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Anatomy and Morphology |
2Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa 50011-1020 USA; 3Department of Genetics, Development and Cell Biology, and Bessey Microscopy Facility, Iowa State University, Ames, Iowa 50011-1020 USA
Received for publication April 6, 2005. Accepted for publication August 1, 2005.
ABSTRACT
Oxalate crystals are very common in angiosperms, but few descriptions of their macropattern (crystal types, their tissue distribution, and development) exist. Because unusually large prismatic crystals and druses, are known from pomegranate, we traced the development of crystal macropattern in various-aged leaf samples from a living plant and from herbarium specimens using unstained whole mounts (some bleached and cleared), stained leaf samples, and leaf and stem cross sections. Preparations were viewed with bright-field light microscopy and with crossed polarizers. Prismatics appear first in the subapical mid-mesophyll layer of a leaf 650 µm long. Additional prismatics form basipetally in the enlarging lamina. A preemptive wave of small prismatics appears basipetally in the midrib. Druses form secondarily acropetally in petiole and midrib, while existing lamina prismatics enlarge and new ones develop among them in mid-mesophyll. Prismatics produced early expand vertically, and many eventually extend from epidermis to epidermis. Later-formed prismatics attain intermediate sizes. No crystals form along lamina veins, but in older leaves, druses occur in spongy mesophyll, mostly near major vein junctions. In the stem, druses are restricted to phloem fibers. No phloem fibers occur in the leaf trace or midvein; therefore, petiolar and midrib druses are only in parenchyma, not in phloem.
Key Words: calcium oxalate • crystals • druses • prismatics • Punica • Punicaceae
Calcium oxalate crystals occur in five morphologically different forms, one or more of which are found in most angiosperm families (Franceschi and Horner, 1980
; Franceschi and Nakata, 2005
). Knowledge of plant crystals consists almost entirely of details of crystal structure, how a crystal forms within a cell, and distribution of crystals in mature organs. We are interested in the ways in which an entire macropattern (crystal types and their specific distribution) develops from leaf inception to leaf death, which is a neglected aspect of crystallization. The cultivated pomegranate seemed to be a desirable species for such a study, for both its scientific interest and its technical convenience.
The single genus Punica (P. granatum and P. protopunica) comprises the eudicot family Punicaceae (Cronquist, 1981
). Punica granatum is the cultivated pomegranate, a shrub or small tree native to warm parts of Asia, but naturalized widely elsewhere.
Calcium oxalate crystals in pomegranate have been described only superficially. Solereder (1908)
merely mentioned that idioblasts with solitary crystals occur in the leaf mesophyll. Metcalfe and Chalk (1950)
reported short septate "crystalliferous fibers" (crystal shape not mentioned) in the wood, abundant "cluster crystals" (druses) in the secondary phloem, and "solitary and cluster crystals present in the lamina and petiole." More specifically for the leaf lamina, they reported (p. 659), "Idioblasts, containing very large solitary crystals, present along the boundary between the palisade and spongy tissues." The compilation of information on angiosperm leaf anatomy by Napp-Zinn (1973, 1974)
added nothing more. The only published illustration shows part of a cleared leaf (Fig. 2 in Turner and Lersten, 1983
) that included incidentally several large prismatic ("solitary") crystals.
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). MATERIALS AND METHODS
A robust pomegranate plant (source unknown), Punica granatum, that regularly flowers and sets fruit in an Iowa State University greenhouse was the source of ample material. In addition, leaf samples collected from two specimens (Punica granatum) in the Iowa State University Ada Hayden Herbarium (ISC) were processed: C.O. Levine 700, Kwang Tung Province, China, 7V1917, and L.H. Pammel s.n., Patterson, California USA (cultivated), 5IX1921.
Young leaves between primordia and 1 cm in length were excised from shoot tips and mounted directly in 50% aqueous glycerin on glass slides and coverslipped. Older stages, ranging from 1 cm to full-grown (about 5 cm in length), were placed whole or cut into smaller pieces into 95% ethanol to remove all or most of the chlorophyll. We selected newly full-grown leaves as well as some that were much older. Leaf samples were then transferred to 50% aqueous ethanol, to distilled water, then to full-strength household bleach. After 10–45 min in bleach, depending on tissue reaction, samples were moved to water (about 15 min), dehydrated in an ethanol series (50, 95, 95, 100, 100%, 1 : 1/100% : xylol—about 10 min at each step). After transfer to xylol (no time limit), the unstained leaf samples were mounted in Permount and a coverslip was added. The bleaching technique was modified from that of Frank (1972)
.
Samples from two mature leaves were also cleared and stained with chlorazol black E by the method of Lersten (1986)
. Two leaf (cross sections) and two stem (longitudinal and cross sections) samples were also cut 50, 70, and 100 µm thick using a Vibratome (Technical Products International, USA), and mounted in 50% aqueous glycerin. All preparations were viewed in bright-field mode or between crossed polarizers, and selected images were captured digitally using a Zeiss MRc digital camera (Zeiss, USA) and processed in PhotoShop 7.0 (Adobe, San Jose, California, USA) and organized into plates with Illustrator 10 software (Adobe).
RESULTS
There are no bud scales or stipules. The oblong to narrowly elliptical mature leaf is about 5 cm long and 1 cm wide, with an entire margin and brochidodromous major venation (Fig. 1). Minor venation encloses various sizes and shapes of areoles, some without included terminal veins, some with one or two unbranched terminal veins, and only a few with branched terminal veins (Figs. 18, 19). Between the two epidermises, the mesophyll consists of a single layer of palisade cells, one mesophyll cell layer parallel to the vasculature and two spongy mesophyll layers (Figs. 14–16). Findings from two herbarium specimens (China and California) cited in Materials and Methods were identical to those of the greenhouse live plant; all figures shown in the following account are from the latter.
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In a 1.2-mm leaf the developing apical nectary is evident, the midvein and some distal main lateral veins are visible, and more prismatics have proliferated basipetally in the lamina (Figs. 4, 5). In the midrib, basipetal formation of prismatics occurs more rapidly than in the mesophyll (Fig. 6), and in a 2-mm leaf a narrow band of midrib crystals extends to druses in the stem, while lamina prismatics are still confined to the distal part of the leaf (Fig. 7). Prismatics comprise most of the initial basipetal wave of crystal formation in the midrib, but subsequent crystals that proliferate in the midrib are almost all druses. These druses appear to form throughout the midrib, so there is no discernible directional pattern once midrib crystals are continuous with those in the petiole. A portion of a slightly older leaf shows the midrib filled with a large number of druses and some scattered prismatics, while basipetal prismatic crystal formation is still proceeding irregularly in the adjacent lamina (Fig. 8).
An enlarged view of a representative area of a 2.5 cm long (about half-grown), bleached leaf shows still-active prismatic crystal formation in the young lamina. Large early-formed prismatics are surrounded by a greater number of scattered small to intermediate-size immature prismatics (Fig. 9). Crystals are not at uniform distances from each other, and they are not associated with veins. A cross section of a leaf at a similar stage (Fig. 10) includes newly initiated prismatics, and one that has begun expanding, all in the middle mesophyll layer. We were unable to determine if neighboring cells become separated, or are instead destroyed, to accommodate the expansion of a crystal idioblast.
Figure 11 is a representative area of an almost full-grown leaf about 4.0 cm long. It shows more large and intermediate sizes of prismatics than in the younger stage leaf of Fig. 10. Prismatics are not closely associated with veins, and druses do not extend from the midrib into main lateral veins and smaller veins (Figs. 10–12). A portion of a bleached leaf at some unknown time after it has reached full size (Fig. 12) shows the enormous number of prismatics in the mature lamina. Leaves remain for a long time on the plant, although they are eventually deciduous, and in older leaves of undetermined age we found druses in some spongy mesophyll cells. Druses were not uniformly distributed, but were instead found in loose clusters in or near the axil formed by the juncture of two large veins. Figure 13 shows a druse cluster adjacent to the midvein, which is also studded with smaller druses. Among the druses in the spongy mesophyll, the downward extensions of several large prismatics can be seen.
Many prismatics attain the maximum possible vertical expansion and extend from upper to lower epidermis; they appear in leaf cross sections as massive crystalline pillars (Figs. 14, 15). In Fig. 15, the twin plane is seen as a vertical line that extends the length of the crystal. Other orientations show prismatics as diamond-shaped (Fig. 16) or cruciform (Fig. 17), and in these the surrounding idioblast cell wall appears noticeably thicker than that of surrounding cells.
Leaves cleared and stained without using bleach show the distribution of prismatic crystal idioblasts (with crystals dissolved) and their large size relative to neighboring cells. Figure 18 is focused at the mid-mesophyll level, where the thicker idioblast cell wall is evident. Focusing at palisade mesophyll level shows the typical distal tapering of idioblasts accompanied by what appears to be an overarching of adjacent palisade cells (Fig. 19; also visible in Figs. 16, 17). Between these large idioblasts would be found many other idioblasts with smaller prismatics, as seen in Fig. 12, that do not show up as larger cells in these cleared preparations.
As mentioned earlier, druses do not occur in association with any veins except the midvein. They predominate in the midrib, along with some prismatics that appear to have been mostly formed in the first basipetal wave of midrib crystallization (Fig. 6). Crystals do not occur in the phloem or xylem of the midvein, only in the surrounding midrib parenchyma cells (Fig. 20); the same configuration occurs in the short petiole (Fig. 21).
All druses seen in leaf and stem are small, without noticeable cores. Highly magnified views of prismatics (Fig. 22), however, show features of interest such as small epitactic crystals (Figs. 23, 24), and periodically nonpolarizing circular and rectangular regions (depending on orientation of prismatic) in the center of one of the large prismatics (Fig. 23, lower right). These latter regions are reminiscent of the nonpolarizing cores observed in druses in other studies (Lersten and Horner, 2000
, 2004
).
In contrast to the macropattern of the leaf, the stem lacks prismatic crystals, but has great numbers of small druses. Druses occur only in the phloem (Fig. 25) in linear files (Fig. 26) that probably indicate they are within long, slender phloem fibers.
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We have described in pomegranate a crystal macropattern development previously unknown among angiosperms. Phloem fiber druses in the stem are discontinuous with parenchyma druses in the petiole and midrib because phloem fibers are eliminated before the leaf trace emerges from the stem. Midrib druses form secondarily after the primary basipetal wave of small prismatics, and they proliferate to greatly dominate the mature midrib. Druses do not extend from the midrib into any other vein orders, but they appear again in older mature leaves in certain areas of the spongy mesophyll. Lamina prismatics arise only in the mid-mesophyll layer near the apex, and from here more prismatics proliferate in a basipetal wave in the young lamina, followed by additional prismatics formed in mid-mesophyll cells between early-formed prismatics. Idioblasts with early-formed prismatics expand aggressively, whereas later prismatic idioblasts originating in the same mid-mesophyll layer stop growing at various intermediate stages of enlargement.
These crystallization events are of intrinsic interest for this species, but a search for possible generalizations about crystal macropatterns requires comparison with crystallization events in choke-cherry (Lersten and Horner, 2004
), the only other species that has been examined in similar detail.
Pomegranate crystallization differs in several ways from choke-cherry. Both species have druses in the stem, but in pomegranate they occur only in the phloem, whereas in choke-cherry they are found only in cortical and pith parenchyma. In the leaf, however, both have druses in the midrib but not in the phloem. Both species have large prismatics restricted to leaf mesophyll, but they form in different strata. Choke-cherry prismatics originate and expand in the palisade mesophyll without causing any evident severe distortion or destruction of surrounding cells. Pomegranate prismatics, in contrast, originate in the subpalisade layer parallel with vascular bundles, then expand in opposite directions, many finally forming a relatively massive crystalline pillar that extends from epidermis to epidermis.
Crystals in pomegranate do not form in association with any veins except the midrib with its midvein, in which small prismatics occur quite early in leaf development, followed by a vastly greater number of druses. In choke-cherry, in contrast, druses in large numbers, and often of quite large size, are in association with all vein orders, but they mostly are present only after the leaf is full-grown. In choke-cherry, midrib druses appear tardily, but they proliferate acropetally from the petiole.
Another species difference is how prismatics progress in the lamina. In choke-cherry, the original apical cluster of crystals long remains isolated, while the lamina grows to 1.0–1.5 cm in length; then prismatics appear almost simultaneously, scattered in all parts of the lamina, with subsequent prismatics filling in the spaces between them. In contrast, pomegranate prismatics form early, in a basipetal wave continuous with the apical cluster; this progression evidently follows lamina maturation, with additional prismatics interspersed later.
Most druses in choke-cherry appear at some time after the leaf reaches full size, populating third-order and smaller-order veins; druses in pomegranate also appear after leaves are mature, but they form in the spongy mesophyll instead of along veins, particularly in clusters near major vein junctions.
The only feature the two species seem to have in common is the initial appearance of a distal cluster of a few crystals in a leaf of approximately the same length (500 µm in choke-cherry and 650 µm in pomegranate). In choke-cherry, these young leaves are still within the closed bud, but the pomegranate leaves have probably already become exposed to outside air. The pomegranate cluster does not occupy the extreme tip, which is still meristematic and will develop later into the crystal-free apical nectary (Turner and Lersten, 1983
).
The differences between these two species are remarkable, and explanations must as yet be mostly speculations. The broadest generalization is that excess ionic calcium must be sequestered in some way because it is left behind during transpiration. Beyond this, it is evident that pomegranate and other species manage and manipulate calcium in highly individual ways. Recent studies of ours support this interpretation. For example, we found that the mature crystal macropattern of Quillaja (Lersten and Horner, 2005
) differs in almost all respects from the mature macropattern of either choke-cherry or pomegranate. In a current study on the formation and mature crystal macropattern of Prunus serotina (Lersten and Horner, unpublished data), a species closely related to choke-cherry (Prunus virginiana), we find a still different story of crystal formation and mature macropattern.
Except for similarity in initial appearance of apical crystals, all else is remarkably different between pomegranate and choke-cherry in both formation and final configuration. In our study of choke-cherry (Lersten and Horner, 2004
), we pointed out several aspects of the crystal macropattern that are probably not responses to transpiration. Because pomegranate leaves do not form within protective bud scales, they are probably exposed to transpiration effects earlier, and one might expect a simpler crystal macropattern. The macropattern is not simpler, but its components could all be ascribed to transpiration. We speculate that the relatively huge prismatics could serve a secondary function, perhaps as a defensive structure of some kind. Franceschi and Horner (1980)
, Horner and Wagner (1995)
, Nakata (2003)
, Mazen et al. (2004)
, and Franceschi and Nakata (2005)
have reviewed the several possibilities for crystal function, most of them still in the realm of speculation. The great accumulation of druses in the phloem of the pomegranate stem possibly provides protection from bark-boring insects, as has been proposed for several species of conifers that have a similar distribution of phloem crystals (Mazen et al., 2004
).
The few detailed studies of crystal macropattern in the literature have already revealed a rich diversity, which will no doubt be greatly expanded as more species are investigated. The crystal macropattern in many species should be considered an integral part of leaf and stem structure, an anatomical component that continues to enlarge long after organ maturity. Whatever generalizations might eventually arise from studies of macropattern diversity, it seems reasonable to predict that crystals will gain considerably more importance for plant anatomy and physiology as more macropattern investigations are conducted. Such studies should also provide guidance as to precisely where subcellular studies of individual crystal cells should be directed.
FOOTNOTES
The authors thank their departments for partial support of this research, the staff of the Bessey Microscopy Facility where the work was done, and the directors of the Richard Pohl Conservatory and the Ada Hayden Herbarium for permission to collect specimens. 
4 Author for correspondence (hth{at}iastate.edu
)
LITERATURE CITED
Cronquist A. 1981 An integrated system of classification of flowering plants. Columbia University Press, New York, New York, USA
Franceschi V. R. H. T. Horner Jr 1980 Calcium oxalate crystals in plants. Botanical Review 46: 361-427
Franceschi V. R. P. A. Nakata 2005 Calcium oxalate in plants: formation and function. Annual Review of Plant Biology 56: 41-71[CrossRef][Medline]
Frank E. 1972 The formation of crystal idioblasts in Canavalia ensiformis DC at different levels of calcium supply. Zeitschrift für Pflanzenphysiologie 67: 350-358[ISI]
Horner H. T. B. L. Wagner 1995 Calcium oxalate crystal formation in higher plants. In S. R. Khan [ed.], Calcium oxalate in biological systems, 53–72. CRC Press, Boca Raton, Florida, USA
Lersten N. R. 1986 Modified clearing method to show sieve tubes in minor veins of leaves. Stain Technology 61: 231-234[ISI][Medline]
Lersten N. R. H. T. Horner 2000 Calcium oxalate crystal types and trends in their distribution patterns in leaves of Prunus (Rosaceae: Prunoideae). Plant Systematics and Evolution 224: 83-96[CrossRef][ISI]
Lersten N. R. H. T. Horner 2004 Calcium oxalate crystal macropattern development during Prunus virginiana (Rosaceae) leaf growth. Canadian Journal of Botany 82: 1800-1808
Lersten N. R. H. T. Horner 2005 Macropattern of styloid and druse crystals in Quillaja (Quillajaceae) bark and leaves. International Journal of Plant Sciences 166: 705–711 [CrossRef][ISI]
Mazen A. H. A. D. Zhang V. R. Franceschi 2004 Calcium oxalate formation in Lemna minor: physiological and ultrastructural aspects of high capacity calcium sequestration. New Phytologist 161: 435-450[CrossRef][ISI]
Metcalfe C. R. L. Chalk 1950 Anatomy of the dicotyledons, 2 vols. Clarendon, Oxford, UK
Nakata P. A. 2003 Advances in our understanding of calcium oxalate formation and function in plants. Plant Science 164: 901-909[CrossRef][ISI]
Napp-Zinn K. 1973, 1974 Anatomie des Blattes II. Blattanatomie der Angiospermen. Handbuch der Pflanzenanatomie, Band VIII, Teil 2A, Lieferung 1, 2. Gebruder Borntraeger, Berlin, Germany
Solereder H. 1908 Systematic anatomy of the dicotyledons, 2 vols. L. A. Boodle and F. E. Fritsch [English translation]. Clarendon, Oxford, UK
Turner G. N. R. Lersten 1983 Apical foliar nectary of pomegranate (Punica granatum: Punicaceae). American Journal of Botany 70: 475-480[CrossRef][ISI]
This article has been cited by other articles:
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  N. R. LERSTEN and H. T. HORNER Crystal Macropattern Development in Prunus serotina (Rosaceae, Prunoideae) Leaves Ann. Bot., May 1, 2006; 97(5): 723 - 729. [Abstract] [Full Text] [PDF]
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500 µm long lacks crystals near tip. Bar = 250 µm. 3. Young leaf primordium 
