A study of the cambial zone and conductive phloem of common beech (Fagus sylvatica L.) using an image analysis method

A study of the cambial zone and conductive phloem of common beech (Fagus sylvatica L.) using an image analysis method
of 7
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  Trees(1994) 9:106-112 9 Springer-Verlag 1994 A study of the cambial zone and conductive phloem of common beech Fagus sylvatica L. using an image analysis method I. Influence of tree age on the structure Pierre Vollenweider, Isabelle Dustin, Rose-Marie Hofer, Pascal Vittoz, Pierre Hainard Institut de Biologie et de Physiologie v~g&ales/Institut de Botanique syst~matique et de G~obotanique, B~timent de Biologie, Universit6 de Lausanne, CH- 1015 Lausanne/VD, Switzerland Received: 7 December 1993/Accepted: 7 April 1994 Abstract. Quantification is a major problem when using histology to study the influence of ecological factors on tree structure. This paper presents a method to prepare and to analyse transverse sections of cambial zone and of con- ductive phloem in bark samples. The following paper (II) presents the automated measurement procedure. Part I here describes and discusses the preparation method, and the influence of tree age on the observed structure. Highly contrasted images of samples extracted at breast height during dormancy were analysed with an automatic image analyser. Between three young (38 years) and three old (147 years) trees, age-related differences were identified by size and shape parameters, at both cell and tissue levels. In the cambial zone, older trees had larger and more rectan- gular fusiform initials. In the phloem, sieve tubes were also larger, but their shape did not change and the area for sap conduction was similar in both categories. Nevertheless, alterations were limited, and demanded statistical analysis to be identified and ascertained. The physiological im- plications of the structural changes are discussed. Key words: Image analysis - Bark structure - Senescence - Fagus sylvatica Introduction Several age-related changes occurring in secondary vas- cular tissues structure have been documented in recent years in various taxa, including trees from all around the world. A general increase in cell size and diameter was observed in the xylem and in the cambial zone (Baas et al. 1986; Iqbal and Ghouse 1990; Aloni 1991; Ajmal and Iqbal 1992; Ridoutt and Sands 1993), as well as in the conductive phloem (Ajmal and Iqbal 1992). At tissue level, a quanti- Correspondence to: R.-M. Hofer tative decrease of wood production is well known (Schweingruber 1988). In the cambial zone, an increase in the tangential area occupied by cambial rays was observed (Ajmal and Iqbal 1992; Ridoutt and Sands 1993). Results about conductive phloem, the least documented vascular tissue, are more contradictory. In Ficus rumphii, the rela- tive transectional area occupied by sieve tubes is increased, and that occupied by axial parenchyma diminishes with tree aging (Ajmal and Iqbal 1992). However, in the stem of Pinus longaeva, quantitative changes in the sieve tube production rate or in the conductive phloem width were not observed (Connor and Lanner 1990). Gradients of auxin from leaves to roots induce and control cambial cells' division and differentiation (Aloni and Zimmermann 1983; Aloni 1987; Aloni 1991). In xylem, large scattered vessels are induced by low amounts of auxin at low heights, and numerous small and densely packed vessels, by high amounts of auxin at higher loca- tions (Aloni and Zimmermann 1983; Aloni 1991). As most studies published on structural age-related changes were realised with samples extracted at different heights, not only age but also the distance to the source of the growth regulators explain the differences described. Many stress-related changes have been observed in the cell and tissue structure of living trees (Verzilov et al. 1979; Keller and Beda-Puta 1981; Fink 1986; Grosser 1986; Shortle and Bauch 1986; Meyer 1987; Sutinen 1987; Ma- tyssek et al. 1990). Some studies have opened new fields in plant research, for example those concerning needle structure in relation to forest decline (Parameswaran et al. 1985; Sutinen 1987; Vogelmann and Rock 1988; Forschner et al. 1989; Holopainen and Nygren 1989; Schmitt and Ruetze 1989; Hasemann and Wild 1990; Hasemann et al. 1990). All indicate that the structure of tissues can be fundamentally changed by stress. Alterations of phloem structure, such as the collapse of sieve tubes, are among the most common features (Parameswaran et al. 1985; Forschner el al. 1989; Schmitt and Ruetze 1989; Hasemann and Wild 1990; Matyssek et al. 1992); this tissue can be considered as one of the most sensitive to physiological changes occurring when a tree declines.  107 Similar trends are observed in aging and in declining trees as well as for primary growth and crown architecture (Roloff 1986), and for secondary growth (Hartmann et al. 1987; Br~iker 1991). For these reasons, the possible influ- ence of tree age on phloem and on cambial zone structure remains of particular interest in such a species as beech, which locally has serious decline problems (Fltickiger and Braun 1989; OFEFP and FNP 1992), and deserves further work at both cell and tissue levels. However, alterations observed in tissue and cell structures are very rarely quantified and most results lack statistical information about the extent of damage in the analysed organ or in the whole plant. In fact, quantification is a major difficulty in histology, yet without it the implications of such changes on tree vitality cannot be understood. Automatic image analysis provides quick and easy quantitative measure- ments of size and shape, and therefore constitutes a useful tool to study tissue and cell structure (Dustin et al. 1994). This paper presents a method using computer-assisted image analysis, to obtain quantitative data on cell size and shape, and on tissue structure in trunk samples of standing beech Fagus sylvatica L.). This study is divided into two parts: I reports the methods of sample preparation and the results obtained about the relationships between structure and age, and II describes the computer procedures (Dustin et al. 1994). The cambial zone and conductive phloem of young and old trees were analysed from the stem, an easy to sample organ providing interesting opportunities for further work in plant physiology. In the phloem, we worked on the last ring, which corresponds to phloem produced during the preceding growing season, and which constitutes the conductive phloem (Esau 1965; Evert 1990). Materials and methods Sample trees. Sampling was carded out in two forests both located between 600 and 700 m of altitude, in the neighbourhood of Lausanne. The chosen trees grew in several stands in mesophilous to slightly acidophilous beechwoods belonging to the alliance of the Galio-Fagion (Moor 1976). This kind of forest constitutes the climatical climax at this altitude and so is one of the most common natural forest types of the Swiss Plateau. Trees grew in several stands slightly to moderately cleared, which can be classified between the high pole stand and the old high forest. Six beeches divided into two groups of three trees each were sampled: the young group averaged 38 years of age and 10 m height and the older group averaged 147 years and 30 m in height. All trees belonged to the canopy, where they were dominant or co-dominant, and all were healthy. Their crowns were normally developed and no symptoms of decline, such as changes in crown architecture, foliar loss or insect and fungal attacks, could be detected. Sampling. Samples were cut out in January and February during dor- mancy in order to have all trees in the same physiological condition. Cuttings containing periderm, phloem, cambial zone and at least the last xylem ring (1-5 tings on average) were taken from the trunk at breast height (1.3 m) on the north and south sides of the trees. The core extraction method developed used two hole saws 25 and 32 mm in diameter, mounted on an electric drill. Cylinders 27 mm for old trees and 21 mm for young ones were sawn between 1 and 3 cm deep in the tree trunk. The saw was cooled steadily by frequent immersion in a bucket full of water, to prevent thermic tissue alterations. Then the samples were removed with a chisel inserted slantwise in trunk and used as a lever. With this method, large samples could be cut out without exerting tractions or pressures on the very fragile tissues of the inner bark. Samples were first put in hermetically closed plastic bags to prevent dehydration, and cooled at 5 ~ C. In the laboratory, they were washed with distilled water and fixed in FAA (ethanol 50%: formal- dehyde 37%: acetic acid 18 : 1 : 11; Jensen 1962); later they were put in a conserving solution (glycerol: ethanol 50%: H20 1:1:1; Schwein- gruber 1978). The size of the samples minimized alterations due to cutting out, but the fixation time thereafter had to be long enough to allow good penetration of the fixative medium. The trunk wounds were treated with a disinfecting paste, which prevents subsequent wood rot and insect attack. Regenerating tissues filled the holes in 2 or 3 years depending on tree vigour. The stem was seldom infected afterwards by fungi and bacteria, as is frequently observed after increment core extractions (Lenz and Oswald 1971). Preparation method. A small truncated pyramid was trimmed out from each cylinder, containing xylem and phloem without periderm if the bark was thick enough. The cutting surface at the top of the pyramid was 6-8 by 3-4 mm. Its length depended on the size of the last xylem growth ring, maintaining a width sufficient for observation, yet the block remained small enough to allow freezing without cracking. Next, the material was infiltrated for at least 48 h with 0.5 M sucrose used as a cryoprotector, before freezing in 0.5 M sucrose in a truncated syringe in liquid nitrogen (-180 ~ C). Then the samples were transferred at -30 ~ C to a Bright cryomicrotome, where they were planed by passes of 20 gm on a Reichert-Jung steel knife with a tungsten carbide blade. Samples were finally stored in a freezer at -20 ~ C to await further handling. Before observation, samples were thawed at +5 ~ C and rinsed wit]~ water for 48 h. The cell protoplasm was then removed with freshly prepared 10% sodium hypochlorite (2 g Na2CO3 in 40 ml H20 and 1 g CaC1202 in 5 ml H20, mixed and filtered). Samples fixed vertically on a wooden block were placed in a Petri dish under rotary agitation (400 rpm for 30 min). In this way the penetration of hypochlorite was improved, reducing the time needed to clear the protoplasm and thus preventing the destruction of middle lamella, which is the wall layer most sensitive to the hypochlorite treatment. The reaction was stopped with acetic acid (acetic acid 5%, 10 min, 400 rpm). Next, samples were dehydrated with acetone passing stepwise from 0 to 100% in 36 h, and were held in the 100% acetone for at least 120 h. They were then critical point dried after 4 h pre-infiltration in liquid CO2. Finally samples were coated with gold for 36 min in a sputter coater (Edwards S150B). They were placed 3 cm under the cathode (0.5 kV, 10 mA) in the metallizing chamber filled with argon (7-8 mbar). Observation method. Samples were observed in reflected light dark field microscopy, with a Leitz Metallux 3 microscope mounted with objectives for metallurgy (5x/0.09 NPL Fluotar DF; 20x/0.35 NPL DF). They were fastened on a support for SEM and placed on a mi- cromanipulator (Fig. 1). This device, which provides ample angles of rotation, was placed directly on the stage of the microscope. By acti- vating the nut wheel, locally observed tissues could be oriented in a plane perpendicular to the incident light, extending the observable field. This was particularly necessary to compensate for the sm~!l depth of field of light microscopy, and for the slight shrinking of cambial zone and conductive phloem, which was observed after crit- ical point drying. Samples were photographed with an Olympus OM2 camera using highly contrasted films (Kodak Technical Pan 2415). A Leitz ocular vario-zoom lens was used with 5x magnification combined with a 5 objective for the last wood ring, and with 8x magnification combined with a 20 objective for the cambial zone and conductive phloem. Image analysis. The computer, the method used for image analysis and the statistical treatment of the variables are described in Dustin et al. (1994). 1 Proportions are by volume at room temperature  108 3 - 3 4 Fig. 1. Micromanipulator composed of a knee-joint (1), mounted on a bush (2), and with four driving bars firmly attached (3). Two of the bars are bound together by a fifth bar (4) preloaded downwards by a spring (5), while the two opposite bars rest under a nut-wheel (6) free to move along a fixed screw (7). The sample (8) is placed on the knee- joint, which can rotate in two opposite directions Resin embedded material. In order to compare digitized images (Fig. 2) with those of resin sections (Fig. 3), samples were dehydrated in ethanol and embedded in resin JB4 (Polysciences). Sections 5 ~tm thick were cut on a Reichert-Jung steel knife with a tungsten carbide blade mounted on a Leitz Minot microtome. Acid fuchsin, astra blue and safranin were used for staining. Results Structure of the cambial zone and conductive phloem Figure 2 shows the digitized image of the cambial zone and conductive phloem. The appearance agrees quite well with that obtained after resin embedding and sectioning (Fig. 3). Calculations based on width of the cambial zone and con- ductive phloem did not show significant differences be- tween the two methods (results not shown). Figures 2 and 3 show typically dormant tissues: in the cambial zone, the thick-walled cells are arranged in a few layers (5 to 6), and present a rather rectangular shape for the stacked fusiform initials, and a more square one for the ray initials. Limits with bordering tissues are particularly well defined, because cell division and differentiation stopped at the end of the vegetative period. Zone cutting during image analysis is made easier by this feature (Dustin et al. 1994). The conductive phloem is limited by the cambial zone and by the previous year s ring of phloem. It contains large living sieve tubes, which allow one to trace the limits with non-conductive phloem where these cells are collapsed and dead. Sieve tubes in conductive phloem can also be dis- tinguished from other cell types by the lighter staining of Figs. 2 and 3. Dormant tissues sampled in the trunk of an old beech. The cambial zone cz) separates the conductive phloem cp) from the xylem (x), and is composed of fusiform 09 and ray (r) initials. The conductive phloem is formed of non collapsed and living sieve tubes (s), of small companion cells (c) and of vertical parenchyma (p). The non-conductive phloem ncp) outside contains only dead and col- lapsed sieve tubes ds). Medullar rays mr) cross anticlinally all tissues. Scale bar: 50 Ixm Fig. 2. Sample sputter coated with gold and photo- graphed in reflected light dark field microscopy Fig. 3. Transverse section of material embedded in resin  Table 1. Quantitative analysis of fusiform initials and cambial zone in the transverse plane Cambium Fusiform initials n = 473e Young trees Old trees Tests f 109 Size parameters Perimeter (Pcb) gm 35.3 • 41.4 _+0.62 *** Area (Acb) ].tin 2 61 • 1.5 76 • 1.9 *** Shape parameters Minimum 2nd moment (minMcb) a I.tm2 15.3 ___0.71 22.9 • *** Maximum 2~d moment (maxMcb) a ~tm 2 112 • 233 • *** Moment ratio (MRcb) - 0.180 • 0.007 0.145 _ 0.006 *** Circularity (Cob) b - 0.612• 0.560___0.008 *** Cambial zone n = 16 e Young trees Old trees Testsf Cell number per stack (CS) c - 5.5 • 6 +0.29 n.s. Width (W~b) Bin 36 • 1.3 42 • 1.4 * Lumen surface ratio (LSR) d - 0.56 • 0.01 0.583-t-0.0098 n.s. a minMcb, maxMcb: principal second moments of area with respect to the mutually perpendicular long and short axis of cell b Ccb= 47~'Acb/Pcb c CS: mean number of fusiform initials per stack of cells d LSR: percentage area of lumen vs cambial zone e n, number of objects analysed f Tests: Student's t-test for fusiform initials, and Wilcoxon matched- pairs signed-ranks test for the cambial zone (mean • SE; ***" P <0.001; *: P <0.05) their lumen on the negative. Indeed the transverse walls (sieve plates) of these long cells are less frequently seen than those of the other cell types. Small companion cells adjacent to sieve tubes, and resulting from the division of the same phloem mother cell, also help to identify the conductive cells. For image analysis, zones containing approximately five stacks of cells are selected from the cambial zone between two medullar rays, as well as from the adjacent conductive phloem. The preparation artefacts are then corrected in both types of tissues. Finally, the corrected images are binarized and analysed, after removal of parenchyma and companion cells in the conductive phloem (Dustin et al. 1994). Quantitative analysis of fusiform initials and of cambial zone In the transverse plane, as indicated in Table 1, fusiform initials have a larger perimeter (Pcb) and area (Acb) in old trees (P <0.001). In anticlinal and periclinal directions, principal second moments of area (minMcb and maxMcb), which are calculated along short and long axes of cells, show that differences are highly significant (P <0.001). Circularity (C~b) shows that cells of old trees have a less rounded shape than cells of young ones (P < 0.001). This is confirmed by the ratio of principal second moments (MRcb), which shows that fusiform initials are significantly more elongated in the periclinal direction (P < 0.001). This Table 2. Quantitative analysis of sieve tubes and of conductive phloem in the transverse plane Phloem Sieve tubes n = 220 f Young trees Old trees Testsg Size parameters Perimeter (Pp) gm 81 _ 1.98 105 _ 2.3 *** Area (Ap) p.m 2 296 • 10.7 499 • 18.5 *** Shape parameters Circularity (Cp) a - 0.596_ 0.013 0.594_ 0.014 n.s. Minimum 2nd moment (minMp)b gm2 154 • 5.9 268 • 11.7 *** Maximum 2 nd moment (maxMp) b gm 2 535 • 28 880 • 38 *** Moment ratio (MRp) - 0.382+ 0.015 0.372___ 0.014 n.s. Conductive phloem n = 16 Young trees Old trees Testsg Width (Wp) Bm 153 ___ 16.5 147 ___ 8.4 n.s. Sieve tubes surface ratio (SSR) c - 0.36 + 0.017 0.40 • 0.024 * Conductivity index (CI) d Bm 56 _ 6.1 59 • 5.2 n.s. Sieve tubes density (SD) e mm -z 1191 • 57.7 817 • 30.9 ** a Cp -- 47~-Ap/Pp b minMp, maxMp: principal second moments of area with respect to the mutually perpendicular long and short axis of cell c SSR: percentage area of sieve tubes vs conductive phloem d CI = Wp.SSR e SD mean number of sieve tubes per square millimeter of conduc- tive phloem f n: number of objects analysed g Tests: Student's t-test for sieve tubes, and Wilcoxon matched- pairs signed-ranks test for the conductive phloem (mean ___ SE; *** P <0.001; ** P <0.01; * P <0.05)  110 indicates that differences in cell size between old and young trees are associated with a change of form, cells of old trees being more rectangular. Cell number per stack (CS), which depends on the rate of cell division and differentiation, remains unchanged between old and young trees when the cambial zone is dormant. Nevertheless, the cambial zone is wider (Web) in old trees (P <0.05), which can be explained by the in- crease in size of fusiform initials. The lumen surface ratio (LSR) indicates that more than 40% of the surface of cambial zone in all trees is occupied by cell walls. This high percentage must be a consequence of dormant con- ditions. Indeed, like the cell number per stack (CS), the thickness of cell walls in the cambial zone is related to cambial activity, and cell walls of initials are thicker in winter (Catesson 1990). This LSR ratio does not vary sig- nificantly between old and young trees in spite of the in- crease in size of fusiform initials of old trees. This implies that the increase in lumen size must be associated with an increase in cell wall thickness, which allows the con- servation of the ratio. Quantitative analysis of sieve tubes and of conductive phloem Table 2 indicates that sieve tubes of old trees have signif- icantly larger perimeters (Pp) and areas (Ap) in the trans- verse plane (P < 0.001). Cell shape is comparable between old and young trees, as shown by the circularity (Cp) and ratio of principal second moments (MRp), which are not significantly different. Minimum and maximum second moment of area (minMp and maxMp) show that the increase in cell size in old trees proceeds in both periclinal and anticlinal directions. Phloem ring widths (Wp) are not significantly different, but a larger surface of conductive phloem (SSR) is occu- pied by sieve tubes in old trees (P < 0.05). Nevertheless, the conductivity index (CI) exhibits no significant differ- ences, which indicates that the useful surface for conduc- tion of sap is the same in both categories. This is due to the sieve tubes density (SD), which is proportionally higher in young trees (P <0.01). It means that in old beeches, sieve tube production is diminished; the possible surface loss for elaborated sap conduction is counterbalanced by an in- crease in Ap, with no change in cell shape. Discussion Justification of the method In the transverse plane, the structure of tissues undergoes little change during dormancy. A comparison with the data of Hagemeyer et al. (1989) shows that the appearance of both cambium and phloem remains practically the same as that existing at the end of the growing season. The cores extracted in winter permit comparative studies between trees, which are at this time of year in the same physio- logical state. The method developed provides pictures which are suitable for automatic image analysis. In comparison with methods using resin embedding, the preparation and ob- servation methods presented here offer several advantages: (a) samples can be observed with both reflected light dark field and SEM (data not shown); (b) metallisation is not sensitive to cell wall chemical composition and so xylem, cambial, and phloem cell walls are uniformly stained; and (c) reflected light dark field microscopy and highly con- trasted films improve resolution and enhance the contrast between the lumen and cell wall. Good quality digitized images reduce the time needed for the manual correction step during image analysis, although this step cannot be suppressed entirely with complex and fragile tissues like the cambial zone and conductive phloem (Dustin et al. 1994). Influence of tree age on tissue structure In beech, age-related size and shape changes can be ob- served and analysed in cell and tissue structure. Most of them are only detectable by image and statistical analysis: differences between old and young beeches are subtle in- deed; they can hardly be visualized directly. In this sense, image analysis can be considered as a sensitive in- vestigative tool. Cell size is the most obvious difference between old and young trees. In both the cambial zone and conductive phloem, the cells of old beeches are significantly larger. As published data on other taxa show that length and periclinal width of fusiform initials and sieve tubes increase with age (Bosshard 1965; Iqbal and Ghouse 1990; Ajmal and Iqbal 1992; Ridoutt and Sands 1993), one can expect that the length of the corresponding beech cells follows the same pattern. With shape parameters, one can see that the direction of size increase is not the same for both tissues: in the cambial zone, it is more periclinal than anticlinal, and so there is a change in cell shape with increasing age. One knows that in old trees the rate of anticlinal divisions diminishes, and that a loss of initials can occur (Iqbal and Ghouse 1990). Consequently, this change in shape together with the in- crease in cell size may reflect a decrease in rate of cell division. The time elapsed between two divisions is longer and so the differentiation phase is longer too. In old beech trees, the unchanged translocation surface, as quantified by the conductivity index (CI), does not imply that the conduction, as compared with young trees, remains the same. Indeed, active transport in phloem is not as strongly dependent on the conduction surface, as passive transport in xylem (Aloni 1991). Shape parameters give some further indications about possible modifications of translocation ability with age. They indicate that the degree of collapse of sieve tubes in conductive phloem, which closely reflects translocation ability (Esau 1965; Zamski and Zimmermann 1979), is comparable between both age classes. With the variables analysed here, no change of this parameter can be observed; accordingly, we can suppose that translocation ability could be conserved as the tree
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks