Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

A rapid fixation/dehydration method of plant specimens for scanning electron microscopy is presented. Prior to critical‐point drying (CPD) the specimens are immersed in pure methanol. Methanol incubation instantly fixes the elastically extended cell walls. Owing to this instant fixation, shrinking of the specimens is prevented, resulting in an improved preservation of cell dimensions comparable to in vivo conditions. The method is most suitable for plant epidermal surfaces. It avoids the time‐consuming fixation/dehydration in routine investigation of plant surfaces prior to CPD, especially for delicate specimens.

The kinetics of inhibition by protein- and RNA-synthesis inhibitors (cycloheximide and cordycepin, respectively) of indole-3-acetic acid (IAA)-induced elongation growth were investigated using abraded coleoptile segments of Zea mays L. Removal of the cuticle - a diffusion barrier for solutes - by mechanical abrasion of the outer epidermal cell wall increased the effectiveness of inhibitors tremendously. In an attempt to elucidate the role of 'growth-limiting protein(s)' (GLP) in the growth mechanism the following results were obtained. The elongation induced by IAA was completely inhibited when cycloheximide (10 μmol·l(-1)) was applied to abraded coleoptile segments as shortly as 10 min before the onset of the growth response (=5 min after administration of IAA). However, when cycloheximide was applied after 60 min of IAA treatment (when a steady-state growth rate is reached), the time required for complete cessation of growth was much longer (about 40 min). Cycloheximide inhibited the incorporation of [(3)H]leucine into protein within about 5 min. Cordycepin (400 μmol·l(-1)) prevented IAA-induced growth when applied as shortly as 25 min before the onset of the growth response (=10 min before administration of IAA) but required more than 60 min for a full inhibition of steady-state growth. The incorporation of [(3)H]adenosine into RNA was inhibited by cordycepin within 10 min. It is concluded that, contrary to previous investigations with nonabraded organ segments, the initiation of growth by IAA depends directly on the synthesis of GLP. Moreover, the apparent lifetime of GLP is at least four times longer than the time required by cycloheximide to inhibit the initiation of growth by IAA. This is interpreted to mean that GLP is not present before IAA starts to act but is synthesized as a consequence of IAA action starting a few minutes before the initiation of growth. Interpreting the kinetics of growth inhibition by cordycepin in a similar way, we further conclude that GLP synthesis is mediated by IAA-induced synthesis of the corresponding mRNA which starts about 10 min before the onset of GLP synthesis. Inhibition by cycloheximide and cordycepin of IAA-induced growth cannot be alleviated by acidifying the cell wall to pH 4-5, indicating that these inhibitors do not act on growth via an inhibition of auxin-mediated proton excretion.

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(2-4, 11, 14, 25). Xyloglucan is thought to serve a structural role within the wall, tethering adjacent microfibrils (12, 15). Modification of xyloglucan by hydrolysis (12,16,18,21) or endotransglycosylation (13, 28) may alter the rheological properties of the wall and thus influence the cell's ability to grow. Considerable interest, therefore, centers on the architectural arrangement of xyloglucan within the wall.Xyloglucan is able to hydrogen bond to cellulose in vitro (1,2,17,30); its binding in the cell wall has been assumed to be due largely to hydrogen bonding to the surfaces of newly formed ('naked') cellulosic microfibrils (2,11,14,17,23 3 Abbreviations: DCB, 2,6-dichlorobenzonitrile; GTC, guanidinium thiocyanate; XG2, a-D-xylopyranosyl-(1-*6)-D-glucose (= isoprimeverose).in the medium instead of binding within the almost cellulosefree wall (27). However, in normal cell walls, the xyloglucan:cellulose ratio often greatly exceeds that which can be achieved during in vitro binding experiments (17). This suggests that hydrogen bonding to the surfaces of microfibrils may only partially account for the immobilization of xyloglucan within the accreting wall.Little information is available concerning the formation of a xyloglucan/cellulose complex under physiological conditions. To study the proposed role of newly formed segments of microfibril in the binding of xyloglucan in vivo, we specifically inhibited cellulose synthesis by use of DCB and analyzed the effect of this inhibition on the wall binding of newly synthesized xyloglucan. We also report the effect of the inhibition on cell expansion. MATERIALS AND METHODS Plant Material, Polishing, and Incubation ConditionsPea seeds (Pisum sativum var Alaska) were soaked in running tap water for 10 h and then were grown in vermiculite in the dark at 25 ± 40C. After 7 to 8 d, the seedlings were brought into normal laboratory lighting and the 1.5-to 2.5-cm long third intemodes were 'polished' by gentle pulling (three times) through a loop of Whatman No. 3MM chromatography paper, held between thumb and forefinger, to remove some of the wax and thus enhance uptake of tracers (9). Segments (9 mm) were cut from the polished internodes and shaken gently for 1 h in distilled water at 250C (<150 segments in 200 mL of water).Ten segments were then removed and gently shaken in a 22-mL plastic vial containing 2 or 4 mL of 38 mg/L nystatin and 0.02% penicillin in distilled water. Where appropriate, DCB (Aldrich) was added as a 100-mM solution in DMSO to a final concentration of 100 ,M.

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The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a 'growth-limiting protein' induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.