Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol82.Issue3-4.Dec1999:
Scientia Horticulturae 82 (1999) 243±254
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: Rubus spp.; 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)
0304-4238/99/$ ± see front matter Published by Elsevier Science B.V.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 5 1 - 5
244
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
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 47 Thornfree (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 -1,2,3-thidiazol-5ylurea (thidiazuron; TDZ). TDZ (100 mM) was prepared in 2.5% dimethyl
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
245
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
(4 28C), and the other groups was kept at alternating 6 0.18C for 16 h and
24 0.18C 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 chromatography (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
Data analysis was calculated by mean separation according to Duncan's
multiple range test and the Fisher's protected least significant difference (LSD)
test at the 5% level of confidence.
246
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
Fig. 1. Effect of CU accumulation (from September to March) and TDZ (100 mM) treatment on
budbreak of `Hull Thornless' and `Triple Crown Thornless' blackberry leaf buds. The data are
presented as mean SE (n 3); error bars that do not appear on graphs are smaller than symbols.
Different letters indicate a significant difference within each month on percent budbreak,
determined with Duncan's multiple range test at p 0.05.
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 were 1600 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
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
247
Fig. 2. Effect of two temperature regimes of 48C and 6/248C (16 h at 68C and 8 h at 248C) on
budbreak of `Triple Crown Thornless' and `Hull Thornless' blackberry cultivars. Budbreak in each
cultivar was measured after 0, 200, 400, 600, 800, and 1000 CU. The data are presented as
mean SE (n 3); error bars that do not appear on graphs are smaller than symbols. Different
letters indicate a significant difference within each temperature regime and each cultivar as
determined with Duncan's multiple ranges test at p 0.05.
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
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: Rubus spp.; 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)
0304-4238/99/$ ± see front matter Published by Elsevier Science B.V.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 5 1 - 5
244
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
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 47 Thornfree (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 -1,2,3-thidiazol-5ylurea (thidiazuron; TDZ). TDZ (100 mM) was prepared in 2.5% dimethyl
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
245
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
(4 28C), and the other groups was kept at alternating 6 0.18C for 16 h and
24 0.18C 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 chromatography (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
Data analysis was calculated by mean separation according to Duncan's
multiple range test and the Fisher's protected least significant difference (LSD)
test at the 5% level of confidence.
246
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
Fig. 1. Effect of CU accumulation (from September to March) and TDZ (100 mM) treatment on
budbreak of `Hull Thornless' and `Triple Crown Thornless' blackberry leaf buds. The data are
presented as mean SE (n 3); error bars that do not appear on graphs are smaller than symbols.
Different letters indicate a significant difference within each month on percent budbreak,
determined with Duncan's multiple range test at p 0.05.
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 were 1600 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
A.B. Izadyar, S.Y. Wang / Scientia Horticulturae 82 (1999) 243±254
247
Fig. 2. Effect of two temperature regimes of 48C and 6/248C (16 h at 68C and 8 h at 248C) on
budbreak of `Triple Crown Thornless' and `Hull Thornless' blackberry cultivars. Budbreak in each
cultivar was measured after 0, 200, 400, 600, 800, and 1000 CU. The data are presented as
mean SE (n 3); error bars that do not appear on graphs are smaller than symbols. Different
letters indicate a significant difference within each temperature regime and each cultivar as
determined with Duncan's multiple ranges test at p 0.05.
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