Integrative Transcriptomic, and Proteomic Analysis of Flavonoid Biosynthesis During Fruit Maturation In Chinese Raspberry “Rubus Hu”

Rubus chingii, is a red-fruited species of Rubus native to China, which is a popular and nutritious fruit in China. However, change in avonoid composition and content during fruit maturation is poorly understood. This study examined avonoids and the genes/proteins during four fruit ripening phases using LC-MS/MS. As a result, six major kinds of anthocyanins were rst identied in R. chingii, which primarily consisted of avanol-anthocyanins, are new to Rubus. Apart from anthocyanins, concentrations of fruit avonoids were much higher than most berries including raspberries, and it is this that contributes to their high phenolic concentrations and antioxidant capabilities. In contrast to other known raspberries, R. chingii had a decline in avonoids during fruit maturation, which was due to down-regulation of genes/proteins involved in phenylpropanoid and avonoid biosynthesis. Surprisingly, anthocyanin continuously decreased during fruit coloration. This suggests that anthocyanins are not responsible for the fruit’s reddish coloration. The biosynthesis of these avanol-anthocyanins consumed two avonoid units both produced through the same upstream pathway. Their presence indicates a reduction in the potential biosynthesis of anthocyanin production. Also, the constantly low expression of RcANS gene down-regulated overall anthocyanin biosynthesis. The lack of RcF3’5’H gene/protein hindered the production of delphinidin glycosides. Flavonoids primarily comprising of quercetin/kaempferol-glycosides were predominately located at fruit epidermal-hair and placentae. The prole and biosynthesis of R. chingii avonoids are unique to Rubus. It could be used to broaden the genetic base of raspberry cultivars and to improve their fruit quality. phenol reagent and react for 5 min at room The reaction was neutralized with 2 mL of 5% saturated Na 2 CO 3 and incubated for 60 min at 30 ℃ . The absorbance was measured at 760 nm. A calibration curve was prepared using gallic acid solution (5-100 µg/mL). TPCs were expressed as gallic acid (Sigma-Aldrich, Shanghai, China) equivalent (mg/g FW). for was using a of (Sigma-Aldrich, China) concentrations (50–900 mol/L). Results were expressed at TEAC (mmol/g FW). three homologs for each Rc3GGT/UGT79 and RcGT1, but they had relatively low expression and did not show a clear trend of change at the gene/protein level. The consistently low level of expression of RcANS suggests a relatively low concentration of anthocyanins. Notably, the genes/proteins of RcLAR and RcANR2 were highly expressed in unripe fruit, resulting in the relative abundance of avan-3-ols, i.e. (epi)catechinand and (epi)afzelechin. These avan-3-ols were interacted with each other or with cyanin/pelargonin to generate proanthocyanins or dimeric anthocyanins respectively. condensed forms, which have not been previously reported in Rubus. The lower avonoid and anthocyanin concentrations in latter phases of R. chingii fruit development is due to the down-regulation of phenylpropanoid, and avonoid biosynthesis. These mechanisms appear to be unique to R. chingii, and have not been reported in other fruit crops. Multiple genes and proteins in these pathways were divergent in function and differently regulated.


Introduction
Rubus chingii Hu (Chinese raspberry), Chinese name "Fu-Pen-Zi", is indigenous to China, and mainly distributed along the lower-middle reaches of the Yangtze River. Like red raspberry (R. idaeus L., subgenus Idaeobatus Focke), blackberry (Rubus sp., subgenus Rubus L.), and black raspberry (subgenus Idaeobatus), it is highly appreciated by consumers not only for its special avor but also for its nutritional properties due to the abundant antioxidants.
Plant avonoids mainly consist of anthocyanins, condensed and hydrolyzable tannins. These avonoids contribute to the taste, avor, and color of food and drink, e.g. astringency and/or color, and have a wide of pharmaceutical uses, such as its astringent action. Prior studies have extensively examined Rubus avonoids. For example, anthocyanin compositions have been identi ed and quanti ed in raspberries. Red and black raspberry share the same pro le of anthocyanins. Their anthocyanins are predominantly cyanidin glycosides (e.g. glucosides, sophorosides, rutinosides, sambubioside and glucosyl-rutinosides), but they only contain low to trace levels of pelargonidin glycosides (Kula et al. 2016;Ludwig et al. 2015;Mazur et al. 2014). Black raspberry has up to ve-fold greater anthocyanin content than red raspberry (Krauze- Baranowska et al. 2014). Flavonols in red and black raspberry are mainly kaempferol/quercetin glycosides with glucosides, rutinoside and coumaroylglucoside (Kula et al. 2016). Moreover, agricultural technologies, elicitors, stimulating agents and plant activators, have been applied to increase Rubus avonoid content (Jin et al. 2012). In R. chingii, previous phytochemical studies have isolated a series of avonoids (Ding 2011;Guo et al. 2005). The avonoids dramatically change as the fruit ripens, which has attracted considerable research attention. However, to our knowledge, few studies have been conducted on avonoid change and their biosynthesis during maturation in R. chingii. This study was undertaken to investigate the change in composition and content of avonoids, and explore potential mechanisms underlying avonoid biosynthesis during fruit maturation based on the genetic and enzymatic components.

Materials And Methods
Plant Material.
R. chingii Hu fruits were collected from ve to six plants at varying maturation phases i.e. mature green (MG), green yellow (GY), Yellow orange (YO) and Red (RE) during the growing season (May, 2019) at LINHAI, Zhejiang, China (Fig. 1a). Fruits were collected and immediately frozen in liquid nitrogen then stored in a -70℃ freezer. Ten fruits were pooled as one replicate. Three biological replicates were used for all experiments. The fruit were ground in liquid nitrogen into powder and subsequently used for analysis of mRNA, protein and metabolites. The content measurements of composition were determined relative to overall fruit weight.
Total anthocyanin content was determined via spectrophotometry. Fruit tissue was ground in liquid nitrogen. Approximately 0.3 g of tissue were weighed and added to 10 mL 1% (v/v) HCl methanol (HCl and methanol: Shanghai Hushi Chemical, Shanghai, China) and incubated for 24 h at room temperature in the dark. After centrifugation for 15 min at 13,000 × g, supernatants were measured for absorbance at 530, 620 and 650 nm. The anthocyanin content was estimated using the following formulas: The anthocyanin content = ∆A×V×M/(ε × m) Where V is the extract volume (mL), ε is the molar extinction coe cient of cyanidin-3-glucoside at 530 nm (29600), M is the molecular weight of cyanidin-3glucoside (449 g mol − 1 ), and m is the mass of the fruit extracted. The results were expressed as cyanidin-3-glucoside equivalents (mg/g FW).
Total content of carotenoid was determined via spectrophotometry. Sample fruit were grounded in liquid nitrogen and approximately 0.3 g tissue (ground powder) was mixed with 10 ml extraction solution (ethanol:acetone = 1:2). The extraction was vortexed and then put in darkness for at least half hr until the residues became colorless. The absorbance of the extractive solvent was measured at 440, 645 and 663nm for carotenoid, and chlorophyll a/b respectively.
Total phenolic content was determined using the Folin-Ciocalteu method following the procedure . Fruit tissue was completely ground with liquid nitrogen. Approximately 0.3g of tissue was weighed, mixed with 10 mL of acidi ed methanol (0.1% hydrochloric acid) for 24 h in the dark and centrifuged for 15 min at 3000 rpm. The extract was appropriately diluted to fall in the range of the standard curve, 2 mL of which was transferred to another colorimetric tube, mixed with 1 mL 0.5 N Folin-Ciocalteu's phenol reagent (Sigma-Aldrich, Shanghai, China), and allowed to react for 5 min at room temperature. The reaction was neutralized with 2 mL of 5% saturated Na 2 CO 3 and incubated for 60 min at 30 ℃. The absorbance was measured at 760 nm. A calibration curve was prepared using gallic acid solution (5-100 µg/mL). TPCs were expressed as gallic acid (Sigma-Aldrich, Shanghai, China) equivalent (mg/g FW).
The free radical-scavenging activity was determined using the ABTS radical cation decolorization method (Jin et al. 2016 Frozen sections and Flavonoid In situ DPBA staining The fresh fruits were chopped into several parts and embedded in SCEM embedding medium (Section-Lab, Hiroshima, Japan). The targeted tissue was embedded until the surface was tightly covered with adhesive lm and then immediately frozen at − 20°C. The frozen samples in embedding medium were trimmed and then carefully sliced to produce 50-80 µm fresh-frozen sections using a CM1850 Cryostat (Leica microsystems, Wetzlar, Germany) set at − 20°C.
The sections of fruit were stained in a freshly prepared aqueous solution of 0.25% (w/v) 2-aminoethyl diphenylborate (DPBA) (Tokyo Chemical industry, Tokyo, Japan) and 0.00375% (v/v) Triton X-100 (Sigma-Aldrich, Shanghai, China) for at least 30 min. A Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss AG, Jena, Germany) was used to excite the roots with 30% maximum laser power at 458 nm, and the uorescence was collected at 475-504 nm for kaempferol and 577 to 619 nm for quercetin (Lewis et al. 2011). Total RNA extraction, library construction, and bioinformatic analysis Fruit RNAs were extracted by CTAB method, their quality was tested with an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) (Agilent, Santa Clara, CA, USA) for RNA concentration, RIN value, 28S/18S ribosomal RNA and the fragment length distribution. The RNA purity was determined using a NanoDrop™ (Thermo Fisher Scienti c, Wilmington, USA).
The libraries construction followed the method described in Li et al. (Li et al. 2013). The mRNAs were isolated from total RNA with oligo(dT) and then fragmented. The rst and second strand of cDNA were synthesized, puri ed and resolved with EB buffer for end repair and adenine (A) addition. After that, the cDNA fragments were connected with adapters and those with suitable size were PCR ampli ed. Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System (Thermo Fisher Scienti c, Rockford, USA) were used to quantify and qualify the libraries.
The read data were processed following the procedure (Li et al. 2013). The low-quality reads (> 20% of the bases with low quality < 10) and reads with adaptors and unknown bases (N > 5%) were ltered to get clean reads. The clean reads were assembled into unigenes using Trinity, for functional annotation and expression estimation. Data are available via NCBI with accession (PRJNA671545). The relative expression was estimated by Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Based on the relative expression, differential expressed unigenes were de ned by threshold (fold Change > 2.00 or < 0.5; adjusted P value < 0.05) and they were subjected to pathway enrichment (FDR < 0.01 are de ned as signi cant enrichment).

Protein extraction, HPLC fractionation, LC-MS/MS assay and bioinformatic analysis
Fruit proteins were extracted by the method described by Li et al. Li et al. 2020). The concentration was determined using a BCA protein assay kit (Thermo Fisher Scienti c, Rockford USA). The extracted proteins were reduced, and then digested by trypsin. After trypsin digestion, peptides were desalted and processed as TMT10plex™ Isobaric Label Reagent (Thermo Fisher Scienti c, Rockford, USA).
The labeled peptides were fractionated by HPLC, and the peptides were divided into 18 fractions. The peptides were loaded into tandem mass spectrometry (MS/MS), Orbitrap Fusion™ Tribrid™ (Thermo Fisher Scienti c, Rockford, USA). These processes were performed as described by Li et al. (Li et al. 2019;Li et al. 2020). The relative expression of isoform was estimated by comparing the intensities of the reporter ions. Compared to the expression pro le at the MG phase, differential expressed isoforms were de ned by threshold change (change fold > 1.5 or < 0.67 and P < 0.05).
The resulting shotgun MS/MS data were processed using a Maxquant search engine (v.1.5.2.8), and searched against the transcriptomic data concatenated with a reverse decoy database . Data are available via ProteomeXchange with identi er (PXD021977). KEGG enrichment of isoforms were determined by a two-tailed Fisher's exact test. The signi cant threshold was set up (p-value < 0.05) for KEGG enrichment.

Analysis of major anthocyanins and avonoids
Anthocyanins were extracted with 1% (v/v) HCl methanol and concentrated by CentriVap refrigerated Centrifugal Concentrators at 8℃ (Models 73100 Series) (Labconco, Kansas City, USA) and then re-dissolved it with 1 mL 1% (v/v) HCl methanol. Flavonoids was extracted with 70% methanol for 2 h at room temperature in the dark, and refrigerated Centrifugal Concentrators at 8℃ (Labconco Models 73100 Series) and then re-dissolved it with 1 mL 70% methanol. The extract was passed through a 0.22-µm microporous membrane lter for LC-MS analysis.
For anthocyanins, the mobile phases were 1% formic acid-water (A) and acetonitrile (B). Gradient conditions were as follows: 0-25 min, 5-35% phase B; 25-37min; 35-95% phase B. The loading volume was 5 µL, the ow rate was 0.4 mL min − 1 ; the column temperature was 50℃, and the UV detector was set at 530 nm. For avonoids, the mobile phases were 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B). The linear gradient programs were as follows, 0-5 min, 5-10% phase B; 5-25min, 10-25% phase B; 25-37min, 25-95% phase B; Sample injection volume was 5µL; Column oven temperature was 50℃; Flow rate was 0.3mL min − 1; and the UV detector was set at 360 nm. Anthocyanins and avonoids separated by UPLC was analyzed using a MS AB Triple TOF 5600 plus System (AB SCIEX, Framingham, USA) in both negative ion (source voltage at -4.5 kV, and source temperature at 550 • C) and positive ion mode (source voltage at + 5.5 kV, and source temperature at 600 • C). Maximum allowed error was set to ± 5 ppm. Declustering potential (DP), 100 V; collision energy (CE), 10 V. For MS/MS acquisition mode, the parameters were almost the same except that the collision energy (CE) was set at 40 ± 20 V, ion release delay (IRD) at 67 and the ion release width (IRW) at 25. The IDA-based auto-MS 2 was performed on the 8 most intense metabolite ions in a cycle of full scan (1 s). The scan range of m/z of precursor ion and product ion were set as 100-2,000 Da and 50 − 2,000 Da. The exact mass calibration was performed automatically before each analysis employing the Automated Calibration Delivery System.

Changes of anthocyanins, avonoids, phenolics and antioxidant capability
Total anthocyanin was relatively low, ranging from 6.92 (mg /100 g FW) in RE to 19.52 in MG. As fruit matured, anthocyanin surprisingly decreased by 19.37%, 36.56%, and 30.72% from MG to GY, to YO, and to RE (Fig. 1c). Based on dry weight, anthocyanin content also showed a trend of decrease (Table 1). Total content of avonoids ranged from 128.50 (mg RE/100 g FW) in RE to 646.20 in MG, and it decreased by 42.05%, 55.26%, and 23.31% from MG to GY, YO, and RE ( Fig. 1e). Similarly, total content of phenolics ranged from 760.83 (mg GAE/100 g FW) in RE to 4,026.25 in MG. Phenolic content dropped by 39.01%, 54.68%, and 31.64% from MG to GY, YO, and RE respectively (Fig. 1d). In contrast, total carotenoids increased by 5.84%, 11.96% and 43.58% from the MG to GY, YO, and RE phases (Fig. 1b).  . 1f). There were high Pearson correlation coe cients between these parameters ranging from 0.968 to 0.999, and averaging 0.986 (Fig. 1g). The results indicate the avonoids are one of the most important phenolic compositions that contribute to total antioxidant capacity.
Fruit avonoids composition, anatomical structure and avonoid staining during fruit maturation In R. chingii, anthocyanin primarily consisted of monomeric, and dimeric anthocyanins (Fig. 2a, b and c; Fig. S3). Of these anthocyanins, four had avanolanthocyanin condensed forms (dimeric anthocyanins) and pelargonidins were the main type of anthocyanin aglycones. All of condensed forms contained a avan-3-ol [either (epi)afzelechin or (epi)catechin] as the upper unit carbon-carbon linked to a lower anthocyanin unit consisting of different anthocyanin derivatives (Fig. S3). (Epi)afzelechins were the common monomeric precursors ( avan-3-ols) of the avanol-anthocyanin. The glycosides of (epi)afzelechin(4α->8)pelargonidin were the most abundant type (Fig. 2d, e and f). These anthocyanins all signi cantly decreased in content during fruit ripening, which was consistent to the change in content of total anthocyanin. Also, major avonoids consisted primarily of glycosides of quercetin and kaempferol. Of these avonoids, kaempferol-3-o-rutinoside was predominant, followed by ellagic acid and kaempferol-3-glucoside. All them signi cantly decreased in content during fruit ripening, which was in accordance with the change in content of total avonoids. A raspberry fruit is an aggregate fruit composed of drupelets (Fig. 3a). Each drupelet contains the pericarp and seed. The pericarp is made up of the exocarp, hypodermis, and mesocarp layers; while the seed consists of the episperm, endosperm, and embryo. The exocarp is attached with a layer of epidermal hair and the seed is surrounded by placentae. In fruit cross-sections, DPBA uorescence showed avonoid accumulation patterns at various stages of fruit maturation (Fig. 3b). Each avonoid-DPBA conjugate was labeled by a unique uorescent color (kaempferol by yellow and quercetin by red). Flavonol-speci c uorescence was mainly observed in the fruit epidermal hair throughout the entire fruit maturation process, but rarely in fruit esh including the exocarp, hypodermis and mesocarp (Fig. 3c, d, e, and f). As fruit matured, fruit epidermal hair became shorter and thinner. In addition, avonol-speci c uorescence was seen in the placentae and seed coats of developing seed (Fig. 3c, d, d, and f). The results suggest that the majority of avonoids are concentrated in fruit epidermal hair and placentae.

Pro ling of genes and proteins involved in avonoid synthesis
Twelve transcriptomics were developed for BG, GY, YO and RE fruits. A total of 89,188 unigenes were obtained, and 49,755 (55.79%) and 37,833 (42.42%) were annotated in non-redundant and KEGG database respectively. The biggest difference in gene expression was between RE/MG (6,502 up-regulated and 5733 down-regulated unigenes) while the smallest difference was between GY/MG (1,965 up-regulated and 1,966 down-regulated unigenes) (Fig. S1a). Accordingly, in twelve proteomes of BG, GY, YO and RE fruits, 141,036 unique peptides corresponding to 9,478 isoforms, and 8,529 quanti ed isoforms were identi ed. In proteomics, the most difference in isoform expression (506 up-regulated and 618 down-regulated isoforms) was observed between RE/MG while the smallest difference was between GY/MG (765 up-regulated and 799 down-regulated isoforms) (Fig. S1b). In metabolomics, 8,218 high quality ions included 7,611 (92.61%) ions with relative standard deviation (RSD) < = 30% in positive ion mode, while 6,150 high quality ions included 4,683 (76.15%) ions with RSD < = 30% in negative ion mode. The largest difference detected (1,656 and 1024 in positive and negative ion modes respectively) was between RE/MG while the smallest difference was between GY/MG (2,151 and 1,468 in positive and negative ion modes respectively) (Fig. S1c). The results suggest that a large number of genes, proteins, and metabolites are involved in fruit maturation, and the biggest difference is between RE and MG while the smallest difference is between GY and MG.
Generally, avonoid products are involved in four pathways, i.e. "phenylpropanoid biosynthesis", " avonoid biosynthesis", " avone and avonol biosynthesis", and "anthocyanin biosynthesis". KEGG enrichment was performed to discern the multivariate pattern of up-and down-regulated unigenes/isoforms. The unigenes involved in "phenylpropanoid biosynthesis" and " avonoid biosynthesis" were signi cantly enriched in GY/MG, YO/MG and RE/MG and most of them were down-regulated (Fig. S2a). Accordingly, the down-regulated isoforms involved in these pathways' biosynthesis were enriched as well (Fig. S2b). However, neither up-regulated or down-regulated unigenes/isoforms were enriched in " avone and avonol", or "anthocyanin" biosynthesis ( Fig. S2). This suggests that "phenylpropanoid biosynthesis" and " avonoid biosynthesis" are more active in green phases than the other three phases, and responsible for biosynthesis of major avonoid products during fruit maturation.

Differentially expressed gene and protein in avonoid and phenylpropanoid biosynthesis
In transcriptomics, 608 unigenes were predicted to be involved in phenylpropanoid biosynthesis, avonoid biosynthesis, avone and avonol biosynthesis, and anthocyanin biosynthesis, and 309 (50.82%) were differentially expressed. A large part of unigenes were maintained at low expression level during fruit maturation. In our proteomics study we identi ed 116 isoforms involved in these pathways and 55 (47.41%) were differentially expressed. These isoforms were all mapped to their corresponding unigenes, but a large number of isoforms corresponding to these unigenes could not be detected. The inconsistencies between unigenes and isoforms may be due to the discrepancy of transcriptomic and proteomic techniques such as differences in detectable threshold.
In conclusion, most of the differently expressed unigenes/isoforms in these pathways shared a similar trend of change in expression, which was consistent with the high correlations seen between them (Pearson correlation = 0.956). However, the phylogenetically different homologs shows different patterns of gene/protein expression. The results suggest the changes observed in gene expression are consistent with those seen in protein expression and the homologs are divergent in function.

Discussion
Changes in avonoids and their localization during fruit maturation It was surprising to see that the total anthocyanin, avonoids, and phenolics all showed a continuous decrease during the fruit maturation process in R. chingii. This pattern was different from any previous report in Rubus species including red/black raspberry. In red raspberry, anthocyanin concentration continuously increases throughout fruit ripening, but the total phenolic concentration decreases from the green to the veraison stage, and then increases until maturity (Wang et al. 2009). Cyanidin-3-O-sophoroside is the most prominent anthocyanin followed by cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside (Stavang et al. 2015). The avonols, hyperoside and quercetin-3-O-glucoside are both constantly present at low concentrations (Stavang et al. 2015). In black raspberry, the content of avonols e.g. quercetin 3-O-rutinoside and quercetin-glucuronide, and anthocyanins e.g. cyanidin 3-O-xylosylrutinoside and cyanidin 3-O-rutinoside increases during ripening, while the content of avanols, (epi)catechin and B type proanthocyanidin dimers decrease (Hyun et al. 2014). The increasing trend of anthocyanins and the "V"-type change of total phenolics are ubiquitous during maturation in many berries, e.g. blueberry , cranberries (VvedenskayaVorsa 2004), strawberry (Song et al. 2015), and grape (Giribaldi et al. 2007), which is mainly due to their substantial increase in anthocyanin pigment concentration after veraison . In R. chingii, the continuous down-regulation of total avonoids (including anthocyanins) was responsible for the continuous decrease of phenolics. These pigments primarily consisting of an anthocyanin linked to a avonol, were rst reported in Rubus species. Interestingly, fruit anthocyanins decreased inversely with its red coloration during maturation, which indicates that its red coloration is caused by something other than anthocyanins. For example, the accumulation of carotenoids as the fruit maturates may be responsible for the red coloration (Fig. 1B).
R. chingii fruit is very high in antioxidants, which is highly correlated with the phenolic compounds including avonoids. In R. chingii, total content of phenolics peaked at 4,026.25 (mg GAE/100 g FW) in MG (Fig. 1b). It is 10 fold higher than mature fruit in red raspberry (357.83 mg GAE/100 g FW), blackberry (850.52), strawberry (621.92), blueberry (305.38) and cherry (314.45) (Chen et al. 2013;De Souza et al. 2014). Even at the lowest concentration in the RE stage, total phenolic content was 760.83 (mg GAE/100g FW), which was much higher than most of the other fruits (Chen et al. 2013;De Souza et al. 2014). Also, total content of avonoids peaked at 646.20 in MG (mg RE/100g FW), which was higher than that in raspberry (Chen et al. 2013) (Fig. 1d). Similarly, ABTS peaked at 41.18 (mmol TEAC/100g FW) or 411.80 (umol TEAC/g FW) in MG and was over 20 fold higher than mature fruit of red raspberry (6.27 umol TEAC/g FW), blackberry (13.23), strawberry (7.87), blueberry (5.88) and cherry (8.83) (De Souza et al. 2014). The lowest content of ABTS in the RE stage of R. Chingii was 100.52 (umol TEAC/g FW), which was still 10-fold higher than the previously mentioned fruits. The extremely high antioxidant capacity could be a valuable natural source of antioxidants for use in the food industry Flavonoid in situ staining shows that the avonoids (i.e. kaempferol and quercetin derivates) predominately accumulate at the same tissues (epidermal hair and placentae) of fruits during the four typical maturation phases. It is highly likely that these avonoids are synthesized in the cells in which they accumulate. The avonoids in epidermal hair might function as antioxidants that protect fruit from pests and pathogens, while the ones in seed may function as endogenous regulators of auxin transport that is responsible for seed maturation. As the fruit maturated the epidermal hairs became thinner and shorter and many of them fell off. The epidermal hair loss could be also related to the decrease of avonoids ( Fig. 2c and d) Down-regulated expression of genes/proteins in the phenylpropanoid pathway caused a decrease in ux from phenylpropanoids to avonoids The genes and enzymes involved with the phenylpropanoid biosynthesis and the avonoid biosynthesis have been extensively studied in many plants. Most of these genes are involved in multigene families. Some members are divergent in function and others are redundant or underutilized (Kim et al. 2004). In Arabidopsis, two redundant PAL genes (AtPAL1 and AtPAL2) are both expressed in vascular tissues. AtPAL3 is primarily expressed in roots and leaves, albeit at low levels, while AtPAL4 is mainly expressed in developing seed tissue (Raes et al. 2003). These divergent PAL genes respond differentially under various developmental events and environmental stresses (Chang et al. 2008;Cochrane et al. 2004;KumarEllis 2001). In tomato, only one PAL transcript is induced by pathogen or wounding (Chang et al. 2008). In red raspberry, RiPAL1 is expressed during early fruit ripening, while RiPAL2 is expressed at later stages of ower and fruit development ( KumarEllis 2001). PAL genes also show tissue speci c patterns of expression. The expression of RiPAL1 transcripts is much higher than that of RiPAL2 in leaves, shoots, roots, young fruits, and ripe fruits. In blueberry, three PAL genes are up-regulated at the gene/protein level as fruit matures . In this study, two phylogenetically close RcPALs were both down-regulated at the gene/protein level as fruit matured ( Fig. S4a; Fig. 4a and b).
4CL isoenzymes exhibit distinct substrate a nities due to their different metabolic functions. In Arabidopsis, four 4CL genes are divergent in functions e.g. At4CL4 exhibits the rare property of activating sinapate and other 4CL substrates (e.g. 4-coumarate, caffeate, and ferulate) (HambergerHahlbrock 2004). In Physcomitrella patens, three 4CLs display the highest catalytic e ciency towards 4-coumarate, which is distinguished from the fourth 4CL (Silber et al. 2008). In blueberry, Vc4CL and Vc4CL-like are both up-regulated as fruit maturates although they are phylogenetically separated . In this study, two phylogenetically distant CL4 homologs (RcCL4 and Rc4CL-like) showed distinct patterns of expression (Table 2). 4CLs were signi cantly down-regulated at the gene/protein level as fruit maturated while 4CL-like genes were expressed at low levels. The results suggest that 4CL rather than 4CL-like functions in down-regulation of the phenylpropanoid pathway in fruit.
C4H belongs to a large group of cytochrome P450 monooxygenases (P450) in plants and exclusively constitute the CYP73 family, a typical group of P450. In citrus, C4H1 and C4H2 are different in both expression patterns and N-termini, suggesting they have speci c functions in organelles (Betz et al. 2001). In