos et al. (2009). MS analysis was performed with a Time-of-Flight mass spectrometer (ToF–MS) (6200 series ToF, Agilent Technologies, Santa Clara, CA, USA). The sample was provided in a 100 µL syringe (Hamilton-Bonaduz, Switzerland), located in a syringe pump (Model 11 Plus, Harvard Apparatus, Hugo Sachs Elektronik, Hugstetten, Germany), which was set to a flow rate of 8 µL/min. Upon injection to the MS electrospray ionization source sample compounds were detected in negative ionization mode. MS conditions were as follows: 80–3200 m/z range; 300 °C gas temperature; 3 L/min gas flow; 15 psig nebulizer operating pressure; 100 °C sheath gas temperature; 3 L/min sheath gas flow; 2000 V capillary voltage; 2000 V nozzle voltage; 65 V skimmer voltage and 100 V fragmentor voltage. Besides the determination of the compounds exact molecular weight, fragmentation was induced using the same MS parameters except for an increase of fragmentor voltage to 200 V.
Determination of the antioxidant activity
The antioxidant activity of methanolic plant extracts was determined according to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method as described by Brand-Williams et al. (1995) with slight modifications. 0.25 g leaves were grinded with 0.25 g quartz sand and 3 mL methanol for 1.5 min. After centrifuging the homogenate for 10 min at 4 °C and 10,000g, the supernatant was transferred in a new reaction tube. 0, 1, 5, 10, 15 and 20 µL methanolic plant extracts were briefly mixed with 2 mL of 0.1 mM methanolic DPPH solution in cuvettes. The absorbance of DPPH was measured after 30 min at 517 nm using methanol as a blank. The values were used for the generation of a linear equation to calculate the amount of methanolic plant extract which leads to a 50 % absorbance decrease. Using a calibration curve with the vitamin E analogon trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as a standard the antiradical power (ARP) in µmol trolox equivalents (TE)/g fresh weight (FW) was calculated.
Enzyme activity measurements
Enzyme preparations from apple leaves were obtained and assays were performed as described by Slatnar et al. (2010). In brief, 0.5 g leaves, 0.25 g quartz sand, and 0.25 g Polyclar AT were homogenized with 3 mL 0.1 MTris/HCl (containing 0.4 % Na-ascorbate, pH 7.25) in a pre-cooled mortar. After centrifuging the homogenate for 10 min at 4 °C and 10,000g, 400 µL of the supernatant were passed through a gel chromatography column (Sephadex G25 medium, (GE Healthcare, Vienna Austria)). In a final volume of 100 µL: the CHS/CHI assay contained 40 µL crude extract, 5 µL [14C]- malonyl-CoA (1.5 nmol), 5 µL [14C]-p-coumaroyl-CoA (1 nmol) and 50 µL 0.1 M KPi buffer (pH 7.0, 0.4 % Na-ascorbate w/v); the FHT assay contained 0.036 nmol [14C]-naringenin, 30 µL crude extract, 5 µL 2-oxoglutarate (1.46 mg/ml H2O), 5 µL FeSO4·7H2O (0.56 mg/ml H2O) and 60 µL 0.1 M Tris/HCl (pH 7.25, 0.4 % Na-ascorbate w/v; the FLS assay contained 0.036 nmol [14C]-dihydrokaempferol, 30 µL crude extract, 5 mL 2-oxoglutarate (1.46 mg/mL H2O), 5 µL FeSO4·7H2O (0.56 mg/ml H2O) and 60 µL buffer 0.1 M Tris/HCl buffer (pH 7.60, 0.4 % Na-ascorbate w/v). In a final volume of 50 µL the DFR assay contained 0.036 nmol [14C]-dihydroquercetin, 20 µL crude extract, 5 µL NADPH (4.18 mg/100 mL H2O) and 25 mL 0.1 M KPi buffer (pH 6.8, 0.4 % Na-ascorbate w/v). The assays were incubated for 15 min at 30 °C. Protein determination was performed according to Lowry modified by Sandermann and Strominger (1972).
Fire blight resistance evaluation
Artificial shoot inoculations were performed using a suspension of E. amylovora, strain 222 (107 cfu/mL) on own rooted transgenic and non-transgenic control plants grown in the greenhouse as described by Flachowsky et al. (2008). Twenty replicates for each transgenic line were inoculated.
Scab resistance evaluation
Resistance to Venturia inaequalis was tested on greenhouse plants of transgenic lines and non-transgenic control plants grown on their own roots as described by Flachowsky et al. (2010). Fourteen (M803), eighteen (PinS) and twenty (M801, M802) replicates per genotype were used for evaluation. The inoculum consisted of a mixture of scab strains which was prepared from scab-infected leaves. Leaves were harvested from trees of different genotypes, which are known to differ in their reaction to different scab races. These trees were grown in a plot not treated with fungicides in the orchard of the JKI (Dresden, Germany). Scab incidence was scored using the scale of Chevalier et al. (1991).
Quantitative data were subjected to statistical analysis (ANOVA and Duncan’s multiple range test) using the SAS® 9.1 software (SAS institute, Cary, N.C.).
Plant transformation and molecular evaluation of transgenic lines
Evaluation of the disease resistance of the transgenic CsCH3H lines
For the evaluation of resistance to scab up to twenty replicates of the transgenic CsCH3H lines and the non-transgenic control were inoculated with Venturia inaequalis as described. On average, three leaves per plant of the non-transgenic genotype PinS were scored with 4 (sporulation) and all replicates showed susceptible reaction. The number of leaves scored with 4 was lower in transgenic lines M801, M802 and M803 with one leaf per plant on average (Fig. 8, right). Nevertheless, six, six and seven replicates of M801, M802 and M803 showed no compatible interaction. The mean incidence was obviously lower in transgenic plant, but statistically significant differences could not been found. Regarding that all transgenic lines showed susceptible interaction with scab but less severity the development of the disease on transgenic plants seemed to be slowlier but resistance was not introduced.
Dihydrochalcones are accumulated in Malus sp. (apple) in such large amounts that it appears unlikely that this should be just a result of polyphenolic waste storage in vacuoles. Knowledge of their biosynthesis is an essential precondition for understanding the physiological role of dihydrochalcones because this allows studying activities of corresponding enzymes and genes as a response to biotic and abiotic stresses. Despite the large amounts of dihydrochalcones present in Malus sp. their formation has been studied only recently. There still remain a lot of open questions such as the identity of the dehydrogenase providing the precursor for the formation of the dihydrochalcone structure (Gosch et al. 2009; Dare et al. 2013; Ibdah et al. 2014) and of the enzymes involved in 3-hydroxyphloretin formation. In the present study we created transgenic apple lines overexpressing the CH3H from the ornamental plant Cosmos sulphureus, which accepts phloretin as substrate in vitro. This allowed us to study the ability of CH3H to hydroxylate dihydrochalcones in planta. In addition, this shed new light on the 3-hydroxyphloridzin biosynthesis in apple and allowed to evaluate the physiological relevance of increased 3-hydroxyphloridzin levels for pathogen defence.
The three transgenic lines accumulated higher amounts of 3-hydroxyphloridzin than the parent line, thereby confirming that CH3H accepts dihydrochalcones as substrates not only in vitro but also in planta. However, a maximal increase of up to 11.5 % of the dihydrochalcone content was observed. This confirmed that dihydrochalcones are not as good substrates for CH3H as chalcones. Only 3-hydroxyphloridzin, but no 3-hydroxyphloretin accumulation was observed, indicating that the highly reactive 3-hydroxyphloretin is immediately converted to hydroxyphloridzin to avoid undesired cell damage and that the glucosyl transferases of apple accept 3-hydroxyphloretin besides phloretin as substrates. The parent line PinS accumulated between 4.5 and 7 mg/g DW 3-hydroxyphloridzin in the leaves. Considering the lower conversion rates of F3′H compared to CH3H observed in vitro with phloretin as substrate, it seems to be possible that F3′H is involved in the biosynthesis of the constitutive 3-hydroxyphloridzin levels in apple.
All transgenic CsCH3H lines generally showed lower concentrations of polyphenols. Flavonol concentrations were less affected which could be explained by the lower DFR activities but almost unchanged FLS activities in transgenic lines compared to the parent line. Because DFR and FLS compete for dihydroflavonols as common substrates it seems likely that a bottleneck at the stage of DFR increases the availability of precursors for flavonol formation and decreases the concentrations of downstream products such as flavan 3-ols. The decrease of dihydrochalcone concentrations could be partially explained by the lower CHS/CHI activities observed. However, as this was a rather decent decline in comparison to DFR activity we assume that other, unknown factors must be responsible.
Apple polyphenols were shown to possess strong antioxidant and radical scavenging activities (Lu and Foo 2000). Highest activities were observed with flavonols and flavan 3-ols, and 3-hydroxyphloridzin showed considerably higher effects than phloridzin (de Bernonville et al. 2010). No striking differences in the antioxidant capacity were observed between the transgenic lines and the parent lines. As the flavonol concentrations remained unchanged and the total polyphenol concentration decreased drastically, it seems likely that the increased 3-hydroxyphloridzin concentrations in the transgenic lines compensated the decrease resulting from the lower polyphenol concentrations. This assumption is supported by the fact that both 3-hydroxyphloridzin concentrations and the antioxidant capacity increased with leaf age.
The transgenic lines showed reduced susceptibility to fire blight and apple scab, which are the main bacterial and fungal diseases of apple. Two of the three lines showed a statistically significant reduced percentage of shoot necrosis after infection with E. amylovora. All lines showed a lower disease severity after infection with V. inaequalis. The most striking changes in the transgenic lines with respect to polyphenol composition, was the increase of 3-hydroxyphloridzin, the higher amounts of monomeric flavan-3-ols at the expense of oligomeric flavan 3-ols and the generally lower polyphenol concentrations. As polyphenols are assumed to have significant roles in disease resistance (Treutter 2006) the reduced concentrations are probably not responsible for the observed effects. The flavan 3-ols could be of relevance however all three lines showed a similar reduction in flavan 3-ols but different levels of susceptibility. Interestingly the 3-hydroxyphloridzin concentrations correlate very well with the observed decrease in disease susceptibility. Both, lines M802 and M803 showed higher 3-hydroxyphloridzin concentrations in the first, the second as well as in the third leaves and a significant lower percentage lesion length after inoculation with fire blight than the parental line PinS, whereas line M801 showed similar 3-hydroxyphloridzin concentrations as parental line PinS and only a slight but not significant reduction of shoot necrosis. Additionally, the mean incidence of scab on the leaves was lower for transgenic lines M802 and M803 than for M801 and PinS.
The transgenic lines harbouring C. sulphureus CH3H have been created as model plants which allowed for the first time to study the physiological relevance of 3-hydroxyphloridzin. The constitutive overexpression of the C. sulphureus CH3H in transgenic apple plants resulted in an increase of 3-hydroxyphloridzin formation in planta which seems to be correlated with reduced susceptibility or a slowlier development of disease symptoms to two economically important apple diseases. However, the transgenic lines unexpectedly showed lower polyphenol concentrations than the parent lines. The effect of such a drastic change in the polyphenol composition on the apple fruit (e.g. quality, taste, shelf life) needs to be tested in the future. In addition our study has demonstrated that 3-hydroxyphloretin can be created by cytochrome P450 dependent monooxygenases from the flavonoid pathway and has enlarged the substrate spectrum known to be accepted by F3′H. If the MalusF3′Hs are involved in the 3-hydroxyphloridzin biosynthesis in apple will be investigated in a future study.
Author contribution statement
OSH, HF, IR, SM, CK, SF, MZA, CG, AP, KR, MVH, DT performed research, OSH, HF, KS and HH wrote the manuscript, HH designed the study.
This work was supported by the Austrian Science Fund FWF (Projects P24331-B16 and P25399-B16). Olly S. Hutabarat gratefully acknowledges Hasanuddin University (Agricultural Engineering Department, Makassar, South Sulawesi, Indonesia), the Ministry of Education and Culture of the Republic of Indonesia (DIKTI), and Austrian Agency for International Cooperation in Education and Research (OeAD-GmbH) for enabling the performance of the PhD studies abroad. Shadab Faramarzi thanks the Ministry of Science, Research and Technology of the Islamic Republic of Iran for providing funding as a visiting researcher.
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