Microtubule-Organizing Centers, Plants, Fern, Electron Microscopy

(The following appeared in poster form at the ASPP 1996 meeting.)

Index of sections (or simply proceed below):
Introduction
Results

Index of Figure references and Figures
(or simply proceed below to Results for more contextual references to figures):
(MT=microtubules)

Fig.1 (87K jpg): ultrastructure of blepharoplasts unexposed to oryzalin (untreated controls).
Fig.2 (36K jpg): continuous oryzalin exposure abolishes blepharoplast- associated MTs, but blepharoplasts persist.
Fig.3 (23K jpg): autonomous blepharoplas replication: two replication cycles transpired, and non-disjunction [of blepharoplasts] is due to oryzalin--i.e., MTs' absence.
Fig.4 (29K jpg): MT depolymerization revealed structures in the blepharoplast cortex (where MTs terminate) that resemble reported putative MT-nucleating sites in the centrosome.
Fig.5 (53K jpg): MT depolymerization appears not to effect redistribution of blepharoplast-resident gamma-tubulin (immunogold).
Fig.6 (78K jpg): the MT-disruptive effects of oryzalin are reversible (i.e., following transient treatment, MTs return and function resumes).
Fig.7 (69K jpg): transient oryzalin exposure was effective (disrupted cell plate, but MTs have returned).

PLANT MICROTUBULE-ORGANIZING CENTERS
Century-old question:
Are the blepharoplasts found in plants with motile gametes homologs of animal centrosomes?

Blepharoplast structure, function, and composition in the penultimate division of sperm development in the fern Ceratopteris.

Neil A. Durso and Kevin C. Vaughn, USDA-ARS Southern Weed Science Laboratory, Stoneville, MS

INTRODUCTION
The microtubule cytoskeleton of typical animal cells originates at the centrosome--the microtubule organizing center, or MTOC, in such cells, primarily comprising distinctly structured centrioles embedded in amorphous centrosome matrix (a.k.a., pericentriolar material). Typical plant cells, however, have no MTOC comparable to centrosomes in size, structure, function, and organization. To study a centrosome analog in plant cells, we selected a MTOC termed the blepharoplast found in less typical plant cells-those which develop into sperm cells in gametophytic antheridia of the fern Ceratopteris.

At particular times during sperm development, the blepharoplast serves as a centrosome-like MTOC throughout the cell cycle. Here we present some details of the blepharoplast's ultrastructure and inferred function that are analogous to animal centrosomes at these developmental stages. Transient or continuous treatments with 1 uM oryzalin (a microtubule disrupting herbicide) were used to investigate blepharoplast function experimentally, and immunogold electron microscopy indicates the localization of gamma-tubulin, a molecule found universally in MTOCs.

TREATMENT
Ceratopteris gametophytes were transferred to, and cultured on, either:
(i) oryzalin-free media continuously (negative controls)
(ii) media containing 1 uM oryzalin continuously (positive controls)
(iii) medium containing 1 uM oryzalin transiently, then oryzalin-free medium for 5-7 h. (experimental group; by inference, "recovered")

THIS PRESENTATION'S FOCUS
Though blepharoplasts have been found exclusively in spermatogenous cells of gametophytic antheridia, their characteristics change with antheridial development and/or cell cycle stage. However, this presentation is limited to the interphase blepharoplast at developmental stages illustrating analogy with centrosomal MTOCs.

RESULTS

UNTREATED CONTROLS
Fig.1 (87K jpg) illustrates the ultrastructure of blepharoplasts unexposed to oryzalin.

BLEPHAROPLASTS

MTOC insofar as a discrete structure (0.5-1.0um) where MTs focus.
Comprises an amorphous matrix at which MTs appear to terminate or originate (though not penetrate).
In the matrix are embedded (many) discrete, grossly cylindrical structures (no tubulin is detected).
Within the cylinder of 9-fold symmetry extends a central "tubule" apparently connected to the cylinder walls via radial spokes.

CENTROSOMES

MTOC: i.e., a discrete structure (0.5-1.0um) where MTs focus.
Comprises centrosome matrix in which MTs appear to terminate or originate.
In the matrix are embedded (typically 2) discrete, grossly cylindrical centrioles (largely of tubulin).
Within the centriole of 9-fold symmetry extends a central "tubule" apparently connected to the centriole walls via radial spokes.

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CONTINUOUS ORYZALIN EXPOSURE
Fig.2 (36K jpg) illustrates that continuous oryzalin exposure abolishes blepharoplast-associated MTs, but blepharoplasts persist. Whether these blepharoplasts are replication sisters present prior to oryzalin exposure cannot be ascertained. However, the non-disjoined tetramer shown in Fig.3 (23K jpg) indicates that two replication cycles have transpired and that the non-disjunction is due to oryzalin--i.e., MTs' absence. In Fig.4 (29K jpg), MT depolymerization has revealed structures in the blepharoplast cortex (where MTs terminate) that resemble reported putative MT-nucleating sites in the centrosome. Fig.5 (53K jpg) indicates that MT depolymerization appears not to effect redistribution of blepharoplast-resident gamma-tubulin---a protein emerging as a requisite MTOC and MT-nucleating complex component (Gamma-tubulin's localization along MTs in untreated controls is typical in plants and is becoming more widely reported outside the plant kingdom as well).

BLEPHAROPLASTS

MTs' absence appears generally not to affect blepharoplast structure
Blepharoplast replication is not MT-dependent
Only in MTs' absence are putative MT- nucleating structures clearly observed in the blepharoplast matrix
Apparent diameter of the putative MT- nucleating structure is 25-30nm, slightly larger than 25nm-wide MTs
Gamma-tubulin appears localized in the blepharoplast matrix's periphery, where MTs appear to terminate

CENTROSOMES

MTs' absence appears generally not to affect centrosome structure
Centrosome replication is not MT-dependent
Only in MTs' absence are putative MT- nucleating structures clearly observed in the centrosome matrix
Apparent diameter of the putative MT- nucleating structure is 25-30nm, slightly larger than 25nm-wide MTs
Gamma-tubulin appears localized in the centrosome matrix's periphery, where MTs appear to terminate

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TRANSIENT ORYZALIN EXPOSURE
(AND APPARENT RECOVERY)
Gamma-tubulin is reported to be involved not only in MTOCs' function as MT-nucleator, but also in the formation of a functional MTOC. Because oryzalin exposure generally appears not to affect blepharoplast ultrastructure, replication, or gamma-tubulin distribution, and if the blepharoplast is a bona-fide MTOC, the MT-disruptive effects of oryzalin should be reversible, as Fig.6 (78K jpg) indicates. The blepharoplast's juxtanuclear position, near a "cleft" into which MTs extend, is typical in controls as well (not shown).

That the transient oryzalin exposure was effective is indicated in Fig.7 (69K jpg).
SPECULATION:
Here, oryzalin may have taken effect around late anaphase: Whereas cytokinesis was disrupted (the incomplete cell plate indicates disrupted phragmoplast MTs prior to recovery), karyokinesis appears to have proceeded unaffected (the typical oryzalin symptom of lobed nuclei due to spindle MT disruption is not evident). Moreover, there are no residual phragmoplast MTs here, and MTs focus at the blepharoplasts-an indication that interphase has resumed, for this MTOC feature normallyappears only after cell plate completion. Apparently in progress here is the blepharoplasts' post-recovery, MT-dependent redistribution (perhaps following post-recovery disjunction as well). These observations indicate that the blepharoplast's functional potential is independent of the progress/completion of normal MT-based cytokinetic processes of the cell cycle.

BLEPHAROPLASTS

Disrupted MTs recover at the blepharoplast.
Interphase blepharoplasts are juxtanuclear.

CENTROSOMES

Disrupted MTs recover at the centrosome.
Interphase centrosomes are juxtanuclear.

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EXTENDED PERSPECTIVE
Frontiers of the Higher Plant Cytoskeleton
Around a century ago, botanists using light microscopy likened blepharoplasts to centrosomes of animal cells. Decades later (1956), Robert Lepper discounted the claims that blepharoplasts were "ontogenetically and phylogenetically centrosomes." Though not strictly centrosome homologs, EM and immuno-techniques do indicate that they share homologous MTOC features.

One favored basis for the early proposal of homology was the observation that the blepharoplast that arises in latter spermatogenous divisions ultimately disperses to serve as flagellar templates (as centrosomes do). Now, ultrastructural evidence that blepharoplast cylinders do develop into templates for basal bodies (analogous to centrosomal centrioles) is available.

Blepharoplasts' presence at mitotic poles was also cited as centrosome homology. Lepper, however, asserted, "The appearance of the blepharoplast at the spindle pole seems to have no more significance than the appearance of any granule at or near this point." But EM later revealed (i) that the blepharoplast is indeed a discrete organelle at which the mitotic polar (not shown) and interphase MTs unambiguously focus, although (ii) around anaphase, the blepharoplast also departs from the poles and its MTOC service. As the earliest microscopists suspected during the blepharoplast's inactive period, it degenerates into a matrix-only-like structure lacking fine features (EM not shown). In sum, when present in its apparently active form, the blepharoplast demands the attention of MTs as an MTOC. What is the broader significance of these observations? Decades ago, Lepper correctly discarded blepharoplast-centrosome homology. However, their homology in terms of being MTOCs (a useful concept broader than the centrosome, perhaps not developed at Lepper's time) is not to be discarded. He and earlier botanists noted broader evolutionary trends. In progressively higher plants, centrosome-like features (discrete focality for MTs in interphase and spindle arrays) are reduced, as is the period during sperm development in which the blepharoplast appears. The blepharoplast's last function to be reduced is as a flagellar template, absent in higher seed plants. In ferns, even though the MT arrays in early sperm development and in non-spermatogenous cells are both typical of higher plants (cortical in interphase, only diffusely polar in mitosis), the blepharoplast appears in the latter stages of sperm development, boldly demanding attention as an exclusive, centrosome-like MTOC.

Currently, the nature of MTOCs in higher plants remains elusive (though recent experimental evidence is affirming observations that some interphase MTOC functionality resides in the nuclear envelope-less tidy than a centrosome-like entity). Today, we are only slightly less troubled than Lepper who asserted:

There has been reported no evidence which might help to explain the loss of centrosomal function, as the development of the pollen tube helps to explain the loss of the blepharoplast and attendant gametic motility in the Coniferae and the Angiospermae...Moreover, nowhere in the plant cell does there appear to be any evidence...related to the probable mode of loss of the centrosomal function.

More informative techniques are slowly elucidating the nature of MT organization in higher plants, and as this frontier of the higher plant cytoskeleton expands, a continued survey of ancestral lands will prove valuable, likely yielding more than old news.