Changes of lipid components during dormancy in
`Hull Thornless' and `Triple Crown Thornless'
blackberry cultivars
Amir B. Izadyar, Shiow Y. Wang
*Fruit Laboratory, Rm. 211, Bldg. 010A, Beltsville Agriculture Research Center, ARS, U.S. Department of Agriculture, Beltsville, MD 20705-2350, USA
Accepted 9 April 1999
Abstract
Chilling requirements and changes in polar lipids of two blackberry cultivars (Rubus spp.), `Triple Crown Thornless' and `Hull Thornless' were determined during dormancy and budbreak. Under field conditions, `Triple Crown Thornless' required lower chilling units (CUs) than `Hull Thornless' to overcome dormancy. `Triple Crown Thornless' and `Hull Thornless' achieved full budbreak after receiving 600 and 1000 CU (chilling units), respectively. Under cold temperature treatments, `Triple Crown Thornless' needed 400 CU, while `Hull Thornless' needed 600 CU at 48C to obtain 100% budbreak. The shoots kept at intermittent 6/248C (68C for 16 h, and 248C for 8 h) did not reach full budbreak even after receiving 1000 CUs. An increase in phospholipids and glycolipids was detected at the end of dormancy. The increase in phospholipids occurred prior to the increase in glycolipids. The percentage of 18 : 2 fatty acid decreased while that of 18 : 3 increased and there was approximately a fivefold increase in the 18 : 3/18 : 2 ratio observed at the time of budbreak. The increase in the 18 : 3/18 : 2 ratio could serve as an indicator of dormancy termination and growth resumption in blackberry. Published by Elsevier Science B.V.
Keywords: Rubusspp.; Chilling units; Dormancy; Glycolipids; Phospholipids; Fatty acids
1. Introduction
Dormancy is a general term defined as any temporary period in which tissue containing a meristem is suspended from growth (Lang et al., 1987) and chilling
* Corresponding author. Tel.: +1-301-504-5776; fax: +1-301-504-5062
E-mail address:[email protected] (S.Y. Wang)
is the major factor that overcomes dormancy in temperate fruit trees (Samish, 1954). Several authors (Doorenbos, 1953; Jacobs et al., 1981) concluded that dormancy is strictly limited to buds. However, others have postulated that other parts of the tree, including roots and cambiums are also involved in the dormancy (Chandler, 1960; Samish, 1954; Westwood and Chestnut, 1964). Chilling temperatures have shown influence on membrane fatty acid composition and unsaturation (Lyons, 1973). Many plants capable of withstanding cold temperatures also exhibit an increase in lipid unsaturation and an increase in the level of phospholipid during cold acclimation (De la Roche, 1979; Sikorska and Kacperska-Palacz, 1979; Willemot, 1975). Low temperatures or thidiazuron, a growth regulator, increased the degree of unsaturation of fatty acids in the membrane lipids of apple buds, changed the polar head group composition, increased membrane phospholipid content, and changed sterol levels and composition. The ratio of sterols to phospholipids decreased during budbreak and bud growth (Wang and Faust, 1988, 1990). Erez et al. (1997) have shown a marked increase in total phospholipid content in both, dormant vegetative and floral peach buds with exposure to chilling temperatures. The relative level of linolenic acid (18 : 3) in phospholipid fraction of peach buds is directly correlated with the accumulation of chilling. Research on dormancy in blackberry is not well documented. This study was undertaken to (1) characterize the chilling requirements of two blackberry cultivars, and (2) determine whether the changes in membrane lipid composition are related to breaking the dormancy of buds in blackberry cultivars.
2. Materials and methods
2.1. Plant material and treatments
Three-year-old `Hull Thornless' and `Triple Crown Thornless' blackberry plants were selected for the experiment at the Agricultural Research Center in Beltsville, MD. `Hull Thornless' was selected in 1968 at Carbondale, Il, by Jack Hull as a result of the cross SIUS 47Thornfree (Galletta et al., 1981) and `Triple Crown Thornless' was selected in 1983 at Beltsville, MD, by G.J. Galletta as a result of the cross SIUS 68-2-5(C-47)Arkansas 545 (Galletta et al., 1998). Two sets of eight primocanes per cultivar were randomly collected on the 22nd of each month from October 1996 to February 1997 for budbreak observation. The leaves, if any, were removed and primocanes were cut to 30± 35 cm segments. The basal end of the shoots were immersed in water. The stem was recut and water was also replenished at three-day intervals. One set of eight primocanes in each cultivar was treated with N-phenyl-N0
sulfoxide (DMSO) plus 0.5% Tween-20 and applied directly to the buds with a brush until it ran off. The shoots of both the treatment were kept at 248C with 85% humidity and a 16-h photoperiod for three weeks to observe budbreak. The percentage of budbreak was evaluated after three weeks forcing.
Chilling requirements of blackberry cultivars were also determined using controlled chilling treatments. Two groups of forty primocanes were collected on October 22, 1996, from each cultivar. The primocanes were placed in two different temperature regimes; one group at a constant low temperature (428C), and the other groups was kept at alternating 60.18C for 16 h and 240.18C for 8 h daily. Each hour at 2±98C was considered as one chilling unit (CU). Eight shoots were taken from each temperature regime of each cultivar after 200, 400, 600, 800 and 1000 CU exposure. Primocanes were vase-cultured as described above to monitor budbreak. Budbreak percentage was recorded at the end of the forcing period.
2.2. Extraction, fractionation, and analysis of lipids
Lipid analysis was performed on primocanes collected from the field on the 22nd of each month from October 1996 to February 1997. Three primocanes per cultivar were collected at each sampling time. Triplicate bud samples of 0.5 g fresh weight were collected during each sampling time. Lipids were extracted, fractionated, and analyzed according to the procedures described by Wang and Faust (1988). Buds were homogenized and extracted with 10 ml isopropanol containing 4 g of 2,6-di-t-butyl-4-methylphenol (BHT)/ml. Total lipids were separated into neutral, glyco- and phospholipid fractions by silicic acid column chromatography on 100- to 200-mesh Bio Sil A (Bio Rad Laboratories, Richmond, CA). Total fatty acids esterified to polar lipids were derivatized to fatty acids methyl esters (FAME) for flame ionization detection-gas chromato-graphy (FID-GC) analysis.N-Heptadecanoic acid was included in all samples as an internal standard, and methyl heptadecanoate was used as an external standard. Individual FAMEs were identified by a comparison of peak areas with those of authentic standards (Supelco, Bellefonte, PA, USA). This tentative identification of major polar lipid fatty acids was corroborated by further analysis of FAME by gas chromatography-mass spectrometry (GC-MS) (Wang and Faust, 1988). Total glycolipids and phospholipids were determined by the spectrophotometric assays of Roughan and Batt (1968) and Ames (1966), respectively.
2.3. Statistical analysis
3. Results
3.1. Budbreak
Field accumulation of CUs was calculated for the entire experimental period at the Beltsville Agriculture Research Station (Fig. 1). There were1600 CUs from September 22, 1996 to March 22, 1997. The highest level of chilling accumulation in any month was found to be 360 CUs in February 1997. Under field conditions, the results showed that `Triple Crown Thornless' required lower CU than `Hull Thornless' for budbreak (Fig. 1). `Triple Crown Thornless' achieved 66% budbreak in October, while `Hull Thornless' did not attain budbreak at that time. `Hull Thornless' gained 80% budbreak in December. Full bud break (100%) was achieved in `Triple Crown Thornless' and `Hull Thornless' with 600 and 1000 CU, respectively. TDZ treatment was effective in the breaking of dormancy at all sampling times (Fig. 1). In chilling exposure experiments, buds of `Triple Crown Thornless' under constant 48C temperature treatment, required 200 and 400 CUs for 80 and 100% budbreak, respectively, while `Hull Thornless' needed 400 and 600 CUs for 80% and 100% budbreak, respectively (Fig. 2). Both the temperature regimes of 48C and 6/248C were effective in breaking dormancy. However, shoots that were kept at intermittent 6/248C, had
Fig. 1. Effect of CU accumulation (from September to March) and TDZ (100mM) treatment on
lower percentages of budbreak and did not reach full budbreak even after receiving 1000 CU.
3.2. Glycolipids and phospholipids
A decrease in total glycolipids and phospholipids of `Triple Crown Thornless' and `Hull Thornless' buds were observed as the season progressed (Table 1). However, the glycolipids began to increase after December in `Hull Thornless' and after January in `Triple Crown Thornless'. Phospholipid content in buds of both the cultivars decreased from October to November, then increased in January followed by a further drop in February (Table 1).
The most prominent components of fatty acids in glycolipid fractions were palmitic (16 : 0), stearic (18 : 0), linoleic (18 : 2), and linolenic (18 : 3) (Fig. 3). The other fatty acids, 12 : 0, 14 : 0, 16 : 3, and 18 : 1, comprised <5% of total fatty acids (data not shown). The proportion of palmitic acid (16 : 0) increased in the buds of both the cultivars from October to November, decreasing thereafter. The relative percentage of stearic and oleic acid remained almost constant from October to late February. An increase in the percentage of linoleic acid (18 : 2) from October to December in bud tissue was paralleled by a decrease of linolenic
acid (18 : 3). A decrease of linoleic acid (18 : 2) and an increase in linolenic acid (18 : 3) was found in buds after December (Fig. 3). The percentages of increase and decrease of 18 : 2 and 18 : 3 were greater in `Triple Crown Thornless' than in `Hull Thornless'. The ratio of 18 : 3 to 18 : 2 fatty acid of glycolipids in both the cultivars decreased from October to December, thereafter increased from January to February (Fig. 4). `Hull Thornless' had a higher ratio of 18 : 3 to 18 : 2 than `Triple Crown Thornless'.
The most prominent components of fatty acids in the phospholipid fraction were 16 : 0, 18 : 2 and 18 : 3 (Fig. 5). The other fatty acids, such as 12 : 0, 16 : 3, 18 : 0 and 18 : 1 comprised <10%. The relative percentages of 16 : 0, 18 : 0, 18 : 1, 18 : 2, and 18 : 3 fatty acid in phospholipids of both the cultivars remained relatively constant from October to January; however, an increase of 18 : 3 was found from January to February at the expense of 18 : 2 and 16 : 0. Both cultivars had the same distribution pattern of 18 : 3/18 : 2 ratio (Fig. 4), which remained relatively constant from October to January, and increased sharply in February.
4. Discussion
Under field conditions, `Triple Crown Thornless' and `Hull Thornless' fulfilled the chilling requirement for budbreak at the end of October and December, respectively (Fig. 1). This equals 600 CU for `Triple Crown Thornless' and 1000 CU for `Hull Thornless'. However, in chilling exposure experiments, full budbreak was found in `Triple Crown Thornless' and `Hull Thornless' after receiving 400 and 600 CU, respectively, at continuous 48C (Fig. 2). This may be related to the negation effect of high temperature in the field on accumulation of
Table 1
Changes of total glycolipids and phospholipids (mg gÿ1fresh wt) in `Triple Crown Thornless' and `Hull Thornless' blackberry leaf buds from October to February
Month Glycolipida Phospholipida
CUs. Erez et al. (1979, 1997) have shown that the intermittent temperature regimes with high cyclic temperatures (approximately >188C) will delay the chilling accumulation process in peach buds. In our experiment, shoots kept at intermittent 6/248C did not reach full budbreak even after receiving 1000 CU (Fig. 2).
The plant bioregulator, TDZ was effective in breaking dormancy at all sampling times (Fig. 1). TDZ was found to be more effective than cytokinins in breaking dormancy in apple buds (Wang et al., 1986). Steffens and Stutte (1989) found that TDZ was more effective in overcoming paradormancy than in providing a stimulus to negate endodormancy. Therefore, TDZ could be used as an exogenous indicator for the end of endodormancy and marks the transition of buds into paradormancy (Steffens and Stutte, 1989). Our experiments revealed that TDZ treatment increased budbreak in both the cultivars before the buds
received full chilling (during October and November) (Fig. 1). TDZ treatment on buds of `Triple Crown Thornless' and `Hull Thornless' after the buds received 200 CU resulted in 100% budbreak indicating that the two cultivars had not fulfilled their chilling requirement at the time. This also indicates that both cultivars of blackberry have a short endodormancy.
Changes in lipid metabolism may be responsible for the ability of plants to adapt to low temperatures. Increases in level of phospholids and lipid unsaturation were found in plants during cold acclimation (De la Roche, 1979; Sikorska and Kacperska-Palacz, 1979; Wang and Faust, 1990; Willemot, 1975). Erez et al. (1997) also reported a significant increase in phospholipid in peach buds during continuous chilling at 48C. This change is probably one of several
factors in maintaining membrane fluidity and in regulating the activity of membrane-associated enzymes (Cronan and Gelmann, 1975; Raison, 1985). Our study showed that total glyco- and phospholipids increased when dormancy was terminated and buds started to expand (Table 1). Increase in phospholipid content occurred before the increase in glycolipid content, suggesting that phospholipids are more responsive to CUs than glycolipids. Glycolipids have been shown to be a chloroplast thylakoid lipid (Williams et al., 1983). Chloroplast development is accompanied by an increased synthesis of glycolipids and linolenic acid. The increase in glycolipids in our study could be an accelerated development of chloroplast in blackberry buds during paradormancy. In `Hull Thornless' and
`Triple Crown Thornless', the decrease of 18 : 3 in glycolipids from October to December may contribute to the increase of 18 : 2. The increase of 18 : 3 after December may occur at the expense of 18 : 2 and 16 : 0. The sharp increases of the 18 : 3 to 18 : 2 ratio (Fig. 4) in late February indicate that high degrees of unsaturation in glycolipids may be associated with budbreak and bud development. A sharp decreases of 18 : 2 and 16 : 0 and an increase of 18 : 3 in phospholipids after January resulted in a fivefold increase of 18 : 3 to 18 : 2 ratio in both these cultivars (Fig. 4). It has been reported that lipase activity greatly increases in buds of apples when the chilling requirement is satisfied (Liu et al., 1991). This implies that the activated lipase may increase the amount of 18 : 3 fatty acids through conversion of 16 : 0 and 18 : 2 fatty acids to 18 : 3. The increase in 18 : 3/ 18 : 2 ratio has also been reported by others in apples and peaches (Wang and Faust, 1990; Erez et al., 1997). All of these changes may contribute to the increase in overall membrane fluidity and the preservation of the physiological bilayer phase for budbreak and bud growth in blackberry. We did not find a significant increase in 18 : 2 fatty acids concomitant with CUs accumulation, as has been reported previously by others (Wang and Faust, 1990; Erez et al., 1997), which may be due to the short endodormancy of both these cultivars. The increase in 18 : 3/18 : 2 ratio can be attributed to dormancy termination and growth resumption in blackberry.
5. Conclusion
`Triple Crown Thornless' and `Hull Thornless' blackberries, both appear to have short endodormancy. `Triple Crown Thornless' requires lower CUs than `Hull Thornless' to overcome the dormancy. `Triple Crown Thornless' has a shorter chilling period; thus, it should be possible to grow it successfully in areas south of North Carolina or northern Georgia. The plant bioregulator thidiazuron can release blackberry buds from the dormancy. An increase in glycolipid and phospholipid content and unsaturation of fatty acids was found to be associated with termination of the dormancy. This indicates that the breaking of dormancy and growth of blackberry buds probably is mediated through changes in membrane lipids. Further study of the relationship between lipids and chilling requirements may help identify transition stages in dormancy and optimum timing for TDZ applications.
Acknowledgements
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