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Intervascular pit membranes in roots of two species of Osmanthus (Oleaceae).

ABSTRACT

Torus-bearing intervascular pit membranes are part of the bordered pit pairs connecting tracheary elements in roots of Osmanthus armatus and Osmanthus americanus. The pit membrane allows water to pass from cell to cell but blocks transmission of air embolisms. The torus is centrally located on the circular pit membrane and is of such a diameter as to occlude an adjoining aperture when the membrane is displaced during the introduction of air. The center of the torus thickening is strengthened by addition of lignin. Torus-bearing pit membranes are present in secondary xylem (wood) and largely or completely absent from primary xylem. Some pit membranes containing elongate rather than circular tori are the result of fusion of adjacent pits during ontogeny. Torus-bearing pit membranes represent a xeromorphic adaptation that is advantageous during times of water stress.

INTRODUCTION

Bordered pit pairs connect water-conducting tracheary elements of vascular plants and allow water transport from one element to the next. Each pit pair consists of a permeable pit membrane inserted between two pit borders, each with an aperture (Dute et at, 2001). Water passes from the lumen of one tracheary element through the pit pair into the lumen of the neighboring element. Key to the success of the bordered pit pair is the pit membrane, which must allow passage of water molecules yet impede movement of air embolisms.

One modification of the angiosperm pit membrane is its demarcation into a central impermeable torus surrounded by a screen-like margo (Ohtani and Ishida, 1978). It is thought that introduction of air into the system causes the pit membrane to be displaced or aspirated so that the torus blocks an aperture and impedes movement of air bubbles (embolisms). The torus thickening is thought to strengthen the pit membrane and keep it from rupturing during aspiration (Wheeler, 1983; Dute and Rushing, 1987).

The number of angiosperm species known to possess pit membranes with tori totals over 90 (Dute et at, 2010a, 2010b, 2011). Among those species are 17 of Osmanthus, a genus of the Oleaceae (Olive Family) (Dute et at, 2010b). Our laboratory has been especially interested in pit membranes of Osmanthus for many years, including their structure (Dute and Rushing, 1987), development (Dute and Rushing, 1988) and chemistry (Coleman et at, 2004). We, along with other laboratories, have investigated the systematic distribution of the torus among genera within the family (Ohtani, 1983; Rabaey et at, 2008; Dute et at, 2008). All studies used stem and branch material. Recently, we began a project to survey other organs of Osmanthus for presence of torus-bearing pit membranes. As a first step in the study, we observed tori in tracheary elements of leaf veins in perennial leaves of 0. armatus. We now report on the presence, distribution and structure of tori in roots of this same species along with another species, 0. americanus.

MATERIALS AND METHODS

Five specimens of Osmanthus armatus Diets used in a previous study (Dute et al, 2012) provided root samples for the present investigation. Plants were potted in a 7:1 pine bark/sand mixture amended with dolomitic limestone, Micromax and PolyOn (17-5-1) and placed in the Alabama Agricultural Experiment Station Greenhouse on the Auburn University campus. Supplementary material was extracted from a core sample of a large root of 0. americanus (L.) Benth. & Hook. ex Gray growing in the Donald E. Davis Arboretum on campus.

Twenty-eight roots were sampled from individuals of 0. armatus. The root segments selected varied in diameter and in possession of an epidermis versus periderm; thus they varied in age. For light microscopy these segments were placed in 3% glutaraldehyde in 0.05 M potassium phosphate buffer (pH 6.8) under vacuum for 1 h, then kept at 4 C overnight. Next, following a brief buffer wash, specimens were dehydrated in a cold ethanol series culminating in two changes of 95% alcohol. Specimens then were infiltrated overnight with JB-4 resin followed by embedment in the same resin. Transverse, radial longitudinal, and tangential longitudinal sections were cut at 3 Ism thickness using a Sorvall MT-2b ultramicrotome. Sections were heat fixed to glass slides, stained with toluidine blue 0 (TBO, Ruzin, 1999), covered with Permount (Fisher Chemicals, New Jersey), and a coverslip was applied.

Mature root samples of 0. armatus were macerated according to the procedure of Wheeler (1983) by placing specimens in a 1:1 mixture of glacial acetic acid and hydrogen peroxide for three days at 50 C. Following a water rinse, cells of the macerate were stained with TBO and mounted in a drop of water on a slide.

Radial longitudinal root samples for scanning electron microscopy (SEM) from both O. armatus and 0. americanus were air-dried, affixed to aluminum stubs with double-stick carbon tape, sputter-coated with gold palladium and viewed with a Zeiss EVO 50 at 20 kV (Carl Zeiss NTS GmbH, Oberkochen, Germany).

RESULTS

Root Morphology

Figure 1 shows a cross section of a root region possessing considerable secondary vascular tissue. Secondary xylem is bounded centripetally by primary xylem and sclerified pith (Figure 2) and centrifugally by vascular cambium and phloem (Figure 3). A ring of sclerenchyma encircles the xylem and phloem (Figures 1 and 3). Detail of the sclerenchyma cells shows them to have pronounced pit canals and multilamellate walls (Figure 4). These cells vary in length but tend to be short and are thus identified as brachysclereids.

Abbreviations used in the figures in this study: 1 = primary xylem; 2 = secondary xylem (wood); A = aperture of bordered pit; C = vascular cambium; M margo; MX = metaxylem; P = pith; PB = pit border; PH = phloem; PM = bordered pit membrane; PX = protoxylem; R = ray; S sclereids; T = torus; TR = tracheid; TY = tyloses; V = vessel member. --Note: Figures 11, 14, 17 and 18 are images of O. americanus; the remaining images are of O. armatus.

Secondary xylem (wood) is either deposited uniformly about the primary xylem and pith or is deposited in an eccentric fashion (compare Figure 1 and Figure 5). Roots are perennial, and in older root segments growth rings are distinct (Figure 2).

Root wood consists of both axial and ray systems. Rays of the ray system generally are narrow, often appearing only one or a few cells in width (Figure 2) in trans-section. Tangential longitudinal sections (TLS) show a more complex situation in that ray width varies along the vertical length of the ray (Figure 6). Rays emanating from the primary xylem ridges become especially wide as diameter of the secondary xylem tissue increases (Figures 3).

Tracheary Elements

Water-conducting cells of the axillary system of the secondary xylem are called tracheary elements and are of two types, tracheids (vascular tracheids) and vessel members (Figure 7). In 0. armatus both cell types are elongate with helical sculpturing at the lumen surface (Figures 7, 8 and 9). This feature is pronounced in narrower diameter tracheary elements (both tracheids and vessel members). In large diameter vessel members of the spring wood, the sculpturing is faint or absent. Lumen surfaces in 0. americanus have less pronounced helical sculpturing in the narrow diameter tracheary elements and none in large diameter vessel members. Lateral walls of both cell types in both species possess bordered pits (Figures 8 and 9). Vessel members have simple perforations on their oblique end walls, whereas tracheids are imperforate (Figures 7 and 9). It is the tracheary elements that possess tori in their pit membranes (Fig. 9). At maturity the tracheary elements are dead and devoid of cytoplasm; however, two examples were found of vessel members whose lumens were occluded by tyloses, ingrowths of surrounding parenchyma cells (Figure 10).

Pit Membranes and Tori

Each pit membrane is sandwiched between two pit borders, each with an aperture providing access to a cell lumen. The apertures are circular in 0. americanus and elliptical in 0. armatus (Figures 11 and 12). Resolution provided by the SEM shows that concentric microfibrils compose the pit border (Figure 11). In face view in longitudinal section, the torus is a circular object centrally located on the pit membrane (Figures 9, 13 & 14). The torus stains purple with toluidine blue 0, but careful observation shows a blue-green spot in its center (Figure 15). When rotated 90 degrees out of the plane, the pit membrane and its torus are seen in sectional view, and the latter appears lens-shaped (Figure 14). Typically, the diameter of the torus is greater than its associated apertures (Figure 14). For example, as mentioned, pit apertures in torus-bearing pits of 0. armatus tended to be elliptical. Measurements of SEM material showed the mean of the long axis of the aperture ellipse to be 1.30 [mu]m (N = 25; range 0.77-1.90 [mu]m), whereas the mean torus diameter was 2.21 [mu]m (N = 25; range 1.68-2.20 [mu]m). Mean torus diameter is greater than that of the aperture. Although the ranges of the two sets of measurements overlap, no air-dried pit membranes were observed in which the torus was of smaller diameter than its associated aperture.

The fibrillar nature of the margo, which surrounds the torus, could

not be visualized adequately using SEM.

Fused Pits

Perhaps the most interesting aspect of this study is the observation of fused pit outlines in tracheary elements of root wood from both 0. armatus and 0. americanus. In some instances it appears as if single pits possess borders, tori, and apertures that are distinctly elliptical rather than circular (Figure 16). However, in other cases it is clear that the elongate nature of the pit is the result of fusion of two neighboring pits during ontogeny. Scanning electron microscopy of an example shows the figure eight outline of the pit in more detail as well as the elongate torus (Figure 17). Fused pits have elongate apertures (Figure 18).

Torus Distribution in O. Armatus

Tori are found between small diameter tracheary elements and between small diameter elements and larger diameter spring wood vessel members. Tori are absent between the larger diameter spring wood members.

Primary Xylem

Primary xylem of O. armatus, which forms and functions before the secondary xylem, is in the shape of a multipointed star (polyarch stele) surrounding a pith (Figure 19). Figures 20, 21 and 19 show the stele of the root at stages of increasing maturity. In Figure 20, the protoxylem is mature and the center of the stele (the pith) contains a parenchymatous tissue (actually, fiber primordia). Figure 21 shows the stele shortly after the initiation of the vascular cambium. Pith cells have become sclerified. Figure 22 provides a more detailed view of primary xylem ridges and vascular cambium at this time. The latter initiates between the ridges and continues to develop circumferentially until the ridges are ensheathed. Figure 19 shows pith and primary xylem enclosed by wood.

Primary xylem consists of both proto- and metaxylem (Figure 23). The latter matures later and in a position centripetal to (inside of) the former. Figure 24 shows both types of primary xylem in longitudinal view. The later a tracheary element of primary xylem matures, the more extensive is the deposition of secondary wall. Thus the element on the lower right is protoxylem and the pitted element to its upper left, metaxylem. Secondary wall thickenings of the metaxylem can take different arrangements, e.g. Figure 25. In one instance, late metaxylem elements had what might be interpreted as tori. However, this observation needs to be confirmed. Generally, tori are absent from both protoxylem and metaxylem tracheary elements.

DISCUSSION

Microtubules are thought to be responsible for orientation of cellulose microfibrils in walls of plant cells according to the Alignment Hypothesis (Baskin, 2001). Presence of circular microfibrils is observed in both hardwood and softwood pit borders (Harada, 1965a, 1965b; Liese,1965; Schmid, 1965) and is correlated with a ring of cortical microtubules in the adjacent cytoplasm (Chaffey et al., 1997). Thus circular outlines in the pit borders of Osmanthus roots as seen with SEM in this study and with atomic force microscopy (AFM) in a previous study of Osmanthus stems (Dute and Elder, 2011) should come as no surprise.

In 0. americanus (and presumably 0. armatus), construction of the pit border is largely complete by the time that torus thickening material is deposited (Dute and Rushing, 1988). The latter process is itself associated with a plexus of microtubules (Dute and Rushing, 1988). We hypothesize that the fused pit borders and elongate apertures observed in this study result from a rearrangement of microtubules associated with cellulose microfibril deposition. Subsequently, shape and dimensions of this elongate aperture affect the nature of the microtubule plexus, leading to formation of an elongate torus. Elongate tori and pit apertures have been observed at the boundary of primary and secondary xylem in petioles of 0. armatus leaves, but fused pits have not been reported from either branches or leaves of Osmanthus (Dute and Rushing, 1987; Dute et aL, 2012), although it is expected that they exist.

Transmission electron microscopy (TEM) indicates that the torus of Osmanthus consists of a compound middle lamella covered on either side by a torus pad or thickening (Dute & Rushing, 1987). Chemical analysis using acriflavine staining and confocal microscopy shows the torus to contain lignin (Coleman et al., 2004), a wall-strengthening substance (Evert, 2006). TEM of [KMnO.sub.4]-stained material indicates that lignin is localized in the torus pads (Coleman et al., 2004). Detailed views of air-dried pit membranes with AFM show the surface of the torus pad to consist of two parts: 1) a pustular zone surrounded by 2) a peripheral corona of microfibrils (Dute and Elder, 2011). Acidified sodium chlorite removes incrusting material from the pustular surface exposing microfibrils beneath (Ohtani and Ishida, 1978; Dute and Elder, 2011). TBO stains lignin blue-green (O'Brien et al., 1964), and the blue-green spot seen in the center of the torus in the present study corresponds to the pustular zone and to the thickest part of the torus pad where most of the lignin is located. A similar blue-green stained deposit has been discovered in tori of the stern of 0. armatus (Dute, unpublished results).

Morphology of the typical circular bordered pit and torus-bearing pit membrane of the root of Osmanthus is the same as that in the branch and leaf (Dute and Rushing, 1987; Dute and Elder, 2011; Dute et al., 2012). All three organs are perennial and develop considerable amounts of secondary xylem (wood). We are presently investigating flowers of 0. americanus to see whether xylem of such transient organs possesses bordered pit pairs with tori.

In a recent study we hypothesized that tori in leaves of 0. armatus represent xeromorphic features which, along with a thick cuticle and sclereids, enable a perennial leaf to survive times of stress (Dute et al., 2012). Picconia, a genus closely related to Osmanthus (Wallander and Albert, 2000), has pit membranes with tori in its branches (Dute et al., 2008; Rabaey et al., 2008). The two species of Picconia, both of which are xerophytic evergreens, grow on the islands of Macaronesia (Caetano Ferreira et al., 2011). One species, at least, (P. azorica) "colonizes dry environments and is resistant to sea spray" (Caetano Fereira et al., in press). We would hypothesize that the evergreen leaves of Picconia possess tori.

ACKNOWLEDGEMENT

The authors wish to thank the Alabama Agricultural Experiment Station for its support.

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Roland R. Dute, Zachary S. Hubbard, and Ronak V. Patel Department of Biological Sciences, Auburn University Auburn, AL

Correspondence: Roland R. Dute (duterol@auburn.edu)
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Author:Dute, Roland R.; Hubbard, Zachary S.; Patel, Ronak V.
Publication:Journal of the Alabama Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2012
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