copyright 2001, David M. Baker, Electron Microscope Lab, University of California, Berkeley
David M. Baker
MCB481C

Description and Interpretation of Stipule Development in Exbucklandia populnea (Hamamelidaceae)

Purpose

Obtaining high-resolution images of the apical region of Exbucklandia populnea to provide a context for interpreting histological sections being generated in a related project studying the branching rhythm of the plant provided the impetus for the following work. The purpose of the work presented below was to observe, describe, and interpret the development of the protective stipular structure exhibited by individuals of Exbucklandia. Technical goals of the project included: becoming competent in the preparation of specimens for viewing by scanning electron microscopy; becoming competent in the operation of the scanning electron microscope; and becoming proficient at recording the images obtained by Polaroid film and digital image capture.

Introduction

Exbucklandia populnea (Hamamelidaceae), is a tree that exhibits a flushing growth habit in which a few new leaves expand at an elongating shoot tip simultaneously between intervals of dormancy. Each shoot is not tipped by an apical bud of scales or small expanding leaves, but by an asymmetrical green rudder-shaped structure comprised of what has been interpreted as fused lateral stipules of the subtending leaf3 (see figures 1 and 2). When dissected, this rudder structure splits into two equal flaps along a median fissure line to reveal a number of unexpanded shoots in a mat of trichomes. Observations and interpretations of the development of this protective stipular structure are presented below. Commentary on the development of the leaf as a whole is also included. As alluded to above, the work presented here is a part of a larger project of describing and morphologically interpreting the development of the shoot apical region, here defined as the protective stipular structure and the enclosed apical meristem with its developing derivatives.

Materials and Methods

Six shoot apices, consisting of a lamina, petiole, and protective stipulate structure, containing developing shoots, were collected from the UC Berkeley Botanical Garden and brought to the UCB Electron Microscope Laboratory. The lamina and petiole of the leaves were removed and the stipulate structure was dissected to expose the developing shoots within. The developing shoots were fixed for two hours in 2% gluteraldehyde in 0.1M sodium cacodylate buffer, pH 7.2. The samples were rinsed for three fifteen-minute intervals with 0.1M sodium cacodylate buffer, pH 7.2. Two of the six samples were treated with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, pH 7.2, for one hour following fixation. Following this treatment, the two osmium tetroxide treated samples were rinsed for three five-minute intervals in cacodylate buffer, pH 7.2. All of the samples were dehydrated in a seven step dehydration series, for 10 minutes per solution with ethanol concentrations of 35%, 50%, 70%, 80%, 95%, 100% and 100%. The samples were then critical point dried in a Samdri-PVT-3B (Tousimis). The chamber was purged a total of four times (the initial bleed + three additional bleeds). Following mounting on stubs with carbon dots, the specimens were sputter coated for 2.5 minutes, producing approximately a 20 nm layer of gold palladium alloy in a Polaron sputter coater. Two of the samples were damaged upon removal from the sputter coater. These two samples were run a second time in the sputter coater for a one-minute run.

Specimens were viewed under standard imaging conditions at 10,000 volts on an ISI DS 130 scanning electron microscope. Images were captured using both a Polaroid image capture system and Semicaps digital imaging system.

Results

Aerial and lateral views of the developing shoots were obtained. See Figures 2 through 4 below. See the following section for a discussion of each figure.

Discussion

Figure 3 is a low magnification aerial view of the contents of the chamber created by the protective stipulate structure. Two developing shoots can be observed in a shroud of trichomes. Although all of the six specimens observed appeared to have two developing shoots, three shoots have been observed frequently in other samples. It is quite possible that a third shoot is present in this sample, obscured by the trichomes. The developing shoots have been interpreted by the author, as well as others1, to consist of one dominate shoot, a continuation of the main axis, along with branches of higher order whose morphological derivation is under investigation. The larger, more developed shoot was interpreted as the continuation of the main axis. This interpretation could be further justified by determining the divergence angle of the plant, and observing which of the shoots within the stipular structure bears a lamina the determined number of degrees from the lamina subtending the stipular structure, defining it as the next leaf in the helical phyllotactic rhythm of the main shoot. The other shoots within the stipular structure may be either axillary buds of the subtending leaf, or a condensed branching system. The developing shoots are peculiar in that they only bear one leaf. Axillary buds often bear numerous leaves in a orientation similar to the plant body of the embryo. In dicotyledons, which bear two oppositely inserted cotyledons in their embryos and early phases of development, there are often two structures analogous to cotyledons in the axillary buds of the mature plant, the prophylls. If after further investigation of the axillary buds, prophylls are not found, it would be of interest to study the early development of the plant and observe the state of the cotyledons.

Developing leaves have been divided into two zones: the upper leaf zone which, in many dicotyledonous plants, gives rise to the lamina and petiole region of the leaf, and the lower leaf zone which, in many dicotyledonous plants, develops into the leaf base region and stipules3. The leaf base is defined as "the widened point of insertion of the leaf on the shoot axis." 4 Stipules are appendages of the of the leaf base3. These concepts will prove useful in understanding the following discussion.

Figure 4 captures a leaf early in its development, before the stipules have begun their expansion. Two edges of the leaf base have partially grown over the meristem of their origin, producing a cleft between them. These structures that form the cleft have been interpreted as leaf base tissue because they are confluent with the leaf base tissue near the boundary of the upper leaf zone with the lower leaf zone. As can be seen in figure 5, "lips", interpreted as stipule primordia, appear to arise on this tissue. Larger stipules at the same positions of these "lips" of more developed shoots (see figure 6) support the interpretation of the lips as stipule primordia. As can be seen in the more developed stipules in figure 6, there is a change in growth direction between leaf base tissue, visible curving over the shoot apex, and the stipules which expand almost perpendicularly to the inferred direction of growth of the leaf base. It appears that leaf base tissue grows to partially dome over the meristem of the shoot, and from this tissue, appendages or stipules, arise. Histological sections will add strong evidence to either support or refute these conclusions.

The "lips" in Figure 5 and the more developed stipules in figure 6, arising on either side of the upper leaf zone, are in the position of lateral stipules, suggesting that the mature stipulate structure is the result of appression of two fused lateral stipules, rather than the proliferation of a median stipule. If the structrue were derived from a median stipule, one would expect to see the stipule primordia arise on the adaxial surface of the leaf near the boundary of the upper leaf zone and the lower leaf zone.

The independent and lateral origin of the stipules is dramatically illustrated in Figure 6. This image also suggests that the leaf base has not only grown to partially cover the meristem that generated it, but that it has grown to encircle the meristem. The leaf base edges have become confluent to form a tube by the processes of "meristem incorporation and meristem fusion."2More evidence is needed to confirm this observation. A dome, which is here interpreted as the apical meristem, is visible in the cleft created by the leaf base tissue.

Figure 7 captures yet a later stage in development in which the stipules have increased markedly in size and appear to be in the process of becoming appressed. It is unknown if the tissues of the stipules intercalate to form pseudoparenchyma, but it is impossible to come to a conclusion about this aspect of stipule development from the electron micrographs obtained, thus the term "appressed" is used instead of "fused". In this figure, the stipules have taken on the gross form of the protective keele-shaped structure, differing from the mature form mostly in size. A zone of tissue damage is evident to the right of the stipular structure because the lamina and petiole of this leaf were broken off after sputter coating as alluded to above.

The organ enclosing the apical meristem and its developing derivatives in Exbucklandia populnea has its origin as two symmetric lateral stipules on a leaf base that grows to encircle its generitive meristem. The stipules originate as what have been termed "lips" on the leaf base tissue, elongate independently and symmetrically, and finally become appressed to form the mature structure.

There was a noticeable difference in the preservation of trichome structure. Compare the trichomes in figure 7 in which they appear flacid and distorted and the trichomes in figure 5, in which they appear more robust. The difference could be an artifact of tissue preparation, the use or non-use of osmium tetroxide, or just differences that were in the trichomes before they were collected. Unfortunately this cannot be determined from data collected at the apices treated with osmium tetroxide were mixed with the untreated shoot apices before sputtercoating. Thus, it was impossible to asses the benefit of using the osmium.

References

  1. Das, Gitasree., A.K. Das. 1984. Ordering of shoots with special reference to Exbucklandia populnea. Current Science. May 5, 1984: 498-499.
  2. Hagemann in Kaplan, D. R. The Principles of Plant Morphology, Volume III. Berkeley: Odin Readers, 1999: Chapter 15.
  3. Kaplan, D. R. The Principles of Plant Morphology, Volume III. Berkeley: Odin Readers, 1999: Chapter 15.
  4. Troll in Kaplan, D. R. The Principles of Plant Morphology, Volume III. Berkeley: Odin Readers, 1999: Chapter 15.

Figures

Figure 1.

 A Leaf of Exbucklandia and a portion of the "stem" on which it is born. The artificial boundary between the leaf and "stem" in this plant is a the insertion point of the stipular structure. Ax, axis; L, lamina; P, petiole; St, stipules.

Figure 2.

 A close-up of the stipular structure. Ax, Axis; P, petiole; St. stipules

Figure 3.

 Aerial view of the main axis apex with the stipular structure removed. Note two developing shoots in a mass of trichomes. The point of insertion of the upper leaf associated with the removed stipules is out of the frame to the top. Note the stipular scars.

Figure 4.

 Aerial view of a shoot in an early stage of development. Note the cleft between the two zones of leaf base expansion. ULZ, upper leaf zone.

Figure 5.

 Aerial view of a shoot apex bearing stipule primordia which are evident as "lips" on the leaf base tissue. ULZ, upper leaf zone.

Figure 6.

 Lateral view of a developing shoot. The stipules are seen expanding independently. The apical meristem is visible in the cleft. Some of the stipular structure that enclosed this shoot is evident in cellular sheet in the background. AM, apical meristem; St, stipules; ULZ, upper leaf zone.

Figure 7.

 Oblique view showing stipules becoming appressed at margin to form the stipular structure. Note the light colored stipule insertion point. P, petiole scar (upper leaf zone broke during processing); SI, stipule insertion point, SA, stipule appression at the margin.