Plant Physiol. (1988) 87, 721-7260032-0889/88/87/0721 /06/$0 1.00/0

Overwintering Periwinkle ( Vinca minor L.) Exhibits IncreasedPhotosystem I Activity'

Received for publication November 6, 1987 and in revised form February 8, 1988

N. P. A. HUNER*, M. KROL, J. P. WILLIAMS, AND E. MAISSANDepartment ofPlant Sciences, University of Western Ontario, London, Ontario N6A 5B7, Canada(N.P.A.H., M.K.); and Department ofBotany, University of Toronto, Toronto, OntarioMSS JAl, Canada(J.P.W., E.M.)

ABSTRACT

The effects of natural, overwintering conditions on photosystem I andphotosystem II activity were examined in isolated thylakoids of periwin-kle (Vinca minor L.), an endemic, cold-tolerant, herbaceous evergreen.DCMU-Insensitive photosystem I activity (ascorbate/dichlorophenolin-dophenol -- methylviologen) exhibited a twofold increase in light-satu-rated rates upon exposure to low temperature and freezing stress with noeffect on the apparent quantum yield of this reaction. DCMU-Sensitivephotosystem II activity (H20-- dichlorlophenolindophenol) exhibitedonly minor fluctuations in light-saturated rates but a 50% decrease in theapparent quantum yield of this reaction upon exposure to overwinteringconditions. This was correlated with a decrease in the 77°K fluorescenceemission at 694 nanometers. These functional changes occurred with nodetectable changes in the relative chlorophyll contents of the chlorophyll-protein complexes or the chlorophyll-thylakoid protein. The chlorophylla/b varied less than 10% during any single growth year. Analyses of totalleaf extracts indicated that all lipid classes exhibited increased levels oflinoleic and linolenic acid. Neither the trans-A3-hexadecenoic acid levelnor the ratio of oligomeric:monomeric light harvesting of photosystem IIwas affected by exposure to winter stress. The content of the majorchloroplast lipids monogalactosyldiacylglycerol, digalactosyldiacylglyc-erol, phosphatidyl-diacyl-glycerol, and sulfoquinovosyldiacylglycerol ex-hibited minor fluctuations, whereas phosphatidylcholine and phosphati-dylethanolamine content doubled on a mole percent or chlorophyll basis.We conclude that the previously reported increase in photosystem Iactivity during controlled, low temperature growth is observed duringexposure to natural overwintering conditions. This appears to occur withminimal changes in the structure and composition of the photosyntheticapparatus of periwinkle.

Recently, Huner (7) reported that thylakoids isolated fromwinter rye (Secale cereale L. cv Puma) exposed to growth at low,cold-hardening temperatures (5°C) under controlled environ-ment conditions exhibited a 1.5-fold increase in the uncoupled,light-saturated rate of whole chain electron transport (H20 -.MV).2 This increase in whole chain activity was attributed totallyto a 1.6-fold increase in the light-saturated rate of PSI activity(Asc/DCPIP -- MV). Low growth temperature did not affectlight-saturated rates of DCMU-sensitive, PSII electron transport

'Supported by Operating Grants and Strategic Grants from the Nat-ural Sciences and Engineering Research Council of Canada.

measured as either H20-+ DCPP or DPC -- DCPIP (7). Krolet al. (9) reported that the assembly pattern for PSI during ryechloroplast biogenesis at 5°C was distinct from the assemblypattern for PSI during chloroplast biogenesis at 20C. Theysuggested that this altered assembly pattern at 5°C results in achange in the organization of PSI which, in turn gives rise to theincreased capacity for PSI electron transport (9). In contrast, thepattern for development of PSII electron transport was similarat both 5 and 20°C (9). Low temperature growth did not affectthe apparent quantum efficiencies of whole chain, PSI, or PSIIelectron transport (7). Other cold-tolerant species such as wheat,spinach, pea, broad bean, and Brassica also exhibit a 1.4- to 2.2-fold increase in light-saturated rates of PSI electron transportafter growth at 5°C under controlled environment conditions(NPA Huner, unpublished data). Thus, an increased capacity toprocess electrons through PSI may be a general characteristic ofcold-tolerant plant species grown at low, nonfreezing tempera-tures under control environment conditions.

In winter rye, this low temperature-induced increase in PSIactivity has been shown to occur with no change in the followingthylakoid characteristics: Chl/P700 (6, 9), Chl a/b, CPl/CPa,Chl/lipid, Chl/protein, lipid/protein (8-10), polypeptide andlipid complements (8, 10). The only compositional change ob-served in isolated thylakoids from low temperature-grown ryewas a 72% decrease in the trans-16:1 content of PG (8). DSC,fluorescence polarization ofDPH, and EPR measurements using16-doxylstearic acid indicated that low temperature growth ofwinter rye had no significant effect on the fluidity of isolatedthylakoid membranes (8). However, it was shown that PG or acertain proportion ofrye thylakoid PG was specifically associatedwith LHCII (11) and that chloroplast biogenesis at low growthtemperature modulates the organization ofrye LHCII by alteringspecifically the trans-16: 1 content of its PG (8, 10, 11).

Periwinkle (Vinca minor L.) is an angiosperm and a cold-tolerant, herbaceous evergreen commonly used as a groundcover. The leaves of this species that develop in the spring andsummer are the same leaves that subsequently are exposed to

2Abbreviations: MV, methylviologen; LHCII, light harvesting Chl a/b pigment protein complex II; CP1, major Chl a pigment-protein com-plex associated with the PSI reaction center; CPa, Chl a pigment-proteincomplex associated with the PSII reaction center; FP, free pigment; Asc,ascorbate; DCPIP, oxidized 2,6-dichlorophenol indophenol; F685, F6g4,F735, 77°K Chl a fluorescence emission maxima at 685 nm, 694, and735, respectively; PG, phosphatidyldiacylglycerol; SL, sulfoquinovos-yldiacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalac-tosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanola-mine; DOC, deoxycholate; DSC, differential scanning calorimetry; DPH,diphenylhexatriene; EPR, electron paramagnetic resonance.

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Plant Physiol. Vol. 87, 1988

low temperature and freezing conditions of autumn and winter.The purpose of this investigation was, first, to determine if anative plant species which normally is exposed to natural lowtemperature and overwintering conditions exhibits the charac-teristic increase in light-saturated rates of PSI electron observedfor plants grown under controlled environment conditions. Sec-ond, we wished to establish whether chloroplast biogenesis at lowtemperature is an absolute requirement for the observed increasein light-saturated rates of PSI electron transport. Finally, wewished to determine if exposure to natural overwintering condi-tions affected the trans-16:1 content of PG and the LHCIIorganization of periwinkle thylakoids.

MATERIALS AND METHODSPlant Material. Two natural stands ofperiwinkle ( Vinca minor

L.) in London, Ontario, were used in this study for comparativepurposes. Stand 1 was located in north London on the campusof the University of Western Ontario and stand 2 was located insouth London about 6 km from the university campus. Bothsites had a northwest exposure. Leaf samples were harvestedfrom either stand during various times of the year as specified inthe text. In all cases, we examined leaf material produced in thespring, cold-acclimated in the fall, and subsequently subjected toone overwintering season. Leaf area was measured with a Li-Corportable leaf area meter (model LI-3000). Leaf area values rep-resent the averages of at least 8 different samplings ± SD. Eachsampling consisted of the average area of 12 different leaves.Thylakoid Membrane Isolation. Periwinkle leaves were har-

vested and immediately ground at 4°C in 50 mM Tricine (pH7.8) buffer containing 0.4 M sorbitol, 10 mM NaCl, 2% (w/v)PVP, and 0.5% (w/v) BSA with two 10 s bursts of a WaringBlendor at high speed. The brei was filtered through two layersof Miracloth (Calbiochem), and the filtrate was centrifuged at4000xg for 5 min at 4°C. The green pellet containing thylakoidmembranes was resuspended and washed in 50 mM Tricine (pH7.8) containing 10 mm NaCl and 5 mM MgCl2 by centrifugationat l0,000g for 5 min at 4°C. The thylakoid membranes wereresuspended in a minimal volume of 50 mM Tricine (pH 7.8)containing 0.1 M sorbitol, 10 mm NaCl, and 5 mM MgCl2 andkept on ice in the dark until used. Only freshly isolated thylakoidmembranes were used in this study.

Photosynthetic Electron Transport Activities. DCMU-insen-sitive PSI-associated electron transport activity (Asc/DCPIP -.MV) was measured polarigraphically at 25°C as the rate of 02consumption (mg Chl)-' h-' using a Clark-type electrode asdescribed in detail elsewhere (7). DCMU-sensitive PSII-associ-ated electron transport activity was measured spectrophotomet-rically at 590 nm and 25°C in a water-jacketed cell as H20DCPIP (7).Low Temperature Fluorescence Emission Spectra. Fluores-

cence emission spectra (77°K) of isolated periwinkle thylakoidmembranes were obtained with a Perkin-Elmer 650-40 fluores-cence spectrophotometer. Excitation was at 440 nm through 4nm slits, and corrected emission spectra were collected through4 nm slits. The data were plotted using Perkin-Elmer suppliedsoftware. Thylakoids were suspended to a final Chl concentrationof 10 ,ug ml-' in 25 mm Tricine (pH 7.8) containing 50% (w/v)glycerol, 2.5 mM MgCl2, and 5 mM NaCl. All spectra werenormalized to the 686 nm peak.

Lipid and Fatty Acid Analyses. Periwinkle leaves were ex-tracted and analyzed for their lipid content and fatty acid com-positions as described in detail elsewhere (22, 23).

Chl-Protein Complexes. Chl-protein complexes were solubi-lized with DOC and SDS in the ratio of 20:10:1 (DOC:SDS:Chl)and were separated in the dark at 4°C on 7.5% (w/v) SDSpolyacrylamide slab gels as described elsewhere (8). Gels werescanned at 671 nm with a Shimadzu UV-250 spectrophotometer.

Chl and Protein Analysis. Chl was measured according toArnon (1). Thylakoid protein was determined according to Brad-ford (3) after extraction of pigments with 90% acetone.

RESULTS

Growth Characteristics. In stands 1 and 2, flowers appearedearly in the spring (mid-April to mid-May) with new leafmaterialbeing observed after the onset of flowering in the spring. Leavesreached full expansion by late June. Periwinkle leaves from stand1 (1984-1985) increased in area from 1.56 cm2 on May 2, 1984,to 7.02 cm2 by June 24, 1984. The average leaf area from June24, 1984 to March 18, 1985, was 6.44 ± 0.38 cm2. For compar-ison, periwinkle leaves from stand 2 (August 20, 1986-March26, 1987) exhibited an average leaf area of 5.54 ± 0.41 cm2.After the completion of leaf initiation and expansion, no newleaf material was observed. Thus, leaves that were produced inthe spring and summer were the same leaves that subsequentlyoverwintered.PSI and PSII Activities. The results summarized in Figure 1A

and Figure 2A represent PSI and PSII light-saturation curves forthylakoids isolated from periwinkle leaves developed during thespring and summer of 1986 and subsequently exposed to lowtemperature and overwintering conditions in 1987. Exposure tothese natural conditions caused two major effects: first, the light-saturated rate of PSI electron transport exhibited a twofoldincrease during fall and winter reaching a maximum in February(Fig. 1B). This was followed by a decrease in the light-saturatedrate that was irreversible even though no visible signs of leafdamage or senescence were evident. The apparent quantumefficiency for PSI electron transport was independent ofexposureto changing climatic conditions (Table I). Second, exposure tofall and overwintering conditions caused a 50% decrease in theapparent quantum efficiency of PSII-associated electron trans-port (Table I; Fig. 2A) with only minor changes in the calculatedVmax for this reaction (Fig. 2B). Similar results were obtained forthylakoids of periwinkle leaves harvested from either stand 1 orstand 2.

Since periwinkle is typically snow covered during the wintermonths in Canada, the effect of the absence of snow cover onPSI and PSII activity was examined. In stand 1 and 2, snowcover during January and February varied from 15 to 30 cmduring the winters of 1984 to 1987. One section of stand 2 wasmaintained snow free for 14 d from January 9 to January 23,1985, by carefully removing snow on a daily basis such thatperiwinkle leaves were fully exposed to freezing temperaturesand sunlight. An adjacent section in stand 2 remained snowcovered as a control. The snow-covered winter sample exhibitedan 86% higher light-saturated rate of PSI electron transport thanthe sample harvested the previous summer (Table II). A 14 dabsence of snow cover during January 1985 only caused an 11%inhibition of PSI activity and had no effect on the Chl a/b. Incontrast, light-saturated rates of PSII activity were inhibited by38% during the same time period. Clearly, PSII activity appearsto be more sensitive to the absence of snow cover than is PSIactivity of periwinkle thylakoids.Low Temperature Fluorescence Emission Spectra. The in situ

organization and the energy distribution within the photosyn-thetic apparatus was monitored by 77°K fluorescence emissionspectra of Chl a (4). The emission spectra of periwinkle thyla-koids isolated at various times during 1986-1987 season fromstand 1 exhibited a reduction in the fluorescence intensity at 694and 735 nm relative to that at 686 nm (Fig. 3) upon exposure tooverwintering conditions. This effect appeared to be irreversiblesince thylakoids isolated in May 1987 that had overwinteredexhibited an emission spectrum characteristic of overwinteringrather than summer leaves that had never been exposed to winter.

722 HUNER ET AL.

OVERWINTERING OF PERIWINKLE THYLAKOIDS

300

2)ffi200

E100

I (rmol photons*m*s )x10-'

B

MJI J SON.J.FMM J J A S O N D J F M A

1986 1987-MONT H

FIG. 1. A, Light-saturation curves for PSI activity of thylakoids isolated from periwinkle leaves at various times during the 1986-1987 growingseason in stand 1. (0), May 23, 1986; (*) August 20, 1986; (0) September 30, 1986; (A) November 18, 1986; (A) February 2, 1987; (O) March 26,1987. Each point represents the average ± SD of 3 replicate measurements. Activity is calculated as Mmol 02 consumed (mg Chl)-' h-'. B, Vmax,calculated from double reciprocal plots of the data in (A), plotted as a function of time during the 1986-1987 growing season.

300

2000.

F 100I

I (mmol photons-mr2- s) x 10

.

M J J A S O N D J F M A1986 -+- 1987---

MONTH

FIG. 2. A, Light-saturation curves for PSII activity(H20 -> DCPIP) of thylakoids isolated from peri-

winkle leaves at various times during the 1986-1987growing season in stand 1. Each point represents theaverage ± SD of 3 replicate measurements. Activityis calculated as qmol DCPIP reduced (mg Chl)-'h-'. B, Vmax, calculated from double reciprocal plotsof the data in (A), plotted as a function of timeduring the 1986-1987 growing season.

Table I. Estimated Quantum Efficienciesfor PSI and PSII

PSI PSIIDate

Initial slope' r2 Initial slope' r2

23/5/8620/8/8630/9/8618/11/862/2/87

26/3/87

0.2220.2060.1900.2380.2140.182

0.9530.9570.9370.9720.9610.931

0.4610.4060.2990.1930.1940.195

0.9920.9900.8490.9980.9990.987

a mol 02 consumed m2- smol photons (mg Chl) * h

mol DCPIP reduced m2 s

mol photons (mg Chl) h-Chl-Protein Complexes. From August 20, 1986 to March 26,

1987, the Chl a/b of isolated thylakoids from periwinkle leavesof stand 1 decreased from 2.59 ± 0.12 to 2.40 ± 0.05. Similarly,from August 16, 1984 to March 18, 1985, the Chl a/b of isolatedperiwinkle thylakoids decreased from 2.39 ± 0.08 to 2.18 ± 0.10.Thus, minimal variation (8-10%) in the Chl a/b was typicallyobserved over the course of one season.The Chl of higher plant chloroplasts is associated with specific

membrane polypeptides that associate to form the characteristicChl-protein complexes of thylakoid membranes (2). Figure 4illustrates comparative scans of Chl-protein complexes separated

Table II. Effect ofSnow Cover on PSI and PSII ActivityAll measurements represent the means of 3 determinations ± SD.

Chl a/b PSI Activitya PSII Activityb

Summerf 2.39 ± 0.08 150 ± 10 261 ± 26Winter + snowd 2.14 ± 0.07 279 ± 8 271 ± 21Winter-snow' 2.16 ± 0.10 251 ± 3 196 ± 12

a Light-saturated rates; pmol 02 consumed (mg Chl)-' h-' at25°C. b Light-saturated rates; Amol DCPIP reduced (mg Chl)-' h-' at25°C. CLeaf samples were harvested from stand 2 on August 16,1984. d Leaf samples were harvested from stand 2 on January 23,1985, with snow cover. e Leaf samples were harvested from stand 2on January 23, 1985, after a 14 d exposure to the absence ofsnow cover.

from isolated thylakoids of periwinkle leaves exposed to summerand winter conditions. Seven major peaks were observed andidentified by their electrophoretic migration and characteristicabsorption spectra (10). Over the course of 3 years, we observedno major changes in the proportions of the various Chl-proteincomplexes in thylakoids from spring and summer periwinkleleaves compared to periwinkle leaves exposed to low temperatureand overwintering conditions.

Lipid and Fatty Acid Composition. The results summarized inTables III and IV represent a comparison of the lipid and fattyacid analyses of total leaf extracts from summer and winter

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Plant Physiol. Vol. 87, 1988

zui

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FIG. 3. 77°K fluorescence emission spectra of periwinkle thylakoidsisolated from leaves in stand 1 at various times during the 1986-1987growing season (August 20, 1986, February 2, March 26, and May 7,1987) and the 1987-1988 growing season (August 20, 1987). Solid linesare emission spectra of thylakoids from leaves subjected to one overwin-tering exposure. Broken lines are emission spectra of thylakoids fromleaves prior to exposure to overwintering conditions.

3

summer-

winter

[0.3

[0.3

FIG. 4. Scans of SDS polyacrylamide slab gels of Chl-protein com-

plexes from periwinkle thylakoids isolated in summer (August 1985) andwinter (January 1986). Thylakoids were isolated from leaves ofthe 1985-1986 growing season in stand 2. Small arrow indicates the top of gel andthe large arrow indicates the direction of migration. The gel was scannedat 671 nm for Chl. Peak 1, CPla; peak 2, CP1; peak 3, oligomeric LHCII;peak 4, dimeric LHCII; peak 5, CPa; peak 6, monomeric LHCII, peak7, free pigment zone.

periwinkle leaves from stand 1. The summer leaves were devel-oped in the spring of 1987 and were harvested after full expansionin late June and July of 1987. The leaves designated as 'over-winter' developed and expanded in the spring and summer of1986 and subsequently were exposed to overwintering conditionsfrom December 1986 to March 1987. Thus, the former sampleswere obtained from fully expanded leaves that were never ex-posed to overwintering conditions. Exposure to low temperaturesand overwintering conditions caused significant alterations inboth the content and fatty composition of the major phospho-lipids, PC and PE, primarily associated with nonphotosyntheticmembranes (18). A 2-fold increase in the PE content and a 40

Table III. Effect ofOverwintering on the Ratio ofLipid:Chl ofLeafExtracts

Lipid:Chl (mol:mol)Lipid

Summera Over-winter"PC 1.32 ± 0.13 2.42 ± 0.23PE 0.58±0.09 1.46±0.10SL 0.43±0.12 0.55±0.11DGDG 2.94 ± 0.43 3.78 ± 0.51PG 0.97 ± 0.07 1.07 ± 0.21MGDG 3.82 ± 0.52 4.36 ± 0.42

a Leaf samples were harvested during late June and early July 1987,from stand 1; n = 3. b Leaf samples were harvested during March1987, from stand 1; n = 3.

to 80% increase in the PC content was observed after overwin-tering when measured on a Chl basis (Table III) or on a mol %basis (Table IV). In addition, PC exhibited a 1.5-fold and 2-foldincrease in the level of 18:3 and 18:2, respectively, whereas PEexhibited about a 1.2-fold increase in 18:3 and a 1.5-fold increasein 18:2. Increases in the levels of these unsaturated fatty acidsoccurred with a concomitant decrease in the levels of 18:1 in PCand PE.

In contrast, minimal changes were observed in the content ofthe major chloroplast lipids SL, DGDG, PG, and MGDG meas-ured on a Chl basis or as mol % (Tables III and IV). However,the fatty acid compositions of these chloroplast lipids did exhibita trend to greater levels ofunsaturation. Again, this was primarilydue to increases in 18:2 and 18:3 with concomitant decreases in18:1.PG of periwinkle exhibited significant levels of the trans-_A3-

hexadecenoic acid (trans-16: 1) (Table IV). However, the contentof this fatty acid was not affected significantly by exposure tolow temperature and overwintering conditions.

DISCUSSION

Exposure to natural low temperature and overwintering con-ditions induced major functional changes in isolated periwinklethylakoids. First, an approximate doubling in light-saturatedrates of PSI was observed (Fig. 1, A and B) with no effect on theapparent quantum efficiency (Table I). The 77°K fluorescenceemission spectra indicate that this increased PSI activity is notassociated with a shift in energy distribution in favor ofPSI sincethe ratio of F735/F686 appeared to decrease during the wintermonths (Fig. 3). Winter rye (7) and other cold-tolerant speciessuch as wheat, spinach, pea, broad bean, and Brassica exhibit asimilar increased capacity for light-saturated electron transportthrough PSI upon development at cold-hardening temperaturesunder controlled environment conditions (NPA Huner, unpub-lished data). Thus, the increased capacity for PSI electron trans-port under cold-hardening conditions appears to be a generalphenomenon observed under natural and controlled environ-ment conditions. Krol et al. (9) recently suggested that theincreased PSI activity observed after growth of winter rye, anannual, at cold-hardening conditions was a consequence of thy-lakoid biogenesis at low temperature. Although periwinkle thy-lakoids exhibit a twofold increase in PSI activity upon exposureto winter conditions, the overwintering leaves had developed andbecome fully expanded during the warm spring and summermonths. Thus, leaf development was complete prior to exposureto overwintering conditions. We conclude that chloroplast bio-genesis at low temperature is not necessary to observe the in-creased rates of light-saturated PSI activity in periwinkle, adicotyledonous evergreen.

Second, the apparent quantum efficiency for PSII electrontransport (H20 -. DCPIP) decreased by 50% from September to

724 HUNER ET AL.

OVERWINTERING OF PERIWINKLE THYLAKOIDS

Table IV. Lipid and Fatty Acid Comoosition of Total LeafExtractsfrom Summer and Overwintering PeriwinkleSummera Over-winterb

Lipid Fatty acid profile (mol %) Lipid Fatty acid profile (mol %)(mol %) 16:0 16:1 18:0 18:1 18:2 18:3 (mol %) 16:0 16:1 18:0 18:1 18:2 18:3

PC 13 ± 1 23 ± 2 tr 42 ± 2 18 ± 2 18 ± 1 18 ± 1 29 ±2 1 ±0 5 ±0 38 ± 1 27 1PE 5± 1 36± 1 1 1 17± 1 21 ±2 24±2 11 ±2 37±2 1 ±0 2±0 32± 1 28 1SL 4 ± 1 50 ± 1 2 ± 1 15 ± 2 7 ± 1 26 ± 1 4 ± 1 48 ± 2 1 ± 0 9 ± 3 10 ± 1 31 3DGDG 29±4 19±2 1 ±0 4± 1 3± 1 73± 1 28± 1 13±0 1 ±0 2±0 6±0 79±0PG 10± 1 29± 1 221 1±0 25±2 8± 1 15± 1 8± 1 38± 1 19±1 1±0 12± 1 14± 1 18±2MGDG 38 ± 3 9 ± 2 tr 9 ± 1 5 ± 0 77 ± 3 32 ± 1 5 ± 1 tr 2 ± 1 10 ± 1 85 1a Leaf samples were harvested during late June and early July 1987 from stand 1; n = 3. bLeaf samples were harvested during March 1987

from stand 1; n = 3.

March while light-saturated rates exhibited minor changes (Fig.2, A and B). In addition, we reported a decrease in 77°Kfluorescence at 694 nm in periwinkle thylakoids during thewinter. The fluorescence band at 694 nm originates from theChl a of the core antennae of PSII (4). The Chl a fluorescenceassociated with PSII core antennae exhibited an irreversibledecrease during the winter. This may reflect a disorganization ofthe PSII core antennae which would cause the reduced quantumefficiency observed for PSII electron transport. Clearly, furtherexperimentation is required to substantiate this hypothesis.

Isolated thylakoids from Pinus silvestris exhibited seasonalvariations in the light-saturated rates of photosynthetic electrontransport (13-17). PSI and PSII electron transport exhibited adecrease after the first frosts of September and October withmaximum inhibition (60-70%) occurring in February andMarch. Senser and Beck (20) also reported that Hill activity inisolated thylakoids from Picea abies was lowest during the falland winter months. These results are in sharp contrast to thosepresented for periwinkle in this report. Under continuous, nat-ural, snow-covered conditions, periwinkle thylakoids exhibited adoubling in the calculated Vm. for PSI electron transport (Fig.1B) and only minor variation in the calculated Vma, for PSIIactivity (Fig. 2B). Thus, not all plant species exhibit an inhibitionof in vitro PSI and PSII electron transport activity upon exposureto natural overwintering conditions. As discussed by Oquist andMartin (17), the study ofthe effects ofwinter stress on thylakoidsfrom conifers exposed to natural conditions is confounded bythe interaction of light, extreme desiccation, and low tempera-ture. However, periwinkle is typically snow covered during ex-posure to overwintering conditions in Canada which shouldminimize the possible damaging effects of high light intensityand extreme desiccation coupled with freezing temperatures.This may, in part, account for the differences between conifersand periwinkle with respect to inhibition of PSI and PSII activityupon exposure to overwintering conditions. This is supported bythe fact that periwinkle thylakoids exhibited significant inhibitionof light-saturated PSII activity only when exposed to overwinter-ing conditions in the absence of snow cover (Table II).

In general, the fatty acid composition of overwintering peri-winkle leaves tended toward greater unsaturation with the accu-mulation of 18:2 and 18:3 apparently at the expense of 18:1(Table IV). This is consistent with reports on overwinteringspruce and white pine (5, 19, 21), but it contrasts the results forP. silvestris which exhibited a decrease in the level of saturationspecifically in MGDG upon exposure to winter conditions (15).Whether these changes in the fatty acid composition of periwin-kle thylakoids are sufficient to alter the membrane fluidity willhave to await further experimentation.

In contrast to the major thylakoid lipids (MGDG, DGDG,PG, SL) (18), the major lipids of nonphotosynthetic membranes(PE and PC) exhibited a twofold increase in lipid content (Table

III). These results are consistent with recent published reports ofincreased membrane content and lipid unsaturation in nonpho-tosynthetic membranes during low temperature acclimation (12).

Recent results with winter rye indicated a specific 70% decreasein the trans-16:1 content in thylakoid PG upon growth anddevelopment at low, cold-hardening temperatures (18). Concom-itantly, the organization of LHCII was affected such that oligo-meric LHCII predominated during development at high temper-ature whereas the monomeric or some intermediate form ofLHCII predominated upon development at cold-hardening tem-peratures. It was concluded that low, developmental temperaturemodulates the organization of LHCII specifically by altering thetrans-16: 1 content of PG (8, 10, 1 1). In contrast, the results ofthis report clearly show that the organization of LHCII (oligo-meric LHCII:monomeric LHCII) in periwinkle thylakoids isstable to environmental changes and that the trans- 16:1 contentof PG in periwinkle thylakoids is not significantly affected byexposure to overwintering conditions. This may reflect speciesdifferences and/or differences in the pattern of leaf developmentbetween a cold-tolerant, dicotyledonous, herbaceous evergreenand a cold-tolerant, annual grass species.

In summary, the increased rate of in vitro, light-saturatedelectron transport through PSI observed upon exposure of peri-winkle, an endemic species, to natural, overwintering conditionsis consistent with the results reported for winter rye developed atlow temperature under controlled environment conditions (7).The increased PSI activity occurred with minimal change in thestructure of the photosynthetic apparatus. This may be a generalphenomenon in cold-tolerant, herbaceous plants. Since theoverwintering periwinkle leaves examined in this study were fullydeveloped prior to winter, chloroplast biogenesis at low temper-ature does not appear to be an absolute requirement to developan increased capacity for PSI electron transport. Prolonged ex-posure of fully expanded leaves to low temperature may accom-plish the same effect in certain species. The regulation and thepossible physiological importance of this unique phenomenonto plant cold-tolerance is forthcoming (our manuscript in prep-aration).

Acknowledgments-We thank Dr. A. Maun for the use of his Li-Cor leaf areameter and Dr. W. Ware for the use of his fluorescence spectrophotometer. NPAHis gratefuil to Erin Huner for her assistance in the snow cover experiment.

LITERATURE CITED

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2. BAKER NR, JP MARKWELL 1985 Pigment-protein complexes and their inter-actions. In J Barber, NR Baker, eds, Photosynthetic Mechanisms and theEnvironment. Elsevier, Amsterdam, pp 49-90

3. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities ofprotein utilizing the principal ofprotein dye binding.Anal Biochem 72: 248-254

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5. DYOE DR, GN BROWN 1979 Glycerolipid and fatty acid changes in Easternwhite pine chloroplast lamellae during the onset of winter. Plant Physiol 64:924-929

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7. HUNER NPA 1985 Acclimation of winter rye to cold-hardening temperaturesresults in an increased capacity for photosynthetic electron transport. Can JBot 63: 506-511

8. HUNER NPA, M KROL, JP WILLIAMS, E MAISSAN, PS Low, D ROBERTS, JETHOMPSON 1987 Low temperature development induces a specific decreasein trans-,A3phexadecenoic acid content which influences LHCII organization.Plant Physiol 84: 12-18

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