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Ann Bot. 2010 December; 106(6): 885–895.
Published online 2010 October 29. doi:  10.1093/aob/mcq185
PMCID: PMC2990657

Changes in the localization and levels of starch and lipids in cambium and phloem during cambial reactivation by artificial heating of main stems of Cryptomeria japonica trees

Abstract

Background and Aims

Cambial reactivation in trees occurs from late winter to early spring when photosynthesis is minimal or almost non-existent. Reserve materials might be important for wood formation in trees. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules in cambium and phloem were examined from cambial dormancy to the start of xylem differentiation in locally heated stems of Cryptomeria japonica trees in winter.

Methods

Electric heating tape was wrapped on one side of the stem of Cryptomeria japonica trees at breast height in winter. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules were determined by image analysis of optical digital images obtained by confocal laser scanning microscopy.

Key Results

Localized heating induced earlier cambial reactivation and xylem differentiation in stems of Cryptomeria japonica, as compared with non-heated stems. There were clear changes in the respective localizations and levels of starch and lipids (as droplets) determined in terms of relative areas on images, from cambial dormancy to the start of xylem differentiation in heated stems. In heated stems, the levels and number of starch granules fell from cambial reactivation to the start of xylem differentiation. There was a significant decrease in the relative area occupied by lipid droplets in the cambium from cambial reactivation to the start of xylem differentiation in heated stems.

Conclusions

The results showed clearly that the levels and number of storage starch granules in cambium and phloem cells and levels of lipids (as droplets) in the cambium decreased from cambial reactivation to the start of xylem differentiation in heated stems during the winter. The observations suggest that starch and lipid droplets might be needed as sources of energy for the initiation of cambial cell division and the differentiation of xylem in Cryptomeria japonica.

Keywords: Cambial reactivation, confocal laser scanning microscopy, Cryptomeria japonica, lipid, starch, xylem differentiation

INTRODUCTION

Wood is the product of the vascular cambium, and wood formation depends on the cambial activity of trees (Catesson, 1994; Larson, 1994; Funada, 2008). In temperate zones, the vascular cambium of tree stems undergoes seasonal cycles of activity and dormancy, a phenomenon known as annual periodicity. This periodicity plays a critical role in the formation of wood. In late winter and early spring, new cell plates are formed in the cambium. The formation of new cell plates in spring is referred to as cambial reactivation (Catesson, 1994; Larson, 1994). Both the quantity and the quality of wood reflect the timing of cambial reactivation in temperate zones. Therefore, it is very important to develop a full understanding of the mechanism of wood formation and, in particular, of cambial reactivation.

Under natural conditions, cambial reactivation in trees occurs from late winter to early spring, when photosynthesis is minimal or almost non-existent. The analysis of 13CO2 pulse-labelled photoassimilates demonstrated that cell walls of earlywood are derived from the previous year's photoassimilates and from those of the current spring (Kagawa et al., 2006a, b). Therefore, it was suggested that stored materials are crucial to radial growth, wood density and the exploitation of the previous year's photoassimilates for formation of the current year's tree ring. There have been several reports of seasonal fluctuations in levels and partitioning of storage carbohydrates and lipids in trees, e.g. Betula pendula (Harms and Sauter, 1992), Pinus sylvestris (Fischer and Höll, 1992), Juglans nigra and hybrid Juglans major × J. regia (Magel et al., 2001), Pinus cembra (Hoch et al., 2002), Quercus petraea and Fagus sylvatica (Barbaroux and Bréda, 2002; Barbaroux et al., 2003). In addition, levels of starch in xylem cells of main stems have been analysed at various phenological stages, namely, at bud break, during build-up of leaves, and during leaf fall in broad-leaved species, such as Populus × canadensis (Sauter and van Cleve, 1994), Salix caprea (Sauter and Wellenkamp, 1998), and during cambial activity and the differentiation of xylem (wood formation; Sauter and Neumann, 1994; Schaberg et al., 2000; Piispanen and Saranpää, 2001; Hoch et al., 2003; Deslauriers et al., 2009). The data in the cited studies indicate that utilization of reserve materials at various stages of growth provides one of the keys to a full understanding of growth, defence against pathogens, and the development of cold tolerance of trees.

Localized heating induces cambial reactivation that occurs earlier than natural cambial reactivation in a variety of conifers (Savidge and Wareing, 1981; Barnett and Miller, 1994; Oribe and Kubo, 1997; Oribe et al., 2001, 2003; Gričar et al., 2006; Begum et al., 2010) and in a hardwood (Begum et al., 2007). However, the patterns of cambial reactivation are almost identical to those in natural systems. Therefore, it was postulated that artificial heating might provide a good model system to investigate the cambial biology of trees. Such a model system would allow cambial activity and xylem differentiation to be compared directly over relatively short periods of time (Oribe et al., 2001; Begum et al., 2007, 2010).

Using a system for local heating of main stems, Oribe et al. (2003) found that, in Abies sachalinensis, cell divisions in the heated reactivated cambium stopped dividing with the disappearance of starch from the storage tissue around the cambium. Therefore, they proposed that the continuation of cambial activity might require a continuous supply of sucrose. In previous studies, it had been observed that the levels of starch around the cambium in heated poplar stems decreased during cambial reactivation and that starch granules disappeared completely from the cambium and phloem immediately after cambial reactivation and re-appeared again with the start of xylem differentiation (Begum et al., 2007). Therefore, it was postulated that starch might be a source of energy for cambial reactivation and the initiation of xylem differentiation. However, while some studies of the localization of storage starch and cambial reactivation have been described, the sources of the energy that is required from cambial reactivation to the start of xylem differentiation remain to be characterized for a full explanation of the correlation between storage materials and reactivation of the cambium.

In the present study, the localization, levels of starch and lipid (as droplets), and number of starch granules in the cambium and phloem of non-heated control stems and locally heated stems of Cryptomeria japonica trees were examined during a relatively brief period of time in winter. The goal was to identify the sources of energy for cambial reactivation and the start of xylem differentiation when leaf photosynthesis is minimal. Cryptomeria japonica was chose for the analysis because of its commercial importance in Japan. Confocal laser scanning microscopy was used because it provides three-dimensional (3-D) digital images of cambium (Kitin et al., 2000; Funada, 2002) and clearly reveals the localization of stored materials in a semi-quantitative manner. The results demonstrate the relationships between the timing of cambial reactivation and the start of xylem differentiation, on the one hand, and changes in levels of starch and lipids (as droplets) and number of starch granules, on the other, in heated stems in winter. A possible mechanism for cambial reactivation in conjunction with the utilization of storage materials is discussed.

MATERIALS AND METHODS

Plant materials

Two adult Cryptomeria japonica trees, 71 and 93 years old, were studied. They were growing in the field nursery of the Tokyo University of Agriculture and Technology in Fuchu, Tokyo (35°40′N, 139°29′E, 40 m a.s.l.), Japan, i.e. in a temperate zone. The two trees were used for sequential observations of the localization, levels of starch and lipids (as droplets) and number of starch granules in the cambium and phloem from dormancy to the start of xylem differentiation.

Heat treatment

Electric heating tape (Silicone–Rubber Heater; O & M Heater, Nagoya, Japan), 50 cm in length and 30 cm in width, was placed on one side of the main stem of each tree at breast height (‘heated stem’) and the opposite side of each heated area was taken as being the ‘non-heated control stem’ (Begum et al., 2007, 2010). An alternating current was passed through the tape at a potential of 100 V to warm the surface of the stem. The temperature between the outer bark and the heating tape was recorded with a thermometer at the site at which the stem was heated. The temperature was adjusted to 20–22 °C with a thermostat. As demonstrated previously, cambial temperature reaches a stationary value within the selected temperature range in 4–6 h in thick-barked conifers (Oribe and Kubo, 1997). Stems were heated from 23 January 2008 to 21 February 2008, with continuous heating until well-developed xylem differentiation was apparent. No abnormal structures were found in the stems after artificial heating.

Specimen collection

Samples were taken from the main stems at breast height on 11 December 2007 and on 16 January 2008. Then, they were taken at approx. 1-week intervals from heated and non-heated control portions of the main stems. A series of small blocks (2 × 2 × 1 cm3), containing phloem, cambium and some xylem was removed with a scalpel and chisel. Samples were removed in a zigzag pattern to eliminate any effects of wounding. Each block was cut into 2-mm thick samples immediately after removal from the tree.

Light microscopy for observations of cambial activity

The samples were fixed in 4 % glutaraldehyde in 0·1 m phosphate buffer (pH 7·3), under a vacuum, for 1 h at room temperature. Fixed samples were washed in 0·1 m phosphate buffer and trimmed to 3 mm in length for subsequent fixation in 1 % osmium tetroxide in 0·1 m phosphate buffer for 2 h at room temperature. After washing in phosphate buffer, specimens were dehydrated in a graded ethanol series and embedded in epoxy resin. Transverse sections were cut at a thickness of approx. 1 µm with a glass knife on an ultramicrotome (Ultracut N; Reichert, Vienna, Austria) for sequential observations of cambial reactivation and xylem differentiation. Sections were stained with a solution of 1 % safranin in water for observations of cambial reactivation (the presence of new cell plates) and xylem differentiation and then examined under a light microscope (Axioscop; Carl Zeiss, Oberkochen, Germany) (Begum et al., 2007, 2008; Nakaba et al., 2008).

Preparation of samples for observations of starch and lipids, as droplets

After samples had been fixed in 4 % glutaraldehyde in 0·1 m phosphate buffer (pH 7·3), under a vacuum, for 1 h at room temperature, radial sections were cut at a thickness of approx. 40 µm with a glass knife on the freezing stage of a sliding microtome (MA-101; Komatsu Electronics, Tokyo, Japan). For light microscopic observations of storage starch, sections were stained with iodine–potassium iodide (I-KI) for 2–3 min (Nakaba et al., 2006; Begum et al., 2007). For visualization of storage starch by confocal laser scanning microscopy, sections were stained with a 0·5 % aqueous solution of safranin for 2–3 min. After staining, sections were washed five times with distilled water. In addition, for visualization of lipid droplets, sections were stained with 1 % Sudan IV in 70 % ethanol for 2–3 min. After staining, both sets of sections were washed five times with distilled water.

Confocal laser scanning microscopy and preparation of 3-D images

Stained radial sections were mounted on glass slides with water and examined with a confocal laser scanning microscope (LSM 310; Carl Zeiss). Excitation by the helium neon laser (wavelength, 543 nm) with a 590-nm long-pass filter and excitation by the argon ion laser (wavelength, 488 nm) with a 590-nm long-pass filter were used for samples stained with safranin and Sudan IV, respectively. Three-dimensional projection or depth-coding digital images of the starch and lipid droplets were prepared in serial 18 optical sections at 1-μm intervals along the z-axis on a computer, using the software provided with the confocal laser scanning microscope.

Analysis of levels of starch and lipids (as droplets) and number of starch granules

The 3-D projection images were used to analyse the levels of starch and lipids (as droplets) and number of starch granules in the cambium and phloem cells of non-heated control stems and heated stems. Two radial-sectional areas of 30 000 µm2 for cambium and of 80 000 µm2 for phloem were selected for calculations of the levels, as percentages per unit area, of starch and lipid droplets in the cambium and phloem cells. The number of starch granules (average number per cell) in ray cambium, phloem ray parenchyma cells and longitudinal phloem parenchyma cells were counted from dormancy to the start of xylem differentiation. Since the length of fusiform cambial cells is very long and it is difficult to monitor stored materials in an entire cell within a single radial section (Kitin et al., 2000, 2002), the number of starch granules were not measured in those cells. These calculations were made with image-analysis software (ImageJ; National Institutes of Health, MD, USA). Each cited percentage per unit area occupied by starch or lipid droplets and number of starch granules per cell in cambium and phloem is the average value obtained from the two trees that were studied.

In the case of non-heated control stem, data were shown on 11 December 2007 and 16 January 2008, 1 week prior to the start of heating. In the case of both non-heated control and heated stems, data were shown on 29 January 2008, after 6 d of heating, and 14 February 2008, after approx. 3 weeks of heating.

Ambient temperature profile

The daily maximum, average, and minimum air temperatures at the experimental site in Fuchu, Tokyo, from 1 December 2007 to 28 February 2008 are shown in Fig. 1. The maximum, average, and minimum daily temperatures ranged from 7 °C to 21 °C, 3 °C to 13 °C and –2 °C to 8 °C, respectively.

Fig. 1.
Records of the maximum, average and minimum daily air temperatures at the experimental site in Fuchu, Tokyo, from 1 December 2007 to 28 February 2008.

Statistical analysis

Statistical analyses were performed with StatGraphics Plus 5·1 (Stat Point, Inc., Warrenton, VA, USA). Fisher's LSD test was used to compare mean levels of starch and lipids (as droplets) and number of starch granules at different times; differences were taken as significant when P < 0·05.

RESULTS

Status of cambial activity and xylem differentiation

The cambial zone cells that were collected on 11 December 2007 and 22 January 2008 consisted of four or five layers of fusiform cambial cells. These cells were arranged very compactly, indicating that the cambium of Cryptomeria japonica was dormant (Fig. 2A, B). In heated stems, the first cell division occurred on the phloem side of cambium on 29 January 2008, after just 6 d of heating (Fig. 2C), and xylem differentiation started on 14 February 2008, after approx. 3 weeks of heating (Fig. 2D). In non-heated control stems, on the same days, 29 January 2008 and 14 February 2008, the cambial cells had no new cell plates, indicating that the cambium was still dormant (Fig. 2E, F).

Fig. 2.
Light micrographs showing transverse views of cambium from heated and non-heated control stems. (A, B) Cambial cells were arranged very compactly and the cambial zone consisted of four or five layers of fusiform cambial cells on 11 December 2007 (A) and ...

Visualization of starch and lipids (as droplets) by confocal laser scanning microscopy

The usual method for visualization of starch and lipid droplets involves conventional microscopy, and the results of such an examination of thin sections of resin-embedded tissue are shown in Fig. 3A. In addition, thick sections were examined by conventional microscopy without resin embedding for visualization of starch and lipid droplets. The preparation of 1-μm-thin sections and staining them with I-KI for conventional light microscopy is time-consuming and requires considerable skill (Fig. 3A). The preparation of relatively thick sections and staining with I-KI and Sudan IV for observations of starch and lipid droplets, respectively, for conventional light microscopy yields images that are out of focus (Fig. 3B, E). In the present study, approx. 40-μm thick sections we stained with 0·5 % safranin for observations of starch granules and with 1 % Sudan IV in 70 % ethanol for observations of lipid droplets. Safranin and Sudan IV yield strong red and green fluorescence, respectively, providing brighter images of starch granules and lipid droplets. Therefore, confocal laser scanning microscopy was used instead of light microscopy to prepare optical digital images with 3-D projection that allowed acquisition of semi-quantitative data (Fig. 3C, F). The depth-coding images show the relatively uniform distribution on the z-axis of starch within cells (Fig. 3D).

Fig. 3.
Light micrographs and confocal laser scanning microscopic images demonstrating the differences between three methods for examining starch and lipid droplets in radial sections of cambium that include phloem in Cryptomeria japonica. (A, B) Light micrographs ...

Localization, levels, number and size of starch granules in the cambium and phloem of non-heated control and heated stems

On 11 December 2007, the level of starch (percentage of unit area) in ray cambial cells was close to 9 % and no storage starch was detected in fusiform cambial cells (Figs 4A and 5A). On 16 January 2008, 1 week prior to the start of heating, the level of starch in ray cambial cells had increased very slightly from 9 % to 11 % and starch granules had appeared in fusiform cambial cells, accounting for approx. 8 % of the unit area (Figs 4B and 5A); the depth-coding digital images of the same sections demonstrated, again, the relatively uniform distribution on the z-axis of the starch in fusiform cambium (Fig. 4C). After 6 d of heating, on 29 January 2008, when cambial reactivation had occurred, the level of starch had fallen from 11 % to 4 % in the ray cambium (Fig. 5A) and from 8 % to 4 % in the fusiform cambium (Figs 4D and 5A). In contrast, in non-heated control stems, on the same day, 29 January 2008, the level of starch had increased very slightly from 11 % to 12 % in the ray cambium and from 8 % to 10 % in the fusiform cambium (Fig. 5A). After approx. 3 weeks of heating, on 14 February 2008, when xylem differentiation had started, the level of starch had fallen to 2 % both in the ray cambium and in the fusiform cambium (Figs 4E and 5A). In non-heated control stems, on the same day, 14 February 2008, the level of starch had increased very slightly from 12 % to 13 % in the ray cambium and from 10 % to 12 % in the fusiform cambium (Fig. 5A). In heated stems, there were major changes in the respective levels of starch in ray and fusiform cambial cells between 11 December 2007 and 14 February 2008 (P < 0·05; Fig. 5A).

Fig. 4.
Confocal laser scanning microscopic images (projection) showing starch granules in the cambium, phloem and xylem of non-heated control stems and of heated stems. (A) Fusiform cambium on 11 December 2007 showing no starch. (B) Cambium and longitudinal ...
Fig. 5.
(A) Percentage of unit area occupied by starch granules and (B) number of starch granules per cell in the cambium and phloem of non-heated control stems and heated stems at different times in winter (n = 4). Columns and bars show mean values ± ...

On 11 December 2007, the levels of starch in the phloem ray parenchyma cells and the longitudinal phloem parenchyma cells were approx. 24 % and 20 % (Fig. 5A). On 16 January 2008, 1 week prior to the start of heating, the level of starch in phloem ray parenchyma cells had risen from 24 % to 32 %, while in longitudinal phloem parenchyma cells it was about 20 % (Figs 4F, H and 5A). After 6 d of heating, on 29 January 2008, when cambial reactivation had occurred, the level of starch in phloem ray parenchyma cells and in longitudinal phloem parenchyma cells had fallen to 19 % and 11 %, respectively. In contrast, in non-heated control stems, on the same day, 29 January 2008, the level of starch had increased slightly from 32 % to 35 % in the phloem ray parenchyma cells and from 20 % to 23 % in the longitudinal phloem parenchyma cells (Fig. 5A). After approx. 3 weeks of heating, on 14 February 2008, when xylem differentiation had started, the level of starch had fallen in phloem ray parenchyma cells and longitudinal phloem parenchyma cells to close to 7 % in each case (Figs 4G, I and 5A). In non-heated control stems, on the same day, 14 February 2008, the level of starch had increased very slightly from 35 % to 36 % in the phloem ray parenchyma cells and from 23 % to 25 % in the longitudinal phloem parenchyma cells (Fig. 5A). In heated stems, there were significant changes in the levels of starch in phloem ray parenchyma cells and longitudinal phloem parenchyma cells between 11 December 2007 and 14 February 2008 (P < 0·05; Fig. 5A).

On 11 December 2007, the number of starch granules (average number per cell) in ray cambial cells, phloem ray parenchyma cells and longitudinal phloem parenchyma cells were 32, 19 and 33, respectively. On 16 January 2008, 1 week prior to the start of heating, the number of starch granules in ray cambial cells, phloem ray parenchyma cells and longitudinal phloem parenchyma cells had increased from 32 to 51, from 19 to 23 and from 33 to 36, respectively. After 6 d of heating, on 29 January 2008, when cambial reactivation had occurred, the number of starch granules had fallen from 51 to 24 in the ray cambium, from 23 to 18 in phloem ray parenchyma cells and from 36 to 19 in longitudinal phloem parenchyma cells (Fig. 5B). In contrast, in non-heated control stems, on the same day, 29 January 2008, the number of starch granules in ray cambial cells, phloem ray parenchyma cells and longitudinal phloem parenchyma cells had increased slightly from 51 to 55, from 23 to 28 and from 36 to 39, respectively (Fig. 5B). After approx. 3 weeks of heating, on 14 February 2008, when xylem differentiation had started, the number of starch granules had fallen to 16 in the ray cambium, 13 in the phloem ray parenchyma cells and 16 in longitudinal phloem parenchyma cells (Fig. 5B). In non-heated control stems, on the same day, 14 February 2008, the number of starch granules in ray cambial cells, phloem ray parenchyma cells and longitudinal phloem parenchyma cells had increased slightly from 55 to 58, from 28 to 32 and from 39 to 40, respectively (Fig. 5B). In heated stems, there were significant decreases in the number of starch granules in cambial cells, phloem ray parenchyma cells and longitudinal phloem parenchyma cells between 11 December 2007 and 14 February 2008 (P < 0·05; Fig. 5B).

In heated stems, on 29 January 2008, when cambial reactivation had occurred, starch granules in fusiform cambium had decreased in size more than those on 16 January 2008, 1 week prior to the start of heating (Fig. 4B–D). After approx. 3 weeks of heating, on 14 February 2008, when xylem differentiation had started, starch granules had decreased in size and were irregular in shape, and they were more sparsely distributed in the phloem ray parenchyma cells and longitudinal phloem parenchyma cells (Fig. 4G, I) than those on 16 January 2008, 1 week prior to the start of heating (Fig. 4F, H). In contrast, in non-heated control stems, on the same day, 14 February 2008, the size of starch granules in fusiform cambium, phloem ray parenchyma cells and longitudinal phloem parenchyma cells was almost the same as that on 16 January 2008 (Fig. 4B, C, F, H, J, K, L).

From the results, it was clear that the level, number and size of starch had decreased in the ray cambium, fusiform cambium, phloem ray parenchyma cells and longitudinal phloem parenchyma cells from cambial reactivation to the start of xylem differentiation in heated stems.

There were no obvious differences in terms of levels, number and size of storage starch granules between the non-heated control stems and heated stems of the two trees that were examined from 11 December 2007 to 14 February 2008.

Localization, levels and size of lipids (as droplets) in cambium and phloem of non-heated control and heated stems

On 11 December 2007, the percentage areas occupied by lipid droplets in the ray and fusiform cambium were approx. 40 % and 32 % (Figs 6A and 7). On 16 January 2008, 1 week prior to the start of heating, percentage areas occupied by lipid droplets in the ray and fusiform cambium had increased slightly to 42 % and 34 % (Figs 6B and 7). After 6 d of heating, on 29 January 2008, when cambial reactivation had occurred, the percentage areas occupied by lipid droplets in the ray and fusiform cambium had decreased to 24 % and 24 %, respectively (Figs 6C and 7). In contrast, in non-heated control stems, on the same day, 29 January 2008, the percentage areas occupied by lipid droplets in the ray and fusiform cambium had increased very slightly to 44 % and 36 % (Fig. 7). After approx. 3 weeks of heating, on 14 February 2008, when xylem differentiation had started, these areas in ray and fusiform cambium had fallen to 14 % and 13 %, respectively (Figs 6D and 7). In non-heated control stems, on the same day, 14 February 2008, the percentage areas occupied by lipid droplets in the ray and fusiform cambium had increased slightly to 48 % and 39 % (Fig. 7). In heated stems, there were significant changes in the percentage areas occupied by lipid droplets in the ray and fusiform cambium between 11 December 2007 and 14 February 2008 (P < 0·05; Fig. 7).

Fig. 6.
Confocal laser scanning microscopic images (projection) showing lipid droplets in the cambium, phloem and xylem of non-heated control stems and heated stems. Lipid droplets are shown in cambium on (A) 11 December 2007 and (B) 16 January 2008, 1 week prior ...
Fig. 7.
Percentage of unit area occupied by lipid droplets in the cambium and phloem of non-heated control stems and heated stems at different times in winter (n = 4). Columns and bars show mean values ± standard deviation. Means with the same letter ...

On 16 January 2008, 1 week prior to the start of heating, the percentage area occupied by lipid droplets in phloem ray parenchyma cells was close to 11 % and in longitudinal phloem parenchyma cells it was about 7 % (Figs 6F, H and 7). In non-heated control stems and heated stems, there were no obvious differences in the respective levels of lipid droplets in phloem ray parenchyma cells and longitudinal phloem parenchyma cells between 11 December 2007 and 14 February 2008 (Figs 6F–I and 7). The levels of lipid droplets were higher in fusiform and ray cambium than in phloem ray parenchyma cells and longitudinal phloem parenchyma cells.

After 6 d of heating, on 29 January 2008, when cambial reactivation had occurred, the size of lipid droplets in cambium was almost the same as that on 16 January 2008, 1 week prior to the start of heating (Fig. 6B, C). After approx. 3 weeks of heating, on 14 February 2008, when xylem differentiation had started, almost all lipid droplets had decreased in size in ray cambial cells (Fig. 6D). In contrast, in non-heated control stems, on the same day, 14 February 2008, the size of lipid droplets in cambium was almost the same as that on 16 January 2008 (Fig. 6B, E). There were no obvious changes in the size of lipid droplets in phloem ray parenchyma cells and longitudinal phloem parenchyma cells between 16 January 2008, 1 week prior to the start of heating, and 14 February 2008, when xylem differentiation had started in heated stems (Fig. 6F–I).

From the results, it was clear that the levels and size of lipid droplets had fallen in ray and fusiform cambium from cambial reactivation to the start of xylem differentiation in heated stems but there were no obvious similar changes in phloem.

There were also no obvious differences, in terms of levels and size of lipid droplets between the non-heated control stems and heated stems of the two trees that were examined from 11 December 2007 to 14 February 2008.

DISCUSSION

Sucrose is derived from photosynthates and is transported basipetally through the phloem in the stems. Low-level photosynthetic activity has been detected in evergreen conifers under mild temperate conditions in winter (Fry and Phillips, 1977; Zarter et al., 2006), while net photosynthesis is close to zero in evergreen conifers under cool temperate winter conditions when the minimum temperature falls below zero (Troeng and Linder, 1982). Thus, in temperate zones, the low rates of photosynthesis in late winter and early spring might limit the supply of sucrose to stems. The present observations showed that, in heated Cryptomeria japonica stems, cambial reactivation occurred at the end of January when photosynthesis by needles was minimal at the site of the experiment. Therefore, the cambium must exploit sources of sugars other than photosynthesis for reactivation.

It has been well established that cambial reactivation involves a progression of events such as thinning of the radial wall and increase in vacuolation (Bailey, 1930; Funada and Catesson 1991; Catesson, 1994; Rensing and Samuels, 2004), including changes in RNA, protein and carbohydrates, as well as in lipids and enzyme activity in Abies balsamea (Riding and Little, 1984, 1986). Druart et al. (2007) reported that, in Populus tremula, the induction of genes for glycolytic enzymes activated the breakdown of starch to meet the increased energy demand of cambial activity. Therefore, they suggested that the breakdown of starch might play a key role in the generation of energy. In the present study, it was observed that levels of starch in cambium and phloem tissues and the relative area occupied by lipid droplets in cambium fell when cambial reactivation occurred in the heated stems. This observation suggests that storage materials, such as starch and lipid droplets, might be utilized as sources of energy for cell division and the biosynthesis of new cell walls in the cambium of Cryptomeria japonica trees. Oribe et al. (2003) reported that, in Abies sachalinensis, cells in heated reactivated cambium stopped dividing upon the disappearance of starch from storage tissues around the cambium. Therefore, they postulated that the continuation of cambial activity might require a continuous supply of sucrose to the cambium. Previously it had been observed that, in heated poplar stems, the level of starch around the cambium fell when cambial reactivation occurred, with starch granules disappearing completely from the cambium and phloem tissues immediately after cambial reactivation (Begum et al., 2007). In the present study, during cambial dormancy in December, a low level of starch was found in ray cambium but there were no starch granules in fusiform cambial cells. In the middle of January, the level of starch in cambium and phloem tissues was higher than in December. It was found that, in Cryptomeria japonica, the cambium was in resting stage of dormancy in December and in quiescent stage of dormancy in January at the experimental site (Begum et al., 2010). Increases in levels of starch in the fusiform cambium in the middle of January might be related to a change in cambial dormancy from resting stage to quiescent stage. However, in heated Cryptomeria japonica stems, the level of starch fell significantly in the ray and fusiform cambium and phloem tissues when cambial reactivation occurred at the end of January. The present results suggest that decreases in levels of starch in cambium and phloem tissues might be due to the enzymatic conversion of starch to sugar during cambial reactivation in heated stems. Therefore, sucrose might be a source of energy for cambial reactivation and the continuous division of cells in the cambium.

A major step in the development of cold hardiness in the cambium occurs when both energy and carbon become limiting, as photosynthesis declines (Keskitalo et al., 2005). It has been reported that, during cambial dormancy, starch granules are present at low levels in ray and fusiform cambial cells in Cryptomeria japonica (Itoh, 1971), are rare in ray cambial cells of Picea abies (Timell, 1986) and are present only occasionally in the cambium of Salix dasyclados (Sennerby-Forsse and Fricks, 1987). These observations suggest that, during cambial dormancy, the level of starch might be low as a consequence of the breakdown of starch for generation of energy for the development of cold hardiness. Oribe et al. (2003) noted that, in the evergreen conifer Abies sachalinensis, there were no starch granules in the fusiform cambium prior to heating, whereas starch granules appeared upon localized heating of stems. In a previous study of deciduous hardwood hybrid poplar, starch granules were detected in the fusiform cambium throughout the heating period (Begum et al., 2007). By contrast, the present study revealed starch granules in the fusiform cambium prior to the start of heating, even though the hybrid poplar and the Cryptomeria japonica trees studied were growing under the same climatic conditions. Therefore, it seems that the levels of storage starch in the fusiform cambium during winter dormancy vary among species and depend on their respective patterns of cambial growth.

An analyses of 13CO2 pulse-labelled photoassimilates demonstrated that the cell walls of earlywood were derived from both the previous year's photoassimilate and from that of the current spring (Kagawa et al., 2006a, b). Therefore, it was suggested that previous year's stored carbon might be important for the current year's radial growth. Piispanen and Saranpää (2001) reported that, in the branchwood of Betula pendula, sucrose is the principal source of energy in the secondary xylem, with the level of sucrose rising from the xylem towards the cambium to provide the energy required for growth. At the beginning of the growing season, in April, there is a marked increase in the level of starch in xylem ray parenchyma cells, known as the ‘springtime starch increase’ and the accumulated starch provides the energy required for growth and for the production of the first layers of earlywood in Picea glauca under natural conditions (Wang and Zwiazek, 1999). By contrast, Fischer and Höll (1992) failed to detect any significant changes in the level of starch in the xylem of Scots pine (Pinus sylvestris) during xylem differentiation under natural conditions in May, when photosynthesis was active in needles. However, storage starch reappeared around the cambium at the start of xylem differentiation in heated hybrid poplar stems at the end of April, when bud burst had already occurred but photosynthesis was not yet active because of the absence of leaves (Begum et al., 2007). In addition, Oribe et al. (2003) observed that, in heated Abies sachalinensis stems, differentiation of the xylem did not begin, as a consequence of the disappearance of storage starch around the cambium, when the rate of photosynthesis in needles was low. The present results show clearly that, in heated stems, the level of starch in cambium and phloem falls significantly at the start of xylem differentiation in mid-February, when minimal photosynthate is supplied by the needles. Therefore, storage starch might be the source of sucrose for the formation of new cell walls when the xylem starts to differentiate in heated stems of Cryptomeria japonica.

The size and number of starch granules in phloem and cambium decreased from cambial reactivation to xylem differentiation in the heated stems of Cryptomeria japonica. A marked change in number and size of starch granules was also observed in phloem ray parenchyma cells and longitudinal phloem parenchyma cells during xylem differentiation. The decreases in number and size of starch granules were closely related to the change in their levels. Therefore, the reduced number and size of starch granules in phloem and cambium might provide energy during cambial reactivation and xylem differentiation.

In Robinia pseudoacacia, lipid droplets accumulated mainly in the cambium and the level of lipids (as droplets) fell steadily as the distance from the cambium to the phloem and xylem increased during cambial dormancy (Hillinger et al., 1996). Similarly, in the present study of Cryptomeria japonica trees, the relative area occupied by lipid droplets was higher in the cambium than in the phloem and this area remained unchanged in phloem ray parenchyma cells and longitudinal phloem parenchyma cells from December to February. Fischer and Höll (1992) and Sauter and van Cleve (1994) proposed that the storage lipids in the secondary xylem ray parenchyma cells might be generated from sugars or from starch and that the small-scale conversion of starch to lipids might occur in the autumn in Scots pine and poplar trees, respectively. In addition, Sauter and van Cleve (1994) reported that, in the secondary xylem ray parenchyma cells that were located close to the cambium of poplar trees (Populus × canadensis), there were clear reductions in levels of lipids (as droplets) from cambial reactivation to the start of xylem differentiation. Therefore, these authors postulated that lipid droplets might be mobilized for the production of new cells in this variety of poplar in spring time. The genes of the glyoxysomal cycle encoded malate synthase and isocitrate lyase were induced during cambial dormancy to provide energy in Populus tremula (Schrader et al., 2004). The present results show clearly that level of lipids (as droplets) in cambium fell from cambial reactivation to the start of xylem differentiation in heated Cryptomeria japonica stems, while there were no obvious changes in these levels in phloem. In addition, the size of lipid droplets in ray cambial cells decreased during xylem differentiation but there was no obvious change in their size in phloem ray parenchyma cells and longitudinal phloem parenchyma cells. Therefore, it seems likely that lipid droplets, as well as starch, in the cambium might be used as a source of energy for cambial activity and the initiation of xylem differentiation in heated stems.

In conclusion, the localization, levels, number and size of starch granules and lipid droplets might be closely related to cambial reactivation and the initiation of xylem differentiation. In heated stems, the level, number and size of starch granules decreased in cambium and phloem from cambial reactivation to the start of xylem differentiation, indicating that an elevated temperature might induce the enzymatic conversion of starch to sugars. Prominent reductions in levels and size of lipid droplets in the cambium, from cambial reactivation to the start of xylem differentiation, indicate that lipid droplets also serve as a source of energy for the biosynthesis of new cell walls during cambial reactivation. It is clear that starch granules are widely distributed in cambium and phloem tissues whereas lipids (as droplets) are mainly localized in the cambium. Three-dimensional optical digital imaging of starch and lipid droplets by confocal laser scanning microscopy is a new and effective method for investigating the relationship between storage materials and cambial activity in trees.

ACKNOWLEDGEMENTS

This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (nos. 17580137, 19580183, 21380107, 20·5659 and 22·00104).

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