Plant cytoskeleton.pdf

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Plant cytoskeletonClive W Lloyd, John Innes Centre, Colney, Norwich, UK

The major filamentous elements of the plant cytoskeleton are microtubules and actin

microfilaments. although essentially similar to those found in animals, they make different

structures that reflect the special features of plant growth.

The cytoskeleton and the cell wallinteract to regulate plant growth

The behaviour of plant cells during development isdominated by the presence of the cell wall and several of the cytoskeletal arrays that are peculiar to plants arededicated to the layingdown of wall. Animaltissue cells aresurrounded by an extracellular matrix, but this does notstop them migrating. By contrast, the plant cell’s extra-cellular matrix (the cellulose-containing cell wall) istougher and plant cells do not exhibit rapid changes of shape or undergo migratory movements. Instead, theshape of plant tissues depends upon the plane in which thenew crosswall is laid down between dividing cells and uponthe direction in which the cell wall slowly stretches duringinterphase.

Microtubules in nondividing cells

Cellulose microfibrils channel turgor pressure

and regulate the direction of cell expansionMost of the division in plants occurs in apical meristems,such as root and shoot tips. As the cells in the apex expandand divide the older cells cease to divide but continue for awhile to expand. This expansion is not multidirectional, asobserved when blowing up a spherical balloon, but isusually channelled in a particular direction to produceelongated roots or shoots. The driving force for cellexpansion is provided by turgor pressure. This is theosmotic imbalance between intracellular and extracellularfluids that causes the protoplast to swell until the pressureis resisted by the cell wall. When the cell wall isexperimentally stripped away with enzymes, the naked

protoplast emerges as a sphere and swells and shrinks as asphere if placed in solutions of different osmotic potential(Figure 1a). however, when the cell regrows a wall,expansion becomes directional once more, demonstratingthat the directional nature of expansion depends on thewall. The cellulose microfibrils, which are wrapped aroundthe protoplast in layers or lamellae, are strong enough toresist stretching under turgor pressure. In each layer thecellulose microfibrils are roughly parallel but their overallorientation can change from layer to layer. It is believed

that the orientation of microfibrils within the layers nearethe plasma membrane has the greatest effect in determininthe direction in which the cell wall will yield to turgopressure. In actively elongating cells the cellulose microfibrils are usually wound transversely around the cell, likhoops around a barrel, thus preventing increases in girt(Figure 1b). However, neighbouring microfibrils within layer can be separated, and so the turgid cell expand

Article Contents

Introductory article

. The cytoskeleton and the cell wall interact to regulat

plant growth

. Microtubules in nondividing cells

. Microtubules in dividing cells

. Actin

=

(a)

(b)

Figure 1 Thecell wallcontrolsthe directionof expansion.(a) Thewall-le

cell (the protoplast) swells as a sphere and does not elongate. (b) Whencell wall is present, transversely wrapped cellulose microfibrils prevent thprotoplast from expanding sideways. Adjacent turns of cellulose within a

wall layer can, however, be separated, so that the cell expandsperpendicularly to the orientation of the cellulose microfibrils.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net



perpendicular to the direction in which the microfibrils arelaid.

Cytoplasmic microtubules parallel thecellulose microfibrils

Immediately beneath the plasma membrane, attached tothe inner face of the plasma membrane, are hundreds of cortical microtubules that encircle the cell. They aregenerally parallel to one another with overlapping ends.In elongating cells these microtubules are transverse to thecell’s long axis, sharing the same alignment as the cellulosemicrofibrils on the opposite side of the plasma membrane.When microtubules are depolymerized with chemicalssuch as colchicine and oryzalin (the latter, like many plantantimicrotubule drugs, is a herbicide), the organization of the inner wall is eventually lost; the cell loses its ability toundergo directional expansion and swells spherically. Thissuggests that microtubules act as a template for guiding the

direction in which cellulose microfibrils are laid down inthe wall. Both microtubules and cellulose microfibrilstherefore have a role in maintaining cell shape – but howcan they influence each other when they are separated bythe plasma membrane?

Enzyme complexes that synthesize celluloseare embedded in the plasma membrane

In higher plants, cellulose is believed to be polymerizedfrommultienzyme complexes that are embedded withinthelipid bilayer of the plasma membrane in the form of sixparticles constituting a hexagonalrosette. Precursors fed in

from the cytoplasmic side of the complex are polymerizedinto long chains that are spun out onto the extracellularface of the membrane. Each enzyme complex makes manylinear polymers, which associate side-by-side to formcrystalline cellulose microfibrils.

Two ideas have been proposed to account for theparallelism between the cytoplasmic microtubules and thewall microfibrils. Findings from animal biology show thatmembrane-spanning proteins are not necessarily em-bedded statically in the membrane but can float along thefluid sea of membrane lipid, although this mobility can berestricted by the underlying cytoskeleton. One hypothesis,therefore, is that the cellulose-synthesizing particles are

propelled across the surface of the membrane by the act of spinning out a linear crystallite. As they move, they areindirectly guided within membrane channels formedbetween microtubules attached to the inner face of theplasma membrane (Figure2). Another hypothesis is thatthesynthesizing particles are not free but are directlyconnected to the underlying cytoskeleton, which providesmotive force. In principle, microtubule motors such asmembers of the kinesin superfamily could be involved inmoving enzymes (or the microtubules themselves) along

cortical microtubules. Alternatively, fine actin filamentrun alongside the cortical microtubules and these mighdrive particle movement by actin–myosin interaction.

Microtubules orientate the bands of secondary wall thickening in redifferentiatingZinnia elegans mesophyll cells

When mesophyll cells of the ornamental plant Z. eleganare released by grinding up leaves, then grown in culture

they undergo a remarkable transformation over severadays into xylem tracheary cells. In freshly isolated cells thmicrotubules are evenly distributed parallel to the celllong axis, probably in response to wounding. Several daylater, when the cells have recovered, the microtubulereorient so that they are wrapped transversely around thcell. Then the microtubules begin to bunch together, witclear gaps between the bunches, to form spirals ointerconnected transverse hoops. By staining the cellulosin the cell wall with Calcofluor (a fluorescent cellulosebinding dye), thick ribs of cellulose areseen to be depositein patterns identical to the underlying microtubules. Latethe complex polymer, lignin, is deposited in these thicken

ings and the cells now resemble tracheary elements that, ithe plant, normally form the files of cells responsible fotransporting water and dissolved minerals. The patterns osecondary wall thickening therefore mirror the patterns iwhich the microtubules bunched up.

When the drug taxol is added to newly isolatemesophyll cells to stabilize microtubules, the microtubuledo not undergo the reorientation from longitudinal ttransverse; they still bunch up but in the form of abnormalongitudinal bands instead of transverse ones. Lignifie

Cellulose microfibril

Plasma membrane(lipid bilayer)

Microtubule

Microtubule–microtubulecrosslink

Microtubule–plasma

membranecrosslink

Cellulose-synthesizincomplex

Figure 2 Model for the influence of microtubules on the orientation ofcellulose biosynthesis. Cortical microtubules are attached to the plasma

membrane by crosslinkingproteins; they arealso bridged to each other bintermicrotubule linkages. Cellulose is synthesized from multienzymecomplexes that sit in the plasma membrane’s lipid bilayer in the form of

hexagonal rosettes. Cellulose microfibrils are extruded from theserosettethis drives the rosettes along the fluid membrane in lanes provided by thunderlying microtubules. Alternatively, the rosettes could somehow be

linked to the microtubules or coaligned actin filaments – either of which

could provide the motive force for movement.

Plant cytoskeleton

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net



cellulose ribs are then laid down in identical patterns. Suchlongitudinal ribs do not occur in plants. Alternatively, if Z.elegans cells are treated in culture with drugs thatdepolymerize the microtubules, the cells become round;when the drug is washed out the microtubules recover inabnormal, random patterns and bunch up to underlie anet-like pattern of secondarily thickened wall. So in both

cases, experimentally induced changes in the pattern of microtubules cause complementary changes in the patternsin which cell wall products are deposited.

Microtubules can shift their orientation

Microtubules are usually arranged perpendicular to thedirection of expansion in elongating cells, but when cellsstop elongating microtubules are seen with increasingfrequency to become oblique or parallel to the growth axis.Similarly, cellulose microfibrils can be transverse, obliqueor longitudinal and it is now clearthat microtubules form a

dynamic template that can reorientate between thesedifferent configurations.

By microinjecting fluorescently tagged brain microtu-bule protein, tubulin, into plant cells, it is possible to labelliving plant microtubules. This shows that microtubulescan exchange subunits with the soluble cytoplasmic pooland it is likely that they do this by dynamic instability – thephenomenon first observed in animal cells wherebyindividual microtubules randomly switch between phasesof shrinkage and growth. In living pea epidermal cellsinjected with fluorescent tubulin, transverse microtubuleshave been seen to disappear over a twenty-or-so minuteperiod to be replaced by a new, steeply oblique set. The

reverse happens when the stem elongation-promotinghormone, gibberellic acid, induces growth of transversemicrotubules at the expense of preexisting longitudinalmicrotubules. Dynamic instability has not been formallyproved in plants but its space-exploring possibilities of shrinkage and regrowth would explain the observations.

Hormones, light and gravity influencemicrotubule orientation

A large variety of internal and external factors affects thedirection of plant growth. Red light and the hormones

gibberellic acid and auxin are known to stimulate theappearance of transverse microtubules and to encouragecell elongation. Conversely, blue light and the hormonesabscisic acid and ethylene stimulate the appearance of longitudinal microtubules and inhibit cell elongation. Theability of these natural factors to switch microtubulesbetween different alignments provides a likely explanationfor the cyclic reorientation of microfibrils that occurs incells possessing a ‘crossed-polylamellate’ wall structure(alternating transverse and longitudinal lamellae).

When cellulose microfibrils are laid down longitudinally, transverse ‘hoop reinforcement’ is removed and so thcell expands sideways insteadof elongating. This affects thstature of the cell and of the plant organ. For instance, mutation in the biosynthetic pathway of gibberellic acicauses peas to grow as dwarfs with short internodecontaining unelongated cells. Addition of gibberellic aci

to the plants switches microtubules from longitudinal ttransverse and stimulates cell elongation. If differenmicrotubule–microfibril alignments occur on oppositflanks of a stem, then the unequal growth should lead ta bending of the organ. This occurs in phototropism, wherepidermal cells adjacent to the light source have longitudinal microtubules and are inhibited from elongatinwhereas cells with transverse microtubules on the shadeside still elongate, causing bending towards the lighGravity has a similar effect.

Microtubules may be arranged differently in

tip-growing cellsThe arrangement of microtubules has beendiscussed so fain tissue cells undergoing intercalary growth, whicinvolves uniform expansion of the lateral walls. Bcontrast, tip-growing cells are free-growing filaments thaseem only to expand at the tip. Precursors for growth artherefore shuttled, not to the rigid flanks of the tubulaoutgrowth, but to a localized region at the very tip. Tigrowthis seen in filamentousorganisms suchas mosses anfungi; examples from higher plants include pollen tubeand root hairs. In support of tip growth, corticamicrotubules are usually parallel to the direction ogrowth; they run into the tip, probably acting as guidefor the delivery of precursors. Actin filaments are alsaligned parallel to the direction of growth and probablfunction together with microtubules in delivering materiato the tip.

Microtubules in dividing cells

The preprophase band predicts the plane of cell division

One of the major differences between plant and anima

division is that the plane of cell division is predicted somtime before mitosis by cytoskeletal elements. As plantissue cells approach division, the cortical microtubuleswhich were evenly distributed in the G1 phase of the cecycle (Figure 3a), form an ever-narrowing band (Figure 3b

This structure, which forms before the chromatin is fullcondensed into chromosomes, is called the preprophasband (PPB). It predicts the plane in which the cell is abouto divide and does so for symmetrical, asymmetricacurved and straight divisions in all planes. By prophase, th

Plant cytoskeleton




Plant cytoskeleton




band is tightly condensed to form a ring (Figure 3c).It is notknown whether the band forms by a bunching up of thepre-existing interphase array or from a newly polymerizedradial array of microtubules that emerges from the surfaceof the nucleus towards the cortex (Figure 3b). Microtubulesare also found upon the nuclear surface forming a‘prophase spindle’. The preprophase band disappears

between prometaphase and metaphase. Mitosis proceedsmuch as described for animal cells except that the spindlesof land plants are often described as barrel-shaped, withbroad instead of tightly focused spindle poles and withoutastral microtubules (Figures 3d,e). This difference betweenplant and animal spindles requires a short digression onmicrotubule organization in plants.

Principles of microtubule organization inplants

In animal tissue cells, cytoplasmic microtubules radiate

from the centrosome, which contains as its focus a pair of centrioles, surrounded by pericentriolar material. Duringdivision, the centrosome is replicated so that a centriolepair, with pericentriolar material, is found at each pole of the spindle – again providing defined points from whichmicrotubules radiate. In both interphase and mitosis, theslow-growing ‘minus’ ends of microtubules are captured atthe centrosome with the ‘plus’ ends radiating distally, andso the organization of the microtubule arrays is clearlyinfluenced by the presence of fixed microtubule nucleationsites. That the plant mitotic spindle is not so focused at thepoles is usually attributed to the fact that higher land plantsdo not have centrioles and therefore lack a focal point from

which microtubules radiate. Similarly, the interphasemicrotubules of plants do not radiate from a fixed pointand the cortical array is comprised of parallel micro-tubules. Exactly where the cortical microtubules arenucleated is still unclear. g-tubulin, which occurs at the‘minus’ ends of microtubules and is therefore concentratedin microtubule nucleation sites, has been detected botharound the nuclear surface and at the cortex in plants.Plant microtubules may therefore be polymerized at bothlocations, but what organizes the microtubules into arrays?

One possibility is that oligomeric kinesins could, by slidinantiparallel microtubules apart, ‘sort out’ microtubuleinto sets of opposite polarity, as in the spindle and in thphragmoplast(see below). This hypothesis downgrades throle of spindle poles as primary organizers of spindbipolarity. As for the cortical array, although microtubulmotors may eventually be proved to be involved in slidin

neighbouring tubules apart, their regular spacing is likelto be due to structural microtubule-associated proteinthat can be seen between microtubules, like the rungs of ladder.

The cytokinetic apparatus, the phragmoplastdeposits a crosswall between recently dividednuclei

At anaphase, new microtubules appear among the spindlmicrotubules in the form of two interdigitating columnThe structure initially resembles a spindle without poles

suggesting that it is a spindle remnant. There are, howeveinstances (e.g. during cellularization of the multinucleatendosperm tissue) in which phragmoplasts form betweenonsister nuclei, where no such spindle remnants coulexist. It is possible that phragmoplast microtubules areinstead, formed from interdigitating microtubules that arfreshly nucleated from opposing sets of anaphase chromosomes. The microtubules are oppositely directed with fastgrowing ‘plus’ ends meeting at the midline (Figures 3f, 4a

This phragmoplast then starts to widen and becomes morclearly visible as two interdigitating, circular palisades omicrotubules. This circular structure moves outwards ‘lika ripple in a pond’ (Figure 4b). New tubulin subunits ar

probably added to the plus ends of the microtubules ansubunits lost from the ‘minus’ ends by the process otreadmilling. The minimal overlap of the two sets omicrotubules may be maintained by plus end-directekinesin-like proteins that drive the antiparallel microtubules apart. Golgi-derived vesicles track along the opposing microtubules towards the overlapping ‘plus’ endThese vesicles fuse in the midline, depositing a disc of thpolysaccharide, callose, which constitutes the immaturcrosswall or cell plate (Figure 4c). This disc, and th

Figure 3 The microtubule cycle of tobacco BY2 suspension cells. The cells were stained with fluorescent antibodies to the microtubule protein, tubul

(courtesy of Sandra McCutcheon and Grant Calder).(a) The interphasemicrotubule array observedduring the G1 phaseof the cell cycle. Microtubules run transversely aroundthe cell cortex, perpendicul

to the direction of cell elongation.

(b)During G2, a radialarray of microtubulesemerges from thenuclear surface andconnectswith thecortex. Theincreased fluorescenceat theleft-anright-hand edges of the cortex indicate the initial stages in the accumulation of the cortical microtubules into the preprophase band.

(c) The preprophase band of microtubules. The cortical microtubules have disappeared from the ends of the cell and are now all concentrated in thmature PPB. Note that microtubules also cover the nuclear envelope and that radial microtubules connect the nucleus to the PPB.

(d ande) Duringmetaphase (d)and anaphase (e), microtubulesare found only in themitotic spindle. ThePPB hasdisappeared. Note thebroadspindpoles and lack of astral microtubules.

(f) The phragmoplast. This ring-like apparatus is composed of two sets of short, interdigitating microtubules. The dark line along the middle of thisstructureis wherethe microtubulesoverlap. Vesicles meet atthis line andfuseto form thecellplate.The cell plate – the newcrosswall – fills thecentreof th

phragmoplast ring; it grows wider and finally fuses with the mother cell’s sidewall at the site once occupied by the PPB.

Plant cytoskeleton




phragmoplast at its boundary, increases its diameter until

it fuseswith the mother wall, wherethe PPB had been. Thisin-to-out centrifugal movement of the growing cell plate isto be contrasted with the centripetal, purse-string cytokin-esis of animal cells.

Actin

Actin microfilaments in interphase anddivision

During interphase, fine actin filaments lie alongside the

cortical microtubules. Deeper in the cortical cytoplasm liesa less regular network of thicker actin cables and, in largecells, transvacuolar strands of actin traverse the cell. Actinmicrofilaments support the movement of nuclei, chlor-oplasts, endoplasmic reticulum as well as the transport of secretory vesicles in tip-growing cells. It is likely that thesestructures are coated with myosin, which, using the energyof ATP hydrolysis, powers movement along microfila-ments. Strands of actin are sometimes seen to be coalignedbetween adjacent cells and it is possible that actin actually

passes from cell to cell through plasmodesmatal poresThere is evidence that viral movement proteins exploit throute for the intercellular transport of viruses.

In vacuolated cells preparing to divide, the nucleus cabe clearly seen to move into a central position supported btransvacuolar strands of cytoplasm containing actifilaments. These strands will also contain the newl

polymerized microtubules that radiate from the surfacof the premitotic nucleus (Figure3b). Gradually, these actiand microtubule-containing strands reorganize from three-dimensional star-like array into a more-or-lescontinuous two-dimensional sheet across the vacuole. Thstructure is called the phragmosome (not to be confusewith the phragmoplast). The phragmosome is completbefore prophase and – since nuclear division and cytokinesis will occur within it – it therefore predicts the plane odivision. The preprophase band of microtubules, whicalso predicts the plane of cell division, marks the corticaboundary of the phragmosome. Originally, the PPB wafound to consist of microtubules, but it is now known t

contain actin filaments as well. The plane of cell division itherefore predicted by the phragmosomal actin anmicrotubules that, like spokes in a bicycle wheel, connecthe cortical ring of actin and microtubules (the PPB) to thnucleus at the hub. It is not yet known what powers thidramatic reorganization of the cytoskeleton but the actifilaments among the PPB microtubules are thought tassist in the tightening of the band. This premitotic disc, thphragmosome, is seen to support the dividing nucleus ivacuolated cells but equivalent structures may also exist ithe more densely cytoplasmic meristematic cells. Aprometaphase cytoplasmic streaming stops and the actifilaments in the PPB depolymerize, along with th

cytoplasmic microtubules. However, actin filaments remain elsewhere in the cell, bridging the nucleus to thcortex. Actin filaments have also been reported among thspindle microtubules, although their function is uncleaDuring cytokinesis, actin filaments are found among thphragmoplast microtubules, where they may also binvolved in moving vesicles to the cell plate (Figure 4c

Actin filaments therefore exist among microtubule-containing structures throughout the cell cycle.

Cell division in plantsand animals is compared in Table

The actin-binding protein, profilin, is a majoallergen

Pollen of the white birch can cause hayfever. to isolate thcausative agent, scientists screened a birch cDNA expression library with antibodies from the serum of hayfevesufferers. This revealed that the allergen was a small (1215 kDa) actin-binding protein, profilin. When microin jected into stamen hair cells, profilin caused the depolymerization of the transvacuolar strands and thdisplacement of the central nucleus, illustrating that F

(a)

(b)

(c)

Nucleus

Phragmoplastmicrotubules

Cell plate

–

+ +

Cell plate

Golgi vesicle

Actin filament

Microtubule

–

Figure 4 The phragmoplast. (a) The phragmoplast is a double circle of short microtubules whose circumference increases as it deposits a centraldisc (the cell plate), which separates the newly divided sister nuclei.

(b)Seen fromabove, the circular phragmoplast growsoutwardsuntil thecell plate fuses with the sidewalls of the mother cell.

(c) Phragmoplast microtubules interdigitate with their fast-growing

(‘plus’) ends at the midline. Actin filaments run parallel with themicrotubules. Vesicles derived from the Golgi apparatus move along these

cytoskeletal elements and fuse at the midline to form the cell plate.

Plant cytoskeleton




actin structurally supports cytoplasmic strands andregulates the position of the nucleus. It is believed that bysequestering monomeric G-actin, profilin changes theequilibrium with filamentous F-actin, causing the latterto dissociate. However, it is thought that when the growing

ends of actin filaments are uncapped profilin can have thequite different effect of stimulating addition of G-actin.

Further Reading

Cyr RJ (1994) Microtubules in plant morphogenesis: the role of the

cortical array. Annual Review of Cell Biology 10: 153–180.

Lambert A-Mand LloydCW (1994)The higherplantmicrotubule cyc

In: Hyams JS and Lloyd CW (eds) Microtubules, pp. 325–341. Ne

York: Wiley-Liss.

LloydCW (ed.) (1991)TheCytoskeletal Basis of Plant Growthand Form

London: Academic Press.

Lloyd CW (1994) Why should stationary plant cells have such dynam

microtubules? Molecular Biology of the Cell 5: 1277–1280.

Shibaoka H (1994)Planthormone-induced changes in theorientation

cortical microtubules: alterations in the cross-linking between micr

tubules andthe plasmamembrane.Annual Review of Physiology Pla

Molecular Biology 45: 527–544.

Staiger CJ, Gibbon BC, Kovar DR and Zonia LE (1997) Profilin an

actin-depolymerizing factor: modulators of actin organization

plants. Trends in Plant Science 2: 275–282.

Table 1 Division in plants and animals compared

Animals Plants

Cytoplasmic actin depolymerizes in mitosis Some cytoplasmic actin persists during division although

cytoplasmic streaming stops

Cells become round during division The cell wall retains cell shape during division

Division plane not predicted before mitosis Preprophase band and phragmosome predict the division

plane in late G2

Astral microtubules involved in spindle alignment No centrioles, no astral microtubules

Contractile ring pulls inwards Phragmoplast grows outwards

Contractile ring based on actin – no microtubules Phragmoplast contains microtubules aswell asactin filament

No crosswall; cell pinched in two Crosswall built by accretion of Golgi vesicles

Plant cytoskeleton


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