The Periderm Development in Quercus Suber

IAWA Journal, Vol. 25 (3), 2004: 325–335

THE PERIDERM DEVELOPMENT IN QUERCUS SUBER

José Graça & Helena PereiraCentro de Estudos Florestais, Instituto Superior de Agronomia, Universidade Técnica de Lisboa,

1349-017 Lisboa, Portugal

SUMMARY

In the cork oak (Quercus suber L.), the phellogen differentiates during the first year of growth in the cell layer immediately under the epidermis and divides to form 3–6 suberized phellem cells. Division of the phellogen only occurs after suberization of the previous divided cell. During the first four years of growth, the phellem cells have tannin-filled lumens and it is only in the 5th to 7th years that they acquire the characteristics of ʻadult ̓cork cells with empty lumens and thin suberized walls. The len-ticels are formed by the lenticular phellogen, which differentiates under the stomata and has a high meristematic activity. In this region, the cells are unsuberized, with a loose arrangement and intercellular voids, consti-tuting the filling or complementary tissue. After three years, the lenticels appear as small protuberances that soon become conspicuous. Inclusions of sclerenchymatous nodules and isolated sclereids occur occasionally mostly in the vicinity of, or in, the lenticels.

Key words: Quercus suber, cork, phellem, epidermis, phellogen, len-ticels.

INTRODUCTION

The cork oak, Quercus suber L., has a phellogen that is formed as a continuous layer surrounding stems and branches and produces an external layer of cork cells with an appreciable thickness. Unlike most species where the phellogen lives only a few years (Esau 1977; Fahn 1990), the phellogen in the cork oak may live as long as the tree (Natividade 1950). The cork cells are small (7 × 107 cells /cm3) with empty lumens and thin walls. They are stacked in the radial direction with a regular arrangement without intercellular voids (Pereira et al. 1987). The cell walls are highly suberized containing approximately 40% suberin (Pereira 1988). When the initial phellogen is destroyed, e.g., by removal of the cork layer, a traumatic phellogen differentiates in the outer phloem, retaining the cylindrical spatial develop-ment and showing an enhanced meristematic activity in the following years (Natividade 1950; Pereira et al. 1992). The successive formation of traumatic phellogens following removal of cork at periodic intervals (e.g. every 9 years) allows for the exploitation of the cork oak on a sustainable basis.

IAWA Journal, Vol. 25 (3), 2004326 327Graça & Pereira — Periderm in Quercus suber

Studies in the origin and development of the phellogen are scarce (Lev-Yadun & Liphschitz 1989) in spite of the physiological importance of the periderm as the protec-tive tissue in tree stems. The amount of research on the formation and activity of the phellogen in the cork oak has not paralleled its economic importance. The cork oak has a periderm with massive fellemic tissue, of widespread use as a sealant for wine bottles, as well as for surfacing and insulating materials. Early works of Machado (1935) and Natividade (1934, 1950) have described the anatomy of phloem and periderm in the cork oak and the formation of the traumatic phellogens. In this paper we report on the development of the first periderm in the cork oak, il-lustrating its differentiation in young shoots and the characteristics of its development and activity during the first seven years of growth. Attention was paid to the formation of lenticels because of the importance of lenticular channels for the commercial cork quality. In fact the cork stoppers of wine bottles with few and small lenticular channels are more than 5 times more expensive than those with larger ̒ pores ̓and the classifica-tion of cork by porosity is one of the important steps in the cork industry (Pereira et al. 1996).

MATERIAL AND METHODS

The formation of the first periderm in Quercus suber L. was studied in the emerging shoots of 2-year-old cork-oak plants grown in the nursery. The shoots were sectioned at different intervals in all foliar internodes from a distance of 2–3 mm from the apex to approximately 10 cm; the diameter of the sections ranged from 0.5 to 2 mm. Periderm development and phellogen activity was also observed in small branches of mature cork-oak trees under cork production in the Alentejo region of Portugal. Various sections were cut along the branches corresponding to ages from 1 to 7 years. After sectioning, the material was fixed in Navashin solution (Jensen 1962) for at least 24 h and washed under running water. Material was embedded in polyethyleneglycol (Carbowax) 2000 DP, starting with a 20% aqueous solution in a 60 ºC ventilated oven until evaporation of the solvent (c. 48 h) and followed by two 12 h immersion periods in the pure medium. A sliding microtome was used to cut transverse and longitudinal thin sections of 6–8 μm and 20 μm thickness, respectively for the younger and older material. The thin sections were glued with Kaiserʼs glycerol gelatin. Staining was made with safranin/fast green, Sudan IV, toluidine blue O and chrysoidine G/pyronyn Y/astra blau. The observations were made with a Nikon Microphot light microscope.

RESULTS AND DISCUSSION

The arrangement of primary tissues in the cork-oak shoots at the beginning of the sec-ondary development is seen in Figure 1; there are central pith parenchyma, vascular bundles, a parenchymatous cortex with collenchyma characteristics at the outside and the epidermis. In the inner cortex, a ring of perivascular fibres starts to differentiate very early around the vascular bundles, even before the formation of the phellogen.


The epidermis The epidermis is a 1-cell-thick layer of tabular cells without intercellular voids with a cutinized cuticular membrane of variable thickness (Fig. 2). In some cases what appears to be the cuticle proper is distinguished in the outer part of the cuticular membrane. In the sections from near the apex, the cuticular membrane is very thin (Fig. 2a), but increases in thickness in the lower parts of the shoots. In the 1-year old branches of adult trees, the cuticular membrane has an appreciable thickness, is strongly cutinized, and penetrates into the radial walls of the epidermal cells (Fig. 2b). The epidermis is interrupted at intervals by stomata, defined by guard cells with a crescent profile and projecting borders into the stomatal cavity (Fig. 3). Stomata appear on the young and green shoots and branches, where abundant chloroplasts are found in the underlying cortex cells. As later discussed, stomata seem to be the site for initiation of the lenticular channels during phellogen formation. The cork-oak epidermis has numerous trichomes. In the shoots of the nursery grown plants, the trichomes are unicellular (Fig. 2a) or branched (Fig. 4). In the epidermis of the first year of growth of branches of adult trees, trichomes are numerous, simple and tufted of a dendroid type, associated to the very thick cuticular membrane. This supports the idea that trichomes may have an insulating function, and act as a barrier against excessive solar radiation in association with thicker cuticles (Esau 1977; Fahn 1990).

Fig. 1. Transverse section of a young shoot of a cork-oak plant at the beginning of secondary growth. The initial divisions establishing the phellogen (Ph) are visible in the subepidermal layer. At this stage the phellogen completely encircles the stem. A ring of perivascular fibres, 2–5 cells thick (white arrow), is seen to the inside of the cortex (Cx). P = pith; Ep = epidermis. — Scale bar = 100 μm.


Fig. 2–5. – 2: Transverse sections of a 1-year-old cork-oak shoot, showing the epidermis. – a: Section near the apex with a thin cuticular membrane; a unicellular hair is shown. – b: Section in the middle region of the shoot with a thicker cuticular membrane that penetrates into the radial walls of the epidermic cells. Scale bars = 50 μm. — 3: Stomata in the epidermis of a young


Phellogen formation Phellogen forms in cork-oak plants during the first year of growth after initiation of vascular cambium activity, as is common in vascular plants (Fahn 1990). In all cork oaks observed, 1-year-old branches have a complete phellogen extending as a continuous layer around the branch and showing already some phellem cells. The beginning of phellogen activity brings about a change in the external appearance of the small 1–2 mm thick branches. They turn from a translucent lively green, some-times with a visible whitish hairiness, into an opaque green colour, frequently with a reddish tonality. The phellogen develops in the cell layer immediately under the epidermis (Fig. 5). The nucleus of the collenchymatous cortex cells in the subepidermic layers increases in volume, followed by a periclinal division and the formation of the wall between the two daughter cells (Fig. 5a). It is the outer daughter cell resulting from this division that constitutes a phellogen cell and initiates new periclinal divisions forming the first layer of phellem cells (Fig. 5b, c). The inner daughter cell differentiates as phelloderm. This process extends rapidly to all of the subepidermic layer (Fig. 1). The division of the phellogen derivatives was not observed. In the 1-year-old branches, the phellogen produced several layers (3–6) of phellem cells, with the outermost cells tangentially distended due to the radial growth stress. Anticlinal divisions of the phellogen already started to allow for this radial growth (Fig. 5d). This observation, that the first phellogen in Quercus suber stems is initiated in the subepidermic layer, differs from that of Fahn (1990), who described phellogen initia-tion in the epidermis as did Eames and McDaniels (1925). However, the location of the formation of the first phellogen in the cell layer immediately below the epidermis is reported as the most common situation (Esau 1977; Fahn 1990), even if phellogens have been seen to form from living parenchyma cells in many different tissues (Mauseth 1988; Lev-Yadun & Liphschitz 1989).

Lenticels In the beginning of the first year of growth, the cortex cells under the stomata start to divide with an intense meristematic activity (Fig. 4, 6a) and constitute a localized precursor of the phellogen. The divided cells proliferate and protrude to the exterior and the dividing cell layer follows a concave line (Fig. 6a). When the phellogen is formed

←cork-oak shoot, showing the guard cells (arrows); the beginning of meristematic activity for for-mation of the lenticular phellogen is already visible. Scale bar = 50 μm. — 4: Section cut through a multiple trichoma in a section of a young cork-oak shoot. Scale bar = 50 μm. — 5: a: First cell division in the subepidermic layer as a precursor of phellogen formation (arrow). – b: Extension of phellogen formation to all the subepidermic layer; in some places the phellogen mother-cell has already divided (arrow). – c: First cells resulting from the division of the phellogen. – d: Layer of 6–8 phellem cells aligned in rows in a 1-year-old cork-oak shoot; the cells are heavily stained and the outermost cells are tangentially stretched and more or less crushed; anticlinal divisions may be seen (arrows). Scale bar of 5a = 30 μm; of 5b & d = 40 μm; of 5c = 20 μm.


Fig. 6. The formation of lenticular phellogen and lenticels. – a: Intense meristematic activity under a stomata before the formation of phellogen in the surrounding areas. – b: The lenticular phellogen follows a concave line and the tissue tore at the outside as the result of the high mer-istematic activity. – c: The cells produced by the lenticular phellogen have a loose arrangement; sclereids are also visible (arrow). – d: Lenticular channel in a 3-year-old cork-oak branch: in the cortex, large sclerenchymatous nodules may be seen. — Scale bar for a = 20 μm; for b & c = 50 μm; for d = 100 μm.


in the remaining periphery, it joins to these regions, and is now named lenticular phel-logen. It is here that the lenticels and the subsequent lenticular channels originate. The activity of the lenticular phellogen is very intense and produces a high number of cells that bulge the periderm upwards resulting in fractures in the surface layer. The borders of the lenticel protrude (Fig. 6b). The cells that differentiate in the region of the lenticular phellogen are different from the phellem cells formed by the remaining phellogen. These complementary or filling cells (Esau 1977) do not stain with Sudan IV and have a loose arrangement with intercellular voids (Fig. 6c). The lenticular phellogen maintains these characteristics and activity during the fol-lowing years, therefore building up lenticular channels that extend radially through the phellem. The channels are filled with more or less loosened complementary tissue. In some cases a closing layer of cells is apparent, defining the end of season of an yearly growth (Fig. 6d). These cells are presumably suberized, since they stain like the non-lenticular phellem cells. This type of complementary tissue is reported for Quercus and other genera (Esau 1977).

Development of the periderm A transverse section through a 1-year-old stem is shown in Figure 7a, and details of the periderm in Figure 7b. At this stage the periderm in the cork oak makes up a con-tinuous ring of uniform thickness, an external layer of 3–6 phellem cells and a one-cell phelloderm interior to the phellogen.The periderm does not include sclerenchymatous cells. The lenticels are few and small. The phellem cells have tannin-filled lumens and are suberized (Fig. 7b). Division of the phellogen only occurs after suberization of the previously divided cell. The com-plete differentiation of the maturing phellem cell seems to precede the division by the phellogen of a new phellem cell, a situation eventually induced by the need for the protection afforded by the suberized cell walls. The epidermal cells, the cuticular membrane, and trichomes are still visible at the outer part of the periderm, although crushed and disrupted at intervals due to the tan-gential growth stress. In 2-year-old branches (Fig. 7c), the periderm increases its thickness. The outer cells of the phellem are stretched and radially collapsed due to the strong tangential stress from the secondary growth beneath them, which induces in some places the radial tear of the phellem layer. Epidermal cells may still be visible but already are very damaged. The phellem shows the first inclusions of sclerenchymatous nodules, especially in the lenticels, in the radial continuation of those formed in the exterior cortex. In the cortex, the continuity of the ring of the primary perivascular fibres is maintained by the dif-ferentiation of cortical parenchyma cells as large sclereids, more or less isodiametric and with thick walls occupying all the lumen (Fig. 7d), a process already initiated in the first year. Large rays develop in the phloem, which become sclerified, building up large areas of lignified sclereids and fibres in the phloem. During the third year of growth (Fig. 7e), the sclerification of the cortex increases. In the phloem, a large area is occupied by phloem fibres and rays. The regular develop-


Fig. 7. – a. Cross section of a 1-year-old branch showing xylem (Xy) and phloem (Ph). – b: At the end of the first year of growth the outer cells of phellem col-lapse and the epidermis is torn away. – c: Cross section of a branch in the sec-ond year of growth, showing the continu-ous periderm layer, the strongly scleri-fied cortex, the phloem with large rays and fibres and the annual rings in xylem (arrow). – d: The sclerification of cortex cells (here in a 2-year-old branch) ensure the continuity of the primary perivascu-

lar fibre ring. – e: Inclusions of sclerenchymatous cells appear in the phellem, especially in the lenticels (arrow). – f: A 4-year-old cork-oak branch, showing the intense sclerification of the cortex and the large voluminous phloemic rays that cross the plane of the vascular cam-bium into the xylem (arrow). — Scale bar for a = 100 μm; for b = 40 μm; for c = 120 μm; for d = 30 μm; for e & f = 150 μm.


Fig. 8. Cross sections of 5-year-old cork-oak branches showing a well developed layer of mature phellem tissue with hollow and thin-walled cork cells. – a: Numerous tissue tears are seen in the microsection, especially at the boundary of tissues (e.g. xylem/phloem, cortex/periderm) as a result of their different physical properties. – b: The radial increase of the branches results into fractures in the cork tissue that may go as deep as the phellogen. — Scale bars = 250 μm.


ment of the periderm starts to be influenced by the increased activity of the lenticular phellogen and shows small protuberances in these areas. The cells in the phellem are still filled with heavily stained materials and some inclusions of the outer cortex could be observed, mostly made up of sclerenchymatous nodules absorbed into the lenticels and of isolated sclerified cells formed near the phellogen and later overgrown by it (Fig. 7e). The irregularities of the periderm increase during the fourth-year of growth due to the dimensional increase of the lenticels, which become conspicuous. The concave development of the lenticular phellogen is also prominent (Fig. 7f). In each transverse section, some 2–3 inclusions of cortical sclerenchymatous nodules were observed as well as some isolated sclereids, almost always in the lenticular tissue. In the tissues interior to the phellogen, the number of sclereids increased sharply and in some cases occupied almost all the remaining cortex region. Fibres and other scle-renchymatous cells are also abundant in the phloem; the phloem rays are voluminous, triangular in transverse section, and extending into the xylem due to their sclerification. (Fig. 7f). It is during the fifth (Fig. 8a) to the seventh years of growth that phellem cells acquire the characteristics typical of ̒ adult ̓cork cells (Pereira et al. 1987). The cells lose most of the stained inclusions and show empty lumens and thin suberized cell walls. The activity of the phellogen increases, producing in one year of growth 10–20 phellem cells with a regular radial arrangement. The annual growth rings are visible. Due to a continued increase in stem circumference, deep fractures develop in the periderm, sometimes nearing the phellogen (Fig. 8b). The external appearance of the branches, now approximately 2 cm in diameter, is typical for the cork oak, showing a greyish colour and deep longitudinally running fissures. The lenticels form lenticular channels that radially cross the phellem; they are in part open and in part filled with the loosened filling tissue (Fig. 8a). The inclusions of sclerified cells continue to be observed, but are mostly restricted to the lenticels, or to their vicinity, or to the outer part of the phellem. In the tissues interior to the phellogen, the secondary phloem and the remaining primary cortex are mostly occupied by the sclerified cells of phloem rays and corti-cal parenchyma. The sclereids constitute large round nodules, grouped as peripheral arches. The phloem rays are very large, divided into two or three parts, with thickened cells of sclerenchyma; they protrude into the xylem, sometimes rather deeply, due to its volume enlargement associated with the extensive sclerification of its cells.

REFERENCES

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Pereira, H., J. Graça & C. Baptista. 1992. The effect of growth rate on the structure and compres-sive properties of cork. IAWA Bull. n.s. 13: 389–396.

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The Periderm Development in Quercus Suber