mesophyll of soybean leaves at various developmental stages1

Plant Physiol. (1991) 97, 1306-13160032-0889/91/97/1306/1 1/$01 .00/0

Received for publication May 8, 1991Accepted June 20, 1991

Protein Compositions of Mesophyll and ParaveinalMesophyll of Soybean Leaves at Various

Developmental Stages1

Stephen F. Klauer, Vincent R. Franceschi*, and Maurice S. B. KuBotany Department, Washington State University, Pullman, Washington 99164-4238

ABSTRACT

Mesophyll and paraveinal mesophyll protoplasts (PVMP) wereisolated from leaves of soybean (Glycine max) at various stagesof physiological development, and protein compositions of thetwo protoplast types were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Polypep-tides of 27, 29 (previously shown to be storage proteins), and 94kilodaltons were found to be PVMP-specific proteins and werepresent in both nodulated and nonnodulated plants. The 27 and94 kilodalton polypeptides were major PVMP constituents. Allthree polypeptides accumulate as early as one-quarter leaf ex-pansion. Immunoblotting and immunocytochemical studies usingantibodies against the 27/29 kilodalton proteins confirmed thatthey are specific to the paraveinal mesophyll (PVM) and that theyare localized in the PVM vacuole. The 27 kilodalton polypeptideincreased significantly by two weeks depodding, and this accu-mulation was restricted to the PVM vacuole. Radiolabeling ex-periments showed that the difference in relative amounts of the27 and 29 kilodalton polypeptides was due to a greater rate ofsynthesis of the 27 kilodalton polypeptide. The 94 kilodaltonpolypeptide accumulated to a maximum at anthesis, but wasabsent at 2 weeks postanthesis in both depodded and poddednodulated plants, probably because they were nitrogen limited.In nonnodulated plants, it was present through 2 weeks postan-thesis. The results confirm that the 27 and 29 kilodalton proteinsof soybean leaf are stored in the PVM vacuole and show thatthey are accumulated early during leaf development while theyare still strong sinks for nitrogen. The 94 kilodalton protein,previously found to accumulate in leaves after depodding, is alsoa PVM protein and is likely a third vegetative storage protein,although its accumulation appears to be more dependent onexcess nitrogen availability. The results further support the hy-pothesis that the PVM is a specialized leaf tissue that functionsin synthesis and compartmentation of storage proteins.

The PVM' is a specialized tissue that occurs in leaves ofsoybean (8-10) and many other legumes (11, 14, 16). It is adistinct layer of many-branched cells that forms a reticulumat the level ofthe phloem and spans the region between veins.

'Supported in part by U.S. Department of Agriculture grant No.84-CRSR-2-2496 to V.R.F. and M.S.B.K.

'Abbreviations: PVM, paraveinal mesophyll; BSP, bundle sheathprotoplasts; GS, goat serum; MP, mesophyll protoplasts; NC, nitro-cellulose; PVMP, paraveinal mesophyll protoplasts; TBS, Tris-buff-ered saline.

Because of its position, anatomy, and ultrastructural charac-teristics, it has been hypothesized to be a collecting or distrib-uting tissue that could play a major role in the movement ofphotoassimilates and nitrogenous compounds from the leaftissues to the vasculature or vice versa (8, 11, 16). It hasconclusively been shown to be involved in protein storage (9-11, 13), and there is evidence for specialized partitioning ofenzymes involved in nitrate and ureide metabolism as well asa function in amino acid and ureide compartmentation (5,7). Its role in photoassimilate movement and metabolism hasbeen explored (6, 10), and there is also evidence that PVMmay play a role in collecting amino acids transported to theleaf by the transpiration stream (3).Wittenbach (21-24) reported earlier that specific soluble

polypeptides (molecular masses of approximately 27, 29, and94 kD) accumulated in soybean leaves during vegetativegrowth and increased under conditions of depodding (de-creased sink demand). Two of these polypeptides (27 and 29kD) were shown to be glycosylated (23), and immunohisto-chemical studies showed that they were specifically synthe-sized and stored in the PVM and the bundle sheath cells (13).The bundle sheath is functionally very similar to the PVMand can be considered part of it (9, 11, 14). These storageproteins were shown to accumulate until anthesis and thendisappeared soon after podfilling began, whereas in plantsthat were depodded, the storage proteins continued to accu-mulate (9, 10). It was presumed that the storage proteins weredegraded and the amino acids remobilized to provide nitrogento developing seeds.The purpose of this study was generally to further charac-

terize the role ofPVM in protein storage through the followinggoals: (a) to establish clearly the cellular and subcellularlocalization of the 27/29 kD storage proteins; cellular/subcel-lular fractionations and high resolution immunocytochemis-try will demonstrate conclusively the distribution of theseproteins within soybean leaf cells; (b) to determine if the 94kD protein that accumulates during depodding is a PVM-specific protein; the previously reported pattern of its accu-mulation after depodding suggests that it may also be a PVMstorage protein; and (c) to characterize further the pattern ofaccumulation and disappearance ofthe PVM storage proteins.Little is known about growth conditions (other than depod-ded) under which the proteins accumulate. Because they areconsidered to be storage proteins, are they only accumulatedlater in plant or leaf development, and does the source ofnitrogen impact their accumulation? Our study demonstrated

1306

PROTEIN COMPOSITION OF SOYBEAN LEAF TISSUES

8

6

4

2

0

3 8 1 3 1 8 23Days After Appearance

Figure 1. Growth curves for the middle leaflet of trifoliolates ofnonnodulated soybean plants. See text for details. All data points arewithin 10% standard error.

that the 27 and 29 kD storage proteins are specifically local-ized in the PVM vacuole, that the 94 kD protein is PVM-specific and shows a similar pattern of accumulation as the27 and 29 kD proteins, that all three proteins accumulateearly during leaf development, and that their accumulation isnot dependent on the form of nitrogen available.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Soybean (Glycine max L. Merr. cv Wye) plants were grownfrom seeds in 14 cm pots. The nonnodulated plants were

grown in potting soil, whereas the seeds of modulated plantswere innoculated with Rhizobium (strain 31B 1 0O) and plantedin perlite. All plants were maintained in controlled-environ-ment chambers programmed for a photoperiod of 16 h at250C and a dark period of 8 h at 18'C and a photon fluxdensity ofapproximately 400 ,E m-2 s-' at leafcanopy height.Plants were alternately watered with deionized water and witha dilute nutrient solution, the contents of which varied asfollows. For nonnodulated plants, the solution contained 1mM NH4H2PO4, 2 mM MgSO4, 1 mm NH4NO3, 4 mm KNO3,4 mm Ca (NO3)2, with minor nutrients in the form of 9.22,uM H3BO3, 0.16 AM CuSO4, 18 ,M NaCl, 4.79 jAM MnSO4,0.02 ,uM (NH4)6Mo7024, 0.77 AM ZnSO4, 3.46 AM CuCl2, and9.22 tiM Fe as ferric-EDTA. Nodulated plants were wateredthe first 2 weeks with a solution containing 1.5 mm Ca(NO3)2,1 mm KH2PO4, 1.5 mM MgSO4, and with minor nutrients asdescribed for nonnodulated plants. Thereafter, nitrate waseliminated by replacing the Ca(NO3)2 with 1.5 mM CaCl2.The roots were found to be heavily modulated under suchtreatment. For plants that were depodded, flowers were re-moved at anthesis daily as they appeared.The mature, recently expanded trifoliolates (usually the

fourth and/or fifth trifoliolates) from 4- to 6-week-old plantswere used for protoplast isolation. For the leaf expansionstudy, protoplasts were obtained from the fourth trifoliolateof nonnodulated plants at one-quarter, one-half, three-quar-ters, and full expansion. Degree of expansion was determinedfrom a study of nonnodulated soybean leaf growth over thecourse of 6 weeks (Fig. 1). The middle leaflet length oftrifoliolates number 1 through 5 was measured daily fromappearance to maximum expansion. Data for 10 plants were

PROOPLAST ISOLATION TECHNIQUE

DIGESTED LEAF TISSUES:PAAIWEINAL MESCMLL PUTCPLASTS(POPA, N E SHEATH PROTCEASTS FILTER THRJH(1SP), MESCIPLL PROTOPLASTS MP 8a MESH NET

(1,; APR EIMATELY 2G. LEAFTISSUE / 201L DIGESTICN fIUIM

DISCARDMATERIALON NET

FILTER LRGE PMP,THROUGH 3SP RETAINET

150 X 6 SUPERNATANT 20y MESH CN NET. COLLECTCONTAINS MOST LY NET; REPEAT * WITH PASTEUR

3 MIN. IN MP, BSP WITH 15L. PIPETTE, RESUON6 TEST TUBES NESH NET IF ANO PURIFY AS(1.5 1(b. NECESSARY FOLLOWS:

PEU.ETCONTAINSMOSTLYPPI sorE POMPPUMP ANDSSP

RESUSPENDPELLET AND

LAYER ASFoLLOWs:

DILUTE WITH EQUAL PARTS0.5 ML MAIITOL-NACL 150 X G _ OF MANNITOL-NACL BUFFER,LFFER _ No CENTRIFLUE 150-200 X G.T.5 Mi MIYXTURE OF 2.5 10 MIN. a* Pwp FOR 7 MIN. TO CONCENTRATE

PARTS SUCROSE-BUFTER AND IF NECESSARY.1.5 PARTS MANNITOL-BUFFER _

3 PARTS SUCPOSE-BUFFERAND 1 PART CRUDE PVMPFROM NET.

MP AND DEBRIS

.75 ms PSNITOL-NCL RLFFER

2 xL MIXTURE OF 2.5 PASTSSUCRESU-WFER AND 1.5PARTS MANNITOL-LFER

4 Mi SUCROSE-EUFER ,'Z9. I, RIlTRN4 TO

MAINMIN.

8 _ JcOND 13' STVN PEIC

BSP AND SMALL PVMP (SAVE) TO OBTAIN PLRE MP'S:REMOVE TOP 2 LAYERS OF

MP (OFTEN CONTAMINATED) _ GRADIENT,ML. OP SUCROSE ANNIT DL

BUFFER, CENTRIFUGE-:300 X G. FOR 10 MIN.

,ANY PP STILL SUSPENDED

_rrP, CEL.S, DEBRIS

DILUTE PURIFIED MP'S WITIFUAL VOLUME OF MANNITOL-NACL BUFFER, CENTRIFUGE-1SO X G. FOR 3-5 MIN.TO PE LET.

12

10

E0

0-i

0-0-10-0

-

0- 1 st leaf2nd leaf

3rd leaf16 4th leaf0 5th leaf

-L

1 307

Plant Physiol. Vol. 97, 1991

a0 ..4i ..

7'004

V .F~~~~~~~~~~~~~

lo :,a

_ .' i,,.6fB M A'f

L}

tl.-}

c

t..i* S X \

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I

FFigure 3. PVM protoplasts isolated from leaves of 1/4, 1/2, 3/4, and full expansion (A-D, respectively) and mesophyll protoplasts from 1/4, 1/2, 3/4,and full expanded leaves (E-H, respectively). PVM protoplasts become more variable in size in mature leaf due to inclusion of bundle sheathcells that have matured. The "mesophyll" at 1/4 leaf expansion (E) is mostly highly cytoplasmic cells with poorly developed chloroplasts, many ofwhich are still undergoing cell divisions. Marker bars equal 50 im.

averaged and used to determine the length that correspondedto the appropriate degree of expansion.

Protoplast Isolation

Protoplasts were isolated as in Franceschi et al. (12) withvarious minor modifications (Fig. 2) depending on the phys-iological condition of the plant or the level of leaf expansion.Because it interfered with protein analysis, BSA was includedonly in the digestion medium, and not in the isolation buffers.Older podded and depodded plant leaves were more difficultto digest, so the pectolyase Y-23 (Seishin Pharmaceutical Co.,LTD., Tokyo, Japan) concentration in the digestion mediumwas increased from 0.5 to 1%. A digestion period of 3 to 4 hwas used. The most frequently used modifications of theisolation procedure are shown in Figure 2. Purified PVMPwere diluted with an equal volume of mannitol-NaCl bufferand centrifuged at 150 to 200g for 7 min to concentrate intoa loose pellet.The MP isolation procedure was also modified. The pellets

obtained from a 3 min centrifugation at 150g were resus-pended in 1.5 x 12 cm test tubes with 8 mL sucrose buffer +13% (w/v) Dextran T35-50 and layered with 4 mL sucrosebuffer + 9.1% (w/v) Dextran T3550 as in the standard isolationprocedure, but this was then overlaid sequentially with 2 mLofa mixture of 1.5 parts mannitol buffer and 2.5 parts sucrosebuffer medium, and then 1 mL of mannitol-NaCl buffer.After centrifugation at 1 50g for 10 min, BSP and small PVMPwere collected from the interface between the mannitol-NaClbuffer and the sucrose/mannitol buffer layer. The MP thatwere collected at the sucrose/mannitol and sucrose buffer/

Dextran interface were often contaminated significantly withPVMP after this step. To obtain pure mesophyll protoplasts,the contaminated MP layer and the top two layers of thegradient were removed and a new layer of sucrose/mannitolbuffer was applied. After centrifuging for 10 min at 300g,pure MP banded at the sucrose/mannitol and the sucrosebuffer/Dextran interface. These were collected, diluted withan equal volume of mannitol-NaCl buffer, and centrifuged at150g for 3 to 5 min to pellet. Both MP and PVMP werefrozen immediately after isolation in liquid N2 and stored at-20'C until samples were prepared for SDS-PAGE analysis.

Gel Electrophoresis

Soluble proteins from whole leaf tissues were extractedaccording to Wittenbach (21). Protein quantity was deter-mined using the methods of Bradford (1). To prepare proteinsamples for SDS-PAGE, whole leaf extracts, MP, and PVMPpreparations were mixed with equal amounts of 50 mM Trisbuffer (pH 7.3) containing 2% (w/v) SDS, 10% (v/v) glycerol,and 10% (v/v) 2-mercaptoethanol, and the sample was im-mersed in boiling water for 2 min.

Proteins were analyzed by SDS-PAGE on a slab gel (0.75mm thick) containing 7.5 to 15% (w/v) linear acrylamidegradient, stabilized by a 5 to 17% linear sucrose gradient (4).The resolving polyacrylamide gel was overlaid with a 6% (w/v) polyacrylamide stacking gel. Electrophoresis was performedat room temperature with a constant current of 15 mA/gelfor 2 to 2.5 h. Gels were stained for 6 to 8 h in a solutioncontaining 0.1% (w/v) Coomassie brilliant blue R-250, 40%(v/v) methanol, and 10% (v/v) glacial acetic acid. Mol wt

A.

MO, t d

F.*i

E 40d

1 308 KLAUER ET AL.

I 0.

I

:I.

REP, N.


S To determine completeness of protein transfer, the gel wasstained with a Coomassie blue solution immediately afterblotting. Duplicate nitrocellulose blots or strips from blotswere also stained immediately after transfer with a solutioncontaining 0.1% (w/v) amido black, 5% (v/v) methanol, and10% (v/v) acetic acid for 5 min and then destained for 15min with 10% (v/v) acetic acid, 5% (v/v) methanol.

Immunostaining of Nitrocellulose Blots

NC blots were immunostained for localization of the 27and 29 kD soybean leaf glycoproteins. The antibody to theseproteins was prepared by Wittenbach (23). NC blots were air-dried for 30 min after removal from the transfer apparatus.

1 1 3S 4 2 4 FULL 5

1 1 34 2f 4

... ...

.F FW- m

FU L L S

Figure 4. Coomassie blue-stained SDS-polyacrylamide gel (A) andglycoprotein-immunostained western blot (B) of protein extracts fromPVMP of nonnodulated soybean leaf expansion study. Lanes repre-sent polypeptide profiles of PVMP from fourth tnfoliolates at 1/4, 1/2,3/4, and full expansion; lane S, protein standards. Approximately 1 10jig protein/lane.

marker proteins were run simultaneously using a premixedsolution.

29

40W

®_

Protoplast Fractionation

Concentrated pellets of PVMP and MP were resuspendedin 300 and 500 ML, respectively, ofbreaking medium contain-ing 0.4 M mannitol buffer (as in isolation procedure [12]), 10mM DTT, and 250 ,Mm PMSF. Each protoplast type was thenruptured completely in a Yeda press at 50 p.s.i. Chloroplastswere separated from the cytosolic fraction by centrifugationat 600g for 7 min, and rinsed three times with the mannitolbuffer by resuspension and centrifugation. This gentle proce-dure of protoplast rupture usually yielded 80 to 90% intactchloroplasts. Proteins from the cytosol and chloroplast frac-tions were extracted and analyzed by SDS-PAGE as previ-ously described.

Electrophoretic Protein Blotting

Immediately after SDS-PAGE, an electroblot "sandwich"was prepared according to Burnette (2), and this was sub-merged in an electrophoretic transfer tank containing bufferaccording to Towbin et al. (19). Protein transfer to NCmembrane was carried out at 200 mA for 18 to 20 h at 4°C.

I 1 3'a 1 A

2927

MP of nonnodulated soybean leaf expansion study. Lanes represent

polypeptide profiles of MP from fourth trifoliolates at to full expan-sion; WLE, whole leaf soluble protein extracted from preanthesisnonnodulated soybean; S, protein standards. In panel A, note BSAcontamination (arrow) at approximately 68 kD in lane 1/2. Slightcontamination with PVMP is visible in lanes 1/4 through 3/4 of panel B.Approximately 120 ,ug protein/lane.

I 1 34 2 4 FULL

9Z4

>. 4

9

27

B

1 309

I

14M.-Aft AL A

("-) lwmmmm. so _.4W= M48wm


PVMP

I- A

4

-r - U

Ws yy rE H- W -.

Figure 6. Coomassie blue-stained SDS-polyacrylamide gel (A) and glycoprotein-immunostained western blot (B) of protein extracts from whole

leaves and protoplasts of nonnodulated soybean. Lanes 1 through 3 and 5 through 7 are polypeptide profiles from PVMP and MP, respectively;lanes 1 and 5, whole protoplast extract (WE); lanes 2 and 6, cytosol fractions (CYT, includes vacuolar contents); lanes 3 and 7, chloroplastfraction (CHL); lane 4, soluble protein extract from whole leaves (WLE). In panel B, lanes 8 through 10 represent protoplast and whole leaf

protein extracts that were separated by SDS-PAGE, transferred to nitrocellulose, and immunostained with antibody directed against the 27 and29 kD glycoproteins. Lane 8, MP; lane 9, whole leaf soluble protein; lane 10, PVMP; lane S, protein standards. LS and SS identify the large andsmall subunits of Rubisco. Approximately 100 to 120 /g protein/lane.

Blots were then subjected to a series of incubations, all ofwhich, with the exception of the primary antibody step, tookplace at room temperature with gentle agitation. The blotswere first soaked for 1 h in a blocking solution containing 1%(v/v) GS (ICN Immunobiologicals, Lisle, IL) in TBS (10 mMTris-HCl, pH 7.40/0.15 M NaCl). They were then incubatedovernight in a solution of primary (immunoglobulin G) an-

tibody directed against the 27 and 29 kD polypeptides, diluted1:500 (v/v) in TBS containing 1% (v/v) GS and 10% (v/v)glycerol. This was followed successively by a 15 min rinse inTBS containing 1% GS, a 30 min rinse in TBS containing1% GS and 0.1% (v/v) Nonidet P-40 (NP-40 [2]), and another30 min rinse in TBS containing 1% GS. The NC blot was

then incubated for 1 h in horseradish peroxidase-conjugatedgoat anti-mouse immunoglobulinG (Cooper Biomedical Inc.,Malvern, PA) used at a dilution of 1:1000 (v/v) in TBScontaining 1% GS and 10% glycerol, followed by a 10 minwash in TBS with 1% GS and another wash in TBS containing1% GS and 0.1% NP-40. Finally, the blot was rinsed in twochanges ofTBS, each for 30 min, before staining with a freshlyprepared solution containing 0.06 g 4-chloro- l-naphthol(Sigma) dissolved in 20 mL methanol, 0.33 mL 3% H202,and 79.67 mL glass-distilled H20 (15). A dark purplish-colored precipitate identifies the antigen-antibody complex.

Labeling Proteins and Autoradiography

The middle leaflets of recently and fully expanded sixthtrifoliolates from nonnodulated soybean plants were excisedunder deionized water and the petiolule kept immersed in a

1.5 mL microfuge tube containing water. Leaflets were pro-

vided with air circulation, supplemental lighting, and were

allowed to transpire until half the water in the microfuge tubewas consumed. An equal volume of a solution containing 2mm CaCl2, 4 mM L-methionine, and 20 ,Ci of trans-35S label(ICN Radiochemicals) was then added so that the final con-

centration was 1 mM CaCl2 and 2 mM L-methionine. Leafletswere allowed to transpire for periods of 1, 2, 8, and 20 h afterintroduction of the radiolabel. A unlabeled chase solution of1 mM CaCl2 and 2 mM L-methionine was added as necessary

to keep the petiolule immersed in liquid. At the end of thedesignated time period, the petiolule was excised, and the leafblade soluble protein was extracted as previously described(21). Extracts were prepared for SDS-PAGE, and denaturingelectrophoresis performed as described above. After SDS-PAGE, polypeptides were transferred to nitrocellulose mem-brane and some blots were stained with amido black to ensure

a complete transfer. Identical blots were sprayed three timeswith an even coating ofEN3HANCE surface autoradiography

MP

M97K4

36'8l1w431346

.20.1i_14,2

~-VMV

1310 KLAUER ET AL.

Aim-ss--

kII f

t


Airw -- x o i - -TV%~ ~.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~'l

Figure 7. Immunocytochemical localization of the 27/29 kD storage proteins in soybean leaf from plants after 2 weeks depodding. A and B arephase contrast light micrographs (x870; bar = 10 Mim). C and D are electron micrographs (x24,400; bar = 0.25 Am). A, Region of a small veinin preimmune control. Bundle sheath cells (B) and PVM are highly vacuolate. B, Section from same vein as A but treated with antibody to the27/29 kD proteins. The vacuoles of the PVM and bundle sheath cells are heavily labeled; mesophyll cell (M) vacuoles are unlabeled. C, Preimmunecontrol showing appearance of wall (W), chloroplast (C), cytoplasm, and vacuole (V) of PVM. Note material in the vacuole. D, Section throughPVM treated with antibody to the 27/29 kD proteins. The vacuolar material is labeled; cytoplasm, chloroplast, and wall are unlabeled.

enhancer (New England Nuclear, DuPont) and placed with asheet of Kodak x-ray blue-sensitive film in a developingcassette that was light tight. The film was exposed in a freezerat -80'C and then developed using standard procedures.

Immunocytochemistry

Leaf samples (2 mm2) were fixed for 6 h in a fresh solutionof 2% (v/v) paraformaldehyde, 1.25% (v/v) glutaraldehyde,50 mm Pipes (pH 7.2), and 2 mm CaCl2. They were thenwashed with Pipes buffer, dehydrated with ethanol, infiltrated,and embedded in L. R. White resin (Ted Pella, Redding, CA).Sections for light microscopy (1 gm thick) were dried onto

gelatin-coated slides and sections for electron microscopy werepicked up onto nickel grids.Immunostaining of both was similar. The sections were

incubated for 1 h with TBS (10 mM Tris, 500 mM NaCl, 0.3%[v/v] Tween 20, pH 7.2) and 1% (w/v) BSA to block nonspe-cific protein binding sites. The sections were then incubatedfor 6 h in mouse antiserum raised against the 27/29 kDsoybean proteins (13) diluted 1:50 with TBS and BSA. Afterextensive washings with TBS + BSA, the sections were incu-bated 1 h with goat anti-mouse antiserum tagged with 15 nmgold (1:50 dilution with TBS + BSA), then washed extensivelyagain with TBS + BSA, TBS, and distilled water. Sections onnickel grids were poststained with aqueous uranyl acetate/

1311


NODS 1 z 3 4

PAPA BSP A D P D-A D

2927

Figure 8. Coomassie blue-stained SDS-polyacrylamide gels (A) and glycoprotein-immunostained western blot (B) of protein extracts from PVMPof recently, fully expanded leaves of modulated and nonnodulated soybean. Lanes 1 through 5, PVMP extracts from modulated soybean (13 ugprotein/lane); lanes 6 through 11, PVMP extracts from nonnodulated soybean (30 Mg protein/lane). NOD, modulated; NON-NOD, non-nodulated;S, protein standards; PA, preanthesis; PA-BSP, preanthesis bundle sheath protoplasts; A, anthesis; DAD, days after depodding; DP dayspodded; WLE, whole leaf soluble protein extract. In panel B, lane S (far right) and one-half of lane 11 were stained for general protein with amidoblack solution as described in "Materials and Methods."

potassium permanganate (0.75% (w/v], 0.25% [w/v]) solutionfollowed by 1% (w/v) aqueous lead citrate, then viewed andphotographed with a Hitachi H-300 transmission electronmicroscope. Sections for light microscopy were silver en-

hanced using the IntenSE M kit (Amersham) according to themanufacturer's recommendations and stained with Safranin0 prior to examination.

RESULTS

Isolated Protoplasts

The isolated PVMP were considerably larger than the MP(Fig. 3), which was an important aid in ourPVMP purificationscheme (Fig. 2). The protoplasts isolated from leaves at var-

ious stages of growth were found to be quite stable and couldbe stored on ice or at 4°C for up to 24 h without appreciablebreakage. The PVMP preparations often contain BSP, whichhave been found to be similar to PVM in most respects,including their ability to produce and store the 27 and 29 kDstorage proteins (9-10, 13). Although BSP could be removed

from the general PVMP preparation and purified by a filtra-tion step, we did not separate them for most experimentsbecause we consider BSP and PVMP to be part of the same

tissue system (9, 11, 14). Visual inspection ofPVMP and MPpreparations under a microscope showed them to be pure,with less than S% cross-contamination. The only exceptionwas with some of the MP samples from the leaf-expansionstudy. MP preparations from leaves one-quarter, one-half,and three-quarters expanded were contaminated with 5 to10% PVMP and BSP, as determined by light microscopyinspection.

Distribution of Storage Proteins in Developing Leaves

The polypeptide profiles of PVMP from the fourth trifo-liolate of soybean at various degrees of expansion give an ideaof when the storage proteins are first expressed. The 27 and94 kD polypeptides were present as major PVM polypeptidesfrom one-quarter leaf expansion to full expansion (Fig. 4A).A 29 kD polypeptide was present as a minor protein at all

s

N ONto N e:

6 8

2-.

PA. ID F

1312 KLAUER ET AL.

tl~ ..; 4 go917 4

60,11-14

o",W2:9q


lhr 2hr 8hr 20 hr

29s271

Figure 9. Autoradiograph of a western blot from an SDS-gel showing incorporation of trans-35S label into soluble protein of recently maturesoybean leaves after 1, 2, 8, and 20 h. Arrows indicate the 27 and 29kD storage glycoproteins. Note the relatively greater accumulation of the27kD polypeptide over time compared with the 29kD polypeptide. SS, small subunit of Rubisco. Approximately 120 Ag protein/lane.

stages of expansion. Only the 27 kD storage protein reactedto immunostaining of a nitrocellulose blot of these PVMPsamples (Fig. 4B). This glycoprotein was also present in MPsamples from one-quarter to three-quarters expanded soybeanleaves (Fig. 5) due to a minor contamination of the MP withPVMP as discussed above. Some noticeable changes in poly-peptides occurred in MP during expansion (Fig. 5). The MPsample from one-half expanded leaves contained BSA as aresidual contaminant from the isolation procedure.

Distribution of Storage Proteins in Mature Leaves

The 27 and 29 kD storage proteins were found to beexclusive to PVMP in mature leaves of preanthesis soybeanplants. This was clearly demonstrated in both the Coomassieblue-stained gels and in the western immunoblots (Fig. 6).SDS-PAGE analysis of the fractionated protoplasts showedthat neither of these glycoproteins were chloroplastic (Fig. 6).Although there was a chloroplast polypeptide similar in sizeto the 27 kD glycoprotein, close examination showed that itis of a lower molecular mass (26 kD). A 94 kD polypeptidewas also found to be a prominent component of the PVMP,but not ofMP (Fig. 6). Like the 27 and 29 kD storage proteins,this protein was also found in the cytosol/vacuole fraction ofPVMP and was not present in the chloroplasts. The large andsmall subunits of Rubisco, prominent proteins in the MP,were present in PVMP but in considerably smaller relativequantity. This is consistent with a rather low CO2 fixationrate for PVMP (6). There were obvious differences in poly-peptides of chloroplast and cytosol fractions from MP, butdiscussion of the significance of these polypeptides is beyondthe scope of this paper.

Immunocytochemical localization studies using antibodyto the 27 and 29 kD proteins verify the fractionation studies.At the light microscope level, the storage proteins were foundto be localized in the PVM and bundle sheath cells (Fig. 7).Mesophyll cells did not contain detectable levels ofthe storageproteins. Transmission electron microscopy of immuno-stained sections demonstrated that the protein was accumu-lated in the PVM vacuole and label was associated with anelectron-dense material within the vacuole (Fig. 7). There wasno labeling ofchloroplasts, mitochondria, nuclei, or cell walls.Cytoplasmic labeling was also low. Preimmune controls werefree of label.

Distribution of Storage Proteins in Leaves ofPostanthesis Plants

Figure 8A compares the polypeptide profiles ofPVMP fromrecently, fully expanded leaves from modulated and nonno-dulated soybean plants at various physiological stages. The27, 29, and 94 kD polypeptides were found in both modulatedand nonnodulated plants; however, the amounts of eachvaried depending on plant status. The 94 kD polypeptideincreased to a maximum in plants at anthesis. In modulatedplants, the 94 kD polypeptide was absent at 2 weeks postan-thesis for both podded and depodded plants. However, innonnodulated plants it was present in small quantities up to2 weeks postanthesis.The 27 kD polypeptide was present as a major band at all

physiological stages examined (Fig. 8A), but appeared toincrease under conditions of depodding, and decrease in thepodded plants. A 29 kD polypeptide was generally seen undermost conditions, but was present as a very faint band as

1313

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DAD DP DAD Pt... DP DAD :,DA.

A BFigure 10. Coomassie blue-stained SDS-polyacrylamide gels (A) and glycoprotein-immunostained western blot (B) of protein extracts from MPof modulated and nonnodulated soybean. Lanes 1 through 5, MP extracts from modulated soybean; lanes 6 through 11, MP extracts fromnonnodulated soybean; lanes S, protein standards. NOD, modulated; NON-NOD, non-nodulated; WLE, whole leaf soluble protein extract; PA,preanthesis; A, anthesis; DP, days podded; DAD, days after depodding. Approximately 27 Ag protein/lane.

compared with the 27 kD polypeptide. Western immunostain-ing using the antibody raised against the 27 and 29 kD storageproteins gave a positive reaction for the 27 kD polypeptide inour gels (Fig. 8B). A 29 kD band did not stain in westernblots of PVMP preparations, but did show up in blots ofpreanthesis and depodded whole leafextracts. This is probablydue to the low amount in the PVMP preparations as com-pared with the very concentrated whole leaf extracts. Earlierreports had shown an accumulation of approximately equalamounts of the 27 and 29 kD polypeptides in leaves ofdepodded soybean (13, 23). To determine if the difference inrelative quantities of the 29 and 27 kD polypeptides observedin our gels was due to different rates of turnover for thesepolypeptides, leaf proteins were labeled in situ with trans-35Slabel (a "5S-methionine substitute), soluble protein was ex-tracted, and turnover analyzed using SDS-PAGE, westernblotting, and autoradiography. The results showed that therewas a greater synthesis of the 27 kD polypeptide over time(1-20 h) compared with the 29 kD polypeptide (Fig. 9). Thisindicates that the consistent difference in amount betweenthe 27 and 29 kD polypeptides seen on our gels is not areflection of greater turnover ofthe 29 kI) polypeptide. Over-all, PVMP from modulated and nonnodulated plants showedsimilar polypeptide profiles on a qualitative basis (Fig. 8).The polypeptide profiles for MP preparations were consid-

erably different from those ofPVMP at every stage examined(compare Figs. 8 and 10). The 27, 29, and 94 kD polypeptides

were absent in MP preparations from all physiological con-ditions (Fig. 1OA). Western immunoblots ofMP preparationsfor the 27 and 29 kD glycoproteins were negative (Fig. lOB).This further demonstrated that these polypeptides are specificto PVM and that cross-contamination of MP with PVMPwas negligible.

Similar profiles for most major polypeptides were exhibitedfor MP from both nonnodulated and modulated soybeanplants at all physiological stages (Fig. 1OA). A major polypep-tide of 80 kD observed in MP was absent from PVMP. Thisprotein was expressed at one-quarter leaf expansion (Fig. 5A).A polypeptide of approximately 62 kD increased from prean-thesis through 2 weeks postanthesis. There were some differ-ences between modulated and nonnodulated plants in poly-peptides ranging from 25 to 29 kD, but these were not majorpolypeptides in terms of quantity.

DISCUSSION

We have developed techniques that allow us to isolate PVMand mesophyll protoplasts from soybean leaves of almost anyage or plant status. Visual inspection, SDS-PAGE, and west-ern immunoblot analysis show our procedures to result inpreparations of different protoplast types with little or nocross-contamination, and we have been able to use thesesamples to further explore the role of the PYM in storageprotein accumulation.

1314 KLAUER ET AL.

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By SDS-PAGE and immunoblotting analysis, we haveclearly shown that the protein composition of PVM of soy-bean leaves is quite different from that of other leaf mesophylltissues. We have shown conclusively that the 27 and 29 kDstorage proteins are associated with PVM and are not accu-mulated by chlorenchymatous mesophyll cells. The presentstudy also shows that, even in depodded plants, the 27 and29 kD storage proteins are synthesized in the PVM and notthe mesophyll, that they are localized in the vacuole, and thatthey are not accumulated in other subcellular compartmentsof the PVM cells. Accumulation of the 27 kD storage proteinbegins very early during leaf expansion and synthesis of thisprotein occurs regardless of nitrogen source. The major factorin accumulation is probably nitrogen availability. The amountof the 27 kD storage protein in PVM cells decreases duringpodfilling, and increases or stays the same in depodded plants.This is consistent with Wittenbach's observation of increasedbuild-up of the 27 kD protein in whole leaf extracts afterdepodding (21-24), and with the structural studies of Fran-ceschi and Giaquinta (9, 10).The 94 kD polypeptide previously found to accumulate in

depodded plants was also shown to be a PVM protein (Fig.4). This polypeptide is probably the same as the 80 kD proteinnoted by Wittenbach (21-24) and the 90 kD protein observedby Staswick (17) in whole leaf extracts, the differences inmolecular mass being due to use of different molecular massstandards and different gel gradients. Like the 27 kD storageprotein, the 94 kD polypeptide is accumulated early duringleaf development. This polypeptide is not chloroplastic inlocation and we suspect that it is a vacuolar storage proteinthat performs a function similar to that proposed for the 27and 29 kD proteins: that is, to ensure an initial nitrogensource for seeds during early podfiuling. The potential impor-tance of this polypeptide as a vegetative storage protein couldbe significant. On our gels it consistently appeared in quan-tities as great as those of the 27 and 29 kD polypeptides,which under some conditions have accounted for up to 50%of the soluble protein in the leaf (18, 22, 23). In our study, noother major proteins were observed to follow the pattern ofaccumulation/degradation demonstrated by the 27 and 94kD polypeptides.

In all of our SDS-PAGE analyses ofPVMP protein, the 29kD storage protein was present as a minor component, or notdetectable, in comparison with the 27 kD glycoprotein. Thisdiffered from earlier reports that suggested that the two pro-teins accumulated, in most cases, in relatively equal amountsin whole leaves (13, 21-24). Originally, it was thought thatthe two polypeptides were the subunits of a larger 47 kDglycoprotein (23). Although it is still not clear if this occurs,autoradiographic evidence from our in situ radiolabeling ofprotein in whole leaflets and other recent studies (18) showedthat the 27 kD protein was synthesized at a greater rate thanthe 29 kD protein. Therefore, the difference in relativeamounts ofthe two proteins observed here was not a reflectionof a higher rate ofturnover for the 29 kD protein. In addition,Staswick (17) sequenced cDNA clones of the two proteinsand found them to be similar but unique glycoproteins thatwere closely matched with respect to sulfur amino acid con-tent. So the difference in band size observed in our autoradi-

ographs was not due to differences in sulfur content for theseglycoproteins.The 29 kD protein did not show in the western immuno-

blots of PVMP preparations, but was detected in the westernimmunoblots ofpreanthesis and depodded whole leafextracts.The reason for this is not clear. It seems possible that the 29kD protein is more susceptible to degradation during theprotoplast isolation period. Its absence from nodulated ascompared with nonnodulated PVMP preparations was in partbecause considerably less protein was applied to the gels fornodulated PVMP analysis (13 and 30 gg, respectively).

In summary, the polypeptide complement of PVM wasconclusively shown to be considerably different from that ofthe other leaf mesophyll tissue. This difference is a reflectionof the highly specialized function of the PVM. The 27 and 94kD proteins represent major components of the PVM thatare not accumulated by mesophyll cells. The 27 and 29 kDproteins are known to be storage proteins and are shown hereto accumulate in the vacuole. Our results suggest that the 94kD protein is also a vacuolar storage protein, and we arecurrently trying to further characterize this protein.

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10. Franceschi VR, Giaquinta RT (1983) The paraveinal mesophyllof soybean leaves in relation to assimilate transfer and com-partmentation. II. Structural, metabolic, and compartmentalchanges during reproductive growth. Planta 157: 422-431

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13. Franceschi VR, Wittenbach VA, Giaquinta RT (1983) Paraveinalmesophyll of soybean leaves in relation to assimilate transferand compartmentation. III. Immunohistochemical localiza-tion of specific glycopeptides in the vacuole after depodding.Plant Physiol 72: 586-589

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procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354

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23. Wittenbach VA (1983) Purification and characterization of asoybean leaf storage glycoprotein. Plant Physiol 73: 125-129

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mesophyll of soybean leaves at various developmental stages1

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