The brassinosteroid (BR) response transcription factor Brassinazole resistant 1 (BZR1)-mediated BR signalling regulates many specific developmental processes including fruit ripening. Here, we report the effect of 2,4-epibrassinolide (EBR) and BZR1-1D overexpression on carotenoid accumulation and quality attributes of tomato (Solanum lycopersicum) fruit. EBR-treated pericarp discs of ethylene-insensitive mutant, Never ripe, accumulated significantly more carotenoid than those of the control. The results suggest that BR seems to be involved in modulating pigments accumulation. When three independent transgenic lines overexpressing the Arabidopsis BZR1-1D were used to evaluate the role of BZR1 in regulating tomato fruit carotenoid accumulation and quality attributes, fruits of all three transgenic lines exhibited enhanced carotenoid accumulation and increased soluble solid, soluble sugar and ascorbic acid contents during fruit ripening. In addition, the fruits of two transgenic lines showed dark green shoulder at mature green stage, in accordance with the up-regulated expression level of SlGLK2, which is involved in chloroplast development. Our results indicate the importance of BZR1-centred BR signalling in regulating carotenoid accumulation and quality attributes of tomato fruit and the potential application of the BZR1-like(s) for improvement of nutritional quality and flavour of tomato through genetic engineering.
Before domestication, fruits and vegetables retained diverse natural taste and flavour. Domestication has had an inadvertently negative effect on tomato taste and flavour, because disease resistance, yield, fruit colour and shape are top priorities in traditional breeding programmes, and selection for yield, fruit size and shelf-life characteristics has particularly resulted in unintended negative consequences on fruit taste and flavour (Goff and Klee, 2006). Recently, much attention has been paid to tomato fruit taste and flavour enhancement because it is not only of agricultural importance, but also of scientific interest in terms of the chemical, biological and genetic regulation. The ripening of fleshy fruits produces a wide range of ‘bioactive’ compounds important for human health including antioxidant carotenoids, such as lycopene, hypothesized to decrease cardiovascular disease and reduce prostate cancer risk (Erdman et al., 2009; Ford et al., 2012; Seymour et al., 2012). One of the main characteristics of tomato fruit ripening is a massive accumulation of carotenoid. More recently, advances have been made in understanding the genetic control of tasteless tomato. Tomato Golden 2-like (SlGLK2), the tomato gene which was inactivated by traditional breeding programmes, plays an important role in producing sugars and aromas and in increasing levels of chlorophyll and carotenoid, which are considered to be the essences of a fragrant and flavourful tomato (Powell et al., 2012).
Fruit ripening is a process influenced by many specific factors (developmental signals, hormones, light, temperature, plant nutrient status, etc.), of which phytohormones play a central role in the signalling networks underlying plant growth and development (Lee et al., 2012). For example, the ethylene (ET)-involved regulatory mechanism on tomato ripening has been well studied. Additionally, other plant hormones have also been documented to control tomato fruit ripening. The abscisic acid (ABA)-deficient tomato mutants, namely high-pigment 3 (hp3), flacca (flc) and sitiens (sit), show an increased plastid number and an enhanced level of lycopene during fruit ripening (Galpaz et al., 2008). Our previous study showed that jasmonic acid (JA)-induced lycopene biosynthesis in tomato fruit might be independent of ET signal transduction (Liu et al., 2012).
Brassinosteroids (BRs) are plant steroid hormones known mainly for their effects on cell expansion and a wide range of developmental and physiological processes that occur ubiquitously in the plant kingdom (Wang et al., 2012). Extensive studies using genetic, molecular and proteomic approaches have identified most of the major BR signalling components, which have been assembled into one signal transduction cascade (Kim and Wang, 2010). Molecular genetic studies in Arabidopsis have elucidated a phosphorylation-mediated signalling pathway that regulates the stability, subcellular localization and DNA binding activity of two highly similar transcription factors (TFs), bri1-EMS-Suppressor 1 (BES1) and Brassinazole Resistant 1 (BZR1) (Kim and Wang, 2010). In the past decade, genetic and biochemical analyses support a central role of BZR1 in regulation of plant development by studies on the brassinazole resistant 1 (bzr1-1D) mutant-derived BZR1. BZR1 is likely to be regulated by BR signals through protein phosphorylation, which could affect the accumulation of BZR1 protein in the nucleus (He et al., 2002; Wang et al., 2002). Protein phosphatase 2A (PP2A) directly interacts with the putative PEST domain containing the site of the bzr1-1D mutation of BZR1, and the dephosphorylation by PP2A is enhanced by the bzr1-1D mutation (Tang et al., 2011).
Unlike Arabidopsis, tomato confers one of the best systems to study BR functions in fleshy fruit development. In this study, we used ET-insensitive mutant, Never ripe (Nr), to prove that BR could promote carotenoid accumulation in tomato fruit. A dominant Arabidopsis mutant bzr1-1D gene, BZR1-1D, was overexpressed in tomato to evaluate the role of BZR1 in tomato fruit ripening process. The fruits of transformed tomato plants showed dark green shoulder at mature green (MG) stage, increased carotenoid accumulation and enhanced soluble sugar and ascorbic acid contents during ripening. Our results indicate that genetic engineering of BZR1 is a potential way for tomato fruit quality improvement.
Enhanced carotenoid accumulation in Nr by 2,4-epibrassinolide application
Previous studies have shown that ET plays a central role in promoting fruit ripening. To obtain further insight into the role of BR in promoting carotenoid accumulation, the ET-insensitive mutant Nr, which carries a semi-dominant mutation in NR (ETR3), was used. The elevation of lycopene levels in control and 3.0 μm 2,4-epibrassinolide (EBR)-treated pericarp discs of Pearson (PSN) and Nr was found with culture time (Figure 1). Nonetheless, significantly higher levels of lycopene and β-carotene were observed in both EBR-treated PSN discs and Nr discs when compared with control (Figure 1). EBR application also promoted lutein accumulation in pericarp discs of Nr plants at 1 and 3 days post-treatment (Figure 1). Furthermore, the carotenoid accumulation in Nr fruit altered dramatically upon exogenous EBR application on half of one whole Nr fruit, which could be visually recognized from the viewing angle shown in Figure S3. The results demonstrate the important role of BR in the induction of carotenoid accumulation during tomato fruit ripening.
Phenotype of BZR1-1D transgenic lines
To better understand the possible functions as well as to identify possible components of BR signalling pathway related to tomato fruit ripening, we utilized designated transgenic lines, in which BZR1-1D gene from Arabidopsis mutant bzr1-1D with increased dephosphorylated BZR1 was transformed into tomato (Jia, 2009; Tang et al., 2011). The general morphology of flowers of transgenic lines was very similar to that of the wild-type flowers, except that the senescence of flowers in BZR1-1D#6 and BZR1-1D#23 was ahead of that of wild-type flowers (Figure 2). Flowers of transgenic lines developed into normal fruits upon pollination.
The fruit phenotypes of wild type (WT) and BZR1-1D lines at various stages of ripening were shown in Figure 2. The fruit size of all three transgenic lines was similar to that of the WT, while the general fruit ripening time of transgenic lines was approximately 1 week earlier than WT (data not shown). In WT and BZR1-1D#3, fruit ripening begins with full-sized MG stage fruit. In the next stage, the breaker (B) stage, a visible colour change just begins to occur. By the pink (P) stage, the entire fruit has a consistent pink colour. In the final stage of ripening, mature red (R), the fruit has a deep red colour and starts to soften. The differences in ripening between the BZR1-1D#6, BZR1-1D#23 and WT fruits were apparent from the MG stage. WT fruit is uniformly light green before ripening, a characteristic promoting even-ripening at the stem end and facilitating maturity determinations (Powell et al., 2012). However, the colour distribution of fruits from BZR1-1D#6 and BZR1-1D#23 lines was patchy with dark green shoulders at the stem end adjacent to the pedicel. The nonuniformity leads to ripening asynchronism until P stage. In other words, WT showed uniform ripening (u) phenotype with light green fruit, while the two transgenic lines, BZR1-1D#6 and BZR1-1D#23, seemed to be Uniform ripening (U) phenotype. U encodes a TF, SlGLK2, which determines chlorophyll accumulation and distribution in developing fruits (Powell et al., 2012). We sequenced GLK in WT to confirm that WT possess SlGLK2 (Figure S4). The chlorophyll contents were elevated by 8.3%, 88.1% and 106.0% in the blossom (stylar) portions of BZR1-1D#3, BZR1-1D#6 and BZR1-1D#23, respectively (Figure 3a). Moreover, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) to compare the expression levels of SlGLK2 in transgenic lines and WT fruits. In the MG fruits of BZR1-1D#3, BZR1-1D#6 and BZR1-1D#23, the expression levels of SlGLK2 were up-regulated to 2-, 8-, and 26-fold of WT, respectively (Figure 3b). Transmission electron microscopy (TEM) revealed that BZR1-1D expression increased the number (twofold) and the size of MG fruit chloroplasts and promoted grana thylakoids accumulation and development (Figure 3c). In addition, the number of plastoglobules per chloroplast profile was also increased in BZR1-1D#23. Thus, BZR1-1D appears to be involved in regulating pigments accumulation in tomato fruits.
BZR1-1D overexpression alters carotenoid levels in fruits
The colour change during the ripening of tomato fruit is largely due to chlorophyll degradation and accumulation of carotenoids, including lycopene, β-carotene and lutein (Karlova et al., 2011). Lycopene is the most abundant carotenoid analysed in tomato fruit. It was found that the fruits of transgenic lines accumulated much more pigments than WT at R stage (Figure 2). To examine the underlying causes of the colour changes observed in ripe fruits between transgenic lines and WT in detail, high-performance liquid chromatography (HPLC) analysis of carotenoid (lycopene, β-carotene and lutein) levels was performed on WT and BZR1-1D overexpressed fruits at four developmental stages (Figure 4). The pericarp of three BZR1-1D transgenic lines showed increased levels of both lycopene and β-carotene at P and R stages except for β-carotene of BZR1-1D#6 and BZR1-1D#23 at P stage and BZR1-1D#23 at R stage, accounting for the darker red of the ripe BZR1-1D fruits (Figure 4a). Transgenic fruits at B stage showed slight decrease in the lutein content, and no difference was observed at P and R stages compared with WT (Figure 4a). Consistent with pericarp, elevated levels of lycopene were observed in the flesh of fruits from BZR1-1D lines. The β-carotene content was also increased in fruit flesh at B stage. No significant difference in lutein content was detected in the fruits between the WT and transgenic lines (Figure 4b).
BZR1-1D regulates lycopene biosynthetic genes
The biosynthetic pathway of carotenoid has largely been elucidated, and nearly all the genes encoding carotenoid biosynthetic enzymes have been identified (DellaPenna and Pogson, 2006; Fraser and Bramley, 2004; Hirschberg, 2001; Römer and Fraser, 2005; Ronen et al., 2000). To test whether the observed alterations in carotenoid content were attributed to changes in expressions of genes involved in the carotenoid biosynthetic pathway, qRT-PCR was used to measure the mRNA levels of SlDXS (Figure 5a), SlGGPS (Figure 5b), SlPSY1 (Figure 5c), SlPDS (Figure 5d), SlZDS (Figure 5e) and SlCYC-B (Figure 5f) in the pericarp of fruits from transgenic lines and their corresponding WT. Interestingly, although genes (SlDXS, SlGGPS, SlPSY1, SlPDS, SlZDS) related to lycopene biosynthesis showed different expression patterns, they were all significantly up-regulated in three transgenic lines at MG and R stages except SlDXS of BZR1-1D#6 at MG stage (Figure 5a–e). Among these, two genes (SlPSY1 and SlZDS) were also markedly up-regulated at P stage (Figure 5c,e). We noted that in pericarp, expression levels of SlPSY1 of three transgenic lines showed a major increase at the MG stage and remained higher than WT thereafter (Figure 5c). As lycopene is accumulated mainly at the R stage and the expression levels of lycopene biosynthetic genes generally reaches a maximum at the P stage, the biosynthetic gene transcripts at the P stage may contribute most to lycopene formation in tomato fruits (Galpaz et al., 2008; Telef et al., 2006). SlCYC-B gene, which encodes the enzyme converting lycopene to β-carotene, shows ripening-specific expression pattern (Hirschberg, 2001) and is thus associated with β-carotene production during ripening (Ronen et al., 2000). In three independent transgenic lines, the mRNA levels of SlCYC-B were increased in fruits at MG, B and R ripening stages (Figure 5f).
Additionally, SlHSP21 encodes chloroplast small heat shock protein HSP21 and plays an important role in developing fruits during the transition of chloroplasts to chromoplasts under normal growth conditions (Neta-Sharir et al., 2005). The transcriptional change of SlHSP21 was investigated, and the expression levels of SlHSP21 were significantly increased by ~12-, 101-, and 961-fold, respectively, in MG stage fruits of all three BZR1-1D transgenic lines compared with the WT (Figure 5g). These results suggest that the accumulation of carotenoid is mainly controlled at the transcript level of the genes encoding carotenoid biosynthetic enzymes, which is consistent with former reports (DellaPenna and Pogson, 2006; Römer and Fraser, 2005), and BZR1 plays an important role in carotenoid accumulation by regulating biosynthetic genes directly or indirectly.
Quality attributes of BZR1-1D lines
Selection of u inadvertently compromised ripe fruit quality in exchange for desirable production traits (Powell et al., 2012). Two transgenic lines BZR1-1D#6 and BZR1-1D#23 showed U phenotype in current survey. Thus, we investigated the soluble solid, organic acid, soluble sugar, ascorbic acid contents and sugar-acid ratios in the three BZR1-1D lines. The contents of soluble solids in BZR1-1D#3, BZR1-1D#6 and BZR1-1D#23 were 8.3%, 11.3% and 26.8% higher, respectively, than those in WT at R stages (Figure 6a). The increases in soluble sugar in transgenic lines compared with that in WT were 4.3%, 34.8% and 38.4% at P stage and 4.1%, 25.3% and 10.8% at R stage, respectively (Figure 6b). Significant higher levels of ascorbic acid were also observed in the fruits of three BZR1-1D lines than in those of WT at both P and R stages (Figure 6c). No significant difference in organic acid content and sugar-acid ratio was observed between the WT and the transgenic lines (data not shown). Our results demonstrate an important role of BZR1 in quality attributes of edible tomato fruits.
Application of BR in agriculture has been expanded along with the research progress on BR signalling and functions. In comparison with yield improvement and stress resistance, very little is known about the role of BR in regulating the biosynthesis of secondary metabolites as well as nutrient quality during fruit ripening. The use of the specific BR biosynthesis inhibitor, Brassinazole (Brz), is an effective way to clarify BR functions (Asami et al., 2001). BR function in tomato fruit ripening was confirmed by exogenous co-application of EBR and Brz and sole application of EBR to tomato fruit at MG stage, which significantly inhibited and promoted tomato fruit ripening, respectively (Figure S1). Discs of epidermal pericarp tissues were used in this study because they not only exhibited physiological changes as the whole fruit, but also conferred many advantages over the whole fruit (Figure S2). Unlike whole fruit, which is composed of nonhomogeneous tissues (e.g. pericarp and locular jelly), pericarp discs are composed of relatively homogenous tissue (Saltveit, 1989). Moreover, the cut surfaces absorbed the applied solutions of 3.0 μm EBR much more rapidly, and a large number of uniform discs could be prepared from the same Nr fruit, thereby reducing the variability within the experiment and also overcome the problem of limited fruit materials. Microbial contamination was easy to be suppressed in the 9-day experiments.
Previous studies have shown that ET plays a central role in promoting fruit ripening. In addition, tomato fruit ripening induced by BR was reported to be associated with increment in ET production (Vidya Vardhini and Rao, 2002). Although BR promotes ET production (Vidya Vardhini and Rao, 2002) and Nr is not impaired in any step of ET biosynthesis (Lanahan et al., 1994), the dramatic colour contrast within one whole Nr fruit (Figure S3) suggests the existence of a BR-induced carotenoid accumulation pathway independent of NR-mediated ET signal transduction. BR may promote the carotenoid biosynthesis and ripening of tomato fruits by either ET-dependent way or ET-independent way. It is acknowledged that climacteric fruits such as tomato are characterized by an increment in the synthesis of the phytohormone ET upon the initiation of ripening (Klee and Giovannoni, 2011). The role of the respiratory climacteric is still unknown, but ET acts to initiate and coordinate ripening in climacteric fruits (Fraser et al., 2007). The climacteric ET leap might arise from other phytohormone facilitation, including BR (Vidya Vardhini and Rao, 2002), JA (Liu et al., 2012) and ABA (Zhang et al., 2009).
Carotenoid is produced by all photosynthetic organisms as well as by some nonphotosynthetic bacteria and fungi and synthesized in both dark- and light-grown tissues, such as endosperm, roots, leaves, flowers and fruits via the general isoprenoid biosynthetic pathway in chromoplasts (Ronen et al., 2000; Shumskaya et al., 2012; Welsch et al., 2008). EBR is previously shown to regulate carotenoid accumulation (Vidya Vardhini and Rao, 2002). Nevertheless, the effect of BR on carotenoid biosynthetic gene expression has not been investigated. In consideration of that, the accumulation of carotenoid is mainly controlled at the transcript level of genes encoding the biosynthetic enzymes (DellaPenna and Pogson, 2006; Römer and Fraser, 2005; Sandmann et al., 2006), and all carotenoids biosynthetic genes in plants and algae are nuclear encoded (Harker and Hirschberg, 1998; Ronen et al., 2000), we used qRT-PCR to measure the mRNA levels of genes related to carotenoid synthesis and catabolism in transgenic lines (Figure 5a–f). Our results indicate that BZR1 promotes carotenoid accumulation by regulating the expression of carotenoid biosynthetic genes. Carotenoid biosynthesis should be viewed as a dynamic process, that the complexes are formed in a dynamic fashion and recruited as needed (Shumskaya et al., 2012). The ET-independent induction of carotenoid in the present work may have other effects besides their classical roles in light-harvesting complexes and photosynthetic reaction centres. A publication by Schwartz et al. (1997) on ABA formation started the way to research on continuative reactions mediated through carotenoid metabolic enzymes, thereby establishing carotenoid as a source of diverse signalling substance in plants (Welsch et al., 2008). Beyond their functions as colourants and nutrients, carotenoids also serve as precursors of important volatile flavour compounds (Vogel et al., 2010). Expression level of SlHSP21, which is not regulated by ET (Lee et al., 2012) was up-regulated, consistent with its role in carotenoid accumulation (Neta-Sharir et al., 2005).
BZR1 was reported to function as a TF itself as well as a cofactor that regulates additional TFs to control secondary BR-responsive genes (Wang et al., 2002). GLK1 and GLK2 are related transcriptional factors that function redundantly to promote chloroplast development (Fitter et al., 2002; Waters et al., 2008, 2009). In our study, two transgenic lines (BZR1-1D#6 and BZR1-1D#23) showed U phenotype (Powell et al., 2012) controlled by SlGLK2, which determines chlorophyll accumulation and distribution in developing fruits (Figure 2). BR signalling directly regulates the common target genes of the phototransduction pathways through the transcriptional activity of BZR1, and whereby BR modulates the plants response to light. In addition, loss of GLK function leads to reduced accumulation of a suite of photosynthetic gene products that are required for light harvesting and chlorophyll biosynthesis (Bravo-Garcia et al., 2009). Interestingly, accumulating physiological evidence also suggests that light is involved in the fruit ripening process, primarily impacting pigmentation (Liu et al., 2004). BZR1-centred BR signalling, SlGLK2 and light may act in cooperation with each other to regulate tomato fruit photomorphosis. Furthermore, soluble solid, soluble sugar, and ascorbic acid contents in fruits of three BZR1-1D lines were up-regulated (Figure 6), consistent with a positively regulatory impact of GLK overexpression, leading to elevated carbohydrates and carotenoid in ripe fruits (Powell et al., 2012).
One of the least-understood knowledge in BR-related research field is probably the regulatory mechanisms of the bioactive levels of BR in response to various developmental and environmental factors (Ronen et al., 2000). As other growth-promoting plant hormones, a very low amount of BR can generally stimulate growth and an excessive amount is usually detrimental to plants. In addition, BRs are not subject to active transportation within the plant as other growth-promoting phytohormones (Montoya et al., 2005; Symons and Reid, 2004; Symons et al., 2008). These observations suggest that plants must have evolved strategic tactics to regulate BR homeostasis and guarantee their optimal growth and development under various conditions. Former studies on BR biosynthesis in tomato have identified CYP85A1 (SlDWARF) and CYP85A3, which encode the enzymes (CYP85A1 and CYP85A3) involved in BR biosynthesis in tomato, catalyse the same reactions as CYP85A1 and CYP85A2 in Arabidopsis (Bishop et al., 1996; Nomura et al., 2005). CYP85A3, encoding CYP85A3 oxidase, which catalyses brassinolide synthesis, is peculiarly expressed in fruits, whereas castasterone, synthesized by CYP85A1, is the major active BR during vegetative growth in this species (Nomura et al., 2005). It was previously found that BZR1 plays dual roles in inducing the expression of thousands of BR response genes to promote growth and suppressing the expression of key BR biosynthetic genes to negatively regulate BR biosynthesis (He et al., 2005). Consistent with Arabidopsis plants overexpressing the mutant bzr1-1D gene, which has a long-petiole phenotype similar to transgenic plants overexpressing the BR biosynthetic enzyme DWF4 (Wang et al., 2002), SlDWARF was up-regulated in tomato fruits overexpressing the mutant bzr1-1D gene (Figure S5a). In contrast to SlDWARF, CYP85A3 showed a different expression pattern despite that it was up-regulated as well. The expression peak of CYP85A3 was postponed to the last stage of two transgenic lines (Figure S5b). Database searches revealed sequence homology between Arabidopsis BZR1 and other plant proteins, including the tomato LAT61 gene previously identified as an anther specific gene (65% identity) (Wang et al., 2002). To determine whether the endogenous tomato BZR1 (LAT61) responds to the presence of ectopic BZR1-1D expression, we analysed SlBZR1 (LAT61) expression levels in the three transgenic lines by qRT-PCR (Figure S5c). The expressions of SlBZR1 genes were up-regulated in three transgenic lines consisting with the elevated expression of BR biosynthetic genes.
In conclusion, elevated carotenoid contents and enhanced quality attributes in fruits of BZR1-1D overexpression lines support the importance of the TF BZR1 activity in controlling tomato secondary metabolism and ripening. Our findings have significant implications for metabolic engineering of carotenoid in plants with the goal to increase the nutritional value. Furthermore, it also provides opportunity for plant breeders to recover and enhance better-tasting tomatoes. The sequenced genomes of several fleshy fruit species, including tomato (Tomato Genome Consortium, 2012), provide the foundation to investigate interactions between TFs and regulatory sequences of downstream effectors influencing colour, texture and flavour of fleshy fruits. Further study will shed light on how BZR1 contributes to the dynamic assembly and organization of the complex ripening process in response to various developmental signals.
Plant materials and growth conditions
Cultivar PSN is the parental line for the ET-insensitive mutant Nr. Our previous work has got three transgenic lines 35S::BZR1-1D::CFP#3 (BZR1-1D#3), 35S::BZR1-1D::CFP#6 (BZR1-1D#6) and 35S::BZR1-1D::CFP#23 (BZR1-1D#23) from tomato cultivar Zhongshusihao (ZS4) (Jia, 2009). Transformation was performed via Agrobacterium-mediated transformation method using cotyledon explants as described (Park et al., 2003). These transgenic lines carry the Arabidopsis mutant bzr1-1D gene driven by the cauliflower mosaic virus (CaMV) 35S promoter and cause increased dephosphorylated BZR1 accumulation (Tang et al., 2011). Seeds from the T2 generation of the BZR1-1D#3, BZR1-1D#6, BZR1-1D#23 tomato lines are available upon request.
Tomato seeds including three transgenic lines of T1 generation were sterilized with sodium hypochlorite (NaOCl), germinated on filter paper and sown in seedling trays filled with a rich soil mixture. Three weeks after germination, the seedlings were transplanted to plastic pots (34 cm in diameter, 37 cm in depth) filled with perlite and turfy soil (3 : 1 by vol.). They were grown in a greenhouse under a 16-h photoperiod at 22 (night) to 28 °C (day). Flowers were tagged at anthesis, and the number of fruits was limited to fewer than four per cluster. The fruits displaying the first sign of colour change were identified as breaker (B) stage. The average days from anthesis to B for ZS4 and transgenic lines were determined to be 46 and 40 on average, respectively. Fruits at 3 days before B were marked as MG. Fruits at 3 and 10 days after B were staged as P (pink) and R (mature red), respectively. For the ET-insensitive Nr mutant and its wild-type PSN, the same sampling standards were used. After harvesting and the soluble solid content measurement, fruits were immediately frozen in liquid nitrogen and stored at −80 °C.
Fruit disc preparation and EBR chemical treatment
Disc preparation was carried out with sterile technique as previously described (Saltveit, 1989; Vidya Vardhini and Rao, 2002) with modifications. Uniformly shaped MG stage fruits were washed with 1% active NaOCl solution and rinsed with sterile distilled water. Pericarp discs cut from equatorial regions with a 1.0-cm-diameter cork borer were separated into individual disc with the thickness of 2 mm. The discs were rinsed in sterile water, blotted dry on sterilized filter paper and kept on 9-cm diameter sterilized Petri plates with filter paper soaked with test solution.
2,4-Epibrassinolide at 3.0 μm was found to be the most significant in promoting lycopene accumulation in tomato (Vidya Vardhini and Rao, 2002). Therefore, 3.0 μm of EBR (Sigma, St. Louis, MO) was selected to study the effect of exogenous BR treatment on carotenoid accumulation. Twenty-four pericarp discs of Nr fruits from four plants were placed epidermis down on each Petri plate supplied with 6 mL of either test solution (viz., filter-sterilized 3.0 μm EBR) or distilled water (control). The Petri plates were kept in a growth chamber maintained at 22 ± 1 °C, 12000 LX. On day 4, another dose of 3 mL solution was added. Lycopene, β-carotene and lutein contents were determined on the 1st, 3rd, 6th and 9th day. We also treated the fruits at MG stage by (i) painting one side of the whole Nr fruits with lanolin containing 3.0 μm EBR and the other side with 0.07% ethanol, (ii) co-treating fruit with 3.0 μm EBR and 5.0 μm Brassinazole (Brz, the highly specific BR biosynthesis inhibitor, provided by Dr. Tadao Asami) and sole treating with 3.0 μm EBR. All treated fruits were kept in a growth chamber with the temperature of 22 ± 1 °C, under the light of 12000 LX. The EBR and Brz solution was prepared by dissolving EBR and Brz in a limited volume of ethanol and bringing to the final ethanol content 0.07% with distilled water. The corresponding control contained the same concentration of ethanol as the EBR and Brz solution.
Transmission electron microscopy
Pericarp specimens were excised from fruit at the MG stage. The specimens were first fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer for 4 h and washed three times in the 0.1 m phosphate buffer for 15 min. Then samples were post-fixed with 1% OsO4, dehydrated in ethanol and embedded in Spurr resin. Ultrathin sections were examined with JEM-1230 transmission electron microscope.
Determination of carotenoid content
Carotenoid was extracted and analysed as previously described (Liu et al., 2012; Olives-Barba et al., 2006) with modifications. Tomato fruits (~0.6 g for disc, pericarp and flesh) were homogenized with 30 mL of hexane/acetone/ethanol (1 : 1 : 1 by vol.) solution and magnetically stirred for 30 min. The extracts were centrifuged at 3000 g at room temperature for 10 min and then 15 mL of water was added. 3 mL of the upper layer for fruits at MG and B stages and 1 mL for the case of P and R fruits were recovered, filtered through 0.45-μm membrane filters and evaporated to dryness in a nitrogen stream. The residue was dissolved with THF/acetonitrile/methanol (15 : 30 : 55, by vol.) solution to a final volume of 1 mL and 20 μL were injected onto HPLC using an autosampler. All the procedures were performed under dim light. The analysis was performed using Shimadzu HPLC (Shimadzu, Kyoto, Japan), consisting of two LC-20AT solvent delivery units, a DGU-20A3 degasser, a CTO-10ASVP column oven, an SIL-20A autosampler and an SPD-M20A diode array detector. The HPLC system was connected to a computer with LC solution version 1.25 software. A Hypersil C18 column (5 μm particle size, 4.6 mm × 250 mm; Elite Analytical Instruments Co., Ltd., Dalian, China) was used with a mobile phase of methanol/acetonitrile (90/10 by vol.) containing 0.05% TEA at a flow rate of 1.2 mL/min. The total retention time was 30 min. Absorbance was detected at 475 nm. Standards (lutein, lycopene and β-carotene; Sigma) were prepared and used to identify and quantify corresponding carotenoid. The carotenoid content was expressed as μg/g fresh weight (FW) of tomato fruits.
RNA extraction and qRT-PCR analysis
Total RNA was isolated from tomato fruit (~100 mg) using Trizol reagent according to manufacturer's instruction (Takara Bio, Otsu, Japan). Genomic DNA was removed using the DNase (Bio-Rad, Hercules, CA). RNA concentration and purity were determined by spectrophotometry. RNA integrity was evaluated on a 1.5% (wt/vol) agarose gel. 5 μg RNA was reverse-transcripted into cDNAs using PrimeScript RT Master Mix (Takara). Each cDNA sample was diluted in 100 μL of water and used as template for quantitative reverse transcription polymerase chain reaction (qRT-PCR). PCR primer sequences were included in Table S1.
qRT-PCR was performed in a total volume of 25 μL, including 2 μL of diluted cDNA, 200 nm for each primer and 12.5 μL of 2× SYBR Green PCR Master Mix (Takara) on an iCycler (Bio-Rad Inc.). The qRT-PCR program included a preliminary step of 30 s at 95 °C, followed by 40 cycles of 95 °C for 10 s and 58 °C for 1 min. Tomato SAND (Sol Genomics Network accession number: SGN-U316474) was used as an internal control to normalize small differences in template amounts (Exposito-Rodriguez et al., 2008). Relative gene expression was calculated according to a 2−ΔΔCT method, in which ΔΔCT = (CT, Target − CT, Sand) Time X − (CT, Target − CT, Sand)Time 0 (Livak and Schmittgen, 2001). Time X is any time point, and Time 0 represents MG tissues of WT. Three PCR replicates were conducted and the fold change in each target gene of time 0 was set to 1. The experiment was repeated twice and similar results were obtained. For each experiment, data were analysed separately. Results of one representative experiment are shown.
Pericarp tissues from MG fruit pericarp (~0.5 g) were ground and extracted in 10 mL of 80% acetone, centrifuged at 1500 g for 10 min at room temperature and then the residue was removed. Total chlorophyll contents were determined by reading the absorbance at 652 nm with a spectrophotometer (UV-2500; Shimadzu Corp., Kyoto, Japan). Total chlorophyll content was estimated as μg/g FW (Yuan et al., 2010).
Soluble solid contents
Soluble solid contents (oBRIX) of total fruit juice from fresh harvested P and R fruits were determined with a hand-held refractometer (Chengdu Optical Instrument Factory, Chengdu, China).
Soluble sugar contents
Tomato (~0.5 g) was homogenized with 25 mL distilled water, heated in a boiling water bath for 20 min and then centrifuged at 4000 g at room temperature for 10 min to collect supernatant. The mixture of 1 mL supernatant and 5 mL 0.1% anthracenone (dissolved in 80% sulphuric acid) was homogenized and heated in a boiling water bath for 10 min. Total soluble sugar content was determined by reading the absorbance at 620 nm with a spectrophotometer (UV-2500; Shimadzu Corp.). Soluble sugar was quantified by external calibration.
Ascorbic acid contents
Ascorbic acid contents were analysed as previously described with minor modifications (Yuan et al., 2010). Five grams frozen pericarp of P and R fruits were ground to a fine powder in liquid nitrogen and extracted twice with 20 mL of 1.0% (w/v) oxalic acid by spinning at 1500 g for 5 min. Each sample was filtered through a 0.45-μm cellulose acetate filter. HPLC analysis was carried out using a Waters instrument with a Model 2996 PDA detector (Waters Inc., Milford, MA). Samples (20 μL/sample) were separated at room temperature on a Waters Spherisorb C18 column (250 × 4.6 mm id; 5 μm particle size), using a solvent of 0.1% oxalic acid at a flow rate of 1.0 mL/min. The amount of ascorbic acid was calculated from absorbance values at 243 nm, using authentic ascorbic acid as a standard. Results were expressed as μg/g FW.
The experiments except qRT-PCR were repeated three times and similar results were obtained. For each experiment, data were analysed separately. Results of one representative experiment are shown. Data were analysed using Statistica (SAS Institute Inc., http://www.statsoft.com). Differences in carotenoids (lycopene, β-carotene and lutein) contents and chlorophyll contents (Figures 1 and 3a) were analysed by one-way analysis of variance (ANOVA); if the ANOVA analysis was significant (P < 0.05), Duncan's multiple range test was used to detect significant differences between groups. Differences between WT and transgenic lines BZR1-1D#3, BZR1-1D#6 and BZR1-1D#23 in each figure were analysed using Student's t-tests. The values were reported as means with standard deviation (SD) for all the results.
We are grateful to Tomato Genetics Resource Center (University of California, Davis, CA) for providing Nr (LA3537) tomato seeds and to Dr. Tadao Asami (University of Tokyo, Japan) for providing Brassinazole. We also thank Dr. Guoqing Song (Michigan State University, East Lansing, MI) for critical reading of the manuscript. This work was supported by National Basic Research Program of China (2009CB119000), National Science Foundation of China (31171951) and Ph.D. Programs Foundation of Ministry of Education of China (20090101110100).