Foliar application of abscisic acid induces dormancy responses in ...

HORTSCIENCE 46(9):1271–1277. 2011.

Foliar Application of Abscisic Acid Induces Dormancy Responses in Greenhouse-grown Grapevines Yi Zhang Department of Horticulture and Crop Science, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691 Tracey Mechlin Department of Horticulture and Crop Science, The Ohio State University, 2021 Coffey Road, Columbus, OH 43210 Imed Dami1 Department of Horticulture and Crop Science, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691 Additional index words. ‘Cabernet Franc’, ‘Chambourcin’, dormancy, growth inhibition, periderm formation, phytotoxicity, senescence Abstract. The purpose of this study was to investigate the influence of foliar application of abscisic acid (ABA) on grapevine dormancy, specifically to: 1) determine the optimum foliar application concentration of ABA and 2) evaluate the morphological and physiological changes of greenhouse-grown grapevines in response to exogenous ABA application. Vitis vinifera ‘Cabernet Franc’ and Vitis spp. ‘Chambourcin’ with different leaf ages (40, 50, 80, 100, 110, and 120 days) were subjected to foliar ABA application at different concentrations (0, 100, 200, 400, 600, 800, 1600, and 3200 mgL–1) and to a coldacclimated regime. Concentrations of 800 mgL–1 or higher were phytotoxic and the optimum concentrations were between 400 and 600 mgL–1. Optimum concentrations of ABA inhibited shoot growth and advanced growth cessation, periderm formation, and leaf senescence, which led to advanced dormancy in both cultivars. In this study, it was concluded that exogenous ABA induced endodormancy because single cuttings (not paradormant) under favorable growing conditions (not ecodormant) were used. Furthermore, grapevine response to ABA was influenced by leaf age and cold treatment. ABA was effective in inhibiting shoot growth and increasing periderm formation in the young vines with 40- to 50-day old leaves and the old grapevines with 80- to 120-day old leaves. However, ABA was effective in inducing early shoot cessation, leaf senescence and abscission, and dormancy in old vines with 100- to 120-day old leaves only. The advanced morphological and physiological changes induced by exogenous ABA mimicked those triggered by environmental cues during the cold acclimation process. It was suggested that advancing the cold acclimation process using foliar ABA application may be beneficial for long-season grape cultivars grown in regions with short growing seasons and early fall frost events.

The grape and wine industries in Midwestern and Eastern states have been expanding rapidly and demand for premium wine grapes has also increased. Unfortunately, several premium cultivars do not ripen properly in those regions as a result of the short growing season.

Received for publication 22 June 2011. Accepted for publication 26 July 2011. We are thankful for the financial support provided by the Department of Horticulture and Crop Science, OARDC/OSU, and the Lonz Foundation. The ABA sample was provided by Valent Bioscience Co. (Libertyville, IL). We also thank Trudi Grant and Dave Scurlock for their technical assistance to grow potted grapevines and Lee Duncan and Kesia Hartzler with environmental control of the greenhouses and growth chambers. 1 To whom reprint requests should be addressed; e-mail [email protected].

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This impacts not only the quality of the fruit and wine, but also the cold acclimation process and ultimately the winter hardiness of grapevines (Zabadal et al., 2007). Freeze protection methods have been developed for grapevines, including active and passive methods. Active protection methods include wind machines, heaters, and over-vine sprinkling (Poling, 2007). Passive methods include site and variety selection (Striegler, 2007; Zabadal et al., 2007) and application of chemical protectants (Dami and Beam, 2004; Dami et al., 2000). Among chemicals, growth regulators such as ABA have been applied to crops including Secale cereale L. (Churchill et al., 1998) and Malus domestica (L.) Borkh. (Guak and Fuchigami, 2001) to improve their cold hardiness. ABA is a plant hormone that plays several physiological roles, including regulation of

shoot and root growth (Saab et al., 1990), stomatal closure (Liang and Zhang, 1999), and leaf senescence (Thomas and Stoddart, 1980). Additionally, ABA has been suggested as a dormancy-inducing hormone because it accumulates in dormant nodes and increases after the tissue is exposed to low temperatures (Dogramaci et al., 2010). The variation of ABA concentration in plant tissues, mostly in nodes and xylem tissues, correlates with the cold acclimation process of plants (Mader et al., 2003; Rinne et al., 1994). Therefore, ABA has been proven to play an important role in the development of cold acclimation and ultimately the increase of freezing tolerance in plant species such as Arabidopsis thaliana (L.) Heynh. (Mantyla et al., 1995), Betula pendula Roth. (Li et al., 2003), Hordeum vulgare L. (Bravo et al., 1998), Secale cereale L. (Churchill et al., 1998), Triticum aestivum L. (Dallaire et al., 1994), Solanum tuberosum L. (Mora-Herrera and Lopez-Delgado, 2007), and Acer saccharum Marsh. (Bertrand et al., 1997). Exogenous application of ABA has also been investigated with grapes. ABA was used to delay budburst for spring frost protection but was not effective on field-grown grapevines (Hellman et al., 2006). ABA has also been used on table grapes to enhance color development and advance fruit maturity (Amiri et al., 2009; Peppi et al., 2007). To our knowledge, there are no published reports on the effect of exogenous ABA on inducing dormancy of grapevines. The purpose of this study was to investigate the influence of foliar application of ABA on grapevine dormancy. Our hypothesis stated that ABA would induce morphological and physiological changes in grapevines that mimic environmental cues including low temperature and/or a short photoperiod that lead to dormancy. The specific objectives were to: 1) determine the optimum foliar application concentration of ABA; and 2) evaluate the morphological and physiological changes of greenhouse-grown grapevines in response to exogenous ABA application. Materials and Methods Plant materials, growing conditions, and abscisic acid treatment In the first year, 1-year-old dormant Vitis vinifera ‘Cabernet Franc’ (CF) grafted on V. riparia · V. rupestris ‘Couderc 3309’ were planted in 7.6-L pots and placed on benches in the greenhouse in Dec. 2008. In the second year, the same potted vines were used after being pruned back to two nodes and stored in a 4 C cooler to satisfy their chilling requirement. In Year 2, 1-year-old Vitis spp. ‘Chambourcin’ (CHB) grapevines were also used. Before applying ABA, the average leaf number and shoot length per vine were 16 and 120 cm, respectively. In the first year, the plants were grown in a climate-controlled greenhouse for the nonacclimated condition and a growth chamber (Conviron, Pembina, ND) for the cold-acclimated regime. The greenhouse conditions were as follows: 22/19 C and 50/50% relative humidity (day/night). The light intensity in the

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greenhouse was maintained at 300 mmolm–2s–1 using 1000-W metal halide and 1000-W high-pressure sodium lights (Sunlight Supply, Woodland, WA) with a 12-h photoperiod. The growth chamber conditions for the cold acclimation experiment were 23.0/15.0 C and 50/50% relative humidity (day/night) in the first week and then 15.0/7.0 C (day/night) for 3 weeks (Grant et al., 2009). The light intensity in the growth chamber was maintained at 300 mmolm–2s–1 with an 8-h photoperiod. All grapevines were watered daily and fertilized every other day with 100 mgL–1 20–20–20 fertilizer (Peters Professional, Marysville, OH). In the second year, the conditions in the greenhouse were the same as in Year 1 with the exception that the growth chamber for the cold acclimation experiment was maintained at 12-h photoperiod. The ABA (VBS 350001) sample was provided by Valent Bioscience (Libertyville, IL). The a.i. was 20.0% (w/w) S-ABA. The ABA sample was dissolved in deionized water with 0.05% Tween20 (Acros Organic, Hampton, NH). Whole vines were sprayed with ABA solutions to runoff with a 7.6-L handheld sprayer (Gilmour Gardening Innovation, Peoria, IL) averaging a spray volume of 0.2 L/vine. Leaf age was determined based on EichornLorenz (EL) stages of shoot development (Eichhorn and Lorenz, 1977) with 1-d leaf age corresponding to the first unfolded leaf (EL stage 7) originating from the third basal node. In Year 1, two experiments were conducted. The first experiment consisted of applying ABA to CF in the greenhouse at a concentration of 0, 100, 200, 400, 800, 1600, and 3200 mgL–1 when leaf age averaged 40 d. The second experiment consisted of applying ABA to CF in the greenhouse at concentrations of 0, 200, 400, and 600 mgL–1 in the greenhouse and growth chamber when leaf age averaged 110 d. In Year 2, experiments were repeated to confirm ABA concentrations and plant response relative to leaf age and growing conditions. The first experiment was conducted at 50- and 100-d leaf age stages in the greenhouse (non-acclimated) and growth chamber (cold-acclimated) with ABA concentrations of 0, 200, 400, and 600 mgL–1. The second experiment was conducted in the greenhouse by applying ABA (0 and 600 mgL–1) at 40, 80, and 120-d leaf age stages. In the Year 1 experiment, a randomized block design was used with five replications per treatment of two vines per replication; in the Year 2 experiment, four replications per treatment were used with one vine per replication. Four vines of uniform growth per cultivar

were selected for ABA treatments. In all experiments in both years, the shoot tips of the grapevines were cut back to 14 to 16 nodes a week before ABA application. Abscisic acid phytotoxicity The phytotoxicity of ABA was evaluated in leaves and nodes. Visual observation of leaf distortion was made 24 h after ABA application and leaf injury was assessed and recorded a week after ABA treatments and based on leaf damage incidence (LDI) and leaf damage severity (LDS). LDI is the ratio of number of damaged leaves to the total number of leaves. Leaves with scorching-like dots and flecks were recorded as damaged leaves. Damaged leaf area larger than 0.1 cm2 was counted in severity assessment. Abscised leaves were counted as damaged leaves as well. LDS assessed the percentage of damaged leaf area using a score rating as follows: 0: 0% (no symptom); 1: 1% to 25%; 2: 26% to 50%; 3: 51% to 75%; and 4: 76% to 100% damaged leaf area. An LSD = 4 was also assigned to abscised leaves. Bud injury assessment was measured by assessing shoot growth resumption 2 months after ABA application. Nodes that did not break were excised and visually evaluated whether they were alive (green) or injured (brown). Vegetative growth Shoot growth. Each shoot was marked at 15 cm from the tip before ABA application. Then the distance between the tip and marker was measured three times a week and the shoot growth rates (cmd–1) were calculated as the total new growth divided by the number of days. Growth cessation. In the first year, all lateral shoots were removed during the course of the experiment. Shoot tip abscission was used to determine growth cessation but it did not occur for CF. Therefore, in the second year, growth cessation was estimated by proxy using the methods by Garris et al. (2009) on both CF and CHB. This method consisted of monitoring the summer lateral shoot emergence. The number of nodes with a lateral shoot having at least one fully expanded leaf was recorded. Growth cessation was determined when no summer laterals were produced (Garris et al., 2009).

brown 4 weeks after ABA application. Periderm formation was expressed as the ratio of number of brown to total number of internodes per shoot. Leaf senescence and abscission. This physiological stage was assessed by monitoring chlorophyll concentration and eventually counting leaf abscission. The measurement of chlorophyll concentration was conducted twice a week using a SPAD-520 chlorophyll meter (Spectrum Technologies, Inc., East Plainfield, IL) for 4 weeks. Five random measurements were taken on the upper surface of the fifth basal leaf. These measurements were conducted on CF and CHB grapevines in the second year. Leaf abscission was determined as the percent of abscised leaves to the total number of leaves 30 d after ABA application (DAA). Dormancy Lignified shoots with periderm formation from the base were excised into one-node cuttings 5 cm long, then inserted into 2.5 cm · 2.5-cm foam medium (Smithers-Oasis, Kent, OH) and placed in 55 cm · 25 cm · 7 cm plastic trays (T.O. Plastics, Clearwater, MN) filled with water. For each replication, 10 to 12 brown cuttings were used with the exception of young grapevines (leaf age = 40 or 50 d), from which green cuttings were used. Trays were then placed under forcing conditions on benches in a growth chamber (Conviron, Pembina, ND) with the following settings: 24-h photoperiod with 300 mmolm–2s–1, 22 C, and 80% relative humidity. Budburst was recorded as EL stage 5 and monitored three times a week for 30 d. Dormancy was estimated as the number of days to 50% budburst (D50BB). The higher D50BB, the more nodes are dormant. An increased D50BB indicated that nodes went into endodormancy. Statistical analysis All data were subjected to analysis of variance using Minitab statistical software (Minitab Inc., State College, PA). The model tested for main effects of application timing and concentration and for potential interaction between timing and concentration. When appropriate, means were separated using least significant difference (a = 0.05). Results

Periderm development and leaf senescence Periderm development. Periderm formation was determined by counting shoot internodes that changed color from green to tan or

Abscisic acid phytotoxicity. Leaf damage symptoms in CF grapevines were observed 24 h after ABA spray application at the concentrations of 800 mgL–1 and above. CF

Fig. 1. Leaf damage shown on ‘Cabernet Franc’ leaves 1 week after abscisic acid (ABA) application. (A–G) ABA concentration of 0, 100, 200, 400, 800, 1600, 3200 mgL–1.

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grapevines treated with ABA concentrations of 100, 200, 400, and 600 mgL–1 showed the same symptoms 6, 5, 3, and 2 d after ABA spray application, respectively (Fig. 1). A week after ABA application, leaf damage was detected on all ABA-treated grapevines in both cultivars. On mature leaves treated with ABA at all concentrations, the damage started with black spots on the lower surface of the blade, then the spots expanded and appeared on the upper surface as yellow (CF) or red (CHB) spots and flecks 0.25 cm2 in size (Fig. 1). At the shoot apical zone, ABA concentrations of 1600 and 3200 mgL–1 burned shoot tips and young leaves. ABA concentrations of 800 mgL–1 and lower caused shoot stunting but growth resumed later. Two to 4 DAA, leaf deformation was apparent on the first unfolded leaf treated with ABA at or above 400 mgL–1. Deformed leaves had abnormal margins and failed to develop the typical five-lobe leaf shape in CF. ABA concentrations of 400, 800, 1600, and 3200 mgL–1 caused a proportional increase of deformed leaves of one, three, five, and five, respectively. The growth of normally shaped leaves resumed after the emergence of the apical five leaves. This demonstrated that ABA concentrations equal to 400 mgL–1 or higher caused a transient disturbance of the normal development of shoot tips, which led to leaf deformation. A week after ABA application, LDI increased proportionally with ABA concentrations. ABA concentrations of 0, 100, 200, 400, 800, 1600, and 3200 mgL–1 led to LDI values of 0%, 8%, 24%, 26%, 52%, 64%, and 72%, respectively. LDS followed a similar pattern as LDI and was concentrationdependent as well (Fig. 2A). ABA concentrations at 1600 mgL–1 or above had LDS values above 1, indicating damage above 25% leaf area (Fig. 2A). Bud injury occurred at ABA concentrations of 800 (28%), 1600 (47%), and 3200 mgL–1 (57%). In Year 2, the highest concentration of ABA application was 600 mgL–1 and resulted in no bud injury (data not shown). Therefore, it was concluded that the treatments with concentrations of 800 mgL–1 or higher caused not only extensive leaf damage, but also bud injury. In the cold acclimation experiment, CF grapevines with 110-d-old leaves were used. LDI and LDS also increased with increasing ABA concentrations, which indicated foliar application of ABA was phytotoxic under both cold- and non-acclimated conditions (Fig. 2B). Therefore, the combination of ABA application and cold treatment increased leaf damage, and the severity of the damage was concentration-dependent (Fig. 2B). LDS values in the grapevines with older leaves (leaf age = 110 d; Fig. 2B) were generally higher than those with younger leaves (leaf age = 40 d; Fig. 2A). This prompted a leaf age experiment to verify whether ABA phytotoxicity increased with leaf age. This experiment demonstrated an increase of LDI (data not shown) and LDS, which were proportional to leaf age (Fig. 2C). Therefore, phytotoxicity of ABA was also leaf agedependent. HORTSCIENCE VOL. 46(9) SEPTEMBER 2011

Fig. 2. (A) Effect of abscisic acid (ABA) and concentration on leaf damage severity (LDS) of potted ‘Cabernet Franc’ grapevines (leaf age = 40 d) a week after ABA foliar application. (B) Effect of ABA and concentration on LDS of potted ‘Cabernet Franc’ grapevines under non-acclimated and coldacclimated conditions (leaf age = 110 d) 1 week after ABA application. (C) Effect of ABA (0 mgL–1 or control, and 600 mgL–1) and leaf age on LDS of potted ‘Cabernet Franc’ grapevines a week after ABA application.

To verify whether these responses were cultivar-dependent, CHB was also used. Generally, results from CHB were similar to those from CF with the exception that the color of CHB leaf damage was red, which was different from the yellow color damage on CF leaves. Additionally, the values of LDI and LDS of CHB were less than those of CF grapevines, which indicates that different cul-

tivars have different concentrations of tolerance to ABA (data not shown). Vegetative growth. Shoot inhibition was observed at the first measurement, 48 h after ABA application. The shoot length was reduced by ABA and was concentration-dependent with 200 mgL–1 having the least inhibition and 600 mgL–1 having the most inhibition (Fig. 3). At 28 DAA, shoot length and growth

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rate were reduced by 50% and 39%, respectively, with 600 mgL–1 as compared with control (Fig. 3; Table 1). The numbers of nodes was not different between ABA-treated vines and control (data not shown), indicating that ABA inhibited shoot growth by shortening the internode length. Furthermore, the growth rate decreased with increased leaf age (Table 1). Under cold-acclimated conditions, shoot length and growth rate were further reduced by ABA as compared with non-acclimated conditions (Table 2). The ABA-treated and cold-acclimated vines had shoot lengths 59% and 66% less than that of acclimated and non-acclimated control, respectively (Fig. 4). Furthermore, control grapevines under coldacclimated condition had similar growth rates as those from 400 to 600 mgL–1 ABA-treated grapevines under non-acclimated conditions (Table 2). In other words, the shoot inhibition in control vines under cold-acclimated conditions was similar to that by ABA-treated vines (400 and 600 mgL–1) under non-acclimated conditions. Shoot tip abscission of CF was monitored to determine the occurrence of growth cessation, but no abscission was observed from either control or ABA-treated grapevines when leaf age averaged 40 to 80 d (Tables 1 and 2). However, growth cessation occurred at leaf age of 100 to 120 d when treated with 600 mgL–1 ABA (Tables 1 and 2). Periderm development and leaf senescence. The increase of periderm formation was observed on ABA-treated grapevines with 50- and 100-d-old leaves (Table 2). The ABA-induced periderm formation was concentration-dependent, with 200 mgL–1 having the least increase and 400 mgL–1 and above having the most increase (Table 2). Periderm formation increased by 86% in treated vines with 600 mgL–1 ABA as compared with control (Table 1). Furthermore, periderm formation was affected by ABA and leaf age and the interaction between ABA and older leaves led to maximum periderm formation (Table 1). There was no difference of leaf chlorophyll concentration between control and ABAtreated vines with 50-d leaf age (Table 2). However, ABA at 600 mgL–1 induced leaf senescence in vines with 100-d-old leaves (Table 2). This was confirmed with the leaf age experiment at which 600 mgL–1 ABA induced leaf senescence in the grapevines with 120-d-old leaves. The decrease of chlorophyll concentration in treated vines started 14 DAA and continued until the end of the experiment (Fig. 5). This indicates that ABA accelerated the leaf senescence process and under these conditions was advanced by a minimum of 14 d as compared with the control. In addition, there was an interaction between concentration and leaf age, indicating that leaf senescence was induced by both ABA and leaf age and was enhanced by higher ABA concentration and older leaves (Table 1). No leaf abscission occurred when leaf age averaged 40 to 50 d (Tables 1 and 2). However, leaf abscission increased with ABA concentra-

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Fig. 3. Effect of abscisic acid (ABA) and concentration on shoot length progression of potted ‘Cabernet Franc’ grapevines (leaf age = 50 d).

Table 1. Effect of abscisic acid (ABA) and leaf age on shoot growth, leaf senescence, periderm formation, and dormancy of potted ‘Cabernet Franc’ grapevines. Leaf Days Periderm Leaf Growth ratez Growth abscission formationw to 50% (cmd–1) cessationy senescencex (%) (%) budburst 0 2.66 18 38 4 35 17 Concentration (mgL–1) 600 1.63 16 29 36 65 20 Leaf age (d)

40 80 120

3.06 a 2.37 a 1.01 b

19 a 18 ab 15 b

38 a 35 ab 28 b

0b 11 b 49 a

6b 62 a 82 a

8c 21 b 27 a

Concentration *** *** *** ** *** *** Leaf age *** *** *** ** *** *** NS NS ** NS *** * Concentration · leaf age z Means with a column followed by the same letter are not significantly different at P # 0.05. y Number of nodes per vine with summer laterals having at least one fully expanded leaf. x Chlorophyll concentration of the upper surface of the fifth basal leaf, 28 d after ABA application. w Percent of shoot internodes that changed color from green to brown to the total shoot internodes, 28 d after ABA application. NS, *, **, ***Non-significant or significant at P # 0.05, 0.01, or 0.001, respectively.

Table 2. Effects of abscisic acid (ABA) and concentrations on shoot growth, leaf senescence, periderm formation, and dormancy of potted ‘Cabernet Franc’ grapevines. Days Leaf Periderm z –1 Leaf abscission formationv to 50% Leaf Concn Growth rate (cmd ) Growth –1 x w NCA CA (%) budburst (%) age (d) (mgL ) cessation senescence 2.84 a 17 41 0 0c 7 50 0 3.52 ay 200 3.16 ab 2.46 b 16 39 0 20 b 8 400 2.48 b 2.25 b 16 42 0 31 a 8 600 1.96 c 1.47 c 16 39 0 28 ab 9 100

0 2.25 a 1.28 a 20 a 32 a 7b 41 b 18 b 200 2.01 ab 0.96 ab 18 ab 30 ab 10 b 66 ab 20 b 400 1.59 bc 0.84 bc 18 ab 28 ab 26 a 80 a 24 a 600 1.09 c 0.69 c 17 b 25.6 b 31 a 85 a 25 a z Growth rate under non-acclimated (NCA) and cold-acclimated (CA) regimes 28 d after ABA application (DAA). y Means within a column followed by the same letter are not significantly different at P # 0.05. x Number of nodes per vine with summer laterals having at least one fully expanded leaf. w Chlorophyll concentration of the upper surface of the fifth basal leaf, 28 DAA. v Percent of shoot internodes that changed color from green to brown to the total shoot internodes, 28 DAA.

tion when leaf age averaged 100 d (Table 2). At equal concentration, leaf abscission in vines with leaf age 120 d increased 4.5-fold as compared with that in vines with leaf age 80 d (Table 1).

Dormancy. All grape cuttings had budburst at the end of the 30-d long dormancy assay (58 DAA). The ABA treatments did not promote bud dormancy on grapevines with leaf age averaging 40 to 50 d (Tables 1 HORTSCIENCE VOL. 46(9) SEPTEMBER 2011

Fig. 4. Effect of abscisic acid (ABA) on shoot length progression of potted ‘Cabernet Franc’ grapevines under cold-acclimated and non-acclimated conditions (leaf age = 50 d).

Fig. 5. Effect of abscisic acid (ABA) (0 mgL–1 or control, and 600 mgL–1) on leaf chlorophyll concentration progression of potted ‘Cabernet Franc’ grapevines (leaf age = 120 d).

and 2). The shoots and nodes from the young vines were still green when placed under forcing condition and100% budburst occurred within 2 weeks. However, ABA concentrations of 400 and 600 mgL–1 delayed budburst in older grapevines with leaf age averaging 80 to 100 d (Tables 1 and 2). The delay of budburst between ABA-treated and control vines indicated that the former entered dormancy earlier than the latter. Under forcing conditions, ABA concentrations of 400 and 600 mgL–1 advanced dormancy by 6 to 7 d as compared with control (Table 2). Furthermore, the leaf age experiment not only confirmed that ABA advanced dormancy, but also demonstrated that ABA effect was enhanced by leaf age (Table 1). Discussion ABA concentrations of 800 mgL–1 or higher caused severe leaf and bud injuries. Therefore, it was concluded that ABA concentrations of 800 mgL–1 and higher were phytotoxic. Phytotoxicity at these concentrations HORTSCIENCE VOL. 46(9) SEPTEMBER 2011

has been linked to oxidative damage. In fact, Jiang and Zhang (2001) reported that ABA is a promoter of the antioxidant defense system when plants are under oxidative stress, but high ABA concentration (1000 mgL–1) induced an excessive production of active oxygen species, which led to an oxidative damage to leaves of maize seedlings. Waterland et al. (2010) demonstrated that application of S-ABA in bedding plants induced phytotoxicity. It was also suspected that phytotoxicity was associated with the low pH of ABA solutions, which varied from pH = 2.8 for 3200 mgL–1 to pH = 3.8 for 200 mgL–1. However, buffered ABA solutions to neutral pH led to more injury at equal concentration (data not shown). Therefore, the extreme low pH was not the cause of injury. In addition, leaves of CHB showed less damage than those of CF at the same ABA concentration, which indicates a differential response among grape genotypes to ABA. The published non-phytotoxic concentrations of ABA applied to clusters in vineyards varied between 100 mgL–1 and 1000 mgL–1 (Amiri et al., 2009; Peppi et al., 2007). It is

possible that ABA phytotoxic concentrations are different between greenhouse- and fieldgrown grapevines as well as between leaves and clusters. Furthermore, deformation of newly grown leaves in treated grapevines is termed heterophylly and has been reported on Marsilea quadrifolia L. after ABA application. This response was confirmed to be ABA-inducible by the upregulation of ABA-responsive heterophylly genes (Lin et al., 2005). The shoot growth of grapevines was consistently inhibited by ABA for both cultivars regardless of leaf age. This resulted in short internode length in ABA-treated grapevines. This observation is consistent with previous reports on ABA reducing the internode length of rice (Hoffmann-Benning and Kende, 1992), tobacco (Steadman and Sequeira, 1970), and apple (Guak and Fuchigami, 2001). Furthermore, the shoot length inhibition by ABA in young and old leaves indicates that this is an early response that is independent of leaf age. Shoot growth inhibition has been linked to ABA in several reports, which proposed mechanisms that ABA interacts with other plant hormones such as cytokinin (Vysotskaya et al., 2009), gibberellin (Hoffmann-Benning and Kende, 1992), and ethylene (Hansen and Grossmann, 2000), which led to promotion or inhibition of growth. The growth cessation is an early step in the senescence process and has been considered an early response of cold acclimation (Williams et al., 1972). During the process of cold acclimation in grapevine, a key change is the progression of periderm formation from shoot base to tip (Fennel and Hoover, 1991; Zabadal et al., 2007). The increased rate of periderm formation in ABAtreated vines may indicate an acceleration of the cold acclimation process. Leaf senescence is another step in the cold acclimation and dormancy processes (Kozlowski and Pallardy, 2002). In this study, advanced leaf senescence was observed in ABA-treated grapevines of both cultivars. Previous research on cotton rootstocks indicated that leaf senescence was closely associated with the ABA accumulation in the roots (Dong et al., 2008). The linkage between ABA and leaf senescence has been demonstrated in Arabidopsis thaliana with upregulation of two senescence-associated mRNA, pSEN4 and pSEN5, after exogenous ABA application (Park et al., 1998). In this study, the exogenous application of ABA may have led to an increase in the concentration of endogenous ABA, which was closely related to the extent of dormancy. In fact, concentrations of endogenous ABA were found to increase as the grape nodes entered dormancy and were the highest at maximum dormancy (Mochioka et al., 1996; Or et al., 2000). ABA was considered a positive regulator of dormancy induction and maintenance (Kucera et al., 2005). Bud dormancy in woody species including grapes is categorized into three different types based on the inhibition sources: 1) paradormancy, the inhibition is from distal organs; 2) endodormancy, the inhibition is from internal bud signals; and 3) ecodormancy, the inhibition is from unfavorable

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environmental conditions (Lang et al., 1987). In this study, it was concluded that exogenous ABA induced endodormancy because we used single cuttings (not paradormant) under favorable growing conditions (not ecodormant). This study also demonstrated that ABA was effective in inducing early responses, including shoot growth inhibition and periderm formation, in the grapevines with young leaves (leaf age 40 to 50 d). However, ABA was ineffective in inducing growth cessation, leaf senescence and abscission, and dormancy in young vines. The latter responses were affected by ABA only with the grapevines with old leaves (leaf age 80 to 120 d), thus deemed agerelated. The age-related effect of ABA has been explained by the function of an ABAinduced, age-induced gene RPK1 in Arabidopsis, which encodes a receptor kinase (Lee et al., 2011). The conditional overexpression of RPK1 at the mature stage accelerated the senescence and cell death and upregulated various ABA inducible genes, which indicated the effect of ABA was age-dependent (Lee et al., 2011). Based on this greenhouse study, the optimum concentration of ABA was between 400 and 600 mgL–1 and its exogenous application led to morphological and physiological changes similar to those induced by environmental cues including low temperature and/or a short photoperiod during the cold acclimation process. Furthermore, this study demonstrated the chronological sequences of morphological and physiological changes in CF and CHB starting with early responses, including shoot inhibition and periderm formation, followed by later responses, including growth cessation and leaf senescence and abscission, which ultimately led to bud dormancy. Therefore, the exogenous application of ABA initiated a cascade of steps that advanced the cold acclimation process in the absence of environmental cues. It was suggested that advancing the cold acclimation process using foliar ABA application may be beneficial for long-season grape cultivars grown in regions with short growing seasons and early fall frost events. Literature Cited Amiri, M.E., E. Fallahi, and M. Mirjalili. 2009. Effects of abscisic acid or ethephon at veraison on the maturity and quality of ‘Beidaneh Ghermez’ grapes. J. Hort. Sci. Biotechnol. 84: 660–664. Bertrand, A., G. Robitaille, Y. Castonguay, P. Nadeau, and R. Boutin. 1997. Changes in ABA and gene expression in cold-acclimated sugar maple. Tree Physiol. 17:31–37. Bravo, L.A., G.E. Zuniga, M. Alberdi, and L.J. Corcuera. 1998. The role of ABA in freezing tolerance and cold acclimation in barley. Physiol. Plant. 103:17–23. Churchill, G.C., M.J.T. Reaney, S.R. Abrams, and L.V. Gusta. 1998. Effects of abscisic acid and abscisic acid analogs on the induction of freezing tolerance of winter rye (Secale cereale L.) seedlings. Plant Growth Regulat. 25:35– 45.

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