Dynamic changes of enzymes involved in sugar and organic acid level modification during blueberry fruit maturation

Journal Pre-proofs Dynamic Changes of Enzymes Involved in Sugar and Organic Acid Level Modification During Blueberry Fruit Maturation Xiaobai Li, Chunnan Li, Jian Sun, Aaron Jackson PII: DOI: Reference:

S0308-8146(19)31742-X https://doi.org/10.1016/j.foodchem.2019.125617 FOCH 125617

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

13 May 2019 27 September 2019 29 September 2019

Please cite this article as: Li, X., Li, C., Sun, J., Jackson, A., Dynamic Changes of Enzymes Involved in Sugar and Organic Acid Level Modification During Blueberry Fruit Maturation, Food Chemistry (2019), doi: https://doi.org/ 10.1016/j.foodchem.2019.125617

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Dynamic Changes of Enzymes Involved in Sugar and Organic Acid Level Modification During Blueberry Fruit Maturation Xiaobai Li*1,2, Chunnan Li3, Jian Sun4, Aaron Jackson5 1Zhejiang

Academy of Agricultural Sciences, Hangzhou 310021, China.

2Key

Laboratory of the Ministry of Agriculture for Creative Agriculture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China 3Hangzhou

Academy of Agricultural Sciences, Hangzhou, Zhejiang 310024

4Zhejiang

Research Institute of Traditional Chinese Medicine Co., Ltd., Hangzhou 310023, China 51904 South Oak, Stuttgart, AR 72160 USA.

*Corresponding author: [email protected]; [email protected]

ACKNOWLEDGEMENTS This research study was supported by the Young Scientist Training Program of ZAAS; the Industrial Project of ZAAS. Development and Demonstration of Horticultural Crop Varieties Based on Creative Agriculture, Science Technology Department of Zhejiang Province (2018C02057)

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Abstract: In blueberry, sugars and organic acids determine fruit organoleptic quality and drastically change during fruit maturation. This study examined enzymes involved in the metabolism of sugars and organic acids during the three maturation phases (green, pink and blue). During maturation, an increase in sugar (mainly fructose and glucose) was

associated

with

up-regulation

of

VcSPP

(CUFF.32787.1),

VcSPS

(CUFF.14989.1), and VcINV (gene.g3367.t1.1, CUFF.8077.1 and CUFF.47310.2). A decrease in citrate was associated with VcACLY (CUFF.27347.1 and CUFF.28772.1) in the acetyl-CoA pathway and with VcGAD (CUFF.15663.1 and CUFF.13757.1) and VcGLT (CUFF.6416.1) in the GABA shunt. A decrease in malate was associated with VcMDH (CUFF.30072.1, CUFF.18332.1 and CUFF.24878.1) involved in malate biosynthesis,

and

with

VcADH

(gene.g1507.t1.1,

CUFF.3210.1

and

gene.g30667.t1.1) as well as VcPDC (CUFF.47532.1) involved in fermentation. Multi-isoforms of enzymes were divergent and differentially regulated, suggesting that they have specialized functions in these pathways. The information will contribute to the understanding of blueberry organoleptic quality.

Key words: Soluble sugar; Organic acid; Enzyme’s regulation; Enzyme’s Phylogeny; Ripening; Vaccinium corymbosum

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1.Introduction Blueberry is a much sought after fruit that is attributed as having incredible nutritional and health benefits. Recent advances in blueberry varieties have allowed for them to be grown across larger environmental extremes while providing an abundant harvest. Generally, commercial highbush blueberry can be subdivided into two types, i.e. northern and southern highbush blueberries. The former usually requires a long period of low temperatures during the winter season to induce flowering the following spring, while the latter requires fewer days of colder temperatures to trigger flowering. Recently, a draft-genome is assembled using RNA-Seq data from five fruit developmental phases of the southern highbush cultivar “O’Neal” (Gupta et al., 2015). The “O’Neal” genome provides a valuable data resource for proteomic studies on fruit quality in southern highbush blueberry. The five phases of fruit development are generally divided into two stages of “expansion” (i.e. pad and cup phases) and three stages of “maturation” (i.e. green, pink, and blue) (Gupta et al., 2015). The maturation stages are characterized by color and quality changes. Sugars and acids are two important components that influence both fruit flavor and taste. Sugars, especially sucrose, glucose, and fructose, are responsible for fruit sweetness, while organic acids, particularly citrate and malate, determine fruit acidity. A moderate concentration of acid can make fruit more palatable, whereas high acid content often reduces fruit quality. The change of sugar and acid over time is a result of synthesis, degradation, and transport processes. In plants, most photosynthates (e.g. sucrose) are transferred from the leaf (i.e. source) to the fruit (i.e. sink), where they undergo metabolism. In citrus, 50% of sucrose is translocated to fruits from leaves throughout fruit development (Katz et al., 2011). In apple, almost all of the sorbitol and half of the sucrose are converted to fructose, and at least 80% of the total carbon flux goes through fructose (M. Li, Feng, & Cheng, 2012). Organic acids are another form of nutrient shortage, which play an essential 3

role in energy generation (Sweetman, Deluc, Cramer, Ford, & Soole, 2009). Additionally, low pH could enhance sink strength of fruit and increase carbohydrate uptake (Hockema & Etxeberria, 2001). The metabolism of sugars and organic acids couples with other pathways. For example, respiration of sugars via glycolysis, oxidative pentose phosphate pathway, and/or the tricarboxylic acid cycle (TCA) provide energy (ATP), reducing power, NAD(P)H and precursors for the synthesis of organic acids, amino acids, anthocyanins, defense and aromatic compounds. A number of key enzymes are involved, and the dynamic changes of the enzymes responsible for sugar and acid accumulation have been studied in multiple plant species. The purpose of this study was to identify enzymes and pathways involved in sugar and organic acid metabolism during the course of fruit maturation in blueberry.

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2 Material and Methods: 2.1 Materials Blueberry fruits were collected from three or more plants of the O‘Neal variety of southern highbush blueberry (V. corymbosum) at green, pink and blue phases during the growing season (May, 2017) at Yangdu, Zhejiang (Figure 1A). Gupta et al. (2015) describes the three phases of blueberry maturation i.e. green, pink and blue. Fruits were frozen in liquid nitrogen as they were harvested from the plant and then stored in a -70℃ freezer. Three biological replicates for each phase were designed and tissue was derived from the entire fruit for both proteomic and quality analysis.

2.2 Protein Extraction, Trypsin Digestion and TMT Labeling The entire fruit was ground in liquid nitrogen, and proteins were extracted using the TCA-acetone extraction method (X. Li, Jin, Pan, Yang, & Guo, 2019). The protein concentration was determined using a BCA protein assay kit (TermoFisher). The extracted proteins were reduced and then digested by trypsin. After trypsin digestion, peptides were desalted using TMT10plex™ Isobaric Label Reagent (TermoFisher).

2.3 HPLC Fractionation, LC-MS/MS Analysis and PRM Assay 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 FusionTM TribridTM (ThermoFisher). These processes were conducted as described in Li et al. (2019). The threshold of the differentially expressed proteins was set (change fold>1.5 or <0.67 and P <0.05). The PRM analyses were performed using a Q-Exactive Plus mass spectrometer (ThermoFisher). Proteins were extracted, digested into peptides and the peptides were separated as described in Li et al. (2019).

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2.4 MS/MS Database Analysis, Protein Annotation, and Functional Enrichment The resulting shotgun MS/MS data (PRIDE, Accession: PXD011815) were processed using a Maxquant search engine (v.1.5.2.8), against a blueberry database concatenated with a reverse decoy database (Gupta et al., 2015). The resulting PRM MS data was processed using Skyline (v.3.6). The parameters were set up as described in Li et al (2019) (X. Li et al., 2019).

2.5 Fruit Anatomical observation Fruits were immersed and stored in FAA solution (10 formaldehyde/5 glacial acetic acid/35 ethyl alcohol) for one month. Radial and Transverse sections were taken and dehydrated in a graded ethanol series (20%, 40%, 60%, 80%, 95%, 100%, 100% for 30 minutes per step) followed by paraffin infiltration and embedding using tert-butyl alcohol as an intermediate solvent. Sections of 12 to 14 μm were obtained using a 0.25-mm steel microtome blade on a rotary microtome and were mounted on glass slides. The mounted sections were deparaffinized and stained with Safranin O and Aniline Blue. Finally, slides were sealed with neutral balsam, observed through a light microscope (Olympus SP 350, Japan) and photographed.

2.6 Determination of Fruit Firmness and Maturity Index Fruit firmness (kg/cm2) was determined using a TA-XT2i texture analyzer (Stable Micro Systems, England) fitted with a 5 mm diameter probe. The penetration rate was 1 mm/s with a final penetration depth of 10 mm. Measurements were conducted on the equator of the fruit. The maturity index (MI) was calculated according to the ratio between the total soluble solids and titratable acidity (TA). Three biological replicates (including 3-5 technical replicates for each) were performed for the measurement.

2.7 Determination of Total Sugars Total sugar content (TSC) in fruit was determined by using the 3,5-dinitrosalicylic acid method (DNS method, Lindsay, 1973). Approximately 2 g of tissue (ground 6

powder) from 5-6 fruits were mixed with 1mL of solution A (500mL: 3.25g 3, 5-dinitrosalicylic acid; 2mol/L NaOH 162.5mL; 22.5g glycerol) and 1mL H2O, and then boiled at 95℃ for 30 min. The slurry was added to 1mL of solution B (500 mL: 2.5g 3,5-dinitrosalicylic acid; 0.5g Phenol; 0.075g Na2SO4; 2.5g NaOH; 50g NaKC4H4O6·4H2O), and then transferred to a 10-mL volumetric tube and centrifuged for 10 min at 8000 g. The absorbance was measured at 540 nm. Three biological replicates (including 3 technical replicates for each) were performed for the measurement.

2.8 Determination of Titratable Acidity Titratable acidity was measured in a weighed aliquot of homogenate (about 2 g ground tissue from 5-6 fruits) using 0.01 N NaOH and a semi-automated titrator (Multi-Dosimat E-415; Metrohm AG, Switzerland), using phenolphthalein as an endpoint indicator. Anthocyanin content was measured by the pH differential method using the extinction coefficient of malvidin-3-glucoside (28,000). Three biological replicates (including 3 technical replicates for each) were performed for the measurement.

2.9 Determination of Organic acid Approximately 2 g ground tissue from 5-6 fruits was mixed with 2 mL water and then exposed to ultrasonic irradiation for 30 min to promote extraction. The resulting slurry was centrifuged at 8,000g and 4 ℃ for 10 min. The supernatant was then transferred to a 10 mL volumetric flask. The residue was added to 1 mL water and centrifuged at 8,000g and 4 ℃ for 10 min. The supernatant was merged together, and the final volume was 3 mL. The extract was passed through a 0.45μm microporous membrane filter. The final filtrate was used for HPLC analysis. Chromatographic separation was performed in a RP-HPLC ACE C18 column, particle size 5 μm (250 mm, 4.6 mm), at 35 ℃ , and 214 nm. The analyses were performed at a flow rate of 0.8 mL/min. The mobile phase consisted of 1000mL 7

0.01M phosphate buffer (pH=2.3) and 20mL methanol. Three biological replicates (including 3 technical replicates for each) were performed.

2.10 Determination of Sugar Approximately 2 g ground tissue from 5-6 fruits were mixed with 2 mL water and exposed to ultrasonic irradiation for 30 min to promote extraction. The resulting slurry was centrifuged at 8,000g and 4 ℃ for 10 min. The supernatant was passed through a 0.45 μm microporousmembrane filter .The final filtrate was used for HPLC analysis. The analysis column was an Agilent Carbohydrate column (5 um, 4.6 mm × 250 mm), the mobile phase was acetonitrile-water (75:25), and the flow rate was 1.0 mL/min; the column temperature was 30 ℃ , and the injection volume was 10 μL. Three biological replicates (including 3 technical replicates for each) were performed.

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3. Results 3.1 Dynamic Change of Fruit Appearance and Texture During maturation, fruit gradually expanded by 29.77% and 11.80% of width, and by 35.93% and 11.13% of length in the transition of green to pink (green/pink) and in the pink to blue phase (pink/blue), respectively (Fig. 1A; Fig. 2A). Maturity index (MI) is used to assess the maturity of many fruit crops. In blueberry, the MI rose by 345.70% in green/pink and by 48.66% in pink/blue (Fig. 2A). In contrast, fruit firmness sharply dropped by 91.77% in green/pink, and then by 17.26% in pink/blue (Fig. 2A). Tissue type of the fruit also underwent changes during maturation. Mesocarp is composed of the parenchyma layer while epicarp is formed by the epidermal and hypodermal layers. Cell size of parenchyma layer was much larger than that of the epidermal and hypodermal layers. As the fruit matured, cell size of parenchyma layer dramatically enlarged in green/pink rather than in pink/blue (Fig. 1B). The enlargement was associated with a sharp drop of firmness in green/pink. This may be due to an increasing likelihood for larger cells to burst rather than separate from neighboring cells. In contrast, a smaller size cell, with an increasing surface area and higher proportion of cell wall material contributed to fruit firmness. Here, the shape and packing of fruit parenchyma cells changed during fruit maturation, which in turn influenced cell wall strength and cell-to-cell adhesion (Fig. 1B). The adhesion between neighboring cells determined the fruit firmness. Fruit appearance and texture changed more in green/pink than in pink/blue (Fig. 1).

3.2 Dynamic Change of Sugars and the Proteins Involved in Their Metabolism During Fruit Maturation During maturation, TSC successively increased by 128.65% in green/pink and by 24.33% in pink/blue (Fig. 2B). Similarly, total soluble solids (TSS) increased by 32.02% in green/pink and by 22.49% in pink/blue. Three major sugars were identified, i.e. sucrose, fructose and glucose. The latter two were similar in content 9

and predominant in blueberry fruit, while sucrose was present in relatively low concentrations (Fig. 2C). Major sugars increased during fruit maturation. Fructose increased by 591.21% in green/pink, and by 24.45% in pink/blue. Glucose increased by 413.82% in green/pink, and by 21.32% in pink/blue. Sucrose increased by 13.62% in green/pink and by 23.32% in pink/blue. Glucose and fructose are produced through starch and sucrose metabolism and catabolized via glycolysis (Fig. 3A and B). In starch and sucrose metabolism, 30 differently expressed proteins corresponding to 16 enzymes were identified (Table 1). During maturation, sucrose-phosphate phosphatase (SPP), VcSPP (CUFF.32787.1) involved in sucrose biosynthesis, as well as sucrose-phosphate synthase (SPS), VcSPS

(CUFF.14989.1),

and

invertase

(INV),

VcINV

(gene.g3367.t1.1,

CUFF.8077.1 and CUFF.47310.2) both involved with fructose and glucose biosynthesis, were successively up-regulated. In contrast, fructokinase (FK), VcFK (CUFF.34746.1 and CUFF.17458.1) involved in the degradation of fructose, and phosphoglucomutase

(PGM),

VcPGM

(CUFF.25219.2,

CUFF.46452.1,

and

CUFF.38786.1) involved in glucose catabolism, were successively down-regulated. Sucrose synthase (SuSy), involved in the conversion of fructose and sucrose, VcSuSy (CUFF.9127.1) was down-regulated. During glycolysis, 33 differently expressed proteins corresponding to 16 enzymes

were

identified.

Fructose-bisphosphate

aldolase

(FBA),

VcFBA

(CUFF.1669.1 and CUFF.48231.1), fructose bisphosphatase (FBP), VcFBP (CUFF.33526.1 and CUFF.46948.1), and phosphoglycerate kinase (PGK), VcPGK (CUFF.6095.1),

pyruvate

kinase

(PK),

VcPK

(CUFF.11308.1)

were

all

down-regulated during maturation (Fig. 3B). Likewise, phosphofructokinase (PFK), VcPFK (CUFF.32791.1 and CUFF.57761.1), phosphoenolpyruvate carboxykinase (PCK) VcPCK (gene.g24276.t1.1), enolase (ENO), VcENO (CUFF.8875.1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), VcGAPDH (CUFF.38881.1 and gene.g31625.t1.1) were all down-regulated. Notably, acetyl-CoA synthetase 10

(ACSS), VcACSS (CUFF.4807.2), alcohol dehydrogenase (ADH), VcADH (gene.g1507.t1.1, CUFF.3210.1, and gene.g30667.t1.1), aldehyde dehydrogenase protein (ALDH), VcALDH (CUFF.36657.1), pyruvate decarboxylase (PDC), PDC (CUFF.47532.1) were all up-regulated, all of which are involved in fermentation and coupled with malate degradation. Additionally, ALDH (CUFF.47450.1), ENO (CUFF.44279.1), and PK (CUFF.14751.1, CUFF.11685.2, CUFF.21999.1, and CUFF.22005.1) were expressed differently than the others and their expressions were at the lowest in the pink phase. Phylogenetic analysis of blueberry proteins (translated sequences) and reference proteins in other species indicated that multi-isoforms were categorized into different groups. INV (CUFF.8077.1 and gene.g3367.t1.1) were categorized into acid invertase, while INV (CUFF.47310.2) classified into alkaline/neutral invertase (Fig. 4A). VcFBP (CUFF.46948.1 and CUFF.33526.1) were chloroplastic FBPI and FBPII respectively (Fig. 4I). VcGlgc (CUFF.21624.1 and CUFF.31855.1) were small and large subunits respectively (Fig. 4Q). VcPGM (CUFF.25219.2 and CUFF.46452.1) were cytosolic while VcPGM (CUFF.38786.1) was plastidic (Fig. 4H). Similarly, VcGAPDH (CUFF.38881.1 and gene.g31625.t1.1) were classified into cytosolic and plastidic groups respectively (Fig. 4K). VcPFK (CUFF.32791.1 and CUFF.57761.1) went into PFK3/6 and PFK2 groups respectively. VcSuSy (CUFF.27618.1 and CUFF.9127.1) were grouped together into SuSyIII. VcFK (CUFF.34746.1 and CUFF.17458.1) were both placed into plastidic groups (Fig. 4B). Interestingly, different isoforms were differentially regulated. Typically, VcADH (gene.g1507.t1.1, CUFF.3210.1,

and

gene.g30667.t1.1),

and

VcADHL

(CUFF.31687.1

and

CUFF.6192.1) were respectively divided into ADH and ADH-like groups (Fig. 4O). VcADHs, and VcADHLs were regulated in opposite directions from each other. VcTPS (CUFF.33913.1) and VcTPS (CUFF.10131.1) were classified into TPS7 and TPS11 groups respectively, and also oppositely regulated. Plastic VcFBA (CUFF.1669.1 and CUFF.48231.1) were down-regulated while cytosolic VcFBA 11

(CUFF.50615.1) was up-regulated (Fig. 4M). VcPK (CUFF.21999.1, CUFF.22005.1, and CUFF.14751.1) were separated from the other VcPK (CUFF.11308.1 and CUFF.11685.2) and were also regulated differently (Fig. 4N). The difference in both category and regulation of these isoforms implied their roles in distinct functions.

3.3 Dynamic Changes of Organic Acids and the Proteins Involved in Their Metabolism During Fruit Maturation In contrast to sugars, TA consistently decreased by 70.38% in green/pink and by 17.60% in pink/blue (Fig. 2B). Five kinds of organic acid were also identified, i.e. citrate, malate, quinate, tartrate, and shikimate (Fig. 2D). Of these, citrate was the predominant acid, followed by quinate and malate. All decreased in content as the fruit matured. Citrate dropped by 53.79% in green/pink, and by 35.70% in pink/blue. Malate dropped by 89.36% in green/pink, and by 4.23% in pink/blue. Quinate dropped by 86.61% in green/pink, and by 74.19% in pink/blue. Metabolism of citriate, malate and quinate was involved in TCA, the pentose phosphate pathway, and the shikimate pathway (Fig. 3C, D, and E). In TCA, 18 differently expressed proteins corresponding to 12 enzymes were identified (Table 1). During

maturation,

malate

biosynthesis/degradation,

dehydrogenase

VcMDH

(MDH)

(CUFF.30072.1,

involved

in

malate

CUFF.18332.1

and

CUFF.24878.1) were all down-regulated, while ATP citrate (pro-S)-lyase (ACL) involved

in

citrate

biosynthesis/degradation,

VcACL

(CUFF.27347.1

and

CUFF.28772.1), were both up-regulated. Additionally, VcACL (CUFF.43438.1), VcOGDH (CUFF.5203.1), phosphoenolpyruvate carboxylase (PEPC), VcPEPC (CUFF.45541.1 and CUFF.8537.1) were all expressed at their lowest level in the pink phase. Notably, in a GABA shunt of TCA for citrate degradation, glutamate decarboxylase (GAD) VcGAD (CUFF.15663.1 and CUFF.13757.1) and glutamate synthase (GLT), VcGLT (CUFF.6416.1) were all up-regulated. In the pentose phosphate pathway which connects to the shikimate pathway, five differentially 12

expressed proteins corresponding to four enzymes were all down-regulated as fruit matured. In the Shikimate pathway, 3-phosphoshikimate 1-carboxyvinyltransferase (EPSPS), VcEPSPS (CUFF.47349.1 and CUFF.27788.1) were both up-regulated while

3-dehydroquinate

dehydratases

(DHQ),

DHQ

(CUFF.35149.1

and

CUFF.35531.1) were expressed the most in the pink phase. Phylogenetic analysis indicated that multi-isoforms of these enzymes were categorized into different groups (Fig. 4). VcMDH (CUFF.18332.1, CUFF.24878.1, and CUFF.30072.1) were categorized into three groups, represented by different organelle origins (glyoxysomal, chloroplastic and cytoplasmic) (Fig. 4T). These VcMDHs were all down-regulated. The various isoforms were also observed to be differently regulated. VcACL (CUFF.28772.1, CUFF.27347.1, and CUFF.43438.1) were respectively categorized into β, α2, and α1 chain groups and their expressions were differentially regulated (Fig. 4R). The separate organelle origins and differential regulation of isoforms indicated that they had specialized functions in the metabolic pathways. A PRM assay was developed for quantitation of target peptides. The comparison of expression of 17 proteins in PRM and shotgun proteomics shows a very high correlation (average of 0.997, Pearson correlation coefficient) (Supplemental Table S1). For example, expressions of plastic VcFBA (CUFF.1669.1) at three phases shows a very high correlation (0.999) between Shotgun and PRM proteomics. The results verified by PRM suggested the data generated using the shotgun proteomics method was reliable.

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4. Discussion In most fruit, glucose and fructose account for the majority of soluble sugars (besides sorbitol). Exceptions include peach, mandarin and litchi with the predominant soluble sugar being sucrose (Desnoues, Gibon, Baldazzi, Signoret, Genard, & Quilot-Turion, 2014; Yang, Wang, Wang, Huang, Qin, & Hu, 2013). In lowbush and several wild blueberry species, glucose and fructose are the two major soluble sugars and are present in similar content, but sucrose occurs at a much lower content and sorbitol is hardly detected (Ayaz, Kadioglu, Bertoft, Acar, & Turna, 2001; Gibson, Rupasinghe, Forney, & Eaton, 2013). Therefore, the three major soluble sugars were investigated during fruit maturation in south highbush blueberry. All three sugars increased as fruit matured, with fructose being the predominant sugar (Fig. 2A). In many fruits, malate and citrate are the two major organic acids. Citrate is the predominant organic acid in citrus (M. Chen et al., 2013) and melon (Tang, Bie, Wu, Yi, & Feng, 2010), while malate is the predominant organic acid in apple (Berüter, 2004) and loquat (F. X. Chen, Liu, & Chen, 2009). In this study, all five organic acids, citrate, malate, quinate, tartrate, and shikimate declined as blueberry fruit matured, Citrate was the most abundant, followed by quinate and malate.

4.1 Sugar Accumulation Associated with Sucrose Metabolism Generally, sucrose is transported into the fruit cells by the symplasmic or apoplastic pathway and accumulates mainly in the vacuole. The sugars transported to the fruit are converted into other forms of soluble sugar. In plant development, INV is of key importance in the generation of hexoses that are subsequently used for metabolism and signaling (Vargas & Salerno, 2010). Sucrose is hydrolyzed by cell-wall INV into glucose and fructose, which may then be taken up via plasmalemma monosaccharide sugar transporters. In fruit e.g. apple and grape, the phloem-unloading is driven by cell-wall INV (Zhang et al., 2006). In the cytosol, sucrose is also hydrolyzed to glucose and fructose by INV. A reversible reaction from 14

sucrose to fructose and UDP-glucose could be catalyzed by SuSy. During blueberry fruit maturation, VcINV (gene.g3367.t1.1 and CUFF.8077.1) were up-regulated, resulting in a sharp increase of glucose and fructose (Fig. 3A, Table 1). FK specifically catalyzes the transfer of a phosphate group from ATP to fructose as the initial step in its utilization. VcFK (CUFF.34746.1 and CUFF.17458.1) were both down-regulated as fruit matured (Fig. 3A, Table 1). Additionally, some sucrose biosynthesis occurs in fruit, even though sucrose is mainly supplied via the phloem. A “futile” cycle of sucrose hydrolysis and re-synthesis could provide greater sensitivity for regulating the net accumulation of sugars and the sucrose/hexose ratio in the cells (Desnoues et al., 2014). SuSy plays an important role in sink strength. In citrus, three non-allelic genes (CitSUS1, CitSUSA and CitSUS2) differ markedly in their molecular structure and potential physiological roles. CitSUS1 and CitSUSA show contrasting expression patterns in edible tissue (Komatsu, Moriguchi, Koyama, Omura, & Akihama, 2002). CitSUS1 supplies materials for cell wall synthesis during the cell division stage, while CitSUSA provides sucrose-phosphate synthase (SPS) which can also be used (UDP-glucose and fructose) for sucrose re-synthesis. In pear, two SuSy isozymes are active during fruit development (Suzuki, Kanayama, & Yamaki, 1996). In this study, VcSuSy (CUFF.9127.1 and CUFF.27618.1) were grouped into SuSyIII together with AthSuSy5/6 of Arabidopsis, OsSuSy5/6 of rice, and MdSUS3 of apple (Fig. 4B). SuSyIII are specifically expressed in the phloem of vascular tissue and is involved in sieve plate callose synthesis (Barratt et al., 2009). VcSuSys were both down-regulated during maturation (Fig. 3A, Table 1), which was consistent with the decrease of MdSUS3 mRNA as apple fruit matured (Tong et al., 2018). SPS catalyzes the synthesis of sucrose-p from the substrates fructose-6p and UDP-glucose, and then sucrose-p is hydrolyzed by SPP to yield free sucrose. Here, up-regulation of VcSPP (CUFF.32787.1, CUFF.14989.1 and CUFF.32787.1) implies a flux of initial products of sugar metabolism towards sucrose re-synthesis and storage (Fig. 3A). 15

PGM facilitates conversion of glucose-1p and glucose-6p, while glucose-6p will enter the pentose phosphate pathway and undergo a series of reactions to yield riboses and/or NADPH. PlasPGM regulates not only photosynthesis through starch synthesis, but also starch degradation. In potato, repression of plasPGM lowers the photosynthesis rate, and changes photosynthetic metabolites in the cytosol and plastid (Lytovchenko, Bieberich, Willmitzer, & Fernie, 2002). CytoPGM participates in sucrose and hexose sugar metabolism and provides intermediates for glycolysis or building blocks for cellular components (Liu, Wu, Fan, Li, & Li, 2006). Here, cytoVcPGM (CUFF.25219.2 and CUFF.46452.1) and plasVocPGM (CUFF.38786.1) were all up-regulated as fruit matured (Fig. 3A), which was consistent with the accumulation of PGM in the ripening fruit of Kiwi (Nardozza et al., 2013) and grape (Niu, Wu, Yang, & Li, 2013). Up-regulation of PGMs in fruit could accelerate the production of hexoses, while down-regulation of FK could control fructose catabolism (Fig. 3A). A dynamic balance is proposed between sucrose re-synthesis and degradation, which results in net products of fructose and glucose in ripening fruit. CytoFBA is a key metabolic enzyme in glycolysis/gluconeogenesis, while plasFBA is involved in the Calvin cycle. During strawberry fruit maturation, cytoFBA continuously increases, while the plasFBA gradually degrades (Schwab, Aharoni, Raab, Pérez, & Sanz, 2001). In green and white stages, plasFBA represents 15 and 8% of the total FBA activity, respectively. In pink and red stages, cytoFBA increases dramatically accounting for 50 and 75% of the total FBA activity, respectively. It was similar to blueberry, plasVcFBA (CUFF.1669.1 and CUFF.48231.1) were down-regulated while cytoVcFBA (CUFF.50615.1) was up-regulated (Fig. 3B; Fig. 4M). PFK phosphorylates fructose-6p into fructose-1,6p, which is a key control step of glycolysis in plants (Mustroph, Sonnewald, & Biemelt, 2007). In blueberry, VcPFK (CUFF.57761.1 and CUFF.32791.1) were up-regulated as fruit matured (Fig. 3B; Table 1), in accordance with an increase in activity of PFK in peach (Desnoues et 16

al., 2014) and citrus (Katz et al., 2011). In addition to PFK, Eno and GAPDH are also two important enzymes in glycolysis. VcENO (CUFF.8875.1 and CUFF.44279.1) and VcGAPDH (CUFF.38881.1 and gene.g31625.t1.1) were up-regulated during maturation. Compared to PFK, FBP catalyzes an interconversion of fructose-6p and fructose-1,6p, which is a reversible step in both glycolysis and gluconeogenesis. However, VcFBP (CUFF.33526.1 and CUFF.46948.1) were both down-regulated as fruit matured (Fig. 3B). The up-regulation of PFKs, cytoFBA, ENO and GAPDH suggests an increasing rate of glycolysis to supply ripening fruit with the necessary energy molecules or intermediates , while down-regulation of FBPs suggests a decreasing rate of gluconeogenesis (a reversible pathway of glycolysis).

4.2 Degradation of Citrate through Acetyl-CoA pathway and GABA shunt Citrate is synthesized via TCA in the mitochondria, and a portion is then transported and stored in the vacuole. Vacuolar citrate is released to the cytosol, and then metabolized into isocitrate by cytosolic aconitase (cytoACO) and into 2-oxoglutarate by NADP isocitrate dehydrogenase (IDH). ACL catalyzes a reversible reaction of citrate to oxaloacetate and acetyl-CoA and controls the shifts between carbohydrates, organic acids, fatty acids, isoprenoids, flavonoids, and other compounds (Fatland et al., 2002). In plants, ACL is a heteromeric enzyme composed of two distinct subunits (ACLα and ACLβ). In lupin, ACLα and ACLβ are encoded by one gene, respectively (Langlade et al., 2002), whereas in Arabidopsis they are encoded by three and two genes, respectively (Fatland et al., 2002). Here, VcACL (CUFF.27347.1, CUFF.28772.1, and CUFF.43438.1) corresponding to α and β subunits were differentially regulated (Fig. 3E; Fig. 4R). Up-regulation during fruit maturation could be associated with citrate degradation through the acetyl-CoA pathway. In the GABA shunt of TCA, glutamate decarboxylase (GAD) catalyzes the 17

reversible conversion of glutamate to GABA and CO2, which is the main route of GABA biosynthesis (Takayama & Ezura, 2015). Glutamate dehydrogenase (GDH) catalyzes the amination of 2-oxoglutarate (synthetic reaction) or the deamination of glutamate (catabolic reaction), and can adjust the direction of the reaction according to the metabolic necessity (Grabowska, Nowicki, & Kwinta, 2011). Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate, ATP and ammonium while glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. In blueberry, both down-regulation of GDH and GGAT could ensure the synthesis of glutamate from 2-oxoglutarate, while down-regulation of GS could block the conversion of glutamate to glutamine and render a flux from glutamate to GABA. Five proteins i.e. AAT (CUFF.17399.1 and CUFF.17397.1), GAD (CUFF.15663.1and CUFF.13757.1), GLT (CUFF.6416.1) involved in the acceleration of the GABA shunt were all up-regulated, while proteins VcGDH (CUFF.17155.1), VcGGAT (CUFF.25529.1) and VcGS (CUFF.20494.2) were all down-regulated (Fig. 3E). Therefore, both the GABA pathway and acetyl-CoA pathway are the main pathways of citrate degradation during blueberry fruit maturation.

4.3 Decrease

of

Malate

Coupling

with

TCA

and

Glycolysis/Gluconeogenesis During the later stages of maturation in grape, sugar metabolism supports the synthesis and accumulation of hexose (Sweetman et al., 2009). Malate has the potential to take over the role of sugar (major carbon source) for energy metabolism and biosynthesis. Malate released from the vacuole fulfills this function through its involvement in gluconeogenesis, respiration, and biosynthesis of secondary compounds (Sweetman et al., 2009). Phosphoenolpyruvate carboxykinase (PCK) requires OAA which is derived from the oxidation of malate by MDH. The cytosolic PCK catalyzes the ATP-dependent 18

decarboxylation of OAA to PEP, which is required for gluconeogenesis in plant (Walker, Battistelli, Moscatello, Técsi, Leegood, & Famiani, 2015). PCK induces a gluconeogenic flux from malate to sugar in the flesh and pericarp of a wide range of fruits. In the cytosol, release of malate leads to change in metabolites and a subsequent rise in gluconeogenesis (Walker et al., 2015). Previous radiolabelling studies have demonstrated that a proportion of the malate in ripening grape pericarp is converted to sugars by gluconeogenesis through the pathway involving PCK (Ruffner, 1982). In tomato fruit, PCK protein and activity appears until the breaker stage (initiation of color change) (Bahrami, Chen, Walker, Leegood, & Gray, 2001). In raspberry, blueberry and red currant fruits, PCK activity is potentially associated with malate degradation (Famiani et al., 2005). In strawberries, the absence of PCK protein and activity is associated with the lack of malate degradation in the fruit (Famiani et al., 2005). In grapes, the up-regulation of PCK indicates a conversion from the use of PEP for respiration pre-veraison to its use for gluconeogenesis in the post-veraison, which coincides with malate degradation (Sweetman et al., 2009). Here, VcPCK (gene.g24276.t1.1) was up-regulated greatly as fruit matured, which suggests that there is a link between malate metabolism and other pathways i.e. gluconeogenesis and respiration (Fig. 3B). Malate is used as a source of carbon for aerobic ethanol fermentation with NAD+ regeneration (Ponce-Valadez & Watkins, 2008). In grape, a large amount of malate released from the vacuoles at veraison may be converted to pyruvate via NAD (P)-ME, and thus could activate a ‘‘PDH bypass” (Sweetman et al., 2009). A “PDH bypass” mediated by PDC and ADH help alleviate cytosol acidity of fruit cells. During fruit maturation, ADH expression is tightly controlled, and increasing ADH activity parallels a shift from a predominance of aldehydes to alcohols (Kalua & Boss, 2009). In grape, fruits produce a higher level of ADH and have increased ADH activity in the late stages of ripening (Sweetman et al., 2009). In peach and apricot, ADH activity and alcohol levels are the highest in an early stage of maturation, and 19

alcohol derivative esters predominate at maturity (Gonzalez-Aguero, Troncoso, Gudenschwager, Campos-Vargas, Moya-Leon, & Defilippi, 2009). In blueberry, ADH

(gene.g1507.t1.1,

CUFF.3210.1,

and

gene.g30667.t1.1)

and

PDC

(CUFF.47532.1) were up-regulated, accompanied by the up-regulation of NADP-ME (CUFF.22988.2) (Fig. 3B). These results suggest a coupling between malate degradation and fermentation which could contribute to flavor development in blueberry. During the initial stage of fruit development, cytosolic malate is transported, and sequestered in the vacuole. Then, cytoMDH synthesizes malate from OAA until the equilibrium is re-established. Mitochondria malate from the TCA cycle is drawn off for accumulation in the vacuole, rather than processed into to OAA by mitoMDH (Sweetman et al., 2009). In peach, MDH activity rises to a high level in early development and gradually declines to lower levels as the fruit matured (Desnoues et al., 2014)

Here, glyoxysomal NAD-MDH (CUFF.18332.1), cytoplasmic

NAD-MDH (CUFF.30072.1), and chloroplastic NADP-MDH (CUFF.24878.1) were all down-regulated (Fig. 3B and E; Fig. 4T). This result suggests that down-regulation of VcMDH reduces the rate of malate synthesis during fruit maturation. During photosynthesis the decarboxylation of malate to pyruvate by NADP dependent chloroplastic ME (NADP-ME) may release CO2 that can then be taken up by RuBisCO of the Calvin Cycle (Drincovich, Casati, & Andreo, 2001). During respiration, the malate can be converted to pyruvate by mitochondria NAD-dependent ME (NAD-ME), or cytosol NADP-ME (Drincovich et al., 2001). In apple, activity of NADP-ME occurs at low levels in the early stage of fruit development, slightly increases in the pre-climacteric stage, and then greatly increases in over-ripe fruit (Berüter, 2004). Thus, NADP-ME is involved in CO2 fixation in young fruit and malate

decarboxylation

during

maturation.

Here,

up-regulation

of

VcME

(CUFF.22988.2) may function in malate decarboxylation and be responsible for a 20

decrease of malate during fruit maturation (Fig. 3B and E).

4.4 Metabolism of Quinate and Shikimate Involved in Shikimate Pahway and Pentose Phosphate Pathway Quinate and shikimate are other major organic acids in blueberry fruit which are synthesized/degraded via the shikimate pathway linked to biosynthesis of the aromatic amino acids (tryptophan, phenylalanine, and tyrosine), and phenolic secondary metabolites and their precursors (Corea et al., 2012). An estimated 20-50% of fixed carbon passes through this pathway in land plants (Corea et al., 2012). DHQ and quinate dehydrogenase (QDH), responsible for biosynthesis of shikimate and quinate respectively, belong to the same gene family, and both diverge into two phylogenetic clades after gene duplication prior to the angiosperm/gymnosperm split. In our study, only two differentially expressed DHQ proteins were identified, and none of the differentially expressed QDH proteins were identified. This was most likely due to the lack of QDH information in the blueberry draft-genome database. The shikimate pathway begins with phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E-4P), followed by seven reactions catalyzed by six enzymes, and finally produces chorismate (Fig. 3C). PEP and E-4P are respectively derived from glycolysis and the non-oxidative branch of the pentose phosphate pathway (Fig. 3), thus connecting the shikimate pathway to central carbon metabolism. In the pentose phosphate pathway of blueberry fruit, five differentially expressed proteins were all down-regulated as fruit matured. The suppressed pathway resulted in the limited E-4P, which subsequently influenced biosynthesis of shikimate and quinate. In the shikimate pathway, four differentially expressed enzymes were up-regulated. However,

DHQ-SDH

(CUFF.35149.1

and

CUFF.35531.1)

were

slightly

down-regulated in the blue phase. Up-regulation of 3-deoxy-7-phosphoheptulonate synthase (DHS) may somewhat stimulate the synthesis of shikimate and quinate. However their accumulation could be largely offset by the up-regulation of 21

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and chorismate synthase (CS) (Fig. 3D). Up-regulation of EPSPS and CS may enhance catabolism of shikimate into other secondary metabolites e.g. amino acids. Hence, a decrease of shikimate and quinate may be due to down-regulation of the pentose phosphate pathway and up-regulation of enzymes involved in their degradation in the shikimate pathway.

5. Conclusion Sugars and organic acids are two of the most important organoleptic traits in fruit and their metabolism is tightly related to the process of fruit maturation. The metabolism and accumulation of sugars and organic acids is linked to sucrose hydrolysis, glycolysis and TCA, as well as the shikimate pathway. During maturation, an increase in sugar was associated with up-regulation of VcSPP, and VcSPS, and VcINV in sucrose metabolism. A decrease in citrate was associated with VcACLY in the acetyl-CoA pathway and with VcGAD and VcGLT in the GABA shunt. A decrease in malate was associated with VcMDH, VcADH and VcPDC in glycolysis and TCA. Additionally, multi-isoforms of enzymes were categorized into different branches and differentially expressed. This result suggests that they have specialized functions in the pathways involved in sugar and acid metabolism. For example, VcADH and VcADHL were respectively divided into ADH and ADH-like groups and oppositely regulated. These enzymes could be developed into novel protein biomarkers for delineating dynamic changes of fruit quality.

22

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26

Caption: Fig. 1. Blueberry fruits at three ripening phases (green, pink and blue) (A) morphologic changes (B) microstructural changes.

Fig. 2. The dynamic changes of the quality traits during fruit maturation. (A) Fruit firmness, size, and maturity index (MI) (B) total sugar content (TSC), total soluble solids (TSS), and titratable acidity (TA) (C) fructose, glucose and sucrose and (D) malate, citrate, quinate, shikimate and tartrate during blueberry maturation. The size of fruit was indicated by its width and height. Bars represented the standard deviations. Compared to the traits in green, those in pink and blue were significantly different (*: P<0.01; **: P<0.001).

Fig. 3. Dynamic regulation of enzymes involved in soluble sugar and organic acid during blueberry fruit maturation. The relative abundance of enzyme proteins is shown in these tables. Accumulation of soluble sugar was involved in (A) Starch and sucrose metabolism and (B) Glycolysis/Gluconeogenesis, while metabolism of organic acid was involved in (C) Pentose phosphate pathway, (D) Shikimate pathway, and (E) Citrate cycle (TCA cycle). Only differentially expressed proteins were shown, and the identified enzymes are indicated by red , while the unidentified enzymes are indicated by violet . 6PGL: 6-phosphogluconolactonase; AAT: Bifunctional aspartate aminotransferase

and

glutamate/aspartate-prephenate

aminotransferase;

ACO:

aconitase; ACL: ATP-citrate synthase; ADH: Alcohol dehydrogenase; INV: Invertase; ALDH: Aldehyde dehydrogenase; DLST: Dihydrolipoyllysine-residue succinyltransferase; ENO: Enolase; FBA or ALDO: Fructose-bisphosphate aldolase; FBP: Fructose-1,6-bisphosphatase; FK: Fructokinase; G6PD3: Glucose-6-phosphate dehydrogenase 3; GAD: Glutamate decarboxylase; GDH: Glutamate dehydrogenase; GS: Glutamine synthetase; Glutamate

synthase

GGAT: Glutamate--glyoxylate aminotransferase; GLT:

(NADPH);

GAPDH: 27

Glyceraldehyde

3-phosphate

dehydrogenase; GPI: Glucose-6-phosphate isomerase; HK: Hexokinase; IDH: Isocitrate dehydrogenase; MDH: Malate dehydrogenase; OGDH: Oxoglutarate dehydrogenase;

PCK:

Phosphoenolpyruvate

carboxykinase;

PDC:

Pyruvate

decarboxylase; PFK: Phosphofructokinase; PGK: Phosphoglycerate kinase; PGM: Phosphoglucomutase; PK: Pyruvate kinase; RPI: Ribose-5-phosphate isomerase; SDH: Sorbitol dehydrogenase; SPP: Sucrose-phosphatase; SPS: sucrose-phosphate synthase; SuSy: Sucrose synthase; TAL: transaldolase; TKT: L-xylulose

5-phosphate

3-epimerase;

SK:

Transketolase; UlaE:

Shikimate

kinase;

DHS:

3-deoxy-7-phosphoheptulonate synthase; DQD: 3-dehydroquinate dehydratase; aroA: 3-phosphoshikimate 1-carboxyvinyltransferase; DHQS: Dehydroquinate Synthase; QDH:

quinate

dehydrogenase;

SDH:

shikimate

phosphoenolpyruvate carboxylase; TRE1: trehalase 1; SS3:

dehydrogenase;

PEPC:

starch synthase 3; PHD:

pyruvate dehydrogenase.

Fig. 4. Phylogenetic analysis of the differently expressed enzyme proteins involved in sugar and acid metabolism. (A) Invertase (INV) (B) Sucrose synthase (SuSy) (C) Hexokinase (HK) (D) Fructokinase (FK) (E) Trehalose-phosphate synthase (TPS) (F) Alpha-glucosidase (AGlu) (G) Beta-glucosidase (BGLU) (H) Phosphoglucomutase (PGM) (I) Fructose-1,6-bisphosphatase (FBP) (J) Phosphofructokinase (PFK) (K) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (L) Enolase (ENO) (M) Fructose-bisphosphate aldolase (FBA) (N) Pyruvate kinase (PK) (O) Alcohol dehydrogenase

(ADH)

Phosphoenolpyruvate

and

Alcohol

carboxylase

dehydrogenase-like

(PEPC)

(Q)

(ADHL)

(P)

Glucose-1-phosphate

adenylyltransferase (GlgC) (R) ATP citrate (pro-S)-lyase (ACL) (S) Aldehyde dehydrogenase

(NAD+)

(ALDH)

(T)

Malate

dehydrogenase

(MDH)

(U)

6-phosphogluconolactonase (6PGL). The proteins from highbush blueberry (V. corymbosum) are shown in bold and followed by the database accessions.

28

Highlights: 

Dynamic changes of enzymes in sugar and acid biosynthesis were investigated.



Association between enzymes and major sugars and organic acids was analyzed.



Divergence and different regulation of multi-isoforms suggested their specific functions.

A

Green

Pink

13.58±1.13

12.22±0.27

8.99±0.29

11,89±0.34

Green

Blue

15.43±0.72

17.25±1.11

A

5.00

Firmness (Kg/cm2) Height (mm)

Width (mm) MI (%)

4.00

3 3.00 2 8.00 2

3.00

3.00 1 8.00

2.00

TSC (mg/g FW)

1 3.00

1.00

**

29

**

0.00 Green

Pink

Blue

8

44.00

3

24.00

.00 .00

2.00 4.00

A 42.00 32.00

Fructose (mg/g FW) Sucrose (mg/g FW)

Glucose (mg/g FW)

** **

22.00

**

Malate (μg/g F Citrate (μg/g F

12400.00

**

9400.00 6400.00

12.00

3400.00

2.00

400.00

0.50

*

190.00 130.00

0.25

70.00 10.00 Green

Pink

C

PtINV(XP_002306166.1) Blue TcINV(EOX97770.1) CsINV(XP_004144831.1) VvINV(XP_002276670.1) VcINV(CUFF.47310.2) TcINV(EOY29921.1) PtINV(XP_002309496.1) VvINV(XP_002279133.2) VvINV(XP_002278880.2) CsINV(XP_004141427.1)

s

a l

l l a a s n t p

p

p n a

p

l

p

E

TPS11

SlTPS(NP_001233896.1)

TPS7

VvTPS7(XP_002268174.1) RcTPS7(XP_002522255.1) VvTPS7(XP_002268174.1) VvTPS(CAN67058.1) VcTPS(CUFF.33913.1) 30 FvTPS11(XP_004309955.1) PpTPS11(EMJ28208.1) CsTPS(AGD98700.1) VcTPS(CUFF.10131.1) GmTPS(XP_003549506.1)

D

HK-like

AtHKL1(NP_001322008.1) AthHKL3(NP_195497.1)

Green

Mitochondrial

AthHXK3(NP_001321728.1) LeHK4(AAY60842.1) AthHK2(NP_001077921.1) AthHK1(NP_194642.1) LeHK3(AY60841.1) VcHK(CUFF.22630.1) VcHK(CUFF.16539.1) LeHK2(AAG35735.1) LeHK1(Q8H0Q2) AtHKL2(NP_188639.2)

Plastid

C

6.000 B 4.000 2.000 0.000

Acid invertase

VcINV(CUFF.8077.1) DcINV(CAA53098.1) DcINV(CAA53099.1) AtINV(P80065.2) CcINV(ABI17894.1) VcINV(gene.g3367.t1.1) RhINV(AFU25742.1)

Alkaline/neutral invertase

A

t

0.00

F

SuSyII SuSyIII

AthSuSy2(Q00917) MdSuSy5L(XP_008362572.1) MdSuSy7L(XP_008353901.1) MdSuSy5(XP_008381393.1) VcSuSy(CUFF.27618.1) AthSuSy6(NP_001318683.1) VcSuSy(CUFF.9127.1) AthSuSy5(NP_177480.1) OsSusy5(H6TFZ4.1) OsSusy6(Q6K973.1)

SuSyI

.

ZmSuSy2 (NP_001105411.2) ZmSuSy1 (NP_001105323.1) AthSuSy3(NP_192137.1) AthSuSy1(NP_001031915.1) CitSusy2(BAA88902.1) CitSusy1(BAA88905.1) PsSusA(O24301) CitSusyA(BAA88904.1) ZmSuSy2 (NP_001105194.2) AthSuSy2(NP_001331823.1)

Plastidic

VcFK(CUFF.34746.1) SlyLeFK4(AY099454) AthFK(NP_001318782.1) SlyLeFK1(U64817.1) VcFK(CUFF.17458.1) SlyLeFK3(AY323226) AthFK(NP_564875.2) AthFK(NP_172093.1) AthFK(NP_172092.1) AthFK(NP_180697.1)

cytosolic

AthFK(NP_191507.1) SlyLeFK2(AAB57734.1) AthFK(NP_564875.2)

BGlu-like g

J

BGlu44 Chloroplastic FBPII Chlorop

VvFBP(CBI38227.3) VvFBP(XP_002270826.2) PtFBP(XP_002323933.3) RcFBP(XP_002532766.1) VcFBP(CUFF.33526.1) 31 TcFBP(EOX98655.1) FrcpFBPII(ABW38331.1) AtcpFBPII(NP_201243.1) OscpFBPII(NP_001058297.1) AtcpFBPI(P22418.2) AtcpFBPI(NP_190973.1) VvFBP(CAN62980.1)

H

AGlu2-like

I

AGlu

VcBGlu(CUFF.908.1) SlBGluL(XP_004247424.1) VvBGlu(XP_002268147.1) GmBGlu44(XP_003534146.1) GmBGlu44(XP_003528968.1) CaBGlu44L(XP_004512738.1) CsBGluL(XP_004161840.1) TcBGlu(EOY21180.1) VcBGlu(CUFF.38011.1)

BGlu

G

TcBGlu(EOY33795.1) TcAGlu(EOY20266.1) TcBGlu(EOY33800.1) VcAGlu(CUFF.39742.1) PtBGlu(XP_002313393.1) VcAGlu(CUFF.39742.2) VcBGlu(CUFF.14636.2) FvAGluL(XP_004300234.1) VcBGluL(CUFF.19253.1) PpAGluL(EMJ11593.1) CsBGluL(XP_004137360.1) VcAGluL(CUFF.39718.1) SlBGluL(XP_004241371.1) VvAGluL(CBI37476.3) VvBGluL(XP_002278377.1) CaAGluL(XP_004512367.1) VvBGluL(XP_002278363.1) MtAGluL(XP_003612579.1) VvBGluL(CBI24412.3)

K

Cytosolic PFK3/6 PFK2

TcPFK3(EOY29818.1) TcPFK3(EOY29820.1) PpPFK(EMJ24581.1) GmPFK6(XP_003526507.1) GmPFK6(XP_003522720.1) VcPFK(CUFF.32791.1) PtPFK(XP_002329965.1) RcPFK(XP_002525025.1) FvPFK2(XP_004293419.1) VvPFK2(XP_003635479.1)

Plastidic

AthPGMpl2(AAP37735.1) VcPGM(CUFF.38786.1) AthPGMpl1(NP_199995.1) AthPGMcyt2(NP_173732.1) AthPGMcyt1(NP_001154464.1) VcPGM(CUFF.25219.2) VcPGM(CUFF.46452.1)

TcPFK2(EOY29039.1) VcPFK(CUFF.57761.1)

Cytosolic Chloroplastic

CrGAPDH(EOA38185.1) VcGAPDH(CUFF.38881.1) VvGAPDH(CBI14856.3) PbGAPDH(AFG28407.1) PpGAPDH(EMJ23673.1) VcGAPDH(gene.g31625.t1.1) RcGAPDH(XP_002513328.1) PtGAPDH(XP_002312235.1) TcGAPDH(EOY14200.1) PpGAPDH(EMJ15917.1) SlGAPCP1(XP_004248314.1)

O

Plastic

AthFBA3(NP_178224)

32

Group1 PlasticGroup2Cytosolic

M

VcENO(CUFF.44279.1) FvENO(XP_004308163.1) PpENO(EMJ19205.1) AtFBA7(NP_194382) VvENO(XP_002267091.1) AthFBA5(NP_194383) GmENO(XP_003548246.1) VcFBA(CUFF.48231.1) GaENO(XP_004514974.1) AthFBA8(NP_190861) VcENO(CUFF.8875.1) AthFBA6(NP_181187) PtENO(XP_002322420.1) AthFBA4(NP_568127) PpENO(EMJ03254.1) VcFBA(CUFF.50615.1) AthFBA2(NP_568049) AthFBA1(NP_565508) VcFBA(CUFF.1669.1)

N

Group3 PEPC3 PEPC4

33

ChPEPC3(ABV80356.1) TcPEPC3(EOY13253.1) VvPEPC3(XP_002285441.1) CsPEPC3(XP_004146028.1) VcPEPC(CUFF.45541.1) AtPEPC2(Q9FV65.1) RcPEPC4(ABR29877.1) RcPEPC4(XP_002524382.1) TcPEPC4(EOY02184.1) VcPEPC(CUFF.8537.1)

Group2

GmPK(XP_003521432.1) CsPK(XP_004143296.1) SlPK(XP_004249465.1) VcPK(CUFF.22005.1) VcPK(CUFF.21999.1) VcPK(CUFF.14751.1) OsPK(ABA96475.1) OgPK(CBX24455.1) RcPK(XP_002528639.1) PsPK(ABR16677.1) VcPK(CUFF.11685.2) TcPK(EOY09026.1) VvPK(XP_003632737.1) RcPK(XP_002529161.1)

Group1

FvPK(XP_004307020.1) CaPK(XP_004491929.1) TcPK(EOY08081.1) PtPK(XP_002326407.1) VvPk(XP_002283911.1) GmPK(XP_003542538.1) GmPK(XP_003535269.1)

ADH

GmADH-like(XP_003545496.1) GmADH-like(XP_003545495.1) AtADH2(P14674.1) AtADH2(P14675.1) SlADH2(NP_001234099.1) NtADH-like(AAT40104.1) PtADH3(AAO74899.1) LsADH(BAA07911.1) PpADH(EMJ01137.1) VcADH(CUFF.3210.1) VcADH(gene.g30667.t1.1) DkADH(AEB71537.1) TcPK(EOY18967.1) DkADH(AGA15793.1) VvPk(XP_002263319.1) VcADH(gene.g1507.t1.1) VcPK(CUFF.11308.1)

ADH-like

VcADH-like(CUFF.31687.1) PtADH-like(XP_002324694.1) VvADH-like(CBI16175.3) VvADH-like(XP_002281275.2) VvADH-like(XP_002281265.1) OeADH(AEQ04839.1) VcADH-like(CUFF.6192.1) RcADH-like(XP_002510634.1) VvADH-like(XP_002285916.1) PpADH(EMJ24243.1)

T

ALDH family3

Vc6PGL(CUFF.41390.1) Sl6PGL1(XP_004239036.1) Pt6PGL(XP_002311348.1) Pt6PGL(XP_002316080.1) Rc6PGL1(XP_002512062.1) Tc6PGL1(EOY01656.1) Pt6PGL(XP_002321375.1) Pt6PGL(XP_002317815.1) Fv6PGL4(XP_004307920.1) SoACLβ1(AFO64345.1) Zm6PGL(ACN33806.1) AthACLβ2(NP_001332247.1) 6PGL1

U

ALDH family2

CaALDH(XP_004502484.1) VcALDH(CUFF.47450.1)

Large subunit

S

LcALDH(AAO72532.1) MtALDH(XP_003608978.1) GmALDH(XP_003549631.1) RcALDH(XP_002511424.1) FvALDH(XP_004298932.1) VcALDH(CUFF.36657.1) CaALDH(XP_004498346.1) MtALDH(ACJ85852.1) TcALDH(EOY16341.1) CaALDH(XP_004502482.1)

Small subunit

Q

R

PtGlgC(XP_002321214.1) VvGlgC(XP_002263255.1) NtGlgC(ABD60582.1) VcGlgc(CUFF.21624.1) VvGlgC(CBI37674.3) VvGlgC(XP_002283855.1) VcGlgC(CUFF.31855.1) PtGlgC(XP_002313036.1) ShGlgC(AAD56405.1) ShGlgC(ABC26923.1)

ACLα

AthACLα1(NP_001184954.1) VcACL(CUFF.27347.1) CitACLα2(Cs7g08950.2) VcACL(CUFF.43438.1) CitACLα1(Cs6g01210.2) AthACLα3(NP_172414.1)

ACLβ

6PGL4

Sl6PGL4(ACN33806.1) VcACL(CUFF.28772.1) Vc6PGL(CUFF.41390.1) CitACLβ1(Cs9g02230)

SoACLα1(AFN22056.1)

Chloroplastic Cytoplasmic

RcMDH(XP_002532626.1) VvMDH(CAN77649.1) VcMDH(CUFF.30072.1) TcMDH(EOX96367.1) CsMDH(XP_004133776.1)

Glyoxysomal

34

VcMDH(CUFF.18332.1) VvMDH(XP_003631692.1) TcMDH(EOX97285.1) PpMDH(EMJ01799.1) RcMDH(XP_002522037.1) VvMDH(XP_002263670.2) FtMDH(AAA87008.1) AtMDH(P46489.1) VcMDH(CUFF.24878.1) Mt(XP_003638165.1)

35

Pathway

Table 1 Dynamic changes of key enzymes involved in metabolism of major sugars and acids during blueberry fruit maturation Unique Enzyme (KO)

Protein accession

Green

Pink

Blue

G/P P/B G/B

VcAMY(CUFF.31900.1)

14.80

20.06

16.20

2

0.90 ± 0.11 0.82 ± 0.05

1.31 ± 0.11

*

VcAGlu(CUFF.39718.1)

21.92

39.35

19.40

5

0.62 ± 0.05 0.83 ± 0.03

1.70 ± 0.40

*

*

VcAGlu(CUFF.39742.1)

29.51

106.63

35.30

9

0.69 ± 0.02 0.90 ± 0.02

1.39 ± 0.02

*

*

VcAGlu(CUFF.39742.2)

17.14

63.79

24.10

4

0.66 ± 0.03 0.91 ± 0.05

1.47 ± 0.08

*

*

VcBAM(CUFF.21062.4)

66.57

323.31

33.70

13

0.65 ± 0.02 1.15 ± 0.03

1.21 ± 0.08 *

VcBGLu(CUFF.908.1)

13.05

17.18

17.60

3

1.30 ± 0.01 0.78 ± 0.02

0.93 ± 0.05 *

VcBGLu(CUFF.14636.2)

32.20

20.72

8.20

2

0.77 ± 0.03 1.02 ± 0.10

1.24 ± 0.09

VcBGLu(CUFF.19253.1)

51.80

108.61

25.10

11

0.76 ± 0.04 0.88 ± 0.03

1.40 ± 0.10

*

VcBGLu(CUFF.38011.1)

47.19

118.69

27.80

15

1.01 ± 0.02 0.80 ± 0.02

1.26 ± 0.06

*

VcFK(CUFF.34746.1)

35.80

6.62

15.30

1

1.29 ± 0.02 1.02 ± 0.04

0.74 ± 0.05

VcFK(CUFF.17458.1)

23.73

15.12

25.40

2

1.33 ± 0.10 1.06 ± 0.02

0.63 ± 0.16

VcGBE1(CUFF.7300.1)

87.85

224.10

25.50

17

1.51 ± 0.05 0.88 ± 0.03

0.71 ± 0.04 *

VcGlgC(CUFF.31855.1)

31.32

8.24

8.00

1

0.62 ± 0.09 0.81 ± 0.04

1.60 ± 0.08

*

VcGlgC(CUFF.21624.1)

45.48

13.25

4.10

2

0.96 ± 0.03 0.81 ± 0.03

1.29 ± 0.12

*

VcHK(CUFF.22630.1)

27.79

148.76

43.30

10

1.01 ± 0.04 0.77 ± 0.04

1.26 ± 0.10

*

*

VcHK(CUFF.16539.1)

53.74

263.85

26.00

9

0.97 ± 0.01 0.69 ± 0.02

1.37 ± 0.03

*

*

VcINV(gene.g3367.t1.1)

51.82

323.31

32.20

12

0.64 ± 0.01 1.11 ± 0.00

1.21 ± 0.01 *

VcINV(CUFF.8077.1)

65.08

127.90

14.70

9

0.72 ± 0.01 0.84 ± 0.03

1.45 ± 0.06

VcINV(CUFF.47310.2)

26.10

80.34

32.20

3

0.68 ± 0.07 1.00 ± 0.05

1.35 ± 0.08

VcISA(CUFF.2309.1)

47.82

8.58

2.20

1

1.27 ± 0.09 1.09 ± 0.09

0.69 ± 0.02

VcPGM(CUFF.25219.2)

27.36

53.60

23.10

2

0.75 ± 0.05 0.87 ± 0.02

1.44 ± 0.08

VcPGM(CUFF.46452.1)

13.51

177.60

69.70

8

0.76 ± 0.03 0.94 ± 0.05

1.32 ± 0.08

VcPGM(CUFF.38786.1)

29.89

5.88

3.70

1

0.41 ± 0.03 0.86 ± 0.06

1.75 ± 0.10 *

Glycogen phosphorylase (PYG) [EC:2.4.1.1]

VcPYG(CUFF.30589.1)

96.56

208.23

27.50

20

1.39 ± 0.03 0.84 ± 0.01

0.86 ± 0.02 *

Sucrose-phosphatase (SPP)

VcSPP(CUFF.32787.1)

48.12

271.12

38.60

19

0.73 ± 0.02 0.98 ± 0.01

1.32 ± 0.01

VcSPS(CUFF.14989.1)

118.51

278.25

22.40

26

0.67 ± 0.01 0.84 ± 0.01

1.51 ± 0.08

VcSuSy(CUFF.9127.1)

103.32

22.52

6.60

4

1.62 ± 0.09 0.73 ± 0.03

0.71 ± 0.20 *

VcSuSy(CUFF.27618.1)

64.17

45.23

12.80

3

1.41 ± 0.12 0.72 ± 0.07

0.92 ± 0.26 *

Trehalose-phosphate synthase (TPS) [EC:2.4.1.15 3.1.3.12]

VcTPS(CUFF.10131.1)

97.59

148.92

20.20

12

0.73 ± 0.02 0.88 ± 0.03

1.43 ± 0.02

*

*

VcTPS(CUFF.33913.1)

94.56

25.81

4.70

3

1.64 ± 0.03 0.80 ± 0.02

0.62 ± 0.08

*

*

Acetyl-CoA synthetase (ACSS) [EC:6.2.1.1]

VcACSS(CUFF.4807.2)

17.71

57.05

42.00

5

0.75 ± 0.06 0.93 ± 0.08

1.35 ± 0.09

Aldehyde dehydrogenase (NAD+) (ALDH) [EC:1.2.1.3]

VcALDH(CUFF.36657.1)

57.41

323.31

49.20

22

0.58 ± 0.01 0.77 ± 0.04

1.70 ± 0.04

*

VcALDH(CUFF.47450.1)

34.42

53.79

16.80

3

0.98 ± 0.04 0.82 ± 0.05

1.24 ± 0.02

*

VcADH(gene.g1507.t1.1)

49.40

44.74

10.70

2

0.68 ± 0.04 0.95 ± 0.01

1.41 ± 0.03

VcADH(CUFF.3210.1)

13.60

27.92

47.50

3

0.67 ± 0.04 0.90 ± 0.06

1.31 ± 0.14

VcADH(gene.g30667.t1.1)

29.41

49.70

26.70

3

0.63 ± 0.04 0.90 ± 0.06

1.49 ± 0.10

*

*

VcADHL(CUFF.31687.1)

37.00

33.56

15.00

5

1.29 ± 0.04 1.07 ± 0.05

0.71 ± 0.06

*

*

VcADHL(CUFF.6192.1)

35.08

32.06

13.10

4

1.28 ± 0.04 1.03 ± 0.00

0.74 ± 0.02

*

*

VcFBA(CUFF.1669.1)

42.59

323.31

49.20

17

1.52 ± 0.01 0.98 ± 0.03

0.53 ± 0.01 *

*

*

Fructose-bisphosphate aldolase (FBA) [EC:4.1.2.13]VcFBA(CUFF.48231.1)

38.40

165.24

31.70

7

1.28 ± 0.06 0.95 ± 0.04

0.77 ± 0.11

*

VcFBA(CUFF.50615.1)

38.63

323.31

64.00

18

0.80 ± 0.02 0.96 ± 0.03

1.28 ± 0.01

*

VcINV(gene.g3367.t1.1)

51.82

323.31

32.20

12

0.64 ± 0.01 1.11 ± 0.00

1.21 ± 0.01 *

VcINV(CUFF.8077.1)

65.08

127.90

14.70

9

0.72 ± 0.01 0.84 ± 0.03

1.45 ± 0.06

VcINV(CUFF.47310.2)

26.10

80.34

32.20

3

0.68 ± 0.07 1.00 ± 0.05

1.35 ± 0.08

VcENO(CUFF.8875.1)

48.26

323.31

66.30

17

0.79 ± 0.01 0.86 ± 0.04

1.39 ± 0.03

*

VcENO(CUFF.44279.1)

40.47

323.31

44.40

7

0.93 ± 0.02 0.84 ± 0.03

1.27 ± 0.10

*

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [EC:1.2.1.12]

VcGAPDH(CUFF.38881.1)

37.09

176.42

54.50

7

0.82 ± 0.01 0.92 ± 0.02

1.31 ± 0.06

VcGAPDH(gene.g31625.t1.1) 22.00

48.60

25.00

3

0.67 ± 0.05 0.84 ± 0.04

1.52 ± 0.16

*

*

Glucose-6-phosphate 1-epimerase [EC:5.1.3.15]

VcG6PE(CUFF.35168.1)

16.81

40.40

20.50

3

1.36 ± 0.05 1.02 ± 0.04

0.65 ± 0.06

*

*

VcFBP(CUFF.33526.1)

43.62

159.90

24.90

8

1.24 ± 0.03 1.02 ± 0.01

0.78 ± 0.02

*

*

VcFBP(CUFF.46948.1)

44.86

321.35

38.00

12

1.53 ± 0.02 0.90 ± 0.04

0.63 ± 0.04 *

*

*

Alpha-glucosidase (AGlu) [EC:3.2.1.20]

Beta-amylase (BAM) [EC:3.2.1.2]

Beta-glucosidase (BGLU) [EC:3.2.1.21]

Fructokinase (FK)

Starch and sucrose metabolism

Coverage [%] peptides

Alpha-amylase (AMY) [EC:3.2.1.1]

[EC:2.7.1.4]

1,4-alpha-glucan branching enzyme (GBE1) [EC:2.4.1.18] Glucose-1-phosphate adenylyltransferase (GlgC) [EC:2.7.7.27] Hexokinase (HK) [EC:2.7.1.1]

Invertase (INV) [EC:3.2.1.26]

Isoamylase (ISA) [EC:3.2.1.68]

Phosphoglucomutase (PGM) [EC:5.4.2.2]

[EC:3.1.3.24]

Sucrose-phosphate synthase (SPS) [EC:2.4.1.14] Sucrose synthase (SuSy) [EC:2.4.1.13]

Alcohol dehydrogenase (ADH)

[EC:1.1.1.1]

Alcohol dehydrogenase-like (ADHL) Glycolysis/Gluconeogenesis

MW [kDa] Score

Invertase (INV) [EC:3.2.1.26]

Enolase (ENO) [EC:4.2.1.11]

Fructose-1,6-bisphosphatase [EC:3.1.3.11]

36

*

* *

* *

* * *

* *

* *

*

* *

*

* * *

*

* * *

* *

* *

* *

* * *

*

VcHK(CUFF.22630.1)

27.79

148.76

43.30

10

1.01 ± 0.04 0.77 ± 0.04

1.26 ± 0.10

*

VcHK(CUFF.16539.1)

53.74

263.85

26.00

9

0.97 ± 0.01 0.69 ± 0.02

1.37 ± 0.03

*

VcPFK(CUFF.32791.1)

23.51

42.16

6.50

1

0.73 ± 0.07 0.99 ± 0.05

1.31 ± 0.09

*

VcPFK(CUFF.57761.1)

23.07

82.12

33.30

8

0.82 ± 0.02 0.96 ± 0.01

1.25 ± 0.05

*

Pyruvate decarboxylase (PDC) [EC:4.1.1.1]

VcPDC(CUFF.47532.1)

64.90

323.31

35.60

21

0.58 ± 0.02 1.09 ± 0.01

1.32 ± 0.07 *

Phosphoenolpyruvate carboxykinase (PCK) [EC:4.1.1.49]

VcPCK(gene.g24276.t1.1)

73.64

323.31

58.30

34

0.41 ± 0.02 0.87 ± 0.05

1.67 ± 0.09 *

*

*

Phosphoglycerate kinase

VcPGK(CUFF.6095.1)

50.51

323.31

61.40

21

1.44 ± 0.01 0.96 ± 0.01

0.65 ± 0.02 *

*

*

VcPK(CUFF.11308.1)

57.63

128.48

28.70

6

1.28 ± 0.07 0.81 ± 0.03

0.93 ± 0.12 *

*

VcPK(CUFF.14751.1)

57.39

246.19

35.70

7

0.86 ± 0.01 0.80 ± 0.02

1.36 ± 0.12

*

*

VcPK(CUFF.11685.2)

52.03

145.97

19.50

6

0.98 ± 0.09 0.78 ± 0.01

1.28 ± 0.10

*

*

VcPK(CUFF.21999.1)

14.95

38.86

44.90

4

0.99 ± 0.02 0.73 ± 0.01

1.21 ± 0.03

*

*

VcPK(CUFF.22005.1)

23.97

61.89

61.00

2

0.81 ± 0.10 0.74 ± 0.11

1.49 ± 0.39

*

*

VcACL(CUFF.27347.1)

46.73

111.02

24.30

5

0.33 ± 0.02 0.50 ± 0.08

2.16 ± 0.47 *

*

*

VcACL(CUFF.28772.1)

65.82

323.31

42.80

13

0.82 ± 0.01 0.94 ± 0.05

1.27 ± 0.10

VcACL(CUFF.43438.1)

46.44

49.97

17.30

5

0.77 ± 0.01 0.60 ± 0.08

1.61 ± 0.16

*

*

Dihydrolipoyllysine-residue succinyltransferase (DLST) [EC:2.3.1.61]

VcDLST(CUFF.48155.1)

51.32

25.04

20.10

2

0.35 ± 0.03 0.68 ± 0.13

2.07 ± 0.91 *

*

*

Isocitrate dehydrogenase (IDH) [EC:1.1.1.42]

VcIDH(CUFF.43700.2)

46.47

323.31

50.70

16

0.72 ± 0.02 0.98 ± 0.03

1.32 ± 0.05

Malate dehydrogenase (MDH) (NAD-MDH) [EC:1.1.1.37]

VcMDH(CUFF.30072.1)

35.85

109.55

22.30

3

1.18 ± 0.08 1.03 ± 0.05

0.78 ± 0.05

VcMDH(CUFF.18332.1)

37.43

128.44

22.80

7

1.44 ± 0.03 0.99 ± 0.04

0.65 ± 0.02

VcMDH(CUFF.24878.1)

7.18

47.61

51.60

5

1.34 ± 0.03 1.01 ± 0.04

0.71 ± 0.05

*

VcME(CUFF.22988.2)

42.22

67.19

26.80

7

0.73 ± 0.04 1.07 ± 0.05

1.20 ± 0.06

*

Hexokinase (HK) [EC:2.7.1.1]

Phosphofructokinase (PFK) [EC:2.7.1.11]

Pyruvate kinase (PK)

(PGK) [EC:2.7.2.3]

[EC:2.7.1.40]

TCA cycle

ATP citrate (pro-S)-lyase (ACL) [EC:2.3.3.8]

Malate dehydrogenase (NADP+) (NADP-MDH)[EC:1.1.1.82] NADP-dependent malic enzyme (NADP-ME)[EC:1.1.1.38] Oxoglutarate dehydrogenase(OGDH)

[EC:1.2.4.2] VcOGDH(CUFF.5203.1)

* * *

115.93

323.31

35.90

18

1.06 ± 0.01 0.70 ± 0.02

1.27 ± 0.02 *

*

73.64

323.31

58.30

34

0.41 ± 0.02 0.87 ± 0.05

1.67 ± 0.09 *

*

Phosphoenolpyruvate carboxylase (PEPC)[EC:4.1.1.31]

VcPEPC(CUFF.45541.1)

110.42

323.31

60.00

22

1.11 ± 0.01 0.71 ± 0.02

1.19 ± 0.05 *

*

VcPEPC(CUFF.8537.1)

51.80

132.16

28.60

7

1.33 ± 0.03 0.70 ± 0.01

0.95 ± 0.07 *

Bifunctional aspartate aminotransferase and glutamate/aspartate-prephenate aminotransferase (PAT;AAT)[EC:2.6.1.1 2.6.1.78 2.6.1.79] Glutamate--glyoxylate aminotransferase (GGAT) [EC:2.6.1.4 2.6.1.2 2.6.1.44]

VcAAT(CUFF.17399.1)

17.46

323.31

52.40

6

0.53 ± 0.01 0.96 ± 0.05

1.53 ± 0.15 *

VcAAT(CUFF.17397.1)

19.58

92.57

33.70

4

0.62 ± 0.03 0.97 ± 0.12

1.42 ± 0.17 *

VcGGAT(CUFF.25529.1)

53.34

158.27

26.00

11

1.70 ± 0.07 0.95 ± 0.10

0.41 ± 0.06

57.26

22.35

3.30

1

0.81 ± 0.09 0.90 ± 0.01

1.33 ± 0.29

30.07

225.87

35.30

8

0.76 ± 0.03 0.92 ± 0.02

1.32 ± 0.07

225.75

323.31

25.70

46

0.46 ± 0.01 0.78 ± 0.05

1.77 ± 0.03 *

44.30

290.75

31.40

11

1.34 ± 0.15 0.92 ± 0.02

0.78 ± 0.05

Glutamate decarboxylase (NADPH/NADH) (GAD) VcGAD(CUFF.15663.1) [EC:4.1.1.15] VcGAD(CUFF.13757.1) Glutamate synthase (NADPH) (GLT) [EC:1.4.1.13 VcGLT(CUFF.6416.1) 1.4.1.14] Glutamate dehydrogenase (NAD(P)+) (GDH) VcGDH(CUFF.17155.1) [EC:1.4.1.3]

*

*

*

* *

* * *

* *

Glutamine synthetase (GS) [EC:6.3.1.2]

VcGS(CUFF.20494.2)

19.68

5.97

4.50

1

1.45 ± 0.09 1.00 ± 0.10

0.60 ± 0.09

Glucose-6-phosphate dehydrogenase (G6PD)[EC:1.1.1.49]

VcG6PD3(CUFF.1720.1)

14.34

6.49

6.90

1

1.21 ± 0.09 1.06 ± 0.05

0.78 ± 0.03

Vc6PGL(CUFF.41390.1)

29.14

13.56

6.00

2

1.48 ± 0.07 1.06 ± 0.11

0.51 ± 0.02

*

*

Vc6PGL(CUFF.32421.1)

28.52

142.22

41.00

10

1.20 ± 0.05 1.13 ± 0.03

0.72 ± 0.03

*

*

22.47

60.03

31.10

6

1.24 ± 0.04 1.05 ± 0.04

0.79 ± 0.05

*

24.13

223.40

50.20

7

1.22 ± 0.02 1.05 ± 0.02

0.77 ± 0.02

*

Pentose phosphate pathway

GABA shunt

*

VcPCK(gene.g24276.t1.1)

Phosphoenolpyruvate carboxykinase (ATP)(PCK) [EC:4.1.1.49]

6-phosphogluconolactonase

(6PGL)[EC:3.1.1.31]

Ribose-5-phosphate isomerase

(RPI) [EC:5.3.1.6] VcRPI(CUFF.32999.1)

Transketolase (TKT) [EC:2.2.1.1]

VcTKT(CUFF.27889.1)

±

Shikimate pathway

*

3-phosphoshikimate 1-carboxyvinyltransferase (EPSPS) [EC:2.5.1.19]

±

*

*

*

±

VcEPSPS(CUFF.47349.1)

55.94

323.31

33.00

8

0.61 ± 0.02 0.89 ± 0.05

1.50 ± 0.03

*

*

VcEPSPS(CUFF.27788.1)

28.01

100.34

55.40

7

0.69 ± 0.06 0.84 ± 0.03

1.49 ± 0.07

*

*

VcDHQ-SDH(CUFF.35149.1) 26.54

116.09

32.80

7

0.58 ± 0.02 1.39 ± 0.02

0.98 ± 0.25 *

*

68.39

55.00

5

0.50 ± 0.03 1.38 ± 0.05

1.11 ± 0.07 *

*

3-dehydroquinate dehydratase / shikimate dehydrogenase (DHQ-SDH)[EC:4.2.1.10 1.1.1.25] VcDHQ-SDH(CUFF.35531.1) 8.87 3-deoxy-7-phosphoheptulonate synthase (DHS) [EC:2.5.1.54]

VcDHS(CUFF.42264.1)

30.88

98.31

40.90

5

0.69 ± 0.05 0.87 ± 0.07

1.44 ± 0.17

Chorismate synthase (Cs) [EC:4.2.3.5]

VcCs(CUFF.7532.1)

45.54

277.86

39.70

17

0.54 ± 0.02 1.00 ± 0.05

1.45 ± 0.04 *

Accession: the unigenes in blueberry transcriptomic database. MW: Theoretical molecular mass. KO: Enzyme ID in KEGG pathway G/P: comparion between green and pink fruit; P/B: comparion between pink and blue fruit; G/B: comparion between green and blue fruit. *: significant difference between two phases on the threshold of P<0.05 and fold change>1.5

37

　

*

Conflict interest The authors declare no conflict of interest.

38