Life without cellulose: Tobacco cell responses to short-term and long-term inhibition of cellulose synthesis

(The following appeared in poster form at a U. Missouri 1997 meeting.)

ABSTRACT (mostly redundant with BODY)
Plant cells establish new cross walls between daughter cells by cytokinesis. Requisite polysaccharides are delivered by golgi-derived vesicles directed, in large part, by the microtubules of the phragmoplast to the developing cell plate between daughter nuclei. Callose deposition appears to have an integral role in the expansion and maturation of the plate, prior to the appearance of cellulose in the mature cross wall. This presents investigators with a process of de novo cellulose synthesis. Our laboratory has reported the effects of hours-long treatments of onion seedlings with the cellulose synthesis inhibitor dichlobenil (DCB). The present report is of a similar treatment on BY2 cells, which are used broadly as a model system. In addition, Delmer and co-workers have reported that other cells of tobacco adapt to long term culture on DCB, so we also report the effects that months of growth in DCB-containing media have on BY2 cells. Untreated suspension cultures of BY-2 cells display finely dispersed, microscopic "colonies" of fewer than a dozen or so cells. A three hour DCB treatment has no gross effects on either the microtubule arrays or growth habit. Our immunofluorescence methods indicate no apparent effect on callose distribution, even in developing cell plates. However, immunogold and transmission electron microscopy (EM) reveal the same effects observed in onion cell plates (20X callose enrichment, distortion, and persistent immaturity). Cell plate pectin is slightly elevated, and cellulase-gold EM also indicates the absence of cellulose in cell plates. The predominant growth habit of cells cultured for months on DCB is radically altered to macroscopic, globular aggregates of dozens of packed cells. Our immunofluorescence methods reveal no notable differences in callose distribution nor in microtubule arrays, versus controls. Moreover, remarkably, EM reveals that cell plate morphology and its callose and pectin distribution are generally similar to controls. However, cellulose, even in mature, parental walls, appeared to be reduced 50X versus untreated cells, and pectin content increased 37X in the parental walls. These walls are distinctly lamellate, with 8-10 layers of pectinaceous material in each wall. These results support prevailing hypotheses and indicate that short term inhibition of cellulose synthesis may involve diversion of precursors, but the long-term adaptation involves synthesis of cell walls by a notably altered pathway in which pectin depositions substitute for cellulose.

LIFE WITHOUT CELLULOSE:
Tobacco cell responses to short-term and long-term inhibition of cellulose synthesis
Neil A. Durso and Kevin C. Vaughn, USDA/ARS/SWSL, PO Box 350, Stoneville MS 38776

BODY
Our laboratory has reported that DCB treatments < 24h disrupt cell plate development in onion root tips, resulting in waviform, callose-enriched, cell plates. Such plates represent an exaggeration of the fenestrated plate stage of the model described by Samuels et al. using cryo-EM and suspension-cultured tobacco BY-2 cells (P.1). We report that short-term DCB-treatments of BY-2 cells also result in cell plate development similar to that described for onion roots. According to our onion results and the model of Samuels et al., callose deposition is initiated in the middle stages of cell plate development, perhaps providing the motive force for plate spreading. Indeed, in early stages in BY-2 cells, a DCB effect is not observed (P.2). However, as in onion, DCB effects are manifest in latter, fenestrated plate stages (P.3). Cellulose is decreased to < 10% of controls (P.4, left; Table 1A), and callose deposition is increased >10X (P.5, Table 1A directly below).

(BODY cont. below)

Table 1: Quantification of immuno-/enzymo-gold in situ EM sections of the indicated specimens were probed with the indicated gold conjugate. Ratio = [ # gold particles / µm2 cell wall or plate in treated specimen ] $fraction divisor$ [# gold particles / µm2 cell wall or plate in untreated controls]
A) 3h, 10µM DCB developing cell plate		B) >6mo., 1µM DCB parental cell wall
anti-callose	11.2	anti-callose	0.7
anti-xyloglucan	6.9	anti-xyloglucan	1.1
anti-PGA*	2.4	anti-PGA	37.2
cellulase-gold	0.06	JIM7, esterified PGA	3.4
* PGA: polygalacturonic acid (pectin backbone)		JIM5, less esterified PGA	5.1
		cellulase-gold	0.02
		anti-extensin	32.0

Delmer and co-workers have reported on the adaptation of (non-BY-2) tobacco cultures to DCB over long terms. No evidence of callose enrichment was reported. However, pectin appeared to replace normal cellulose-xyloglucan. We have similarly produced DCB-habituated BY-2 cells by months-long suspension culture in media containing 1.0uM DCB.
Short-term (3h, 10uM) DCB treatments produce no macroscopic alterations in the macroscopic growth habit of BY-2 suspension cultures. But within 7 days on 1.0 uM DCB, the finely dispersed, typical growth habit of BY-2 cells is converted to globular aggregates (P.6). Our non-embedding immunofluorescence microscopy methods reveal no aberrations in callose distribution or in microtubule arrays in either short or long-term treatments (P.7). In all cases, callose appears similarly: in developing cell plates (a, b); as fibrils (and some small, random deposits) on parental walls (b, c, e, f2); and in cross walls (all of P.7).
For DCB-adapted cultures, Delmer and co-workers reported a 5X increase of total pectin using classical chemical analyses on extracts of isolated walls, and a 2X increase in relatively unesterified pectin using immunogold on wall-enriched fractions. No changes in callose content were detected, and our in situ analyses on DCB-habituated BY-2 cells are corroborative (Table 1B), in marked contrast to short-term DCB treatments (Table 1A & P.5). Our in situ analyses also indicate a much greater increase in total pectin (P.8, Table 1B), as well as an increase in both relatively unesterified and esterified pectin (P.9, Table 1B). Unlike the ultrastructure of parental cell walls in untreated BY-2 cells, those of DCB-habituated cells have a distinctly, finely fibrous network of electron opaque strands with which anti-pectin associates primarily (P8&9, habituated). Finally, enzymo-gold indicates cellulose levels at only 2% of controls (Table 1B), localized rarely but consistently along the plasma membrane (P.4, right).
These results indicate that, in the short term, DCB inhibits cellulose biosynthesis, diverting the UDP-glucose substrate into the callose (and xyloglucan) pathway(s). On the other hand, long-term DCB-habituation appears to modify cell wall synthesizing mechanisms such that a more extensive pectin network viably substitutes for the normal cellulose-xyloglucan network (P.10).