YANG Wen, FENG Fu-Ying, HOU Hai-Tong, JIANG Gui-Zhen, XU Yi-Nong *, KUANG Ting-Yun

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1 Acta Botanica Sinica 2004, 46 (2): Alternation in Lipid Composition of Wheat Leaves Induced by Phosphate Deficiency Is Related to Both Lipid Biosynthesis and Phosphatidylglycerol Degradation YANG Wen, FENG Fu-Ying, HOU Hai-Tong, JIANG Gui-Zhen, XU Yi-Nong *, KUANG Ting-Yun (Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing , China) Abstract: In this study, the causes of the changes in lipid composition induced by different phosphate nutrient levels were investigated. Wheat plants were grown in phosphate-deficient and phosphatesufficient conditions, respectively, and lipid compositions in the leaves of 9-day-old and 16-day-old plants were analyzed. We found that phosphate deficiency induced a dramatic change at the lipid levels in photosynthetic membranes of wheat leaves and the extent of changes in lipid composition depended on the leaf ages. Phosphate deficiency induced a gradual decrease in PG and MGDG and a concomitant increase in DGDG and SQDG from the first leaf to the second and the third leaf on 16-day-old plants. In addition, as compared to leaves grown under phosphate sufficient solution, PG content in the first leaf of 16-day-old plants was significantly lower than that of 9-day-old leaf with 2.5 mol% versus 5.5 mol% when these plants were grown under phosphate deficient condition. From these results, it is suggested that the alternation in lipid composition in wheat leaves induced by phosphate deficiency is related to both lipid biosynthesis and PG degradation. PG decrease in younger leaves is mainly due to insufficient phosphate supply for PG biosynthesis, while PG degradation mainly resulted in the PG decrease in older leaves. Key words: phosphate transport; phosphate deficiency; thylakoid membrane lipids; phosphatidylglycerol; wheat leaf The reaction process of photosynthesis depends on highly organized pigment protein complexes that are embedded in the polar glycerolipid matrix of thylakoid membranes inside chloroplasts in the higher plants (Essigmann et al., 1998). Therefore, glycerolipids play an important role in maintaining the structure and function of protein complexes. Thylakoid membranes consist of four glycerolipids: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), phosphatidylglycerol (PG) and sulfoquinovosyl diacylglycerol (SQDG) (Siegenthaler and Murata, 1998a). In general, lipid composition is highly conserved in higher plant photosynthetic membranes (Benning, 1998). It has been shown that phosphate deficiency strongly affected lipid composition of the thylakoid membrane by decreasing the relative content of PG and increasing that of DGDG and SQDG in both photosynthetic bacteria (Benning et al., 1993; Güler et al., 1996) and Arabidopsis thaliana (Essigmann et al., 1998). In addition, the culture condition of phosphate deficiency resulted in an increase of SQDG and a decrease of PG in Chlamydomonas reinharditti (Sato et al., 2000a). These results corroborated that plants were able to respond to phosphate deficiency by a selective accumulation of SQDG and DGDG to a lesser extent (Härtel et al., 1998). Phosphate is a key compound in metabolic processes in plants, including energy transfer, photosynthesis, respiration and lipid biosynthesis (Raghothama, 2000). Higher plants possessed two distinct pathways for the synthesis of glycerolipids in photosynthetic membranes, the prokaryotic pathway in inner envelope of the chloroplasts and the eukaryotic pathway in endoplasmic reticulum (Ohlrogge and Brows, 1995). PG in photosynthetic membranes is the only product of the prokaryotic pathway, and the remaining chloroplast lipids MGDG, DGDG and SQDG in many plants including wheat, are synthesized entirely by the eukaryotic pathway. In addition to enzymes, some components containing phosphate are used as substrates in glycerolipid biosynthesis through both pathways. For example, both pathways begin with the synthesis of phosphatidic acid (PA) and phosphatidylcholine (PC) are the substrate for MGDG and SQDG synthesis in the eukaryotic pathway (Siegenthaler and Murata, 1998b). Therefore, changes in the lipid composition induced by phosphate Received 6 Jun Accepted 1 Sept Supported by the State Key Basic Research and Development Plan of China (G ) and the National Natural Science Foundation of China ( A). * Author for correspondence. Fax: +86 (0) ; <yinongxu@ns.ibcas.ac.cn>.

2 deficiency are involved in lipid biosynthesis. It is well known that under phosphate deficiency the phosphate in older leaves can be transported to younger leaves (Mimura, 1995). PG is the sole phospholipid in thylakoid membranes. A lot of studies have demonstrated that PG plays an important role in the structure and function of thylakoid membranes (Hagio et al., 2000; Kruse et al., 2000; Sato et al., 2000b; Hagio et al., 2002; Xu et al., 2002a; Gombos et al., 2002). So far, there is little knowledge about whether phosphate in PG is able to remobilize from older leaves to younger ones. In the present study, the causes of the changes in lipid composition of wheat leaves induced by phosphate deficiency were investigated. We found that phosphate deficiency induced drastic changes at level of lipids in wheat leaves, which are not only involved in lipid biosynthesis but also in PG degradation. 1 Materials and Methods 1.1 Materials Seeds of winter wheat (Triticum aestivum var. Zhongyou 9507) were soaked for 5 h in distilled water and were germinated at 25. Seedlings were cultured in Hoagland solutions containing 0.01 mmol/l and 1.0 mmol/l KH 2 PO 4, respectively, in a controlled environment chamber under a 14 h photoperiod (400 µmol. m 2.s 1 ) at 25 /20 (day/ night). Nutrient solutions in the hydroponic systems were changed every two days. The first fully expanded leaves of 9-day-old plants and the first, the second and the third expanded leaves of 16-day-old plants were used in this investigation. 1.2 Lipid extraction and separation Wheat leaves were ground into powder in liquid nitrogen. Lipids were extracted according to the method of Bligh and Dyer (Bligh and Dyer, 1959). Lipid extracts were separated into individual lipid classes by two-dimensional silica gel TLC (G model, 10 cm 10 cm, Qingdao Oceanic Chemical Plant). TLC plates were developed with acetone/ toluene/water (91:30:8, by volume) in the first direction and with chloroform/methanol/isopropylamine/concentrated ammonia (65:35:0.5:5, by volume) in the second direction. Spots were visualized by spraying the plates with 0.01% primuline in acetone/water (60:40, by volume) under UV (360 nm). 1.3 Fatty acid analysis The fatty acid analysis was carried out according to the method of Xu et al. (2002b). Individual lipids separated by TLC were transesterified to the fatty acid methyl esters with 5% H 2 SO 4 in methanol at 85 for 1 h. The fatty acid methyl esters were extracted with hexane and were separated on a Hewlett-Packard 6890 gas chromatography supplied with a hydrogen flame ionization detector and a capillary column HP INNOWAX (30 m; i.d mm). The column was run at (5 /min). Heptacanoic acid (17:0, from Sigma) was used as the internal standard. The relative content of individual lipids was demonstrated by molar percent (mol/%). 2 Results The major polar glycerolipids detected in leaves of higher plants were MGDG, DGDG, PG, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) (Siegenthaler and Murata, 1998b). Phosphate deficiency induced a decrease in the relative content of all phospholipids and a concomitant increase in the relative content of other lipids (data not shown). In higher plant leaves, MGDG, DGDG, SQDG and PG are mainly distributed in thylakoid membranes (Siegenthaler and Murata, 1998a). Therefore, it is generally considered that the levels of these four kinds of glycerolipids in the leaves may represent those in thylakoid membranes. In this study, we focused on the effect of phosphate deficiency on the composition of these four lipids. The relative amount of PG in leaves of seedlings grown in phosphate-deficient solution decreased approximately 45 mol% and those of SQDG and DGDG increased about 23 mol% and 10 mol%, respectively, as compared to those of PG, SQDG and DGDG in plants grown in phosphate-sufficient solution (Fig.1). The phosphate in older leaves may be transported to younger leaves under phosphate deficiency conditions (Mimura, 1995). To investigate whether the phosphate in PG can be reutilized under the phosphate-deficient condition, it is necessary to know the changes in lipid composition among different leaves during wheat plant growth. Fig.1. Composition of four lipid classes in the leaves of the 9- day-old wheat grown in Hoagland solutions containing 1.0 mmol/ L (solid bars) and 0.01 mmol/l (blank bars) phosphate. Values represent the means of three independent measurements. Error bars are indicated.

3 YANG Wen et al.: Alternation in Lipid Composition of Wheat Leaves Induced by Phosphate Deficiency Is Related to Both Lipid Biosynthesis and Phosphatidylglycerol Degradation Therefore, we analyzed the relative amounts of lipids in the different leaves on 16-day-old wheat plants, among which the third leaf was just fully expended and the second and first leaves had been expended for 3 and 7 d, respectively. When wheat plants were grown in phosphate-sufficient solution, no difference in lipid composition was found among different leaves (data not shown). In this culture condition, the relative amounts of PG, MGDG, DGDG and SQDG were about 10 mol%, 51 mol%, 33 mol%, and 6 mol%, respectively (Fig.2). It was very possible that in phosphatesufficient solution there was phosphate available sufficing for plant growth and development. When wheat plants were grown in phosphate-deficient solution, the levels of individual lipid classes were apparently different among leaves. Phosphate deficiency induced a gradual decrease in PG and MGDG and a concomitant increase in DGDG and SQDG from the first leaf to the third leaf (Fig.2). Among these four lipid classes, the most obvious change was the PG content which decreased by approximately 19 mol%, 58 mol% and 75 mol% for the third, second and first leaf, respectively, grown in phosphate-deficient solution as compared to that grown in phosphate-sufficient solution. Despite phosphate-deficient condition affected significantly the composition of lipids among the leaves, the level of individual lipids extracted from all leaves in 16-day-old plants was almost the same as those from leaves on 9-dayold plants (Fig.3). This means that the relative higher level of PG in younger leaves on 16-day-old plants induced by phosphate deficiency is the results of the PG re-distribution among leaves, i.e. the phosphate in PG can transport from older leaves to younger ones. Fig.2. Composition of four lipid classes extracted from leaves in 16-day-old wheat plants grown in different solutions. Lipid content was the same among leaves when plants were grown in phosphate-sufficient solutions and the means of lipid composition in three leaves are shown here (solid bars). The individual lipid contents in the third leaf (cross-hatched bars), the second leaf (hatched bars) and the first leaf (dot bars) were shown under phosphatedeficient conditions. Values represent the means of three independent measurements. Error bars are indicated. DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyldiacylglycerol; PG, phosphatidylglycerol; SQDG, sulfoquinovosyl diacyglycerol. Fig.3. The levels of individual lipids extracted from all leaves in 16-day-old (solid bars) and 9-day-old plants (blank bars) grown in phosphate-deficient culturing solution. Values represent the means of three independent measurements. Error bars are indicated. Abbreviations are the same as in Fig.1. The abbreviations are the same as in Fig.2. 3 Discussion It has been reported that phosphate was able to be remobilized from older leaves to younger leaves to ensure the preferential growth of younger leaves (Mimura, 1995). Our results suggest that in phosphate-deficient wheat plants, phosphate in PG can be also remobilized from older leaves to younger leaves. First, the relative content of PG in the first leaf on 16-day-old plants was significantly lower than that on 9-day-old plants with 2.5 mol% versus 5.5 mol% (Figs.1, 2). Second, the relative content of PG in the third leaf (7.9 mol%) was higher than that in the second leaf (4.1 mol%) and the first leaf (2.5 mol%) (Fig.2). Third, the level of individual lipids from all leaves on 16-day-old plants was almost the same as that from the leaves on 9-day-old plants (Fig.3). These results suggest that phosphate in PG can be transported from older leaves to younger leaves when phosphate was a limiting factor for wheat plant growth. About 85% of PG in higher plant leaves is mainly present in chloroplast and the remains are located in extraplastidic membranes (Browse et al., 1986). Someone may argue that phosphate remobilization in PG may be not involved in the photosynthetic membrane PG. However, under phosphatedeficient condition PG in the first leaf on 16-day-old plants decreased by 54.9 mol%, as compared to that in the same leaf on 9-day-old plants (Figs.1, 2), which was far much higher than the proportion of extraplastidic PG. This means that the photosynthetic membrane PG mainly contributes to the loss of PG in older leaves Phosphate deficiency induced a decrease in the amounts of both PG and MGDG, with a concomitant increase in those of DGDG and SQDG (Figs.1, 2). The similar phenomena were also observed in photosynthetic bacteria (Benning et al., 1993; Güler et al., 1996), Chlamydomonas reinharditti (Sato et al., 2000b) and A. thaliana (Essigmann et al., 1998).

4 Our results suggest that the changes in lipid composition induced by phosphate deficiency are related to both lipid biosynthesis and PG degradation. There was only one leaf on 9-day-old plants, and in this case, little phosphate of PG could be transported from this leaf to the new leaves. In addition, phosphate deficiency resulted in 45 mol% of decrease in PG, as compared to phosphate-sufficient condition (Fig.1). These results indicate that phosphate was insufficient for PG biosynthesis under phosphate-deficient condition. On 16-day-old wheat plants, there were three fully expended leaves, among which the first leaf was the oldest one and its PG level was the lowest. The third leaf was the youngest one and has just expended. When plants were grown in 0.01 mmol/l phosphate solution for 16 d, PG level in the third leaves was 48 mol% and 69 mol% higher than that in the second and first leaves, respectively, and only 19% lower than that in leaves on plants grown in 1.0 mmol/l phosphate solution (Fig.2). These results suggest that PG decrease induced by phosphate deficiency in different leaves results from different mechanism. PG decrease in the younger leaves is mainly due to insufficient phosphate available for PG biosynthesis, while PG decrease in the older leaves mainly results from PG degradation. If phosphate levels in culture solutions only affected PG biosynthesis and degradation, PG decrease induced by phosphate deficiency should be accompanied with an increase in other three lipids. However, phosphate-deficient condition only induced the increase in DGDG and SQDG, in contrast, the level of MGDG decreased (Figs.1, 2). These results suggest that phosphate levels also affect at least the biosynthesis process of MGDG and DGDG. Some compounds containing phosphate, such as PA and PC, are involved in the biosynthesis of MGDG and SQDG (Ohlrogge and Brows, 1995). SQDG is the final product of photosynthetic lipid biosynthesis, whilst some molecules of MGDG are used to synthesize DGDG (Siegenthaler and Murata, 1998b). This latter process does not need any compound containing phosphate. Therefore, if phosphate is only a limiting factor for MGDG synthesis but not for DGDG synthesis from MGDG, as a result, MGDG should decrease whilst DGDG increase. References: Benning C, Beatty J T, Prince R C, Somerville C R The sulfolipid SQDG is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation. Proc Natl Acad Sci USA, 90: Benning C Biosynthesis and function of the sulfolipid sulfoquinovosyl diacylglycerol. Annu Rev Plant Physiol Plant Mol Biol, 49: Bligh E G Dyer W J A rapid method of total lipid extraction and purification. Can J Biochem Physiol, 37: Browse J, Warwick N, Somerville C R, Slack C R Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the 16:3 plant Arabidopsis thaliana. Biochem J, 235: Essigmann B, Güler S, Narang R A, Linke D, Benning C Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Aarabidopsis thaliana. Proc Nat Acad Sci USA, 95: Gombos Z, Varkonyi Z, Hagio M, Iwaki M, Kov cs L, Masamoto K, Itoh S, Wada H Phosphatidylglycerol requirement for the function of electron acceptor plantoquinone Q B in the photosystym reaction center. Biochemistry, 41: Güler S, Seeliger A, Härtel H, Renger G, Benning C A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid SQDG. J Biol Chem, 271: Hagio M, Gombos Z, V rkonyi Z, Masamoto K, Sato N, Tsuzuki M, Wada H Direct evidence for requirement of phosphatidylglycerol in photosystem of photosynthesis. Plant Physiol, 124: Hagio M, Sakurai I, Sato S, Kato T, Tabata S, Wada H Phosphatidylglycerol is essential for the development of thylakoid membranes in Arabidopsis thaliana. Plant Cell Physiol, 43: Härtel H, Essigmann B, Lokstein H, Hoffmann-Benning S, Peters-Kottig M, Benning C The phopholipid-deficient pho1 mutant of Arabidopsis thalina is affected in the organization, but not in the light acclimation, of the thylakoid membrane. Biochim Biophys Acta, 1415: Kruse O, Hankamer B, Konczak C, Gerle C, Morris E, Radunz A, Schmid G H, Barber J Phosphatidylglycerol in involved in the dimerization of photosystym. J Biol Chem, 275: Mimura T Homeostasis and transport of inorganic phosphate in plants. Plant Cell Physiol, 36:1 7. Ohlrogge J, Brows J Lipid biosynthesis. Plant Cell, 7: Raghothama K G Phosphate transport and signaling. Curr Opin Plant Biol, 3: Sato N, Hagio M, Wada H, Tsuzuki M. 2000a. Enviromental effects on acidic lipids of thylakoid membranes. Biochem Soc Trans, 28: Sato N, Hagio M, Wada H, Tsuzuki M. 2000b. Requirement of phosphatidylglycerol for photosynthetic function in thyla-

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