Development of epidermal crystals in leaflets of Stylosanthes guianensis (Leguminosae; Papilionoideae)

CURT L. BRUBAKER' AND HARRY T. HORNER Department of Botany, Iowa State University, Ames, IA 50011-1020, U.S.A.

Received August 19, 1988

BRUBAKER, C. L,, and HORNER, H. T. 1989. Development of epidermal crystals in leaflets of Stylosanthes guianensis (Leguminosae; Papilionoideae). Can. J. Bot. 67: 1664 - 1670.

In developing leaflets of Sfylosanthes guianensis (Aubl.) Sw., twin prismatic calcium oxalate crystals form in adaxial and abaxial epidermal crystal idioblasts. These cells eventually die and collapse, leaving the crystals embedded in a matrix of cutin and cell-wall materials. Adaxial crystal idioblasts develop above large conical cells that, in turn, are interspersed among smaller, multiple-layered palisade parenchyma. Abaxial crystal idioblasts develop beneath a uniseriate layer of large hori- zontally branched cells abutting the abaxial epidermis. Spongy parenchyma occupies the middle mesophyll above the layer of branched cells. The abaxial crystals and the branched cells of the lowerniost mesophyll develop simultaneously. Adaxial crystals and the conical cells develop later and in conjunction with each other. In mature leaflets, the adaxial and abaxial crystals and their associated collapsed crystal idioblasts form networks, the interstices of which are occupied by either single stomates and accompanying epidermal cells (adaxial) or clusters of stomates and accompanying epidermal cells (abaxial). Epidermal crystals are known from other Leguminosae; however, to our knowledge this is the first report where epidermal crystal development involving cell death and collapse is correlated with two types of specialized mesophyll cells.

BRUBAKER, C. L., et HORNER, H. T. 1989. Development of epidermal crystals in leaflets of Sfylosanthes guianensis (Leguminosae; Papilionoideae). Can. J. Bot. 67 : 1664 - 1670.

Dans les folioles incomplktement diffCrenciCes du Sfylosanthes guianensis (Aubl.) Sw., des cristaux gCminCs prismatiques d'oxalate de calcium sont formCs dans des idioblastes Cpidermiques adaxial et abaxial. Ces cellules Cventuellement meurent et s'affaissent, laissant les cristaux noyCs dans une matrice de cutine et de materiel pariCtal. Les idioblastes adaxiaux se differencient au-dessus de cellules grandes et coniques qui, 2 leur tour, sont dispersCes parmi de plus petites cellules d'un parenchyme palissadique multi-sCriC. Les idioblastes adaxiaux se differencient au-dessous d'une couche uni-sCriCe de grandes cellules horizontalement ramifiCes et contigues 2 1'Cpiderme abaxiale. Le parenchyme lacuneux occupe le mCsophylle moyen au-dessus de la couche de cellules ramifiCes. Les cristaux abaxiaux et les cellules ramifiCes du mCsophylle infirieur sont formCs simultanCment. Les cristaux adaxiaux et les cellules coniques sont formCs plus tard et conjointement. Chez les folioles complbtement diffCrenciCes, les cristaux adaxiaux et abaxiaux et les idioblastes dCgonflCs qui y sont associCs forment des rCseaux dont les interstices sont occupCs par soit des stomates isolCs et des cellules Cpidermiques annexes (adaxial), soit des groupes de stomates et des cellules Cpidermiques annexes (abaxial). Les cristaux Cpidermiques sont connus chez d'autres 1Cgumineuses; cependant, autant que nous le sachions, la prCsente constitue une premibre mention oh le dCveloppement des cristaux Cpidermiques impliquant la mort et l'affaissement est corrClC avec deux types de cellules spCcialisCes du mCsophylle.

[Traduit par la revue]

Introduction

The presence of calcium oxalate crystals as intracellular inclusions is widespread in Leguminosae (Solereder 1908; Metcalfe and Chalk 1950; Zindler-Frank 1987). In contrast, the most current review of calcium oxalate crystals in Legumi- nosae (Zindler-Frank 1987) mentions only three reports of crystals that could be interpreted as being extracellular: scleri- fied epidermal cells of Acacia senegal twigs (Wattendorff 1974), adaxial epidermal cells of Gleditsia triacanthos leaflets (Borchert 1984), and epidermal cells of Stylosanthes leaflets (Borodine 1885).

Reports of extracellular crystals in angiosperms are, in general, few and often disputed (Franceschi and Horner 1980). Franceschi and Horner suggested that the extracellular crystals in many species actually form intracellularly and are displaced to the apoplast during development. In Legumi- nosae, and in angiosperms as a whole, very few developmental studies have been done to determine whether what appear to be extracellular crystals actually form outside the protoplast.

In Acacia senegal, the crystals are small (usually less than 0.5 pm) and are embedded in the secondary wall layer closest to the primary wall of the sclerified epidermal cells of twigs

(Wattendorff 1974). Wattendorff (1974) studied only peri- derm development and did not include observations on the development of the crystals and the epidermal cells with which they are associated. It remains to be determined whether the crystals form in the wall or intracellularly and are later moved to the apoplast.

Citing Borchert (1984), Zindler-Frank (1987) listed Gledit- sia triacanthos as another taxon with what might be considered extracellular crystals. Borchert, however, described the epi- dermal crystals in G. triacanthos as "confined to the prox- imity of the cell wall." Because the epidermal crystals were incidental to his study, he made no further comments, so it is not known whether or not these crystals are truly extracellular.

The nature of the extracellular crystals in Stylosanthes has long been controversial. Borodine (1885) first described S. elatior Sw. and S. procumbens Sw. as unique among papilionoid legumes because of a single discontinuous layer of calcium oxalate crystals embedded below the cuticle in the outer wall of leaflet epidermal cells. According to Borodine, the abaxial epidermis, when viewed under crossed polarizers, consisted of a network of crystals whose interstices were each occupied by a single stomate.

Vogelsberger (1893) studied nine species of Stylosanthes and provided a more detailed but conflicting description.

'Author to whom all correspondence should be addressed. According to him, the crystals are found in numerous small

Printed in Canada 1 ImprimC au Canada

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

y.

BRUBAKER AND HORNER 1665

polygonal epidermal cells with thickened anticlinal walls. The crystals are not extracellular. The crystal idioblasts form a network whose interstices are each occupied by one or two stomates. Underneath the crystal idioblasts of the adaxial epi- dermis are large conical cells containing "slime." The abax~al crystal idioblasts underlie cells, each with a large lumen filled with a brownish material, that comprise a hypodermal spongy tissue. Vogelsberger attributed Borodine's (1885) description of the Stylosanthes crystals as being embedded in the outer wall of leaflet epidermal cells to a mistaken impression that the conical cells were epidermal cells. However, Borodine (1 885) did not describe the internal leaflet anatomy, and Vogels- berger's comments must be considered speculative.

Solereder (1908) provided the first illustration of the leaflet epidermal crystals in Stylosanthes. He reiterated the observa- tions of Vogelsberger (1 893) and dismissed Borodine's (1885) description as erroneous. Solereder's (1908) line drawing of the leaflet epidermis of Stylosanthes procumbens Sw. shows a network of small crystal idioblasts, each containing a kinked or straight crystal. The interstices of the network are occupied by clusters of stomates. Metcalfe and Chalk (1950) included a reproduction of Solereder's (1908) figure of the S. procum- bens epidermis but merely listed Stylosanthes as a genus with epidermal crystals and mentioned parenthetically the "curious distribution of the crystals."

In a scanning electron micrograph of the leaflet epidermis of Stylosanthes humilis, the crystals are shown embedded in cuplike structures interspersed among larger epidermal cells (Troughton and Donaldson 1972). The figure caption described the crystals as embedded in the outer cutinized walls of epidermal cells. Many of the crystals are exposed externally and none appear to be enclosed within cells.

More recently, Leelavathi et al. (1984), in the only develop- mental study of Stylosanthes leaflets, described the devel- opment of the crystal idioblasts of the leaflet epidermises of S. fruticosa as follows: polygonal "crystalliferous meri- stemoids" with dense cytoplasms and large nuclei form a net- work around the larger "stomata1 meristemoids"; the solitary crystals form next to the nuclei and as the crystals enlarge, the nuclei are displaced and shrink, disappearing at maturity. They did not discuss the fate of an idioblast after the nucleus had disappeared, and at no point in their description are Stylosanthes crystals described as extracellular.

Zindler-Frank (1987), who cited Borodine (1885; cited by Zindler-Frank and others as Borodine 1884) and Solereder (1908; Zindler-Frank used the earlier 1899 German edition), attempted to resolve the conflicting reports in Stylosanthes by suggesting that the crystals formed intracellularly but were displaced to the apoplast by fusion of a crystal membrane with the cell membrane.

All of these studies and the fact that there is generally a paucity of information about calcium oxalate crystal develop- ment in plants suggest that further research is needed. The present study is directed at resolving the question of the extracellular crystals in Stylosanthes leaflets.

Materials and methods A small shrub of Stylosanthes guianensis (Aubl.) Sw. was grown

from seed in the Iowa State University Department of Botany green- house (Ames, IA 50011). A voucher has been deposited in the Ada Hayden Herbarium of Iowa State University (ISC: Brubaker H108).

For light and scanning electron microscopy, portions of developing and mature leaflets were fixed in 2% paraformaldehyde and 4%

glutaraldehyde in a 0.1 M phosphate buffer (pH 7.2) for 12 h at 4OC, postfixed in 1 % osmium tetroxide in the same buffer at room temper- ature for 2 h, and dehydrated in a graded ethanol series to absolute ethanol (Glauert 1975).

For scanning electron microscopy (SEM), some of the samples in absolute ethanol were either cryofractured or left whole, critical- point dried and mounted on copper stubs with double-stick tape, coated with gold-palladium, and viewed using a JEOL-.ISM 35 SEM. Polaroid Type 665 film was used to record the images.

For light microscopy, the remaining fixed samples in absolute ethanol were embedded in Spurr's resin (Spurr 1969), sectioned at 1-2 pm, placed on glass slides, and stained with toluidine blue 0 . For whole leaflet clearings, excised mature and developing leaflets were fixed in 95 % ethanol, processed according to Shobe and Lersten (1967), and stained in chlorazol black E. Both sections and clearings were viewed and photographed using bright-field and polarizing optics. Techpan film and Kodak D-19 or D-76 developer were used to record and develop the images.

Observations Early in development, Stylosanthes guianensis leaflets con-

sist of two protodermal layers and four internal layers of cells (Fig. 1). The majority of cells in the internal layer abutting the adaxial protoderm become large conical cells with few chloro- plasts that, in the mature leaflet, will protrude into the palisade parenchyma. The remaining cells of this layer differentiate as normal palisade parenchyma. We have designated this layer the conical cell layer (CCL; Fig. 1). The lowermost internal cell layer abutting the abaxial epidermis develops into a single layer of large, horizontally branched cells with few chloro- plasts. We have designated this layer the submesophyll layer (SML; Fig. 1). The two middle layers give rise to the vein procambia, the majority of palisade parenchyma, and the spongy parenchyma. In Fig. 1, only the enlarged SML cells show any evident differentiation.

Clearings (Figs. 2-5) provide paradermal views of the layers shown in Fig. 1. The adaxial (Fig. 2) and abaxial (Fig. 4) protoderms and the CCL (Fig. 3) consist of small, densely packed cells without evident intercellular spaces. The SML cells, however, are enlarged and intercellular spaces are present (Fig. 5).

As leaflets continue to develop (Fig. 6), the early leaflet organization changes, including the greatly enlarged SML. The enlarged conical cells can now be seen beneath small adaxial epidermal cells (paired arrows), whereas larger adaxial epidermal cells overlay partially developed palisade parenchyma. The small adaxial epidermal cells are incipient crystal idioblasts. Although there is little difference in size among the abaxial epidermal cells, some are more darkly stained and each displays a solitary calcium oxalate crystal at this stage (Fig. 6; double-headed arrows).

Clearings of slightly older leaflets show that the incipient crystal idioblasts of the adaxial epidermis have developed thick anticlinal walls, in comparison with other epidermal cells (Fig. 7). The incipient crystal idioblasts form a network whose interstices are each occupied by a single stomate and larger epidermal cells. A comparison of Figs. 7 and 8 shows the net- work of incipient crystal idioblasts (Fig. 7) directly above the enlarged conical cells (Fig. 8). The spaces underneath the stomates are occupied by substomatal chambers and some pali- sade parenchymal

The abaxial epidermal crystal idioblasts, shown at low mag- nification in Fig. 9, also have thickened anticlinal walls. In contrast with the adaxial crystal idioblasts, the abaxial crystal

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

y.

1666 CAN. J . BOT. VOL. 67, 1989

FIGS. 1 - 11. Cross sections and clearings of Stylosanthes guianensis leaflets in successive stages of development. Bars = 20 pm. Fig. 1. Cross section of leaflet early in development. Only enlarged cells of the submesophyll layer (SML) show any evident differentiation. Conical cell layer (CCL) and remaining cell layers of leaflet consist of small, densely packed cells. Figs. 2 - 5 . Four optical planes of focus from a cleared leaflet the same age as that shown in Fig. 1. Fig. 2. Focus on adaxial protoderm. Cells are small and densely packed with no intercellular spaces. Fig. 3. Focus on conical cell layer underlying protoderm shown in Fig. 2. Cells are small and densely packed with no intercellular spaces. Fig. 4. Focus on abaxial protoderm. Cells are small and densely packed with no intercellular spaces. Fig. 5. Focus on submesophyll layer overlying abaxial protoderm shown in Fig. 4. Cells are enlarged and intercellular spaces are present. Fig. 6. Cross section of developing leaflet. Enlarged vacuolate conical cells are visible underneath small adaxial epidermal cells (paired arrows). Cells of the submesophyll layer are enlarged and highly vacuolate. Top and bottom insets show abaxial and adaxial epidermises as seen between crossed polarizers. Only abaxial

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

y.

BRUBAKER AND HORNER 1667

idioblasts already contain crystals (Fig. 9). The SML cells are slightly larger than shown previously and have separated farther from each other causing intercellular spaces to become larger (Fig. 10). A comparison of Figs. 9 and 10 shows the network of abaxial crystal idioblasts (Fig. 9) directly beneath the SML cells; the clustered stomates in the interstices of the network of the abaxial epidermis lie below the intercellular spaces of the SML.

Crystals also form in the adaxial crystal idioblasts (Fig. 11; double-headed arrow). In all the leaflets we examined, crystals formed in the abaxial crystal idioblasts first, followed by their appearance in adaxial crystal idioblasts. An immature crystal can be see inside a crystal idioblast in Fig. 12.

Up to this point, the leaflets have been folded conduplicately and concealed within the clasping stipules of an older leaf. As each leaflet begins to expand beyond the protective stipules of the older leaf, the crystals are full size and the crystal idio- blasts begin to die and collapse. This sequence of development is illustrated in Figs. 13 - 18.

In Fig. 13, the adaxial epidermal crystal idioblasts have not yet collapsed. The thickened anticlinal walls and the single stomates characteristic of the adaxial epidermis are evident. In Fig. 14, each outer idioblast wall is partially collapsed and beginning to drape around the crystal. A large area of the adaxial epidermis at this stage shows the network of collapsing crystal idioblasts with single stomates in the interstices (Fig. 15). A highly magnified view of epidermal idioblasts in various stages of collapse is shown in Fig. 16. The outer walls of the crystal idioblasts in Fig. 17 have completely collapsed and are draped around the crystals. The thickened anticlinal walls of these cells do not collapse but form cuplike structures in which the crystals are located. The thickened, uncollapsed anticlinal walls are shown at higher magnification in Fig. 18. The outer cell wall appears to have broken and pulled away from the crystals in some instances. Each crystal is embedded in a matrix of cutin and other cell-wall materials. It appears to be exposed externally, but this may be an artifact of pro- cessing. As shown in Fig. 18, the crystals are twinned pris- matics that may be kinked or straight. Some of the twin planes of the crystals are indicated by arrows (Fig. 18).

Mature leaflets are illustrated in Figs. 19-25. In a leaflet cross section (Fig. 19), the crystals are seen embedded in a matrix of cutin and other cell-wall materials. The conical cells are easily distinguished from the palisade and spongy paren- chyma because of their large size and the presence of few chloroplasts. The conical cells protrude into the palisade parenchyma. The cells of the SML, abutting the abaxial epidermis, also have few chloroplasts. Both the conical cells and the SML cells enlarge and become highly vacuolate early in development. The SML cells develop sooner and are typi- cally larger at maturity. The majority of vacuoles in both types of cells usually have no evident inclusions, but occasionally

the vacuoles are filled with an unknown granular substance. The mature abaxial and adaxial networks of crystals and col-

lapsed crystal idioblasts are visible in mature leaflet clearings (Figs. 20 and 22). The mature conical cells (Fig. 21) underlie the network of adaxial crystals (Fig. 20), whereas the SML cells (Fig. 23) overlie the network of abaxial crystals (Fig. 22). The thickened anticlinal walls of the collapsed crys- tal idioblasts are readily apparent on both leaflet surfaces (Figs. 24 and 25). Figure 24 is oriented upside down for easier viewing and shows many abaxial crystals above a large SML cell. On the adaxial epidermis (Fig. 25), a solitary stomate is surrounded by collapsed crystal idioblasts, each containing a twinned prismatic crystal.

Discussion This is the first report in Leguminosae where development

of epidermal crystals involving death and collapse of crystal idioblasts is associated with two types of specialized meso- phyll cells. We suspect that this developmental process occurs in all Stylosanthes species. The conflicting reports in the litera- ture probably result from previous investigators examining leaflets at different developmental ages. Vogelsberger (1893) described the crystals as intracellular, whereas Borodine (1885) described the crystals as embedded in the outer wall of epidermal cells. Vogelsberger could have been looking at leaflets prior to the collapse of the crystal idioblasts, whereas Borodine examined leaflets after their collapse.

All other reports of what might be interpreted as extracellu- lar crystals in Leguminosae are not comparable. In the study of Acacia senegal periderm formation, Wattendorff (1974) listed Stylosanthes as another taxon where calcium oxalate crystals were observed in epidermal cell walls. However, at maturity any similarities between Stylosanthes and Acacia are superficial. In contrast to the larger twinned prismatic crystals of Stylosanthes guianensis, which are embedded in a matrix of cutin and the cell-wall remnants of collapsed crystal idioblasts, the smaller crystals of Acacia are associated with living epidermal cells that are sloughed off during periderm devel- opment.

In Gleditsia (Borchert 1984), the crystals are also smaller than the Stylosanthes epidermal crystals. The Gleditsia epi- dermal crystal cells show no evidence of collapse even at maturity. In Acacia and Gleditsia, crystal cell development probably occurs differently than in Stylosanthes guianensis.

Intracellular deposition of crystals in leaflet epidermal crys- tal idioblasts, followed by death and collapse of the crystal idioblasts, may occur in other Leguminosae genera without the developmental and spatial association with specialized meso- phyll cells observed in Stylosanthes guianensis.

Vogelsberger (1893) observed epidermal crystals in Arachis leaflets, a genus closely related to Stylosanthes, that were

crystal idioblasts contain crystals. Double-headed arrows indicate position of two crystals. Figs. 7- 10. Four optical planes of focus in a cleared leaflet slightly older than that shown in Fig. 6. Fig. 7. Focus on adaxial epidermis. Single stomates and thin-walled epidermal cells occupy interstices of network of crystal idioblasts with thickened anticlinal walls. Fig. 8. Focus on conical cell layer underlying adaxial epidermis shown in Fig. 7. Enlarged conical cells lie directly underneath adaxial crystal idioblasts seen in Fig. 7. Fig. 9. Focus on abaxial epidermis. Clusters of stomates and thin-walled epidermal cells occupy interstices of network of crystal idioblasts with thickened anticlinal walls. Most idioblasts contain crystals. Fig. 10. Focus on submesophyll layer overlying abaxial epidermis shown in Fig. 9. Submesophyll cells have been stretched and the size of intercellular spaces is larger than shown in Fig. 5. Fig. 11. Cross section of leaflet nearing maturity. Enlarged conical cells protrude into palisade parenchyma. Branched cells of the submesophyll layer are separated by large intercellular spaces. Top and bottom insets show abaxial and adaxial epidermises as seen between crossed polarizers. Adaxial and abaxial crystal idioblasts contain crystals. Double- headed arrows correlate positions of crystals in light micrograph with their positions in insets.

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

y.

1668 CAN. J . BOT. VOL. 67, 1989

FIGS. 12- 18. Scanning electron micrographs of successive stages of crystal idioblast collapse. Fig. 12. Developing leaflet. An immature crystal is visible in epidermal cell. Bar = 6 pm. Fig. 13. Adaxial epidermis. Crystal idioblasts with thickened anticlinal walls form a network around single stomates. Bar = 10 pm. Fig. 14. Adaxial epidermis. Outer walls of crystal idioblasts are partially collapsed. Bar = 2.5 pm. Fig. 15. Adaxial epidermis. Single stomates occupy interstices of network of partially collapsed crystal idioblasts. Bar = 20 pm. Fig. 16. Adax- ial epidermis. Outer walls of crystal idioblasts in various stages of collapse. Bar = 10 pm. Fig. 17. Abaxial epidermis. Clustered stomates occupy interstices of network of fully collapsed crystal idioblasts. Outer idioblast walls are draped around twinned prismatic crystals. Bar = 100 pm. Fig. 18. Collapsed crystal idioblasts. Each collapsed idioblast contains a single twinned prismatic crystal (arrows indicate twin planes). Outer walls appear broken; crystals are exposed externally. Bar = 5 pm.

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

y.

BRUBAKER AND HORNER

FIGS. 19-25. Scanning electron micrographs of mature whole and cryofractured leaflets and light micrographs of mature whole leaflet clear- ings and leaflet cross sections. Fig. 19. Cross section of mature leaflet. Large conical cells protrude into palisade parenchyma. Submesophyll layer occupies lowermost layer of leaflet mesophyll abutting abaxial epidermis. Conical cells and horizontally branched cells of the sub- mesophyll layer have thin peripheral cytoplasms surrounding large central vacuoles. Top and bottom insets show adaxial and abaxial epider- mises as seen between crossed polarizers. Both epidermises contain crystals embedded in a matrix of cutin and other cell-wall materials (double-headed arrows). Bar = 50 pm. Figs. 20-23. Four optical planes of focus from a mature, cleared leaflet. Fig. 20. Focus on adaxial epidermis. Single stomates occupy interstices of network of collapsed crystal idioblasts. Bar = 40 p m Fig. 21. Focus on conical cell layer underlying abaxial epidermis shown in Fig. 20. Bar = 40 pm. Fig. 22. Focus on abaxial epidermis. Clusters of stomates occupy interstices of network of collapsed crystal idioblasts. Bar = 40 pm. Fig. 23. Focus on submesophyll layer overlying abaxial epidermis shown in Fig. 22. Bar = 40 pm. Fig. 24. Scanning electron micrograph of cryofractured leaflet. Leaflet is oriented upside down for viewing ease. Abaxial crystals are located above a large submesophyll cell. Bar = 20 pm. Fig. 25. Scanning electron micrograph of adaxial epidermis. Solitary twinned pris- matic crystals are each located in a cup formed by its collapsed crystal idioblast. Bar = 20 pm.

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

y.

1670 CAN. J . BOT. VOL. 67, 1989

similar to the crystals he observed in Stylosanthes. The crystal idioblasts were described as cells with thickened walls that were slightly smaller than those he observed in Stylosanthes. Each crystal idioblast contains a single crystal. These crystal idioblasts are located above and below leaflet veins rather than large specialized mesophyll cells as in Stylosanthes.

Waldron's (1919) description of the epidermal crystal idio- blasts in Arachis leaflets conflicts with that of Vogelsberger (1893). According to Waldron, each of the young crystal idio- blasts, which are found in both epidermises, contains a single rounded crystal. With continued development, the crystal idio- blasts fuse to form "epidermal vessels" (possibly coenocytic idioblasts), which are irregular in shape and may include up to 30 crystals each. His drawing of the crystals indicates they are twinned prismatics.

More recent reports also describe and (or) illustrate epi- dermal crystals in Arachis (Kothari and Shah 1975; Khrzhan- ovskii and Mamontov 1972; Burba r t and Collins 1941; Leelavathi et al. 1984; Yarbrough 1957) but add little addi- tional information. As in Stylosanthes, a developmental study of the leaflet epidermal crystals in Arachis will be necessary to resolve the conflicting reports in the literature.

Leaflet mesophyll cells that resemble the conical cells and (or) submesophyll layer cells have been reported or illustrated in other legume taxa such as Arachis (Waldron 1919; Khrzhanovskii and Mamontov 1972; Yarbrough 1957; Kumari et al. 1983; Reed 1924), Onobrychis (Lees et al. 1982; Alexandrov 1925), and Arthrocarpum, Chapmannia, and Pachecoa (Gillet 1966). These cells are described as storage sites for water and (or) tannin. In all of these genera, the enlarged mesophyll cells are not associated with epidermal crystals and only superficial comparisons can be made with Stylosanthes guianensis.

This present study resolves the question of the intracellular origin of the leaflet crystals in Stylosanthes. It further associ- ates these crystals with two specialized types of cells that occur in other legume taxa. However, the functional significance of this association in Stylosanthes, if any, is not known.

Acknowledgements

Light and scanning electron microscopy, photomicro- graphy, and darkroom procedures were carried out in the Bessey Microscopy Facility, Department of Botany, Iowa State University. We thank Dr. N. R. Lersten and Dr. D. R. Farrar for reviewing the manuscript.

ALEXANDROV, W. 1925. ~ b e r ein neues Beispiel einer besonderen Art des Wassergewebes in den Blattern. Ber. Dtsch. Bot. Ges. 43: 418-426.

BORCHERT, R. 1984. Functional anatomy of the calcium-excreting sys- tem of Gleditsia triacanthos L. Bot. Gaz. 145: 474-482.

BORODINE, J. 1885. Sur la ripartition des cristaux d'oxalate de chaux dans les feuilles des LCgumineuses et des RosacCes. In Bulletin du congrks international de botanique et d'horticulture (riuni i St. Pktersbourg, le 5- 15 mai 1884). Imprimerie de l'acadimie impCriale des sciences, St. Pitersbourg. pp. 69 -77.

BURKHART, L., and COLLINS, E. R. 1941. Mineral nutrients in peanut plant growth. Soil Sci. Soc. Am. Proc. 6: 272-280.

FRANCESCHI, V. R., and HORNER, H. T., JR. 1980. Calcium oxalate crystals in plants. Bot. Rev. 46: 361 -427.

GILLETT, J. B. 1966. The species of Ormocarpum Beauv. and Arthrocarpum Ba1f.f. (Leguminosae) in south-western Asia and Africa (excluding Madagascar). Kew Bull. 20: 323 - 355.

GLAUERT, A. M. 1975. Fixation, dehydration, and embedding of bio- logical specimens. North-Holland Publishing Co., Amsterdam.

KHRZHANOVSKII, V. G., and MAMONTOV, Y. G. 1972. Comparative morphological study of Arachis leaves. Izv. Timiryazev. Skh. Akad. 1972(6): 219-223.

KOTHARI, M. J., and SHAH, G. L. 1975. Epidermal structures and ontogeny of stomata in the Papilionaceae (tribe Hedysareae). Bot. Gaz. 136: 372-379.

KUMARI, D. S., SESHAVATHARAM, V., and MURTY, U. R. 1983. Com- parative leaf anatomy of the wild species and cultivated varieties of the genus Arachis. Oliagineux, 38: 27-37.

LEELAVATHI, P., PRABHAKAR, M., and RAMAYYA, N. 1984. Structure, distribution, ontogeny and taxonomic significance of crystalliferous cells in Leguminosae. Indian J. Bot. 7: 125 - 13 1.

LEES, G. L., HOWARTH, R. E., and GOPLEN, B. P. 1982. Morphologi- cal characteristics of leaves from some legume forages: relation to digestion and mechanical strength. Can. J. Bot. 60: 2126 -2132.

METCALFE, C. R., and CHALK, L. 1950. Anatomy of the dicotyle- dons. Clarendon Press, Oxford.

REED, E. L. 1924. Anatomy, embryology, and ecology of Arachis hypogea [sic]. Bot. Gaz. 78: 289-310.

SHOBE, W. R., and LERSTEN, N. R. 1967. A technique for clearing and staining gymnosperm leaves. Bot. Gaz. 128: 150- 152.

SOLEREDER, H. 1908. Systematic anatomy of the dicotyledons. Clarendon Press, Oxford.

SPURR, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: 31 -43.

TROUGHTON, J., and DONALDSON, L. A. 1972. Probing plant struc- ture. McGraw-Hill, New York.

VOGELSBERGER, A. 1893. Ueber die systematische Bedeutung der anatomischen Charaktere der Hedysareen. Diss. K. B. Friedrich- Alexander-Universitat, Erlangen.

WALDRON, R. A. 1919. The peanut (Arachis hypogaea)-its history, histology, physiology, and utility. Contrib. Bot. Lab. Morris Arbor. Univ. Pa. 4: 301 -338.

WATTENDORFF, J. 1974. The formation of cork cells in the periderm of Acacia senegal Willd. and their ultrastructure during suberin deposition. Z. Pflanzenphysiol. 72: 1 19 - 134.

YARBROUGH, J. A. 1957. Arachis hypogaea. The seedling, its epicotyl and foliar organs. Am. J. Bot. 44: 19-30.

ZINDLER-FRANK, E. 1987. Calcium oxalate crystals in legumes. In Advances in legume systematics. Part 3. Edited by C. H. Stirton. Royal Botanic Gardens, Kew, England. pp. 279 -3 16.

Can

. J. B

ot. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

y.