Seasonal Variation of Western White Pine (Pinus monticola D. Don

Plant CetlPhysioi. 37(2): 189-199 (1996)JSPP © 1996

Seasonal Variation of Western White Pine (Pinus monticola D. Don) FoliageProteins

Abul K.M. Ekramoddoullah' and Doug W. TaylorCanadian Forest Service, Pacific Forestry Centre, 506 West Burnside Road, Victoria, British Columbia, Canada

Recently, a western white pine protein, Pin m III, wasshown to be associated with overwintering and frost hardi-ness of western white pine foliage. To examine whether Pinm III is directly involved in frost hardiness by functioningas an antifreeze protein, work is underway to clone thegene encoding this protein and to assess the function of thisgene in freezing tolerance by incorporating the gene in atest plant, such as tobacco. Here, we examined in moredetail, by SDS-PAGE and also by two dimensional gel elec-trophoresis, the seasonal variation of additional proteins inwestern pine foliage. SDS-PAGE analysis of three seedlotsshowed that different proteins reached a maximum level indifferent months, although most proteins (5 to 11) reacheda maximum level in winter months (December, Januaryand February). The 2-D gel analysis of foliage sampled onthree harvest dates (October, January and April) of oneseedlot revealed a seasonal variation of a large number pro-teins (76 to 184). Of the seasonally varied proteins, theamino terminal sequence of several proteins including Pinm III was determined. One of the sequences was identifiedby homology to that of the small subunit of ribulose bi-phosphate carboxylase, whose level increased substantiallyfrom fall to spring. The amino terminal sequence of Pin mIII had 89% homology to a sugar pine protein, Pin 11. Theanti-photosystem II antibody was used to monitor the an-nual variation of the extrinsic 23-kDa photosystem II pro-tein. The level of the extrinsic 23-kDa photosystem II pro-tein decreased slowly as fall progressed and reached itslowest level in December and then increased in early springindicating that this variation is due to photosynthetic activ-ity of the foliage during the season.

Key words: Amino acid sequence — Electrophoresis —Frost hardiness — Phostosystem II — RuBisCo small sub-unit — Western-immunoblot.

White pine blister rust is a disease of five-needle pines,Pinus monticola, Pinus strobus and Pinus lambertiana,caused by the fungus blister rust fungus, Cronartium ribi-cola. We have been studying proteins involved in host-pathogen interaction of this white pine-blister rust patho-system (Ekramoddoullah and Hunt 1993). During this

To whom correspondence should be addressed.

ongoing investigation on protein biosynthesis in white pinefoliage following infection with the pathogen, it was ob-served that environmental factors also contributed to thechanges in the synthesis of proteins.

Most temperate-zone perennial plants have an annualcycle with a growing phase in summer and a dormant phasein winter (Lavender 1985). Following environmental cuessuch as low temperature and short photoperiod, plantsundergo several physiological changes including the devel-opment of frost hardiness. Frost hardiness is a mechanismby which plants attain adequate freezing tolerance duringcold weather and resume growth when the risk of freezingis over (Weiser 1970, Guy 1990).

A positive correlation of the expression of cold-regu-lated genes with freezing tolerance has been observed in sev-eral plants (Mohapatra et al. 1989, Binh and Oono 1992,Houde et al. 1992). Consistent with these findings, changesin protein synthesis also have been positively correlatedwith frost hardiness (Pomeroy and Siminovitch 1970, Kangand Titus 1980). Cold acclimation was also closely parallel-ed by an accumulation of membrane-bound proteins in theleaves of Korean boxwood (Gusta and Wesser 1972). Crudeprotein extracts obtained from cold-acclimated leaves ofspinach were shown to protect isolated thylakoid mem-branes against freeze-thaw damage (Hincha et al. 1989,1990). Removal of apoplastic proteins accumulated duringcold acclimation of leaves of winter rye increased the levelof injury to the leaves caused by freezing (Marentes et al.1993) and these workers suggested that the extracellular pro-teins might have ice-nucleating and antifreeze propertieswhich control extracellular ice formation in leaf tissues, asis thought to occur in insects and marine invertebrates(Storey and Storey 1988, Duman et al. 1991). Kurkela andFranck (1990) reported that a cold-regulated gene froma plant, Arabidopsis thaliana, encodes a protein havingamino acid sequence homology with certain fish antifreezeproteins.

Some polypeptides encoded by cold-regulated geneshave low molecular weights (Johnson-Flanagen and Singh1987) and have unusual properties of remaining solubleupon boiling (Lin et al. 1990, Lin and Thomashow 1992).During cold acclimation and decrease in photoperiod,there are also changes in the synthesis of other proteins(Weiser 1970, Wetzel et al. 1989, Wetzel and Greenwood1989, 1991, Weiser et al. 1990, Thomashow et al. 1990,Roberts et al. 1991, Voinikov and Korytov 1991, Coleman

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190 A.K.M. Ekramoddoullah and D.W. Taylor

et al. 1991, 1992, Sauter and Cleve 1993, Saez-Vasquez etal. 1993, Zhu et al. 1993, Coleman and Chen 1993), someof which are storage proteins, storing nitrogen over the win-ter which will then be mobilized for new growth in thespring (Wetzel et al. 1989, Wetzel and Greenwood 1989,1991, Roberts et al. 1991). Cold acclimation has also beenassociated with increased levels of antioxidant enzyme sys-tems of some plant species (Esterbauer and Grill 1978,Esterbauer et al. 1980, de Kok and Oosterhuis 1983, Naka-gawara and Sagisaka 1984, Guy and Carter 1984, Sagi-saka 1985, Asada and Takahasi 1987, Schoner et al.1989, Anderson et al. 1992). It was also shown that thelevel of certain heat-shock proteins increased with coldacclimation (Neven et al. 1992). The induction of simi-lar stress-related proteins has also been shown in someplant species during exposure to heat (Vierling and Nguyen1992), salinity (Hofner et al. 1987, Hanson 1992), anddrought (Close et al. 1989), and in seeds during maturationdrying (Bewley and Oliver 1992).

Recently, a sugar pine (Pinus lambertiana) protein,Pin I I was detected in increasing amounts in the fall(Ekramoddoullah et al. 1995). The homologue of this fallprotein, named as Pin m III, was also identified in westernwhite pine (Pinus monitcold). Pin m III was shown to be as-sociated with overwintering and frost hardiness of westernwhite pine foliage (Ekramoddoullah et al. 1995). To exam-ine whether Pin m III is directly involved in frost hardinessby functioning as an antifreeze protein, work is underwayto clone the gene encoding this protein and to assess thefunction of this gene in terms of freezing tolerance by incor-porating the gene in a test plant, eg. tobacco. In this study,we examined in more detail the seasonal variation of addi-tional proteins in western pine protein foliage. The studywas also aimed at the partial amino acid sequencing ofthese proteins so that appropriate antibody/DNA probescould be developed and used to further our understandingof the relationship of environmental stress with host-patho-gen interaction and also in our ongoing study of the an-tifreeze function of Pin m III.

Materials and Methods

Pinus monticola—Seedlings from seedlots 3159 [IngersollCreek (5O°8'N, 118°4.8'W; elevation: 793 m) British Columbia,Canada], 2881 [Eagle River (SQ"51.5'N, 118°45"W; elevation: 366m) British Columbia, Canada], and 2888 [Angus Creek (49°32'N,123°44'W; elevation 198 m) British Columbia, Canada] weregrown as described earlier (Hunt 1988) and kept under natural daylength and temperature in a shelter house at the Pacific ForestryCentre (48°25'N, 123°W; elevation: 30 m). They were fertilizedwith N : P : K (20 : 20 : 20, 0.5 g liter"1) twice a week and withFeSO4 (0.155 g liter"1) supplement once every two weeks (June 30to October 31). On October 23 and October 31, the fertilizer usedwas N : P : K (4 : 25 : 35, 0.5 g liter"1). No fertilizer was used be-tween Oct. 31 and March 25. Seedlings were watered twice a weekduring the entire experiment. From March 25 to Aug 30, seedlings

were again fertilized weekly with N : P : K (20 : 20 : 20, 0.5 gliter"1). Seedlings were 4 months old when first sampled. One nee-dle from each seedling was collected monthly for a year (Aug,1992 to July 1993). Needles were pooled by seedlot (36 seedlings/seedlot) for each collection.

Extraction of proteins—Proteins were extracted as describedelsewhere (Ekramoddoullah 1991, 1993) with minor modifica-tions. Foliar samples were lyophilized and ground to powder in liq-uid nitrogen with a mortar and pestle, after which 50 mg of needlepowder was extracted with 0.7 ml of extraction solution (ES) (4%SDS, 5% sucrose, 5% mercaptoethanol) for lOmin at roomtemperature with gentle stirring. The extract was centrifuged at10,000 xg for 15min, and the clear supernatant was heated to100°C for 3 min and then cooled to room temperature. Proteinswere precipitated by adding cold (—20°C) acetone (8 x volume ofthe supernatant); precipitation was allowed to continue for 1 h,(at -20°C) after which the sample was centrifuged at 10,000 xg. The pellet was resuspended in 0.2 ml of ES, centrifuged at10,000 xg for 15 min, and the protein content of the supernatewas determined (Ekramoddoullah and Davidson 1995) usingbovine serum albumin as a standard. Briefly, the protein solutionand standard were spotted on a polyvinylidene difluoride mem-brane (Immobilon-P, Millipore Canada Ltd., Toronto, Canada).The membrane was stained with 0.1% Coomassie blue R-250(Bio-Rad Laboratories, Richmond, CA, U.S.A.) in 50% metha-nol for 8 min, and then destained in 50% methanol: 10% aceticacid for 8 min at room temperature. The membrane was thenrinsed with water for 10 min and scanned using a laser scanner(Molecular Dynamics, model 110A, Sunnyvale, CA, U.S.A.) inter-faced with a workstation (SPARK 1, Sun Microsystems of CanadaInc. Vancouver, B.C., Canada) and PDI (Protein + dna imageWare systems, Huntington Station, NY, U.S.A.) for membraneblot processing with the software program ONED™. Scanning,detection and quantification were performed according to the PDIinstruction manual.

One-dimensional gel electrophoresis—SDS-PAGE was car-ried out in a protein slab cell apparatus (Bio-Rad) utilizing 0.75-mm thick 12% gel and the Laemmli buffer system (Laemmli 1970).A sample volume of 25 //I (containing 3 ng, protein) was applied ineach well. To calibrate the gel, low molecular weight (range: 14.4-97.4 kDa) standard markers (Bio-Rad) were used. The gel wasstained with silver (Hochstrasser et al. 1988), scanned and ana-lyzed with the software programme ONED. The band quantita-tion is calculated based on the optical density (O.D.) of all the pix-els within the band boundary and is expressed in O.D. units permm.

Two-dimensional gel electrophoresis—Two-dimensional elec-trophoresis was carried out using a Millipore Investigator System.Isoelectrofocusing (IEF) gels were prepared to the level of 170 mmhigh using ampolytes (pH 3-10) in long and narrow tubes (190 x 1mm). Fifty micrograms of protein in a 20 ̂ 1 volume [ 10 /il of ES +10//1 of solution F (0.1 g dithiothreitol, 0.4 g of cholamidopropyl-dimethylhydroxyprpanesulfonate, 54 g of urea, 0.5 ml ampholine,pH3-10 and 6.5 ml water)] was loaded onto each IEF gel. IEFwas carried out overnight. The gels were then extruded and laid ontop of the second-dimension SDS-PAGE gel (12%). Gels werestained with silver according to the Millipore Investigators Sys-tem's manual. Each run was replicated twice. Gel calibration wasdone using the following standard molecular weight and pimarkers (2-D gel standards; Bio-Rad): conalbumin, albumin, ac-tin, rabbit muscle glyceraldehyde 3-phosphate dehydrogenase, car-bonic anhydrase, trypsin inhibitor and myoglobin.

Scanning of gels and computer analysis of separated proteins


Seasonal variation of proteins 191

—Stained gels were scanned by a laser scanner (Molecular Dynam-ics) interfaced by PDI with ONED™ software (version 2.0)for processing one-dimensional gels and blots and PDQUEST™software (version 5.0) for processing two-dimensional gels. Anoriginal version of the software program was described by Garrelset al. (1984). Scanning, detection, estimation of molecular weightand pi, and quantitation of protein bands (one-dimensional gelsand Western immunoblots) or spots (two-dimensional gels) wereperformed according to the PDI instruction manual. The scannerwas calibrated with an optical density photographic step tablethaving 21 steps with density range of 0.05 to 3.05 (Eastman KodakCompany, Rochester, N.Y.). The band quantitation of 1-D gelsand blots is calculated based on the optical density of all the pixelswithin the band boundary and is expressed in O.D. units per mm.Two-dimensional gel scans obtained from gels were converted togel images, which were then converted into gel spots by an auto-detection method. Gel spots detected were carefully examinedagainst the original gels. Each pixel of a gel scan is originallyassigned an optical density (O.D.) value based upon the step tabletcalibration of the scanner, and linear interpolations of opticaldensity are used for spot quantitation. The quantitation of spotswas performed automatically employing a 2-dimensional Gaus-sian model. The spot quantitation is the Gaussian volume and isexpressed in protein data units (PDU). A matchset consisting of 2-D gel spot data obtained from October 1992, January 1993, April1993 samples of seedlot 2888 and a reference gel was prepared.The reference gel was constructed whereby all spots detected in allsamples of three harvest dates were put together electronically bya process of landmarking (i.e. relating to the obvious common pro-tein spots) and automatic matching. The matchset was also editedby re-checking for match offsets, partial matches, and erratic re-sponses. The data were evaluated by a Student t-test provided withthe software. If a protein spot is significant by this t-test, then theobserved difference between the mean quantitation of a spot in asample of a given collection date and its mean quantitation in thesample of another collection date cannot, with a probabilitygreater than 95%, be attributed to chance.

Amino acid sequence analyses—Pine proteins separated onSDS-PAGE or on two-dimensional gels were electrophoreticallytransferred to Immobilon-P membrane (Matsudaira 1987) usinga semi-dry transblot apparatus (Millipore). The stained proteinbands or spots were cut from the membrane and placed directlyinto the sequencer (Model 470A, Applied Biosystems, Foster City,CA, U.S.A.) for N-terminal sequence analysis (University ofVictoria microsequencing facility, Victoria, B.C., Canada). Theamino acid sequence comparison of the N-terminal peptide wasperformed at PIR (Protein Identification Resource Centre,Washington, DC, U.S.A.) and at NCBI (National Centre for Bio-technology Information, Bethesda, MD, U.S.A.) using FASTA(Pearson and Lipman 1988) and BLAST (Altschul et al. 1990) net-work services.

Synthesis of N-terminal peptide and production of rabbitanti-Photosystem II antibody—The synthesis of the N-terminalpeptide and production of rabbit antibody were carried out undera contract by Multiple Peptide Systems (MPS). The peptideAYGEAANVFGAPKKNTDFITC was synthesized using a varia-tion of Merrifield's original solid phase procedures (Merrifield1963) in conjunction with the method of simultaneous peptide syn-thesis (SMPS) (Houghten 1985). The first twenty amino acids werea part of the N-terminal amino acid sequence of an extrinsic PSIIpolypeptide (23 kDa) characterized in western white pine (Ekra-moddoullah 1993) and the twenty-first amino acid, cysteine,was added at the C-terminal end of the peptide to facilitate the cou-

pling of 5 mg of purified peptide to a carrier protein (Kehole lym-phet hemocyanin) (KLH).

The peptide-KLH was suspended in PBS buffer (3.1 mgml"1), emulsified by mixing with an equal volume of Freund'scomplete adjuvant, and injected into five to six subcutaneous dor-sal sites, for a total volume of 0.6 ml (1.0 mg of conjugate, 0.50mg peptide) per immunization. Rabbits were repeatedly (over 60days) injected with the immunogen in Freund's incomplete adju-vant under a proprietary immunization schedule.

Antibody purification was carried out with an immuno-amni-ty purification kit Proton ™Kit #1 (MPS). Purity of the antibodywas checked by SDS-PAGE. The titer of the affinity-purified rab-bit anti-Photosystem II antibody was 3200.

Western immunoblot—Proteins separated by SDS-PAGE(5/^g lane"1) were electrophoretically transferred (Towbin et al.1979) from the gel onto immobilon-P membrane. Following trans-fer of the separated proteins, the membrane was probed with anti-body as described elsewhere (Ekramoddoullah et al. 1995).

Results

SDS-PAGE protein patterns of the three seedlots—There were 22-24 protein bands well resolved in SDS-PAGE gels of the three seedlots. A typical gel pattern ofseedlot 2888 is shown in Fig. 1. The quantity of each pro-tein band in all three seedlots is shown in Tables 1 to 3.Different proteins reached their maximum level in differentmonths, although most reached a maximum level in thewinter months (i.e. December, January and February).Thus, five proteins (86.3 kDa, 77.1 kDa, 29.0 kDa, 27.8kDa and 23.7 kDa) in seedlot 3159 (Table 1), 8 proteins(72.9 kDa, 44.3 kDa, 38.7 kDa, 37.9 kDa, 36.2 kDa, 20.7kDa, 18.4 kDa and 13.8 kDa) in seedlot 2888 (Table 2) and11 proteins (73.9 kDa, 45.0 kDa, 38.3 kDa, 36.6 kDa, 31.0kDa, 29.3 kDa, 28.3 kDa, 27.1 kDa, 24.6 kDa, 21.0 kDaand 18.8 kDa) in seedlot 2881 (Table 3) were abundant inwinter months.

Two-dimensional gel electrophoretic pattern of pro-teins of seedlot 2888—Foliar samples from seedlot 2888 har-vested in October, 1992, January 1993, and April 1993were analyzed (Fig. 2-4). On average, 603 proteins perharvest date were detected in 2-D gels. Significant proteindifferences were found between October and January; 141proteins were enhanced or induced while 43 proteins de-creased or disappeared. Comparison between January andApril samples showed 29 proteins were either enhanced ornewly synthesized while 47 proteins were reduced or disap-peared. Between October and April, 74 proteins were eithersignificantly enhanced or newly synthesized while 64 pro-teins decreased or disappeared. A few of these proteins arelisted in Table 4 and their gel locations are indicated inFigures 2-4.

Amino acid sequence analysis—From 2-D gels data(Table 4), seven proteins (ssp# 1111, 3428, 4101, 4302,4619, 5103, 6219) which had relatively high PDU values inJanuary samples and a protein (ssp# 8109) from the April



"-23(Photosyittml)

14.4- if*w'

m •-?*

•cit JMD NbU HwU April

Fig. 1 SDS-PAGE analysis of western white pine foliar proteins. This electrophorogram was obtained from seedlings of seedlot2888. Lanes are standard (std) molecular mass markers and samples of different harvest dates. Positions of proteins, whose N-terminalamino acid sequences were established (Figure 5), are shown on the right.

Table 1 Seasonal variation in the SDS-PAGE protein pattern of western white pine seedlings (seedlot 3159)

Proteinband(kDa)

86.377.154.146.042.539.237.635.333.230.029.027.827.025.124.123.720.920.018.517.216.514.712.7

Aug '92

0.0870.5043.2151.0860.9651.4310.1410.0991.0752.5011.3711.0360.5530.2780.9610.2561.0530.4941.3982.0410.6294.5371.732

Sep'92

0.0530.3942.7880.8000.8771.1560.0730.0530.8982.6461.4890.6450.4040.1450.7240.1890.7310.4560.7691.6150.5984.1661.419

Oct'92

0.1700.5191.9030.8160.6080.7740.1650.1060.9392.3621.5871.0380.5210.1870.8710.2730.7360.6571.5161.8860.6744.4242.243

Nov '92

0.1640.5101.9940.8000.5230.5750.1830.0930.8012.3701.6000.8480.4190.1980.7640.1820.7030.8131.0661.7040.6314.2022.030

Protein amount (O.

Dec '92

0.3280.8352.1931.0180.7650.4700.1780.1160.7012.4221.4070.6280.2900.1110.6960.1860.5910.8440.8711.3280.4973.5831.649

Harvest dateJan '93

0.1200.2390.6890.8100.6561.0420.2540.0520.4841.8031.3610.4810.4190.1810.5570.1630.4870.9940.7721.7270.5003.4952.243

Feb '93

0.3980.6670.7820.6230.5770.6670.1730.0270.8652.2181.7011.2530.7530.3090.9980.2770.8560.5571.8661.5700.4211.3882.066

D.)

Mar'93

0.1670.1910.6780.9180.7551.0690.2450.1190.6201.7431.3780.4940.6640.1510.8930.1360.6490.5170.6441.5760.1501.8741.652

Apr '93

0.1400.2520.7420.9210.9510.9680.1760.1670.5931.7251.4020.4250.5990.1530.9520.1160.6300.6040.7841.5480.4622.2400.727

May '93

0.1260.1771.3951.1511.3661.1140.2170.1940.6251.8971.4440.6711.0530.2341.1770.1561.0140.6641.4452.5610.3572.2031.276

Jun '93

0.0160.0300.7200.4230.6020.6180.1310.2580.3800.9691.0330.3630.6420.1730.7930.0730.4980.4770.4601.5900.4372.8670.974

Jul '93

0.1100.1030.8300.8800.9480.8000.2420.6940.4691.2771.3261.0620.6600.3101.0010.2340.9101.0072.6002.4051.2735.5973.729




Proteinband(kDa)

72.955.753.744.341.440.338.737.936.234.132.030.328.026.726.225.724.423.720.719.418.416.014.613.8

Aug '92

0.4550.3841.5550.6360.2810.3070.2460.5380.0860.1231.1612.0971.2650.6620.2490.1080.1670.5660.2750.7930.5162.8614.1592.059

Sep'92

0.4040.5191.5500.5300.2980.3260.2300.4280.1130.1381.0161.6551.3050.5470.2380.0820.1240.5050.2220.7410.5412.2123.3702.196

Oct '92

0.5520.5251.3860.5070.2570.2280.1940.3190.0890.1000.8871.7571.3310.5790.2280.1100.1090.5020.1480.5521.2022.2084.0601.974

Nov'92

1.0751.1322.2091.0410.5860.4620.3330.5120.1370.1061.0552.3811.3340.6930.2710.1330.1080.5750.3760.8042.0162.0843.8871.919

Protein amount (O.

Dec '92

1.3710.8851.0871.0520.5540.6330.6220.4730.1650.1211.0152.6391.4820.8490.2850.1500.1000.7820.4671.0052.3742.5634.0332.777

Harvest date

Jan '93

0.2800.6630.6671.0270.7280.5550.6061.1200.2240.1340.8281.7431.1570.7710.3600.3800.1290.8260.6151.4593.3664.7885.9423.762

Feb '93

0.2570.4260.8560.4950.3380.1580.2410.5410.0860.0500.8012.1061.3120.8390.2420.2470.1340.5390.4211.0732.2603.0373.6683.124

D.)

Mar'93

0.2910.9170.8001.0031.0300.7830.5280.9030.1740.2380.9021.5051.3700.8390.3650.3890.1200.9280.5541.4522.2524.9065.9911.344

Apr'93

0.2661.2301.0590.8131.0500.7260.4090.6610.1360.2640.7501.0121.2250.7520.4330.3040.1330.8820.5501.3421.7034.9156.4960.545

May '93

0.2290.9370.8680.7480.9520.6780.4490.7220.1410.3430.6531.2811.1690.7520.4300.3430.1840.8820.3621.4311.1005.6796.0360.578

Jun '93

0.2201.0921.2690.7291.1240.5040.3540.6270.1680.5770.6671.3541.1850.7790.4980.1650.2530.8220.1631.1261.3264.8675.8042.335

Jul '93

0.0680.9280.6780.4940.7760.3700.2390.3140.1120.6450.4070.9550.8220.7550.3290.1760.0820.7300.5381.5891.3574.9087.4652.012

sample of the seedlot 2888 were selected for N-terminalamino acid sequence analysis. Only ssp #8109 yielded se-quence data (Fig. 5), the others were blocked at N-terminalgroup or there was insufficient quantity. The sequence ofssp# 8109 had 78% homology (p = 0.00015) with ribulose bi-phosphate carboxylase oxygenase (RuBisCo) small subunitfrom Japanese black pine (Pinus thunbergiana) (Proteindata bank accession #gp/X13408/PIRBCS3). From SDS-PAGE gel data (Table 2) eight proteins (13.8 kDa, 14.6kDa, 16.0 kDa, 18.4 kDa, 20.7 kDa, 26.7 kDa, 30.3 kDaand 36.2 kDa) were selected for amino acid sequence analy-sis. The N-terminal amino acid sequence of the 13.8-kDa,14.6-kDa, 16.0-kDa, 18.4-kDa and the 20.7-kDa proteinswas obtained (Fig. 5). The sequences of the other proteinswere not obtained due to blocked N-terminal amino acids.No significant homology was found for N-terminal aminoacid sequences of the 13.8-kDa, 16.0-kDa and 20.7-kDaproteins. One major and minor sequence were obtained forthe 16.0-kDa protein. The first fifteen amino acids of N-ter-minal amino acid sequence of the 14.6-kDa protein wereidentical to those of ssp# 8109 which had significant homol-ogy with RuBisCo small subunit. A significant homology

(89%) of the N-terminal sequence of the 18.4-kDa proteinwas found with sugar pine protein Pin I I (protein databank accession PIR#A40451).

Seasonal variation of photosystem II in three seedlots—Western immunoblot was used to establish the specificityof anti-photosystem II antibody which bound to a singleprotein band corresponding to a 23-kDa protein. The levelof the 23-kDa photosystem II protein slowly decreased inthe fall and reached a minimum in December and then slow-ly increased as spring approached, reaching a maximum inearly summer (Fig. 6).

Discussion

During our investigations of the molecular analysis ofthe host pathogen interaction of the white pine blister rustpathosystem (Ekramoddoullah and Hunt 1993), a sugarpine protein, Pin 11, was detected in increasing amounts inthe fall (Ekramoddoullah et al. 1995). Using anti-Pw / Iantibody, its homologue Pin m III was detected in westernwhite pine. Furthermore, the concentration of Pin m HI,which reached its maximum in winter months, was signifi-




Proteinband(kDa)

73.956.553.845.041.940.838.336.634.532.331.029.328.327.124.623.823.021.019.718.817.216.1

Aug '92

0.5690.7022.6010.6650.4630.3420.6040.0620.0791.2030.3441.9011.1830.8880.0770.5250.2000.2061.1060.0900.8012.521

Sep '92

0.6980.8372.1890.7920.3800.3530.5790.0920.1211.2530.3662.1201.4820.9080.1370.5470.2320.2491.0120.7330.7212.497

Oct '92

0.4290.6441.6760.4980.2970.1860.3200.1020.0911.1260.4902.1131.5960.7680.1470.5900.2850.2840.8581.1760.8322.424

Nov '92

0.3460.6451.5080.4760.2610.1160.2680.1050.0960.9680.6842.0931.5120.7100.1160.4960.1910.3510.8911.1360.5581.773

Protein amount (O.

Dec '92

0.7150.8061.7150.6830.5980.3210.5480.1570.1411.2210.9183.0281.8730.9850.1260.8470.2250.5101.0052.2851.6452.077

Harvest date

Jan '93

0.3750.7331.1790.7930.9010.3730.8880.2640.1531.0551.0911.6961.5420.8810.1770.8490.1450.6441.1822.8951.5664.100

Feb '93

0.6790.6641.4330.4940.5950.2250.7380.1530.0841.2230.8482.9462.0511.3770.2890.7940.1880.3881.2962.3501.5943.319

D.)

Mar '93

0.3070.8341.1370.6500.9040.5930.8370.1590.2520.8960.9641.5741.3510.8380.1240.8630.1130.3350.9401.3790.9762.392

Apr '93

0.2691.0731.1330.6661.0470.6560.7950.1810.2700.6330.9311.4291.1360.7220.1190.7430.1050.3101.1281.0081.4422.961

May '93

0.2411.1671.1660.7751.2060.6170.6430.1990.5150.6860.8751.6781.0470.8180.2310.7750.1070.2251.4620.9421.9163.625

Jun '93

0.2060.9801.3860.6301.1480.6420.4600.2320.5980.6160.8061.1271.0400.9200.2060.6350.1030.2081.2621.6453.7154.738

Jul '93

0.0450.9541.1920.5880.8330.5530.3650.2300.8110.3940.9570.7280.8500.8440.1950.6830.2550.5191.3191.4252.2553.889

cantly correlated with frost hardiness of western white pinefoliage. The present study showed that, in addition to Pinm III, other proteins also increase during winter in westernwhite pine foliage. A number of proteins in all three seed-lots increased in amount. One of the major proteins wasPin m III which was previously identified immunochemical-ly (Ekramoddoullah et al. 1995). Although the number ofproteins varied from seedlot to seedlot, this variation wasindependent of the origin of the seed sources. Since thefoliage from seedlings within these seedlots was pooled, thedata did not provide any insight into variation within seed-lots.

Since only a limited number of proteins could be re-solved in SDS-PAGE, we used 2-D gel electrophoresis to an-alyze these proteins more thoroughly. This analysis waslimited to samples of seedlot 2888 that were harvested in Oc-tober 1992, January 1993, and April 1993, since the level ofPin m III (which was shown to increase in October 1992, toreach a maximum in January 1993, and then decrease byApril 1993 in this seedlot) could be used a reference point.The study clearly showed that a large number of proteinsare being synthesized and metabolized as a tree enters intothe dormant phase. Although the function of these pro-teins is unknown, they may belong to several group of pro-

teins such as storage proteins, proteins associated withfrost hardiness, anti-oxidant systems and general stress pro-teins (Close et al. 1989, Schoner et al. 1989, Coleman et al.1991, Ekramoddoullah et al. 1995). Because of our interestin proteins associated with frost hardiness, we attempted tosequence a few of these proteins that were increased duringdormancy (Table 4, January). No attempt was made to se-quence ssp# 6212 because this protein was identified immu-nochemically as one of the isoforms of Pin m III (un-published data). In the sequence analysis, ssp# 8109 fromthe April sample was included because of a dramatic changein the quantity of this protein among three harvest dates.With the exception of SSP# 8109, none yielded any se-quence data from 2-D gels. Since the molecular weights ofthese proteins were known, proteins corresponding to thesemolecular weights in the January sample of seedlot 2888,which were resolved on SDS-PAGE (Table 2), were sub-jected to amino acid sequence analysis. The underlyingassumption was that any amino acid sequence obtainedfrom SDS-PAGE would be the major sequence of thoseidentified in 2-D gels. In support of this assumption, theamino sequence of the 14.6-kDa protein obtained fromSDS-PAGE was shown to be identical to the RuBisCosmall subunit (ssp# 8109). However, the amino acid se-



4.6I

5.0I

pH5.4 6.0 6.6 7.2 7.8 8.4I I I I I I

kDa

76.0 —

43.0 —

36 .0 -

31.0 —

21.5 —

17.5 —

Fig. 2 Two-dimensional gel electrophoresis of western pine foliar protein sample of seedlings of seedlot 2888 collected on October,1992. Positions of standard molecular mass markers are indicated on the left side and pH is indicated on the top. Positions of 11 pro-minent proteins (Table 4) are indicated.

kDa

76.0 —

66.2 —

43.0 —

36.0— '

31.0 —

17 S —

4.6I

5.4 6.0 6.6 7.2 7.8 8.4

-Itn

ijFig. 3 Two-dimensional gel electrophoresis of western pine foliar protein sample of seedlings of seedlot 2888 collected on January,1993. Positions of standard molecular mass markers are indicated on the left side and pH is indicated on the top. Positions of 15 pro-minent proteins (Table 4) are indicated.


196

kDa

A.K.M. Ekramoddoullah and D.W. Taylor

pH

ma ' 5103

Fig. 4 Two-dimensional gel electrophoresis of western pine foliar protein sample of seedlings of seedlot 2888 collected on April,1993. Positions of standard molecular mass markers are indicated on the left side and pH is indicated on the top. Positions of 14 pro-minent proteins (Table 4) are indicated.

quences (Fig. 5) of proteins that were obtained from SDS- could be produced to the N-terminal peptide of these pro-PAGE do not necessarily reflect those identified in 2-D gels teins and measuring them immunochemically—as washaving increased synthesis during winter months. This done in this study with photosystem II and previously withwould require further work in which specific antibodies Pin m III (Ekramoddoullah et al. 1995). The N-terminal

1 S 10Aln-ClrvVol-Glu-PrcHArgJ-Vnl-Gln-Asn-Asn-Ata

1 5 10 15 20 25

Met l̂n-VaiHTiodAA-Pro-PTO-TyKSty-Asn-Als-Lys-Phe-Glu-'nir-t.eu-Ser-Tyr-l-eu-Pro-Arg-Leu-Sor-Gln-Glu-Gln-Leu

14.6 kDa (RuBIsCo small subunlt)

1 5 10

Ajp-Glu-Ar3-Ser-l!eu-Cly-t.eu-Asp-Gly-Prc>-Glu-Leu

16.0 kDa (major)

1 5 10

Asp-Alc-Vd-Leu-Met-Lyt-Se^Ala-iyr-Ala-Pro-GIn

18.0 kDa (minor)

1 5 10 15 20VakSer^3ly-Thr-Ser-Ser-Thr^5lu-Glu-V»l-V«l-Gln-VaH3lii-Ala-Arg-Ar9-Uu-Trp-Asn-Ala-TTir

ia.4 kDa (Pfnm III)

1 5Asp-lls-Pho-Tnr-VaKSIn-Val

20.7 kDa

1 5 10 15Met-Gln-VaHHis)-Pro-Pro-Tyi-Gly-Asn-A]a-Aln-<LysH>he-G1u-Thr-Leu

SSP*8109 (RuBIsCo small subunlt)

Fig. 5 Amino terminal amino acid sequence of six proteins obtained from January samples of seedlot 2888 that were resolved by SDS-PAGE and protein ssp# 8109 (Table 4).



Table 4 Significant" differences in the quantity of a fewproteins between harvest dates of seedlot 2888

SSp*

1,1111,3083,1103,2133,4284,1014,3024,6194,7145,1035,7415,5105,6346,2126,2197,3218,109

Mr c

16.2

pl«

4.624.6 <4.513.821.730.314.627.236.554.417.243.433.539.018.820.325.314.3

" P<0.05, student t-test* sample spot number.c molecular id isoelectric i

nass (kDa).point.

4.94.95.05.05.05.05.05.15.25.25.25.4'5.36.77.4

Protein amount expressed inprotein i

Oct '92

188127

0530195

0182138

0128991

69175

1,192345

0194

data units (PDU)Jan '93

991767879810

1,537883

1,341984866

1,0480

106644

1,2551,120

197686

Apr '93

6301,177

7321,4051,053

478307

72904607

01,099

987178945

1,0911,802

amino acid sequence of the 18.4-kDa protein (i.e. Pin mIII), as expected, showed significant homology to sugarpine protein Pin 11. The N-terminal amino acid sequenceof the 14.6-kDa protein showed a significant homology tothe RuBisCo small subunit of Japanese black pine.

Of interest is the variation observed in the level ofPSII which may be a reflection of the low photosynthetic ac-tivity of the foliage in December (Chevone et al. 1989.

3 °-5i§•0.45^i 0.4^S 0.35S. 0.3-j8 0.25-8 0.2-

5 0.05-i

Seedlot

« pi O> O) O>

i > -£ o _-

Apr to

Date of harvest

Fig. 6 Seasonal variation in the level of the 23-kDa extrinsic pho-tosystem II polypeptide of 2-year-old western white pine seed-lings.

Jurik et al. 1988). This indicated that the efficiency of pho-tosystem II decreased in cold-acclimated conifers (Schoneret al. 1989). This protein could be an indicator of photosyn-thetic activity of the foliage.

In view of the seasonal variation of many proteins inwhite pine foliage, any study on the host-pathogen intera-ction of white pine blister rust pathosystem should takeinto consideration the possible impact of the pathogen onthese environmentally regulated proteins of the host. Thepartial characterization of a few of these proteins wouldallow us to produce antibody probes and thereby study thepossible role of at least some of these protein in host-patho-gen interactions. Moreover, the amino acid sequence infor-mation would be useful in designing appropriate primersfor cloning genes encoding these proteins and examiningtheir possible function as antifreeze proteins.

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(Received July 29, 1995; Accepted January 5, 1996)



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Seasonal Variation of Western White Pine (Pinus monticola D. Don