LIPID COMPOSITION OF PEA (Pisum sativum L.) AND MAIZE (Zea mays L.) ROOT PLASMA MEMBRANE AND MEMBRANE–BOUND PEROXIDASE AND SUPEROXIDE DISMUTASE

Plasma membrane was isolated from roots of pea and maize plants and used to analyze POD and ��OD isoforms, as well as lipid composition� Among lipids, phospholipids were the main lipid class, with phosphatidylcho� line being the most abundant individual component in both pea and maize plasma membranes� ��ignificant differences between the two plant species were found in the contents of cerebrosides, free sterols, and steryl glycosides� Most maize POD isoforms were with neutral and anionic pI values, but the opposite was observed in pea� While both anionic and cationic ��OD isoforms were isolated from maize, only two anionic ��OD isoforms were detected in pea�


INTRODUCTION
The plasma membrane (PM) of root cells has numerous physiological roles comprising cell wall biosynthesis, hormone action, and signalling proc� esses during disease, plant development, and pro� grammed cell death (N e i l et al�, ������ M i �� a and N e i l et al�, ������ M i �� a and M i �� a and L ü t h j e, ���3�� V u l e t i ć et al�, ���5�� �� c h o p f e r �� c h o p f e r and L i s z �� a y, ���6)� In all of these processes, reac� � In all of these processes, reac� tive oxygen species (RO��) such as superoxide anion radicals (O � •-), H � O � , and hydroxyl radicals ( .OH) play a pivotal role� A number of environmental stresses lead to enhanced production of RO�� within PM that may cause oxidative damage� Hydroxyl radicals can initiate lipid peroxidation in a radical chain reaction leading to increased membrane lea��� age and cell death� Proteins embedded in the mem� brane may also be damaged by RO��, leading to loss of enzyme activity and transport processes� The role of extracellular antioxidant enzymes in regulation of RO�� concentrations in the apoplast is important� Evidence for PM�bound POD and ��OD activity in higher plants has been reported (K a r p i n s �� a et al�, ���1�� H a d ž i�T a š �� o v i ć Š u �� a l o v i ć et al�, ���3�� V u l e t i ć et al�, ���3�� K u �� a v i c a et al�, ���5)� It has been shown that protein-lipid interaction is crucial for localization of membrane proteins and, consequently, their function (E s c r i b a et al�, 1997�� v a n K l o m p e n b u r g et al�, 1997�� B e n f e n a t i et al�, 1998�� B e r g l u n d et al�, ������ v a n Vo o r s t and K r u i j f f, ����)� In the present study, we compared the IEF profile of PM�bound POD and ��OD isoforms of pea and maize roots with the lipid composition of these two membranes� POD and ��OD coexistence in PM of pea and maize roots would implicate their specific role in the antioxida� tive protection of membrane constituents, as well as in the redox communication between apoplast and symplast, which is part of signalling processes�

Plant growth
Pea (Pisum sativum L�) and maize (Zea mays L�) seedlings were grown in hydroponic culture with continuous aeration in a growth chamber with day/night temperatures of �1°C/16°C, a 16�h photoperiod, a photon flux density of 4�� µmol m �� s �1 , and 7� to 75% relative humidity� Light was provided by fluorescent tubes (Osram L14�W/��) and incandescent lamps (Philips �5�W)� ��eeds were pre�germinated on moistened paper and then placed in plastic pots filled with a half�strength aerated Hoagland's No� � solution that was renewed every 3 days (H o a g l a n d and A r n o n, 195�)� At day 14, roots of intact plants were washed with distilled water and collected for PM isolation and biochemi� cal analyses�

Isolation of plasma membrane
PM was isolated using a two�phase partition system� Roots were cut into pieces and immediately ground using a Braun blender in � volumes of an extraction medium consisting of 5� mM TRI���HCl, pH 7�5, ���5 M sucrose, 3 mM Na � EDTA, 1� mM ascorbic acid, and 5 mM diethyldithiocarbamic acid� The homogenate was filtered through four layers of a nylon cloth and centrifuged at 1�,���g for 1� min� The supernatant was further centrifuged at 65,���g for 3� min to yield a microsomal pellet, which was resuspended in � ml of a resuspension buffer (5 mM K�phosphate, pH 7�8, ���5 M sucrose and 3 mM KCl)� The PM was isolated by loading micro� somal suspension (1 g) onto an aqueous two phase polymer system to give a final composition of 6�5% (w/w) Dextran T 5��, 6�5 % (w/w) polyethylene gly� col, 5 mM K�phosphate (pH 7�8), ���5 M sucrose, and 3 mM KCl� The PM was further purified using a two�step batch procedure� The resulting upper phase was diluted fourfold with 5� mM TRI��� HCl, pH 7�5, containing ���5 M sucrose, and centrifuged for 3� min at 1��,���g� The resultant PM pellet was resuspended in the same buffer containing 3� % eth� ylene glycol and stored at -8� o C for lipid analyses� All steps of the isolation procedure were carried out at 4 o C� In order to chec�� the purity of the PM of maize and pea roots, the activity of the vanadate�sensi� tive ATPase as a mar��er enzyme was determined (N a v a r i � I z z o et al�, 1993)� Cytochrome c oxi� dase, NADH cytochrome c reductase, and NO 3 � � sensitive ATPase activities were used as mar��ers of mitochondria, endoplasmic reticulum, and tono� plast, respectively (N a v a r i�I z z o et al�, 1993)� Tests with the mar��ers showed that, as a mean value of the isolations performed, ATPase specific activity in both maize and pea was 66% higher in the PM than in the microsomal fraction�� vanadate inhibited ATPase activity by 88% in the PM fractions and by 35% in the microsomal ones� The addition of KNO 3 negligibly reduced ATPase activity in the PM frac� tions (6 and 4% inhibition in maize and pea, respec� tively)� The specific activities of mar��er enzymes such as cytochrome c oxidase and NADH cyto� chrome c reductase in the upper phase of both PM were 4 and 8%, respectively, of those determined in the lower phase�

Fatty acid analysis
Fatty acid methyl ester derivatives from PL were obtained as previously described (Q u a r t a c c i et al�, 1997) and separated by GLC on a Dani 86�1� HT gas chromatograph equipped with a 6��m x ��3��mm ��P��34� fused silica capillary column (��upelco ��igma�Aldrich, U��A) coupled to a flame ionization detector (column temperature of 175 o C)� Both the injector and detector were maintained at �5� o C� Nitrogen was used as the carrier gas at ��9 ml min �1 with a split injector system (split ratio 1:1��)� Heptadecanoic acid was used as the internal standard�

Determination of POD and SOD isoforms
For determination of POD and ��OD isoforms on native gel, the same amounts of PM proteins were loaded� Native electrophoresis was performed on 5% stac��ing and 1�% running gel with a reservoir buffer consisting of ����5 M TRI�� and ��19� M Gly (pH 8�3) at �4 mA for 1�� min� IEF was carried out in 7�5% polyacrylamide gel with 3% ampholite in a pH gradient from 3 to 9� Mar��ers for isoelectrofocusing with pI range of 3�6�9�3 (��igma) were used to deter� mine pI values of POD and ��OD isoforms� To assay POD activity, gels were incubated with 1�% 4�chloro�α�naphthol and ���3% H � O � in 1�� mM K�phosphate buffer (pH 6�5)� Determination of ��OD activity on gels was performed according to B e a u c h a m p and F r i d o v i c h (1971)� After in� cubation in a reaction mixture (��1 M EDTA, ���98 mM nitroblue tetrazolium, ���3� mM riboflavin, and � mM TEMED in K�phosphate buffer, pH 7�8) for 3� min in the dar��, gels were washed in distilled water and illuminated with white light� Band density, ex� pressed in relative units, of different POD and ��OD isoforms after separation on IEF gel was determined using TotalLab software (Nonlinear Dynamics, UK)� Protein content was measured by the method of B r a d f o r d (1976), with bovine serum albumin as a standard�

Statistical analysis
Results, unless differently specified, are the  means of three replicates of three independent experiments (n = 3)� Values for means followed by different letters are significantly different at P ≤ ���5 (Mann�Whitney test)� RE��ULT�� Native PAGE of PM proteins isolated from 14�day�old pea and maize roots showed one POD isoform with low mobility in pea and two POD isoforms in maize (Fig� 1A)� ��everal wea�� bands were noticed in both species as well (Fig� 1A)� The IEF profile of pea POD showed that the low mobile isoform included three cationic isoforms (pI 8�3, 8�8 and 9��) and one neutral POD isoform� Two wea�� anionic (pI 4�5) isoforms were also detected on gels� In maize, PM several isoforms with pI from 6�8 to 5�6 and two wea�� bands with pI 6�8 were detected (Fig� 1B)� ��eparation of PM�bound ��OD isoforms by IEF from pea roots showed the presence of two anionic isoforms (pI of about 5�5) (Fig� �)� Maize PM con� tained several bands, both anionic and cationic: in addition to one strong anionic band with pI of about 5, three wea�� bands (pI of about 5�6), one neutral (pI of about 7��) and two cationic ��OD isoforms (with pI of about 8�6) were observed (Fig� �)� Total POD and ��OD activities calculated as the sum of indi� vidual band densities showed that POD activity was higher in pea (346 relative units) than in maize (�77 relative units)� On the contrary, total ��OD activity in pea PM was three times lower (8� relative units) than in maize (�4� relative units)� Total lipid contents of maize and pea PM, cal� culated as the sum of each lipid class detected and expressed on a protein basis, were not significantly different (Table 1)� Phospholipids were the major lipid class in both species and accounted for about half of the total lipids, with no difference in their amounts� In pea and maize, steryl lipids repre� sented �8 and 33%, respectively, of total PM lipids, and among them F�� accounted for 49% and 54%, respectively� Plasma membrane isolated from pea roots contained cerebrosides in a quantity of ���77 μmol/mg of protein, a value 3�% lower than the con� tent detected in maize (��1�9 μmol/mg of protein)� However, their molar percentage was similar in pea and maize (19 and �1% of total lipids, respectively) (Table 1)� Among PL, phosphatidylcholine (PC) represented the main compound in both plants, fol� lowed by phosphatidylethanolamine (PE) (Fig� 3)� As for cerebrosides, their percentage did not differ between the two species� Fatty acid compositions of PM total lipids showed that linoleic (18:�) and palmitic (16:�) acids were the most abundant fatty acids in both pea and maize, the unsaturation degree being similar (Table �)� A significant difference in the amount of 18:3 and 14:� was found between species� DI��CU����ION A wide variation in lipid composition of isolated Peroxidase isoforms from pea and maize had different mobility on native PAGE gels and different pI values (Fig� 1)� While pea PM contained highly cationic POD isoforms, maize PM contained neutral and slightly anionic isoforms� We detected several ��OD isoforms in maize PM and two anionic ��OD isoforms in pea (Fig� �)� Two anionic (pI 5�5) and two cationic (pI 8�6) ��OD isoenzymes were detected in maize PM by K u �� a v i c a et al� (���5)� Analy� sis of the extracellular and cytosolic ��OD isoforms from ��cotch pine showed that the extracellular iso� forms have distinctly higher isoelectric points than those reported for cytosolic ��OD (�� c h i n �� e l et al�, 1998)� K a r p i n s �� a et al� (���1) found CuZn���OD with high isoelectric points (1���) in PM of the same species� The significant differences found in lipid composition of pea and maize (PC to PE and F�� to PL molar ratios, cerebroside and conjugate sterol amounts, Table 1) indicate that lipids may affect binding of differentially charged POD and ��OD isoforms to PM� In vivo functioning of a particular enzyme is li��ely to be determined by its location in a particular tissue or compartment (cellular or subcellular), which in turn may depend in part on its ionic nature, as was shown for cell wall (P e n e l et al�, ������ C a r p i n et al�, ���1)�  Table 2. Fatty acid composition of pea and maize plasma membrane total lipids� 14:��myristic acid, 16:��palmitic acid, 16:1�palmitoleic acid, 18:��stearic acid, 18:1�oleic acid, 18:��linoleic acid, 18:3�linolenic acid, ��:��behenic acid� M i �� a and L ü t h j e, ���3�� M o j o v i ć et al�, ���4�� V u l e t i ć et al�, ���5)� Although most of these func� tions were also demonstrated for soluble and cell wall-bound POD (H i r a g a et al�, ���1�� K a w a n o, ���3), there may be some functions specific to mem� brane�bound POD, namely the membrane�protec� tive function� For CuZn���OD with high isoelectric points, it has been proposed that in the phloem it acts as a regulator of H � O � pulses, being involved in transmission of systemic signals in wounding or pathogen responses (K a r p i n s �� a et al�, ���1)� Our results showed that POD and ��OD activities are intrinsic to pea and maize root PM (Figs� 1 and �)� Total POD activity, calculated as the sum of individ� ual band densities on IEF gel, was higher in pea than in maize PM� On the other hand maize root PM had cationic ��OD isoforms and higher total ��OD activity than in pea PM� POD and ��OD coexistence in the PM would provide a complementary enzymatic system in ad� dition to NADPH oxidase, their role being to defend against RO�� and maintain the H � O � /O � .�/ .OH bal� ance�