ABA and its signaling pathway are involved in the cold acclimation and deacclimation of grapevine buds

Scientia Horticulturae 256 (2019) 108565

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ABA and its signaling pathway are involved in the cold acclimation and deacclimation of grapevine buds

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Sebastián Rubio, Francisco J. Pérez

Universidad de Chile, Fac. Ciencias, Lab. de Bioquímica Vegetal, Casilla 653, Santiago, Chile

A R T I C LE I N FO

A B S T R A C T

Keywords: Abscisic acid (ABA) Cold-hardiness Grapevine buds Uniconazole-P

The grapevine (Vitis vinifera L.) buds as well as the buds of other deciduous fruit trees acclimate and deacclimate to the cold during the autumn-winter season. Cold acclimation (CA) is characterized by an increase in bud coldhardiness, while deacclimation (DA) by a loss of it. The mechanisms underlying the transition of the buds from CA to DA are not well understood. In this study, the effect of ABA, LT and ABA + LT on the expression of the genes of the ABA signaling pathway, and the role that these genes and ABA play in the transition of the buds from the CA to the DA was investigated. In vitro experiments carried-out with single-bud cuttings were used to analyze the effect of ABA, LT and ABA + LT treatments on the expression of ABA signaling genes. To study the role of ABA and its signaling pathway in the transition of grapevine buds from CA to DA, applications of ABA and Uniconazole-P (Uni-P), an inhibitor of the enzyme that catabolizes ABA, were performed to the grapevine buds during their period of CA and DA. The results show that : a) VvPP2Cs were the only ABA signaling genes which were induced synergistically by ABA + LT, b) ABA improved the cold-hardiness of the buds only during its period of CA c) Uni-P advanced the appearance of the second peak of ABA in the buds and its transition from the CA to the DA.

1. Introduction Cold-hardiness or freezing-tolerance describes the ability of plants to withstand subfreezing temperatures (Kalberer et al., 2006; Wisniewski et al., 2018). The grapevine buds as well as the buds of other deciduous fruit trees acquire cold-hardiness before the arrival of winter, once they have entered into endodormancy and the temperatures begin to drop (Rubio et al., 2016; Gragin et al., 2017). Endodormancy, is a physiological state in which the activity of the bud meristem is inhibited by intrinsic factors located within the bud, and in grapevines is triggered by the SD-photoperiod (Rohde and Bahlerao, 2007; Kühn et al., 2009; Gragin et al., 2017). The development of coldhardiness in grapevine buds follows a well-defined curve of cold acclimation (CA) and deacclimation (DA). During the period of CA, endodormant grapevine buds gain cold-hardiness as the temperature decreases, while during the subsequent DA period, they loss coldhardiness (Ferguson et al., 2011, 2014; Londo and Kovaleski, 2017). The grapevine buds are cold acclimated during endodormancy, while they deacclimate during ecodormancy (Rubio et al., 2016; Gragin et al., 2017). However, the end of the endodormancy does not always coincide with the beginning of the DA. For example, in the southern hemisphere, the end of the endodormancy coincides with the beginning

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of the DA (Rubio et al., 2016), while in the northern hemisphere, where temperatures are cooler, the DA begins long after the buds have been released from the endodormancy (Gragin et al., 2017), and therefore, there is a period in which the buds are in the state of ecodormancy, but continue cold-acclimated, which suggests that under these circumstances, it is necessary that the temperature increase to start the DA of the buds. Recently, it has been shown that the DA rate of grapevine buds depends on the temperature and on the chilling accumulated by the buds. Thus, for the same DA temperature, buds that have been exposed to 360 chilling hours (CH) showed a lower DA rate than those exposed to 860 and 1580 CH (Kovaleski et al., 2018). Because the chilling accumulated by the buds is directly related to its endodormancy status, and this, in turn with its content of ABA (Zheng et al., 2015; Rubio et al., 2019b), the rate of DA of the buds could depend on its content of ABA. Previous studies, showed that the ABA levels increase as the grapevine buds enter endodormancy, and decrease, during the winter month towards the release of endodormancy (During and Bachmann, 1975; Emmerson and Powell, 1978; Koussa et al., 1994; Or et al., 2000). Recently, it has been reported that severe water deficit during berry development shortened the dormancy cycle of grapevines by advancing the release of buds from endodormancy (Shellie et al., 2018). This is interesting, since the transcriptomic

Corresponding author. E-mail address: [email protected] (F.J. Pérez).

https://doi.org/10.1016/j.scienta.2019.108565 Received 21 March 2019; Received in revised form 3 June 2019; Accepted 7 June 2019 Available online 14 June 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

Scientia Horticulturae 256 (2019) 108565

S. Rubio and F.J. Pérez

LTE, the lower temperature that the grapevine bud can resist without damage corresponds to its LTE value which is a measure of its coldhardiness (Ferguson et al., 2011; Mills et al., 2006; Pierquet and Stushnoff, 1980). Each value corresponds to the average of 16 biological replicates of single bud.

analysis of the response of grapevine leaves to dehydration, identified the differential expression of ABA signaling genes in three species that differ in drought tolerance (Hopper et al., 2016). On the other hand, it has been shown that ABA and LT synergistically increase the coldhardiness of single-bud cuttings of grapevines (Rubio et al., 2019a). This synergistic effect was mediated by the CBF/DREB1 transcription factors and was reverted by the dormancy breaking compound, hydrogen cyanamide (Rubio et al., 2019a), which reduce the ABA content in the buds by up-regulating the expression of the ABA catabolizing enzyme VvAH8´-hydroxylase (Vergara et al., 2017). All these facts indicate that ABA plays an important role in the maintenance and release of the bud from the endodormacy, as well as in its transition from the CA to the DA. The signaling and regulatory mechanisms that underlie the transition of the buds from the CA to the DA are complex and poorly understood. Here, we studied the effect of ABA and LT on the expression of ABA signaling genes, and the role that ABA and its signaling pathway play in the transition of the grapevine buds from the CA to the DA.

2.3. ABA determinations Fresh plant material (ten buds per harvest time) was washed with cold water before grounded with liquid nitrogen. The samples were extracted in a shaker for 1 h at 4 °C and for 10 min by ultra-sonication with 3 mL of 80% methanol containing 1% acetic acid and 3 ng of 2,3,5triiodobenzoic acid (TIBA) as an internal standard (Sigma-Aldrich, USA). The extracts were centrifuged at 3000 g for 10 min, and the supernatant was filtered through glass wool and a Sep-Pack C18 cartridge (Waters Assoc., Milford, MA, USA) that had been prewashed with 5 mL of 80% methanol. The procedure was repeated twice, and the filtrate was evaporated to dryness. After evaporation, the residue was dissolved in 1.2 mL of ethyl acetate, and 1.2 mL of 0.5 M KH2PO4 pH 3.0 was added. The mixture was agitated with a vortex and centrifuged at 3000 g for 3 min. After centrifugation, the ethyl acetate layer was removed and the aqueous phase was extracted twice with 1.2 mL of ethyl acetate. The collected ethyl acetate layers were evaporated to dryness. The dry sample was dissolved in 1 mL of ethyl acetate, and an ABA derivative was formed by reaction with pentafluorobenzyl bromide (PFB) (SigmaAldrich, USA) which allowed highly sensitive detection by an electron capture detector (Michler et al., 1986). A Shimadzu gas chromatograph (model GC-2014) equipped with an electron capture detector (ECD2014, Shimadzu Corporation, Kyoto, Japan) and computer integrator was used for ABA determinations. A CBP1 capillary column (25 m x0.25 mm internal diameter) with helium as the carrier gas at a flux of 1.5 mL min−1 was used. The temperature of the column was initially 80 °C and after 1 min was raised to 270 °C at a rate of 20 °C min−1 and maintained there for 5 min. The injector was operated in the splitless mode at 225 °C, and the temperature of the detector was 300 °C. A calibration curve for ABA-PFB derivative was constructed.

2. Material and methods 2.1. Plant material, experimental design and treatments Thompson seedless grapevines (Vitis vinifera L.) grown in a commercial vineyard at the Elqui valley (30° 02´ S elevation 643 m asl) in Chile was used in this study. The vines were planted on its own roots in 1979 and were drip irrigated and trained in an overhead training system. In 2017, five treatments were assigned to the vines in four completely randomized blocks, in which the experimental unit was 5 plants and the observation unit corresponds to the three central plants, and of each of these, 2 loaders with 8 buds each were sampled weekly. The treatments were as follow, I Control (sprayed only with deionized water), II (ABA 53 mg L−1sprayed on 13 June during the CA period), III (Uniconazole-P 50 mg L−1 sprayed on 13 June during the CA period), IV (ABA 53 mg L−1 sprayed on 4 July during the DA period), V (Uniconazole-P 50 mg/L sprayed on 4 July during the DA period). The vines were sprayed to runoff with a 15 L back sprayer averaging a spray volume of 0.5 L vine−1. For gene expression analysis, detached canes each carrying ten buds at position 5–14 were collected from the vineyard and transferred to the laboratory and excised in single-bud cuttings. The cuttings were mounted on propylene sheet and floated in tap water in a plastic container and exposed to the following treatments: (1) LT: buds were placed in a refrigerator at 4 °C in the dark, (2) ABA treatment: buds were sprayed with 100 μM ABA solution and placed in a growth chamber at 14 °C in the dark; (3) LT + ABA treatment: buds were sprayed with 100 μM ABA solution and placed in the refrigerator at 4 °C in the dark.

2.4. RNA purification and cDNA synthesis For gene expression analysis, total RNA was isolated and purified from grapevine buds (0.5 g FW) of V. vinifera Thompson seedless. In all cases, total RNA was extracted and purified using a modification of the method of (Chang et al., 1993), as described by Noriega et al. (2007). DNA was removed by treatment with RNase-free DNase (1 U μg−1 (Thermo Scientific, USA) at 37 °C for 30 min. First-strand cDNA synthesis was performed using the Superscript ® II RT system (Invitrogen, CA, USA). A 1 μg aliquot of purified RNA with 1 μL of oligo (dT)12-18 (0.5 μg x μL−1) was used as a primer and 1 μL of dNTP mix (10 mM) was used for cDNA synthesis. RNA quality and quantity were assessed with a Qubit 2.0 fluorometer (Invitrogen, USA).

2.2. Bud cold hardiness Bud cold-hardiness or freezing tolerance was measured in grape dormant buds by low temperature exotherm (LTE) detection using differential thermal analysis following the method described in Mills et al. (2006), in which the signals were recorded every 2 s, and a decrease in temperature of 4 °C per h starting at 10 °C and ending at -30 °C was programmed. The differential thermal analysis was performed with a Kryoscan, a freezing and data acquisition device that uses Peltier elements for the cooling and detection modules (Badulescu and Ernst, 2006). Bud cold hardiness was measured weekly in the field, and invitro in single-bud cuttings one week post-treatment. Generally two peaks were observed, one corresponded to the high temperature exotherm (HTE) which was assigned to the freezing point of extracellular (apoplast) water, which is non-lethal (Burke et al., 1976) and the other corresponded to the LTE which was assigned to the freezing point of intracellular water, which is lethal (Burke et al., 1976). Because lethal damage to the grapevine buds occurs at temperatures equal or less than

2.5. Quantitative real-time PCR Quantitative real-time PCR (RT-qPCR) was carried out in an Eco Real-Time PCR system (Illumina, San Diego, USA) using KAPA SYBR FAST (KK 4602) qPCR Master Mix (2 x). Design of specific primers for “ABA receptors” VvPYR1a (GSVIVT01013161001), VvPYR1b (GSVIVT01035362001), VvPYR1c (GSVIVT01032747001), “SnRK2-kinases”: VvSnRK2.4 (GSVIVTO1022427001), VvSnRK2.5 (GSVIVT01003554001), VvSnRK2.6 (GSVIVT01031806001) “Phosphatase 2C”: VvPP2C1 (GSVIVT01015156001), VvPP2C2 (GSVIVT01035420001), VvPP2C3 (GSVIVT01016816001), VvPP2C4 (GSVIVT01015308001) and VvPP2C7 (GSVIVT01018464001) and “transcription factors” :VvABI3 (GSVIVT01028540001), VvABI4 (GSVIVT01032785001) VvABI5 (GSVIVT01033832001), VvABF1 (GSVIVT01009485001) and VvABF2 (GSVIVT01031730001) genes was 2

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to the period of CA and was characterized by a gradual decrease in LTE values until reaching a minimum value of -16.4 °C on July 10. Subsequently, the LTE values increased although there was no increase in temperature. This second phase of the curve corresponds to the DA period of the buds. Additionally, the LTE of Thompson seedless buds was monitored during the autumn-winter seasons for four years (2014–2017) in the Elqui valley (Table 1), and for five years (2012–2016) in the Maipo valley (Table 2). In all cases a biphasic CA/ DA curve was observed with a transition point that occurred regularly during mid-July (Tables 1,2). 3.2. Effect of ABA, LT and ABA + LT on genes encoding VvRCAR/PYR/ PYL ABA receptors and VvSnRK2-kinases in grapevine dormant buds In the grape genome, 8 putative ABA receptors (Boneh et al., 2012a) and 6 SnRK2-kinases were identified (Boneh et al., 2012b). VvPYR1c and VvSnRK2.6 were the most responsive ABA receptors and SnRK2kinases to dehydration in leaves and roots of grapevines (Hopper et al., 2016). Here, the effect of LT, ABA and ABA + LT on the expression of 3 paralogs of ABA receptors VvPYR1a, VvPYR1b, VvPYR1c and 3 SnRK2kinases VvSnRK2.4, VvSnRK2.5 and VvSnRK2.6 were analyzed by RTqPCR in grapevine dormant buds after 1 week of treatment (Fig. 2). The results showed that the expression of VvPYR1a was significantly induced by LT and ABA + LT, while the expression of VvPYR1b was only induced by LT. On the contrary, the expression of VvPYR1c and the paralogs of SnRK2-kinases were not induced by any of the treatments (Fig. 2).

Fig. 1. (a) Daily mean temperatures (°C) (b) CA/DA curve of Thompson seedless buds grown in the Elqui valley during the fall-winter of 2017. Coldhardiness was determined weekly by measuring the LTE of grapevine buds by differential thermal analysis between 6 June to 30 July 2017. Bars represents s.d (n = 16).

carried-out using the primer3 program (Rozen and Skaletsky, 2000). cDNA was amplified under the following conditions: denaturation at 94 °C for 2 min and 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s. Relative changes in gene expression levels were determined using the 2−ΔΔCT method (Livak and Schmittgen, 2001). Each reaction was performed in at least three biological replicates, each with three technical replicates. “VvUBIQUITIN” and “VvACTIN” were used as a reference genes for normalization.

3.3. Effect of ABA, LT and ABA + LT on the expression of VvPP2Cs genes In the grapevine genome six PP2Cs paralogs were identified and VvPP2C4 is the major PP2C involved in ABA perception in leaves and roots (Boneh et al., 2012b). It has also been reported that transcript levels of VvPP2C4 and VvPP2C9 are inhibited by the dormancy breaking compound, hydrogen cyanamide, while that of VvPP2C2 is increased (Zheng et al., 2015). Here, the effect of LT, ABA and LT + ABA on the expression of VvPP2C1, VvPP2C2, VvPP2C3,VvPP2C4 and VvPP2C7 paralogs were studied by RT-qPCR in grapevine dormant buds after 1 week of treatment (Fig. 3). Although the combined effect of LT + ABA increased the expression of all VvPP2Cs genes as a general trend, only the expression of VvPP2C2 was increased significantly (Fig. 3).

3. Results 3.1. CA/DA curve in Thompson seedless grapevine buds Variations in the cold-hardiness of Thompson seedless buds grown in the Elqui valley in Chile over the autumn-winter season of 2017 and daily mean temperatures are shown in Fig. 1. The bud cold-hardiness was determined weekly by measuring the LTE of buds through differential thermal analysis. As a result, a biphasic curve with an inflection point on July 10 was observed, the first phase of the curve corresponds

3.4. Effect of ABA, LT and ABA + LT on the expression of AREB/ABF and ABI genes The expression pattern of ABA responsive genes is regulated mainly

Table 1 Bud LTE of Thompson seedless grapevines during grown in the Elqui valley during the years 2014–2017. The bud LTE was determined during the dormancy period by differential thermal analysis. 2014 Date

Bud LTE*

s.d+

2015 Date

Bud LTE

s.d

21 Apr 6 May 22 May 7 Jun 20 Jun 1 Jul 15 Jul 31 Jul 14 Aug 27 Aug

−10,3 −10,1 −10,9 −13,6 −14,3 −16,4 −13,0 −15,6 −14,5 −9,8

1,9 1,4 0,9 0,6 1,5 0,6 1,3 1,0 1,2 0,9

7 Apr 9 Jun 22 Jun 8 Jul 14 Jul 22 Jul 30 Jul 13 Aug 20 Aug 26 Aug

−11,2 −15,0 −15,9 −15,2 −16,4 −17,2 −16,4 −16,8 −15,8 −13,8

1,1 0,9 0,9 1,2 0,9 0,8 0,8 1,0 0,8 1,5

* Bud-LTE, low thermal exotherm of grapevine buds. + s.d standard deviation. 3

2016 Date

Bud LTE

s.d

11 Apr 18 Apr 9 May 23 May 6 Jun 30 Jun 6 Jul 12 Jul 19 Jul 15 Jul 9 Aug 20 Aug

−11,4 −11,9 −13,8 −14,8 −15,3 −15,9 −14,4 −15,0 −14,4 −12,3 −12,6 −11,0

1,2 1,3 1,1 1,1 1,6 1,1 1,5 0,8 1,7 1,5 1,3 1,1

2017 Date

Bud LTE

s.d

6 Jun 13 jun 20 Jun 27 Jun 3 Jul 10 Jul 17 Jul 24 JuL 30 Jul

−13,3 −13,4 −14,4 −14,8 −15,5 −16,4 −14,9 −14,6 −13,8

0,8 0,7 0,5 0,9 0,5 0,7 0,9 1,0 1,4

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Table 2 Bud LTE of Thompson seedless grapevine buds grown in the Maipo valley during the years 2012–2016. The bud LTE was determined during the dormancy period by differential thermal analysis. 2012 Date

*

+

22 May 4 Jun 18 Jun 26 Jun 3 Jul 10 Jul 17 Jul 24 Jul 31 Jul 6 Aug 13 Aug 21 Aug 28 Aug

−10,0 −13,4 −13,2 −11,0 −10,7 −12,8 −17,7 −13,8 −10,6 −13,3 −13,4 −11,1 −7,5

0,5 0,4 0,9 1,6 0,6 1,2 1,7 1,5 0,5 1,3 1,1 1,0 1,2

Bud LTE

s.d

2013 Date

Bud LTE

s.d

10 Apr 16 Apr 7 May 14 May 22 May 28 May 3 Jun 11 Jun 19 Jun 26 Jun 10 Jul 18 Jul 16 Aug 29 Aug 3 Sep

−7,7 −9,6 −9,9 −12,6 −9,5 −13,6 −14,5 −14,7 −16,8 −16,4 −17,2 −16,6 −15,7 −11,3 −11,3

1,2 0,9 1,2 1,0 1,3 0,7 1,6 0,9 1,6 1,1 1,5 1,6 1,4 1,7 1,2

2014 Date

Bud LTE

s.d

9 Apr 12 May 28 May 16 Jun 1 Jul 10 Jul 23 Jul 7 Aug 13 Aug 21 Aug 29 Aug

−6,9 −11,5 −14,9 −15,1 −16,2 −18,5 −16,5 −14,6 −11,9 −10,6 −9,6

1,4 1,2 0,7 1,4 0,8 0,8 1,5 0,9 1,7 1,5 1,7

2015 Date

Bud LTE

s.d

2016 Date

Bud LTE

s.d

7 Apr 4 May 13 May 27 May 15 Jun 22 Jun 8 Jul 14 Jul 22 Jul 30 Jul 14 Aug 22 Aug 28 Aug

−10,5 −13,5 −13,0 −13,4 −16,7 −15,1 −17,0 −17,2 −18,2 −17,1 −16,3 −15,0 −11,8

1,6 1,4 0,6 0,5 0,8 1,4 1,1 1,1 0,9 0,8 1,4 1,4 1,8

11 Apr 27 Apr 9 May 23 May 6 Jun 20 Jun 4 Jul 14 Jul 18 Jul 27 Jul 4 Aug 11 Aug 25 Aug

−11,6 −11,8 −13,4 −15,0 −15,6 −15,6 −19,7 −15,2 −14,9 −13,6 −13,0 −14,2 −9,0

1,4 1,3 1,3 1,4 1,8 1,1 1,6 1,3 1,5 1,3 1,5 1,4 1,2

* Bud-LTE, low thermal exotherm of grapevine buds. + s.d Standard deviation.

In grapevines, VvABI5 and VvABF2 have been linked to drought resistance (Hopper et al., 2016), and hydrogen cyanamide inhibits the expression of VvABF1 and VvABF2 in grapevine buds (Zheng et al., 2015). Here, the effect of LT, ABA and ABA + LT on the expression of VvABF1, VvABF2, VvABI3, VvABI4 and VvABI5 were studied by RTqPCR in grapevine dormant buds after 1 week of treatment (Fig. 4). None of the transcription factors belonging to the ABA signaling pathway was synergistically increased by the combined effect of ABA + LT as were the CBF/DREB1 transcription factors (Rubio et al., 2019a). However, VvABI5 was induced by LT, by ABA and by LT + ABA but not in a synergistically way. VvABI4 was not affected by any of the treatments, while VvABI3 was only induced by LT. On the contrary, VvABF1 and VvABF2 were repressed by LT and ABA (Fig. 4). 3.5. ABA and UNI-P increased cold-hardiness of grapevine buds only during their CA period To test whether the timing of the application of ABA and UNI-P is a relevant factor for the acquisition of bud cold-hardiness. ABA and UNIP solutions were applied to the buds at the beginning (13 June) and at the end (4 July) of their CA period, and the bud LTE was measured weekly until bud-break. The results showed that both ABA and UNI-P applications increased the cold-hardiness of grapevine buds when they were applied at the beginning of the CA period; however, when applied at the end of the CA period, no significant effects on bud cold-hardiness were observed (Fig. 5a, b). Although both ABA and UNI-P increased the cold-hardiness of grapevine buds when they were applied during the CA period, the application of UNI-P advanced in one week the transition of the buds from the CA to the DA period, whereas, application of ABA did not alter the transition date with respect to control buds (Fig. 5c). 3.6. ABA and UNI-P increased endogenous ABA content in grapevine buds regardless of the time of application Fig. 2. Effect of LT, ABA+LT and ABA on the expression of ABA receptors VvPYR1a, VvPYR1b and VvPYR1c and SnRK2-kinases VvSnRK2.4, VvSnRK2.5 and VvSnRK2.6 in grapevine dormant buds collected on 14 May 2017 after 1 week of treatment. Transcript levels were determined by RT-qPCR and normalized against VvUBIQUITIN and VvACTIN. Values are averages of three biological replicates each with three technical repetitions. Bar represent ± s.d. * P ≤ 0.05; ** P ≤ 0.01 (Dunnett´s multiple comparison test).

The exogenous applications of ABA and UNI-P increased the endogenous content of ABA in grapevine buds regardless of the date of its application (Fig. 6 a, b). Early applications (June 13) of ABA to grapevine buds increased their endogenous ABA content, and the maximum concentration of ABA in the buds was reached two weeks after the application, later, no differences were detected with respect to the control buds (Fig. 6 a). Although the early applications of Uni-P to the grapevine buds also significantly increased the endogenous content of ABA, the maximum concentration of ABA in the buds was reached one week earlier than in the control buds and those treated with ABA

by two different families of bZip transcription factors: one that is active in seeds (ABI) and the other in vegetative tissues (AREB/ABF) (Yamaguchi-Shinozaki and Shinozaki., 2006; Nakashima et al., 2014). 4

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Fig. 3. Effect of LT, ABA+LT and ABA on the expression of PP2C-kinases VvPP2C1, VvPP2C2, VvPP2C3, VvPP2C4 and VvPP2C7 in grapevine dormant buds collected on 14 May 2017 after 1 week of treatment. Transcript levels were determined by RT-qPCR and normalized against VvUBIQUITIN and VvACTIN. Values are averages of three biological replicates each with three technical repetitions. Bar represent ± s.d. * P ≤ 0.05(Dunnett´s multiple comparison test).

correlation between the content of endogenous ABA in the buds and bud cold-hardiness was observed during the CA period when data from control and treated buds were considered (Fig. 7b), however, later during the DA period, no correlation was detected between these two parameters (Fig. 7c).

(Fig. 6 a). The late applications (4 July) of ABA and UNI-P also increased the content of endogenous ABA in the buds, but in all the cases, the maximum concentration of ABA was reached one week after the application (Fig. 6 b). 3.7. Relationship between endogenous ABA content and bud cold-hardiness

3.8. Correlation between the chilling accumulated and bud LTE during the CA period

To investigate the relationships between ABA content and coldhardiness in grapevine buds through the CA and DA period, the coldhardiness and the content of endogenous ABA were determined weekly between early June and the end of July in Thompson seedless buds grown in the Elqui valley. The results showed that the content of endogenous ABA in the buds remained high during the CA period and decreased during the DA period (Fig. 7a). Moreover, a positive

A correlation between the accumulated chilling measured as chilling hours (CH) (Weinberger, 1950), and the cold-hardiness of grapevine buds was observed throughout their CA period. The data correspond to measurements carried-out for 4 years in the Elqui valley and for 5 years in the Maipo valley. A correlation with an r2 = 0.63 was 5

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Fig. 4. Effect of LT, ABA+LT and ABA on the expression of ABA signaling transcription factors VvABI3, VvABI4 and VvABI5 and AREB/ABFs VvABF1 and VvABF2 in grapevine dormant buds collected on 14 May 2017 after 1 week of treatment. Transcript levels were determined by RT-qPCR and normalized against VvUBIQUITIN and VvACTIN. Values are averages of three biological replicates each with three technical repetitions. Bar represent ± s.d. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 (Dunnett´s multiple comparison test).

valleys over several years gave us as a result a biphasic CA/DA curve. During the phase of CA, the bud cold-hardiness begin to increase in mid-April, when the buds are endodormant and temperature begins to drop (Rubio et al., 2016). This phase of CA, in which the buds become more tolerant to freezing lasts until July, after which, there is a loss in bud cold-hardiness, initiating their DA (Fig. 1). This phenomenon was thoroughly repeated throughout the years in the Elqui and Maipo valleys in Chile (Table 1,2).

obtained when all data were plotted (Fig. 8a), while when only data from the Maipo valley were plotted an r2 = 0.75 was obtained (Fig. 8b).

4. Discussion 4.1. Cold acclimation and deacclimation of grapevine buds grown in the Elqui and Maipo valleys The monitoring of the bud cold-hardiness in Thompson seedless grapevines during the autumn-winter season in the Elqui and Maipo 6

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Fig. 5. Effect of the application time of (a) ABA, (b) UNI-P on the cold-hardiness of Thompson seedless grapevine buds (c) Comparison of the maximum cold-hardiness of control buds with those treated with ABA and UNI-P. The applications of ABA and UNI-P were carried-out on 13 June (red arrow) and 4 July (blue arrow) of 2017 respectively, and the bud cold hardiness was determined weekly by measuring the LTE of grapevine buds by differential thermal analysis. Bars represents ± s.d * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 (Student´s test) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 6. Exogenous applications of ABA and UNI-P increased the content of endogenous ABA in grapevine buds regardless of the application time. Thompson seedless buds were sprayed (a) on 13 June with ABA and UNI-P (b) and on 14 July 2017 with ABA and UNI-P. ABA was determined weekly after the treatments with a gas chromatograph equipped with an electron capture detector. Values are the average of three biological replicates. Bars represents ± s.d * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001(Dunnett´s multiple comparison test).

since ABA applications to the leaves of field-grown grapevines increase their bud cold-hardiness (Zhang and Dami, 2012). Additionally, it has been shown that ABA and LT synergistically increase the cold-hardiness of single-bud cuttings of grapevines, and this effect was mediated by the CBF/DREB1 transcription factors (Rubio et al., 2019a). In this artificial system, the endogenous content of ABA in the buds correlated positively with the bud cold-hardiness (Rubio et al., 2019a). Interestingly, the same positive correlation between bud cold-hardiness and ABA content was detected here in field grown grapevines during the CA

4.2. The role of ABA and its signaling pathway in the transition of grapevine buds from CA to DA Despite the annual regularity with which the buds move from CA to DA in both valleys, the factors that regulate this transition are poorly understood. It has been suggested that the transition from CA to DA is driven by temperature increases (Ferguson et al., 2011, 2014; Londo and Kovaleski, 2017). However, it also seems likely that the plant hormone abscisic acid (ABA), plays an important role in this transition, 7

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Fig. 7. (a) Relationship between bud cold-hardiness and ABA content in Thompson seedless buds throughout the CA/DA period, (b) Correlation between bud coldhardiness and ABA content during the CA period, (c) Correlation between bud cold-hardiness and ABA content during the DA period. Cold-hardiness was determined by measuring the LTE of grapevine buds by differential thermal analysis. The content of endogenous ABA in the buds was measured with a gas chromatograph equipped with an electron capture detector. Bars represents s.d (n=16) for bud LTE and (n =3) for ABA determinations.

However, additional research revealed that some CBF/DREB1 genes were induced by ABA and drought (Xiao et al., 2006). Recently, we demonstrated that VvCBF/DREB1 genes are synergistically induced by ABA + LT and that they mediates the cold-hardiness response in grapevine buds (Rubio et al., 2019a). Among the ABA signaling genes only the ABA negative regulators VvPP2C2 was induced synergistically by ABA + LT (Fig. 3), whereas genes encoding for the ABA receptors VvPYR/RCAR, the VvSnRK2s kinases and the VvAREB/ABF transcription factors which are positive regulators of ABA were not synergistically induced by ABA + LT. (Fig. 2,4). These results indicate that at least at the transcriptional level only the CBF/DREB1 transcription factors regulate the acquisition of cold-hardiness in grapevine buds, while the ABA signaling transcription factors (AREB/ABF) do not. Interestingly, because ABA increased the cold-hardiness of the buds only during their CA period, which in our case coincides with the period of endodormancy (Rubio et al., 2016), it is likely that the expression of the VvCBF/DREB1 genes are induced synergistically by ABA + LT only during the endodormancy period. This suggest that during endodormancy, a special situation must occur in the grapevine bud that causes the CBF/DREB genes to respond to ABA + LT in a synergistic manner, and explains why the grapevine buds acclimate to the cold only during their period of endodormancy (Rubio et al., 2016; Gragin et al., 2017). Bud cold-hardiness and the chilling accumulated correlates only

period (Fig. 7a). However, despite the fact that ABA and UNI-P applications increased the ABA content during the DA of the buds, their coldhardiness did not vary with respect to the control, and no correlation was detected between them (Fig. 7b). These results suggest that the insensitivity of the buds to ABA in the acquisition of cold-hardiness during the DA period, could be due to a blockage of the ABA signaling pathway. Progress in understanding ABA perception and signal transduction has been made recently (Yamagushi-Shinozaki and Shinozaki, 2006; Cutler et al., 2010; Raghavendra et al., 2010; Weiner et al., 2010). It was revealed that SnRK2, group A PP2Cs, and RCAR/PYR/PYL ABA receptors control the ABA signaling pathway including AREB/ ABFs transcription factors. The phosphorylation of AREB/ABFs by SnRK2 is critical in the ABA-dependent signaling network (Fujita et al., 2013; Nakashima et al., 2009). The promoter regions of ABA-responsive genes contains a conserved cis-element named ABRE, which controls gene expression and is bind by AREB/ABFs transcription factors (Nakashima et al., 2014). Analysis of the promoter regions of genes showing ABA-independent expression in stress response, show a ciselement designated DRE/CRT. A group of AP2/ERF transcription factors were identified as CBF/DREB1 in Arabidopsis which specifically interact with the DRE/CRT and control the expression of a large number of stress-responsive genes (Liu et al., 1998). Originally, it was thought that the genes encoding the CBF/DREB1 transcription factors were only induced by low temperatures (Gilmour et al., 2004). 8

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maximum cold hardiness occurred one week earlier than in the control and ABA-treated buds (Fig. 5,6), suggesting that the second peak of ABA and the maximum cold-hardiness are synchronized. 5. Conclusions 5.1.- In the grapevine buds, only the VvCBF/DREB1 transcription factors are involved in the development of cold-hardiness, while the AREB/ABF belonging to the ABA signaling pathway do not. 5.2.- ABA increase cold-hardiness only during the CA period of the buds, which in our case, coincides with their endodormancy period. 5.3.- The appearance of a second peak of ABA during the endodormancy is synchronized with the maximum cold hardiness reached by the buds. Acknowledgements The logistic support of Martinez & Valdivieso S.A in the field design, treatments, sample collection and financial support to SR is gratefully acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2019.108565. References Badulescu, R., Ernst, M., 2006. Changes of temperature exotherms and soluble sugar in grapevine (Vitis vinifera L) buds during winter. J Appl. Bot Food Qual. Angew. Bot. 80, 165–170. Boneh, U., Biton, I., Zheng, C., Schwartz, A., Ben-Ari, G., 2012a. Characterization of potential ABA receptors in Vitis vinifera. Plant Cell Rep. 31, 311–321. Boneh, U., Biton, I., Schwartz, A., Ben-Ari, G., 2012b. Characterization of the ABA signal transduction pathway in Vitis vinifera. Plant Sci. 187, 89–96. Burke, M.J., Gusta, L.V., Quamme, H.A., Weiser, C.J., Li, P.H., 1976. Freezing and injury in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 27, 507–528. Chang, S., Puryear, J., Cairney, J., 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116. Cutler, S.R., Rodríguez, P.R., Finkelstein, R.R., Abrahams, S.R., 2010. Abscisic acid: emergence of a core signaling network. Ann. Rev. Plant Biol. 61, 651–679. During, H., Bachmann, O., 1975. Abscisic acid analysis in Vitis vinifera in the period of endogenous bud dormancy by high pressure liquid chromatography. Physiol. Plant. 34, 201–203. Emmerson, J.G., Powell, L.E., 1978. Endogenous abscisic acid in relation to rest and bud burst in three Vitis species. J. Am. Soc. Hortic. Sci. 103, 677–680. Ferguson, J.C., Tarara, J.M., Mills, L.J., Grove, G.G., Keller, M., 2011. Dynamic thermal time model of cold hardiness for dormant grapevine buds. Ann. Bot. 107, 389–396. Ferguson, J.C., Moyer, M.M., Mills, L.J., Hoogenboom, G., Keller, M., 2014. Modeling dormant bud cold hardiness and budbreak in twenty three Vitis genotypes reveals variation by region of origin. Am. J. Enol. Vitic. 65, 59–71. Fujita, Y., Yoshida, T., Yamaguchi-Shinozaki, K., 2013. Pivotal role of the AREB/ABFSnRK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants. Physiol. Plant. 147, 15–27. Gragin, J., Serpe, M., Keller, M., Shellie, K., 2017. Dormancy and cold hardiness transition in wine grape cultivars Chardonnay and Cabernet Sauvignon. Am. J. Enol. Vitic. 68, 195–202. Gilmour, S.J., Fowler, S.G., Thomashow, M.F., 2004. Arabidopsis transcriptional activator CBF1, CBF2 and CBF3 have matching functional activities. Plant Mol. Biol. 54, 761–781. Hopper, D.W., Ghan, R., Schlauch, K.A., Cramer, G.R., 2016. Transcriptomic network analyses of leaf dehydration responses identify highly connected ABA and ethylene signaling hubs in three grapevine species differing in drought tolerance. BMC Plant Biol. 16, 118. Kalberer, R.S., Wisniewski, M., Arora, R., 2006. Deacclimation and reacclimation of cold hardy plants: current understanding and emerging concepts. Plant Sci. 171, 3–16. Kovaleski, A.P., Reisch, B.I., Londo, J.P., 2018. Deacclimation kinetics as a quantitative phenotype for delineating the dormancy transition and thermal efficiency for budbreak in Vitis species. AoB Plants 10 ply066. Koussa, T., Broquedis, M., Bouard, J., 1994. Changes of abscisic acid level during the development of grapevine latent buds, particularly in the phase of dormancy break. Vitis 33, 63–67. Kühn, N., Ormeño-Nuñez, J., Jaque-Zamora, G., Pérez, F.J., 2009. Photoperiod modifiethe diurnal expression profile of VvPHYA and VvPHYB transcripts in field grown grapevine leaves. J. Plant Physiol. 166, 1172–1180. Londo, J.P., Kovaleski, A.P., 2017. Characterization of wild North America grapevine cold hardiness using differential thermal analysis. Am. J. Enol. Vitic. 68, 203–212.

Fig. 8. Correlation between accumulated chilling and bud cold-hardiness during the CA period. The accumulated chilling was measured as chilling hours. The data correspond to measurements carried-out for 4 years in the Elqui valley and for 5 years in the Maipo valley. (a) Correlation with an r2 = 0.63 was obtained when all data were plotted, (b) Correlation with an r2 = 0.75 was obtained only with data from the Maipo valley.

during the CA period As with the content of ABA in the buds, bud cold-hardiness correlated with the accumulated cold only during the CA period (Fig. 8 a, b). This seems reasonable, since the VvCBF/DREB1 genes and cold-hardiness would be induced synergistically by ABA + LT only during endodormancy. Therefore, when the grapevine buds are released from the endodormancy, the synergistic effect of ABA + LT on the cold-hardiness is lost, and the bud will not continue to increase it, even if it continues to accumulate cold. From these results, an important practical consequence is derived, since only during the period of endodormancy, the accumulated cold will influence the cold-hardiness of the buds.

4.3. Relationship between ABA peak and maximum bud cold-hardiness Recently, it has been reported that two peaks of ABA are produced during the development of the endodormancy in Thompson seedless buds grown in the Elqui and Maipo valleys of Chile (Rubio et al., 2019b). The biggest one is produced after the buds enter into endodormancy (middle of April) and a second one is produced at the end of June (Rubio et al., 2019b). The second peak was the one observed in this study, and apparently it is related to the maximum cold-hardiness reached by the buds, since the larger its size, the greater the coldhardiness of the buds. Interestingly, ABA reached its maximum concentration in the control buds and in those treated with ABA at the same time. However, in the buds treated with Uni-P, the peak of ABA and the 9

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endodormancy and cold-hardiness in grapevine buds. J. Plant Growth Regulation 35, 266–275. Rubio, S., Noriega, X., Pérez, F.J., 2019a. Abscisic acid (ABA) and low temperatures synergistically increase the expression of CBF/DREB1 transcription factors and cold hardiness in grapevine dormant buds. Ann. Bot. 123, 68–689. Rubio, S., Noriega, X., Pérez, F.J., 2019b. ABA promotes starch synthesis and storage metabolism in dormant grapevine buds. J. of Plant Physiology. 235, 1–8. Shellie, K., Kovaleski, A.P., Londo, J.P., 2018. Water deficit severity during berry development alters timing of dormancy transition in wine grape cultivars Malbec. Sci. Hortic. 232, 226–230. Vergara, R., Noriega, X., Pérez, F.J., 2017. ABA represses the expression of cell cycle genes and may modulate the development of endodormancy in grapevine buds. Front. Plant Sci. 8, 812. Weinberger, J.H., 1950. Chilling requirements of peach varieties. Proc. Am. Soc. Hortic. Sci. 56. Weiner, J.J., Peterson, F.C., Volkman, B.F., Cutler, S.R., 2010. Structural and functional insight into core ABA signaling. Curr. Opin. Plant Biol. 13, 495–502. Wisniewski, M., Nassuth, A., Arora, R., 2018. Cold hardiness in trees: a mini review. Front. Plant Sci. 9, 1394. Xiao, H., Siddiqua, M., Braybrook, S., Nassuth, A., 2006. Three grape CBF/DREB1 genes respond to low temperatures, drought and abscisic acid. Plant Cell Environ. 29, 1410–1421. Yamaguchi-Shinozaki, K., Shinozaki, 2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Ann. Rev. Plant Biol. 57, 781–803. Zhang, Y., Dami, I.E., 2012. Foliar application of abscisic acid increases freezing tolerance of field-grown Vitis vinifera Cabernet franc grapevines. Am. J Enol Viti. 63, 377–384. Zheng, C.H., Halaly, T., Acheampong, A.K., Takebayashi, Y., Jikumaru, Y., Kamiya, Y., Or, E., 2015. Abscisic acid (ABA) regulates grape bud dormancy, and dormancy release stimuli may act through modification of ABA metabolism. J. Exp. Bot. 66, 1527–1542.

Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-shinozaki, K., 1998. Two transcription factors, DREB1 and DREB2, with an ERBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low temperatureresponsive gene expression, respectively, in Arabidopsis. Plant Cell 10, 1391–1406. Michler, C.H., Lineberger, R.D., Chism, G.W., 1986. A highly sensitive method for quantitative determination of Abscisic acid. Plant Physiol. 82, 600–603. Mills, L.J., Ferguson, J.C., Keller, M., 2006. Cold-hardiness evaluation of grapevine buds and cane tissues. Am.J. Enol. Vitic. 57, 194–200. Nakashima, K., Fujita, Y., Kanamori, N., Katagiri, T., Umezawa, T., Kidokoro, S., 2009. Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OsT1 and SRK2I/SnRk2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol. 50, 1345–1363 tissues. Am. J Enol Viti. 57, 194–200. Nakashima, K., Yamaguchi-Shinozaki, K., Shinozaki, K., 2014. The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress response including drought, cold and heat. Front. Plant Sci. 5, 170. Noriega, X., Burgos, B., Pérez, F.J., 2007. Short-day photoperiod triggers and low temperatures increase expression of peroxidase RNA transcripts and basic peroxidase isoenzyme activity in grapevine-buds. Phytochemitry. 68, 1376–1383. Or, E., Belausov, E., Popilevski, I., Tal, Y.B., 2000. Changes in endogenous ABA level in relation to the dormancy cycle in grapevines grown in a hot climate. J. Hortic. Sci. Biotechnol. 75, 190–194. Pierquet, P., Stushnoff, C., 1980. Relationship of low temperature exotherms to cold injury in Vitis Riparia michx Am. J. Enol. Vitic. 31, 1–6. Raghavendra, A.S., Gonugunta, V.K., Christmann, A., Grill, E., 2010. ABA perception and signaling. Trends Plant Sci. 15, 395–401. Rohde, A., Bahlerao, R.P., 2007. Plant dormancy in the perennial context. Trends Plant Sci. 12, 217. Rozen, S., Skaletsky, H., 2000. Primer3 on the www for general users and for biologist Programmers. Methods Mol. Biol. 132, 365–386. Rubio, S., Dantas, D., Bressan-Smith, R., Pérez, F.J., 2016. Relationship between

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