Tree Physiology 17, 81--87
© 1997 Heron Publishing----Victoria, Canada
Effect of heat on cambial reactivation during winter dormancy in
evergreen and deciduous conifers
YUICHIRO ORIBE and TAKAFUMI KUBO
1
Wood Technology Division, Forestry and Forest Products Research Institute, P.O. Box 16, Tsukuba City, Norin Kenkyu Danchi-nai, Ibaraki 305,
Japan
2
Laboratory of Plant Morphology and Bio-Materials Physics, Department of Environmental and Natural Resource Science, Tokyo University of
Agriculture and Technology, 3-5-8 Saiwai-cho Fuchu City, Tokyo 183, Japan
Received July 14, 1995
Summary Responses of cambium to warming were recorded three times (December 14--27, 1990, January 18--February 3 and February 27--March 13, 1991) on 14-year-old
Cryptomeria japonica D. Don and four times (December 12-26, 1990, January 18--February 2, February 26--March 12 and
March 28--April 13, 1991) on 27-year-old Larix leptolepis
Gord., during a period of winter cambial dormancy. Stem
surfaces at breast height, mid-tree height and the crown base
were warmed to 25--30 °C for 2 weeks. After heat treatment,
cambia in the treated regions and in untreated regions 1 m
above each treated area were examined by optical and transmission electron microscopy (TEM). In C. japonica, heat
treatment often resulted in cambial reactivation in the treated
regions, and this response to heat gradually increased as the
dormant season passed from winter to spring. Conversely, in
L. leptolepis, no cell division was observed in the cambial
region of warmed stems until natural resumption of cambial
activity, which occurred after bud break.
Keywords: Cryptomeria japonica, heat treatment, Larix leptolepis, winter cambial dormancy.
Introduction
In the Northern Temperate Zone of Japan, vascular cambia in
conifers usually have annual periods of activity and dormancy.
Cambial activity is terminated in autumn when cambial zone
cells become dormant, and is resumed in spring when the cells
divide and differentiate into xylem and phloem.
The resumption of cambial activity in spring is brought
about both by internal chemical factors and external conditions
(Savidge and Wareing 1981). We have previously shown that,
in the evergreen conifer, Cryptomeria japonica D. Don, cambial reactivation occurs simultaneously throughout the stem
before bud break, whereas in the deciduous conifer, Larix
leptolepis Gord., cambial reactivation occurs a few weeks after
bud flushing in stem portions of the crown, and then progresses
basipetally down the stem (Oribe et al. 1993). We hypothesized
from these results that factors involved in bud and leaf production regulate cambial reactivation in deciduous conifers, but
not in evergreen species. In an earlier study, Savidge and
Wareing (1981) also suggested that temperature was a limiting
factor for cambial reactivation in Pinus contorta Dougl. ex
Loud., an evergreen conifer.
The objective of this study was to determine the cause of
differences in cambial reactivation between evergreen and
deciduous conifers. Specifically, cambia in stem portions of
C. japonica and L. leptolepis were warmed for two-week periods between winter and spring, and the response of cambia to
this heat treatment was recorded.
Materials and methods
Plant materials included three 14-year-old sugi (Cryptomeria
japonica) trees in the Karasawa Experimental Forest of Tokyo
University of Agriculture and Technology, and four 27-yearold karamatsu (Larix leptolepis) trees in a plantation in
Yamanashi Prefecture (Table 1). Between December 12, 1990
and April 13, 1991, heat treatments were carried out three
times on C. japonica (December 14--27, 1990, January 18-February 3 and February 27--March 13, 1991) and four times
on L. leptolepis (December 12--26, 1990, January 18--February 2, February 26--March 12, and March 28--April 13, 1991).
One tree of C. japonica or L. leptolepis was selected on each
occasion (Table 1). Maximum and minimum air temperatures
during the experiments were obtained from the Sano meteorological observatory located 5 km from Karasawa, and at Katsunuma located 10 km from the L. leptolepis plantation
(Figure 1).
Three pliable, silicone-rubber electric heat tapes, 15 cm in
width, were wrapped around the main stem circumference at
breast height, mid-tree height and the crown base of each tree
(Table 1). An alternating current of 30 V was passed through
the tapes to warm the stem surfaces. The temperature between
the outer bark and the heat tape was recorded by a thermometer
at each location, and was adjusted to 25--30 °C. This temperature range was selected to cover the range of summer temperatures that prevail during maximum tracheid production in
conifers.
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ORIBE AND KUBO
Table 1. Heat period and mensurational characteristics of the sample trees (n = 7).
Height at warmed portion (m)
1
Heat period
Height (m)
Percent crown (%)
DBH (cm)
Upper
Middle
Lower
Cryptomeria japonica
Dec 14--Dec 27
Jan 18--Feb 3
Feb 27--Mar 13
13.8
13.0
14.3
48.4
44.4
58.6
13.8
14.0
14.1
8.2
7.4
nm2
5.1
4.2
nm
1.9
1.6
1.2
Larix leptolepis
Dec 12--Dec 26
Jan 18--Feb 2
Feb 26--Mar 12
Mar 28--Apr 13
18.4
19.5
17.9
15.6
54.3
59.7
57.4
56.6
13.3
13.7
12.6
13.0
7.5
8.2
7.8
7.8
4.5
5.3
4.7
4.7
1.4
2.2
2.1
1.8
1
2
DBH = Diameter at breast height (measured at 1.2m).
nm = Not measured.
were washed in 0.1 mol l −1 cacodylate buffer and trimmed to
3 mm in length before postfixation in 1% osmium tetroxide in
0.1 mol l −1 cacodylate buffer for 1 h at room temperature.
Specimens were then washed in 0.1 mol l −1 cacodylate buffer,
immersed in a graded ethanol--propylene oxide series followed
by a propylene oxide--Araldite resin series, and embedded in
Araldite resin. Glass and diamond knives on an ULTRACUT N microtome (Nissei Sangyo. Co., Ltd., Tokyo, Japan) were used to obtain transverse sections of cambia.
Semi-thin sections for optical microscopy were double stained
with safranin and gentian violet. Ultra-thin sections, stained
with 2% uranyl acetate and lead citrate (Raynolds 1963), were
observed with a JEM-100C electron microscope (JEOL, Akishima City, Tokyo, Japan) at 80 kV.
Results
Cambial reactivation in C. japonica
Figure 1. Maximum and minimum air temperatures at Sano, 5 km
from Karasawa, and at Katsunuma, 10 km from the plantation in
Yamanashi Prefecture, during the experiments.
Immediately after each two-week heat treatment, stem disks
were obtained for microscopic observation from both warmed
portions and untreated portions, 1 m above the treated areas. In
addition, on July 7, 1990, a disk from the breast height stem
portion of a C. japonica tree was obtained to examine morphological characteristics of growing cambium.
One-cm blocks containing cambium and some xylem and
phloem tissues, were taken from the disks and fixed in 4%
glutaraldehyde in 0.1 mol l −1 cacodylate buffer pH 7.2 for
15 min at room temperature in a vacuum. After removal of
floating blocks, the remaining sinking material was fixed in
glutaraldehyde for 90 min at room temperature. Specimens
During active growth of cambial zone cells in C. japonica,
cells with a cell plate, a large central vacuole and thin tangential walls were observed in the cambial region (Figure 2).
During the same period, cambial derivatives were radially
expanding and undergoing secondary wall thickening (Figure 3).
No dividing cells were observed in untreated samples collected on December 27, February 3 or March 13, indicating
that cambia were dormant (Figure 4). Dormant cambial cells
had relatively thick tangential walls and numerous subdivided
vacuoles (Figure 4).
Cambial reactivation was often induced by heat treatment of
the stem during the dormant period. On December 27, heat
treatment of the lower portion of the stem had no effect on
cambial reactivation (Figures 5 and 6), but heat treatment of
the middle and upper portions of the stem induced division of
previously dormant cells (Figure 7). Transverse sections of
stems treated from January 18 to February 3 showed newly
divided and radially expanding cells in the cambia (Figures
8--11); secondary wall thickening was observed only in cambial cells from the warmed, mid-tree height stem portion (Fig-
EFFECT OF HEAT ON CAMBIAL DORMANCY IN CONIFERS
Figures 2 and 3. Transverse sections of
active cambium in C. japonica collected July 7, 1990. (2) A TEM micrograph of cambial cells with cell
plates (arrowheads) and large central
vacuoles (v), ×2240. (3) Optical micrograph showing cell division and differentiation, ×264. Note: cz = cambial
zone, ex = expanding cells, and th =
wall thickening cells.
Figure 4. A TEM micrograph of numerous small vacuoles (v) and thick
tangential cell walls (arrowheads) of
dormant cambium in an untreated upper portion of stem of C. japonica on
February 3, 1991, ×2240.
Figures 5 and 6. Transverse sections of
cambium in the lower stem portion of
C. japonica warmed December 14-27, 1990. (5) Optical micrograph,
×528. (6) A TEM micrograph, ×2240.
Note: cz = cambial zone.
Figure 7. A TEM micrograph of cambium with new cell walls (ncw) in an
upper stem portion of C. japonica
warmed December 14--27, 1990,
×2240.
83
84
ORIBE AND KUBO
Figures 8 and 9. Transverse
sections of cambium in the
lower stem portion of C. japonica warmed January 18-February 3, 1991. (8) Optical
micrograph, ×429. (9) A TEM
micrograph of new cell walls
(ncw), ×1820. Note: cz = cambial zone, and ex = expanding
cells.
Figure 10. Optical micrograph
of cells undergoing division, radial expansion and secondary
wall thickening in the middle
stem portion of C. japonica,
warmed January 18--February
3, 1991, ×429. Note: cz = cambial zone, ex = expanding cells,
and th = wall thickening cells.
Figure 11. Optical micrograph
of cell divisions and radial expansion in the upper stem
portion of C. japonica,
warmed January 18--February
3, 1991, ×429. Note: cz = cambial zone, and ex = expanding
cells.
Figure 12. Optical micrograph
of cells undergoing division, radial expansion and secondary
wall thickening in the lower
stem portion of C. japonica,
warmed February 27--March
13, 1991, ×429. Note: cz =
cambial zone, ex = expanding
cells, and th = wall thickening
cells.
Figures 13 and 14. Two TEM
micrographs of (13) untreated
and (14) warmed cambia, collected on December 26, 1990
from the middle stem portion of
L. leptolepis, ×1560. Note:
cambial zone cells have numerous small vacuoles (v) and
thick tangential cell walls (arrowheads) and show no cell division.
Figures 15 and 16. Two TEM micrographs of (15) untreated and (16) warmed cambia, collected March 12, 1991 from the middle stem portion of
L. leptolepis, ×1560. Note: cambial zone cells have numerous small vacuoles (v) and thick tangential cell walls (arrowheads) and show no cell division.
Figures 17 and 18. A TEM micrograph of (17) untreated cambium with new cell walls (ncw), × 1560. (18) Optical micrograph of warmed cambium, ×429, collected April 13, 1991 from the middle stem portion of L. leptolepis. Note: cz = cambial zone, and ex = expanding cells. Current
annual rings have four cambial cells in the untreated portion, and six cambial cells and five radially expanding tracheids in the warmed portion.
Number of cells shown is the average of 10 radial files.
EFFECT OF HEAT ON CAMBIAL DORMANCY IN CONIFERS
ure 10). By the third heat treatment, which was applied before
natural release of cambial dormancy in spring, cambial derivatives producing secondary walls were also observed in the
heat-treated lower portion of the stem (Figure 12).
Cambial dormancy in L. leptolepis
In L. leptolepis, no dividing cells were observed in untreated
cambia during the experimental period from December 12 to
March 12 (Figures 13 and 15), indicating that cambial cells
were dormant. Transmission electron microscope (TEM) micrographs indicated that these cambial cells had relatively thick
tangential walls and numerous subdivided vacuoles; similar
cell characteristics were also observed in dormant cambia of
C. japonica. By April 13, when buds had burst and cambial
dormancy had been released, newly formed cell plates were
frequently observed throughout the untreated regions of the
stem (Figures 17 and 19). Moreover, radially expanding cells
were observed in the upper untreated part of the stem.
Figure 19. A TEM micrograph of an untreated cambium with new cell
wall (ncw), collected on April 13, 1991 from the upper stem portion
of L. leptolepis, ×1920. Note: current annual ring contains five cambial
cells and one radially expanding tracheid (not all shown in figure).
Number of cells shown is the average of 10 radial files.
85
The effect of heat treatments on dormant cambium differed
in L. leptolepis from that in evergreen C. japonica. Heating
stems of L. leptolepis trees failed to reactivate dormant cambial
cells. Thick cell walls and numerous small vacuoles were
present in warmed cambia between mid-December and midMarch (Figures 14 and 16). By the fourth heat treatment from
March 28 to April 13, cambial growth was more conspicuous
in heat-treated portions than in untreated portions; however,
cambial reactivation had already occurred naturally by
April 13 (cf. Figures 17 and 18).
Discussion
Actively growing cambial cells were characterized by thin
tangential walls and large central vacuoles. In contrast, dormant cambial cells of both C. japonica and L. leptolepis were
characterized by thick tangential walls and numerous subdivided vacuoles. Similar cell structures, considered morphological characteristics of dormant cambium, have previously
been observed in Pinus spp., C. japonica and Salix spp., (Itoh
1971, Murmanis 1971, Lisbeth 1986).
During the dormant period, cambial growth often resumed
in heat-treated portions of C. japonica stems (Table 2); similar
findings have been reported for Pinus contorta (Savidge and
Wareing 1981). These results suggest that, in evergreen conifers, cambial dormancy is imposed by low air temperatures,
and that cambial reactivation is directly triggered by a rise in
temperature. This conclusion implies that evergreen conifer
cambia may reactivate in the absence of new shoot and leaf
growth.
However, the extent of cambial activity in heat-treated stem
regions of C. japonica varied on each occasion. The response
to heat treatment, as measured by cambial reactivation and
xylem formation, tended to increase as the period of cambial
dormancy progressed. Thus, there was no cambial response to
heat treatment of the lower portion of the stem in December
(cf. values on December 27, February 3 and March 13 in
Table 2. Extent of cambial activity in both warmed and untreated portions of evergreen and deciduous conifer stems.
Collection date
Upper portion
Middle portion
Lower portion
Untreated
Warmed
Untreated
Warmed
Untreated
Warmed
Cryptomeria japonica
December 27
February 3
March 13
D1
D
nm5
CD2
CD, RE3
nm
D
D
nm
CD
CD, RE, SWT4
nm
D
D
D
D
CD, RE
CD, RE, SWT
Larix leptolepis
December 26
February 2
March 12
April 13
D
D
D
CD, RE
D
D
D
CD, RE
D
D
D
CD
D
D
D
CD, RE
D
D
D
--
D
D
D
CD, RE
1
2
3
4
5
D = Dormancy.
CD = Cell division.
CD, RE = Cell division and radial expansion.
CD, RE, SWT = Cell division, radial expansion and secondary wall thickening.
nm = Not measured.
86
ORIBE AND KUBO
Table 2). These data indicate that, in addition to temperature,
other factors affect cambial dormancy.
The role of plant growth regulators in cambial activity and
dormancy has been demonstrated. It is well known that indol3-ylacetic acid (IAA) is required by the vascular cambium to
promote cambial cell division, radial enlargement and secondary wall thickening of cambial derivatives (Savidge and
Wareing 1981, Little and Savidge 1987). Absence of cambial
reactivation in the lower heat-treated portion of the stem in
December might be a result of IAA deficiency in cambium
during the early stage of dormancy. Seasonal variation in
endogenous auxin concentrations may account for observed
differences in cambial responses to heat treatment in C. japonica; however, this explanation is not supported by other observations. In Abies balsamea (L.) Mill (Sundberg et al. 1987),
not only was cessation of tracheid production poorly correlated
with decline in IAA concentrations, but exogenous IAA,
which induces similar anatomical and histochemical responses
in dormant cambium as endogenous IAA (Riding and Little
1984), did not prevent cessation of cambial growth during the
early stage of cambial dormancy. Furthermore, in A. balsamea
(Sundberg et al. 1987), Picea sitchensis (Bong.) Carrière (Little and Wareing 1981), and P. contorta (Savidge and Wareing
1984), cambial cells retain relatively high endogenous IAA
concentrations throughout dormancy.
Differences in the extent of cambial activity in heat-treated
portions of the evergreen conifer stem during winter months
may also be associated with a changeover between phases of
dormancy. In A. balsamea, cambia that are one-year-old or
more have a rest phase of dormancy followed by a quiescent
phase (Little and Bonga 1974, Riding and Little 1984, 1986).
During the rest phase, cambial dormancy is maintained by
internal agents (Little and Bonga 1974), whereas during the
quiescence phase, dormancy is imposed by external factors,
such as chilling. Under favorable growing conditions, application of exogenous IAA to A. balsamea can break cambial
quiescence, but it cannot break the rest phase of dormancy
(Little and Bonga 1974, Riding and Little 1984, Sundberg et
al. 1987). In C. japonica, cambial response to heat treatment
may reflect a dormancy stage of cambium. Thus, in December,
cambium in the lower portion of the stem may have been in the
rest phase of dormancy.
In L. leptolepis, lack of cambial response to heat treatments
before occurrence of natural cambial reactivation suggests that
low air temperature is not a limiting factor for cambial reactivation in this species (Table 2). Previously, we found that, in
L. leptolepis, cambial reactivation occurred after budbreak in
stem portions located in the living crown, and progressed
basipetally down the stem (Oribe et al. 1993). Based on these
findings together with the fact that IAA is synthesized in young
expanding leaves and transported basipetally down the stem
(Savidge and Wareing 1981, Little and Savidge 1987), we
postulate that cambial reactivation in L. leptolepis is regulated
by IAA in the cambial region. Although it is possible that
cambium remained dormant in heat-treated portions of the
L. leptolepis stem because cambium is IAA deficient until bud
flushing occurs, this conclusion is not supported by the study
of Savidge and Wareing (1982), showing high concentrations
of IAA in dormant cambia from the main stem of L. decidua
Mill.
Differences in cambial response to heat treatment between
C. japonica and L. leptolepis may result from temperature
differences between the bark surface and heat tape arising from
variation in bark thickness and species-specific anatomy. To
avoid cambial injury, cambial temperatures in heat-treated
portions of stems were not directly recorded during the experimental period. Instead, the temperature between cambium and
xylem under the heat tape was measured with copper-constantan thermocouples at breast height in both a C. japonica tree
and a L. leptolepis tree in February. In both species, cambial
temperatures gradually increased and reached stationary values within the selected temperature range in 4--6 h, implying
that cambia of both conifers were warmed at similar rates.
We conclude, therefore, that a rise in air temperature directly
triggers breaking of cambial dormancy in the evergreen conifer, C. japonica, but not in the deciduous conifer, L. leptolepis.
Cambial dormancy in L. leptolepis is broken by several factors
associated with bud flushing.
Acknowledgments
We thank Mr. S. Matsuzaki, Karasawa Experimental Forest of Tokyo
University of Agriculture and Technology for his technical assistance.
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