High temperature reduces apple fruit colour via ... - Wiley Online Library

Plant, Cell and Environment (2011) 34, 1176–1190

doi: 10.1111/j.1365-3040.2011.02316.x

High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex

pce_2316

1176..1190

KUI LIN-WANG1, DIEGO MICHELETTI6, JOHN PALMER3, RICHARD VOLZ4, LIDIA LOZANO7, RICHARD ESPLEY1, ROGER P. HELLENS1, DAVID CHAGNÈ5, DARYL D. ROWAN5, MICHELA TROGGIO6, IGNASI IGLESIAS7 & ANDREW C. ALLAN1,2 1 The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research) Private Bag 92 169, 2School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, 3Plant & Food Research, Motueka Research Centre, Motueka 7198, 4Plant & Food Research Hawke’s Bay Research Centre, Havelock North 4157, 5Plant & Food Research, Palmerston North Research Centre, Palmerston North 4442, 6IASMA Research and Innovation Centre, Foundation Edmund Mach, San Michele all’Adige, Trento, Italy and 7IRTA, Estació Experimental de Lleida, Avenue Alcalde Rovira Roure, 191. E-25198 Lleida, Spain

ABSTRACT The biosynthesis of anthocyanin in many plants is affected by environmental conditions. In apple (Malus ¥ domestica Borkh.), concentrations of fruit anthocyanins are lower under hot climatic conditions. We examined the anthocyanin accumulation in the peel of maturing ‘Mondial Gala’ and ‘Royal Gala’ apples, grown in both temperate and hot climates, and using artificial heating of on-tree fruit. Heat caused a dramatic reduction of both peel anthocyanin concentration and transcripts of the genes of the anthocyanin biosynthetic pathway. Heating fruit rapidly reduced expression of the R2R3 MYB transcription factor (MYB10) responsible for coordinative regulation for red skin colour, as well as expression of other genes in the transcriptional activation complex. A single night of low temperatures is sufficient to elicit a large increase in transcription of MYB10 and consequently the biosynthetic pathway. Candidate genes that can repress anthocyanin biosynthesis did not appear to be responsible for reductions in anthocyanin content. We propose that temperature-induced regulation of anthocyanin biosynthesis is primarily caused by altered transcript levels of the activating anthocyanin regulatory complex. Key-words: regulation.

Malus ¥ domestica;

MYB;

transcriptional

INTRODUCTION In the majority of plant species, pigmentation is controlled by the relative concentrations of anthocyanin, chlorophyll and carotenoid pigments. These pigments are essential for plant performance, but are also all considered as phytonutrients or markers of food health (Mayne 1996; Harborne & Williams 2000; Kirsh et al. 2006). The accumulation of these pigments is affected by the environment. Chlorophyll and Correspondence: A. C. Allan. E-mail: andrew.allan@plantandfood. co.nz 1176

carotenoid concentrations are controlled by light, via several photoreceptors (Reinbothe & Reinbothe 1996), developmental and tissue-specific signals (AmpomahDwamena et al. 2009; Cazzonelli & Pogson 2010) and biotic and abiotic stress including temperature (Li et al. 2008). Anthocyanin accumulation is also developmentally controlled and affected by both biotic and abiotic factors, such as nutrients (nitrogen and phosphate), sucrose, wounding, pathogen infection, methyl jasmonate, water stress, and UV, visible and far-red light (Dixon & Paiva 1995; Chalker-Scott 1999). Temperature has a major effect on anthocyanin synthesis in a diverse range of species, including grape (Vitis vinifera) (Mori et al. 2007), petunia (Petunia hybrida) (Shvarts, Borochov & Weiss 1997), red orange (Citrus sinensis) (Lo Piero et al. 2005) and rose (Rosa hybrida) (Dela et al. 2003). In Arabidopsis, anthocyanins are induced by low temperatures (Leyva et al. 1995) and reduced by high temperatures (Rowan et al. 2009). In apple (Malus ¥ domestica Borkh.) and pear (Pyrus communis L.), low temperatures increase both anthocyanin content and the expression of genes of the anthocyanin biosynthetic pathway (Steyn et al. 2005, 2009; Ubi et al. 2006). In apple, high temperatures prevent the accumulation of cyanidin and UDP-sugars (Ban et al. 2009), resulting in a rapid reduction in anthocyanins, followed by renewed synthesis with cooler temperatures, causing fluctuations in skin colour (Steyn et al. 2005). This response to fluctuating daily temperatures has been postulated to protect the sensitive fruit skin from light stressinduced photoinhibition, by acting as a screen to shade chloroplasts from blue-green light (Steyn et al. 2009). The biosynthesis of anthocyanin pigments and the gene networks that regulate synthesis have been well studied (Grotewold 2006; Allan, Hellens & Laing 2008). Anthocyanin accumulation in Arabidopsis is regulated at the level of transcription by the MYB-bHLH-WD40 (MBW) transcription factor complex (Zhang et al. 2003). The first isolated MYB member of this complex was the PRODUCTION OF ANTHOCYANIN PIGMENTS 1 (PAP1) gene (or MYB75; © 2011 Blackwell Publishing Ltd

Temperature and the anthocyanin regulatory complex 1177 At1g56650), identified by activation tagging (Borevitz et al. 2000). Over-expression of PAP1 in Arabidopsis results in purple-coloured leaves containing increased levels of anthocyanins and quercetin glycosides (Tohge et al. 2005), as a result of coordinative up-regulation of genes of the general phenylpropanid and anthocyanin-specific pathways (Tohge et al. 2005). Many of these up-regulated genes have a common cis-regulatory motif required for PAP1dependent transactivation (Dare et al. 2008). Expression of PAP1 is induced by light (Cominelli et al. 2007), sugars (Pourtau et al. 2006; Solfanelli et al. 2006; Teng et al. 2007) and nutrient deficiencies (Lillo, Lea & Ruoff 2008). Therefore, PAP1 has a central role in mediating the environmental regulation of anthocyanin biosynthesis in Arabidopsis. The transcriptional regulation of anthocyanin biosynthesis is complicated by redundancies and interactions between transcription factors. PAP1 shares high sequence homology to three other Arabidopsis MYBs: MYB113, MYB114 and MYB90/PAP2 (Gonzalez et al. 2008). The MBW transcription factor complex may include PAP1, PAP2, MYB113 and MYB114, as well as three potential bHLH partners [TRANSPARENT TESTA8 (TT8), GLABRA (GL3) and ENHANCED GLABRA (EGL3)] and the WD40 protein TRANSPARENT TESTA GLABRA 1 (TTG1) (Gonzalez et al. 2008). Furthermore, the MBW complex influences the expression of its own genes: TT8 enhances its own expression (Baudry, Caboche & Lepiniec 2006), and is also up-regulated by PAP1 (Tohge et al. 2005; Rowan et al. 2009). Transcription factors of the MYB class that suppress phenylpropanoid/anthocyanin accumulation have been described. These include an R2R3 MYB repressor from strawberry, MYB1 (Aharoni et al. 2001), MYB3, MYB4 and MYB6 from Arabidopsis (Jin et al. 2000) and Antirrhinum MYB308 (Tamagnone et al. 1998). Also in Arabidopsis, the one-repeat MYB protein, MYBL2 (Dubos et al. 2008; Matsui, Umemura & Ohme-Takagi 2008) and CPC (Zhu et al. 2009) repress phenylpropanoid concentrations. In strawberry (Fragaria ¥ ananassa), MYB1 plays a key role in down-regulating the biosynthesis of anthocyanins and flavonols (Aharoni et al. 2001). How the repressor MYBs interact with the MBW transcriptional complex is beginning to be elucidated (Dubos et al. 2008; Matsui et al. 2008). These repressor MYBs all contain C-terminal EAR motifs (named after ethylene-responsive element binding factorassociated amphiphilic repression domain). As well as MYBs, other transcription factor families have EAR motifs and roles in negatively regulating genes involved in developmental, hormonal and stress responses. Recently, 219 proteins containing these motifs were carefully analysed in the Arabidopsis genome (Kagale, Links & Rozwadowski 2010). The flavonoid repressor MYBL2 (Dubos et al. 2008; Matsui et al. 2008) has both an EAR motif and a C-terminal TFLLF motif that is required for repressive activity (Matsui et al. 2008). In Arabidopsis, elevated temperature causes a decline in expression of several genes that encode the anthocyanin activating transcriptional complex [TT8, TTG1–WD40,

EGL3, TTG2–WRK44 (Rowan et al. 2009)]. Upon an increase in temperatures to 32 °C, expression of at least three MYB–EAR repressors is elevated (MYB3, MYB6, MYBL2). One of these repressors, MYBL2, appears to be regulated by TT8 and indirectly by PAP1, via PAP1 elevating expression of TT8. In 35S:PAP1 plants, high temperatures do not further elevate this gene, while in wild-type plants the expression of MYBL2 is elevated by high temperatures. These results suggest that, in Arabidopsis, MYB repressors interfere with the activity of MYB activators under high-temperature conditions where anthocyanin biosynthesis slows (Rowan et al. 2009). The expression of the core anthocyanin pathway genes is reduced because of temperature-induced down-regulation of the transcriptional activator complex. In fruits and flowers, the loss of anthocyanic colour is of commercial importance. However, little is known of the mechanisms of anthocyanin turnover and degradation. Plant leaves, flowers and fruits show differing degrees of anthocyanin degradation during development. Using radiolabelled precursors, a turnover rate of anthocyanin in the range of 3–8% loss per day was seen in seedlings of mustard (Sinapsis alba) (Zenner & Bopp 1987), while a daily turnover rate of 50% or higher can occur in flowers of Brunfelsia, in grapes and in Arabidopsis (Mori, Sugaya & Gemma 2005; Vaknin et al. 2005; Olsen et al. 2009). In Arabidopsis, the fluxes of anthocyanin and flavonol degradation were found to be temperature independent (Olsen et al. 2009). In Brunfelsia calycina (the yesterday–today–tomorrow plant), the flowers change colour from dark purple to white within 2–3 d after flower opening, but before senescence occurs (Bar-Akiva et al. 2010). This process has been extensively studied at the metabolite, transcript and protein levels, and has been found to involve active degradation of anthocyanins, dependent on induced mRNAs and proteins (Vaknin et al. 2005; Bar-Akiva et al. 2010). The degradation of anthocyanins in fruits and berries has been studied in only a limited set of species. In pear (Pyrus communis), net anthocyanin degradation occurs with maturity so that red colour fades especially in response to higher temperature and light (Steyn et al. 2005). High temperature also reduces total anthocyanin concentration in grape (Vitis vinifera Cabernet Sauvignon) (Mori et al. 2007), with the exception of malvidin derivatives. Increased chemical or enzymatic degradation was postulated, with the more derivatized malvidin resisting degradation. Unlike other species, the transcript levels of the biosynthetic genes were not inhibited by heat (Mori et al. 2007). In this study, we examine the biochemical and genetic basis for the reduced red skin colouration of apple fruit in hot climates. We show that hot temperatures result in a down-regulation of the transcriptional activator complex (MYB10, bHLH300). We identify apple MYB repressors that can potentially reduce expression of MYB10 or repress the activity of MYB10 on the promoters of anthocyanin biosynthetic genes. As elevated temperatures did not significantly increase the expression of these MYB repressors in apple, the primary effect of temperature appears to be on

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

1178 K. Lin-Wang et al. the expression of the genes of the activation complex, rather than by induction of repressors. This has implications for the management of temperate crops and for the selection of cultivars that show greater colouration in hotter climates.

MATERIALS AND METHODS

acid/acetonitrile gradient; Hawke’s Bay). Total anthocyanin concentration was calculated using a molar extinction coefficient of 3.43 ¥ 104 as nmol cm-2. For RNA extraction, apple peel from the reddest part of the fruit on the equator was peeled and frozen in liquid nitrogen. Peels from five fruits were bulked together to give two peel replicates at each sampling date, and transferred to a -80 °C freezer.

Plant material and growth conditions Mature trees (10–12 years) of ‘Mondial Gala’ (a red sport of ‘Gala’) growing in blocks at the Institut de Recerca I Technolgia Agroalimentaries (IRTA) Estació Experimental de Lleida, Mollerussa, Spain (altitude 260 m above sea level; 46°09′ N 3°22′ E), and at the Plant & Food Research (PFR) orchard, Havelock North, Hawke’s Bay, New Zealand (altitude 260 m, 39°39′S 176°53′E), and young trees (3 years) of ‘Royal Gala’ (another red sport of ‘Gala’) growing in an orchard block at the PFR orchard, Motueka, Nelson, New Zealand (altitude 16 m, 41°07′S 172°59′E) were chosen for the study. The trees were trained as a vertical axis on ‘M.9’ rootstock (at Lleida and Nelson) at 1.3 ¥ 4.0 m (Lleida), or 1.5 ¥ 3.7 m spacing (Nelson), or as a centre leader pyramid on ‘M.26’ rootstock at 2.0 ¥ 4.0 m spacing (Hawke’s Bay). In Lleida, summer pruning was applied during June, removing the most vigorous shoots to improve light penetration into the canopy. Otherwise, all trees were managed according to standard commercial practice recommended for each region.

Harvesting and sample preparation for regional comparisons During the middle of the apple growing season in 2008, up to 20 well-exposed ‘Mondial Gala’ fruits were marked for sampling on moderate cropping trees (5–10 trees). Four marked fruits per tree (20 fruits in total) were randomly sampled at each of five dates (always at 1100 h local time) at approximately 14 d intervals. The sampling was begun at 75 d after full bloom (DAFB) (16 June, Lleida) or 86 DAFB (7 January, Hawke’s Bay), and continued until fruits were eaten ripe (138 DAFB, Lleida; 143 DAFB, Hawke’s Bay). At each harvest date, the fruits were divided into one set of 10 for anthocyanin analysis, and the other for RNA extraction. For anthocyanin analysis, individual apples were sampled by taking a peel disc of 10 mm diameter from the reddest part of each fruit at the equator. Each disc was placed in 10 mL of methanol:water: 35% hydrochloric acid (50:49:1 v/v/v) (Lleida) or 1 mL of 96% ethanol:water:formic acid (80:20:1 v/v/v) (Hawke’s Bay). Extractions were carried out overnight and in the dark at 4 °C. Anthocyanin was measured as extract absorbance at 532 nm with a Cecil series 1000 spectrophotometer (Cecil Instruments, Cambridge, England) (Lleida) or using a Waters Alliance 2690 highperformance liquid chromatograph (Waters Corp., Milford, MA, USA) with a Zorbax SB-C18 4.6 ¥ 150 mm column (1.8 mm particle size, separation achieved using a formic

Harvesting and sample preparation for heating experiments An air delivery system designed for the measurement of whole-tree gas exchange (Wunsche & Palmer 1997) was modified to include an in-line heater, so that warm air could be directed selectively onto a group of fruits held approximately horizontally over the opening of the 0.3 m diameter flexible delivery tube. Three air delivery systems enabled one group of five ‘Royal Gala’ fruit to be heated on each of six trees located in Nelson, New Zealand. This enabled specific and selective modification of the temperature of the fruit on a tree with minimal disturbance to the fruit light environment and to surrounding unheated fruit on the same tree, which were used as unheated controls. Fruit skin temperatures were monitored using 42 SWG copper/ constantan thermocouples connected to a DeltaT data logger (Type DL2; Delta-T Devices, Burwell, Cambridge, UK). Each group of heated and unheated fruits had one thermocouple inserted just under the fruit skin. Heating was applied continuously over two 7 d periods (14–22 January and 11–18 February 2009), starting approximately 39 and 11 d, respectively, before the normal commercial harvesting of the fruits. The fruits were sampled at 1300 h each day just before switching on the heaters (day 0), and again after 1, 2, 5 and 7 d. Each sampling consisted of one heated and one control fruit from each of the six trees. Fruit peel was immediately sampled for anthocyanin concentration and RNA extraction as previously described.

Real-time quantitative PCR (qPCR) analysis RNA was isolated from fruit peel by modification of the method of Chang, Puryear & Cairney (1993). First-strand cDNA synthesis was carried out by using oligo dT according to the manufacturer’s instructions (SuperScript III; Invitrogen, Carlsbad, CA, USA). To remove genomic DNA contamination, all the cDNA samples were DNase treated (DNA-free Kit; Ambion, Austin, TX, USA). Candidate genes for expression analysis were chosen based on literature reports: apple TTG1, coding for a WD40 protein (Brueggemann, Weisshaar & Sagasser 2010), apple MYB10 (Takos et al. 2006; Espley et al. 2007), bHLH3 and bHLH33 (Espley et al. 2007). Because of the availability of the apple genome (Velasco et al. 2010), other candidates were chosen by BLAST search to published anthocyanin activators or repressors. Apple bHLH300 (EB120313) was identified as having good blast match to Arabidopsis TT8. Arabidopsis

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

Temperature and the anthocyanin regulatory complex 1179 TTG2 (WRKY44, AT2G37260) has several possible candidates by best BLAST match, which are well expressed in apple fruit: WRKY44 (EB153781) and WRKY10 (HM122713). Three other candidates were also good BLAST matches: WRKY9 (EB149602), WRKY13 (HM122716) and WRKY7 (HM122726), but expression of these genes was low in apple peel samples. Candidate apple MYB repressors were chosen if they had an identity cut-off of 1e-20 and contained an EAR repression domain.A phylogenetic tree of R2R3 MYBs and R3MYBs was built with PhyML (Guindon et al. 2005), based on the alignment of the repressor MYBs with Mafft (Ronquist & Huelsenbeck 2003), using default parameters and based on the LG substitution method using maximum likelihood with 100 bootstrap (Le & Gascuel 2008). Real-time qPCR DNA amplification and analysis were carried out using the LightCycler 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany), with LightCycler 480 software version 1.5. The LightCycler 480 SYBR Green I Master Mix (Roche) was used and the 10 mL of total reaction volume was applied in all the reactions following the manufacturer’s method.The qPCR conditions were 5 min at 95 °C, followed by 40 cycles of 5 s at 95 °C, 5 s at 60 °C and 10 s at 72 °C, followed by 65–95 °C melting curve detection. The qPCR efficiency of each gene was obtained by analyzing the standard curve of a cDNA serial dilution of that gene. Primers were designed to the region that could easily distinguish genes from one another in the same phylogeny branch.The apple actin primers were based on the actin sequence of apple (Actin, accession number CN938023). Actin was selected as a reference gene because of its consistent transcript level throughout fruits, with a crossing threshold (Ct) change of less than two cycles. To confirm the amplification of the expected DNA sequence, qPCR amplicons were sequenced. Analysis of variance (anova) tables and least significant differences (LSDs) were calculated from summary statistics using standard formulas.

Transient assay of gene function in tobacco leaves Candidate transcription factors were tested using the dual luciferase assay of transiently transformed Nicotiana benthamiana leaves (Hellens et al. 2005). The promoter of apple DFR (1.3 kb upstream of DFR, EB151303) was isolated from apple genomic DNA and inserted into the cloning site of pGreenII 0800–LUC (Hellens et al. 2005) and modified to introduce a Nco1 site at the 3′ end of the sequence, allowing the promoter to be cloned as a transcriptional fusion with the firefly luciferase gene (LUC). This promoter, along with previously published promoters from Arabidopsis (AtDFR–LUC; Hellens et al. 2005, and two versions of the apple MYB10 promoter, pR1–MYB10 and pR6–MYB10; Espley et al. 2009), were used in transient transformation of N. benthamiana leaves. Agrobacterium strain GV3101 (MP90) was transformed with the reporter cassette, while other Agrobacterium cultures were transformed with cassettes containing an MYB activator gene, or

bHLH TF gene, or MYB repressor gene fused to the 35S promoter, respectively, in either pART27 (Gleave 1992) or pHex2 (Hellens et al. 2005). These bacterial strains were cultured as previously reported, then suspended in infiltration buffer (10 mm MgCl2 and 0.5 mm acetosyringone) before infiltration into young leaves of N. benthamiana grown under glasshouse conditions. Three days after inoculation, leaf discs were assayed for luminescence using the Orion Microplate Luminometer (Berthold Detection Systems, Pforzheim, Germany) as previously reported (Espley et al. 2009). To assay the induction of anthocyanins (Espley et al. 2007), Nicotiana tabacum ‘Samsun’ plants were grown under glasshouse conditions until reaching at least 10 cm in height. Agrobacterium cultures were prepared as for the dual luciferase assay. Two or three Agrobacterium cultures containing the MYB activator gene, or bHLH TF gene, or MYB repressor gene, all fused to the 35S promoter, were mixed (300 mL each) and infiltrated into the abaxial leaf surface of N. tabacum as for the dual luciferase assay.

RESULTS High-temperature climate reduces anthocyanin concentration in peel of maturing apples The fruit-growing region of Lleida in Spain is frequently subject to periods of high summer temperatures (>30 °C), while New Zealand fruit-growing regions have more moderate summer temperatures. As previously reported for ‘Royal Gala’ apples (Steyn et al. 2009), the ‘Mondial Gala’ fruits show a marked decline in skin anthocyanin content (Fig. 1a) when grown under hot orchard conditions. During the sampling period, maximum mean temperatures (averaged over 10 d intervals) were 23–24 °C in Hawke’s Bay, and 29–34 °C for Lleida. Mean minimums were 11–15 °C in Hawke’s Bay, and 13–16 °C for Lleida. Mean solar radiation was also different: 16–21 MJ m-2 in Hawke’s Bay, and 27–30 MJ m-2 in Lleida. The different growing conditions had a dramatic effect on skin anthocyanin concentrations. At the final tree ripe timepoint, Spanish ‘Mondial Gala’ accumulated anthocyanin at under 20 nmol cm-2, while New Zealand ‘Mondial Gala’ showed a fivefold greater accumulation to reach over 100 nmol cm-2 (Fig. 1b). The low rate of anthocyanin accumulation resulted in many Spanish-grown apples appearing green/yellow with a slight blush (Fig. 1a,ii), while others showed low pigmentation. In contrast, the least pigmented New Zealand-grown apples were still darker than the most highly coloured apples from Spain (Fig. 1a,i). This markedly lower anthocyanin concentration over the 65 d of sampling in Spain could be caused by one or a combination of reduced synthesis, increased degradation or increased photo-oxidation of the phenylpropanoid intermediates produced by an otherwise active pathway. When the transcript abundance of the phenylpropanoid pathway biosynthetic genes was measured over fruit development, a dramatic reduction in gene expression was observed

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

1180 K. Lin-Wang et al.

(b)

Anthocyanin concentration (nmol cm–2)

(a)

110 100 90 80 70 60

New Zealand Spain

50 40 30 20 10 80

90

100

110

120

130

140

Days from full bloom

Relative expression

(c)

Figure 1. Occurrence and biosynthesis of anthocyanins in apple peel under two climatic conditions. Mature ‘Mondial Gala’ apples orchard grown in New Zealand (a, i) or in Spain (a, ii) showing extremes in skin anthocyanin content during the last 10 weeks of maturity in the two climatic areas (b), and gene expression analysis of early (CHS, chalcone synthase) and late (LDOX, leucoanthocyanidin dioxygenase) anthocyanin biosynthetic genes over the same time period (c). Error bars for all time-points represent the SEM of three biological replicates.

(Fig. 1c, P < 0.001) for the samples from Spain compared with those from New Zealand. This reduction applied to all the pathway genes tested, from chalcone synthase (CHS) through to UDP-glucose:flavonoid-3-O-glycosyltranferase (UFGT) (Fig. 1c & Supporting Information Fig. S1), and suggests coordinative down-regulation of the expression of these genes at higher temperatures.

Down-regulation of the anthocyanin pathway via a reduction in transcriptional activation complex The coordinated reduction in the biosynthetic pathway may be caused by transcriptional regulation by the MBW complex. These apple transcription factors were measured

and included the MYB activators, MYB10 (also known as MYB1; Takos et al. 2006; Espley et al. 2007; Lin-Wang et al. 2010), WD40-TTG1 (Brueggemann et al. 2010), bHLH3 and bHLH33 (Espley et al. 2007), as well as new candidates available from the recently released apple genome (Velasco et al. 2010), WRKY10 and WRKY44, and bHLH300 (Fig. 2). Many potential members of the MBW complex showed reduced expression (MYB10, bHLH300 and WRKY10, P < 0.001; WD40, P = 0.08) under Spanish growth conditions, suggesting that there could be a reduced transcription factor abundance resulting in a reduced ability to trans-activate the apple anthocyanin pathway genes. The bHLH candidates known to function in enhancing anthocyanin biosynthesis, apple bHLH3 and bHLH33 (Espley et al. 2007), showed no significant

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

Relative expression

Temperature and the anthocyanin regulatory complex 1181

Figure 2. Relative expression levels of candidate genes of the apple anthocyanin transcriptional activation complex in peel of ‘Mondial Gala’ apples orchard grown under either New Zealand or Spanish climatic conditions. A reduced expression in Spanish fruit of MYB10 and candidate members of the MWB complex, bHLH300, TTG1-WD40 and WRKY10, is seen over a 65 d time-course. Error bars represent mean ⫾ SEM of three biological replicates.

difference in transcript profile between growth regions (not shown).

On-tree heating of maturing apples mimics climatic effects on apple colour The reduced anthocyanin concentrations in the peel of ‘Mondial Gala’ observed in our experiment could be caused by many factors. Apart from the temperature difference (mean difference of 6–10 °C maximum temperatures, 1–2 °C minimum temperature), other factors could include hours of sunshine, rainfall, soil nutrients, day length, orchard

management and slight genetic differences among trees within the ‘Mondial Gala’ strain. To control for these factors, an on-tree experiment was conducted in New Zealand (Nelson region, mid-February) in which individual apple clusters were heated to approximately 8 °C above ambient orchard temperatures (Fig. 3a,b). This was achieved using heated air pumped over the apples (Fig. 3a). Apples, including unheated control fruits from the same trees, were sampled at five time-points for analysis of anthocyanins and gene expression. Localized air heating was used to raise both day and night-time temperatures of selected fruits, which ranged from 7 to 22 °C for control fruits, and 17–35 °C for heated fruits (Fig. 3b). Night temperatures stayed above 17 °C for heated apples, while control fruits experienced temperatures as low as 7 °C. During this experiment, anthocyanin concentrations were reduced after 1 d of heating (Fig. 3c). This appeared more as a failure to synthesize anthocyanins, as control fruits were experiencing a phase of dramatic increase in red colouration, while heated fruits remained at 20–25 nmol cm-1, before declining by day 5 of the treatment. This experiment suggests that maintenance of temperatures above 20 °C results in reduced anthocyanin concentrations and poor red apple colour. When the gene transcript profiles of the anthocyanin biosynthetic genes and associated regulatory complex were examined, the effect of heating was immediately apparent (Fig. 4). While control fruits were experiencing a drop in temperature below 20 °C, heated fruits remained above this temperature. Transcripts of all the biosynthetic genes (Fig. 4, P < 0.001; Supporting Information Fig. S2) and MYB10 (P < 0.001) increased with anthocyanin biosynthesis (Fig. 3c) in unheated control fruits, but failed to increase, or actually declined (e.g. MYB10, LDOX) in heated fruits. The transcripts of bHLH300 (Fig. 4), TTG1 and WRKY10 (data not shown) did not decrease during this experiment.

Apple R2R3 MYB transcription factors, which function to inhibit the anthocyanin pathway It is apparent that heat, or perhaps the absence of cold, affects the apple anthocyanin transcriptional activation complex and therefore expression of the biosynthetic pathway genes. The anthocyanin biosynthetic pathway and complex are also affected by transcriptional MYB repressors, such as Arabidopsis MYBL2 (Dubos et al. 2008; Matsui et al. 2008), which has been shown to be unregulated when Arabidopsis is grown under higher temperatures (Rowan et al. 2009). We utilized BLAST match of the known Arabidopsis EAR motif containing repressor MYBs, MYBL2, MYB3, MYB4, MYB6, against the apple genome (Velasco et al. 2010) to identify apple homologs of these genes (Fig. 5). The resulting phylogenetic tree is well supported and clusters eight apple C-terminal EAR motif MYBs with known repressors including strawberry MYB1 (Aharoni et al. 2001). Of note is the finding that the closest matches to Arabidopsis MYBL2 (apple MYB17, MYB27, MYB28, MYB50, MYB111) are all R2R3MYBs (see

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

1182 K. Lin-Wang et al. Functional assays of the repression activity of these MYBs were performed using a transient luciferase assay system (Hellens et al. 2005), as well as transient induction of anthocyanic biosynthesis in leaf patches in tobacco (Espley et al. 2007). Tested MYBs that harboured an EAR repression domain (MYB16, MYB17, MYB111) inhibited the activation of the Arabidopsis and apple DFR promoters by apple activators MYB10–bHLH3 (Fig. 6a). MYB8, a possible stress-related R2R3 MYB in a related clade, but without an EAR motif, had no effect on the activation assay (Fig. 6a,b). It has been reported that MYB10 can autoregulate its own promoter (Espley et al. 2009). By fusing the MYB10 promoter (versions with and without the

(a)

(b)

Air temperature Mean unheated fruit temperature Mean heated fruit temperature

40

30 25 20 15 10

1

0 5

5

2

Sample no.

7

11 Feb 12 Feb 13 Feb 14 Feb 15 Feb 16 Feb 17 Feb 18 Feb

Date

Anthocyanin concentration (nmol cm–2)

(c)

55 50 45 40

Relative expression

Temperature (oC)

35

35 30 25 20 15 Unheated Heated

10 5

0

2

4

6

8

Days from beginning of experiment

Figure 3. Orchard-based apple heating system designed to raise the day and night temperatures of selected ‘Royal Gala’ apple fruits on-tree (a) to mimic hotter climatic conditions (b) and to reduce peel anthocyanin content (c). Apples were sampled for anthocyanin analysis at points shown by arrows (b). Error bars represent the SEM of three biological replicates.

alignment in Supporting Information Fig. S3), while the Arabidopsis gene is an R3MYB. MYB111 also contains a C-terminal TLVLFR motif, shown to add to the repression activity of Arabidopsis MYBL2 (Dubos et al. 2008; Matsui et al. 2008).

Unheated

Heated

Figure 4. Relative expression of the early (CHS, chalcone synthase) and late (LDOX, leucoanthocyanidin dioxygenase) biosynthetic genes and of MYB10, and its potential bHLH partner bHLH300, in apple peel from ‘Royal Gala’ fruits subjected to an orchard-based on-tree heating system designed to raise the day and night temperatures of selected on-tree apple fruits to mimic hotter climatic conditions. Error bars for all time-points represent the SEM of three biological replicates.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

Temperature and the anthocyanin regulatory complex 1183

C-terminal EAR motif

Figure 5. A rooted phylogeny of R2R3MYB transcription factors of apples, and of other anthocyanin-repressing MYBs of other plant species. Genes containing known EAR or other repression domains indicated.

auto-regulatory domain; R6 and R1, respectively) with luciferase, it was shown that the repressor MYBs could prevent this auto-regulation (Fig. 6a). When tobacco leaves are infiltrated with Agrobacteriumexpressing apple MYB10 and bHLH3 genes, anthocyanin

biosynthesis is initiated and a coloured ‘anthocyanic patch’ forms within 7 d (Espley et al. 2007). Three of the eight apple C-terminal EAR motif MYBs, from the phylogeny in Fig. 5, were cloned into expression vectors and included in the transient assay. The inclusion of apple R2R3 EAR

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

1184 K. Lin-Wang et al. indication of involvement in this environmental response. We performed qPCR expression analysis for all the MYBs in Fig. 5 and Table 1, and found little evidence of elevation of the repressor class under hotter climatic conditions or with on-tree heating of specific fruit clusters on a tree (Fig. 7, MYBs with a significant fruit expression shown). Expression levels for most of these MYBs were higher under New Zealand (temperate) conditions, where anthocyanin accumulation is highest (Fig. 7a; MYB16 and MYB17 P < 0.001; MYB111 P = 0.04). Only one repressor, MYB15, had a slightly higher profile (P = 0.48) with Spanish conditions and at the last time-point of the heating experiment (P = 0.07, Fig. 7a,b). The primary effect of temperature therefore appeared to be on the expression of the genes of the activation complex, rather than by induction of a MYB repressor.

1.4

(a)

1.2

Luc/Ren

1 0.8 0.6 0.4 0.2

+buf f er +MYB8 +MYB111 +MYB17 +MYB16 +buf f er +MYB8 +MYB111 +MYB17 +MYB16 +buf f er +MYB8 +MYB111 +MYB17 +MYB16 +buf f er +MYB8 +MYB111 +MYB17 +MYB16

0

MYB10 + bHLH3

MYB10 + bHLH3

MYB10 + bHLH3

MYB10 + bHLH3

DFR (At)

DFR (Md)

R1 (Md)

R6 (Md)

A single night of low temperature is sufficient to induce anthocyanin biosynthesis and a significant response in gene transcription

(b)

MYB10 + bHLH3 (+MYB8)

MYB10 + bHLH3 (+MYB16)

Figure 6. R2R3 MYB repressors of apple (a) prevent the activation of the promoters AtDFR–LUC, MdDFR–LUC, MdR6MYB10, by MYB10 and bHLH3 in transient assays in tobacco, and (b) inhibit anthocyanin biosynthesis in tobacco leaves. Error bars represent the SEM of three biological replicates.

An on-tree heating experiment was performed on maturing ‘Royal Gala’ fruits (in New Zealand, Nelson region, midJanuary), using the same heating system and sampling points as previously (Fig. 2). However, just before the last sample date (day 7), heating was interrupted, allowing all fruits (treated and control) to reach temperatures as low as 7 °C (Fig. 8a). The heated fruits showed a significant recovery in anthocyanin content (Fig. 8b) and gene transcripts of both the anthocyanin biosynthetic system, and the transcriptional activation complex increased (Fig. 8c, P < 0.01). Even the transcription of bHLH300, which had not altered significantly with heat (Fig. 4), showed an increase in expression (P < 0.001) in the heated fruits after this one cold night. Other biosynthetic genes followed a similar pattern (Supporting Information Fig. S4). No repressor MYBs showed any down-regulation, and expression of MYB17 was up-regulated (not shown).

DISCUSSION

repression domain MYBs to this assay system completely inhibited this anthocyanin response (Fig. 6b, shown for MYB16). Inclusion of MYB8, which has no EAR motif, in the tobacco leaf assay did not affect the anthocyanin response. This relatively simple assay therefore shows these candidate MYBs are potent repressors of anthocyanin biosynthesis.

The transcriptional profile of repressor MYBs suggests they are not induced by high temperatures An elevation in transcript level of any apple R2R3 EAR repression domain MYBs, during heat-induced reduction of apple skin anthocyanin biosynthesis, would be a good

The biosynthesis of pigments in many plants is affected by environmental conditions, often with important economic consequences. In apples and pears, there are reports of fast changes in fruit peel colour with temperature (Lancaster et al. 2000; Steyn et al. 2004, 2009; Ubi et al. 2006). We have confirmed the speed of this response and performed on-tree manipulations to test the effect of temperature in isolation from other environmental influences. Our results suggest that a lack of transcription of the phenylpropanoid pathway genes is responsible for reduced anthocyanin biosynthesis at higher temperatures. Our findings support the hypothesis that temperature affects anthocyanin biosynthesis via MYB10. The apple anthocyanin pathway has been shown to be controlled by the MYB transcription factor, MYB10 (Takos et al. 2006; Ban et al. 2007; Espley et al. 2007), which is

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

Temperature and the anthocyanin regulatory complex 1185 Table 1. Apple R2R3 MYBs shown in Fig. 5

Name

EST count

Linkage group

mRNA Libraries

Closest AtMYB

Possible function

GenBank number

Apple skin peel, 150 d after full bloom (DAFB) Apple peel and cortex 150 DAFB, senescing leaf Fruit cortex only

AT1G66370 MYB113

Anthocyanin elevation

EU518249

AT1G68320 MYB62

Phosphate and GA response Phosphate and GA response Regulator of trichome branching

DQ074462

AT1G22640 MYB3 AT4G38620 MYB4 AT4G38620 MYB4 AT3G13540 MYB5

Repressor Repressor Repressor Trichome branching

EG631227 HM122617 HM122618 EB128346

AT1G22640 MYB3 AT4G09460 MYB6

Repressor Repressor

GO562864 EB142177

AT3G01140 MYB106 AT1G09540 MYB61

CO867489 EB126674

MYB1/10

5

9, 32.8M

MYB7

8

16, 10M

MYB8

4

6, 28M

MYB12

17

3, 0.96M

MYB15 MYB16 MYB17 MYB26

4 19 6 11

8, 32M 2, 30M 14 33M 7, 20M

MYB27 MYB28

2 5

6, 28M 13, 1.6M

MYB29 MYB40

4 6

9, 34M 12, 31M

Root tips and xylem, cold-stored fruit, heat-treated leaf Spur buds trees only Flower, veg. bud ‘M.9’ phloem only Young fruit, leaves, young root, buds Fruit Young fruit, leaves, buds, internodes Buds, shoot, fruitlet Young fruit, tips, buds

MYB48 MYB49 MYB50 MYB51 MYB52 MYB53 MYB54 MYB55 MYB56

4 3 2 2 1 0 5 0 0

13, 13M 8, 10M 13, 1.6M 9, 15M 10, 27M 5, 10.9M 17, 22M 17, 26M 13, 2.3M

Roots, flowers Flowers, fruit Leaves, shoots Young roots Pooled cDNA None Flowers, buds None None

AT3G06490, MYB108 AT4G38620 MYB4 AT4G09460 MYB6 AT5G26660 MYB86 AT3G12720 MYB67 AT3G12720 MYB67 AT5G40350 MYB24 AT2G47190 MYB2 AT1G68320 MYB62

MYB111

19

9, 32M

AT4G09460 MYB6

MYB110

2

17, 24.8M

Fruit cortex, seed, buds, leaves, flowers Flowers, buds

Trichome branching Vascular,roots, stomates and flowering Pathogen response Response to UV-B Repressor C4H repressor Not known Not known Jasmonate response Salt and water stress Phosphate and GA response Repressor

AT1G66380 MYB114

Anthocyanin elevation

AT1G68320 MYB62 AT3G13540 MYB5

DQ267899 EG631283

DT001438 EB156950 DR992317 GO515747 Genome only Genome only EB710142 Genome only Genome only EB137767 CN993940

An approximate EST count (Newcomb et al. 2006), the tissue library from which the ESTs were sequenced, and the location in the apple genome (Velasco et al. 2010) are shown, as well as the best Arabidopsis hit from BLAST match and its possible function, along with an apple GenBank deposit if available.

potentially auto-regulated (Espley et al. 2009). In apple, MYB1, MYBA and MYB10 genes may be allelic to one another (Lin-Wang et al. 2010). In addition, the chromosomal location of this MYB in the recently published apple genome (Velasco et al. 2010) has only one gene, indicating that in ‘Golden Delicious’, these genes are not linked paralogues. Genetic and mapping evidence suggests this gene is the major controller of apple skin colour (Takos et al. 2006; Zhu, Evans & Peace 2010), and flesh and foliage colour (Chagné et al. 2007). Therefore, information on environmental effects on expression of this major gene is important for breeding programs and orchard management practices. We found that MYB10 expression was reduced in a hot climate and that heating on-tree fruit rapidly lowered transcript levels of this MYB. Lower temperatures also stimulate MYB10 expression (Fig. 8) (Ban et al. 2007). Other members of the MBW transcriptional complex are affected by temperature; one potential bHLH partner, and the TTG1 and TTG2-like genes all declined in expression with higher temperatures.

The effect of temperature on MYB10 expression may be caused by several processes (Fig. 9). There may be direct temperature-induced changes in activity of the promoter of MYB10. Recent studies have shown that heat is perceived directly by the plant nucleus via specific thermosensing nucleosomes that allow DNA unwrapping (Kumar & Wigge 2010). The MYB10 locus may be in such a thermo-sensitive region. Alternatively, temperature-sensitive transcription factors may modulate the transcription of MYB10. In Arabidopsis, high light and heat stress elicits a NAC transcription factor, which influences flavonoid biosynthesis and accumulation of anthocyanins (Morishita et al. 2009). Heat shock factors are a class of transcription factor, represented by 21 genes in Arabidopsis, and have been shown to regulate a number of heat shock genes via transcriptional activation at TATA-proximal heat shock elements (HSE) (Kotak et al. 2007; Von Koskull-Doring, Scharf & Nover 2007; Nishizawa-Yokoi et al. 2009). These HSFs may themselves be induced by AP2-domain TFs, such as DREB2A (Schramm et al. 2008), or interact as HSF–AP2 complexes

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

1186 K. Lin-Wang et al.

Relative expression

(b)

Relative expression

(a)

New Zealand

Spain

Unheated

Heated

Figure 7. Relative expression of candidate MYB repressors (MYB15, MYB16, MYB17 and MYB111) in peel of (a) ‘Mondial Gala’ apples grown in two different climates over a 65 d time-course. (b) ‘Royal Gala’ apples subjected to orchard-based on-tree heating over 7 d. Error bars for all time-points represent the SEM of three biological replicates.

on target promoters (Almoguera et al. 2009). DREBs are members of the large AP2-domain class of transcription factors (also termed CBF) and are integral to plant temperature response (Chinnusamy, Zhu & Zhu 2007). These factors interact at cis-acting dehydration-responsive element (DRE)/C-repeat (CRT) sequences in gene promoters. Finally, a number of WRKY TFs involved in biotic and abiotic stress response are heat induced (Li et al. 2010), so our observations that apple WRKY10 is down-regulated by heat may be a further link between heat and anthocyanin concentrations. In apples, we have found that high temperature reduces MYB10 expression, while previously the expression of apple MYBA (which shares the same nucleotide sequence as MYB1) was shown to be induced by low temperature and UV-B (Ban et al. 2007). The promoter of MYB10 may be activated by cold sensing TFs, such as CBF and ICE1 (Chinnusamy et al. 2007), which is prevented at elevated

temperatures. Finally, repressor TFs, such as EAR motif MYBs, could interfere with the expression of MYB10. We have shown that alleles of the MYB10 gene that are autoregulated by MYB10 (the R6 promoter mutation present in red foliage, red-fleshed apples; Espley et al. 2009) are repressed by EAR motif MYBs, some of which may be affected by temperature. This study has taken advantage of the extensive apple EST database (Newcomb et al. 2006) and the recently released apple genome (Velasco et al. 2010), to isolate and study new transcription factors, such as apple bHLH300, WRKY44, WRKY10, as well as the family of EAR motif R2R3MYB repressors. Several novel observations were made of the R2R3MYB repressors. Firstly, these repressors can efficiently inhibit anthocyanin production in a tobacco heterologous system. In addition, all these family members belong to the R2R3 class of MYB, with no apparent single repeat R3 MYBs, as observed in Arabidopsis (Dubos et al.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

Temperature and the anthocyanin regulatory complex 1187

(b)

Air temperature Mean unheated fruit temperature Mean heated fruit temperature

40

Temperature (°C)

35 30 25 20 15 10 5

0

1

2

14 Jan

15 Jan

16 Jan

5 17 Jan

18 Jan

19 Jan

7 Sample no. 20 Jan

21 Jan

Anthocyanin concentration (nmol cm–2)

(a)

Unheated Heated

55 50 45 40 35 30 25 20 15

0

2

4

6

8

Days from beginning of experiment

Date

Relative expression

Relative expression

(c)

Unheated

Heated

Unheated

Heated

Figure 8. Effect of orchard-based apple heating with a single night of low temperatures on anthocyanin concentration and anthocyanin-related gene expression of ‘Royal Gala’ fruits. A log of the heating system, which raised the day and night temperatures of selected on-tree fruits, but was turned off at day 6 of the experiment (red line on all panels). (a) Apples were then sampled (arrows in a) and peel tissue measured for anthocyanin analysis (b), and for expression of biosynthetic (CHS and LDOX) and regulatory (MYB10 and bHLH300) genes (c). Error bars for all time-points represent the SEM of three biological replicates.

2008; Matsui et al. 2008). However, during growth in hot climates and on-tree heating, there is little or no increase in expression of these MYB repressors. Moreover, for most R2R3 MYB repressors, expression declines in hot temperatures. This may be caused by their regulation by MYB10 itself, either directly or via a bHLH functioning in an analogous mechanism to Arabidopsis TT8/EGL3. This is consistent with results showing the anthocyanin activation complex up-regulates the inhibitors of the complex (Dubos et al. 2008; Matsui et al. 2008), suggesting that their function may limit the MBW activator complex. However, there is one apple repressor (MYB15) that increases slightly in both hot climates and in orchard heating experiments. Genetic resources may provide more evidence as to what effect this family has on fruit skin pigmentation.

We have used transient transformation assays to show that these apple R2R3 MYB repressors can potentially interfere with the activation of both the anthocyanin biosynthetic steps and with MYB10 stimulation of its own expression. This has implications for understanding transcriptional control of anthocyanins in many other species: if feed-forward cascades of pigmentation are prevented by active repressors, then mutations in these genes may be responsible for high-coloured varieties. There are already reports of auto-regulation of MYB expression (Espley et al. 2009) and TT8 expression (Baudry et al. 2006), as well as Arabidopsis MYBL2 involvement in preventing the regulatory loop (Dubos et al. 2008; Matsui et al. 2008). Our work suggests this complexity of regulation could occur in maturing fruit peel tissues.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

1188 K. Lin-Wang et al.

Heat Cold

bHLH

MdMYB10

Repressor MYBs

Regulation of anthocyanin biosynthesis

Figure 9. Schematic showing the major interaction of the high temperatures with the anthocyanin pathway via inhibition of expression of MYB10. Dotted lines may indicate other levels of control.

The sensitivity and responsiveness of anthocyanin biosynthesis to temperature was demonstrated in an on-tree experiment where a single night of lower temperature was sufficient to elicit a large increase in transcription of MYB10 and consequently the biosynthetic pathway. Further experiments are required to determine the threshold temperatures that regulate MYB10, which may provide answers as to whether it is heat, or an absence of cold, that primarily affects the activation complex in apples. There is also the possibility that large fluctuations between daily minimum and maximum temperatures trigger activation. We conclude that high temperatures directly affect the transcriptional levels of MYB10, and maybe other members of the activation complex. This results in a decline in the expression of genes of the anthocyanin biosynthetic pathway and less red colour in apple peel. Most candidate apple MYB repressors also decline in expression under high temperatures, possibly because their expression is coordinated by MYB10. However, their repressor activity against promoters of the anthocyanin pathway, and on the promoter of MYB10 itself, suggests a key role in modulating the apple anthocyanin pathway.

ACKNOWLEDGMENTS This work is part of an EU-funded IRSES project & Marie Curie, and was funded also by the New Zealand Foundation for Research Science and Technology C06X0812 ‘Exploiting Opportunities from Horticultural Genomics’ and MSI & PREVAR. The authors thank Tim Holmes and Martin Heffer for the pictures, Robert Schaffer for comments on the manuscript and Duncan Hedderley for statistical advice.

REFERENCES Aharoni A., Ric de Vos C.H., Wein M., Sun Z., Greco R., Kroon A., Mol J.N.M. & O’Connell A.P. (2001) The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal 28, 319– 332.

Allan A.C., Hellens R.P. & Laing W.A. (2008) MYB transcription factors that colour our fruit. Trends in Plant Science 13, 99–102. Almoguera C., Prieto-Dapena P., Diaz-Martin J., Espinosa J.M., Carranco R. & Jordano J. (2009) The HaDREB2 transcription factor enhances basal thermotolerance and longevity of seeds through functional interaction with HaHSFA9. BMC Plant Biology 9, 75. Ampomah-Dwamena C., McGhie T., Wibisono R., Montefiori M., Hellens R.P. & Allan A.C. (2009) The kiwifruit lycopene betacyclase plays a significant role in carotenoid accumulation in fruit. Journal of Experimental Botany 60, 3765–3779. Ban Y., Honda C., Hatsuyama Y., Igarashi M., Bessho H. & Moriguchi T. (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant & Cell Physiology 48, 958–970. Ban Y., Kondo S., Ubi B.E., Honda C., Bessho H. & Moriguchi T. (2009) UDP-sugar biosynthetic pathway: contribution to cyanidin 3-galactoside biosynthesis in apple skin. Planta 230, 871–881. Bar-Akiva A., Ovadia R., Rogachev I., et al. (2010) Metabolic networking in Brunfelsia calycina petals after flower opening. Journal of Experimental Botany 61, 1393–1403. Baudry A., Caboche M. & Lepiniec L. (2006) TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors, allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana. The Plant Journal 46, 768–779. Borevitz J.O., Xia Y., Blount J., Dixon R.A. & Lamb C. (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. The Plant Cell 12, 2383–2393. Brueggemann J., Weisshaar B. & Sagasser M. (2010) A WD40repeat gene from Malus ¥ domestica is a functional homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1. Plant Cell Reports 29, 285–294. Cazzonelli C.I. & Pogson B.J. (2010) Source to sink: regulation of carotenoid biosynthesis in plants. Trends in Plant Science 15, 266–274. Chagné D., Carlisle C.M., Blond C., et al. (2007) Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple. BMC Genomics 8, 212. Chalker-Scott L. (1999) Environmental significance of anthocyanins in plant stress responses. Photochemistry and Photobiology 70, 1–9. Chang S., Puryear J. & Cairney J. (1993) A simple and efficient method for isolation RNA from pine tree. Plant Molecular Biology Reporter 11, 113–116. Chinnusamy V., Zhu J. & Zhu J.K. (2007) Cold stress regulation of gene expression in plants. Trends in Plant Science 12, 444–451. Cominelli E., Gusmaroli G., Allegra D., Galbiati M., Wade H.K., Jenkins G.I. & Tonelli C. (2007) Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. Journal of Plant Physiology 165, 886–894. Dare A.P., Schaffer R.J., Lin-Wang K., Allan A.C. & Hellens R.P. (2008) Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes. Plant Methods 4, 17. Dela G., Or E., Ovadia R., Nissim-Levi A., Weiss D. & OrenShamir M. (2003) Changes in anthocyanin concentration and composition in ‘Jaguar’ rose flowers due to transient hightemperature conditions. Plant Science 164, 333–340. Dixon R.A. & Paiva N.L. (1995) Stress-induced phenylpropanoid metabolism. The Plant Cell 7, 1085–1097. Dubos C., Le Gourrierec J., Baudry A., Huep G., Lanet E., Debeaujon I., Routaboul J.M., Alboresi A., Weisshaar B. & Lepiniec L. (2008) MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. The Plant Journal 55, 940– 953.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

Temperature and the anthocyanin regulatory complex 1189 Espley R.V., Hellens R.P., Putterill J., Stevenson D.E., Kutty-Amma S. & Allan A.C. (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. The Plant Journal 49, 414–427. Espley R.V., Brendolise C., Chagné D., et al. (2009) Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. The Plant Cell 21, 168–183. Gleave A.P. (1992) A versatile binary vector system with a T-DNA organizational-structure conducive to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology 20, 1203–1207. Gonzalez A., Zhao M., Leavitt J.M. & Lloyd A.M. (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/ bHLH/Myb transcriptional complex in Arabidopsis seedlings. The Plant Journal 53, 814–827. Grotewold E. (2006) The genetics and biochemistry of floral pigments. Annual Review of Plant Biology 57, 761–780. Guindon S., Lethiec F., Duroux P. & Gascuel O. (2005) PHYML Online – a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Research 33, W557–W559. Harborne J.B. & Williams C.A. (2000) Advances in flavonoid research since 1992. Phytochemistry 55, 481–504. Hellens R.P., Allan A.C., Friel E.N., Bolitho K., Grafton K., Templeton M.D., Karunairetnam S., Gleave A.P. & Laing W.A. (2005) Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13. Jin H., Cominelli E., Bailey P., Parr A., Mehrtens F., Jones J., Tonelli C., Weisshaar B. & Martin C. (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO Journal 19, 6150–6161. Kagale S., Links M.G. & Rozwadowski K. (2010) Genome-wide analysis of ethylene-responsive element binding factorassociated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis. Plant Physiology 152, 1109– 1134. Kirsh V.A., Hayes R.B., Mayne S.T., Chatterjee N., Subar A.F., Dixon L.B., Albanes D., Andriole G.L., Urban D.A. & Peters U. (2006) Supplemental and dietary vitamin E, beta-carotene, and vitamin C intakes and prostate cancer risk. Journal of the National Cancer Institute 98, 245–254. Kotak S., Larkindale J., Lee U., von Koskull-Doring P., Vierling E. & Scharf K.D. (2007) Complexity of the heat stress response in plants. Current Opinion in Plant Biology 10, 310–316. Kumar S.V. & Wigge P.A. (2010) H2A.Z-containing nucleosomes mediate the thermosensory response in Arabidopsis. Cell 140, 136–147. Lancaster J.E., Reay P.F., Norris J. & Butler R.C. (2000) Induction of flavonoids and phenolic acids in apple by UV-B and temperature. Journal of Horticultural Science & Biotechnology 75, 142– 148. Le S.Q. & Gascuel O. (2008) An improved general amino acid replacement matrix. Molecular Biology and Evolution 25, 1307– 1320. Leyva A., Jarillo J.A., Salinas J. & Martinez-Zapater J.M. (1995) Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light-dependent manner. Plant Physiology 108, 39–46. Li F., Vallabhaneni R., Yu J., Rocheford T. & Wurtzel E.T. (2008) The maize phytoene synthase gene family: overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and thermal stress tolerance. Plant Physiology 147, 1334–1346. Li S.J., Zhou X., Chen L.G., Huang W.D. & Yu D.Q. (2010) Functional characterization of Arabidopsis thaliana WRKY39 in heat stress. Molecules and Cells 29, 475–483.

Lillo C., Lea U.S. & Ruoff P. (2008) Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway. Plant, Cell & Environment 31, 587–601. Lin-Wang K., Bolitho K., Grafton K., Kortstee A., Karunairetnam S., McGhie T.K., Espley R.V., Hellens R.P. & Allan A.C. (2010) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biology 10, 50. Lo Piero A.R., Puglisi I., Rapisarda P. & Petrone G. (2005) Anthocyanins accumulation and related gene expression in red orange fruit induced by low temperature storage. Journal of Agricultural and Food Chemistry 53, 9083–9088. Matsui K., Umemura Y. & Ohme-Takagi M. (2008) AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. The Plant Journal 55, 954–967. Mayne S.T. (1996) Beta-carotene, carotenoids, and disease prevention in humans. FASEB Journal 10, 690–701. Mori K., Sugaya S. & Gemma H. (2005) Decreased anthocyanin biosynthesis in grape berries grown under elevated night temperature condition. Scientia Horticulturae 105, 319– 330. Mori K., Goto-Yamamoto N., Kitayama M. & Hashizume K. (2007) Loss of anthocyanins in red-wine grape under high temperatures. Journal of Experimental Botany 58, 1935–1945. Morishita T., Kojima Y., Maruta T., Nishizawa-Yokoi A., Yabuta Y. & Shigeoka S. (2009) Arabidopsis NAC transcription factor, ANAC078, regulates flavonoid biosynthesis under high-light. Plant & Cell Physiology 50, 2210–2222. Newcomb R.D., Crowhurst R.N., Gleave A.P., et al. (2006) Analyses of expressed sequence tags from apple. Plant Physiology 141, 147–166. Nishizawa-Yokoi A., Yoshida E., Yabuta Y. & Shigeoka S. (2009) Analysis of the regulation of target genes by an Arabidopsis heat shock transcription factor, HsfA2. Bioscience, Biotechnology, and Biochemistry 73, 890–895. Olsen K.M., Slimestad R., Lea U.S., Brede C., Lovdal T., Ruoff P., Verheul M. & Lillo C. (2009) Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies. Plant, Cell & Environment 32, 286–299. Pourtau N., Jennings R., Pelzer E., Pallas J. & Wingler A. (2006) Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta 224, 556–568. Reinbothe S. & Reinbothe C. (1996) Regulation of chlorophyll biosynthesis in angiosperms. Plant Physiology 111, 1–7. Ronquist F. & Huelsenbeck J.P. (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rowan D.D., Cao M., Lin-Wang K., et al. (2009) Environmental regulation of leaf colour in red 35S:PAP1 Arabidopsis thaliana. New Phytologist 182, 102–115. Schramm F., Larkindale J., Kiehlmann E., Ganguli A., Englich G., Vierling E. & von Koskull-Doring P. (2008) A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis. The Plant Journal 53, 264–274. Shvarts M., Borochov A. & Weiss D. (1997) Low temperature enhances petunia flower pigmentation and induces chalcone synthase gene expression. Physiologia Plantarum 99, 67– 72. Solfanelli C., Poggi A., Loreti E., Alpi A. & Perata P. (2006) Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis. Plant Physiology 140, 637–646.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190

1190 K. Lin-Wang et al. Steyn W.J., Holcroft D.M., Wand S.J.E. & Jacobs G. (2004) Regulation of pear color development in relation to activity of flavonoid enzymes. Journal of the American Society for Horticultural Science 129, 6–12. Steyn W., Holcroft D., Wand S. & Jacobs G. (2005) Red colour development and loss in pears. Acta Horticulturae 671, 79–85. Steyn W.J., Wand S.J., Jacobs G., Rosecrance R.C. & Roberts S.C. (2009) Evidence for a photoprotective function of low-temperature-induced anthocyanin accumulation in apple and pear peel. Physiologia Plantarum 136, 461–472. Takos A.M., Jaffe F.W., Jacob S.R., Bogs J., Robinson S.P. & Walker A.R. (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiology 142, 1216–1232. Tamagnone L., Merida A., Parr A., Mackay S., Culianez-Macia F.A., Roberts K. & Martin C. (1998) The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10, 135–154. Teng S., Keurentjes J., Bentsink L., Koorneet M. & Smeekens S. (2007) Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene. Plant Physiology 139, 1840–1852. Tohge T., Nishiyama Y., Hirai M.Y., et al. (2005) Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. The Plant Journal 42, 218–235. Ubi B., Honda C., Bessho H., Kondo S., Wada M., Kobayashi S. & Moriguchi T. (2006) Expression analysis of anthocyanin biosynthetic genes in apple skin: effect of UV-B and temperature. Plant Science 170, 571–578. Vaknin H., Bar-Akiva A., Ovadia R., Nissim-Levi A., Forer I., Weiss D. & Oren-Shamir M. (2005) Active anthocyanin degradation in Brunsfelsia calycina (yesterday–today–tomorrow) flowers. Planta 222, 19–26. Velasco R., Zharkikh A., Affourtit J., et al. (2010) The genome of the domesticated apple (Malus ¥ domestica Borkh.). Nature Genetics 42, 833–839. Von Koskull-Doring P., Scharf K.D. & Nover L. (2007) The diversity of plant heat stress transcription factors. Trends in Plant Science 12, 452–457. Wunsche J.N. & Palmer J.W. (1997) Portable through-flow cuvette system for measuring whole-canopy gas exchange of apple trees in the field. HortScience 32, 653–658. Zenner K. & Bopp M. (1987) Anthocyanin turnover in Sinapis alba L. Journal of Plant Physiology 126, 475–482. Zhang F., Gonzalez A., Zhao M., Payne C.T. & Lloyd A. (2003) A network of redundant bHLH proteins functions in all TTG1dependent pathways of Arabidopsis. Development 130, 4859– 4869.

Zhu H.F., Fitzsimmons K., Khandelwal A. & Kranz R.G. (2009) CPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis. Molecular Plant 2, 790–802. Zhu Y., Evans K. & Peace C. (2010) Utility testing of an apple skin color MdMYB1 marker in two progenies. Molecular Breeding 27, 525–532. Received 27 December 2010; received in revised form 3 March 2011; accepted for publication 7 March 2011

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Gene expression analysis of the apple anthocyanin biosynthetic pathway during the last 10 weeks of maturity in ‘Gala’ apples in the two climatic areas shows coordinative down-regulation of all the anthocyanin steps (CHS and LDOX are shown in Fig. 1). Error bars for all time-points represent the SEM of three biological replicates. Figure S2. Gene expression analysis of the apple anthocyanin biosynthetic pathway during orchard-based on-tree apple heating rapidly reduces expression of apple anthocyanin-related genes and shows coordinative downregulation of all the anthocyanin steps (CHS and LDOX are shown in Fig. 4). Error bars for all time-points represent the SEM of three biological replicates. Figure S3. Protein line-up of MYB transcription factors of apples and known anthocyanin-promoting and repressing MYBs of other plant species. Figure S4. Orchard-based apple heating with a single night of very low temperatures (red line in all panels) caused coordinative elevation of gene expression of the apple anthocyanin biosynthetic pathway (CHS and LDOX are shown in Fig. 5). Error bars for all time-points represent the SEM of three biological replicates. Table S1. Oligonucleotide primer sequences used for qPCR analysis. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 34, 1176–1190