Alteration Physical Chemical Structure the Cell Wall of ...Plant Physiol. Vol. 91,1989 water...

Plant Physiol. (1989) 91, 39-470032-0889/89/91/0039/09/$01 .00/0

Received for publication June 13, 1988and in revised form March 3, 1989

Alteration of the Physical and Chemical Structure of thePrimary Cell Wall of Growth-Limited Plant Cells Adapted to

Osmotic Stress1

Naim M. Iraki2, Ray A. Bressan, P. M. Hasegawa, and Nicholas C. Carpita*

Department of Botany and Plant Pathology (N.M.I., N.C.C.) and Department of Horticulture (R.A.B., P.M.H.),Purdue University, West Lafayette, Indiana 47907

ABSTRACT

Cells of tobacco (Nicotiana tabacum L.) adapted to grow insevere osmotic stress of 428 millimolar NaCI (-23 bar) or 30%polyethylene glycol 8000 (-28 bar) exhibit a drastically alteredgrowth physiology that results in slower cell expansion and fullyexpanded cells with volumes only one-fifth to one-eighth thoseof unadapted cells. This reduced cell volume occurs despitemaintenance of turgor pressures sometimes severalfold higherthan those of unadapted cells. This report and others (NM Irakiet al [1989] Plant Physiol 90: 000-000 and 000-000) documentphysical and biochemical alterations of the cell walls which mightexplain how adapted cells decrease the ability of the wall toexpand despite diversion of carbon used for osmotic adjustmentaway from synthesis of cell wall polysaccharides. Tensilestrength measured by a gas decompression technique showedempirically that walls of NaCI-adapted cells are much weakerthan those of unadapted cells. Correlated with this weakeningwas a substantial decrease in the proportion of crystalline cellu-lose in the primary cell wall. Even though the amount of insolubleprotein associated with the wall was increased relative to otherwall components, the amount of hydroxyproline in the insolubleprotein of the wall was only about 10% that of unadapted cells.These results indicate that a cellulosic-extensin framework is aprimary determinant of absolute wall tensile strength, but com-plete formation of this framework apparently is sacrificed to divertcarbon to substances needed for osmotic adjustment. We pro-pose that the absolute mass of this framework is not a principaldeterminant of the ability of the cell wall to extend.

Growth and differentiation of higher plants under condi-tions of severe osmotic stress are modified at two levels ofcomplexity. Cells must use significant amounts of carbon toadjust osmotically but still obtain and use sufficient additionalcarbon to divide, grow, and build, among other things, func-tional cell walls that are compatible with the altered metabo-lism required for survival. Under conditions of saline stress,each cell must expend additional energy to either regulate salt

' Supported by grant US-535-82 from the United States-IsraelBinational Research and Development Fund (BARD) and a fellow-ship from America-Mideast Educational and Training Foundation toN.M.I. Journal paper No. 11,733 of the Purdue University Agricul-tural Research Station.

2 Present address: Department of Biology, Bethlehem University,Bethlehem, Israel.

uptake and accumulate cellular osmotica or include andpartition the salt primarily in the vacuolar compartment. Atthe second level of complexity, the multicellular organismundergoes a severely altered morphogenesis, proceeding be-yond osmotic adjustment, to produce a plant capable ofsurvival under continued stress conditions. While the latterlevel is difficult to examine experimentally, the basic cellularphysiology associated with the adaptation to osmotic stresscan be readily investigated with cell populations in culture.Cells of tobacco (Nicotiana tabacum L., cv W38) in liquidculture adjust osmotically to severe saline stress by absorbingconsiderable amounts of NaCl from the medium and parti-tioning much of it in the vacuole (3-5, 9, 10). These cellsaccumulate sugars and amino acids as additional osmoticawhich presumably osmotically balance the cytoplasm withthe vacuole. Similar cell lines of tobacco and tomato cellshave adapted to impermeant PEG,3 but, unlike cells adaptedto NaCl, cannot use the osmotic agent for adjustment. Cellsadapted to PEG-induced water stress also accumulate sub-stantial amounts of sugars and amino acids (8, 18, 19), butdo this at the expense of the synthesis of cell wall polysaccha-rides ( 18).The growth physiology is also altered considerably during

adaptation to either saline or PEG stress. Cell volume isreduced to as little as one-fifth to one-eighth that ofunadaptedcells (3-5, 10). This reduced volume occurs despite mainte-nance of more than adequate turgor needed to drive cellexpansion under normal conditions. Indeed, turgor pressureshave been found to be severalfold higher than those of una-dapted cells (3-5, 9, 10, 18). Hence, we have indicated that areduction of wall extensibility likely is responsible for thesmall cells (3-5, 9, 10, 18). This is a surprising observationconsidering the reduced proportion ofcarbon utilized for wallsynthesis. The slow cell expansion character of the salt-adapted cells is genetically stable, and tobacco plants regen-erated from these cells, even in the absence of stress, havereduced internode elongation and smaller and thicker leaveswhich expand at slower rates (9, 10). Boyer and coworkers (7)have presented substantial data to support the hypothesis thatlow conductivity of water in growing tissues results in a waterpotential gradient between the expanding cells and watersource. Decreasing the supply of water to the growing regionsof plant tissues could then limit growth by decreasing the

3PEG 8000.39

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Plant Physiol. Vol. 91,1989

water potential gradient even in the presence of adequateturgor pressures. In suspension-cultured cells it is difficult toimagine sufficient restriction to the flow of water to establisha water potential gradient which could limit cell enlargementrate in the presence of high turgor. Therefore, we have pro-moted the concept that reduced ability for the wall to expandlimits growth in these cells (3, 8-10, 18). This is at least partlytrue in growing cells of whole plant tissues even consideringBoyer's elegant data and well-presented arguments for restric-tion of water flow (7). In fact, recent work by Boyer andcoworkers (6) has focused on the possibility that cell wallmetabolism is involved in growth reduction induced by os-motic stress.The present study was initiated to investigate common

physical and chemical modifications in wall structure of cellsadapted to NaCl or PEG stress which could explain theiraltered growth physiology. We investigated changes in thephysical ability ofthe walls to withstand turgor pressures uponadaptation to stress and then examined the chemical changesassociated with this change in physical structure. We haveestablished that ability of the cell wall to expand is governedby chemical determinants unrelated directly to the actualtensile strength of the wall. While this and the followingreports provide basic information ofhow cell wall metabolismis altered upon adaptation to osmotic stress, our results alsoprovide important insight into the biochemical determinantslikely responsible for cell expansion in dicots.

MATERIALS AND METHODS

Cell Cultures

Cells of tobacco (Nicotiana tabacum L. cv W38) weremaintained in liquid culture in medium of pH 5 containingMurashige and Skoog salts (24) (prepared commercially byGibco) and (per liter): 30 g sucrose, 1.0 g casein hydrolysate(Sigma; enzymic), 100 mg inositol, 3 mg 2,4-D, 0.1 mgkinetin, and 0.4 mg thiamine-HCl. Cells were subculturedbiweekly at 0.8 to 2.0 g fresh weight per 100 mL of freshmedium in 500-mL Erlenmeyer flasks. Cultures were incu-bated at 26°C on a gyratory shaker at 110 rpm with a 2.5-cmdisplacement. Cells were adapted to grow in medium supple-mented with either 30% PEG (-28 bar) or various concentra-tions of NaCl as was described for tomato and tobacco (3, 8)and have been maintained for several years prior to use inexperiments described here.

Determination of Cell Volumes during Growth ofSuspension-Cultured Cells

Cell volumes were estimated by determining the freshweight minus dry weight of culture samples after collectingcells by vacuum filtration and dividing by the total numberof cells in samples of equivalent weight (3). Cell number wasdetermined by counting cells with a hemacytometer after cellclumps were separated by treatment with 15% chromic acidat 65°C for 30 min. This method results in an overestimateof cell volume because there is no correction for extracellularvolume including cell free space. However, with proper filtra-tion of the cells we have found that extracellular volumes are

quite constant from sample to sample. Values also correlatedwell with empirical measurements of cell length and widthand calculation ofvolume using simple equations for sphericalor ellipsoid solids (4, 5). Some differences in extracellularvolume between adapted and unadapted cells would be ex-pected because of cell size differences. However, this effectalso should be relatively constant as cell samples are takenover time.

Measurements of Tensile Strength

General theoretical and practical aspects of these determi-nations were reported earlier (1 1). Only unadapted and NaCl-adapted cells were compared in these experiments because oftechnical problems encountered with cells in the viscous PEGmedium. Cells in logarithmic phase of the culture cycle weretransferred to fresh medium, and 20 mL ofthe cell suspensionwere placed in a small beaker with gentle stirring in a nitrogengas decompression bomb (Parr Instruments). Nitrogen gaswas introduced slowly (to avoid heating) to the desired pres-sure. After equilibration for 15 min, the suspension wasjettisoned into a large cylinder. An aliquot was mixed withone-half volume of 20% chromic acid in a 3-mL Reacti-vial(Pierce) and stirred gently for 1 h. The number of cells thatsurvived decompression was visualized by bright-field micros-copy and counted with a hemacytometer. Samples of thesame fresh cell suspension were also taken for determinationof incipient plasmolysis in a graded series of NaCl solutions,and turgor pressure was calculated as the difference betweenwater potential of the growth medium (determined by vaporpressure osmometry) and the osmotic potential causing incip-ient plasmolysis of 50% of the cell population. We found thismethod to be the most reliable method for determination ofosmotic potential. Measurements were made in less than 30min. The amount of NaCl uptake during this time wasnegligible, and at least 1 h further incubation was requiredbefore there was noticeable deplasmolysis resulting from up-take of the exogenous NaCl. As with any method for deter-mination of osmotic potential, there are potential problemswhich could affect the accuracy ofthese measurements. Thesepotential problems have been addressed at length (3-5, 8, 18,19), and alternative procedures resulted in comparable values.Cells at this phase ofculture growth were spherical to ellipsoid,and the cell radii were estimated from cell dimensions(4, 5). Wall thickness was about 0.1 zIm for both unadaptedand adapted cells based on calculations from electronmicrographs.

Isolation of Cell Wall Material for Carbohydrate Analysis

Cells in logarithmic and stationary growth phase were fil-tered on coarse sintered-glass funnels, rinsed briefly in iso-osmolar mannitol, and frozen in liquid N2. Acetone powderswere prepared of the total material to precipitate solublepolysaccharides as well as insoluble components of the wall.This treatment, of course, resulted also in precipitation ofsubstantial amounts of soluble protein, but the presence ofthis protein did not interfere with carbohydrate analyses.Samples of dry powders (1.5 g) were extracted sequentially asfollows: once with 125 mL of ice cold 5 mM EDTA for 12 h

40 IRAKI ET AL.



CELL WALLS AND OSMOTIC STRESS

with constant stirring; twice with 125 mL of0.5% ammoniumoxalate (pH 6.5) at 100°C for 1 h each; once with 50 mL of0.1 M KOH for 4 h; and twice with 4 M KOH overnight each.The EDTA and ammonium oxalate solutions each extractedpectic substances by chelation of Ca2+ crosslinking the galac-tosyluronic acid units, and the 0.1 M KOH removed additionalpectic substances, perhaps by hydrolysis of ester linkages orother weak alkali labile bonds (15, 21). The 4 M KOH ex-tracted hemicelluloses through disruption of hydrogen bond-ing or breakage of yet unidentified covalent linkages (2). TheKOH solutions contained NaBH4 (3 mg/mL) to prevent endpeeling (1), and the extraction was carried out under N2 withcontinuous stirring. The KOH extracts were neutralized withglacial acetic acid. All fractions were filtered through What-man GF/F glass fiber filters, dialyzed against running deion-ized water overnight at room temperature, and lyophilized.The remaining material containing hemicellulose and cellu-lose was washed with deionized water and lyophilized. Cel-lulose content in samples of the material was determinedaccording to Updegraff (33). All results expressed representthe average values from usually three, but sometimes two,different experimental samples. Variance was always less than+ 5%.

Preparation of Cell Walls for Amino Acid Analysis

NaCl-adapted and unadapted cells at various times duringculture were collected by filtration through a nylon mesh filterand homogenized with a Tekmar tissuemizer in 6 volumes ofeither 50 mm potassium phosphate (pH 7.0) or water. Thedebris was collected again by filtration and washed sequen-tially with at least three volumes of the following: chloroformmethanol (1:1 v/v) at 80C for 3 h, water, 1% SDS at 100°Cfor 3 h, water, methanol, and acetone. The acetone-washedwall material was then dried and stored at room temperature.

Amino Acid Analysis and Determination of ProteinContent of Washed Cell Walls

Washed cell walls were hydrolyzed in 6 M HCI at 120°C for24 h. The hydrolyzed wall material was filtered over glassfiber to remove charred debris and dried under an air stream.This material was dissolved in water and partially purified byDowex 50 cation exchange chromatography (27). The aminoacids were separated, derivatized, and quantified by capillarygas chromatography as described by Rhodes et al. (27). Totalprotein content of the walls was calculated by summation ofthe amounts of each of the amino acids measured.

RESULTS

Cell Enlargement of Cultured Cells Adapted to HighLevels of NaCI

Tobacco cells adapted to either 428 mM NaCl or 30% PEGhad a greatly reduced ability to gain fresh weight, althoughtheir rates of dry weight accumulation were very similar tounadapted cells (Fig. 1). Adapted cells have measurablysmaller dimensions (Table I) and a reduced rate of cell en-

largement (Fig. 2). Maximum cell volume of cells in 171 mM

Culture Age, daysFigure 1. Accumulation of dry weight (A), fresh weight (B), and freshweight/dry weight ratio (C) during the cell culture cycle of unadaptedcells (0), cells adapted to 428 mM NaCI (@), and cells adapted to30% PEG (A).

NaCl is less than one-half that of unadapted cells, and cellvolume is lowered further in cells adapted to higher concen-

trations of NaCl (Fig. 3).

Tensile Strength

Pressures required to break 50% of the population of cellswere measured empirically (Table I). However, tensilestrength is the ability to withstand the tangential force percross-sectional area required for breakage (26). For steel rods,plant fibers, and cylinders cut from wood, N m2 are typicalunits of tensile strength and reflect the external force exertedin a single dimension required to break the material. Our

a)

L-

L I

-..

0

-C

('D

D

LL

41




Table I. Calculation of the Tensile Strength of Unadapted and NaCI-Adapted Tobacco Cells

Turgor Breaking Cell Sizec Wall TensileCell Line Pressurea Pressureb Length Width Thicknessd Strengthe

bar bar AM Am bar

SO 4 20 179 71 0.1 4,260-10,740S25 14 3 54 46 0.1 1,960-2,300

a Calculated from incipient plasmolysis of 50% of the cells in a graded series of NaCI and the waterpotential of the incubation medium. b Pressure in excess of turgor pressure required to burst 50%of the population of cells by nitrogen gas decompression. Values represent means of at least threeindependent experiments. c Length and width of ellipsoid and spherical cells were measured empir-ically in a population of cells at mid-logarithmic stage of growth. Values represent the mean of at least36 samples. d Thickness was estimated empirically from electron micrographs. e Tensile strengthwas estimated using the equation (P.r)/2t, which reflects the difference in area of the cell and cell wallupon which the force is applied, P is the sum of turgor and breaking pressure, r is the radius estimatedfrom length and width, and t is wall thickness. The range reflects the difference in length and width.

0co

C)x

E

z

0

0

LL

I-0

x

E

0

0

0 0o 20 30

Culture Age, days

Figure 2. Changes in cell volume during growth of unadapted cells(0) and cells adapted to 428 mm NaCI (0). Volume was estimatedfrom determination of fresh weight minus dry weight of an aliquot ofthe cell culture and calculations of cell number in that aliquot. Enoughcells were counted in each aliquot to provide ± 2% error.

measurements are based on the internal pressures required toburst the cell wall. Values obtained from stretching large cellsor pieces of tissue are difficult to compare to those estimatedfor living cells, especially considering that the cell wall is nota homogeneous material of defined thickness, but a polyla-mellate matrix of cellulose microfibrils cross-linked with non-

cellulosic polysaccharides and protein. We define tensilestrength as the ability to withstand the tangential force perunit wall thickness resulting from the cell's internal pressure,and this force can only be estimated from breaking pressure,cell diameter, and wall thickness (11). Turgor pressures ex-

erted by the cells correspond to enormous tensions becausetangential stresses imposed by the pressure of large cells are

borne by the extremely thin cell walls. Breaking pressures are20 bar for walls of unadapted cells but only 3 bar for walls ofNaCl-adapted cells (Table I). Accounting for the contributionof turgor pressure, 24 bar is required to break the walls of

Level of Adaptation, mM NaCIFigure 3. Cell volume of cells adapted to increasing levels of NaCI.Cell volumes were determined as in Figure 1 with cells adapted(grown for more than 200 generations) to the levels of NaCI indicated.Values are maximum cell volumes of cell populations at stationarygrowth phase.

unadapted cells and 17 bar is required for NaCl-adapted cells,(Table I). Wall thickness is about 0.1 tm, but even thoughtotal pressures contributing to the actual breaking pressureare only about 40% higher in unadapted cells, the estimatedtensile strengths are 2- to 5-fold lower in NaCl-adapted cellsbecause of their smaller size (Table I). For comparison, me-chanical properties of differentiated cells in tissues must com-prise a broader range of determinants than strictly those ofthe cell walls of isolated, spherical cells. For example, thetensile properties are related also to the orientation and or-ganization of the cells in the tissue, the cementing of adjacentcells by the middle lamellae, and the orientation of the cellu-lose microfibrils in the individual cells (26). For tissues, tensilestrengths range from 800 to 5,000 bar for sisal fibers depend-ing on relative humidity (29) and 1300 to 3300 bar for Pinusradiata late wood depending on the orientation of the fibercells in the wood to the stress axis (35). Despite such markeddifferences in the determinants of strength, these values aresurprisingly comparable to those we estimate for the primarywalls of the tobacco cells (Table I).

IRAKI ET AL.42




The apparent contradiction that adapted cells have muchhigher turgor pressures and decreased ability to expand yetweaker cell walls is explained readily in that breaking pressuresof the wall are about an order of magnitude higher thannormal turgor. Tensile strength was lower in adapted cells,but elevated turgor pressures exhibited by these cells rose tovalues that only approach these breaking pressures (Table I).These data well illustrate that the chemical determinants ofwall expansion and wall tensile strength are different. TheNaCl-adapted cells have greatly reduced ability to expand andhave higher turgor pressures even though tensile strength issubstantially lower (Table I). We initiated studies of thechemical composition of the cell walls of adapted and una-dapted cells to reconcile these marked differences.

Cell Wall Polysaccharide Structure

The sequential extraction of pectic substances from theacetone powders yielded insoluble material corresponding to70, 31, and 42% of the total dry weight of the unadapted,PEG-, and NaCl-adapted cells, respectively (Fig. 4). The lowrecovery in both lines of adapted cells is a result of loss of lowmolecular weight material, principally reducing sugars andamino acids precipitated by the acetone, during dialysis ofthefirst extract. The lower proportion of the total amount of wallrecovered from both NaCl- and PEG-adapted cells in theacetone powders reflects this enhanced accumulation of sol-utes at the expense of wall synthesis (Fig. 4).The NaCl- and PEG-adapted cells have only about half the

amount of cell wall polysaccharides per g total dry wt asunadapted cells (Fig. 4). Not only is the total amount of cellwall greatly reduced upon adaptation to saline or water stress,but there are also differences between adapted and unadaptedcells in the chemical composition of the wall as well. Cellwalls of adapted cells have much lower proportions of cellu-lose and, consequently, higher proportions of hemicellulose

0.1 M KO"-sol

SO: (I)i nso iu k. ( kumon. Ox4sEDTA-sal

0.125ImOH-d Ceil

EDTA-s

P30: {

and protein than those of unadapted cells (Fig. 4). The pro-portions of total pectin in walls of adapted and unadaptedcells are about the same, but the organization and composi-tion of this material differs markedly. Only a small portion ofpectin from unadapted cells is extracted by cold EDTA, whilemost of the pectin is extracted sequentially with either hotammonium oxalate or dilute KOH (Fig. 4). In contrast, alarger proportion of the pectin of the adapted cells was ex-tracted by cold EDTA. These results demonstrate that waterand saline stress induced changes in the organization of thepectins whereby a fraction of the Ca2+-insolubilized pectin ismore loosely bound and, hence, more easily extracted fromwalls of adapted cells.

Protein Content and Amino Acid Composition of the CellWall

The insoluble protein content of the cell wall increases asthe cells adapt to higher levels of NaCl (Fig. 5). Cells adaptedto 727 mm NaCl have about 3-fold more protein per mg ofcell wall than unadapted cells. Except for the hydroxyprolinecontent, the amino acid composition of wall protein does notchange much as cells adapt to salt. As a percent of the totalamino acids, hydroxyproline decreases about 10-fold at thehigher level of adaptation (Table II) and amounts appearcorrelated with maximum cell volume (Fig. 6). The propor-tion of insoluble protein in the wall varies slightly duringculture but is always 2- to 3-fold higher in the NaCl-adaptedcells (Fig. 7A). The gross difference between unadapted andsaline adapted cells in the proportion of hydroxyproline is aresult of accumulation of hydroxyproline during the laterstages of culture of unadapted cells, and this stage is charac-terized by cell expansion (Fig. 7, B and C). Cell walls ofunadapted cells contain high amounts of hydroxyproline atthe end oftheir growth cycle after cell elongation has occurred(Fig. 7, B and C). Salt-adapted cells elongate negligibly (Fig.

Figure 4. Fractionation of the acetone powders preparedfrom unadapted cells (SO) and cells adapted to 428 mmNaCI (S25) and 30% PEG (P30). Proportions (by weight)of insoluble material remaining after dialysis of the acetonepowders are represented to the left. The insoluble mate-rials remaining were then fractionated further to give rep-resentative pectic substances, hemicellulose and cellulose,and are shown on the right. Values are weight percent oftotal insoluble material.

0.1I KOHC4d C

AIIImxLOI-4F

43




CZ 300 -

E

200 - 0

0D 0

0" 200 400 600

Level of Adaptation, mM NaCIFigure 5. Amount of insoluble protein in cell walls of stationary phasecells adapted to increasing levels of NaCI. Cells were adapted as inFigure 3; insoluble protein was measured from amounts of aminoacids released from 6 M HCI hydrolysis of purified cell walls.

2), and the hydroxyproline content of these walls does notincrease at the end of the culture cycle (Fig. 7, B and C).

DISCUSSION

Tensile Strength and the Cellulose-Extensin Network

Measurements of tensile strength by the nitrogen gas de-compression technique (11) demonstrated empirically thatthe cell walls of adapted cells have reduced capacity for

withstanding pressure (Table I). This finding was surprisingconsidering that the walls ofthe adapted cells were clearly lessextensible based on failure of the walls of adapted cells toexpand even though they developed higher turgor pressuresthan unadapted cells. Measurements of tensile strength inother single cells, however, also revealed that forces requiredto physically expand and rupture the wall instantaneouslywere much higher than turgor pressures normally developedby such cells. Carpita (1 1) demonstrated that about 40 bar ofpressure are required to rupture 50% of the population ofcarrot cells, yet turgor pressures were only about 5 bar.Unadapted cells of tobacco were similar, but turgor pressuresof the salt-adapted cells more closely approached the tensilestrength (Table I). Many reports have proposed mathematicalmodels to describe the relationship between turgor pressure

and the resistance of the wall in controlling the rate of cellexpansion (for review, see refs 12 and 31). The data presentedhere clearly demonstrate that biochemical modifications indiscrete, load-bearing regions of the wall are responsiblefor growth and that growth does not result merely fromturgor pressure high enough to pry the microfibrils apartmechanically.More recent investigations have focused on the biochemical

bases for alterations in the wall which drive growth. As a

result of more sophisticated determinations of linkage struc-ture of many of the polysaccharide and protein componentsof the wall, several models of the organization of the wallhave been proposed (2, 21, 23). In general, the wall of dicotsis viewed as framework of cellulose microfibrils with xylo-glucans, principally, and arabinoxylans hydrogen-bonded tothe surface ofthe microfibrils and spanning in milieu between

Table II. Amino Acid Composition (as Percent of Total Amino Acids) of Insoluble Protein from CellWalls of Stationary Phase Cells Adapted to Increasing Concentrations of NaCI from 0 to 727 mM

Nomenclature for cells adapted to levels of salt in the medium was established previously (3). SO areunadapted cells; Si 0 are cells adapted to medium supplemented with 10 g/L NaCI, and so forth. Molarconcentrations are S10, 171 mM; S20, 342 mM; S25, 428 mM; S30, 513 mm, S35, 599 mm; S40 684mM; S42.5, 727 mM.

Cell TypeAmino Acid

So S10 S20 S25 S30 S35 S40 S42.5

mol %Alanine 8.0 9.7 9.5 9.7 9.8 10.4 9.5 9.8Glycine 7.9 8.8 9.8 9.8 9.8 9.7 9.8 7.2Valine 7.5 8.4 8.2 8.2 7.4 5.6 8.0 5.8Threonine 5.8 6.0 6.3 6.0 5.8 7.1 6.1 6.1Serine 8.5 7.0 7.7 7.5 7.3 7.4 7.1 7.6Leucine 9.5 10.4 10.5 10.4 10.8 11.0 10.2 10.6Isoleucine 5.5 6.0 6.2 6.2 5.3 6.3 6.1 6.2Proline 5.1 5.1 5.3 5.2 5.4 4.2 5.2 5.8Hydroxyproline 6.9 3.1 1.3 1.8 0.9 1.2 0.6 0.8Methionine 0.7 0.7 0.9 0.9 0.2 0.6 0.8 0.5Asp + Asn 9.5 9.9 10.0 9.7 10.0 10.8 9.9 10.3Phenylalanine 4.5 4.4 4.5 4.6 4.9 4.9 4.5 4.6Gin + Glu 9.5 9.9 9.5 9.7 10.2 10.8 9.8 10.7Lysine 7.6 5.0 5.7 7.6 6.2 3.7 4.8 6.2Tyrosine 2.0 1.8 1.1 1.5 0.9 2.8 2.7 2.3Arginine 3.3 2.6 1.9 1.4 2.8 2.1 2.5 3.8Histidine 0.4 0.2 0.9 0.6 1.3 1.1 0.7 0.6Cystine 0.2 0.4 0.1 0.4 0.2 0.2 0.2

IRAKI ET AL.44




8

a)

0~x0

-o

I

6

4

2

0 1 . * j

0 1.0 2.0 3.0

Cell Volume, ml x 107Figure 6. Hydroxyproline content as mol % of insoluble cell wallprotein as a function of maximum cell volume. Cell volumes are ofcells adapted to various levels of NaCI as in Figure 3, and hydroxy-proline was measured as in Figure 5.

them (2). This reinforced framework is then embedded in a

matrix of pectic polysaccharides, including polygalacturonanand rhamnogalacturonan (2, 21). The polyuronans compris-ing the gel are organized via Ca2' electrostatic interactionsand phenolic ester and ether linkages (15, 21). Extensin com-prises a HRGP4 with intramolecular isodityrosine linkageswhich reinforce a "polyproline II-like" helix into a stiff rod-like molecule (13, 30). Through proposed intermolecularisodityrosine linkages, or yet unknown covalent linkages, theextensin network can form a "warp-weft" arrangement, en-

veloping the individual cellulose microfibrils and establishinga rigid matrix (23). Such an interaction was suggested as adeterminant of wall expansion during growth through revers-ible changes in the organization of the cellulose-extensinnetwork (23), but others (28) have proposed that incorpora-tion of the HRGPs into the wall matrix signals irreversiblecessation of growth concomitant with further differentiation.Alternatively, Fry (14) has suggested that suppression of per-oxidase secretion by growth regulators might delay polymer-ization of the extensin network during cell expansion.The HRGP content of unadapted cells is higher than that

of adapted cells even during the active division and growthphase of the cells and increases dramatically when unadaptedcells rapidly expand and enter stationary phase (Fig. 7, B andC). There is expression of the elongation phase of cell devel-opment exhibited by unadapted cells that is characterized byincreased deposition ofHRGPs into the cell wall. An apparentinverse correlation between growth rate and HRGP contenthas been reported elsewhere (23), but the phenomena maynot be connected causally. The accumulation ofHRGPs mayoccur in unadapted cells to increase the ability ofthe enlargingcells to withstand increasing tensile forces. Adapted cells donot enlarge appreciably nor do they accumulate HRGP in thecell wall (Fig. 7, B and C). This developmental alterationeasily explains why tensile strength is lower (Table I). The

'Abbreviation: HRGP, hydroxyproline-rich glycoprotein.

c300 - . A

NaCI-adapted Cells

0M 200c

E 0

..Unadapted Cells

0~~~~~.4§ 10-o

2I0)

E-o 0.3 -

CD

0.2 \jUnadapted Cells

-c1I 0.1 NaCI-adapted Cells

8 -

CDC:~~~~~~~~~el

/.UnadaptedCelr2- ~~~~NaCl-adapted Cells

1 A A-/ A

O0 10 20 30 40

Culture Age, days

Figure 7. Variation in total insoluble protein and hydroxyprolinecontent of cell walls of unadapted and adapted (428 mm NaCI) cellsover the entire cell culture cycle. A, Comparison of total insolubleprotein per mg cell wall from unadapted and adapted cells. B, Freshweight accumulation by unadapted and adapted cells through theculture cycle. C, Comparison of the mol % hydroxyproline in theinsoluble protein of the cell walls of unadapted and adapted cellsthrough the cell culture cycle.

absolute mass of the walls of both NaCl- and PEG-adaptedcells is only about half that of unadapted cells and, further,the proportion of cellulose in the wall itself is only half of thatof unadapted cells (Fig. 4). The principal HRGP in theinsoluble protein of the wall is extensin ( 13, 23) and, hence,the mass of the cellulose-extensin matrix of the walls ofadapted cells is grossly reduced compared to that ofunadaptedcells (Fig. 4). Reduction of the mass of the cellulose-extensinmatrix can readily explain loss of tensile strength (Table I),

45



Plant Physiol. Vol. 91, 1989

but decrease in the ability ofthe wall to expand in the adaptedcells despite loss of the absolute strength indicates that morediscrete determinants of expansion exist. Therefore, accu-mulation of the HRGPs and the extent of formation of thecellulose-extensin network is not likely a direct determinantof the ability of the wall to extend but, rather, represents astage of differentiation which results in increased mechanicalstrength of the wall. Expression of this developmental stage isprevented during adaptation to osmotic stress (Fig. 7C). Wehave not ruled out the possibility that a small population ofcross-linking proteins, including HRGPs, participate in aload-bearing function that reduces the ability of the wall toextend. It is possible also that the degree or kinds (intra versusintermolecular linkage) of isodityrosine between HRGPs isaltered during adaptation. Finally, other types ofprotein cross-links may form during adaptation and limit the ability of thewall to extend. There are also numerous, discrete changes inthe pectin and hemicellulosic constitutes that could affectcarbohydrate-protein interactions or cell wall metabolism andthese possible alterations are discussed in the companionpapers.

In a separate line of reasoning, the xyloglucan-cellulosenetwork represents a more plausible determinant ofthe abilityof the cell wall to expand. In elongating cells, cellulose micro-fibrils are oriented nearly transverse to the plane ofelongation(2). This orientation easily reinforces axial expansion of thedeveloping cylindrical cells, but with virtually no reinforce-ment of the forces generated in wall in the longitudinal plane.Xyloglucans and other hemicelluloses that hydrogen bond tothe microfibrils and span the distance between adjacent mi-crofibrils may comprise this reinforcement. Control of en-zymic cleavage of the xyloglucan polymers may constitute adirect control of the rate of expansion, and there is consider-able evidence in support ofthis concept. Release ofxyloglucanfragments from cell wall preparations is increased by pretreat-ment of seedlings with auxin or acid (16, 22), and Terry andcolleagues (32) have confirmed these data in living tissues byuse of an innovative centrifugation technique. Wall extensi-bility has often been suspected of being under enzymaticcontrol, and a considerable body of data directly implicateendo-glycosidases that participate in the digestion specificallyof xyloglucans comprising the load-bearing function (for re-view, see refs. 12 and 31). Reduction in molecular size ofxyloglucans is induced in vivo by auxin or acid (25), but theexact role of the xyloglucans and their hydrolysis in growth isyet unclear. Terry et al. (32) also found that auxin increasesthe solubility ofpectic polysaccharides, and so more extensivereorganization of the wall matrix must accompany increasesin the rate of wall expansion. To gain insight into how suchorganizational changes occur upon adaptation to saline ordrought stress, the chemical structure of the polysaccharidescomprising the walls of adapted and unadapted cells wasexplored.

Why Do Plant Cells Which Are Adapted to OsmoticStress Fail to Expand Normally?

Reduction in growth induced by desiccation stress is not asimple consequence of turgor loss (7, 8, 20). We have indi-cated that this growth reduction involves another mechanism

which is most likely actively regulated by controlling cell wallextensibility (3-5, 9, 10, 18) through some signal/inductionprocess involving the perception of a desiccating environ-ment. We have pointed out that such an active growth reduc-tion response to disiccation could logically contribute to asurvival strategy of the plant to conserve water (3, 8, 10).Restoration of normal growth rates after osmotic adjustmentunder desiccating conditions would jeopardize the survival ofthe plant by accelerating the depletion of a limited amount ofsoil moisture. This would be especially true considering anuninhibited growth rate would greatly increase the transpiringleaf surface.We reported that cultured cells adapted to NaCl also exhib-

ited a reduced growth rate after osmotic adjustment hadresulted in increased levels of turgor (3-5). This was a some-what surprising result since a saline environment should notimpose a constraint on water usage (as a desiccating environ-ment would) as long as adjustment of cell water relationsallows for sufficient water uptake. In other words, in contrastto the desiccation environment, where a limited water supplyis essentially "mined" by the plant, the saline environmentoffers an unlimited supply of "hard to extract" water. There-fore, once osmotic adjustment is achieved there should be noneed to initiate a reduced growth rate to conserve water. Infact, a reduced growth rate in a saline environment mightactually be detrimental. Because the uptake of ions which aretoxic to cytoplasmic function is involved in the osmoticadjustment of plant cells to salt (3), these ions are compart-mentalized into the cell vacuole (5). In transpiring plants, ionsare continuously supplied to the tissues. Uptake and com-partmentation of these ions would result in increasing iongradients unless the ions could be secreted outside the cells orbe diluted by cell expansion. Inhibition ofgrowth would morerapidly expose the cells to injurious salt concentrations andrequire more strict regulation of ion uptake by the roots.Thus, halophytes, which have evolved in saline environments,have much less growth inhibition in response to external saltand may actually exhibit growth stimulation in response tomoderate concentrations of salt (17, 34). It is important forhalophytes to avoid growth inhibition by salt as a survivalmechanism which helps prevent cytoplasm/vacuole ion gra-dients from becoming too large to prevent accumulation oftoxic levels of ions in the cytoplasm.Why then do glycophytes respond to salt exposure by

reducing growth? This reduction in growth most likely resultsbecause glycophytes have not evolved a mechanism for distin-guishing desiccation environments from salt environments.Elimination of the ability ofglycophytes to respond to salt byreducing their growth may actually allow them to toleratemore salt, but less desiccation. However, an important impli-cation of this hypothesis is that a genetic alteration whicheliminates reduction in growth in response to salt might allowplants under saline irrigation to adapt to the salt environmentwithout reductions in growth (yield). Therefore, if salt-in-duced reduction in growth involves an alteration in someproperty of the cell wall, identification of the alteration andthe eventual genetic manipulation of this cell wall character-istic would have important consequences for the success ofsaline agriculture. We found some drastic alterations in the

46 IRAKI ET AL.




properties of cell walls of cells adapted to osmotic stress. Wepropose that some of these properties are involved in con-trolling the mechanical strength of the wall while others areinvolved in the control of cell expansion and growth. In oursubsequent studies of the chemical constituents of the cellwall medium, we have uncovered more likely determinantsof cell expansion. The results of these studies are summarizedin the companion reports.

ACKNOWLEDGMENTS

We wish to thank Jean Clithero and Glenda McClatchey forexcellent technical assistance, Marla Binzel for cell morphometrymeasurements, and Drs. David Rhodes and Larry Dunkle for criticalreview of the manuscript.

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