ABSTRACT
To understand the precise process of wood formation, it is necessary to identify the factors that regulate cambial activity and development of cambial derivatives. Here, we investigated the combined effects of localized-heating and auxin on cambial reactivation and the formation of earlywood tracheids in seedlings of the evergreen conifer Abies homolepis in winter. Three treatments were applied, namely heating (artificial increase in temperature 20–22 °C), heating-plus-auxin transport inhibitor N-(1-naphthyl) phthalamic acid (NPA) and heating-plus-defoliation (removal of needles and buds), with an approximate control, for investigations of cambial activity by light microscopy. After one week of heating, cambial reactivation occurred in the heating, heating-plus-NPA and heating-plus-defoliation treatments. In untreated controls, cambial reactivation occurred later than in heated stems. Earlywood tracheids were formed after three and six weeks of heating in the heating and heating-plus-NPA treatments, respectively. No tracheids were formed after eight weeks of heating in heated-defoliated seedlings. Numbers of new tracheids were reduced in heated stems by NPA. Our results suggest that an increase in the temperature of the stem is one of the most important limiting factors in cambial reactivation, which is independent of needles and buds and of the polar transport of auxin from apical sources. However, after cambial reactivation, initiation and continuous formation of earlywood tracheids require basipetally transported auxin and other endogenous factors originating in mature needles and buds.
INTRODUCTION
Environmental factors and endogenous components of trees interact to influence the formation of wood. The activity of the vascular cambium (cambium) produces wood and increases the radial diameter of tree stems. In temperate-zone trees, cambial activity and dormancy exhibit annual periodicity (Catesson 1994; Larson 1994; Prislan et al. 2013; De Micco et al. 2016; Funada et al. 2016; Kitin & Funada 2016; Schmitt et al. 2016). An increase in temperature in winter or early spring is closely associated with the resumption of cambial activity (cambial reactivation) and the induction of xylem differentiation (Oribe & Kubo 1997; Begum et al. 2013, 2018). However, the involvement of additional factors that might control cambial reactivation and xylem differentiation in evergreen conifers and deciduous hardwood trees has been proposed (Savidge & Wareing 1981a, b; Savidge & Barnett 1993; Oribe et al. 2003; Begum et al. 2013, 2018; Kudo et al. 2014; Funada et al. 2016; Oribe & Funada 2017). It is necessary to identify the factors that influence cambial growth if we are to understand the process of wood formation and enhance the production of bio-mass.
Auxin is produced in young needles and/or elongating shoots and is transported basipetally to the cambium. It appears to be an essential stimulant of cambial cell division and the formation of earlywood tracheids (Larson 1964; Odani 1975; Little & Savidge 1987; Little & Sundberg 1991; Sundberg et al. 2000; Funada et al. 2001; Ursache et al. 2013; Campbell & Turner 2017). Application of exogenous indole-3-acetic acid (IAA) to disbudded and defoliated stems or cuttings causes the resumption of cambial activity and the radial expansion of cambial derivatives (Little & Bonga 1974; Sundberg & Little 1987, 1990; Mellerowicz et al. 1992). In contrast, removal of apical sources and inhibition of the supply of IAA to the cambium decreases levels of IAA in cambial regions (Sandberg & Ericsson 1987; Sundberg & Little 1987, 1990; Sundberg et al. 1993; Funada et al. 2001). Decreases in levels of IAA in cambial regions are associated with the inhibition of cambial activity and xylem differentiation. In addition, auxin-transport inhibitors, such as N-(1-naphthyl) phthalamic acid (NPA), methyl-2-chloro-9-hydroxyfluorene-9-carboxylic acid (CF) and morphactin, inhibit the polar transport of auxin and drastically decrease tracheid production below the corresponding treated portions of conifer stems (Phelps et al. 1977; Yamaguchi et al. 1980, 1983; Sundberg et al. 1994). Moreover, the application of NPA on the stem inhibits the formation of tension wood in hardwood hybrid poplar (Populus deltoides × Populus nigra) (Yu et al. 2017). The cited studies suggest that the basipetal polar transport of auxin to the cambium is crucial for wood formation.
Auxin regulates the number of dividing cambial cells and plays an important role in positional signaling (Uggla et al. 1996, 1998). In evergreen and deciduous conifers, levels of IAA in cambial regions vary seasonally during the active cambial season but are relatively constant during cambial dormancy in winter (Savidge et al. 1982; Savidge & Wareing 1984; Sandberg & Ericsson 1987; Sundberg et al. 1987, 1990; Savidge 1991; Funada et al. 2001, 2002). A rapid increase in cambial activity is associated with an increase in the total amount of IAA in the cambial region (Sundberg et al. 1991; Funada et al. 2001, 2002). In addition, Baba et al. (2011) showed that, in the deciduous hardwood Populus tremula × tremuloides, the responsiveness of the cambium to auxin decreases during cambial dormancy. However, no clear relationship has been found between levels of endogenous auxin and the timing of cambial reactivation (Sundberg et al. 1991; Funada et al. 2001, 2002, 2016). Thus, other factors might be important in the physiological regulation of cambial reactivation.
In several conifers, the dormant cambium is reactivated by localized heating of stems in wintertime (Savidge & Wareing 1981a; Barnett & Miller 1994; Oribe & Kubo 1997; Oribe et al. 2001, 2003; Gričar et al. 2006; Begum et al. 2010b; Rahman et al. 2016; Oribe & Funada 2017), as it is in diffuse-porous (Begum et al. 2007) and ring-porous (Kudo et al. 2014) hardwoods. The pattern of cambial reactivation and the formation of earlywood tracheids in the locally heated stems of conifers are similar to those observed under natural conditions. Therefore, it is clear that an increase in temperature is a direct trigger for cambial reactivation in evergreen and deciduous species (Begum et al. 2013, 2018). The response of the cambium in locally heated regions of stems can be analyzed directly without affecting the physiology of the whole tree. Therefore, artificial heating of stems provides a good model for investigations of cambial reactivation (Begum et al. 2013, 2018; Funada et al. 2016).
After cambial reactivation, cambial cells begin to differentiate, with the elongation or expansion of cells and the thickening of cell walls. The application of IAA to defoliated isolated stems of Pinus contorta initiated cambial cell division and the radial expansion of cambial derivatives but secondary walls were not formed during such differentiation (Savidge & Wareing 1981b; Savidge & Barnett 1993). Cambial cells differentiated into tracheids and secondary walls were formed in the presence of just one pair of needles, with or without the application of IAA. The cited studies suggest that some specific tracheid differentiation factor(s) [TDF(s)] from needles might regulate the differentiation of xylem. However, no such TDFs have yet been identified.
The present study was designed to identify factors that might be involved in cambial reactivation and the formation of earlywood tracheids in the evergreen conifer Abies homolepis. In this study, localized heating of stems was applied in winter. Just above locally heated regions of stems, NPA was applied to inhibit the basipetal polar transportation of auxin from needles and buds. In addition, localized heating was applied to stems of defoliated seedlings to inhibit the supply of factors from needles and buds. We investigated the timing of cambial reactivation, the position of formation of the first new cell plates, the timing of initiation of earlywood tracheids, and the number of layers of earlywood formed in the variously treated and control stems.
MATERIALS AND METHODS
Plant materials
This experiment was designed around the localized heating of stems of seedlings of the evergreen conifer Abies homolepis (age approximately 2 years; height approximately 20 cm; average diameter of stems in the sample-collection area, 2 mm). The seedlings were grown in pots in the field nursery of Tokyo University of Agriculture and Technology in Fuchu, Tokyo (35.684 °N, 139.479 °E). The seedlings were subjected to sequential observations of cambial activity and the differentiation of xylem for studies of the role of auxin during the transition from dormant to active cambium.
Three treatments were applied to a total of 50 seedlings, including controls. These treatments are shown schematically in Figure 1.
Schematic representation of the treatment of seedlings, namely control, heating (artificial increase in temperature to 20–22 °C), heating plus treatment with the auxin transport inhibitor NPA and heating plus defoliation (removal of needles and buds). Electric heating wire was wrapped around stems 2 to 4 cm above the base of seedlings below the site of NPA treatment and defoliation.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
Localized heat treatment
Electric heating wire (Nippon Heater Co., Ltd, Tokyo, Japan; 6 m long and 0.5 cm wide) was wrapped around the stem of individual seedlings, starting 2 to 3 cm above soil level (Begum et al. 2012). The heating wire covered 3 to 4 cm of the stem of each seedling and this region was taken as the locally heated region of the stem. An alternating current was passed through the heating wire at a potential of 100 V to warm the surface of the stems. The temperature between the outer bark and the heating tape was recorded with a thermometer and, at the site at which each stem was heated, the temperature was adjusted to between 20 and 22 °C with a thermostat (TC-1NP; As One Co., Osaka, Japan) and recorded with a data logger (Ondotori Jr. TR-52; T&D Co., Matsumoto, Japan). Seedlings without electric heating wire were used as non-heated control stems. Localized heat treatment was initiated on 6 February 2015 and continued until 3 April 2015.
Treatment of stems with NPA
The auxin transport inhibitor N-(1-naphthyl) phthalamic acid (NPA; Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) was applied to main stems at a site 3 to 4 cm above each locally heated region on 3 February 2015 (three days before the start of heating). NPA was applied, at a concentration of 1% (10 mg in 1 gm lanolin), around the circumference and over a distance of 3 cm of each respective stem. Each NPA-treated region was covered with aluminum foil. The NPA and lanolin mixture was replaced with a fresh mixture on a weekly basis.
Defoliation
For defoliation, all needles and terminal buds were removed from the respective seedlings on 3 February 2015 (three days before the start of localized heating). Cut surfaces were coated with lanolin to prevent dehydration.
Collection of samples
Samples were collected from 2 to 3 cm above the soil, where the heating wire had been wrapped around stems and from similar regions of stems of non-heated controls. Localized heating of stems was started three days after the application of NPA and defoliation. Samples were collected at weekly intervals from 6 February to 6 March 2015 and then at biweekly intervals until 3 April 2015. On each sampling date, samples were collected from two seedlings subjected to each condition namely, no heating, heating, heating plus NPA and heating plus defoliation. There was no obvious difference in the date of onset of cambial reactivation and progression of cambial activity between the two studied seedlings of each treatment.
Preparation for light microscopy
Samples were immediately 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. After trimming, specimens were dehydrated in a graded ethanol series and embedded in epoxy resin. Transverse sections were cut from embedded samples at a thickness of approximately 1 μm with a glass knife on an ultramicrotome (Ultracut N; Reichert, Vienna, Austria). The sections were stained with a solution of 1% safranin in water for 30 min with five or six subsequent washes with water for visualization of cambial cell division and xylem differentiation.
All sections were examined under a light microscope (Axioscope; Carl Zeiss, Oberkochen, Germany) as described by Begum et al. (2007, 2016) and Nakaba et al. (2015, 2016).
Temperature profiles and cambial reactivation
Meteorological data for 2015 for the duration of our experiment was obtained from the Japan Meteorological Agency in Fuchu, Tokyo. The daily maximum, average and minimum temperatures from 20 January to 30 April 2015 are shown in Figure 2.
Meteorological data showing the maximum, average and minimum daily air temperatures at the experimental site in Fuchu, Tokyo, from 20 January to 30 April 2015.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
From 20 January to 28 February 2015, the daily maximum temperature was between 3 and 18 °C and the minimum temperature fell below freezing (0 °C) several times. In March and April, the daily maximum temperature was between 6 and 27 °C and the daily minimum temperature occasionally fell below 0 °C.
RESULTS
Cambial dormancy
In stems of Abies homolepis seedlings, before the start of heating in winter, the cambium consisted of four or five radial layers of radially narrow and compactly arranged fusiform cells (6 February 2015; Fig. 3). There was no evidence of cell division in the fusiform cambial cells and ray cambial cells. The cambium was located between the phloem cells and the narrow-diameter thick-walled latewood tracheids that had been formed during the previous growing season. These observations confirmed the dormancy in samples of cambium collected from seedlings before the start of localized heating.
Light micrograph showing transverse view of cambium collected before the start of heating on 6 February 2015. Four or five layers of fusiform cambial cells were arranged compactly with no evidence of cell division, confirming cambial dormancy. C = cambium, Ph = phloem, Xy = xylem. Scale bar = 50 μm.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
Light micrographs showing transverse views of cambium collected from the seedlings on 13 February 2015 after one week of heating. – a: Under natural conditions, in the non-heated control seedling, three to five layers of fusiform cambial cells were compactly arranged with no evidence of cell division, confirming cambial dormancy. – b: In the heat-treated seedling, new cell plates (arrowhead) in the cambium were observed in the locally heated region of the stem. – c & d: Cambial cell division was also evident (arrowhead) in the locally heated region of the stem of the heat- plus NPA-treated seedling (c) and the heated and defoliated seedling (arrowhead indicates new cell plate; d). – C = cambium, Ph = phloem, Xy = xylem. – Scale bars = 50 μm.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
Cambial reactivation and formation of earlywood tracheids
On 13 February 2015, there was no evidence of cell division in the cambium of stems of non-heated control seedlings (Fig. 4a). Three to five layers of fusiform cambial cells were arranged compactly, providing evidence of dormancy (Fig. 4a). In heat-treated seedlings, by contrast, new cell plates with thin cell walls were formed after just one week of heating in locally heated regions of stems, providing evidence of cambial reactivation (Fig. 4b). Cambial cell division was also apparent on the same day in locally heated regions of stems treated with NPA and in heated and defoliated seedlings, indicating that neither the application of NPA nor defoliation inhibited cambial reactivation in locally heated regions of stems (Fig. 4c & d). The pattern of initiation of cell division in the cambium was similar and new cell plates were formed on the phloem side of the cambium in locally heated regions of stems of heated, heated and NPA-treated and heated and defoliated seedlings (Fig. 4b, c & d).
Light micrographs showing transverse views of cambium collected from seedlings on 20 February 2015 after two weeks of heating. – a: Under natural conditions, in the non-heated control seedling, cambium was dormant. – b: In the heat-treated seedling, cambial cell division had continued (arrowheads) but no cambial cells had differentiated into tracheids in the locally heated region of the stem. – c & d: Cambial cell division had also continued (arrowhead) in the locally heated region of the stem of the heat- and NPA-treated seedling (c) and of the heated and defoliated seedling (arrowhead indicates new cell plate; d). – C = cambium, Ph = phloem, Xy = xylem. – Scale bars = 50 μm.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
On 20 February 2015, the cambium was still dormant in samples collected from non-heated control seedlings (Fig. 5a). In heat-treated seedlings on 20 February 2015, division of cells in the cambium had continued but cambial cells had not differentiated into tracheids after two weeks of heating in locally heated regions of stems (Fig. 5b). On the same date, similar results were observed in samples of locally heated stems treated with NPA and in locally heated stems subjected to defoliation, indicating the presence of dividing cambial cells and the absence of differentiating cells (Fig. 5c & d).
On 27 February 2015, the cambium was still dormant in samples of non-heated control seedlings (Fig. 6a). In the heat-treated seedlings on this date, differentiation of xylem had been initiated and one to two layers of expanding tracheids were formed after three weeks of heating in locally heated regions of stems (Fig. 6b). The newly formed tracheids had large radial diameters and thin cell walls, resembling earlywood (Fig. 6b). On the same date, division of fusiform cells and ray cells was seen to have continued but cambial cells had not differentiated into tracheids in locally heated regions of stems exposed to NPA and to heating plus defoliation (Fig. 6c & d). Only dividing cambial cells, with five or six layers of fusiform cells, were observed, indicating that the application of NPA and defoliation had suppressed the initiation of earlywood tracheids in locally heated regions of stems (Fig. 6c & d).
Light micrographs showing transverse views of cambium collected from seedlings on 27 February 2015 after three weeks of heating. – a: Under natural conditions, in the non-heated control seedling, three or four layers of fusiform cambial cells were compactly arranged, with no evidence of cell division. – b: In the heat-treated seedling, one or two layers of earlywood tracheids (arrowheads) with large diameters and thin cell walls were formed in the locally heated region of the stem. – c: In the heat- and NPA-treated seedling, cambial cell division had continued (arrowhead) but no earlywood tracheids were formed in the locally heated region of the stem. – d: In the heated and defoliated seedling, new cell plates with thin cell walls were observed (arrowhead) but there were no cells that were differentiating into tracheids in the locally heated region of the stem. – C = cambium, Ph = phloem, NXy = new xylem, Xy = xylem. – Scale bars = 50 μm.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
By 6 March 2015, fusiform cambial cells had started to divide in the stems of non-heated control seedlings, indicating that cambial reactivation had occurred under natural conditions (Fig. 7a). Cambial reactivation under natural conditions in non-heated control seedlings started three weeks later than cambial reactivation in locally heated regions of stems with or without NPA and with or without defoliation (Table 1). Cell division was initiated on the phloem side of the cambium of non-heated control seedlings. This pattern of cambial cell division was similar to the pattern of initiation of cell division in the cambium of heated, heated and NPA-treated, and heated and defoliated seedlings.
The timing of cambial reactivation and earlywood formation in seedlings of the evergreen conifer Abies homolepis subjected to various treatments. On every sampling, two seedlings were used for each treatment.
In heat-treated seedlings on 6 March 2015, some cambial cells had differentiated into xylem cells, and three or four layers of earlywood tracheids with large radial diameters and thin cell walls were formed after four weeks of heating in locally heated regions of stems (Fig. 7b). On the same date, in the heated and NPA-treated, and in heated and defoliated seedlings, some new cell plates were seen in the cambium but no cambial cells had differentiated into tracheids after four weeks of heating in locally heated regions of stems (Fig. 7c & d).
By 20 March 2015, a few cambial cells had differentiated into earlywood tracheids with large radial diameters and thin cell walls in stems of non-heated control seedlings (Fig. 8a). In heat-treated seedlings on 20 March 2015, we observed that eight or nine layers of earlywood tracheids with large radial diameters and thin cell walls were formed after six weeks of heating in locally heated regions of stems (Fig. 8b). On the same date, in heat- and NPA-treated seedlings, xylem had begun to differentiate and one or two layers of earlywood tracheids had been induced after six weeks of heating in locally heated regions of stems (Fig. 8c). The differentiation of tracheids in locally heated regions of stems in heat- plus NPA-treated stems began three weeks later than in heat-treated stems, indicating that the application of NPA had delayed the initiation of earlywood tracheids. On the same day, in the heated and defoliated seedlings, we found new cell plates and thin cell walls in the cambium but no newly differentiating earlywood tracheids after six weeks of heating in locally heated regions of stems (Fig. 8d).
Light micrographs showing transverse views of cambium collected from seedlings on 6 March 2015 after four weeks of heating. – a: Under natural conditions, in the non-heated control seedling, new cell plates were seen in the cambium (arrowheads) on the phloem side of the cambium. – b: In the heat-treated seedling, three or four layers of earlywood tracheids were formed in the locally heated region of the stem. – c: In the heat- and NPA-treated seedling, cambial cell division had continued (arrowhead) but there were no cambial cells that had differentiated into tracheids in the locally heated region of the stem. – d: New cell plates with thin cell walls were also observed (arrowhead) in the cambium of the locally heated region of the stem of the heated and defoliated seedling. – C = cambium, Ph = phloem, NXy = new xylem, Xy = xylem. – Scale bars = 50 μm.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
On the last day of observations on 3 April 2015, we found an increase in cambial activity in stems of non-heated control seedlings (Fig. 9a). Cambial cell division and the differentiation of cambial derivatives into xylem had continued and three or four layers of earlywood tracheids were formed in stems of non-heated control seedlings (Fig. 9a). In heat-treated seedlings, fifteen or sixteen layers of differentiated earlywood cells were observed after eight weeks of heating in locally heated regions of stems (Fig. 9b). On the same day, in the heat- and NPA-treated seedlings, three or four layers of tracheids were evident in locally heated regions of stems (Fig. 9c). In the heated and defoliated seedlings, we found some new cell plates in the cambium but no differentiated xylem cells after eight weeks of heating. Thus, the formation of earlywood tracheids had been completely inhibited by defoliation after the start of cambial cell division in the locally heated region of stems (Fig. 9d).
Light micrographs showing transverse views of cambium collected from seedlings on 20 March 2015 after six weeks of heating. – a: Under natural conditions, in the non-heated control seedling and with the continuation of cambial cell division, xylem differentiation had been initiated (arrowheads) and tracheids had started to form. – b: In the heat-treated seedling, eight or nine layers of earlywood tracheids with large diameters and thin cell walls were visible in the locally heated region of the stem. – c: In the heat- and NPA-treated seedling, cambial cells had differentiated into tracheids (arrowheads) and one or two layers of earlywood tracheids were formed in the locally heated region of the stem. – d: In the heated and defoliated seedling, cambial cells had not differentiated into tracheids in the locally heated region of the stem (arrowhead indicates new cell plate). – C = cambium, Ph = phloem, NXy = new xylem, Xy = xylem. – Scale bars = 50 μm.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
The cambial reactivation occurred in locally heated regions of stems when the maximum, average and minimum temperatures were approximately 10 °C, 4 °C and -2.8 °C, respectively. Daily temperatures started to rise consistently from mid-March to the beginning of April. The timing of the initiation of cambial cell division and the formation of earlywood tracheids in locally heated regions and non-heated control regions of stems of Abies homolepis seedlings is summarized, for the various treatments in Table 1.
Light micrographs showing transverse views of cambium collected from seedlings on 3 April 2015 after eight weeks of heating. – a: Under natural conditions, in the non-heated control seedling, three or four layers of earlywood tracheids were formed. – b: In the heat-treated seedling, fifteen or sixteen layers of earlywood tracheids were formed in the locally heated region of stem. – c: In the heat- and NPA-treated seedling, three or four layers of tracheids were formed in the locally heated region of the stem. – d: In the heated and defoliated seedling, no cambial cells had differentiated into tracheids in the locally heated region of the stem (arrowhead indicates new cell plate). – C = cambium, Ph = phloem, NXy = new xylem, Xy = xylem. – Scale bars = 50 μm.
Citation: IAWA Journal 39, 4 (2018) ; 10.1163/22941932-20170211
DISCUSSION
Localized heating of stems induces earlier division of cells in the cambium than that observed in non-heated control stems under natural conditions in seedlings of the evergreen conifer Abies homolepis. Cambial reactivation occurred after one week of localized heating, as observed on 13 February 2015, while cambium of non-heated control stems remained dormant. In the non-heated control stems, cambial cell division had resumed by 6 March 2015, with a three-week delay as compared to locally heated stems. An artificial increase in the temperature of stems during cambial dormancy in winter induces cambial reactivation in conifers (Savidge & Wareing 1981a; Barnett & Miller 1994; Oribe & Kubo 1997; Oribe et al. 2001, 2003; Gričar et al. 2006; Begum et al. 2010a, b, 2012; Rahman et al. 2016; Oribe & Funada 2017) and in ring-porous (Kudo et al. 2014) and diffuse-porous (Begum et al. 2007) hardwoods. In addition, under natural conditions, cambial reactivation occurs when the ambient air temperature starts to rise from late winter to early spring in the evergreen conifer Cryptomeria japonica and in the deciduous diffuse-porous hardwood Populus (Begum et al. 2008, 2010a). Our present results support the hypothesis that an increase in the temperature of stems is a direct trigger for cambial reactivation when the cambium is dormant.
Levels of endogenous IAA in cambial regions vary seasonally during the active growth of cambium in conifers such as Pinus contorta, Abies balsamea and Pinus densiflora (Savidge & Wareing 1984; Sandberg & Ericsson 1987; Sundberg et al. 1987, 1990; Funada et al. 2001). By contrast, during cambial dormancy, levels of IAA in cambial regions in late winter are relatively low and remain unchanged until the growth of buds and needles is initiated in conifers (Sundberg et al. 1987; Funada et al. 1987, 2001, 2002). In the present study, we applied NPA to stems to inhibit the polar transport of auxin from apical sources when levels of IAA in cambial regions were assumed to be relatively low. Cambial reactivation occurred below the NPA-treated locally heated regions of stems after one week of heating. The timing of cambial reactivation in locally heated regions of NPA-treated seedlings was the same as that in locally heated regions of heat-treated seedlings without NPA treatment. These results indicate that a new supply of auxin and an increase in levels of auxin in cambial regions, via polar transport from needles and buds, is not necessary for cambial reactivation and that, while the level of endogenous auxin in the cambial region during dormancy might be low, this is sufficient for the maintenance of cambial cells and for cambial reactivation in Abies homolepis seedlings.
Levels of IAA in the cambial region of stems fall after removal of needles and buds (Sundberg & Little 1987, 1990; Sundberg et al. 1993; Funada et al. 2001). In a deciduous ring-porous hardwood, Quercus serrata, cambial reactivation occurred in locally heated stems during cambial dormancy in winter after removal of buds (Kudo et al. 2014). Thus, they postulated that cambial reactivation might be independent of the growth of buds, new shoots and leaves and, as a consequence, of the supply of auxin. In the present study on the evergreen conifer Abies homolepis, cambial activity resumed in locally heated regions of stems after one week of heating in heated and in heated and defoliated seedlings during cambial dormancy in winter. There were no obvious differences, in terms of the timing of cambial reactivation in locally heated regions of stems, between heated and heated and defoliated seedlings. Therefore, our results suggest that, in seedlings of the evergreen conifer Abies homolepis, the endogenous components essential for cambial reactivation might be present in the dormant cambium and, thus, defoliation has no effect on the initiation of cambial reactivation.
The cambium reactivated by localized heating during cambial dormancy in winter produced wide layers of earlywood tracheids in Picea abies (Gričar et al. 2006), Cryptomeria japonica and Abies firma (Oribe & Kubo 1997; Begum et al. 2012). Moreover, the formation of earlywood was initiated after only two weeks of cambial reactivation in heated regions of stems of two evergreen conifers, namely, Cryptomeria japonica and Chamaecyparis pisifera (Begum et al. 2010b; Rahman et al. 2016). In the present study, we found that earlywood formation was initiated two weeks after cambial reactivation in locally heated regions of heat-treated seedlings. One or two layers of earlywood tracheids with large diameters and thin walls were formed after three weeks of heating (two weeks after cambial reactivation) in locally heated regions of stems of heat-treated seedlings when cambium of control seedlings remained dormant. Further, we observed the continuation of earlywood tracheid production after four weeks, six weeks and eight weeks of heating in locally heated regions of stems of heat-treated seedlings. However, in heat- and NPA-treated seedlings, cambial activity resumed after one week of heating but we found no earlywood tracheids in the locally heated region of the stems after three weeks of heating. The formation of earlywood tracheids was apparent after six weeks of heating in locally heated regions of stems of heat- and NPA-treated seedlings. Under the heated condition, the initiation of formation of earlywood tracheids after cambial reactivation was delayed when we applied NPA to the stems. The application of NPA to stems probably decreased the supply of auxin considerably below the treated region and, thus, the initiation of formation of earlywood tracheids was delayed. A supply of adequate auxin and an increase in levels of auxin in cambial regions, via polar transport from buds and needles, might be important for the induction of earlywood tracheids early in the growing season.
In evergreen conifers, needle-generated components support the formation of xylem cells in stems (Savidge & Wareing 1981a, b; Savidge 2000). Removal of leading shoots and branches (pruning) significantly decreased amounts of xylem cells in Pinus sylvestris and Pinus densiflora (Sandberg & Ericsson 1987; Funada et al. 1989, 1990, 2001; Sundberg et al. 1993). In isolated stem segments of Pinus contorta, differentiation of xylem cells was suppressed after cambial reactivation in the locally heated region of stems after needles and buds had been removed (Savidge & Wareing 1981a). However, cambial cells differentiated into tracheids and secondary walls were formed in the presence of only a single pair of needles (Savidge & Wareing 1981b; Savidge & Barnett 1993). Therefore, it was suggested that unidentified tracheid differentiation factors (TDFs), originating in needles, might be essential for the differentiation of cambial cells into tracheids. In the present study we observed that cell division in the cambium was initiated but cambial cells did not differentiate into tracheids, even after eight weeks of heating, in the locally heated region of stems after defoliation. The failure of cambial cells to differentiate into tracheids after cambial reactivation in locally heated regions of the stem of defoliated seedlings might have been due to the complete absence of a supply of essential factors from apical sources to the cambium.
The initiation of differentiation of cambial derivatives into earlywood tracheids after cambial reactivation in late winter or early spring involves a series of events. After cambial cells have divided, cells expand and cell walls thicken, with the formation of bordered pits (Funada et al. 2016). Cell division in the cambium and events during the differentiation of cambial derivatives into tracheids are maintained by auxin, which provides positional information (Uggla et al. 1996, 1998). The transportation of auxin from intact buds and needles to the cambium is suppressed by treatment of stems with NPA while the supply of other factors from apical sources to the cambium might be expected to remain unchanged (Yamaguchi et al. 1980; Sundberg et al. 1994). By contrast, when needles and buds are removed, the supply of all essential factors, including auxin, from buds and needles to the cambium is suppressed (Savidge & Wareing 1981a, b; Savidge 2000). In the present study, after cambial reactivation, the initiation of differentiation of cambial derivatives into tracheids was delayed when transportation of auxin from buds and needles was inhibited by NPA. However, no cambial cells differentiated into tracheids after defoliation. The results of these two treatments suggest that a new supply of auxin and an increase in levels of auxin from buds and needles, via polar transport, is essential for the initiation of formation of earlywood tracheids. In addition, some factor(s) from buds and needles, other than auxin, might play a regulatory role in the differentiation of earlywood tracheids in the evergreen conifer Abies homolepis.
In conclusion, our results suggest that an increase in the temperature of the stem in winter can induce cambial reactivation in seedlings of the evergreen conifer Abies homolepis. A new supply of auxin and an increase in levels of auxin in the cambium via polar transport from apical sources and, also, the presence of needles and buds are not essential for the breaking of winter cambial dormancy and the initiation of cambial reactivation. The initiation of cell division in the cambium might utilize a limited amount of auxin that is available in the cambial region, but the absence of a continuous supply of auxin from needles and buds to the cambium appears to limit cambial growth and the initiation of formation of earlywood tracheids in locally heated regions of stems. In addition, factors other than auxin that are supplied from apical sources, such as buds and needles, to the cambium appear to be essential for the formation of earlywood tracheids.
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. 20120009, 20-5659, 21380107, 22-00104, 24380090, 15K07508, 15H04527, 16K14954 and 18H02251).
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Corresponding author; e-mail: funada@cc.tuat.ac.jp
