Low Temperature Development Induces a Specific Decrease in trans- 3-Hexadecenoic Acid Content which Influences LHCII Organization

Plant Physiol. (1987) 84, 12-18 0032-0889/87/84/0012/07/$01.00/0

Low Temperature Development Induces a Specific Decrease in trans-A3-Hexadecenoic Acid Content which Influences LHCII Organization1 Received for publication July 2, 1986 and in revised form December 1, 1986

NORMAN P. A. HUNER*, MARIANNA KROL, JOHN P. WILLIAMS, ELLEN MAISSAN, PHILLIP S. Low, DANE ROBERTS, AND JOHN E. THOMPSON

Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7 (N.P.A.H., M.K.); Botany Department, University of Toronto, Toronto, Canada M5S JA] (J.P.W., E.M.); Chemistry Department, Purdue University, West Lafayette, Indiana 47907 (P.S.L.); and Biology Department, University of Waterloo, Waterloo, Canada N2L 3GJ (D.R., J.E.T.) ABSTRACT Lipid and fatty acid analyses were perfornmed on whole leaf extracts and isolated thylakoids from winter rye (Secalk cereak L. cv Punia) grown at 5°C cold-hardened rye (RH) and 20°C nonhardened rye (RNH). Although no significant c ge in total lipid content was observed, growth at low, cold-hardening temperature resulted in a specific 67% (thylakoids) to 74% (whole leaves) decrease in the trans-A3-hexadecenoic acid (trans16:1) level associated with phosphatidyldiacylglycerol (PG). Electron spin resonance and differential scanning calorimetry (DSC) indicated no significant difference in the fluidity of RH and RNH thylakoids. Sepa-

ing temperatures (5C) (RH)2 exhibited a differential extractability to the nonionic detergents, Triton X-100 and-,-octylglucoside, compared to Chl b of rye thylakoids developed at warm temperatures (20C) (RNH). Autoradiograms of RH and RNH thylakoids extracted with SDS indicated that the LHCII polypeptides were phosphorylated in vitro to the same extent by a lightdependent thylakoid protein kinase. However, solubilization of labeled RH and RNH thylakoids with B-octylglucoside and subsequent electrophoresis indicated that minimal amounts of labeled LHCII could be extracted from RH thylakoids compared to the copious amounts of labeled LHCII extracted from RNH thylakoids. Labeled RH LHCII polypeptides were reported to be associated with a nonpigmented protein complex with a molecular mass greater than that of CPl upon solubilization of RH thylakoids with ,3-octylglucoside. Griffith el al. (13) concluded that growth and development of winter rye at low temperature results in an alteration in protein-protein interactions associated with LHCII. This is consistent with previous reports which indicated that RH thylakoids exhibited a decrease in particle size on the EF fracture face ( 17), an increase in the F685/F742 but did not exhibit a concomitant increase in PSII activity at the expense of PSI activity nor any detectable change in photosynthetic unit size (11, 15-17). However, Huner et al. (10, 17) have also reported that no significant differences between RH and RNH thylakoids exist with respect to pigment or polypeptide composition. In this report, we examine the lipid and fatty acid compositions of RH and RNH thylakoids in order to determine if the observed changes in the structure and function of RH LHCII

ration of chlorophyll-protein complexes by sodium dodecyl sulfate-poly-

acrylamide gel electrophoresis indicated that the ratio of oligomeric light harvesting complex:monomeric light harvesting complex

(LHCII,:LHCII3) was 2-fold higher in RNH than RH thylakoids. The

ratio of CP1a:CP1 was also 1.5-fold higher in RNH than RH thylakoids. Analyses of winter rye grown at 20, 15, 10, and 5C indicated that both, the tras-16:1 acid levels in PG and the LHCII,:LHCII3 decreased concomitantly with a decrease in growth temperature. Above 40C, differential scanning calorimetry of RNH thylakoids indicated the presence of five major endotherms (47, 60, 67, 73, and 86C). Although the general features of the temperature transitions observed above 40°C in RH thylakoids were similar to those observed for RNH thylakoids, the transitions at 60 and 73°C were resolved as inflections only and RH thylakoids exhibited trasitions at 45 and 84°C which were 2°C lower than those observed in RNH thylakoids. Since polypeptide and lipid compositions of RH and RNH thylakoids were very similar, we suggest that these differences reflect alterations in thylakoid membrane organization. Specifically, it is suggested that low developmental temperature modulates LHCII organization such that oligomeric LHCHI predominates in RNH thylakoids whereas a monomeric or an intermediate form of LHCII predominates in RH thylakoids. Furthermore, we conclude that low developmental temperature modulates LHCH organization by specifically altering the fatty composition of thylakoid PG.

2Abbreviations: RH, cold-hardened rye; RNH, nonhardened rye: LHCII, light harvesting Chl a/b protein associated with photosystem II; LHCII,, oligomeric form of LHCII; LHCII2, dimeric form of LHCII; LHCII3, monomeric form of LHCII; CPla, oligomeric form of CPI; CPI, Chl a-protein complex associated with the reaction center of photosystem I; CPa, Chl a-protein complex associated with the reaction center for PSII; FP, free pigment; F6g5, 77'K Chl a fluorescence emission maximum at 685 nm; F742, 77°K Chl a fluorescence emission maximum at 742 nm; DOC, deoxycholate; DPH, 1,6-diphenyl-1,3,5-hexatriene; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SL, sulfoquinovosyldiacylglycerol; PG, phosphatidyldiacylglycerol; MGDG, monogalactosyldiacyl glycerol; DGDG, digalactosyldiacyl glycerol; trans-16:1, trans-A3-hexadecenoic acid; 16:0, palmitic acid; 18:3, linolenic acid; DSC, differential scanning calorimetry; DCMU, 343,4-dichlorophenyl)1,1 dimethyl urea; EF, exoplasmic fracture surface; 1(1,14), 16-doxylstearic acid.

Recently, Griffith, et al. (13) reported that Chl b of thylakoid membranes isolated from winter rye grown at low, cold-harden'

Supported by the Natural Sciences and Engineering Research Coun-

cil of Canada. 12

LOW TEMPERATURE DEVELOPMENT AND trans-A3-HEXADECENOIC ACID are related to alterations in thylakoid lipid composition and content. MATERIALS AND METHODS Plant Materials. Winter rye (Secale cereale L. cv Puma) was grown in vermiculite watered with Hoagland nutrient solution. Seedlings were initially grown in controlled environment growth chambers at a 20°C/16°C (day/night) temperature regime and a 16 h photoperiod with a light intensity of 200 Mmol photons m 2 s-' (PAR) for 7 d. After this time, the primary leaf had fully expanded whereas the second leaf had only partially expanded (18). Seedlings (7-d-old) were then shifted for an additional 7 to 8 weeks to a temperature regime of 5°C/5°C (day/night) with all other conditions kept constant. These plants are referred to as RH. Control seedlings remained at the 20°C temperature regime for an additional 2.5 to 3 weeks. These plants are referred to as RNH. Krol et al. (18) have shown that RH and RNH plants were of comparable physiological age when grown under these conditions. Isolation of Thylakoid Membranes. Uppermost, fully expanded leaves of RH and RNH plants were macerated at 4°C and 50 mm Tricine (pH 7.8) containing 0.4 M sorbitol and 10 mM NaCl with two 5 s bursts of a Waring Blendor. The brei was filtered through two layers of Miracloth and the filtrate was subsequently centrifuged at 3000g for 5 min. The pellet was washed in 50 mM Tricine (pH 7.8) containing 10 mM NaCl and 5 mM MgCl2 and subsequently resuspended in 50 mM Tricine (pH 7.8) containing 0.1 M sorbitol, 10 mm NaCl, and 5 mM MgCI2 and kept on ice in the dark. Lipid and Fatty Acid Analyses. Uppermost fully expanded leaves of RH and RNH plants as well as isolated RH and RNH thylakoids were extracted and analyzed for their lipid and fatty acid compositions as previously described (27, 28). Separation of Chlorophyll-Protein Complexes. Thylakoid membranes were isolated as described above except that they were washed once in cold, double-distilled H20, once in 1 mM EDTA (pH 8.0), and twice in 50 mM Tricine (pH 8.0). The thylakoid membranes were then resuspended in 0.3 M Tris (pH 8.8) containing 13% (v/v) glycerol and 1% (w/v) SDS (SDS:Chl=10:1) and then 2% (w/v) DOC in 0.3 M Tris (pH 8.8) was added to give a DOC:SDS:Chl of 20:10:1. The solubilized membranes were immediately subjected to SDS polyacrylamide slab gel electrophoresis at 4°C in the dark according to Waldron and Anderson (26) to separate the pigment-protein complexes. Room temperature scans of the pigmented complexes and their characteristic absorption spectra were obtained using a Shimadzu UV-250 spectrophotometer. The relative Chl contents of the individual pigmented complexes were determined by relative peak areas. Differential Scanning Calorimetry of Thylakoid Membranes. RH and RNH thylakoids were isolated as described above and then resuspended in a high ionic strength medium containing 50 mM Tricine (pH 6.8), 0.1 M sorbitol, 10 mm NaCl, 70 mM KCI, 5 mM MgCl2, and 20 mM (-mercaptoethanol to a final Chl concentration of 2 to 3 mg ml-'. Heat capacity measurements were made in a Microcal-1 differential scanning calorimeter (Microcal Inc., Amherst, MA) employing matched 1 ml platinum cells. A 1 ml aliquot of freshly prepared thylakoid membranes was added to one cell and an equal volume of buffer in the other cell. The heating rate was 1°C min-'. Heat capacity was determined from the integrated peak area derived from a calibrated heat pulse delivered to the reference cell of the calorimeter. Electron Spin Resonance. A stock solution (2 mM) of I(1,14) was prepared in absolute ethanol and stored at -20°C. To minimize the reduction of spin label partitioned into thylakoids, the membranes were first taken up in resuspension buffer (50 mM Tricine [pH 7.8], 0.1 M sorbitol, 10 mM NaCl, and 5 mM

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MgC12) containing 10 mM hydroxylamine and kept on ice in the dark for 5 min. At the end of this treatment, the suspension was centrifuged at 4000g for 5 min, and the membranes were taken up in resuspension buffer to a concentration of 2 mg Chl/ml. For incorporation of spin label, 20 1l of I(1,14) stock solution were evaporated onto the bottom and sides of a 15 x 75 mm disposable culture tube, and 200 Al of thylakoid membrane suspension were added to the same tube. The tube was vortexed intermittently for 5 min, and the spin labeled membranes were then taken up into a 100 Al capillary tube, which was sealed at one end with capillary tube sealant (Miniseal) and placed in the cavity of a Varian E-12 ESR spectrometer. Spectra were recorded at 5 and 25°C, and rotational correlation time (Tc) were calculated according to the following equation: Tc = 6.5 x 10-`0 w,[hI/h- )'-l]s where w, and hi are the width and height of the low-field spectral line and h_, is the height of the high-field spectral line (24). Previous studies with thylakoid membranes have indicated that there is no change in the value of Tc when twice the normal concentration of spin probe is used (i.e. 40 Ml rather than 20 Ml of stock solution) (22). This can be interpreted as indicating that perturbation effects attributable to the probe itself are minimal. As well, identical Tc values were obtained in the presence and absence of 16 mM chromium oxalate, a spin-broadening agent that eliminates the water signal seen in the scans (5). This serves as a control on how well the h_, value is measured.

RESULTS

Lipid and Fatty Acid Composition. The results of Table I indicate that, in general, there were no significant differences in the lipid content of rye leaves developed at either 5 or 20C. Fatty acid analysis of the various lipid classes indicated that linolenic acid (18:3) is the major unsaturated fatty acid present. However, RH leaves exhibited only a minimal increase (2-9 mol %) in the total 18:3 content of the various lipids. The most significant change in fatty acid composition was the 74% decrease in the trans-16:1 level and the accompanying 48% increase in the palmitic acid level (16:0) associated with PG. Since trans16:1 is thought to be specifically associated with chloroplast photosynthetic membranes (14), the lipid and fatty acid composition of isolated RH and RNH thylakoids was examined (Table II). Comparison of the results of Tables I and II indicate that the lipid composition of isolated rye thylakoids exhibited a significant decrease in PC and PE content and a concomitant increase in the content of DGDG and MGDG relative to that observed for total leaf extracts. This was expected since DGDG and MGDG are the major lipid components of photosynthetic membranes from higher plants (14). As was observed for total leaf extracts, there were no significant differences in lipid content between RH and RNH thylakoids. However, low temperature development appeared to result specifically in a 67% decrease in trans- 16:1 levels with a 61% increase in 16:0 levels associated with PG. This resulted in a trans-16:1/16:0 of about 1.3 in RNH thylakoids in contrast to a trans-16:1/16:0 of 0.3 in RH thylakoids. Thus, growth and development of rye leaves at low temperatures appears to result in a major and specific decrease in the trans- 16:1 level associated with thylakoids. The minor increases (2-8 mol %) in the 18:3 content of RH thylakoid, MGDG, DGDG, and PG may lead to changes in the fluidity of the thylakoid membranes. Therefore, the microenvironment of the RH and RNH lipid bilayer was compared by electron spin resonance using the spin label I(1,14). The results presented in Table III indicate that there was no significant difference in the rotational correlation time (Tc) for RH and RNH thylakoids measured at either 25 or 5°C. As expected, both

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Plant Physiol. Vol. 84, 1987

HUNE,R ET AL.

Table I. Lipid and Fatty Acid Compositions of Total Leaf Extracts All values were calculated as mol % of the total. The data represent the mean of five different isolations ± SD. RNH Leaves RH Leaves Fatty acid profile

Lipid

16:0

16:1

18:0

18:1

Lipid

18:3 mol % PC 15±2 24±2 2±0.5 2± 1 39±2 33±4 19±3 PE 5 ± 1 31 ± 5 1 ± 0.4 1 ± 0.4 39 ± 4 28 ± 4 5 ± 2 SL 6±2 29±3 1 ±0 1 ±0 7±2 62±5 5± 1 DGDG 25±4 8±0.5 1 ±0 1 ±0 3±0.4 87± 1 26± 1 PG 11±1 23±3 23±2 1±0 1±0 7±1 45 ± 4 10±1 MGDG 38±2 1 ±0.5 tia tr 3± 1 95± 1 34±3 a tr, trace amounts only (