From: Boyer, J.S. 1995.Measuring the water status ofplants and soils. AcademicPress, San Diego. 178 p.

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Chapte! 2

Pressure Chambet

Pr€ssure chambers are the most widely used field inshumentstor measuring pla.rt water status. They ar€ porrable, allow rapldmeasur€ments, and ar€ shrdy. T€mperature needs little conhol and nocomplex insbumenhtior is required. The ti$ue simply is s€aled intoth€ top of the chamber in such a vray that mo6t is insld€ afld or y asmall amount extends outside th.ough the top (Fig. 2.1A). The s€atgiv€s an airtight ba.iie! b€tw€€n the int€rior and th€ atmosphericpr€ssu.e outside (Flg. 2.18, c). This allows the tissu€ to be pr€ssurizedlnsid€, forcing water toward the outside. The pressure flecessary tohold the warer at the outside sufface measur€s rhe water status of thetissue- The more dehydrated the tissue, the mor€ pr€ssure is r€quired.

"_dEFiguE 2.1. PEsuE .hamb€r d6ig.. A) Easic Lyour of clEmber and tiesu€duin8 pE$qiatim, B) Enla€€d view of a sl rhar is tiSht€ned by gaspEsrF; the *at .FEd the l$su€ beoms righter as pEsure driv€s thedbber stopp{ (G'n!r.hed) de?e. iito the me betow the dbhb€r top. c)Enl.rEEd vtew of a sl that i! dShth€d hdMlly; tightenin8 the Hss ioresthe.ul'b€r *al acailst dE ti$ue,

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The method has ben used successtully wiih leaves, bra.ches,and roots and can provide information about all of the components ofthe water potential {Scholander ct ai., 1955, Tyr€€ and Hamel, 1972).Becaus€ the chambe! ls portable, it can be moved to the expelin€ntalsite, and conditions ih the plant can be left undisturbed until rhemoment of sanpling. However, b€caus€ plants us€ larg€ pressures tomov€ water/ large pressurcs are needed during measuiements.Therefor€, prelsure chambers must b€ built strongly and the seal musthold the tissue in place against large for.ee. Early €fforrs wfth plessurechambers were plagued by explosions that prevented the method Iromb€ing developed (Dixon, 1914). After Scholande.r and his coll€agu€sbuilt a safer unit (S.holand€r ei al., 1964, 1965), the merhod becamebetter understood and Boy€r (1957a) show€d that ft could be used romeasure the water potential. Commercial velsions are now availabte(App€ndix 2.1),

Principles of the MethodThe method is based on th€ conc€pt that the wat€r porenrial in

cells creat€s a tenslon (negativ€ pr€csure) in the cell wals thai Frllswater toward trc cells from the xylem, th€ root c€lls, and finally the soil(Fig. 2.2A). Exclsing a plant part causes the xylem water to pull backinto th€ xylem (Ftg.2.2B). Appllng prelsure to the tissue rais€s thecell vrater potential and forces water out and inro the xyld which isop€n to the atmosphere outside of th€ chamber. Xylem solutioneventually appears at this surface when the applied plelsure tuliyoppos€s the tension odginally in the sample [Fig.22C). Scholander er,1. (1965) consid€red the pressur€ ro be a dire.t measur€ of rhe rmsionin the xyt€m because of the continuous liquid phase €xtending into thecell walls.

The liquid moving in the walls and rylen is not pure ware!.Roots absorb salts from the soil and deltvef them to the shoor via ihexylem. Together with certain organtc constituenrs traveting in lhexylem, th€ xyl€m and cell wall solution contain sufficient solut€ io haveosmotic potentials as low as {.4 MPa (Boy€r, 1957a). Boy€t (1967a)slEwed that if solute effects were taken into accounr, the Dressurechsmb€r could be us€d to measure ihe tissue waler poren al. Sincethen, the pressure chamber has been wtdely us€d lor measuring tissue

Fia!re lt" D'a8ah of the waEr barspon sy3Em h . pt,nt, A) Uguid.@tinuiry cuB berwq the eit eturion and rhe elL inrU" ,f," Uf L.

-rne cn wds dd xyleh Gpopta5t, stippted spac*) arc al$ waEFf t€d. The@er h rhe apoplasr is ontinwuj kth w.rer in rhe sojl ek?t thar jn r@ryiEtan.6 rh€r .c wdy sbsEree (caspdian sFips, nor shM) in the waltsor @ er elr3 rh.t fore water to now ttrcuah the @r prcroptasts, tatpr,otop,larB,trffnit low w.Der porhri.ts tr ra'sioE 0o he apoptasr and the{L D, E:asha. le} opero tE xyts b dE ahncph€E. IE rylq $luti6

lehacts b.@s! elts in rhe ve$ets where 3uffi.i€nqy shau pores e{3r,o

lI:I'j-:lu::l fmm rcE!.hir fdriher. c) Mourinr dl€ ref in a preeuF

Ijlr.rrr@i!@s pF*u€ ro tE.ppued thar eturu rhe ryl€m loturis !o itsp6Eon n he inla.t planr in A. ThG presuE p.". 6hr€,.<ts rhe teroio(n%atik plsuE) qerr.d on rhe xyte;/apoplasrgludon in the intact pletam hus 's a masuF of he E6ion

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pressure Chamber TheorvTh€s€ conceprs can be formalized ;y considering thecolnponenrs that conrribute ro rh€ wal€r potential. tn cetts and dissues,rne major on€s are soluL€, pressure, sotid: (po.ous rotids atso r€rmed

; rt7M..surinE Wetd Stattr

matric$), and gravity. The distinction beiw€en th€m sometimesbecomes blu-lr€d becaus€ of th€ difficulty in categorizhg the forc€s,esp€cially those arising fiom solutes and solids. ln cells,macromolecules can dissolve or som€times precipitate to form a g€l(porous mahix) or form aggregales that may or may not be in solution.Derpite this compl€xity, w€ will consider aggregates to be in solutionunlers they pr€cipitate, a practice also followed by J. Willard cibbs(1931). We wtll distinguish belween pressure gen€rat€d in pores ardPressure aPplied externally (Cibbs, 1931). Also, as discuss€d in Chap.1, we will describe th€ forces on a unit area basis (prersufe) 1,'hich isproportional to the iree €n€rgy per mole of molecul€s (see Chap. 1).Accordingly, the components of the water potential de

Y 0 = Y , + Y p + Y , + Y g ( 2 . 1 )

where the subscripts r, /, m. and g represeni the €f{ects of solute,pre8sure, matrlx, and glavity, respectively, and Y- is the warerpot€ntial. Each poteniial refers to the same point in the solution, andeach componmt ts addittve algebraically according to wheihe! itlncreas€s (positive) or d€creas€s (n€gative) the Y- at lhat point. Theincreas€ or decrease is always relative to pure water at atsnospherlcpressur€/ at th€ same temperature as the solution (s€e Chap- 1).

The components affect Yu in specific ways. Solute lowe$ thechemical pot€ntial of nater by reducing the tendmcy of watermol€cul€s to €scape from each other compared to pure wate! b€causesome of the solution volume is occupied by solute molecd€s that dilurethe water molecules/ decr€asing the number .ble to escap€. h a similarfashion, porous solids that ar€ wettable occupy volum€ and causesurface eftucts that r€duce the escaping tendecy of the water in thematrix. Ext€rnal pressures appli€d to the liquid increase th€ escapingtendency of the water if they ar€ above aknospheric but reduce thees.aplng t€ndency if th€y are below atrnospheric. Clavi9 afecrspressures because of the weight of the water, and th€ escaping tendencyof water is incr€ased or decreased d€p€nding on whether glavityircleases or decreases the local pr€ssure relative to atrnosphericpressure. Pressures ar€ high at the bottom of the ocean for ihts r€aso.but low at the top of a tall tre€. Each €omponeni decreases Y, (is

rcgative in Eq. 2.1) except pressure and gravty above the atmosphertclevel, which inci€as€ Yo (are positve in Eq. 2.r).

For most of ou. purpos€s, gravuational pot€ntials will b€ignored because they become significant only at hetghts great€r than Imeter in v€rtical water columns. In th€se €as€r Eq. 2.1 reduces to:

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Water in plants gmerally has a n€gativ€ Ys be.ause y6 and ly,ar€ negativ€ and Yp do€s not fully comp€nsate for them. Wat€r wfllmove toward more n€gative Y, or more negative components of lydand plants use this principl€ ro extract water from rhe soil.

We may further conceptualize plant water by recognizing tharit is located in two compartments separated by a diff€renriallypermeable membrane (Fig. 23). The first compartment is the interiorof ihe cells (the p.otoplasts which coll€ctively are the symptast) and rhes€cond is the cell walls and xylen outsid€ of the protoplasts(co ectively the apoplast). The m€mbrane separating the comparrmentsls theplasrnal€mma ofeachcell, and it aliows water to move fre€ly butliltle solut€ (i.€,, the nenbrane is differentially permeable and refl€cts

Figure 2.4 shows thatin the protoplasrcomparrment (Fig.2.4A),there is a conc€nkated solution (%r,t and usually a pressur€ aboveatmosph€ric (the hrrgo!, Yplpt so thit ihe water potential (lg&ft, is

Vwe) = Yst + vptp), (2.3)

wher€ the subs.ript f) deno[es the protoplast conpartrhent. The mahicpotential is g€nerally negligibl€ ln the protoplast compartment (y,0)= q Boye4 r96h).

In the apoplast compartment (Fig. 2.48), rhere is a d utesolution (Ys(., ed no turgor. lnstead, th€re are surlaces arising fromthe porous mahix of th€ cell walls (Fig. 23, ins€r) and thes€ g€nerate amabic potential Yur.l which is €xpress€d nosdy as a tension, l€.,negauve pressure when th€ por€s are waleFnlbd. These compon€ntsf l

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which sho$/s that the components of the water potentiat in rheprotoplasts are diff€rert kom those in th€ apoplast but they balance€ach ottler lo.alty. Nore that the rugor tn rhe c€Us is posltiv€ (\P"/",)and tne water in the apoplast is under tension (v,rdt. This caus6;large pressur€ differenc€ across the ptasmalemma. i,\ibre it nor for theconskaining effect of the c€I watt, rhe plasmalemma would burst.

Upon pressurization in a prBsu.€ chamber, as in Fig.2.ZC, thewater in the cells is uniformly expos€d ro an exr€rnal Dr€ssur€ jnaddiHon to th€ turgor Cig. 2.4D). This r.is€s the ce wat;r porenrial

FiAuE 2,3. EnL.ted vi€w of ohparthentatim in plant tissues. The fnst@mPartrnent ls iside dE €U! (pDi.'plast, open sp.€) .nd tE *ond is in rhecell waus and xyleh (apoplast, srippled spaes). The h'o obp.rrrsis desepaEbd by the plastulet'W (plMa rembhne in magnified i@t). TheprctopLsts arc water-fill€d. Ihe waui Mrain warer in rhe poEs held by Eiehydrcplillc surraes of the poes dd by surfae reroim at €ch airlwate.maiscs. The wall poFs are s 3null ttEt they withstand hi8h teciotu

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lisuE 2,4. Pot ndal diagmm 3h@ina w.!e! potenaEl .nt ib .omponqrsiEide ells (4 4d nrwalb and ryl€m(B). the diEd6 of the aw indiqteuwheths ihe potential ie Ei*d or low€red by e.h @mpondi. llE <ell int€.io.A is esstially in eqo ibnu widl its w.tl B e th.r the d,agrans in A ed Bq be €quled s in (c). prsft (pJ applied r,o tissue in rhe pte3suchar$€r his rhe porential of rhe cll in-lerio,,s in (D). Watet moks out qdhydEEs the wall, raisint 3 porenri.t, Wh€n pr! Lutflcs y-(,r as shom inD, riquid app€6 .t rhe ot sudae md dc ml hove ,t equ fnum,Y o G ) = Y , k ) + Y B k ) ,

where the subscript (d denotes the apoplast compattmentThe water potential in €ach protoplast is alnost always ihe

same as in its own c€il wall (Molz and Ferrier, 1982) as shown in lig.2.4C:

Yu{a) = Yu(p).

Substituting Eq. 2.3 and 2.4 in Eq. 2.5 giv€s

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large enough, water noves into the plant. When the rate of uPtakeequals the rate of evaporation, th€ water potential be.omes constant.Anythif,g that changes the rate of loss or uptak€ will altet the wat€rpot€ntial of the cells, and the potential can chang€ quickly. Variationsin wind speed or brief periods of cloudy weath€r can cause espec'allylaig€ changes. and taking the pressure chamber to the field is a Soodway to convinc€ yours€lf oa this.

As a cons€quence, the pr€ssures in th€ xylem vaiy malkedlyduring th€ day. B€for€ sun up when kanspira[on is low, Pressur€soft€n are above ahnospheric. The roots absorb salls from the soil and,b€caus€ they exhtbit active m€chanisms to kansfer saltr to the xylem,they Geate a sufffomt concentration in the xylem to attract waterosmoti€ally florn the soil (Fig. 2.2). Pr€ssures build uP in the xyl€m justas they do in c€lls and are termed root Pr€ssures. Root prelsures of0.05 !o 0.4 MPa are ftequent in rapldly growing plantt and occasionallythey are even tugh€r (to 0.6 MPa). The p.€ssur€ chamber cannotmeasue these posidve pressures or the osmotic potential in the xylem.

After sun up, transpiration b€gins and the pressurc falls in thexylen, fi€quefldy readdng t€nsions ol -1 to -2 MPA as the cellsdehyd:ate. Water is pulled from the soil much as a wick pulls liquidfrom its surroundings, except that cells are emb€dd€d in the wick andthe tensions ar€ conholled by the c€lls. Tog€ther wtth th€ xyl€m, thewick-like action can move water ov€t la?ge distances v€ry rapidly. Thepre$ure chambef can measu!€ thes€ t€nsions.

The pores in the c€ll walls are in contact with the wat€r in therylem but they do not drain b€caus€ water molecules adhef€ to themol€cules in the walls and to each other, and ar€ athacted to the solut€sadsorbed to the walls. Th€ w?ll porcs have small dlamet€rs (about 5 to8 nm), and sslface tension keeps the pores wat€r-filled against largetmsions (Figs. 22 and 2.3). The xylem vessels have larger internaldiameiers but they generally remain wat€r-fflled unless t€nsions becomemor€ fl€gative than about -r.0 to -2.0 MPa. In that case/ xylem watercan drain beca6e the water column tends to br€ak (cavitate), but th€small pores ln the surrounding walls remain water'fill€d. Breaks in th€xylem watef can diminish transport in the vascular system.

Pressure applied with the pr€ssure chamber is external and thegas peneEat€s the Essue th.ough the interc€llular air spac€s, thus

above that of the xyl€m, and water flows idto the xylen. By adjusiingthe pressure, the flow can b€ stopped when water just fills the xyl€m.At this balancing pressur€ Pd,, the water has rcturned to its originalposiHon in th€ intact plant'where it forms a stationary nat film withoutany exces! on the cut surfac€ (Flg. 2.2C). This pressure exactly relievesthe t€nsion that had be€n a€ting on th€ xylem solution (Fig.2.4D). Thenegative of PG thus measures the tension in the apoplast, i.e., thematric potential of the apoplast:

-Pg* = Y^1oy QX)

Substituting 8q.2.7 in Eq.2.4, it can b€ seen thar the water porential ofth€ apopiast is the sum of -Prc and the osmohc porential of theapoplast sotution Y{,). From Eq.25,

%101 Pgas'Y,p; = Y.1pv e8)

and the wate. potential o{ the tissue is determined flon %(,1 - Ps,(Boyer, 1967a). The Y,rd is measur€d by werpresswing the tissie,couecting a small amount ol exudate ftom the rylem, and deteoiningtts osmotic potmtial in an osmorneter (s€€ Chap.3).

Thre€ basic principles are dernonstrated by these r€lationships.First, the pressurc chamber measures the tension in the xylem and cellwalls becarse the appli€d pr€ssure reliev€s th€ tension and the ryl€nis directly obs€rved. S€cond, the measurements require an equilibiumbetween the pressure and the xylem solution (hence the €qual signs inEqs.2.7 and 2.8). One must make the measurements at equilibtiMpress'ires (no water moves in or out of the tissue) to have a validmeasurement. Thid, the applied pressure rajses the ware! pot€nrial inthe tissue, During the measurement, the tissue does nor have the snmepotential as in the intaet plant (Fig. 24D).

SIGNIFICANCE OF TT{E ]IIEORYTh€ ability of water to move depends on ils water potential or

the components of the water potentlal, specifically rhe osmotic porential,pr€sstrr€. or matrtc potential. In planr!, low potedtials ale frequentbecaus€ the shoot tissues become dehydrated on a daily basis. The lowpotenrials create a t€nsion on wate. in ihe xylem and, when rhe pu is

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s$rounding each cell with unifom Prersure (Figs. 2.2 and 2.3) Onty

the cell to cell contact ar€as are not in contact with the gas, and liqutd

can flow ftom cell to cell through thes€ ar€as. Thus, the Pressure"squeezes" the liquid in the cell toward any region wher€ the cell io cell

contact is at a lower Potenual.The temp€rature doe! not enter any of the above equations

because il is uniform in lhe sample. How€ver, Pressurevoluhe work

depends on the Kelvin temperature, and the waGt Poimtial of ilre

tissue becomes less negative as the temPeratute decreas€s within ihe

blological rang€. You will se€ this aB a slight decrease in the balanongpr€rsure as a leaf sample becomes colder evm though th€re is no

change in the wate! content of the samPle. Th€ theoretical basis fo! lhe

t€mperature depend€nce is tleated in Kramet and Boyer (195).

Types of Pressure ChambersAll pressure chanbers are constructed limilarly excePt for ihe

seal in the toP. Figure 2.rB shows a Pressue-activated seal ihat lelies

on th€ slippag€ of the s€aling matqial, usually r!bbe!, in a conshaPed.avtty built Into ih€ underside of the chambd toP. As Pressueinceases tnside tlrc chamber, the outward force Pushes the seallngmaterial into ttle cone. The degeasing dtamet€! of the cone tlghtens the

seal around the plant Part.Pressuredriven seals hav€ the advantage that they are quick

and altomahc. The surface b€tw€en the seal and the cone needs to be

lubricated but otherwire no maintenanc€ is required. The disadvdlageis that there ls no conhol ov€r the force aPPlied to the tissue and thes€als must be long to align the movement tn the cone The long s€alrestricts samPl€s to long-stemmed branches and leaves with longp€ttoles. B€cause the force apPlied to the tissue can become very latge

at high pressur€s, it can damage soft tissue6 or evm intertuPt flow.

Thus, the derign ls best for woody st€ms.Another type of seal involves a rlbber Packlng gland whose

tightness can b€ adjust€d by the operator (Fig. 21C). The rubber is

enclos€d in a well on toP of th€ chamber. A Packing Plate siis on toP

of the rubb€r and can be pushed down. Because the rubb€r .annotdeform outward, it deforms into the cote!, filling it according to theforce on the packing plate. The deformation s€als the stem or Petiola

Manually tightened s€als hav€ the advantage that a minimumof force is applied to the plani mat€riat. The operator listens forescapingeas and tighteB just enough to prev€nt audible lsakage. Theseal is small so that short-stemned samPles can be used. This 9"e ofseal is preferr€d ov€r the pressur€-drlven s€al becaus€ it is less likely todamage the tissue.

S€als can vary in diameter to allow large diameter stems to beus€d but the stem must be €specially secute becaus€ Pressures €x€rt aforce per unit area, and doobling the radius of the s€aled tissueincreai€s the fore fourfold. tn thb sibation, s€als must exert a muchlarg€r forc€ on the tissue to hold it in the pressure chamber.

How to Make Measurements

PRELMINARY CHECKSThe static presure insid€ a confining v€ssel is the same on all

the walls, and the pressure gaug€ may be mounted on the gas feed linera*rer than on the chambe. its€lf. B€fore uslng th€ Instsument for thetust ttmg the gaug€ may r€€d to be che{ked for accuracy. caug€s areavailable at thr€e lev€ls of accuracy: standard, test, and master test. Thestandard gaug€ is us€d on equiPment requiring moderately accuratepr€Asure readings. T€st gaug€s are used to calibrate standard gaugesand give mor€ reproducible readings. Master test ga'rges have acalibration kaceable to ttle National Buleau of Standards and aletypically used to calibrate test gauges. Of the three, test gauges arepiefelred for pressure chamb€rs and their a.curacy can be aBsum€d. Ifsfdndard gaug€s are !s€d, they should be calibrat€d at least with a testgauge.

Befor€ pressurizing a pressut€ €hamber, t€st for its ability towithstand high pressu.es- Measurements with Plants usually do not€xce€d 6 M?a buL what€v€r the haximum, tests should be at Pressuresat l€ast twice th€ marimum. For th€ test, fill the chamber wlth waterso that there is no air. Seal a rnetal rod s€curely in the toP in place ofthe dssue. Pr€ssuriz€ the water and che.k for leaks. Theincompr€ssibility of the water ensur€s that any failure wlll not be

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PROCEDURE1) After checking that the pressure chamber and seal are in qoodcondition, clean, rnd dry, check thdr Lhe incoming gas wilt enler closeto the bottom. Cov€r rhe bonom v/ith a tayer of warer so that theincoming Bas pass€s through the water. Make a baffle h Dr€vent waterfrom splashing onto lhe tissue (Fig- 2s). rjne rhe wa s;iih wer turerpaper. Connect a cylind€r of compress€d at ro rhe sas line.

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FiAur. 2.s. Waier in the botion of a presure d.mb€r rcdues overhetina oIthe enlerint Eas and humid'Iies rhe ar abud t e ledf. A vcp.r, ba-ffteprcvenrs splashins on rhe lear. Notmlty, rh. damber w,u s aGoL.d qur

2) Sdect the sample/ avoiding damaged tissue whenever possibl€.Ercis€ the tissue with a razor blade, ins€rt it swiftty into the s€at in thechambe. top, and assemble the chanber. Th€ time fiom €xcision tosealtng the chamb€r should be no longef ihan 10 sec ro avoialdehydradng the tissue.

If the time is longer than 10 sec, us€ a humidified slove box toload the sample in the s€al and ass€mbte rhe chamber (see Chap.3).Work in low light ro avoid h€ating and dehydraring the ti;rueAlternately, imm€diately before excjsion, enclose the tissue to bepr€ssudz€d in a fl€xible plasti. bag to rerard evaporarion Ourne! andlrng, 1980). S€aL the bag enough to i.nibit 4aporation but alow gas

Fi8lre 2.6. Mssurin8 the xyleE leruion w h a pressure chamb€r. Note lh.t

il";trffi""]5ffi"::...rT.sd" or rh" ap";tu. ed *"*. *r.ty sr",*"

iiffiirlLti,x.jT,i,ij:rhade th€ ba8, €xcis€ the sampre, and road

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_,h" rissue has been praced in the chamos, appry a ltnaamount of pressure and check for leaks. r air is reaUnj irrrougrr amanualy s€aled unit, stowly righten rhe s€at unrit "uatr"

r"oi"g"stops. Rais€ th€ pressu.e slowly;nd ,n smal steps.4) Obs€rve the cut surface of rhe tissue as pressur€ is being apptied.ALwAys OBSERVE FRoM rrft sloe narirEn. IHAN ABovE THE

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calcdahd according to the osmometer instructions (se ChaP. 3). The

water potential of the tissue is the sum of the aPoplast osmotic Potentialand the negative of the balancing Pressure (E+ 23).

Working with Plant TissueTher€ are giadients in waber potential in Plants. As a result,

tissue samples ar€ not uniforn, and large sarnPles us€d with plessure

chambers may have signiffcantly different wat€. Potentials in differ€ntpalts. The rylem is the source of water for nany plant organs andusuarly is the wetter part of the 8tadient. After som€ ttme elaPs€s in

lhe preisur€ chamber, the water in the tissue equillbrates and thechamb€r indicates an average. Usually, the averaS€ is aPProachedwithin 10 min which is the tim€ required for a prcssur€ chambermeasurement. However, in some fleshy samPles, tim€6 can b€ muchlonger (hours or days).

Tyr€e and Hammel (1972) showed that the average isdetermined by how much wate. is Pres€nt in each Part of th€ samPleas well d by th€ pot€nttal of the wat€r (also see Fig.3.19) according to

27M.eiling Wta Statut

CHAMBIR IN CASE ]}IE TISSUE IS BIOWN OUT OF THE SEAL(FIg. 2.6). Do not tecut the tissue becaus€ the initial dcision is th€ref€r€nce PoBttion marking the location of watet in the xylem in the

lntact plant. It is to this Posrtion that the xyld soluHon must berehrrned by the Pr€sslre. Increase the Pr€ssure lntil llquld i! sianditg

5) As pr€ssur€ increases, it is nomal to obsese gas bubblPr on thesu.race, tut ttrey shoUa U (orming slowly enough not io obs.ure thearrival of lhe llqurd. After Iiquid aPpears, reduce the Pressure andallow the liquid to be Pulled into the tissue until a wet film is aU thatremains on the cut su.fac€. This js the Position of the xylen solutiorbefore excieing lhe sanple, and it should require a balancing Plelsurethat exactly opposes the tension in th€ xylen b.tore ex.ision. Th€meniscus is flat lndicating that the water is not constrained by tensionsthat would otherwis€ be oPeratins. Adjust the Pressure so that thewater filn r€mains at the ort surtuce. For vour first measuiements,satisfy yours€lf that the liquid film is maturtained for 30 to 60 minwithout changlng the final balancing p.essne. During this time,evaporanon ftom the film car be Prevented by lining a vtal with wetfilter paper and inverting it over th€ cut surface. For routinemeasurements, it will sufffce to obses€ th€ meniscus for only 1 o! 2mtn. The balancing Pressure is the negauve of the tension in the xylem

\8q,2.7).6) After determining the balancing Pr€ssure, tinse the surface withwater, dry the surface, overPressure, and collect a small (1G20 Pl)sample of xylen solution in a miqoliter syring€ for a metsuremeni ofthe xylem osmodc Pot€ntial Measure th€ osmotic PotenHal with amicrollter osmomets or psychroneter (s€€ ChaP. 3) If th€ osmoticpobenBal is sufficiently close to z€ro, this steP can be omitted insubsequ€nt determtna$onsz) Releas€ the air tn the chamber and remove the tissue. InsPect for

damage ftom pressurizing and s€aling

CATTTJLATING WATER I'OTENTIAIS FROMPRESSURE CTIAMBER DATA

Gauge presswes are converted to megaPascals (MPa) or ba'sac.ordlns to 1 MPa = 10bars = ldn€wtonsm'3 = ld dvnescm_'= 145lb i^'2 = ,.87 afirospheres. The apoplast osEroHc Potefltial (negativ€) is

tT

ttI

r

!

. . i - i

A w a g e v , = ' V ' ,

(2.e)

wh€!€ / is the watq votume in the protoplasn of cell i, Y; is th€water pot€fltial of cell i, and v is th€ total water volume ln theprotoplasm of all th€ c€tls (the symPlasm). The symbol r adds lhecontibution of all the cell Ir and Y; in the tissue. Dividing by v givesa volumeweighted average. The volume of water in the aPoPlast is notconsideled In this calculatton becaus€ the xylem and wall matrix are€onsideled to be incomPr€ssible Out see s€ction on Changes in XylemDimensions}

Equadon2J shows that those parts ofth€ tissue containing thelarg€st water volume mak€ the largest conkibution to th€ wat€rpot€ntial measured with a pressur€ thamber. The cells iar from thexylem accomt for a larger volum€ than the few (ells next to th€ xylem,and the pot€ntial of the far cells will dominate the volume av€rage ina pr€ssure chamber. You can obs€rve this by raPidly Pre$urizing asampie, which often forces a kansient show of rylem solution at a low

292a M.a*ing Wet r Stdte.

rL

!-IL

II

pr€ssure b€cause of the releas€ of water from the wetter cellsimmediately around th€ xylen. However, the solution disapp€ars andthe nnal stable r€ading is always at a higher prEsure renecting th€eventual contributron of the far cells to the volume average. Thevolum€-averaaina conept applies to the componsls of Y as well as toany ofter c€Il param€ters d€p€nding on Y and m€asured at the ti$uelevel. With care, the volume av€rage measurd with the pr€ssurechamber should be the same as the volume average ln the intact plantbefore samplir'8.

Other factors can chanSe the Yd and a particular problem forthe pressur€ chamber is the inherently d€hydrating natut€ of themeasuement. Only o€lsed tissue can be us€d, which eltmlnat€s wat€ruptake. The gas entering the pressure chamb€r is dry and warm as ar€sult of comptession, which favors evaporation. Waber also evaporatsfrom the cut surface on the outside of the chambe!- Steps need to betaken to minirniz€ these probl€ms, specifically by loaali.g the chamberrapidly, humidifying the chamb€r, and raising the pressule slowly. Lraddition, you should plan to tak€ the chamb€r to the plant to besampled and avoid the temptaEon to carry the plant or exosed sampleto the pr€ssur€ chamb€r. Not onty is loading morc rapid, but theplantenvironment is uchanged and th€ water potenHal of the intact systemis more r€adily pr€s€rved in the sample.

LEA\'EsMost leav€s €quillbrat€ npidly with the applied pressute and

ar€ favored material for pressure chamber measur€ments. Enclose asmuch of the leaf as possibl€ insid€ the chamber because pressurechambels measure tensions that extend thloughout the sample andpressure must similarly €xtend over the whole sample tnsofar as tspractical. This ensures that any deformation caused by t€nsions in thelntact plant will be reproduc€d by the pressure in th€ chamber.

laaves having petiol€s require a round seal but Fasses mdcertatn conifer needl€s requlr€ a sllt s€al, Sampling ls sinilar for bothexc€pt, for wtde gnss leaves having a large rnidrib, the blade issampled on one side of the midrib. In tlus casq us€ a sharp razor bladeto cut toward the mldrib p€ip€ndicularly (Fig.2.7A). Grip tn€ tissue onthe apical slde of the cut and t€ar toward the leaf tip (Fig. 2.78). This

r - lL J

t 1

{-Tom Edsecur>l-+

Figlr. ?z Sampling a wid€ Abs lef, CDt a.ross the laf alnost ro L\emidvein (A), rear the l€.I toward rhe .pex rrom the .ut (B), ud ifte.t rltesple in the pe$w .hdbei (c). obere rhe .ppearme of the ryiemshiion at the dt sulace during pressuriztion but i6noE elurion .ppearinA

gives a triangular sampl€ with a danaged edgeparall€ling the midrib.Obserye th€ xylsn solution at tt€ cut end but ignore the eartyappearance in the v€lns cloest to the darnaged edge (Fig. 2.7C).

Because leaf tissue is soA aad easily crushed, avoid pressurechamb€r designs with pressur€"activated seals whele ther€ is no controlov€r how much force th€ seal applies to the tissue. A slight amount ofcushing g€nerally occu6 and do€s not affect the balancing pr€ssure butprolongs the time nec€ssary to make the measurement. If .rushingappears s€v€re, resr whether the rylem has be€n affecbed by leaving lhetissue in the sea I and exising the edg€ of the leaf ro erpose the endr of

\

30 M.e*Ang W,rar St htt

the veins. Apply pressur€ to the leaf ln a water-filled pr€isurechamb€r, The flow ttuough the Ussue in the seat should be much fasterthan that obs€rv€d vrlth the intact sample, indtcating that the xylem wasnot conskict€d by the seil.

Crushlng the tissue also can releas€ solution ftom the c.ushedcells, This adds liqutd to the rylem soluliorr wl dr appears at the cuts$rlace earlier than lf the tbsue had not been crushed. Te3t this €ft€€tby loadlng a leaf tnto the seal in the usual way, but excise th€ bladebefore pressurlzing the earnple. Pressurlze and tighten the s€al. Anyreleased liquid will appear on the cut sutface and rnust be ftomcrushing by the seal because there is no oiher tissue in ihe chamber.CrushinS must be consid€red whenever the flow of water in or out ofthe leaf is impoltmt esp€€ially in pressure activated seals. Sometimespressur€ chambers are used to study the rate of wate! r€leas€ froml€av€s (Boy€r, 1974 Koide, 1985, Tyre€ el rt., 1981) and care should b€taken to avold pressure activated seals in such studi€s.

BRANCHESFor woody branches, lt rnay be d€sirable to strip away tl€

ttssues outside of the xyl€m for a short length so that only woody tissueis ins€rt€d into the eeal. R€gardless of the length of the b.affh, €ndoseas much as possible tn the F€ssute chambe!.

Brancll€s contain a significant amount of nonleaf tissu€s suchas ptth, codex, cambium, and so on. These oftm equilibraie slowlywith the vas.ular tissue and pessure r€adings may be too rapid torcomplete equilibration. The eff€ct can be demonstrated by m€asuringth€ water potential of the intact branch, tn€n lenovlng €ach leaf/placing Vaselhe ovef the cut surfaces to retard gas €ntry, and repealinglhe measutemht ff the branch without leav€s giv€s a water potentiardifferent from that of the intact b.anch, equilibration of the entu€branch did not occur during the intact measurement One must thenchoos€ whether a "l€af balancing pressure" or 'branch balancingpressure' is deshed. The leaf balancing pressure is usually achiev€d inminute! but the whole sample balancing pressure may requi.e hours or

ttttT

t

ilril r

l ,l ,

I1

l

ROOTSRoots are often too fragil€ tomeasure individually unless there

is €xtetrive s{ondary thickening. Therefore, one usually uses a wholeroot sysiem afte. deta.hing the shoot and s€als the stump of thedetopped stem in the chamber. Remove the root mediun by placinglhe root system in a water vapoFsaturated glove box and gendyallowing the medim to fall a ay. Ass€mble the chamber withoutexposing th€ roots to dry ak. Be sure that the chamber has b€enpr€humidified and note the balancing press'ire in the same fashion aswith leves or branches.

ROOTS IN SO[5Itis possibl€ toobiainan averagewater potential for a rooFsoll

complex by leaving the soil attach€d to the root system. Pressurize thesampl€ in the same way as with other tissues.

Significant gradients in wat€r potential can b€ pr€s€nt in th€root-soil compl€x, particulady next to the root surface. The movem€ntof water during pressurization collapses these gradients by forcingwat€t fron the bulk soil to the root surfac€. Thus, pressur€ readingstend to b€ w€ighted toward the potential of the bulk soll. Also,pressurizing wet soils can force water into the intercellular spaces of theroot tissues qdth unpredictable eff€cts (Passioura, 1984). There nay besalt gradients next to roots when transpiration is rapid (Kramer andBoyer, 1995) and thes€ can aftuct the pressure chamb€r readings.

As with l€aves and brancher, the pressure chamber measuresthe tensior arising ftom the mahic potential in the apoplast (Eq. 24).However, an efldodermis s€parate! th€ stel€ from the cortex and hasCasparim skips that create a hydraulic barrier, and water probablyflows mosdy throrgh the protoplasm at this barrier. Thus,lhe apoplastt€nsion measur€d with the presslre chamber nay €xtend only lnto thest€le, and the cortical apoFlast may be under much less tension.

Becaus€ soil conhins solutes that affect the watff potential ofthe roots, the selectivity of the root system for *at€r is important.Assuming completely s€lectiv€ ioots, the watei polential of the root tsobtained by determining the osmoti€ potential of lhe solution in theroot xylem and adding the makic potential of the stele of the roots (Eq.2.4). If the Casparian ships are not completely s€lective, however, a

t

I

-

i

-!

I

3332

i - tI I

: l _l r lt l- t -r ; ri ; ri , li l r

M.asting Weter Star$

corre.tton may need to b€ appli€d (s€€ Significance of ReflectionCo€fffcients, Chap. 4).

Roots in hydrated soils often will exude liquid onto ihe cutsurface without any pressure appli€aHon. This is a normal expressionof root pressurg and pr€ssure chamber measulernents .amoi be made.In dry soil, no exudation takes Place and Pressur€ measur€ments

Measrr.ring the Components of the Water PotentialWith th€ pr€ssure chamber, the components of the water

potenHal can be measured in th€ tissues su olnding the aylenprovid€d lt is ensured that th€re is equilibrium b€fwe€n the ryl€m andthe rest of the tissu€s. The nethod allows lhe Hssues io iemaincompletely intact in the eicised sampl€, which is an advanta8e.

O6MOIIC POTENTIALWhen solute is added to waler, the fr€e enelgy of the water

decreases becaus€ the solute occupies space otheMise occupied bywater, diluting the water and d€creasing ils chemical potential. Asdiscussed in Kramer and Boy€r (1995), Yj approximates -RTCS= -R'In lVfor dilute ideal solutions of notdissociating solut€s. The Cs is the molarconcmtration of solute Siven as r/v (mol n'3 of water), R is the 8asconstant (m3.MPa.mol1K1), and T is the iemp€rature (K). Thisrelationehip shows that Ys is propornonal to th€ solute concenkatio&and the pressure chanber can be ued to removewater from the celltl€aving the solutes b€hind and making the cell soluHon moreconc€ntrabed. As long as temperature is constant and the number ofmoles of solute n is a constant inside the cells, -R?n is a conrtant (i):

V.vs = -RTtr = k, (2.10)

Rearranging Eq. 2.10 gives th€ equation of a line with a slope of (1/k):

The pressure chamber removes water fron the cells byoveryressuring th€in md/ as the wate! mov€s out/ th€ sotution in thewalls is diluted and its osmotic pot€nrial approaches zero. Equation 2.8b€comes -Pj,s = Yae, add, with water loss from the cells, turgorbecome6 z€ro so rhat -p$, =yu4 =v,tpt Replacing vsin Eq.2,r I with

- 1 = 1 . v .Pgos *

(2.12\

T I! i

Thus, in turgorless tissue, the $motic potential of the cells is dir€ctlym€aslred with the pr€ssure charnb€r, and a plot of -llp@ versus ygiv€s a skaight line of slope (l/t) because of th€ ioncentrariondependmce of the osmoric porential (Fis. 2.8).

PROCEDURE1) Appry an overpr€ssure to the tissue, drive out a small volume ofwater, collect the volume in a syringg and note the votume.2) Adiust the pressu€ to th€ new batance, and nore rhe pr€ssule_ Thisgiv€s tlle pressue at the n€w warer volume in the celts after removins

3) Repeat 1) and 2) for about lO warer contenrs.4) Determtne the tinal water cont€nr of the tissue by reteasing thepr€.surc/ r€rnoving the tissue, excising the velns and any stem,weighina the interv€inal tissue, and ov€n drying the tissue.5) The diff€rence bena'€en th€ weighr of the inrerveinal ttssue befor€and afier oven drying is the volume of war€r in rhe lissu6 a h€ end ofthe prsssure s€ries (th€ veif,s and stem are usually removed becausetlcir water content is considered to be retativety consrant).5) Add €ach removed volum€ to the volume tn the tissue at the end ofthe pr€sswe s€ries and expr€ss €ach sum as th€ r€larive water cont€ntat each balancing pr€sure (Rtchre., 19%). The relative water contenris r€lative to the maximum wat€r conrent in fully hydrared tissueeJeressed as a percenrag€ (s€e Fig.2.E). The relativ€ warer content isa measur€ of Y in Eq. 2.12.7) PIot -1lPr!s versus the relative warer content (Fig. 2.8). The initialpari 0ow pressur€s) is not shaighr, but th€ finat part (high pressures)

a -

(2.17)

34 Meefui,g w4r2, StdtttTttttrL

tt

35

Fiarr€ r.3. PF$ua voluDe deEmimtion for ! Tda bFnch in a pn$u.e.himber, The relative warsr ontenr b the volume of warer in the iissueRlative to tlBt in a tully hyddted $mple, OvelPre$@s r6ore warer froEtlhe lef in sieps, and he baldcing prcsure dd rehoved volutu arc nobd ateach step Gndividual data points). Ihe rctative warer dror is derldired bymesu.in6 rhe volum€ Mainiry afrer a[ ovetPE$urd are finished dd a.ldingthe volumes that were rdov€d by ea.h ov€rpre$uc. lh€ dashed ]ine showsthe_liner rel.iiffihip g@ehed by the osmoric potenti.r (y,@/ ks,2.11 ad2.12). Exkapolation to rhe arG o! rhe nshr Eives the oshoric iorendal at 1mZ,rclative waler @nleni, and to the ais above Eive, the apopjasi votume, Ihe.udilirear p..t of the pre$ue.vohme Elation (on the tithi at hiSh .etarivewale. @ntents) shows the eff€cr of L\e osmotic poiendat plus rhe tu€orprsuF (Yr{d + Yprp, iside the elL. Il|e rPse, delermbed hom lhe dashedline is subF.cted from the vJ''r + vd, ro ave rhe tur8or Ge Fi8, z9). DaEfrom l. S. Boyd (upublished).

indicat€s the osmotic pot€ntial at any oth€r water content (Fig. 2.&dashed line). Extrapolatlng the straight line to the X axis shows theeolume of waer remaining after all the protoplast water has beenremoved (P@ becomes in6nit€, Fig.23). The remaining water is thevolurne of the v,all and xyl€m water (apoplast volume), considered to

ASSL'MPTIONSOther ways of analyzing P.v daia hav€ be€n sugS€sted but

these are generaly less satisfactory than the approach in Eq.2.12 Gyreeand Richter, 1982). All m€thods r€st on the assumpdon of a constanrsolut€ cont€nt in the cells, and Kikuta and Richter {1988) point out thatthe cont€nt may not b€ constant if solutes are generated by th€ cellsduring pressaization. This is not a probl€m with most tissu€s butwheat leaves appear capable of enough soluie A€neration to cause anerro! (Kikuta and Richter, 1988). In g€neral, th€ cell solutions areassumed to be so dilute that conc€ntrations can be used Instead ofactiviti€s to expreGs solution properti€s (Tyre€ and Richt€r, 1981).

Caution needs to be us€d in the extrapolation of the straightline. The extrapolarions in Fig.2.8 assum€ that all thewater is releasedfrom the cells and none from the walls and rylem. The wall pores andxyl€m usually are r€asonably rigtd except in young tissue (Iyre€, 1976),but stgniffcant wat€r releas€d by thes€ structlres can causeextrapolation erlors. In particular, the extrapolation to the x axi s is longdd thus there is a considsabl€ degr€e of uncertainty (s€€ Changes InXylem Dinensiois).

The method also assumes that the c€ll walls around the xyl€mare not rigid ard wili collapse into th€ cell compartmmt withoutlesiste.e as the tissue dehydrate.. In tissu€s with stiff walls, thecollapse may be resisted and .P@r may not equal Y,arr, nece5sitating acorrection (see Nesauv€ Pressuie! Inside Protoplasts).

You can te6t wh€ther any of th€se problems aff€ct you. data bycalcrdating YjGtV at various V, that is, Yro) multiplied by the relativewatet content at atry r€lative wat€. content. This i! a measure oI thesolute conbent of the tissue (Eq. 2.10) and establish€s whether the solutecontent has .emain€d constant over a wide range of water contents, asrequired by th€ theory. l! also shows whether the xylem and cells

Il

r r

I _ t; 1

f 11 l

Lt! l

l tb€comes shaight. The srraight po.tion represents rhe region *here Eqs.2.11 and 2.12 are followed and -r/P@s- 1/ys!D) or, in o$er words,wh€re .Pr,, dir€rtly measures the ismotic pdlentiat ot the rissue.Extrapolating th€ skaight porhon of the Iine to orher wat€r conrenE

.- \

-_Lp@ropt[r

ApoptDt

t36 M..fl'ing lAlaa.r Stats

solute next to the surfac€s. The wettability attracts water from the airand any :iquid wat€r and, b€cause water forms skong bonds with otherwater molecul€s/ the pores in the maktx tend to Rll. Eleckicallyconstrain€d ions n€xt to the pore surfaces also move water into thematdx with osmotic-like force. As a consequence, the water content ofth€ matsix can b€come very large. Pr€ssures are generated next to thesu aces and the whole matrix can swell.

37

Ifollow solutlon b€havlor. The Y5O)v should flotskalght lin€ portion of Fig. 24. It is vrise to mak€m€asuremeds of vf

Another fuatur€ of the method requiring

vary alonS tl€this test on all

caution ls lhe

: iL .

Il

lII

b 0.8

F

l

I

.t

..

t

tfL

titI

corciderable flne needed for making a series of overpreisur6. It istmporlant to us€ compressed air so ihat oxygm is available to the dssuedurtng th€ m€asurements and to minimize evaporatlon by humidilyingthe chamber, collecting the !€mov€d liquid quickt, and deLeminingeach n€w balanclng pre$ur€ quickly Out be sure to walt loflg enoughfor a t ue balance). Avoid pr€ssures above 3 to { MPa if possiblebecaus€ cell membranes can be disrupted and release cell solutes to thevasculai s'€iern/ which will cause the plot to deviate toln llnearity. Ifyou have difficully achi€vlng ltnearity, measure the osmotic pot€ndalof th€ soluBon exuding from the cut surtnce. An osmotic pot€ntialsigniflcantly below zelo at high presslres means rhat c€ll membraneshav€ broken and cell solutes arc being released to the apoplast In tltrssltuadon, the measurement must be abandomd.

TURGORTurgor results in pfessure on the cell solution, and the

balanctng pressue3 in a pressure chmber are l€ss than in comparableturgorl€ss tissue. When nilrgor is pres€nt, the balancing pr€ssuies donot follow Eqs. 2.11 and 2.12, and a plot of Eq. 2.12 curves downwald(on the right close to th€ Y axis, Fig. 23). By comparing thedownwardly deviating line (wtuch d€scribes -1lPF = 1/ (Y4o1+ Yno)wlth lhe linear exEapoladon (which describes -I7P@ = 1,i %(,t, iheturgor can be determined by difference (Eq.2J), andthe turgor can befoond at any dssue water content (F19.2.9).

MATRIC POTENIIALMatric potentials occur b€caus€ the surface of a liquid tlas

ProPeitiec that dtfi€r from those in the interior. In any porous medillmwettable by wat€r, solids extend the surface so that a large! share of themolecules hav€ surface prop€rties. The wettability results mosrly fromhydrogen bonding berer€en water and OH groups on the surfaces andfrom surface rharg€s that attsact the watef dipole. The surfu€e chargesalso attract ions ln the water. The total effect is to consrraln warer and

I I

water volume (cm3)5.2 5.4

e2 94 96 9A

Relal;ve Wat€r Conient (%)

EigsE ze. Th€ tu8or (Y,.,j) dd voluhe (D of water h the eUs of lbe TantbEnd, shoh in fu. 24.

,llc slop€ drrp{p/dv was msru€d ar ea.h vplp, and

v. dd the elastic modulus (.) als wB @ltulat€d. In th€ eEmple shoM, theslope b dia@ tt'touth . point and e = (0.380 MPa/0.180 cmr).s.32 cnr = 11.2Mla. Data fbm l. s. Boys {upublish€d).

For most plant cells, the walls are the majof site of the matrlcpotential (Boyer, 196ft). Th€ s'rrfaces are highly wettable, and waterfills th€ por€s. Because of the small por€ diameter/ tenslons (negative

E-11.2M

!

F:i!

ti\

,iilti,;l. l

l1

I

38 M.eerina Wata Stetws- ; ,

i I

r:1-t- ' l r

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t

pressures) to about -56 MPa can be prese.t wthout draining the water.Of course th€ tension on po.e water varies berw€€n zero and -S8 Mpa,and the variou! pr€ssures neasur€d with a pressure chambe!demonshate thls principle. Accordingly, pressure chambers give adirect measure of the makic por€ntial in the wals of rhe livine tissue(Y,f,r in Eqs. 2.4 and 2.4.

The pr€ssure chamber also can measure th€ matric Doiential inleaves killed by freezing and rhawjng (Boyer, r967b); which issometimes ls€tul(s€e Figs.2.10 and 2.11). In this situatioo th€r€ isnoturgor and the osmotic potential is virtually without effe.r bftause ther€are no membranes. The only force holding warer in the tissue is thematric potential r€sultlng from surface jnteradions. As pressure isapPlied to the system,water and solutemove out of the cels andexrdefrom the cut end of the petiole. The walls r€nd to collapse inro the cellcompartment and resist collaps€ according to rhe strength of rhe wall,but the measur€d matric porential is accurate r€gardless ofhow muchthe walls collapse.

The forc€s holding water in rhe dead matrix arc of rhe samephysical nature as thos€ holding warer in ahe apoplasr of living iissue.How€ver, fr€€zing and thawing iood rhe wals with p.otoptast solurionand ahange the matric polential of the apoplasr to a higher value (Iessnegative) than in the living tissue (Boyer, 196 ). Only when the waterconlent in the frozenlrhawed tissue is the same as in the wa s oftivinEtls.ue do the matric potenriats approach rho* in the living Hssue, a;pointed out by Boyer (1967b) and passioura (1930).

With frozenlthar^,ed tissue, it is usua y desirable ro ke€p thepetiole ative so that ir retains its usuat shengrh in lhe s€al in lne;p ofthe chamb€r. Wrap the periole in wet cotton while the leaf blade isbeing fro"€n and, with carc, a freezi.g tim€ can be found thai allowsthe blade to be froz€n but not the petiole. Mount the petiole h the s€alof th€ pr$sure chamber in the usual way and pressurize the tislueslightly. Indeasing pressures will caus€ the cell solurion to €xude onrothe cut surfac€ and decreasing pressures witl cause the €xudare to moveback into the tissue. Because the matin is flooded 1'ith cell solurlonreleased by freee,/thawing, rhe pressures afe small and rates of€xudahon are slow.

ELASTIC MODULUS OF PLANT TISSUEAs turgor is generat€d by the walts pressing on the ceu

cont€nts, the walls ar€ under shain much as th€ cover of a ball comesund€r shaln at high inr€rnal pressures. The strain 's etasric andrcv€rsible. The elasticity of rhe sttain€d cell wa can be m€asu.ed atvalious water conlents using a pressur€ chamber {Fig. 2.9). B€causepressure applies a force 'n tlue€ dim€nsions, th€ elasdcity is det€rminedas the bulk modulus of elasticity (e, Mpa) defined by

(2.13)

(2.14J

I" = o * # ' ,

av^, = ".{.

Th€ bulk modulus is a proportionatihy conshnt indicating how muchchange occurs in th€ relative c€lt volum€ dyly when the Dressureinstde the ceu chaflges by an amount d\y,rD,. The more elas6; thp cetlwall, the smalls is the value of e. The J is marrmum wnen turqor isat its man'num but becomes zero r,rhen the turgor is zero for; cetlhaving thin elastic walls. Figure 2.9 shows this effect usinq data fromFig. 2.8. Because rhe pressure chamber measures votu;e-averasediissue Yerrl rhe r is a volume-averaged paramepr.

precautions

SAIETY

. Pressur€ rhambe.s can be dangerous because of the large gasvolumes involved. There can be explosive release of the tissue from ihes€al or failur€ of a chamber compon€nr Atways work at rhe side of thechambef and n€ver view rhe cut surface from overhead (Fig. 2,6). Toavoid the hil(e of a chamb€! componenr, prcssure test rhe seat€d andwater-filled chanber before the firct us€ as described earlier. A[componenis should withstand pr€ssurcs at l€ast doubl€ the maximunexpecled to be used.

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DE}IYDRATION DURING I,OADINCExcised tissu€ €ontinually dehydrates. ln normal air, its

potendal de!'reas€s significandy in a few s€conds. It is generallyimpos5tble to prevent this completely but piacing th€ tissue in aBaurated atmosphe€ and ke€ping it as isothermal as possibleminimiz€s the probl€m @lant tissue produces small amounts of heatmetabollcally and thus los€s water slowly even in sahrated air).

wth thes€ principles in mind, one mustwork swifdy and placethe pressure chamber next to the plant to minimize *le tirne betw€eflexcision and chamber loading. Becaus€ th€chamber contains saturatedair,loading reduces the rate of€vaporation. If loading tim€! are longerthan 10 s€c, quickly place the tissue jnto a glove box with a saturatedatmospher€ so that loading can be extended without dehydratirg ihettssu€. A conv€nient glove box can be consEffted from a Sttrofoambox with a Phnghs sheet on top (see Chap. 3). An alternative is toqutckly place a thin polyethylene bag around the sanple inmediatelyprior to excision (Turner and l-on& 1980). Seal the bag loos€ly, sbade,excise the tissue, and ins€rt the sample into the seal of rh€ rhambe! top.Apply pr€ssure to the tissue in the bag.

DEFIYDRATION DURINC PRESSURZ,\TIONAfter seali.g in the chamber, the rissue is exposed to

dehydration hom the dry gas used to pressurize the sample. Pressur€chambers should b€ humidified by bubbling the incoming gas throughwater in the bottom and pasi walls lined with wet filter paper. Thewat€r in the bottom not only humidifies bul also cools the incomingcas.

A ba{fle can be inserted in the prersur€ chamberjust above theliquid to prev€nt water droplets fron splashing onto the tissue (Fig.2.5), A sinple baffl€ can be made fiom the bottom of a sultably sizedpolyethylene bottle which is forced into the pressure chanb€' and h€tdby the walls just above th€ surface of the water. Place ofle or two smallholes in this fals€ bottom to allow compress€d air to mov€ into themain body of the crhamber. From anothet plastic bottle, cut a nat discthat is slighdy smalle. than the inside diarn€ter of the chamber. Placethis disc on top of the false bottom. The incoming air will bubbtethrough th€ wat€r, pass lhrough the small holes in the fals€ bottom, andgo around the disr to €nter the main portion of the chamber. The disc

breaks up any water droPtets that ar€ blown through th€ holes. If yourprcssur€ chanber is not de.iSned for gas to enter the bottom, ins€d atube insid€ the chamber to lead the gas from the inl€t to the bottom

MEASURINC AT EQUIUBRIUMPressure chambers genenlly use large tisEue samPles A€caus€

there are wat€r potentlal gradi€nts in Plants and soils {s€€ Boy€r el 4i.,1980, for an examPle), it is imPortant to make m€asurem€nts slowlyenough to allow the Fadients ln the samPle to equllibrate and form avolum€-aveiaged Pot€ntial. Fo! the most part, gradient! in leaf samPleswi! equllibrate adequat€ly in 10 min. In larger samPl€s (e.9., branches),

Pressure chanbers are usetul for making water Potentialm€asurements rapidly, and it is tempting not to wait for a truebalancing pr€ssue. Ind€€d, some Pressure chambe$ are designed toallow air to enter at a st€ady ral€ and be shut off at th€ filst sign ofliquid on the cul surface. This method ts not reiommended b€caus€ itdo€s nol allow balancing pressur€s to be achiev€d- Mor€over, the tlssuecan be heat€d by raPid air edtry lnto the chamber (Puribch and Turner,1973). To ensure that equilibrium occurs, the pr€ssure should beadjusted to give a stable, flat water film at the cut xvlem surface. Thefflm shoutd not gow or sh.ink, indicating that water is neither exitingnor entering ihe tissue.

OVERHEA'TINGWh€n pressur€ chambers are used continuouslv at hLgh ambient

temp€ratures, sPurious readings can result. The problem is most often€ncountered at t€mperatures above 3Cr"C in the fi€ld when larg€amounts of hst are produced by frequent €omPresston of the incominggas (Puritch and Turn€r, 1973). The errors in the readings are causedinitially by €xcessive d€hydration of the tissue and ultimately byb'€akdown of the menbran€s b€cause of the heat. The breakdowncauses readings to be ioo low. The eff€ct can be minimized bywrapping a wet paPer towel around the outside of th€ chambef toaltow evaporative cooling of th€ charnber walls. Also. Pressurizingslowly and bubbiing the gas through water on the.hamber bottom wtllhelD keeD the chamb€r cool.

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AVOIDING TI5SUE HYDRATION BY SURFACE WATERPlant tissue sometimes may be coated with dew or have

dloplels of water on the surface. Wh€n prelsurized, the liquid waterls forced into tfu tissue and rais€s its wat€r potmtial. If water ispr€sent on the tissue to be pressurized, blot the Hssue dry. If possible,allor", some rine for th€ tissu€ to dry complet€ly b€fo'e sampling.Avoid tissue contact wtth w€t filter paper on the walls by fotding iheleaf loos€ly afld holdtng the folds in place with a rubber band or tape.Water splashlng ftom th€ bottom of the chamber can be aeoided byconskucting a baffle in the botton (Fig. 25).

ANATOMICAL ERRORPressure chamb€rs use the cut surfac€ of the sanple as a

r€ference posltion for the measurement. Therefore, never r€cut thetissue after r€moving the sample f.on the plant. Tissu€s complising thecut surface can have hollows o. pith thar allow the xylem solution tospill over and be trapped after u ffrst appears on the cut surhce (Boy€r,1967a). More prdsure is necessa.y ro maintain the liquid at the suriacein this situahon ard the reading will be spuriously high. In rhis case,pr€ssure readings are consid€red to be relative rarher than absolut€measurements (Boyer, 1967a).

B1JBBLING ]N XYLEM SOLUTIONIn a 9pical sample/ pressurtzanon does not cals much

movement of gas tfuough the tissue. Of that appendng at ih€ cutsurface, most trav€ls through the intercellular spaces. A small amounimoves tkough the rylem probably becaus€ gas has been forced intosolution at high pr€ssure and bubbles out of solution as armosphericPressur€ is €ncountered.

Wouflding of p.essurized samples can allow gas to mter th€vas.ular tissue more rapidly. Liquid in the xylen appears premabrelyat th€ cut surface and bubbling can be so severe thafth€ water films atedifffculi to obs€rve or are dissipated as a fin€ spray. wlle|ever possible,avoid using wound€d tissue for m€asurements. lf worhds cannot beavoided, it is sometimes possible to coat the wounded area wirhpetrolatum {Vaseline) and d€crease th€ rate of gas mrry.

Xylem solutions som€times contain compounds that havesurfactant prop€rtier/ and the solutior foams on the cur surface. If

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foaming is excessive, rhe m€asurement must be abandoned. tf tt ismoderate, imr€ase the plessure until liquid accumulates u.d€rneath thefoam m the cut surface. Then withdraw th€ liquid into rhe tissu€ anddetermine th€ balane pr€ssur€. If the foam {ails io break up durtngiNs pro.edui€. touch it with your ing€r.

CIIANCES IN XYLEM DMEMIONSMatl1re plant xylem in s€condary tiisues withstands large

pre6sures. Immature xylem/ protoxylem, and metaxylem do not nor dotissues around the rylem, md th€ir volune decreas€s as warer flowsout of the cells. The xylem is oft€n und€r large bensions, and when thetissue is excis€d and pressu.iad, som€ of the xylem can changedimensioff or can €xchang€ wate. with the surroundi.g tissues,particularly if the sample contains g.owing tissues. To avoid erorcalsed by thes€ effects, the xylen should b€ pre.suriz€d atong as muchof its lmgth as possible to force rhe surrounding ttssue ro th€ samewat€f cont€nt during the measurement as tt had when the xylem wasunder iension in the plant. You can t€st wh€th€r these effects ar€ aproblem by changing th€ posirion o{ the sanple in th€ seal. Usealt€lnating measur€m€nts with most of the rylem pressurized (most ofst€in insid€ of chamber) or the least anount of rylem pressuriz€d (mosrot st€m outside). If balancing pressures are l€ss when mosr of rhexylem is pressurized, stem tissues are *ns ive to prcssure and need !o

POTB{TIAL GRADIENTSln addition to the need to let potential gradientg equilibrate in

the sample, there ar€ long{istance gradienrs thar need to b€ considered(e.*, Boyet et d.,79AOl. Th€ two largest cof,tributors to gradients arethe distance of ihe sample ftom the wat€r supply and the deg.€e ofillumination of the sample. In most cases, the variability behveensampl€s can be naik€dly reduced by knowing where gradients €xisrand by sampling in the same part of th€ gladienr. Comparisons orpot€ntials between planrs under thes€ conditions requlre samples fromthe same pa( of rhe canop, similarly iltuminated, and at a simlarstag€ of development

Depending on th€ res€arch qu€stion, one should sample ar anappropriate position in the piant gradient. For exampte, to meajure the

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44 M.es!;tg Wat t Stats

water potential of leav€s that are doing nost of the phorosyntheric workfor the plant, sample at the top of the canopy using leaves that arerecently fully expanded and oriented p€rp€ndicular to the incominglight.

Enclosing leaves in gas exchange clvettes poses specialproblems becaus€ the enclosure generally changes the l€af wate!potential. Always measure the waler potential of the leaf in the cuvefteif you wish to relate leaf performance in the cuvette to the wate! statlsof the leaf. Avoid sampling leaves outside the cuvette and assumingthat tlle watq potential is the same as inside rhe cuv€tte. If you l:ffiorsacrifi€e th€ leaf ln the cuv€tte for the pressure chamb€r m€asurem€ni,considd uslng a thernocouple psy€h.romets on a small leaf sample(s€e Chap.3).

NEGATIVE PRESSURES IN5IDE PROTOPLASTSPressu.es rise and fall in cells according to changes in cetl water

cont€nts. Turgor is high in the protoplasts wh€n ware. contents arehiglr and tensions a.e small in the apoplasr. A! water contentsdecrease, turgor diminishes and tmsions become grarer. A questionthen aris€s: if water cont€nts continue to decrease, do€s turgordisappear and tenslon begin to €xtend into the protoplasts and, if so,do€s thjs affect pressure chamber measurem€nts? The answ€r appearsto be yes for both qu$tions under particular conditions.

The eff€ct depends on how much the cell wals resist collaps€under tension. In most tissues, the cell walls are thin and follow iheshrinkage of the cell solution withour significant resistance as watercontents decrease. In sunflower, for example, the walls occupy only 9-12% of the total cell volum€ (Boyer, 1967b), and they tmd to fold afldfollow the shrlnklng protoplasis as the cells dehydrate, which is cl€nrlyvisible under the €lectron microscope (Fellows and Boyer, 1978). Thissuggests that they do not exert a significant counierfoice ro rheshrinkag€, and Fig. 2.r0 shows that the volume changes read:ly infrozenlthawed tissue, and the matric potenrial (Yhl-, is smatt whocompared to that in the living tissue (Y,rdr). The frozen/thawed tissuehas no turgor and the smail Y,i, indicat€s that rhe watls do not resistsfuinkage as water is lost from the matrix. This tack of muchcounterforce continues until r€lative waler conr€nts tattbelow 1Gm%.Accordingly, at a higher wat€rcontent of 60%, the free energy diagram

on the righr of Fig. 2.lO shows that \P,r,r is a very sma componentcompared lo PsG in the tiving tissue. Ther€fore, in living sunflower, arrnes€ rugner watercontents, rensions in proroptasrs can be n€gteded forprcssure chamber measurements.

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Earzlo. CornFris t erwen pls@vollftdab i.livin8 4d fioalrhawedreive orsuntower. h hvina tissue, rhe presrure ch.hber i_p-.J mesuB fiem.hj. potentjal ttrr!) of rhe apoplast (Eq. 2.A. In frcrnl6';;ed bsque. thePr6$E_dEmber tudws rhe tuEic pollntint y,{lj of rhe 668 rcnlivhSti$@. The Y.rl, is hostly ou!€d by the eU waus. Not€ ihat betwq aO a;]ry.

RwC: v-ll) chdS6 tiid€ hd Lr8€ anour! or hrubo q be rcmvedhd@trt that tE hus €lt p* into rhe shnr*ingeU @mp{brents. Theellwa,rs GUpy 9-l2olo of the lotat cU volume dd thci haE ljtrle rcsi3tan e io theshnnla8e. Th! diaEEh o the nAl giveq th omponenB measuEd by lhepGsu e cnqber h I ivinS ri5sue .i 6elo RWC ta kd lrom $e gr.ph 6 rlP leA.m nvhE Esu.,a 6wo water onrent is uable tosmerure nl4or, ad rhe.p

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How€ver, dssues having thick cetl watls behave difi€rmrly.Figure 2.11 shows thar in rhododendron, (he apopldsr votume was i6to 28% of th€ total water volume jn the tissue (Boyer 196 ). The wallswer€ r€latively rlgid and resisled cotlaps€ and in consequence rh€frozenlthawed tbsue showed a substantiat lr,(, at all water contenrs.Therefor€, when turgor disappeared in livins leaves, rhe teruion in thexylem extended into the su ounding protoptasts.

A simple rsr for thjs effe.t is ro d€t€rmine wheth€r ly./_,.y iscollslant over the linear.dge ofvoluftes in Eq. z.tz (see Mei[iringtt€ Componmrs of the Water pot€ntial, Osmoti€ potential). If it is noa

l lossPle caus: E.: tension in rhe proroplasr comparrmenr caus€d by

lhe resishnce of ell walls to coltaps€ doring dehydration ofthe rissu;.rne Fnsion can be subtracted from the pFs {o give a more accurateosmotic potential (Fig.2.r r). Free2€/rhaw-a comparable s.mpte and,from measurem€nts of y_t] at various relative warcr contents as shownin Fig.2.11, subrract the pre$de u*d ro m€.sure y_il from p-. in theliving tissue (Fig.2. on the righr). rnis subtractiii'n snour.d gtve acor€cted V,f).y that is constant and rhus provide a true V"1r) ai each

. It should be recognized {har in the living tissue, the rension

Deg'ns to*tend into the protoptasts onlyafrer hrrgo, decreases lo zero.ryree (ryl6) recogni4d rhar this r€nsion coutd be pr€s€nt butconsid€red it to b€ a "n€gativ€ tu.gor" (atr unfo(unate misnomer) tharwas negligible in most cas$. His test was bas€d on the stsaightness ofthe slope of the line B€nerat€d by the data, as in Fiq. 2.8. H€recognized that a bener test would have b€€n yrpr. y = k to indtcate tharthe solution in the celts behav€d idealty. Ho;;er, th€ l€lr coutd notbe made because th€ rcquir€d dara w€re not availabte. For rhem€asuemenrs shown in Figure 2.& how€ver, the t€st could b€ madeand Yj(Ptv = & at all relarive water contents showing that neAarivepressures we.e not a factor.

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figlre 2.11. Sae cmparien .s in FiE. 2.10 bur {or rhododmdrn iGrlail ofsunfl@e. Rhododendrm leaves lEve stilfer eI walls {25-28% ot eII vol@]thd sutflo*r l9-12% otetl volume), dd rp.r' ir. $gnifi@nrcodponot of-r'3,s ,t mst wahr qbnts (e.t., diagEn on riSht firr 60% RWC). A, shorho the n8h! tP-a,) onhibutEs e huch l,o the me.suMsr ot -p-. rhir ly-/,,must be NbE{l.d f6m - Prs ro obt in a valid nersure of v,,o, i" li"-6 ti.*".This be@me ihpo.tanr f; hosuRmmB of t6j h dy ti;u" havinS ,igidcex warb. ror d test to deFmim wherher rhe rubtracrion is nees.w, e. thetext Data ftom l. s. Boyef (upublished)_ I

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