Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison

Accepted Manuscript Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison Fan Xu, Zhu-mei Xi, Hui Zhang, Cheng-jun Zhang, Zhen-wen Zhang PII:

S0981-9428(15)30035-8

DOI:

10.1016/j.plaphy.2015.06.005

Reference:

PLAPHY 4206

To appear in:

Plant Physiology and Biochemistry

Received Date: 22 March 2015 Revised Date:

25 May 2015

Accepted Date: 8 June 2015

Please cite this article as: F. Xu, Z.-m. Xi, H. Zhang, C.-j. Zhang, Z.-w. Zhang, Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison, Plant Physiology et Biochemistry (2015), doi: 10.1016/j.plaphy.2015.06.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT The EBR and Brz + EBR treatments significantly increased the reducing sugars in ‘Cabernet

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Sauvignon’ (Vitis vinifera L.) grape berries.

Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison

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Brassinosteroids are involved in controlling sugar unloading in Vitis

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vinifera ‘Cabernet Sauvignon’ berries during véraison

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Fan Xu1, Zhu-mei Xi1,2,*, Hui Zhang1, Cheng-jun Zhang1, Zhen-wen Zhang1,2

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College of Enology, Northwest A&F University, Yangling 712100, China;

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Shaanxi Engineering Research Center for Viti-Viniculture, Yangling 712100, China;

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E-Mails: [email protected] (F. X.); [email protected] (H. Z.); zhangcj @sjtu.edu.cn (C.-J. Z.);

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[email protected] (Z.-W.Z.);

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* Author to whom correspondence should be addressed; E-Mail: [email protected]

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Tel: +86-29-8709-1874 Fax: +86-29-8709-2107

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Abbreviations BRs: Brassinosteroids; EBR: 24-epibrassinolide; Brz: brassinzole; INVs: sucrose

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metabolic enzymes invertases; SuSyn: sucrose synthases; ABA: abscisic acid; CS: castastarone;

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6-DeoxoCS: 6-deoxocastastarone; BL: brassinolide; -1-

Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison

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Abstract:

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Sugar unloading in grape berries is a crucial step in the long-distance transport of carbohydrates from

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grapevine leaves to berries. Brassinosteroids (BRs) mediate many physiological processes in plants

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including carbohydrate metabolism. Here, ‘Cabernet Sauvignon’ (Vitis vinifera L.) grape berries

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cultivated in clay loam fields were treated with an exogenous BR (24-epibrassinolide; EBR), a BR

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synthesis inhibitor (brassinazole; Brz), Brz + EBR (sprayed with EBR 24 h after a Brz treatment), and

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deionized water (control) at the onset of véraison. The EBR treatment sharply increased the soluble

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sugars content in the berries, but decreased it in the skins. The EBR and Brz+EBR treatments

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significantly promoted the activities of both invertases (acidic and neutral) and sucrose synthase

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(sucrolytic) at various stages of ripening. The mRNA levels of genes encoding sucrose metabolic

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invertase (VvcwINV), and monosaccharide (VvHT3, 4, 5 and 6) and disaccharide (VvSUC12 and 27)

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transporters were increased by the EBR and/or Brz + EBR treatments. Generally, the effects of the Brz

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treatment on the measured targets contrasted with the effects of the EBR treatments. The EBR and Brz

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treatments inhibited the biosynthesis of the endogenous BRs 6-deoxocastastarone and castasterone.

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Both EBR and Brz+EBR treatments increased the brassinolide contents, down-regulated the expression

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of genes encoding BRs biosynthetic enzymes BRASSINOSTEROID-6-OXIDASE and DWARF1,

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(VvBR6OX1 and VvDWF1) and induced BR receptor gene BRASSINOSTEROID INSENSITIVE 1

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(VvBRI1) expression in deseeded berries. Together, these results show that BRs are involved in

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controlling sugar unloading in grape berries during véraison.

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Key words Cabernet Sauvignon, Grape berries, Sugar transportation, Endogenous brassinosteroids,

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Véraison

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1. Introduction

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As a primary metabolite and energy source, sugars play important roles in the development and quality

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of grape (Vitis vinifera L.) berries (Agasse et al., 2009). Sugars accumulate to very high levels in grape

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berries during ripening, and modulate a range of vital processes such as the synthesis and accumulation

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of anthocyanins and aroma compounds (Conde et al., 2007).

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Sugar transportation is a fundamental process in which leaves (the sources) provide carbohydrates to

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the fruit (the sink). During this process, hydrostatic pressure drives the movement of photoassimilates

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including sucrose, glucose, and fructose, toward sink tissues through the carpellary vascular bundles,

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comprising peripheral (in skin) and central bundles (Zhang et al., 2006). Sucrose, which is synthesized

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in mesophyll cells via photosynthesis, is the predominant metabolite for carbon transport. Glucose and

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fructose are the main metabolites taken up into sink cells and the major soluble sugars in grape berries

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(Agasse et al., 2009). In the long-distance transport of carbohydrates between sources and sinks, the

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pathway in which sucrose is released from the vascular system to the sink cells is the crucial and final

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step. During this step, sucrose can be imported into fruits from the apoplast by direct sucrose

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transporters. Alternatively, it can be hydrolyzed into glucose and fructose by the sucrose metabolic

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enzymes invertases (INVs) or sucrose synthases (SuSyn), and then taken up via monosaccharide

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transporters (Agasse et al., 2009; Wang et al., 2014; Zhang et al., 2006).

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Zhang et al. (2006) reported that the shift of phloem unloading from the symplasmic to apoplasmic

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pathway occured at or just before the onset of ripening in Kyoho grape. Recently, the enzymes and

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genes involved in sugar unloading in grape berry have received considerable attention. Acidic

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invertases (INVs), are located in the cell wall (cwINV) and vacuole (vINV), while the neutral form

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(nINV) is localized in the cytoplasm. The cDNA sequence of a cwINV (AY538262) and its promoter

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region (EF122148), the complete cDNA sequence of a nINV (NIN1, EU016365) as well as three

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incomplete genomic sequences, and two vINVs cDNAs (VvGIN1 and VvGIN2) have been cloned from

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grapes (Hayes et al., 2007). Grape appears to have several monosaccharide and disaccharide

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transporters that coordinate sugar transport in diverse tissues, at different developmental stages and

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under varying environmental conditions. Six monosaccharide transporters (VvHT1–6) and three

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disaccharide transporters (VvSUC11, 12 and 27) were shown to be associated with the site at which

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sugars unloaded from the apoplast were imported into grape berries.

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The physiological and biochemical mechanisms of sugar transport into berries have been studied

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widely, but less is known about the regulation of sugar unloading in grape berries. Plant hormones may

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be key compounds in regulating the production of biomass and movement in grape (Wheeler et al.,

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2009). Such hormones include gibberellins (GA3) (Moreno et al., 2011), abscisic acid (ABA) (Moreno

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et al., 2011; Wheeler et al., 2009), and ethylene (Conde et al., 2006; Deluc et al., 2007). In an

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experiment in which exogenous GA3, PBZ (paclobutrazolan, a GA biosynthesis inhibitor), and ABA

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were sprayed onto grape leaves, GA3 promoted carbon allocation to the roots and berries of grapevines

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(Moreno et al., 2011). ABA is involved in plants’ responses to stress and possibly controls berry

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development by triggering the initiation of ripening (Wheeler et al., 2009). There is also some evidence

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that ABA regulates grape hexose transporters HTs (Çakir et al., 2003; Hayes et al., 2010). For example,

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VvHT1 was shown to be transcriptionally regulated by VvMSA, a member of the ASR (for ABA-,

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stress-, and ripening-induced) family (Çakir et al., 2003), whereas ABA induced VvMSA expression in

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the presence of sucrose (Çakir et al., 2003). VvHT5 expression was shown to be regulated by ABA

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during the transition from source to sink in response to infection by a biotrophic pathogens.

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Previously, we reported that brassinosteroids (BRs) could enhance the accumulation of reducing sugar

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in ‘Cabernet Sauvignon’ and ‘Yan 73’ grapes (Xi et al., 2013; Xu et al., 2015). BRs, a group of

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steroidal plant hormones, are essential for normal plant development. They have been well studied and

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widely applied to increase the yields (Janeczko et al., 2010; Shahid et al., 2014) and enhance protection

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against abiotic stress (Kang et al., 2009; Shahid et al., 2014; Yuan et al., 2014). These compouds are

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involved in the ripening and development of fleshly fruits such as tomato (Li et al. 2008), cucumber

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(Kang et al., 2009; Yuan et al., 2014), and strawberry (Chai et al., 2013). In grape, exogenous

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application of BRs was shown to significantly promote ripening (Symons et al., 2006) and increase the

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concentrations of phenolics (Xi et al., 2013) such as proanthocyanidin (Xu et al., 2015) in grape berries

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and wines (Xu et al., 2014; Xu et al., 2015).

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There have been reports regarding carbohydrate metabolism promotion in several species following the

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application of 24-epibrassinolide (EBR), an exogenous BR. EBR treatments increased the enzyme

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activities of INVs and SuSyn and/or their mRNA levels (Li et al., 2008) in tomato (Li et al., 2008),

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wheat (Liu et al., 2006), cucumber (Yu et al., 2004; Yuan et al., 2014), cotton (Bibi et al., 2014) and

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pea (Shahid et al., 2014), as well as the sugar content (sucrose, fructose or total soluble sugars) in

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tomato (Li et al., 2008), wheat (Liu et al., 2006), cucumber (Kang et al., 2009; Yuan et al., 2014),

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oilseed rape calli (Janeczko et al., 2009), Wolffia arrhiza (Bajguz and Asami 2005) and pea (Shahid et

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al., 2014). In contrast, application of a BR synthesis inhibitor, brassinazole (Brz), to W. arrhiza cultures

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decreased their sugar content (Bajguz and Asami 2005). However, little is known about BRs’ effects on

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sugar transport and unloading in grape. In our previous studies, EBR was applied by spraying the entire

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surface area of the berries in the cluster (Xi et al., 2013; Xu et al., 2014; Xu et al., 2015). The berries

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were the only organ affected by exogenous EBR because BRs cannot be transported over long

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distances (Symons and Reid 2004). Instead, they exert their effects in a paracrine manner at their

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synthesis sites. Therefore, we hypothesized that EBR may affect the sugars’ transport, and especially

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sugar unloading, in grape berries.

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In Arabidopsis, the availability and diversity of mutants has stimulated extensive studies on plant

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hormone signal transduction and the related physiological effects. However, there are no BR-deficient

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grape mutants. Considering this difficulty, in addition to the EBR treatment, we included a treatment

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with the BR biosynthesis inhibitor, brassinazole (Brz), and a Brz + EBR treatment in which EBR

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solution was spray onto berries 24 h after the Brz treatment. The latter treatment was designed to

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determine whether the exogenous BR could rescue the effect of the loss of endogenous BRs. In order to

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examine whether BRs are involved in controlling sugar unloading in grape berries during véraison, we

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analyzed berries of ‘Cabernet Sauvignon’ subjected to the above treatments at five different periods.

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We determined the distribution of sugars (sucrose, fructose, and glucose) and reducing sugars in the

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grape berries. We also measured the berry skin weight, the activities of sucrose hydrolytic enzymes

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(SuSyn and acidic and neutral INVs), the transcript levels of genes encoding HTs, sucrose transporters

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and INVs, and the contents of endogenous BRs.

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2. Materials and Methods

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2.1 Experimental design and sample collection

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‘Cabernet Sauvignon’ (V. vinifera L.) is one of the most commonly used wine grape varieties. Berries

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of ‘Cabernet Sauvignon’ were sampled from the vineyard of Rongzi Chateau, located in Xiangning

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County, Linfen City, Shanxi Province, China (110°82′E, 37°36′N; 1100 m above mean sea level). Soil

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analysis results showed that the soil was clay loam and alkaline (pH 8.52) (Supplemental Table 1).

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Each year, before bud break, a mixture of 15 kg P2O5 ha−1 (triple super phosphate), 50 kg N ha−1 (urea)

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and 50 kg K2O ha−1 (potassium sulfate) was applied to the experimental area as fertilizer. The vineyard

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was planted in 2007 and was maintained using the single cordon pruning method. The vines, trained on

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a vertical shoot-positioning system with a pair of wires, were planted in West–East oriented rows with

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0.8-m and 2.5-m spaces within and between rows, respectively. The vines were trained onto a bilateral

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cordon at 1 m above the ground, in which shoots were trained upwards and each vine carried

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approximately 20 grape clusters. All viticulture practices, such as hedging and soil work, were

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performed according to standard commercial vineyard practices and were identical for all experimental

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modalities. The experimental design consisted of completely randomized blocks, each with 20

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self-rooted grapevines of similar vigor. Four independent blocks were randomly selected within one

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field, with each block incorporating two neighboring rows, with 10 plants per row. Each block received

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a different spray treatment: deionized water (control), 0.4 mg/l 24-epibrassinolide ([EBR], TRC,

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Toronto, Canada) only once, 1.31mg/l (0.4 µM) brassinazole ([Brz], Sigma Chemical Co., St. Louis,

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MO, USA), 1.31 mg/l Brz + 0.40 mg/l EBR (Brz+EBR, with the EBR sprayed 24 h after spraying with

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Brz). The stock solutions of EBR and Brz were prepared by dissolving each compound in 1 ml ethanol

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(98%). The control stock solution contained 1 ml 98% ethanol without EBR or Brz. Each solution was

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mixed with 1 ml Tween 80 (Xi’an Chemical Factory, Xi’an, China) diluted to 1 L with sterilized

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deionized water. The solutions were sprayed onto grape clusters at a rate of 10 ml/cluster, and the

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solution covered the entire surface area of the berries in each cluster. The application dates were 8

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August for the control, EBR, and Brz, and 8 and 9 August for Brz + EBR. All spray applications were

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carried out at sunset. Grape clusters were collected in 2013 at several phenological stages at the five

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stages defined by Eichhorn and Lorenz (1977) (Supplemental Table 2). Eight clusters per treatment

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were randomly selected from four vines per block at the five stages. Two hundred berries were

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randomly sampled from each replicate. The samples were stored at −80°C until use. For each replicate,

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the physicochemical indices described below were assayed.

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2.2 Determination of the physicochemical indices of grape berries

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Grape juice was collected and used to determine reducing sugars and titratable acids contents,

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according to methods proposed by OIV (2012). To quantify reducing sugars in the skins, triplicate

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samples of freeze-dried skin powder (0.50 g, dry weight) were weighed into 50-mL centrifuge tubes

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with 10 mL distilled water, and then shaken at 300 rpm/min for 100 min at 25°C. The supernatant was

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collected and analyzed using by the same method used to analyze the juice. The skins of 100 berries

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collected randomly from three replicates for each group were weighed, freeze-dried for 24 h at −40°C

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and 100 Pa, and then reweighed. The fresh and dry weights were measured by electronic scale.

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Seeds were removed from the samples, then each sample was homogenized in a mortar with liquid

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nitrogen. A 2.0-g sample of the powdered tissue was placed into a 15-mL microfuge tube. Then, 10 mL

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80:20 (v/v) ethanol: twice-distilled water was added, and the mixture was heated for 3 min at 80°C.

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The samples were centrifuged for 10 min at 10000 rpm/min using a Sorvall RC-5C Plus centrifuge

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(Kendro Laboratory Products, Newton, CT, USA). The supernatant was collected, and the pellets were

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extracted twice with 10mL of 80:20 (v/v) ethanol: twice-distilled water for 20 min at 80°C, and then

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centrifuged for 10 min at 10000 rpm/min. The three supernatants were combined in conical flask and

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then the flask was placed in boiling water to allow the supernatant to evaporate. The dried extracts were

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made up tp 25mL with twice-distilled water. Finally, the mixture was filtered through a 0.45-µm

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membrane filter (Iwaki Glass, Shearwater Polymers Inc., Huntville, AL, USA) and then a 25-µL

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aliquot of the mixture was injected into a high performance liquid chromatography (HPLC) (Model

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1525, Waters Corp., MA, USA) equipped with a 7725i manual injector. Sugars were analyzed using an

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Inertsil NH2 column (4.6 mm × 250 mm, particle size 5 µm) at the column temperature of 35 °C. The

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HPLC system was equipped with a 2414 differential refractive index detector. The mobile phase was an

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acetonitrile solution (80%, v/v) with a flow rate of 1.4 mL/min. Glucose, fructose and sucrose

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identification and quantification were based on the peak area of D-glucose, D-fructose and sucrose

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standards, repectively (Sigma Chemical Co.). For the quantification of glucose, fructose, and sucrose in

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the samples, standard curves were obtained (ca.R2=0.99) by successive injections of increasing

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amounts of sugar from a stock solution of sugar (0.1, 0.5, 1, 5, and 10 ng mL-1).

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2.3 Measurement of enzyme activities

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The skins were separated from the berries, and then 1 g powdered skin tissue was homogenized in a

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mortar with liquid nitrogen with 10 ml 200 mM Hepes–NaOH (pH 7.5). The samples were centrifuged

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at 10,000 rpm/min for 20 min, and the supernatant was used as the crude enzyme solution. The SuSyn

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activity was measured in an assay mixture consisting of 50 µL 100 mM UDPG, 20 µL 50 mM MgCl2,

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50 mM Hepes-NaOH (pH 7.5), 15 µL 100mM fructose, and 10 µL distilled water. The mixture was -9-

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incubated in a water bath at 30ºC for 30 min and then 200 µL 2 M NaOH was added to stop the

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reaction. Then, 0.5 ml 0.1% hydroquinone and 1.5 ml concentrated hydrochloric acid were added, and

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the mixture was incubated in a water bath at 30ºC for 10 min. The SuSyn activity was calculated from

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the absorbance value at 480 nm. As described above, AI activity was assayed by adding 50 µL reaction

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buffer (1% sucrose and 0.1 M pH 5.5 acetic acid buffer) to 50 µL crude enzyme solution, and then

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incubating the mixture at 34°C for 30 min. For the inactivated-enzyme control 50 µL crude enzyme

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solution was incubated at 100°C for 30 min and then assayed as described above. The reaction was

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stopped by adding 1.5 ml 3,5-dinitrosalicylic acid and incubating the mixture in a water bath at 100°C

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for 5 min. The mixture was diluted to 25 ml with distilled water. The NI activity assay was similar to

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the AI activity assay, except that the reaction was performed in phosphate buffer (pH 7.5). Both AI and

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NI activities were calculated from the absorbance value at 540 nm.

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2.4 Determination of endogenous BRs content

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Endogenous BRs were quantified as described by Chai et al. (2013) with minor modifications. To

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extract BRs, 1.0 g each of deseeded berries and seeds were ground in a mortar and homogenized in

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PBS buffer extraction solution (pH 7.3). The extracts were centrifuged at 10, 000rmp for 20 min, and

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then the supernatant was collected and stored at 4°C until enzyme-linked immunosorbent assays

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(ELISAs). Three steroid compounds (castastarone, 6-deoxocastastarone and brassinolide, abbreviated

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as CS, 6-deoxoCS, and BL, respectively) were measured using different kits. The ELISA procedures

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were those recommended by the manufacturer (Shanghai Meilian Biotechnology Co, Ltd. Shanghai,

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China). The ELISA plates were analyzed using a Mul-tiskan MK3 microplate reader (Thermo

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Labsystems, Vantaa, Finland).

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221 2.5 Real-time quantitative PCR analysis

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The relative transcript levels of 16 genes encoding monosaccharide (6) and disaccharide transporters

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(3), MSA protein (1), sucrose metabolic enzymes and INVs (3), and BRs biosynthesis (2) and signaling

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pathway (1) proteins in developing berry skin were determined in this study. Total RNAs were

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extracted and purified from grape skins using the modified SDS/phenol method. The purified RNAs

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were used immediately or stored at −80ºC. We prepared cDNA from the purified total RNA as

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described by Xu et al. (2015). The cDNA synthesis was controlled by PCR using 1 µL cDNA in a

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20-µL reaction mixture. As the control, the Actin gene was amplified with VvActin primers. Real-time

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quantitative PCR (RTqPCR) analysis was carried out according to the method of Xu et al. (2015) with

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minor modifications. The annealing sequences of the primers used for real-time PCR are shown in

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Supplemental Table 3. The transcript levels of each gene were normalized to that of VvActin (182-bp

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products), and relative transcript levels were calculated using the equation 2−∆∆Ct, where ∆∆Ct=

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(Ct,target – Ct,VvActin)treated − (average Ct,target – average Ct,VvActin)control (Livak and Schmittgen

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2001). For all RTqPCR reactions, three replicates per sample were analyzed.

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2.6 Statistical analysis

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Data were analyzed using SPSS 17.0 software (SPSS, Chicago, IL, USA). The significance of

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differences between each treatment was determined by one-way analysis of variance (ANOVA) and

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Duncan’s new multiple range test at the 0.05 and 0.01 significance level. The gene transcript level data

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subjected to two-way ANOVA were calculated using the 2−∆∆Ct method, which calculates the relative

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3. Results

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3.1 Physicochemical indices of grape berries

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The reducing sugars contents in grape berry skin and juice, titratable acids content in juice, and fresh

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and dry weight of skins of ‘Cabernet Sauvignon’ grape berries are shown in Fig. 1. The EBR treatment

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resulted in significant or highly significant increases in the reducing sugars content in juice during

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berry ripening, especially at 60 days after anthesis (DAA) when it increased by 65.02% (Fig. 1a). This

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enhancing effect decreased at later stages. In contrast, the Brz treatment decreased the reducing sugars

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content in juice at 66 and 86 DAA. The reducing sugars content in juice was higher in Brz +

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EBR-treated grapes than in control grapes at 93 and 100 DAA (Fig. 1a). The titratable acids content in

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juice was higher in the Brz-treated grapes than in grapes receiving other treatments from 66 to 100

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DAA. In contrast, the titratable acids content in juice was lower in EBR-treated grapes than in grapes

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receiving other treatments at 60 and 66 DAA (Fig. 1b). The reducing sugars content in skin was

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significantly decreased by EBR treatment from véraison to maturity, but increased by Brz treatment at

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66 DAA. The Brz + EBR treatment decreased the reducing sugars content in skin from 86 to 100 DAA

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(Fig. 1c). Interestingly, the EBR treatment significantly decreased the dry and fresh weight of skins at

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60 DAA (Fig. 1d, e). The Brz + EBR treatment significantly increased the skin weight at harvest.

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The glucose, fructose, and sucrose contents in deseeded berries were analyzed using HPLC. The

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glucose and fructose contents were significantly higher, by 55.40% and 78.08% higher, respectively, in

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deseeded EBR-treated berries than in control berries at 60 DAA (Fig. 2 a, b). These enhancements in

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the glucose and fructose contents decreased gradually in the later stages. The Brz + EBR treatment also

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increased the hexose content at 66 and 100 DAA. The Brz treatment resulted in the decreased glucose

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content in deseeded berries during the period of rapid sugar accumulation (from 86 to 93 DAA). The

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hexoses contents were much higher than sucrose contents throughout berry development. Sucrose was

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only detected in the deseeded berries near the harvest date (Fig. 2 c-g). These results demonstrated that

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exogenous EBR increased the soluble sugars content in grape berries.

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268 3.2 INV and Susyn activities

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The activities of acidic and neutral INVs, and SuSyn, in berry skin are shown in Fig. 3a–c. In the

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control, the acidic INV activity remained relatively stable from véraison to maturity, whereas the

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neutral INV peaked at 86 DAA and then declined slightly. The SuSyn activity showed lower levels at

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60 and 66 DAA, and then increased rapidly from 86 to 100 DAA. The EBR treatment significantly

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increased the activities of acidic and neutral INVs from 86 to 100 DAA, compared with those in the

275

control. Similarly, the Brz + EBR treatment increased acidic INV activity at 93 and 100 DAA, and

276

neutral INV activity from 86 to 100 DAA. The EBR treatment significantly increased SuSyn activity at

277

60 DAA (Fig. 3c). The Brz treatment significantly decreases acidic INV activity at 60 DAA and neutral

278

INV activity at 66 DAA, relative to those in the control (Fig. 3a, b). Collectively, these results showed

279

that BRs regulated the activities of acidic and neutral INVs, and SuSyn, but the effects varied

280

depending on the stage of ripening.

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3.3 Expression patterns of sugar transporter genes and VvMSA

283

The relative transcript levels of the genes encoding monosaccharide (VvHT1-6) and disaccharide

284

(VvSUC11, 12 and 27) transporters in developing berry skin were determined (Fig. 4,5). A two-fold

285

change cutoff was used to define differentially expressed genes between the EBR- or Brz-treated group

286

and the corresponding control group. The EBR treatment resulted in increases in VvHT3, 4, 5 and 6 - 13 -

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transcript levels, to varying extents, during different phases of berry development, but had almost no

288

effect on VvHT1 and VvHT2 transcript levels (Fig. 4a, b). The Brz and Brz + EBR treatments

289

down-regulated the VvHT1 transcript level at 66 DAA (Fig. 4a). The expression of VvHT3 was induced

290

at 86 DAA by EBR and Brz + EBR treatments, but was repressed from 60 to 86 DAA by the Brz

291

treatment (Fig. 4c). Except at 93 DAA, the mRNA levels of VvHT4 were up-regulated by the EBR and

292

Brz + EBR treatments (Fig. 4d), but strongly down-regulated by the Brz treatment from 66 to 100 DAA.

293

From 60 to 66 DAA, the transcript level of VvHT5 remained very low. The EBR treatment increased

294

the transcript level of VvHT5 from 86 to 93 DAA (Fig. 4e). The EBR treatment increased the transcript

295

level of VvHT6 at 60 and 66 DAA, and the Brz + EBR treatment increased its level at 66 DAA.

296

However, these two treatments barely affected the transcript level of VvHT6 from 86 to 100 DAA

297

compared with that in the control (Fig. 4f).

298

The transcript levels of VvSUC11 and VvSUC12 markedly increased during ripening. In contrast, the

299

transcript level of VvSUC27 remained low. After véraison, the transcript level of VvSUC27 in berry

300

skin decreased dramatically until 86 DAA, and subsequently increased slightly until maturity (data not

301

shown). The EBR, Brz + EBR and Brz treatments had different effects on the three disaccharide

302

transporter genes (VvSUC11, 12 and 27) (Fig. 5a–c). The Brz + EBR treatment decreased the transcript

303

levels of VvSUC11 from 66 to 100 DAA, whereas the Brz treatment increased the VvSUC11 transcript

304

levels from 60 to 93 DAA, especially at 60 DAA (Fig. 5a). The transcript level of VvSUC12 during

305

berry development was decreased by the Brz treatment, but increased at 86 DAA with the EBR

306

treatment (Fig. 5b). From 60 to 93 DAA, the transcript level of VvSUC27 in grape berries was

307

significantly higher in EBR and Brz + EBR treated samples than those in the Brz (Fig. 5c).

308

The MSA protein is a component of the transcription-regulating complex involved in sugar signaling.

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The target of VvMSA is the proximal promoter of VvHT1 (Çakir et al., 2003). Surprisingly, all three

310

treatments inhibited the transcript levels of VvMSA from 60 to 66 DAA, but did not affect its transcript

311

levels from 83 to 100 DAA (Fig. 4g)

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312 3.4 Expression patterns of genes encoding sucrose metabolic enzymes and INVs

314

VvcwINV, VvGIN1 and VvGIN2 encode INVs. As shown in Fig. 5d–f, the EBR treatment increased the

315

transcript level of VvcwINV in grape skin at 60 and 66 DAA, and the Brz + EBR treatment increased its

316

transcript level at 60 DAA, compared with the control. However, EBR treatment results in a drastic

317

decline in the transcript level of VvcwINV during the later ripening stages. The Brz treatment resulted

318

in decreased VvcwINV transcript levels in grape skin at 60 DAA and 93 DAA, compared with in the

319

control (Fig. 5d). The transcript level of VvGIN1 during berry development was down-regulated by the

320

EBR treatment, but up-regulated at 66 DAA by the Brz + EBR treatment (Fig. 5e). The Brz treatment

321

increased the transcript level of VvGIN1 at 86 DAA but decreased it at 60, 93 and 100 DAA. The EBR

322

and Brz treatments barely affected the transcript levels of VvGIN2, except at 86 DAA (Fig. 5f). Overall,

323

the structural genes VvcwINV, VvGIN1 and VvGIN2 had different transcriptional patterns in response to

324

EBR and Brz + EBR treatments, and the transcriptional activation of these structural genes depended

325

on the stage of maturity.

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3.5 Concentrations of endogenous BRs, and expression patterns of genes encoding BRs biosynthetic

328

enzymes and a BR receptor

329

To further explore the role of BRs in sugar unloading, we analyzed the contents of endogenous BRs

330

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The contents of CS, 6-deoxoCS and BL in deseeded berries and seeds were measured using ELISA. In

332

both deseeded berries and seeds, the three endogenous BRs were detected in fruit at all five stages of

333

development. The most abundant BR was BL, followed by CS and then 6-deoxoCS. There were higher

334

levels of the three BRs precursors in seeds than in deseeded berries. In the deseeded berry, the

335

6-deoxoCS and BL concentrations peaked at 93 DAA and then declined slightly until harvest (Fig. 6a),

336

whereas the CS concentration remained relatively stable. In seeds, the 6-deoxoCS concentration

337

showed a similar trend to that observed in deseeded berries (Fig. 6b). The concentration of CS was low

338

at 60 DAA and 66 DAA, and then peaked at 93 DAA. The BL concentration remained relatively stable,

339

with a slight decrease from 86 DAA to maturity (Fig. 6a). The EBR treatment resulted in significant

340

decreases in the 6-deoxoCS and CS levels in deseeded berries from 60 to 66 DAA. However, the BL

341

concentration in the deseeded berries was higher in EBR and Brz + EBR treated samples than in the

342

control. In the Brz treatment, the BL concentration in seeds decreased at 60 DAA and increased at

343

harvest, compared with in the control level. Surprisingly, the EBR treatment decreased the BL

344

concentration in seeds at 60 and 66 DAA.

345

The EBR treatment significantly decreased the mRNA levels of VvBR6OX1 at 66 and 86 DAA.

346

Similarly, the Brz + EBR treatment decreased its mRNA level from 66 to 93 DAA. The Brz treatment

347

resulted in decreased VvBR6OX1 transcript level during berry development (Fig. 6c). All three

348

treatments slightly decreased the mRNA level of VvDWF1 during berry development (Fig. 6d).

349

However, the EBR treatment increased the transcript level of VvBRI1 from 60 to 93 DAA (Fig. 6e).

350

The Brz + EBR treatment also increased the transcript level of VvBRI1 from 86 to 93 DAA. Conversely,

351

the Brz treatment significantly decreased the transcript level of VvBRI1 during berry ripening. Thus,

352

the exogenous EBR application affected the expressions levels of genes related to the biosynthesis

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353

and/or signal transduction of endogenous BRs.

354 3.6 Correlations among the sugar and total endogenous BR contents, skin weights, and activities of

356

INVs and SuSyn

357

There were strong correlations between the contents of reducing sugars, glucose and fructose in the

358

deseeded berries, and the dry and fresh weight of berry skins (Table 1). There was also a positive

359

correlation (p < 0.01) between the total BRs (6-deoxoCS + CS + BL) contents in deseeded berries and

360

seeds. However, the reducing sugars content in skin was more weakly correlated with the total

361

endogenous BRs and sugars content in deseeded berries. The activity of SuSyn was strongly positively

362

correlated (p < 0.01) with the reducing sugars, glucose and fructose contents in deseeded berries, with

363

the dry and fresh weights of skin, and with the total BRs contents in deseeded berries. These

364

correlations were weaker for acid and neutral INVs. There was no significant correlation between the

365

sugars content in berries or deseeded berries and the acidic INV activity. These results confirmed that

366

the endogenous BRs contents in grape berries were highly correlated with sugar unloading during

367

ripening.

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4. Discussion

376

BRs are involved in the ripening of ‘Cabernet Sauvignon’ grapes (Symons et al., 2006). Our previous

377

study showed that exogenous EBR enhanced the phenolic content and antioxidant capacity in

378

‘Cabernet Sauvignon’ and ‘Yan 73’ grapes berries. An interesting and unexpected result was that the

379

EBR treatment also significantly increased the reducing sugars content in grape berries (Xi et al., 2013;

380

Xu et al., 2015). In the present study, the EBR treatment strikingly and rapidly enhanced the soluble

381

sugars content in grape juice (Fig. 1, Fig. 2), but the extent of the increase gradually decreased during

382

the later stages of ripening. The Brz treatment decreased the reducing sugars in the juice. These results

383

are in agreement with recent reports that an EBR treatment can increase the sugars content (sucrose,

384

fructose or total soluble sugars) in tomato (Li et al., 2008), wheat (Liu et al., 2006), cucumber (Kang et

385

al., 2009; Yuan et al., 2014), oilseed rape calli (Janeczko et al., 2009), W. arrhiza (Bajguz and Asami

386

2005) and pea (Shahid et al., 2014), and that conversely, the application of Brz to W. arrhiza cultures

387

decreased their sugars content (Bajguz and Asami 2005). Grape berry skin is the most important tissue

388

for unloading sugar into the pulp. The skin contains peripheral bundles, a kind of carpillary vascular

389

bundle, which channel sugars into the developing grape berry (Zhang at al., 2006). The EBR treatment

390

resulted in an increase in the reducing sugars content in juice and a decrease in the reducing sugars

391

content, and the dry and fresh weights of skins (Fig. 1a). These results indicated that more

392

carbohydrates were unloaded from skin cells into pulp cells in the EBR treatment than in the control

393

and Brz treatments. In our previous studies, after the EBR treatment, secondary metabolism pathways

394

such as anthocyanin (Xi at al., 2013) and proanthocyanidin biosynthesis (Xu at al., 2015) were more

395

active than primary metabolic pathways during the period of rapid sugar accumulation. That is, less

396

carbohydrate was used for cell expansion or division. Therefore, BRs may have different physiological

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functions in grapes depending on the developmental stage. They may affect the primary metabolism at

398

earlier stages of berry ripening and the secondary metabolism at later stages.

399

Like in other higher plants, the sugar unloading activity in grape depends on the sink strength (Zhang et

400

al., 2006). The activities of sucrose-metabolizing enzymes, including INVs and SuSyn, are key indexes

401

of sink strength (Li et al., 2008; Liu et al., 2006). The INVs hydrolyze sucrose to the hexose monomers

402

glucose and fructose, and support phloem unloading at sink organs by maintaining a sucrose gradient

403

between the end of the phloem path and the unloading site (Agasse at al., 2009; Hayes et al. 2007). In

404

this study, sucrose was not detected in grape berries at the onset of ripening (Fig. 2), indicating that

405

sucrose, once unloaded from phloem to sink cells, might be rapidly hydrolyzed into glucose and

406

fructose by INVs or SuSyn, and then stored in vacuoles. To investigate the capacity for sucrose

407

hydrolysis in grape skin, we measured the activities of SuSyn and acidic and neutral INVs. At 24 h

408

after the treatment, only the SuSyn activity had increased significantly in response to exogenous EBR

409

(Fig. 3). Previously, in tomato (Li et al., 2008), wheat (Liu et al., 2006), cucumber (Yu et al., 2004;

410

Yuan et al., 2014), cotton (Bibi et al., 2014) and pea (Shahid et al., 2014), increases in the SuSyn

411

activity after EBR treatments were observed. In addition, the correlation between SuSyn activity and

412

the reducing sugars concentration in juice was highly significant (Table 1). These results suggested that

413

SuSyn may be the major enzyme that hydrolyzes sucrose at this stage, and that this enzyme plays a

414

major role in the rapid increase of the sugars content in juice in response to EBR treatment.

415

The enzymatic hydrolysis of sucrose in grape skin depends on the total activities of INVs and SuSyn

416

rather than the sucrolytic activity of SuSyn alone, because these enzymes play different roles in the

417

hydrolytic reaction. In this study, the activity of acidic INV was higher than that of neutral INV during

418

berry development. During the periods of rapid sugar accumulation and maturation (86, 93 and 100

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DAA), both types of INVs showed higher activities in EBR- and Brz + EBR-treated berries than in

420

control berries. This result is consistent with recent reports that EBR treatments could also enhance

421

INV activities in tomato (Li et al., 2008), cucumber (Yu et al., 2004; Yuan et al., 2014), and cotton

422

(Bibi et al., 2014). There were no significant effects of EBR and Brz + EBR treatments on SuSyn

423

activity from 93 to 100 DAA. Thus, EBR changed the conversion rate from sucrose to hexoses by

424

regulating INV activities in the skins at latter ripening stages. In summary, the EBR and Brz + EBR

425

treatments increased the enzymatic hydrolysis of sucrose by regulating the activities of SuSyn and

426

INVs, but the effects depended on the developmental stage and the specific enzyme.

427

The expression patterns of VvcwINV, VvGIN1 and VvGIN2 differed during ripening, and these genes

428

showed different sensitivities to BRs. The EBR and Brz + EBR treatments caused rapid increases in the

429

levels of VvcwINV at véraison, and the Brz treatment decreased its transcript levels during the period of

430

rapid sugar accumulation (93 DAA). The transcript abundance of GIN1 and GIN2 decreased during

431

berry development, consistent with the results of a previous report (Deluc et al., 2007). The VvGIN1

432

transcript level was significantly up-regulated at 60 DAA by the Brz + EBR treatment, and strongly

433

down-regulated during berry development by EBR treatment. The Brz treatment increased the

434

transcript level of VvGIN1 at 86 DAA but decreased it at other stages. These results indicated that

435

endogenous BRs may inhibit VvGIN1 expression during berry ripening, promote the cell-wall

436

catabolism of sucrose to fructose and glucose, and negatively regulate sucrose hydrolysis in vacuoles.

437

Monosaccharide and disaccharide transporters mediate the uptake of sugar across the plasma

438

membrane and its compartmentalization into the tonoplasts of flesh cells. In a previous study, the

439

expression and activities of some sugar transporters, including monosaccharide and disaccharide

440

transporters, were coordinated with cell wall INV activation and sugar accumulation (Conde et al.,

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2007). In this study, the expressions of VvSUC11 and VvSUC12 were up-regulated in grape berries

442

during ripening (Fig. 5a, b), similar to the results reported previously for Shiraz (Davies et al., 1999)

443

and Carmenere (Pastenes et al., 2014) grape berries. The transcript level of VvSUC27 declined until 86

444

DAA and then increased slightly from 93 to 100 DAA (data not shown), which is different from

445

Davies’ report (1999) but similar to Pastenes’ study (Pastenes et al., 2014). This might be due to

446

varietal differences and/or the cultivation conditions. VvSUC27 may be responsible for phloem loading

447

and sugar retrieval during long-distance transport (Afoufa-Bastien et al., 2010). The EBR and Brz +

448

EBR treatments resulted in significant increases in the VvSUC27 transcript level during earlier stages

449

of véraison. VvSUC12 may also be involved in either phloem unloading or sucrose import into berry

450

tissues, and VvSUC11 might be responsible for sucrose accumulation in berry vacuoles

451

(Afoufa-Bastien et al., 2010). The mRNA level of VvSUC12 decreased during berry development in

452

response to Brz and increased in response to EBR at 86 DAA. The VvSUC11 transcript level decreased

453

in response to the EBR treatment, but increased at 60 or 66 DAA in response to the Brz treatment.

454

These differences in expression patterns may be related to the different roles of VvSUC11, 12 and 27 in

455

berry development. In Ugni Blanc and Carmenere grapevines, the VvSUC11, 12 and 27 expression

456

levels under water deficient or deficit irrigation conditions responded to ABA (Medici et al., 2014;

457

Pastenes et al., 2014). Additionally, Arabidopsis AtSUT2, ATSUC4 and ATSUC2 genes are

458

orthologs of the grapevine VvSUC11, 12 and 27, respectively (Reinders et al., 2012). Gong et al.

459

(2015) found that AtSUC2 and AtSUC4 are important regulators of plant abiotic stress tolerance that

460

use the ABA signaling pathway, which may cross with sucrose signaling. These results suggested that

461

the changes in the VvSUC11, 12 and 27 transcript levels after a treatment may be caused by the

462

interactions between ABA and the BRs.

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In addition to the disaccharide transporter-containing pathway, sucrose can also be hydrolyzed to

464

glucose and fructose by INVs and then taken up by monosaccharide transporters (Agasse et al., 2009;

465

Zhang et al., 2006; Wang et al., 2014). In this study, the VvHT1, VvHT2 and VvHT3 transcript levels

466

were higher than those of other HT genes, consistent with the results of a previous study (Hayes et al.,

467

2007). The HT genes showed different expression patterns in grape berries around véraison. The

468

VvHT3transcript level was high in young berries, but decreased around véraison and increased again

469

during the sugar storage phase (Hayes et al., 2007). However, in the Carmenere grape, there was a

470

lasting reduction in VvHT3 expression from véraison until the season’s end (Pastenes et al., 2014). This

471

difference might be due to varietal differences and/or cultivation conditions. The accumulation of

472

VvHT5 mRNA, although weak, was mostly associated with the late ripening period (Conde et al., 2006).

473

The transcript levels of VvHT6 increased during véraison and at the beginning of ripening (Deluc et al.,

474

2007). In this study, the transcript levels of VvHT3, VvHT4, VvHT5 and VvHT6 were increased by EBR

475

and Brz + EBR treatments. These results suggested that the EBR and Brz + EBR treatments promoted

476

the expression of HT genes, which contributed to the sudden and sharp import of sugars into the berries.

477

Among the up-regulated genes, VvHT5 appears to have a specific role in enhancing sink strength under

478

stress conditions (Medici et al., 2014; Pastenes et al., 2014). This gene is controlled by ABA in

479

grapevine leaves in response to biotrophic fungal infection and water-deficit stress (Medici et al., 2014;

480

Pastenes et al., 2014). The ABA-induced expression of VvHT5 was shown to be mediated through

481

ABRE motifs (Hayes et al., 2010; Medici et al., 2014). We identified BR regulatory motifs in the

482

VvHT1-6 promoter using similar methods to those reported by Hayes et al. (2010), but no BR response

483

elements (BRREs) were found (data not shown), consistent with the results of Afoufa-Bastien et al.

484

(2010). The absence of BRREs from HT gene promoters suggests that BRs may regulate VvHT genes

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via an indirect pathway, such as by changing the concentrations of sugars or plant hormones. This

486

hypothesis is supported by the differential responses of VvHT genes to changes in sugar concentrations

487

(Conde et al., 2006; Deluc et al., 2007).

488

VvMSA, the upstream gene to regulating VvHT1 expression, was induced by ABA but only in the

489

presence of sucrose (Çakir et al., 2003). Medici et al. (2014) also reported that VvMSA at least partly

490

orchestrates the different but complementary functions of VvHT1, VvSUC11 and VvGIN2 in

491

grapevine leaves response to water-deficit stress. In the present study, the EBR and Brz + EBR

492

treatments down-regulated VvMSA close to véraison (Fig. 4g), which may explain why these treatments

493

did not significantly effect VvHT1 (Fig. 4a) and VvGIN2 (Fig. 5f) expression levels. Interestingly, the

494

Brz treatment also inhibited VvMSA expression from 60 to 66 DAA (Fig. 4g). These effects may be

495

related to the similar decreases in 6-deoxoCS and CS contents in deseeded berries caused by the three

496

treatments (Fig. 6).

497

The results of this study showed that exogenous EBR enhanced sugar unloading in grape berries during

498

véraison. To determine whether exogenous EBR affects BRs biosynthesis in grape berries, we analyzed

499

the endogenous BRs contents. The conversion of 6-deoxoCS to CS, catalyzed by the VvBR6OX1 gene

500

product, is an important BR activation step in grape. In other plants, BL is the BR with the highest

501

bioactivity. In both deseeded berries and seeds, there were higher concentrations of BL than

502

6-deoxoCS and CS. This was inconsistent with the results of a previous study, in which CS was the

503

only bioactive BR detected in grape berries (Symons et al., 2006). This discrepancy might be due to

504

varietal differences, different analytical methods or differences in growth conditions at the vineyards.

505

The levels of the three endogenous BRs in both deseeded berries and seeds remained relatively stable

506

from véraison to maturity, consistent with the results reported by Symons et al. (2006). Currently, the

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site of BR synthesis and accumulation in grape berries is unknown. The seed might be the synthesis

508

and/or storage tissue for BRs, based on the distribution of the three endogenous BRs observed in the

509

present study. Unlike auxin, ABA, and GA, BRs are not transported over long distances (Symons and

510

Reid 2004). Instead, they are synthesized in the seeds and transported to other target cells in the fruit

511

pulp or skin.

512

Surprisingly, the EBR treatment decreased the 6-deoxoCS and CS contents in deseeded berries at 24 h

513

after treatment, and so did the Brz treatment, albeit with a slower effect (Fig. 6). This result is

514

consistent with a previous study in which the EBR treatment slightly decreased the CS content in wheat

515

(Janeczko et al. 2010). It also suggested that both exogenous EBR and Brz may disequilibrate the

516

endogenous BRs biosynthesis system via different pathways. We speculated that there may be a

517

feedback mechanism in BRs biosynthesis in grape. Such a mechanism would maintain the endogenous

518

BRs’ balance, but it could be quickly disrupted by exogenous BRs or excessive BL. Brz may have

519

directly inhibited the endogenous BRs biosynthesis pathway through its effects on the transcription of

520

VvBR6OX1, and VvDWF1 or other key genes. There were no significant changes in the endogenous

521

BRs content from 86 to 100 DAA, regardless of the treatment. Thus, the three types of treatments have

522

short-lived effects on the biosynthesis of endogenous BRs. In both the EBR and Brz + EBR treatment,

523

the BL content in deseeded berries was significantly higher than that in control, which is in line with

524

recent reports that EBR treatment can enhance the BL content in wheat (Janeczko et al., 2010;

525

Janeczko and Swaczynová 2010). The exogenous EBR may have penetrated through the skin and

526

accumulated inside the grape. Alternatively, the exogenous EBR may have triggered the conversion

527

from CS to BL. The VvBR6OX1 and VvDWF1 transcript levels in grape skin were significantly lower

528

in the EBR and Brz + EBR treatments than in the control (Fig.7 C), similar to the 6-deoxoCS and CS

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contents. The increase in the VvBRI1 mRNA level and BL content in the grape skins in the

530

EBR-treated samples suggested that the grape skin cells became more sensitive to BRs. The total

531

endogenous BRs content in seeds or deseeded berries were strongly correlated with the contents of

532

reducing sugars, glucose, and fructose in juice, and with the dry and fresh weights of the skin (Table 1).

533

This result suggested that exogenous EBR affected the unloading by transporting BRs into skin cells.

534

The effects of the three treatments on endogenous BRs during later stages (86-100 DAA) were

535

relatively negligible. This was most likely due to the field conditions and/or the manner in which EBR

536

was applied. In wheat, EBR’s impact on the amounts of carbohydrates, proteins, fats and minerals

537

contained in the grains of field-cultivated plants was smaller than in plants grown in pots (Janeczko et

538

al. 2010). Additionally, a higher EBR content in the leaf tissue was found when the hormone was

539

applied by soaking or drenching rather than by spraying (Janeczko and Swaczynová 2010).

540

The application of Brz to plants resulted in growth inhibition or dwarfism, but exogenous BL reversed

541

this effect. This indicates that BRs’ functions are essential to plant growth and development. In this

542

work, most of the targets analyzed (reducing sugars and titratable acids in berries, reducing sugars in

543

the skin, skin thickness, neutral and acidic INVs, VvSUC11, VvSUC12, VvSUC27, VvGIN1, VvHT3,

544

VvHT4, VvcwINV, and VvBRI1 transcript levels, and the 6-deoxoCS content in seeds) showed that an

545

EBR treatment applied 24 h after a Brz treatment neutralized the inhibitory effect of Brz on sugar

546

unloading. Our findings are consistent with a previous report in which the growth inhibition caused

547

by Brz was reversed by the addition of EBR in W. arrhiza (Bajguz and Asami 2005). The results

548

provide further evidence that endogenous BRs are involved in controlling sugar unloading in grape

549

berries during véraison.

550

Besides BRs, some other plant hormones such as GA3 and ABA, promote carbon allocation to grape

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Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison

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berries (Moreno et al., 2011). Genetic analyses showed that signaling in plants is closely associated

552

with ABA biosynthesis and signaling (Dekkers and Smeekens 2007). Cross-talk between hormone

553

pathways is a well-explored phenomenon, and there are many examples of hormone pathways that

554

modify, impinge upon, or promote the signaling of other hormones. As mentioned above, BRs may

555

regulate VvHTs genes, which are regulated by ABA under biotic stress (Hayes et al., 2010) via an

556

indirect pathway. Recent work has revealed that BRs and ABA have similar physiological function;

557

that is, both control stomatal opening and repress stomatal development in cotyledons and leaves in at

558

least some plants (Serna 2014). However, it is unknown whether there is crosstalk between BRs and

559

ABA or other hormones in sugar unloading in ripening grape berries. These topics are among our

560

future research interest.

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Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison

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Author's contribution

574

F. Xu designed the experiments, made all the measurements and the manuscript preparation. Z.M. Xi

575

was the supervisor of the whole experiment and has contributed to the design of the experiment and to

576

manuscript draft. C.J. Zhang has contributed to determine the enzyme activities. H. Zhang has

577

contributed to collect samples and to measure the physicochemical indices of the grape berries. Z.W.

578

Zhang critically revised and edited the manuscript.

579

Acknowledgements

580

This study was supported by the National Technology System for Grape Industry (CARS-30-zp-9), the

581

Natural Science Foundation of Shaanxi Province (2011JM3004). Thanks for the Key Laboratory of

582

Horticultural Plant Biology and Germplasm Innovation in Northwest, Ministry of Agriculture of China.

583

Thanks for Rongzi Chateau, Xiangning County, Linfen City, Shanxi Province, China.

584

Conflicts of Interest

585

The authors declare no conflict of interest.

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595 Reference

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secondary metabolism in grape berry using exogenous 24-epibrassinolide for enhanced phenolics

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695

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stress. Acta Physiol Plant. 36:2845-2852.

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Zhang, X.Y., Wang, X.L., Wang, X.F., Xia, G.H., Pan, Q.H., Fan, R.C., Wu, F.Q., Yu, X.C., Zhang, D.P.,

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705

Figure captions

707

Fig.1 Reducing sugars in juice (a), titratable acids in juice (b), reducing sugars in skin (c), fresh (d) and

708

dry weight of skins (e) of developing grape in response to the EBR, Brz, and Brz + EBR treatments.

709

The asterisk on a bar represents the content of each index in the three treated groups is significantly

710

higher or lower than the corresponding control at 1% level (for two asterisks) and 5% level (for one

711

asterisk), respectively. Statistically significant difference between the treated groups and the control is

712

calculated by Duncan’s new multiple range test. The bars indicate the mean of three values and their

713

standard deviation. Reducing sugar as glucose; Titratable acid as tartaric acid. DAA: day after anthesis.

714

Fig.2 The contents of glucose (a) and fructose (b) and sugars distribution (c-g) in deseeded berries of

715

developing grape in response to the EBR, Brz, and Brz + EBR treatments. The asterisk on a bar

716

represents the content of each index in the three treated groups is significantly higher or lower than the

717

corresponding control at 1% level (for two asterisks) and 5% level (for one asterisk), respectively.

718

Statistically significant difference between the treated groups and the control is calculated by Duncan’s

719

new multiple range test. The bars indicate the mean of three values and their standard deviation. DAA:

720

day after anthesis.

721

Fig.3 Acid invertase (a), netural invertase (b) and sucrose synthase (c) activities in the skin of

722

developing grape in response to the EBR, Brz, and Brz + EBR treatments. The asterisk on a bar

723

represents the content of each index in the three treated groups is significantly higher or lower than the

724

corresponding control at 1% level (for two asterisks) and 5% level (for one asterisk), respectively.

725

Statistically significant difference between the treated groups and the control is calculated by Duncan’s

726

new multiple range test. The bars indicate the mean of three values and their standard deviation. DAA:

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day after anthesis

728

Fig.4 Fold changes of gene (VvHT1-6 and VvMSA) expression between the treated group and the

729

corresponding control in the skin of developing grape in response to the EBR, Brz, and Brz + EBR

730

treatments. The berries that were treated with deionized water were taken as the control. The vertical

731

axis refers to the fold change in expression of the target gene in the three treated berries relative to that

732

in the control, which is calculated using the 2-∆∆Ct method (Livak and Schmittgen 2001). The bars

733

indicate the mean of three values and their standard deviation. DAA: day after anthesis.

734

Fig.5 Fold changes of gene (VvSUC11,12,27, VvcwINV and VvGIN1,2 ) expression between the treated

735

group and the corresponding control in the skin of developing grape in response to the EBR, Brz, and

736

Brz + EBR treatments. The berries that were treated with deionized water were taken as the control.

737

The vertical axis refers to the fold change in expression of the target gene in the three treated berries

738

relative to that in the control, which is calculated using the 2-∆∆Ct method (Livak and Schmittgen 2001).

739

The bars indicate the mean of three values and their standard deviation. DAA: day after anthesis.

740

Fig.6 Contents of endogenous BRs (6-DeoxoCS, CS and BL) in deseeded berries (a) and seeds (b) and

741

fold changes of gene (VvBR6OX1, VvDWF1 and VvBRI1) expression between the treated group and the

742

corresponding control in the skin (c-e) of developing grape in response to the EBR, Brz, and Brz +

743

EBR treatments. The berries that were treated with deionized water were taken as the control. The

744

vertical axis refers to the fold change in expression of the target gene in the three treated berries relative

745

to that in the control, which is calculated using the 2--∆∆Ct method (Livak and Schmittgen 2001). The

746

asterisk on a bar represents the content of each index in the three treated groups is significantly higher

747

or lower than the corresponding control at 1% level (for two asterisks) and 5% level (for one asterisk),

748

respectively. Statistically significant difference between the treated groups and the control is calculated

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Brassinosteroids are involved in controlling sugar unloading in Vitis vinifera ‘Cabernet Sauvignon’ berries during véraison

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by Duncan’s new multiple range test. The bars indicate the mean of three values and their standard

750

deviation. DAA: day after anthesis.

751

Table.1 Correlations among reducing sugars (J and S), glucose(J), fructose(J), dry weight (S), fresh

752

weight (S), Acidic INV activity (S), Neutral INV activity (S), SuSyn activity (S) and total BRs (DB and

753

SE) of grape berries measured at five stages (n =19).

754

Note: J: juice, S: skin, DB: deseeded berries, SE: seeds. Total BRs = castastarone +

755

6-deoxocastastarone + brassinolide. * Correlation is significant at the 0.05 level. ** Correlation is

756

significant at the 0.01 level.

757

Supplemental Table 1 Physicochemical properties of the soil from the vineyard in this study.

758

Supplemental Table 2 Spraying and Sampling time. Note: DAA: Number of days after anthesis; DAW:

759

Number of weeks after anthesis; RR: red ripe; BPC: berries per cluster; * The phenological stages

760

according to Eichhorn and Lorenz (1977).

761

Supplemental Table 3 List of primers for real-time PCR in this study.

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Table.1 Correlations among reducing sugars (J and S), glucose(J), fructose(J), dry weight (S), fresh weight (S), Acidic INV activity (S), Neutral INV activity (S), SuSyn

Reducing sugar (J)

Reducing sugar (S)

Glucose (DB)

Fructose (DB)

Dry weight (S)

Fresh weight (S)

RI PT

activity (S) and total BRs (DB and SE) of grape berries measured at five stages (n =19) Acidic INV

Neutral

SuSyn

activity (S)

INV

activity (S)

Total BRs (DB)

Total BRs (SE)

activity (S)

1

Reducing sugars(S)

0.758**

1

Glucose (DB)

0.993**

0.735**

1

Fructose (DB)

0.995**

0.755**

0.992**

1

Dry weight (S)

0.973**

0.744**

0.977**

0.981**

1

Fresh weight(S)

0.888**

0.501*

0.901**

0.902**

0.927**

0.388

0.138

0.392

0.399

0.391

0.338

1

0.655**

0.304

0.686**

0.642**

0.650**

0.664**

0.675**

1

SuSyn activity (S)

0.925**

0.619**

0.920**

Total BRs (DB)

0.697**

0.267

0.713**

Total BRs (SE)

0.717**

0.755**

activity (S)

0.728**

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Neutral INV

EP

activity (S)

1

0.939**

0.907**

0.897**

0.340

0.572*

1

0.663**

0.612**

0.620**

0.246

0.598**

0.591**

1

0.723**

0.785**

0.645**

0.024

0.467*

0.570*

0.351

AC C

Acidic INV

SC

Reducing sugars(J)

1

J: juice, S: skin, DB: deseeded berries, SE: seeds. Total BRs = castastarone + 6-deoxocastastarone + brassinolide. * Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level.

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a

**

160 140

**

*

**

**

**

**

**

120 60 40 20 0

**

60

66

86

DAA 6

**

16

**

14 12 10 8

**

100

6 4 2 0

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66

86

DAA

93

100

**

5

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Dry weight of skins (g/100 berries)

e

93

18

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Reducing sugar in skin (mg/g)

180

SC

200

Fresh weight of skins (g/100 berries)

d

c

4 3

**

EP

2 1

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0

60

66

86

DAA

93

100

Fig.1 Reducing sugars in juice (a), titratable acids in juice (b), reducing sugars in skin (c), fresh (d) and dry weight of skins (e) of developing grape in response to the EBR, Brz, and Brz + EBR treatments. The asterisk on a bar represents the content of each index in the three treated groups is significantly higher or lower than the corresponding control at 1% level (for two asterisks) and 5% level (for one asterisk), respectively. Statistically significant difference between the treated groups and the control is calculated by Duncan’s new multiple range test. The bars indicate the mean of three values and their standard deviation. Reducing sugars as glucose; Titratable acids as tartaric acid. DAA: day after anthesis.

ACCEPTED MANUSCRIPT

a

b

CK

70

EBR

60

Brz+EBR

80

** *

Brz

**

** *

70

**

*

60

40

* * **

30

**

20 10

50 40

**

*

30 20

**

10

0

60

66

86

93

100

0

60

66

86

DAA

DAA

c

d

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50

Fructose (mg/g)

Glucose (mg/g)

80

100

g

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Fig.2 The contents of glucose (a) and fructose (b) and sugars distribution (c-g) in deseeded berries of developing grape in response to the EBR, Brz, and Brz + EBR treatments. The asterisk on a bar represents the content of each index in the three treated groups is significantly higher or lower than the corresponding control at 1% level (for two asterisks) and 5% level (for one asterisk), respectively. Statistically significant difference between the treated groups and the control is calculated by Duncan’s new multiple range test. The bars indicate the mean of three values and their standard deviation. DAA: day after anthesis.

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a

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Fig. 3 Acidic invertase (a), netural invertase (b) and sucrose synthase (c) activities in the skin of developing grape in response to the EBR, Brz, and Brz + EBR treatments. The asterisk on a bar

TE D

represents the content of each index in the three treated groups is significantly higher or lower than the corresponding control at 1% level (for two asterisks) and 5% level (for one asterisk), respectively. Statistically significant difference between the treated groups and the control is calculated by Duncan’s new multiple range test. The bars indicate the mean of three values and their standard deviation. DAA:

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day after anthesis.

ACCEPTED MANUSCRIPT b

a

c

4

Fold change of gene expression

EBR

Brz+EBR 3

VvHT1

VvHT2

4

2

2 1

0.5 1

0

0.0 86

93

100

60

66

86

93

f

e

12

VvHT4

10

60

100

VvHT5

3

10

8

8 2

6

6

4 1

4

Trace

2

2

0

0 60

66

86

93

100

0 60

66

DAA

g 2.0

VvMSA

1.0

0.5

0.0 66

86

93

100

DAA

93

100

86

93

100

VvHT6

60

66

86

93

100

DAA

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60

86 DAA

66

12

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Fold change of gene expression

3

1.0

60

Fold change of gene expression

VvHT3

5

1.5

0

d

6

2.0

Brz

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Fig.4 Fold changes of gene (VvHT1-6 and VvMSA) expression between the treated group and the corresponding control in the skin of developing grape in response to the EBR, Brz, and Brz + EBR treatments. The berries that were treated with deionized water were taken as the control. The vertical axis refers to the fold change in expression of the target gene in the three treated berries relative to that

EP

in the control, which is calculated using the 2-∆∆Ct method (Livak and Schmittgen 2001). The bars

AC C

indicate the mean of three values and their standard deviation. DAA: day after anthesis.

ACCEPTED MANUSCRIPT b

a

c

VvSUC11

VvSUC12

Brz Brz+EBR

300

30

10

1

4

VvSUC27

35 2

6

5

2 60

66

86

93

100

d

0

60

66

86

93

100

e 20 EBR

VvcwINV

15

Brz Brz+EBR

0

60

5.5

VvGIN1

5.0

6

4.5

3.5 4

3.0

1.0 1.5 2

1.0 0.5 0.5

0.0

0.0 60

66

86

93

100

86

93

100

f

6.0

4.0 10

66

RI PT

0

Fold change of gene expression

40

EBR

350

VvGIN2

SC

Fold change of gene expression

3

0

60

66

86 DAA

100

60

66

86

93

100

DAA

M AN U

DAA

93

Fig.5 Fold changes of gene (VvSUC11,12,27, VvcwINV and VvGIN1,2 ) expression between the treated group and the corresponding control in the skin of developing grape in response to the EBR, Brz, and Brz + EBR treatments. The berries that were treated with deionized water were taken as the control. The vertical axis refers to the fold change in expression of the target gene in the three treated berries

TE D

relative to that in the control, which is calculated using the 2-∆∆Ct method (Livak and Schmittgen 2001).

AC C

EP

The bars indicate the mean of three values and their standard deviation. DAA: day after anthesis.

ACCEPTED MANUSCRIPT b

M AN U

SC

RI PT

a

c

d

Brz+EBR

1.0

0.8

0.6

0.4

0.2

0.0

66

86

AC C

60

93

Fold change of gene expression

TE D

Brz

EP

Fold change of gene expression

1.2

1.2

VvBR6OX1

EBR

VvDWF1

1.0

0.8

0.6

0.4

0.2

0.0 100

60

66

86 DAA

93

100

DAA

VvBRI1 Fold change of gene expression

e

6

4

2

0 60

66

86

93

100

DAA

Fig.6 Contents of endogenous BRs (6-deoxocastastarone, castastarone, and brassinolide) in deseeded berries (a) and seeds (b) and fold changes of gene (VvBR6OX1, VvDWF1 and VvBRI1) expression

ACCEPTED MANUSCRIPT between the treated group and the corresponding control in the skin (c-e) of developing grape in response to the EBR, Brz, and Brz + EBR treatments. The berries that were treated with deionized water were taken as the control. The vertical axis refers to the fold change in expression of the target gene in the three treated berries relative to that in the control, which is calculated using the 2--∆∆Ct

RI PT

method (Livak and Schmittgen 2001). The asterisk on a bar represents the content of each index in the three treated groups is significantly higher or lower than the corresponding control at 1% level (for two asterisks) and 5% level (for one asterisk), respectively. Statistically significant difference between the treated groups and the control is calculated by Duncan’s new multiple range test. The bars indicate the

AC C

EP

TE D

M AN U

SC

mean of three values and their standard deviation. DAA: day after anthesis.

ACCEPTED MANUSCRIPT Highlights (1) 24-Epibrassinolide treatment rapidly increased the sugars content in grape berries. (2) 24-Epibrassinolide treatment increased sucrose synthase and invertase activities.

(4)

24-Epibrassinolide

treatment

altered

biosynthesis

of

AC C

EP

TE D

M AN U

SC

brassinosteroids.

the

RI PT

(3) 24-Epibrassinolide treatment altered the mRNA levels of sugar transporter genes. endogenous

ACCEPTED MANUSCRIPT Author's contribution F. Xu designed the experiments, made all the measurements and the manuscript preparation. Z.M. Xi was the supervisor of the whole experiment and has contributed to the design of the experiment and to

RI PT

manuscript draft. C.J. Zhang has contributed to determine the enzyme activities. H. Zhang has contributed to collect samples and to measure the physicochemical indices of the grape berries. Z.W.

AC C

EP

TE D

M AN U

SC

Zhang critically revised and edited the manuscript.

ACCEPTED MANUSCRIPT

Sand

Clay

Saturation Silt (%)

Physical

RI PT

Supplemental Table 1 Physicochemical properties of the soil from the vineyard in this study. Texture

(%)

(%)

(%)

49.06

28.40

22.54

Clay loam

53.0

Organic

Total

Total

Total

Nitrate

Ammonium

Available

Available

Available

matter

nitrogen

phosphorus

potassium

nitrogen

nitrogen

nitrogen

phosphorus

potassium

(g/kg)

(g/kg)

(g/kg)

(g/kg)

(mg/kg)

(mg/kg)

(mg/kg)

(mg/kg)

(mg/kg)

12.6

0.765

0.631

17.42

19

5.8

24.8

10.47

102

M AN U

8.52

TE D

properties

EP

pH

AC C

Chemical

SC

properties

ACCEPTED MANUSCRIPT Supplemental Table 2 Spraying and Sampling time. First

Second

Third

Fourth

Fifth

Sampling

Sampling

Sampling

Sampling

Sampling

August 9th

August 15th

September 4th

September 11th

September 18th

Spraying time August 8th or Date (2013) 9th 59

60

66

86

93

100

DAW

9

9

10

13

14

15

35

35

37

37

Phenological stages Stage of

10% RR 10% RR BPC

100% RR BPC 100% RR BPC

BPC

development

+3 weeks

RI PT

DAA

37

38

100% RR BPC

Maturity or

+4 weeks

harvest

SC

Note: DAA: Number of days after anthesis; DAW: Number of weeks after anthesis; RR: red ripe; BPC:

AC C

EP

TE D

M AN U

berries per cluster; * The phenological stages according to Eichhorn and Lorenz (1977).

ACCEPTED MANUSCRIPT Supplemental Table 3 List of primers for real-time PCR in this study. Accession number

Primer

VvActin

BN000705

F

CTTGCATCCCTCAGCACCTT

R

TCCTGTGGACAATGGATGGA

F

GTCTATGTTTCAGGGTTTG

R

AAGAAGATTTGGGCTATG

F

GTTGCCGTCAACTTCGCAAC

R

GAAGGAATTTAGCTATGGCAGAG

AY538259 and

F

AGAGGAACTATGGAGGTGG

AY854146

R

AACAAGGCAAGCAACGAC

AY538260

F

CTGATGTTGCAGCGTGTTC

R

GGAGGCCATACCAACTACG

F

GACCTCCACTTTCTTCGC

R

AAGCACCACTCCCACAAT

F

ACCCTTAGTCTTGTGCCT

AJ001061

VvHT2

AY663846

VvHT3 VvHT4 VvHT5

AY538261

VvHT6

AY861386

VvMSA

AF281656

VvcwINV

VvSUC11 VvSUC12 VvSUC27

AF021808

AF021809

AF021810

NM_001280960.1

AC C

VvBR6OX1

AAB47172.1

TE D

VvGIN2

AAB47171.1

EP

VvGIN1

AY538262

VvDWF1 VvBRI1

XM_002284610.1

TTCGCATACTTCCCGTTT

M AN U

R

SC

VvHT1

Sequence (5'→3')

RI PT

Gene name

F

AGTCGGAGAAAGACCCTG

R

CTCGTGATGCTCGTGGAA

F

ATGAATCATCTAGYGTGGAGCAC

R

CTTAAACGATATCTCCACATCTGC

F

CCATCTCCATCCCATCGTAACC

R

GGCTATCCAAGTTTCCAACCAACC

F

GAGCACAGTTCCAGTAATCAAAGG

R

GTGAGGCGTAGTTTTAGGACTCC

F

CCATGGGATCAACTTTTTG

R

CATTTTACCACCCATATTGAT

F

CTTCATCCATTCCTCATCCA

R

GCGGCAATCATACAACTG

F

TGTAGTGGGTGCGTTTGC

R

GATGACCGTGGGCTCTACA

F

GACAAGAGCTTAGAGTCCCAAAAC

R

GAAAATTATTGTACATCCATATTGCTT

F

ACCGAGAAGGAAGTGCAGGAG

R

ACCATCACATTCGTTGAGCAGG

F

AAGGTAGCGTGTGCCTGTTT

R

GTTTCCCTGCTACTGCTTGC