Genotypic Variation in Lysophospholipids of Milled Rice - Journal of

Sep 3, 2014 - In this study, 20 rice accessions were grown over two seasons at the same location to explore diversity in LPLs of milled rice. Levels o...
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Genotypic Variation in Lysophospholipids of Milled Rice Chuan Tong,† Lei Liu,§ Daniel L. E. Waters,§ Terry J. Rose,§,⊗ Jinsong Bao,*,† and Graham J. King§ †

Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology, Zhejiang University, Huajiachi Campus, Hangzhou 310029, China § Southern Cross Plant Science and ⊗Southern Cross Geoscience, Southern Cross University, Lismore, NSW 2480, Australia ABSTRACT: Phospholipids (PLs) play a prominent role in both grain cellular structure and nutritional function of cereal crops. Their lyso forms (lysophospholipids, LPLs) often combine with cereal starch to form an amylose−lipid complex (ALC), which may influence starch properties. In this study, 20 rice accessions were grown over two seasons at the same location to explore diversity in LPLs of milled rice. Levels of specific LPLs differed significantly among rice genotypes, demonstrating there is a wide diversity in LPLs in rice grain. The main LPL components were lysophosphatidylcholine (LPC) 16:0 (ranging from 3009.7 to 4697.8 μg/g), LPC18:2 (836.6−2182.3 μg/g), lysophosphatidylethanolamine (LPE) 16:0 (625.7−1139.8 μg/g), and LPE18:2 (170.6−481.6 μg/g). Total LPC, total LPE, and total LPL ranged from 4727.1 to 7685.2 μg/g, from 882.8 to 1809.5 μg/g, and from 5609.8 to 9401.1 μg/g, respectively. Although significant (P < 0.001) environment and genotype × environment (G × E) interactions were detected by analysis of variance (ANOVA), these effects accounted for only 0.7−38.9 and 1.8−6.6% of the total variance, respectively. Correlation analysis between LPL components provided insight into the possible LPL biosynthesis pathway in plants. Hierarchical cluster analysis suggested that the 20 rice accessions could be classified into three groups, whereas principal component analysis also identified three groups, with the first two components explaining 57.7 and 16.2% of the total variance. Further genetic studies are needed to identify genes or quantitative trait loci (QTLs) underlying the genetic control of LPLs in rice grain. KEYWORDS: rice, phospholipids, lysophosphatidylcholine, lysophosphatidylethanolamine, genotype, environment

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and 14.3% lysophosphatidylethanolamine (LPE).8 Analysis of five rice genotypes found brown rice contains 7−9% PLs, comprising PC14:0 (0−1.0%), PC16:0 (19.6−21.1%), PC18:1 (36.0−49.3%), PC18:2 (26.9−43.0%), PC18:3 (0.6−1.4%), PE14:0 (0−1.3%), PE16:0 (20.9−23.3%), PE18:1 (22.0− 36.8%), PE18:2 (37.9−52.0%), and PE18:3 (0.8−3.0%).9 In cereal endosperm, lysophospholipids (LPLs) often integrate with starch to form an amylose−lipid complex (ALC),10−12 which may influence starch pasting viscosity and swelling properties13,14 and reduce amylose digestibility.15,16 Similarly, the complex of lysophosphatidylcholine (LPC) with amylose may decrease swelling power, solubility, and starch digestibility,15,17 whereas increasing ALC decreases water solubility and increases pasting temperature and peak viscosity.18 Several mechanisms may explain these observations. ALC blocking amylose leaching may reduce the velocity of thermal gelatinization,19 whereas retrogradation could be affected by a transition from an extended to a V-amylose structure due to lipid.20 Additionally, starch granule surface lipids could decrease amylose swelling, which may have an impact on pasting and gelatinization behavior.21 Extensive investigations of LPLs have been conducted recently in recognition of their importance in grain quality. Highperformance liquid chromatographic−mass spectrometric (HPLC-MS) quantification and molecular characterization of PLs and LPLs have been conducted in Arabidopsis,22 oats,23

INTRODUCTION Rice (Oryza sativa L.) is an indispensable food crop, providing essential nutrition and energy for more than half the world’s population. Before farmers grow new high-yielding rice varieties, consumers must first accept their grain quality, and so rice grain quality is a critical consideration for rice breeders. Rice grain quality is assessed according to milling performance, visual appearance, cooking and eating quality (primarily determined by grain physicochemical properties), and nutritional content. The properties also contribute to the rice storage characteristics and processing properties.1−3 The relative contribution of a range of chemical components, including starch, proteins, lipids, vitamins, and many trace elements,2 determines rice grain eating and nutritional quality. If rice genetic improvement programs are to deliver new high-quality and high-yielding rice cultivars efficiently, a more complete understanding of the genetic and environmental control of individual components of the rice grain and their interactions is required. Lipids play a key role in determining the storage, processing, and cooking quality of rice4,5 and are generally divided into three broad groups of simple, compound, and derived lipids, consisting primarily of neutral triacylglycerols, free fatty acids, and phospholipids (PLs). Although PLs account for a small proportion of the rice grain by mass, they may play a requisite and beneficial role in human nutrition by interfering with neutral sterol absorption and by stimulating bile acid and cholesterol secretion.6 The rice bran lipid fraction contains 5.5−6.7% PLs, which is composed of 31.8−46.8% phosphatidylcholine (PC) and 25.0− 38.9% phosphatidylethanol (PE),7 whereas the embryo contains 2.0−2.5% of PLs comprising 42.9−66.7% PC, 42.9−66.7% PE, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 9353

July 6, 2014 August 19, 2014 September 3, 2014 September 3, 2014 dx.doi.org/10.1021/jf503213p | J. Agric. Food Chem. 2014, 62, 9353−9361

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(MSD, 6120). An Agilent Eclipse Plus C18 RRHD column with 1.8 μm, 50 × 2.1 mm internal diameter, was used to separate PLs. The linear gradient elution program consisted of a mobile phase of acetonitrile with 0.005% TFA and water with 0.005% TFA. The solvent gradient was programmed from 10 to 99% acetonitrile in 10 min at a flow rate of 0.3 mL/min and held at 99% acetonitrile for 1.5 min. An electrospray ionization (ESI) source was used for MSD; the MS parameters were as follows: drying nitrogen gas flow, 12.0 L/min; nebulizer pressure, 35 psig; SIM mode same as our previous study;26 fragmentor, 150; capillary voltage, 3000 V (positive); drying gas temperature, 350 °C; vaporizer temperature, 350 °C. Control of the HPLC-MS system was by Agilent ChemStation software. An external standard was applied, and each sample was measured in triplicate. Statistical Analysis. Data analyses were conducted using SAS program version 9 (SAS Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was used with the general linear model procedure (Proc glm). PROC means was performed for means of genotypes and environments, followed by Duncan’s multiple-comparison tests (P < 0.05). Correlation between phenotypes was calculated with PROC CORR, and principal component analysis was conducted with PRINCOMP. Hierarchical cluster analysis was computed using Ward’s method in SPSS version 19.0.

maize,24,25 and rice.9,26 Although some progress has been made in defining PL synthesis and metabolism in plants,27 our understanding of the PL synthesis pathway is limited to knowledge of several genes in Arabidopsis,28 with single nucleotide polymorphisms identified in seed-expressed phospholipase D, an important enzyme in PL biosynthesis.29,30 There are few reports of the extent of genotypic diversity in specific PLs in rice grain due to the limited capacity of existing extraction and quantification assays.3,26 Rice grain lipids are influenced by both genetic and environmental factors.31 During rice grain ripening, linoleic and linolenic acid content appear to decrease with increasing growing temperature, whereas the myristic, palmitic, and oleic acid content rise, although there is negligible seasonal and cultivar variation of these LPL precursors.32 Sowing date and field location also appear to play a greater role in accounting for variation in rice LPL levels than genetic factors.33 Although these studies suggest genotype and environment have a significant influence on rice PL components and accumulation, data that reveal the extent of genotype × environment (G × E) interaction on rice LPLs are still lacking. The objective of this study was to quantify the variation in LPLs among 20 diverse rice accessions originating from a wide geographical distribution over two growing seasons. The results will contribute to our understanding of the factors that may contribute to selecting and managing rice grain LPL composition, either through breeding or agronomic manipulation.

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RESULTS

Of the 20 rice genotypes studied, eight represented the japonica subspecies, eight the indica subspecies, and four belonged to the aus group. A total of 10 individual LPLs were identified in the classes of lysophosphatidylcholine (LPC14:0, LPC16:0, LPC18:1, LPC18:2, LPC18:3) and lysophosphatidylethanolamine (LPE14:0, LPE16:0, LPE18:1, LPE18:2, LPE18:3). Rice Lysophosphatidylcholine. The effects of genotype by season (year) interaction for rice LPC components LPC14:0, LPC16:0, LPC18:1, LPC18:2, LPC18:3, and total LPC (TLPC) (where TLPC = LPC14:0 + LPC16:0 + LPC18:1 + LPC18:2 + LPC18:3) were significant (P < 0.001) (Table 2). In particular, genotypic variance of LPC14:0, LPC16:0, LPC18:1, LPC18:2, LPC18:3, and TLPC accounted for 79.9, 59.5, 93.2, 61.6, 83.2, and 59.5% of the total variance, respectively, whereas year accounted for 18.1, 33.9, 3.7, 36.6, 14.0, and 38.9% of the total variance, respectively. Genotype × season interaction also affected rice LPCs (P < 0.001), although they accounted for only 1.8−6.6% of the total variance (Table 2). Extensive variation in LPC composition was observed among the 20 rice accessions (Table 1). The main LPC components were LPC16:0 and LPC18:2, which accounted for 54.7−64.7 and 19.5−31.0% of total LPCs, respectively. Minor components included LPC18:1, LPC14:0, and LPC18:3, which represented 5.6−14.8, 4.6−11.1, and 0.3−1.1% of total LPCs, respectively (Table 1; Figure 1). Mean individual LPC content was similar across the two seasons, ranging from 406.2 to 436.0 μg/g for LPC14:0, from 3752.0 to 3918.6 μg/g for LPC16:0, from 562.8 to 576.2 μg/g for LPC18:1, from 1464.2 to 1581.5 μg/g for LPC18:2, and from 37.0 to 39.9 μg/g for LPC18:3 (Table 1; Figure 1). Significant difference between the two years (P < 0.001) was observed only for mean TLPC of the 20 accessions (Table 1). Among the three groups of rice accessions, the mean LPC18:1 content across two years in the japonica rice group was higher than those in indica and aus rice groups (Table 3). Rice Lysophosphatidylethanolamine. Although genotype, season, and genotype × season interactions were significant (ANOVA; P < 0.001) for LPE14:0, LPE16:0, LPE18:1, LPE18:2, LPE18:3, and total LPE (TLPE) (where TLPE = LPE14:0 + LPE16:0 + LPE18:1 + LPE18:2 + LPE18:3) (P < 0.001; Table 2),

MATERIALS AND METHODS

Plant Materials. The 20 rice accessions (Table1) from the OryzaSNP project (http://www.oryzasnp.org/) were used in this study and represent a wide range of geographical origin and genetic diversity.34 All were nonwaxy cultivars with apparent amylose content ranging from 11 to 26%.35 These cultivars were grown in the field over two consecutive seasons (2010−2011 and 2011−2012) in a randomized block design with two replications in the same field in Lingshui, Hainan province, China (18.48°N).35,36 Sowing was in early December and harvest in April. Associated metrological data (mean temperature, sunshine duration, and precipitation) were reported previously.35 Grain was stored at 4 °C prior to milling to white rice using a Satake rice machine (Satake Corp., Japan) and grinding to flour in a Cyclone sample mill (UDY, Fort Collins, CO, USA) in the laboratory. When some accessions were of insufficient sample weights for analysis, mixed samples from two replications in the same year were used in this study. Chemicals. All solvents and reagents were of HPLC grade. Four LPL standard samples, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC16:0), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC18:1), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPE16:0), and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPE18:1), were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Starch Phospholipid Extraction. A rapid single-step LPL extraction method26 was used. Briefly, LPLs were extracted directly from triplicate milled rice flour samples with 75% n-propanol (v/v, 8 mL/0.16 g sample) at 100 °C in Pyrex culture tubes (capped) for 2 h. The weight of the capped culture tube before and after extraction was measured to ensure an accurate extraction solvent/sample ratio (m/m) was used for subsequent calculations of LPL content. A 1 mL aliquot of the extract was used for quantitative HPLC-MS analysis of LPLs. Determination of LPLs by HPLC-MS. Quantitative analysis of LPLs was carried out with three technical replications according to the method of Liu et al.26 using an Agilent HPLC-MS series 1290 (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, binary pump, autoinjector, and an Agilent quadrupole mass detector 9354

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a

Azucena Dom Sufid Dular FR13A IR64-21 LTH M202 Minghui63 Moroberekan N22 Nipponbare Pokkali Sadu-Cho SHZ-2 Swarna Tainung67 Zhenshan97B Aswina Cypress Rayada

G01 G02 G03 G04 G05 G06 G07 G08 G09 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20

411.0 d 532.3 c 331.5 fg 496.3 c 504.1 c 530.3 c 371.0 e 238.5 j 256.0 ij 604.9 b 430.8 d 300.6 gh 442.5 d 305.7 gh 279.6 hi 294.1 h 353.0 ef 811.2 a 512.3 c 416.6 d

japonica japonica aus aus indica japonica japonica indica japonica aus japonica indica indica indica indica japonica indica indica japonica aus

436.0 a 406.2 a

LPC14:0

group

3918.6 a 3752.0 a

3846.5 fg 4270.9 cde 4697.8 a 4431.6 bcd 3758.0 gh 3760.9 gh 3279.9 kl 3009.7 m 3572.4 hij 4541.9 ab 3909.3 fg 3345.6 jkl 4452.7 abc 3195.8 lm 3500.5 ijk 3190.6 lm 4189.3 de 4083.6 ef 3934.9 fg 3734.1 ghi

LPC16:0

576.2 a 562.8 a

894.5 a 893.8 a 427.2 h 465.3 g 398.2 hi 500.4 efg 600.0 d 537.1 e 496.7 fg 593.8 d 680.6 c 473.8 fg 492.5 fg 468.4 fg 506.7 ef 755.2 b 415.9 h 576.7 d 746.5 b 466.8 fg

LPC18:1

1581.5 a 1464.2 a

1621.6 d 1828.1 c 2182.3 a 1437.9 fg 1584.1 de 1464.3 f 1335.6 gh 923.4 j 1243.8 h 1949.7 b 1503.1 ef 1115.7 i 1808.2 c 1813.6 c 1248.8 h 836.6 j 1608.0 de 1737.7 c 1761.3 c 1452.7 f

LPC18:2

39.9 a 37.0 a

33.4 e 66.5 a 46.5 d 23.0 h 58.2 b 28.0 fg 29.9 efg 18.4 i 32.4 ef 27.7 g 34.4 e 27.0 gh 43.6 d 64.0 a 31.3 efg 17.8 i 33.0 e 53.6 c 67.8 a 33.1 e

LPC18:3

Different letters in the same column represent significance at the 0.05 level. Units: μg/g.

year 2011 2012

variety

genotype

Table 1. Mean of Rice Lysophospholipids of Different Genotypes at Different Yearsa

6552.2 a 6222.2 b

6807.0 de 7591.6 ab 7685.2 a 6854.1 de 6302.7 fg 6284.0 fg 5616.4 ij 4727.1 l 5601.4 ij 7718.0 a 6558.1 ef 5262.7 jk 7239.5 bc 5847.4 gi 5566.8 ij 5094.3 k 6599.0 ef 7262.8 bc 7022.8 cd 6103.3 gh

TLPC

56.4 a 52.0 a

60.8 de 77.7 b 41.3 j 61.7 cde 61.6 cde 72.6 b 41.9 j 27.9 m 34.3 kl 67.3 c 52.4 gh 43.3 ij 59.7 ef 38.3 jk 32.3 lm 39.9 j 48.1 hi 102.4 a 65.8 cd 55.3 gh

LPE14:0

921.8 a 896.2 a

1041.6 b 1139.8 a 971.3 cd 977.6 c 890.1 efg 974.2 cd 749.8 i 625.7 j 895.1 efg 941.2 cde 861.3 fgh 820.3 h 1123.9 a 841.5 gh 733.1 i 812.1 h 915.9 def 940.2 cde 985.3 c 939.0 cde

LPE16:0

77.1 a 76.2 a

128.5 a 134.0 a 54.2 k 63.2 ghi 56.0 jk 67.6 efgh 70.7 ef 58.6 ijk 72.7 e 65.6 fgh 82.3 d 71.5 ef 63.8 ghi 69.1 efg 66.8 efgh 116.3 b 56.4 jk 80.6 d 92.4 c 62.2 hij

LPE18:1

372.1 a 361.4 a

462.6 b 444.4 c 396.5 e 361.7 fg 386.3 e 378.9 ef 311.5 h 170.6 k 356.3 g 379.4 ef 353.6 g 284.1 i 421.2 d 481.6 a 293.8 i 250.3 j 395.2 e 415.1 d 438.8 c 353.8 g

LPE18:2

7.0 a 6.6 a

6.8 fg 13.7 b 7.0 f 3.0 kl 12.4 c 3.6 jk 4.3 ij ∼0 n 6.1 fg 2.3 lm 6.2 fg 3.6 jk 8.5 e 15.7 a 5.0 hi 1.8 m 5.9 gh 9.6 d 15.1 a 5.8 gh

LPE18:3

1433.5 a 1393.2 a

1700.3 b 1809.5 a 1470.3 ef 1467.2 ef 1406.4 fg 1496.9 de 1178.1 hi 882.8 j 1364.4 g 1455.7 ef 1355.8 g 1222.7 h 1677.1 b 1446.1 ef 1130.7 i 1220.4 h 1421.5 efg 1547.9 cd 1597.4 c 1416.1 fg

TLPE

7985.6 a 7615.5 a

8507.4 cd 9401.1 a 9155.5 ab 8321.3 de 7709.0 fgh 7780.9 fg 6794.5 jk 5609.8 m 6965.8 ij 9173.7 ab 7913.9 efg 6485.3 kl 8916.5 bc 7293.5 hi 6697.5 jkl 6314.7 l 8020.5 ef 8810.6 bc 8620.2 cd 7519.5 gh

TLPL

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1483.0 249.7 49.7

4812061.5 3265383.2 317542.1

1960.3 569.4 69.3

94686.5 19668.5 5756.7

3317.3 24.8 130.6

34425.3 3447.6 792.2

118.5 6.2 2.8

275660.8 48622.0 12118.0

6966212.0 4110997.0 413046.9

they were mainly affected by genotype, accounting for 75.4− 95.5%, and season, accounting for 0.7−21.9% of the total variance. Genotype × season interactions accounted for only 2.1−4.8% of the total variance (Table 2). The main LPE components were LPE16:0 and LPE18:2, accounting for 58.2−70.9 and 19.3−33.3% of total LPEs, respectively (Table 1 and Figure 1). LPE16:0 content ranged from 625.7 μg/g in genotype G08 to 1139.8 μg/g in G02. LPE18:2 varied from 170.6 μg/g in G08 to 481.6 μg/g in G14 (range = 311.0 μg/g). The mean LPE16:0 and LPE18:2 values were relatively stable over the two years, being 921.8 and 372.1 μg/g in 2011 versus 896.2 and 361.4 μg/g in 2012, suggesting environment had little effect on LPE16:0 and LPE18:2 contents (Table 2). TLPE varied from 882.8 μg/g in G08 to 1809.5 μg/g in G02 among the 20 different accessions (Table 1). There was little variation in mean TLPE between years, suggesting environmental influence on TLPE was minor (Table 2). The japonica rice group had higher mean LPE18:1 content than those in the indica and aus rice groups (Table 3). Rice Total Lysophospholipids (TLPL). TLPL (where TLPL = TLPC + TLPE) content varied extensively among the 20 genotypes (ANOVA; P < 0.001) such that genotype accounted for 60.6% of the total variation (Table 2). Environment and genotype × environment interaction accounted for 35.8 and 3.6% of the total variance of TLPL, respectively, representing a small but significant effect (P < 0.001). The lowest TLPL content was found in indica cultivar Minghui63 (G08, 5609.8 μg/g), whereas the highest was found in japonica cultivar Dom Sufid (G02, 9401.1 μg/g) (Table 1; Figure 2). Mean TLPL levels were similar between years, 7985.6 μg/g in 2011 and 7615.5 μg/g in 2012, reflecting the relatively stable response to environmental effects (Table 2). Correlation Analysis of Lysophospholipids. LPL composition between years was significantly correlated (P < 0.01), suggesting rice LPLs were little affected by environment (Table 4). The lowest correlation coefficient between years was 0.810 for LPC16:0 (P < 0.01), whereas the highest was 0.967 for LPC14:0 (P < 0.01). Correlations between LPL composition were all positive (P < 0.01). TLPL was correlated with its major components, that is, LPC16:0, LPC18:2, LPE16:0, LPE18:2 (r > 0.723, P < 0.01) (Table 4; Figure 1B,D,G,I). Additionally, the correlation between LPC and LPE components with the same fatty acid composition, for example, LPC14:0 and LPE14:0, LPC16:0 and LPE16:0, and so on, were higher than those between different fatty acid compositions. Cluster Analysis of Lysophospholipids. Hierarchical cluster analysis of the 20 rice genotypes was based on individual LPLs. Two groups separated at a relative distance of 13 were identified, whereas there were three groups at a distance of approximately 11 (Figure 3). Genotypes G07, G08, G09, G12, G15, and G16 formed the first group, G01, G02, and G19 formed the second group, and the remaining genotypes formed the third group. Principal component analysis suggested that the first three components accounted for 51.8, 19.8, and 14.8% of the total variance, respectively. The three groups of 20 accessions were also clearly defined by the first two principal components (Figure 4). The mean LPL components were lowest in group 1, except for LPC18:1 and LPE18:1, and highest in group 2, whereas those of group 3 were intermediate to groups 1 and 2 (Table 5).

All mean square values were significant at the P < 0.001 level.

694767.3 412755.2 19860.7

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DISCUSSION Rice grain PLs are of significance for storage, consumption, processing, and eating quality3 and, as with starch properties, may be

a

134367.4 5352.0 4466.2 117337.2 26602.5 2884.6 genotype (G) year (E) G×E

19 1 19

1462034.7 832716.8 161989.2

TLPC LPC18:3 LPC18:2 LPC18:1 LPC16:0 LPC14:0 df source

Table 2. Mean Square Values of Analysis of Variance (ANOVA) for Rice Lysophospholipidsa

LPE14:0

LPE16:0

LPE18:1

LPE18:2

LPE18:3

TLPE

TLPL

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Figure 1. Individual rice LPL component content in two years: (A) LPC14:0 content; (B) LPC16:0 content; (C) LPC18:1 content; (D) LPC18:2 content; (E) LPC18:3 content; (F) LPE14:0 content; (G) LPE16:0 content; (H) LPE18:1 content; (I) LPE18:2 content; (J) LPE18:3 content. See Table 1 for rice accession information.

Table 3. Mean of Lysophospholipids among Different Rice Subspeciesa

a

group

LPC14:0

LPC16:0

LPC18:1

LPC18:2

LPC18:3

TLPC

LPE14:0

LPE16:0

LPE18:1

LPE18:2

LPE18:3

TLPE

TLPL

aus indica japonica

462.3 a 404.4 a 417.2 a

4351.4 a 3691.9 a 3720.7 a

488.3 b 483.7 b 696.0 a

1755.7 a 1479.9 a 1449.3 a

32.6 a 41.1 a 38.8 a

7090.2 a 6101.0 a 6322.0 a

56.4 a 51.7 a 55.7 a

957.3 a 861.3 a 932.4 a

61.3 b 65.4 b 95.6 a

372.9 a 356.0 a 374.6 a

4.5 a 7.6 a 7.2 a

1452.3 a 1341.9 a 1465.4 a

8542.5 a 7442.8 a 7787.3 a

Different letters in the same column represent significance at the 0.05 level. See Table 1 for rice accession information. Units: μg/g.

affected by both genotype and environment.37 In the past decade, several reports of quantitative PL analysis by HPLC coupled with ESI-MS, silica column, or thin layer chromatography have

been published.25,38−40 Although these methods have demonstrated LPLs differ between rice accessions, the influence of environment was not investigated.41,42 In the present study, the 9357

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0.600** 0.823** 0.138 0.827** 0.420 0.899** 0.630** 0.738** 0.093 0.683** 0.334 0.730** 0.890** 0.616** 0.736** 0.260 0.720** 0.504* 0.831** 0.712** 0.923** 0.297 0.812** 0.790* 0.914** 0.875** a

0.967** 0.584** 0.075 0.481* 0.307 0.670** 0.893** 0.482* 0.018 0.440 0.245 0.506* 0.456** LPC14:0 LPC16:0 LPC18:1 LPC18:2 LPC18:3 TLPC LPE14:0 LPE16:0 LPE18:1 LPE18:2 LPE18:3 TLPE TLPL

* and ** indicate significance at P < 0.05 and 0.01 levels, respectively. 2011, above diagonal; 2012, below diagonal; and 2011 and 2012, in diagonal.

0.333 0.247 0.180 0.608** 0.931** 0.470* 0.411 0.521* 0.265 0.794** 0.958** 0.658** 0.525* 0.502* 0.567** 0.163 0.806** 0.669** 0.739** 0.578** 0.774** 0.217 0.957** 0.678** 0.863** 0.789** 0.248 0.045 0.911** −0.064 0.161 0.206 0.435 0.461* 0.942** 0.226 0.192 0.490* 0.273 0.520* 0.768** 0.154 0.632** 0.330 0.779** 0.601** 0.878** 0.178 0.638** 0.286 0.802** 0.808** 0.957** 0.613** 0.121 0.468* 0.332 0.689** 0.940** 0.597** 0.101 0.482* 0.280 0.601** 0.691** 0.614** 0.816** 0.104 0.819** 0.840 0.883** 0.591** 0.674** 0.043 0.631** 0.287 0.664** 0.863** 0.435 0.327 0.148 0.662** 0.935** 0.544 0.489 0.498 0.208 0.743** 0.927** 0.625** 0.578** 0.485* 0.684** −0.007 0.947** 0.592 0.823** 0.464* 0.597** −0.042 0.776** 0.513* 0.655** 0.813**

LPC18:3 LPC18:2 LPC18:1 LPC16:0 LPC14:0 correlation

Table 4. Correlation Analysis of Rice Lysophospholipids between Yearsa

TLPC

LPE14:0

LPE16:0

LPE18:1

TLPL content varied from 5609.8 to 9401.1 μg/g in the 20 nonwaxy rice genotypes, which is similar to results from rice planted in the Philippines and Portugal, where TLPL concentration ranged from 5520 to 8310 μg/g in 12 nonwaxy rice cultivars.41 LPC accounted for 80.0−84.3% of TLPLs, whereas LPE accounted for only 15.7−20.0%. We identified the main components of LPLs as including LPC16:0 43.7−54.0% (Figure 1B), LPC18:2 12.4−24% (Figure 1D), LPE16:0 10.2−13.0% (Figure 1G), and LPE18:2 4.6−12.4% (Figure 1C) in agreement with Majumder et al.42 and Liu et al.26 These four LPLs accounted for the TLPL content of Dom Sufid (G02) representing the highest among the genotypes in both years, and was consistently 1.68 times higher than Minghui63 (G08), the lowest in both years. This suggests a difference primarily attributable to genetic factors. Further investigation of the genetic control of LPLs may contribute to breeding superior rice varieties for human consumption or industrial use. Although genetic factors accounted for the primary component of LPL variation, environmental factors may also affect grain lipid accumulation and concentration.9,34,43 For example, cold can stimulate synthesis of unsaturated lipids during rice grain filling,5,42 and the concentration of PL18:2 in rice planted in cold environments was higher than that in mild and tropical environments (37.2−40.0% compared to 30.3−33.1%). In contrast, the PL18:1 was lower in cold conditions than in warm conditions.11 LPL content in nonwaxy rice cultivars accounted for 48−67% of starch lipids depending on growing location (Asia or Europe), highlighting the role of environment in controlling LPL content in rice.41 In the present study, genotype, environment, and genotype × environment interaction effects on rice LPLs were significant at P < 0.001 (ANOVA) (Table 2). For individual LPC and LPE, genotype accounted for 57.3−95.5%, environment (season) for 0.7−38.9%, and genotype × environment interaction for 1.8−6.6% of the total variation. Thus, genotype appears to be the major factor determining the difference in rice individual LPL concentration in this study. Year of cultivation was a minor but significant factor influencing LPC18:2, LPC16:0, TLPC, and TLPL composition and variation. The extent and timing of how environmental factors such as temperature during grain filling, soil type, and water availability affect LPL content in rice need to be further investigated. A previous study of brown rice found negative correlations between myristic (C14:0) and linoleic (C18:2), between C14:0 and linolenic (C18:3), between palmitic (C16:0) and oleic (C18:1), between C16:0 and C18:2, between C18:1 and C18:2, and between C18:1 and C18:3 acids, whereas positive correlations existed between C14:0 and C16:0 and between C18:2

0.273 0.052 0.938** −0.011 0.135 0.238 0.401 0.365 0.860** 0.176 0.146 0.404 0.281

LPE18:2

Figure 2. Total LPL content of 20 rice accession in two years. See Table 1 for rice accession information.

0.417 0.810** −0.085 0.683** 0.147 0.759** 0.375 0.557** −0.136 0.421 0.042 0.483* 0.723**

LPE18:3

TLPE

TLPL

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LPC16:0 and its dominant role in synthesis and metabolism. The high correlations between LPC18:1 and LPE18:1 and between LPC18:2 and LPE18:2 as well as between LPC18:3 and LPE18:3 (P < 0.01) support the results of an earlier study,9 suggesting there might be a relationship between unsaturated LPLs in rice. Positive correlations between LPC14:0 and LPE14:0, between LPC16:0 and LPE16:0, and between TLPC and TLPE are also observed in this study. The results from the correlation analysis may provide insight into the putative biosynthesis pathways of LPLs in rice grain. PLs may be synthesized via three pathways in plants, the cytidinediphosphate-diacylglycerol pathway, the 1,2-diacylglycerol pathway, and the headgroup through exchange reactions with other PLs pathway.44 As the main starch lipids, LPLs are probably the hydrolysis products of diacylphospholipid transferred from amyloplast membrane3,45 or catalyzed by phospholipase A2 from PLs.46 The fatty acid composition (C14:0, C16:0, C18:1, C18:2, and C18:3) accounted for a similar percentage in LPC and LPE among different rice accessions (Table 1). If the LPLs were synthesized from PL, fatty acid distributions (C14:0, C16:0, C18:1, C18:2, and C18:3) in PLs were possibly in certain percentage, too. Previous study indicated that the fatty acids distributions were very similar to each other between PC and PE in rice endosperm26 and bran.7,40 These results may imply that each fatty acid composition reacted with choline or ethanolamine at an equal percentage to produce PC and PE, respectively. This possible relationship may provide a useful interpretation of detailed LPL biosynthesis and regulation in metabolism, although the biosynthetic pathway of LPL synthesis in grain endosperm is unclear, particularly the integration of headgroup and fatty acids. Cluster analysis and principal component analysis demonstrated the 20 rice genotypes could be divided into 3 groups according to LPL composition (Figures 3 and 4; Table 5). Variation in LPL composition among the three groups may reflect their genetic relatedness because rice LPL concentration is primarily dependent on genotype.3,5,9,41 Further genetic analyses such as quantitative trait locus (QTL) mapping and genome wide associations studies (GWAS) are warranted to identify the genes responsible for controlling LPLs in rice grain. In summary, broad ranges of LPL composition and content were identified among the 20 rice genotypes over two years, and these differences arose primarily due to genotype differences with environment (season) and genotype × environment interaction effects playing a minor role. Correlation analysis between LPL components provided insight into the possible LPL biosynthesis pathway in plants. Further genetic studies are needed to characterize the genes or QTLs underlying LPLs in milled rice.

Figure 3. Hierarchical cluster analysis dendrogram (Ward’s method) based on individual LPL content of 20 accessions. The vertical dashed line indicated all 20 accessions could be divided into 3 groups at a relative distance of 11. See Table 1 for rice accession information.

Figure 4. Principal component analysis for the genotypes based on their individual LPL content. PC1 and PC2 accounted for 73.9% of total variation, and a plot of PC1 and PC2 separates all 20 genotypes into 3 groups. See Table 1 for rice accession information.

and C18:3.34 Several different relationships were discovered in this study. LPC16:0 was correlated with LPC18:2, TLPC, TLPE, and TLPL (P < 0.01), which may be caused by the abundance of Table 5. Mean Rice Lysophospholipid Content in Three Groupsa groupb

LPC14:0

LPC16:0

LPC18:1

LPC18:2

LPC18:3

TLPC

LPE14:0

LPE16:0

LPE18:1

LPE18:2

LPE18:3

TLPE

TLPL

1 2 3

290.0 b 485.2 a 475.2 a

3316.5 b 4017.4 a 4068.6 a

561.6 b 844.9 a 498.7 b

1117.3 b 1737.0 a 1685.6 a

26.1 b 55.9 a 40.5 ab

5311.5 b 7140.5 a 6768.6 a

36.6 b 68.1 a 60.1 a

772.7 c 1055.6 a 943.3 b

76.1 b 118.3 a 65.5 b

277.8 b 448.6 a 393.0 a

3.5 b 11.9 a 7.3 ab

1166.5 c 1702.4 a 1469.2 b

6477.9 b 8842.9 a 8237.7 a

a Different letters in the same column represent significance at the 0.05 level. Units: μg/g. bGroup 1 consists of M202 (G07), Minghui63 (G08), Moroberekan (G09), Pokkali (G12), Swarna (G15), and Tainung (G16). Group 2 consists of Azucena (G01), Dom Sufid (G02), and Cypress (G19). Group 3 consists of the remaining genotypes.

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AUTHOR INFORMATION

Corresponding Author

*(J.B.) Phone: +86-571-86971932. Fax: +86-571-86971421. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ALC, amylose lipids complex ANOVA, analysis of variance HPLC-MS, high-performance liquid chromatographic-mass spectrometric LPC 14:0, 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC) LPC16:0, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC) LPC18:1, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (OHPC) LPC18:2, 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LHPC) LPC18:3, 1-linolenoyl-2-hydroxy-sn-glycero-3-phosphocholine (LnHPC) LPE14:0, 1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (MHPE) LPE16:0, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PHPE) LPE18:1, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (OHPE) LPE18:2, 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LHPE) LPE18:3, 1-linolenoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LnHPE) LPC, lysophosphatidylcholine LPE, lysophosphatidylethanolamine LPLs, lysophospholipids PLs, phospholipids

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