Article pubs.acs.org/JAFC

Lipid Partitioning in Maize (Zea mays L.) Endosperm Highlights Relationships among Starch Lipids, Amylose, and Vitreousness Mathieu Gayral,† Bénédicte Bakan,† Michele Dalgalarrondo,† Khalil Elmorjani,† Caroline Delluc,‡ Sylvie Brunet,§ Laurent Linossier,§ Marie-Hélène Morel,∥ and Didier Marion*,† †

INRA, Biopolymers, Interactions, Assemblies Research Unit, La Géraudière 44316 Nantes Cedex 3, France Limagrain Europe, Domaine de Mons, 63260 Aubiat, France § Limagrain Cereal Ingredients ZAC Les Portes de Riom, Avenue George Gershwin 63200 RIOM Cedex, France ∥ INRA, Agropolymers Engineering and Emerging Technologies, 2 place Pierre Viala, 34060 Montpellier Cedex 02, France ‡

S Supporting Information *

ABSTRACT: Content and composition of maize endosperm lipids and their partition in the floury and vitreous regions were determined for a set of inbred lines. Neutral lipids, i.e., triglycerides and free fatty acids, accounted for more than 80% of endosperm lipids and are almost 2 times higher in the floury than in the vitreous regions. The composition of endosperm lipids, including their fatty acid unsaturation levels, as well as their distribution may be related to metabolic specificities of the floury and vitreous regions in carbon and nitrogen storage and to the management of stress responses during endosperm cell development. Remarkably, the highest contents of starch lipids were observed systematically within the vitreous endosperm. These high amounts of starch lipids were mainly due to lysophosphatidylcholine and were tightly linked to the highest amylose content. Consequently, the formation of amylose−lysophosphatidylcholine complexes has to be considered as an outstanding mechanism affecting endosperm vitreousness. KEYWORDS: maize, endosperm, vitreousness, lipids

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INTRODUCTION The texture of the maize (Zea mays L.) kernel is determined by the amount of vitreous and floury endosperm. Grain vitreousness is a key agronomic trait that influences postharvest resistance to insects and fungi, starch digestibility for livestock feeding, and semolina yield for food uses such as cornflakes production. The research performed on maize vitreousness has been indirectly driven by a strategic nutritional demand for improving the lysine content of maize proteins. Indeed, since the discovery of opaque2 mutation that led to maize with satisfactory grain lysine content but low agronomic value, many genetic studies and breeding programs have been conducted on opaque2 (o2) mutants. This led to the creation of quality protein maize (QPM) with higher nutritional quality than normal maize, satisfactory agronomic traits, and higher vitreousness.1,2 Owing to the numerous loci associated with this trait,3,4 improving kernel vitreousness is a big breeding challenge. Furthermore, the mechanisms that lead to the floury and vitreous endosperm partitioning in normal maize are not known. It is generally admitted that the floury and vitreous textures are the result of differences in the interactions between the major storage products, i.e., proteins and starch. Indeed, numerous mutants with the floury/opaque endosperm phenotype are affected in the protein and starch biosynthesis. The storage proteins, i.e., zeins, are synthesized in the endoplasmic reticulum (ER) and finally stored as protein bodies. Thus, a floury endosperm is closely related to mutations that alter zein structure and synthesis5−8 or ER function.9,10 These mutations induce the formation of irregularly shaped © 2015 American Chemical Society

protein bodies and decrease the endosperm storage protein content. In these mutants, the production of unfolded/ misfolded proteins induces ER stress and the unfolded protein response (UPR).11 UPR is a ubiquitous mechanism of eukaryote cells which controls protein synthesis and protein folding. UPR strongly impacts cell metabolism, including carbohydrate and lipid metabolism, which is responsible for metabolic diseases in humans.12 In QPM endosperm, an increase of γ-zein content is observed13 accompanied by a modification of the pyrophosphate-dependent fructose-6phosphate 1-phosphotransferase activity involved in glycolysis.14 Similarly, the pyruvate orthophosphate dikinase, which is under o2 control, could play a role in endosperm starch/ protein balance15,16 and can influence endosperm vitreousness. Other floury phenotypes are related to mutated genes involved in carbohydrate and lipid metabolism. For example, opaque9 is disrupted in the starch synthesis pathway17 and opaque5 (o5) in the galactolipid synthesis pathway.18 o5 encodes a monogalactosyldiacylglycerol synthase that affects galactolipid composition of amyloplast membranes leading to abnormal starch granules. Furthermore, in the Fl2 mutant altered in the posttranslational maturation of a 22-kD-zein storage protein, membranes of protein bodies contained a higher phosphatidylinositol (PI) content and a lower phosphatidic acid (PA) content, while the storage lipid content, i.e., triacylglycerol Received: Revised: Accepted: Published: 3551

January 16, 2015 March 12, 2015 March 20, 2015 March 20, 2015 DOI: 10.1021/acs.jafc.5b00293 J. Agric. Food Chem. 2015, 63, 3551−3558

Article

Journal of Agricultural and Food Chemistry

We added water up to 50 mL, and a diluted aliquot (10 X, 66 μL) was placed in the 96-well plate with 200 μL of GOPOD buffer and incubated for 20 min at 50 °C. Absorbance was read at 510 nm, and the subsequent glucose contents were determined using a standard curve. Glucose content was further converted to starch contents. Amylose was quantified according to a modification of the Megazyme amylose/amylopectin assay procedure. Briefly, zeins were extracted from 25 mg of flour with 70% ethanol for 1 h. Starch was subsequently dispersed in 1 mL of DMSO for 15 min at 100 °C, and then we added 2 mL of 95% ethanol and 4 mL of absolute ethanol. After starch precipitation, the supernatant was removed. Starch was dispersed in 2 mL of DMSO for 15 min at 100 °C, then 0.6 M sodium acetate at pH 6.4 was added up to 25 mL. An aliquot was used for starch quantification, and another one was used for amylose quantification, after amylopectin and dextrin precipitation with concanavalin-A. Aliquots were hydrolyzed with α-amylase and amyloglucosidase in 100 mM sodium acetate at pH 4.5, then 40 μL was placed in 96-well plate with 200 μL of GOPOD buffer and incubated for 20 min at 50 °C. The amylose content was determined from the absorbance ratio at 510 nm. Total Starch and Nonstarch Lipid Contents. Endosperm flour lipids were extracted by adding 1 mL of 2-propanol in 20 mg of flour. After 1 h at room temperature and centrifugation (3000g, 5 min), the pellet was extracted twice with 1 mL of chloroform/methanol 2:1 (v/ v) for 1 h. The three supernatants containing nonstarch lipids were pooled and dried under nitrogen flux. Nonstarch lipids were methylesterified with 1 mL of 14% BF3 in methanol at 70 °C for 1 h in the presence of heptadecanoic acid (Sigma-Aldrich, France) as internal standard. To quantify starch lipids, the residual defatted flour was dried and incubated in 5 mL of 2% sulfuric acid in methanol at 50 °C for 12 h in the presence of heptadecanoic acid as internal standard.24 The resulting fatty acid methyl esters (starch and nonstarch) were analyzed by coupling gas chromatography with flame ionization detection (GCFID Clarus680, PerkinElmer, France) using a DB225 (Agilent technologies, France) capillary column (30m × 0.32 mm, 0.25 μm) and a temperature gradient from 50 to 220 °C. Starch and Nonstarch Lipid Composition. To determine the composition of nonstarch lipids, 1 g of endosperm flour (about 20 dissected grains) was extracted in 10 mL of 2-propanol for 1 h and centrifuged (3000g, 5 min). The pellet was extracted three times in chloroform−methanol (2/1) for 1 h. The supernatants were pooled and dried on a rotary evaporator. To remove nonlipid material, lipids were solubilized in 8 mL of chloroform−methanol (2/1), to which 2 mL of 0.9% NaCl in water were added.25 The lower organic phase was collected and dried under nitrogen flux. Then, lipids were solubilized in 1 mL of chloroform containing 1% acetic acid and loaded on a silica (100 mg) cartridge (SupelClean LC-Si, Supelco, USA). Neutral lipids, galactolipids, and phospholipids were eluted with 1% chloroformacetic acid, acetone, and methanol, respectively. Lipids were dried under nitrogen flux and solubilized in chloroform before UPLC analysis. An aliquot was taken for quantification by GC-FID as described above. To analyze the starch lipid composition, 100 mg of thoroughly delipidated flour was dispersed in 1 mL of water during 5 min at 100 °C in water. Then, 4 mL of 1-propanol was added and mixed for 5 min at 100 °C. After centrifugation (3000g, 5 min), the pellet was washed with 80% 1-propanol in water. After centrifugation, the supernatants were pooled and dried under nitrogen flux, and the lipids were purified by phase partitioning in chloroform−methanol−0.9% NaCl as described above. Dried lipids were solubilized in 0.4 mL of chloroform before UPLC analysis. Lipids were analyzed on Dionex Ultimate 3000 UPLC equipped with an Uptisphere Strategy 2.2 μm 100 Å column (4.6 × 150 mm) and an evaporative light-scattering detector (Sedex80, Sedere, France). Lipids were eluted using a gradient from solvent A, chloroform, to solvent B, methanol−ammoniac 30%−chloroform 460:35:5 (v/v). The gradient started from 100% A to 20% B in 3 min, and then increased to 100% B in 9 min. After a plateau at 100% B for 3 min, the column was equilibrated in 100% A for 3 min. The analysis was carried out at 30 °C with a flow rate of 1.5 mL/min. Lipid content

(TAG), increased.19 Therefore, many mutants are impacted in the structure of subcellular organelles, e.g., ER, protein bodies, and amyloplasts, delimited by lipoprotein membranes. Surprisingly, no research was conducted on the relationships between endosperm lipids and maize vitreousness, especially by using normal maize inbred lines. It is also important to consider endosperm programmed cell death (PCD). PCD starts at the center of the grain in the floury area to finally affect the entire endosperm.20 Moreover, 10 days after pollination, endosperm faces hypoxia stress (DAP)21 and desiccation at the end of development.22 To delineate the biochemical basis of endosperm vitreousness, it is therefore more judicious to consider separately the floury and vitreous regions than the whole endosperm as was outlined for proteins.23 In this context, we report here the lipid composition of the vitreous and floury mature endosperms of dent and flint maize inbred lines. The results highlight a close relationship among starch lipids, amylose, and vitreousness. The differences observed in the composition and content of nonstarch lipids of vitreous and floury endosperm might be related to differences in their respective metabolic specificities and stress responses occurring along the PCD of developing endosperm.

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MATERIALS AND METHODS

Plant Material, Vitreousness, and Milling Properties. Thirteen maize inbred lines were grown in the Limagrain station in Mons (France). Maize kernels from self-pollinated homozygote plants were collected in the middle third of the ear. For lipid analyses, grains were frozen in liquid nitrogen and stored at −80 °C. For vitreous area determination, 10 grains were cut vertically along the germ center. Sections were scanned, and pictures were analyzed with ImageJ software to determine total endosperm and vitreous areas. Vitreous endosperm mass percentage was measured after hand-dissection of 3 series of 4 grains. For semolina yield determination, a lab degerminator (Satake, Satake Europe LTD) was used in order to remove germ and bran, and to separate the vitreous from the other part of the grain. Vitreous areas and vitreous endosperm weight have a close ranking and are strongly correlated with the milling properties, i.e., semolina yield (Table S1, Supporting Information) R = 0.79 (p < 0.01) and R = 0.82 (p < 0.001), respectively. Principal component analysis of these texture parameters highlighted different groups of vitreousness that correspond to flint (L1 to L4) and dent (L05 to L13) phenotypes. In these inbred lines, L11 is a markedly different dent variety with a very low vitreousness (Figure S1 and Table S1, Supporting Information). The genetic diversity of the 13 inbred lines was characterized by the Nei-Li genetic distance matrix calculated with 3000 SNPs distributed on 10 chromosomes (Table S2, Supporting Information). The floury and vitreous fractions were isolated using hand dissection after the removal of peripheral layers and the aleurone. The fractions were lyophilized and ground under liquid nitrogen. The protein content of isolated dried endosperm was determined by the Kjeldahl nitrogen determination method using a 5.7 factor for protein content. Vitreous/floury statistical analyses were performed using Student’s paired t-test. Error bars are standard deviations and represent differences between vitreous/floury fractions, as far as differences between maize inbred lines. Starch and Amylose Content. Starch was quantified according to a modification of the Megazyme Total starch assay procedure. Briefly, 50 mg of flour was washed in 5 mL of 80% ethanol at 80 °C for 5 min. Starch was subsequently dispersed in 2 mL of DMSO for 10 min at 100 °C. Samples were hydrolyzed 6 min at 100 °C with 3 mL of a 50 mM, pH 7 thermostable α-amylase solution (100U/mL) in MOPS (3(N-morpholino)propanesulfonic acid), before adding 4 mL of 200 mM ammonium acetate at pH 4.5 and 100 μL of amyloglucosidase (3300U/mL). The hydrolysis was conducted during 30 min at 50 °C. 3552

DOI: 10.1021/acs.jafc.5b00293 J. Agric. Food Chem. 2015, 63, 3551−3558

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Journal of Agricultural and Food Chemistry was calculated in regard to a calibration curve obtained with lipid standards. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) were from soybean (Avanti Polar Lipids Inc., USA). Bovine brain phosphatidylserine (PS), egg yolk phosphatidic acid (PA), 1-palmitoyl-lysophosphatidylcholine (C16 lysoPC), and linoleic acid were from Sigma-Aldrich Chimie (Lyon, France). 1-Heptanoyl-lysophosphatidylethanolamine (C17 lysoPE) was provided by Avanti Polar Lipids (Alabaster, Alabama, USA). Galactolipids, i.e., digalactosyldiacylglycerol (DGDG), and monogalactosyldiacylglycerol (MGDG) standards were purified from wheat flour as previously described.26 Standard TAG were purified from the corresponding commercial sunflower oil on SiOH cartridges using stepwise elution with hexane−diethyl ether 99:1 and 95:5 (v/v).

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RESULTS Sequential Extraction Procedure Enabling Quantification and Characterization of Starch and Nonstarch Lipids of Maize Endosperm. The selected sample of 13 maize inbred lines displays a wide range of vitreousness and protein content (from 8.8 to 12.3% DW). The starch content varies from 71.2 to 74.5% DW (Table S1, Supporting Information). In maize endosperm, a part of the lipids are embedded in starch granules and are mainly composed by FFA and lysophospholipids.27 They are not extractible using cold solvents, being only released after starch hydrolysis or by using hot water−alcohol solvents. Therefore, we developed a sequential extraction procedure where lipids are first thoroughly extracted with solvents at room temperature, i.e., nonstarch lipids, while the residual unextracted lipids, i.e., starch lipids, were extracted with hot water−1-propanol or released by acid methanolysis as fatty acid methyl esters (FAME). Compared to nonstarch lipids, starch lipids are characterized by a higher proportion of palmitic acid (about 40% vs about 18%). Conversely, nonstarch lipids contained a higher proportion of unsaturated fatty acids, namely, linoleic (18:2 n-6), linolenic (18:3 n-3) and oleic acids (18:1 n-9), than starch lipids (Figure 1a). These fatty acid compositions are in agreement with previous data obtained for purified maize starch and isolated maize endosperm.28 Finally, when delipidated maize endosperms were extracted with hot aqueous 1-propanol, lipid analysis by normal-phase UPLC showed that these lipids are mainly composed of FFA and lysophospholipids, lysoPE, and especially lysoPC (Figure 1b and Figure S2a, Supporting Information) that specifies starch lipids.27 Starch lipid contents vary from 0.27 to 0.35% of endosperm DW (Table S3, Supporting Information). Therefore, about a third of endosperm lipids occur inside the starch granules. Related to starch contents, these values correspond to 0.37 and 0.49% of starch DW, respectively. These amounts are close to the value found for purified starch from a commercial maize hybrid, i.e., about 0.5%.28 In contrast, nonstarch lipid contents vary from 0.47% to 0.83% of endosperm DW. In all inbred lines, nonstarch lipids are mainly composed of neutral lipids (79 to 95% of nonstarch lipids) (Table S3, Supporting Information) as previously observed in hybrids as well as in amylo- and waxy maize.28 TAG and FFA compose the neutral lipids where only very small quantities of monoacyl (MAG)and diacylglycerol (DAG) can be detected (Figure S2b, Supporting Information, and Figure 3b). This contrasts with the results obtained by Tan and Morrison28 who found very low TAG levels, high amounts of FFA, and measurable levels of DAG and MAG. In regard to the results reported by Tan and Morisson,28 the higher proportion of TAG vs FFA observed herein is probably due to our primary extraction with 2-

Figure 1. (a) Fatty acid composition of starch lipids and nonstarch lipids expressed as percentage of total FAME. Values are the average of two independent biological replicates for the 13 lines (error bar, SD; n = 26) (asterisks indicate significant difference; Student’s t test; *P < 0.05; ***P < 0.001). (b) Starch lipid composition of total endosperm expressed as percent of total starch lipids. FFA, free fatty acid; LysoPE, lysophosphatidylethanolamine; LysoPC, lysophosphatidylcholine. Mean values for the 13 lines (error bar, SD; n = 13).

propanol, which is known to inhibit lipases.25 Finally, membrane lipids, i.e., galactolipids and phospholipids, were minor compounds and represented from 1 to 7.6% and from 2.5 to 13.6% of nonstarch lipids, respectively (Table S3, Supporting Information). Therefore, our sequential extraction procedure is a simple way to accurately determine the starch and nonstarch lipid contents and composition. It does not need extraction of starch, avoiding contamination by residual nonstarch lipids present inevitably on the surface of granules28,29 FFA to Lysophospholipid Ratio of Starch Lipids Is a Marker of Vitreous and Floury Endosperm. In order to explore whether any relationship between biochemical composition and vitreousness can be assessed, the vitreous and floury regions were isolated by hand dissection. As previously shown in opaque and inbred maize lines,23,30 protein contents were higher in the vitreous area, whereas higher starch contents were observed in the floury one (Table S4, Supporting Information). Similarly, slight but significant higher amylose levels were systematically evidenced in the starch of the vitreous endosperm when compared with the starch of the floury area (Table S4, Supporting Information). This result is in accordance with the higher amylose contents measured in maize hybrids and a QPM population with increased vitreousness.31,32 Accordingly, starch lipids and 3553

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Journal of Agricultural and Food Chemistry nonstarch lipids of vitreous and floury endosperm were quantified as FAME. When lipid contents are related to endosperm dry weight, the starch lipid content is not significantly different between the floury and vitreous regions. However, when lipid contents are related to starch dry weight content, a clear and significant trend for higher lipid content in the starch granules of the vitreous fraction than in the starch granules of the floury one is evidenced (Figure 2a).

Figure 2. (a) Starch lipid content in vitreous and floury endosperm from the 13 lines expressed on endosperm and starch dry weight and expressed in dry weight percent. Values are averages of the 13 lines (error bar, SD; n = 13) (asterisks indicate significant difference; Student’s t test; *P < 0.05). (b) Lipid content in vitreous and floury starch from the 13 lines expressed as percent of total starch lipid. FFA, free fatty acid; LysoPE, lysophosphatidylethanolamine; LysoPC, lysophosphatidylcholine. Values are averages of the 13 lines (error bar, SD; n = 13). (Asterisks indicate significant difference; Student’s t test; **P < 0.01; ***P < 0.001.)

Figure 3. (a) Nonstarch lipid content in vitreous and floury endosperm from the 13 lines, expressed in dry weight percent of endosperm. Mean values for 13 lines (error bar, SD; n = 13) (asterisks indicate significant difference; Student’s t test; **P < 0.01). (b) Vitreous and floury endosperm neutral lipid composition expressed as percent of total neutral lipids. TAG, triacylglycerol; FFA, free fatty acid. Values are averages of the 13 lines (error bar, SD; n = 13) (asterisks indicate significant difference; Student’s t test; ***P < 0.001; ns, nonsignificant). (c) Polar lipid contents in vitreous and floury endosperm from the 13 lines expressed in dry weight percent of endosperm. Mean values for the 13 lines (error bar, SD; n = 13).

Interestingly, this increase of lipid contents comes with a major shift in the FFA to lysophosphopholipid ratio due to an increase of the lysoPC content, the major starch phospholipid (Figure 2b and Figure S2a, Supporting Information). Partitioning of Storage and Membrane Lipids in Vitreous and Floury Endosperm. The highest nonstarch lipid contents are observed in the floury endosperm for each inbred line. Indeed, the mean nonstarch lipid amounts of the floury endosperms are almost twice those observed in the case of the vitreous endosperm, i.e., 1 and 0.6% endosperm DW, respectively (Figure 3a). This difference is mainly due to neutral lipids, the main lipid fraction of maize endosperm (Table S3 and Figure S2b, Supporting Information). Indeed, the amounts of membrane (polar) lipids are quite low in all inbred lines, and no significant differences are observed

between the polar lipid contents of the vitreous and floury endosperms (Figure 3c). While the vitreous endosperm is significantly richer in FFA than in TAG, there is no significant difference between the FFA and TAG contents of floury endosperm (Figure 3b). Therefore, in regard to the vitreous endosperm, the higher amounts of neutral lipids in the floury endosperm are mainly due to TAG (Figure 3b). Remarkably, the partition of nonstarch polar lipids between the vitreous and floury areas follows a different pattern. While the levels of total membrane lipids are not significantly different 3554

DOI: 10.1021/acs.jafc.5b00293 J. Agric. Food Chem. 2015, 63, 3551−3558

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Journal of Agricultural and Food Chemistry between vitreous and floury endosperm (Figure 3c), their compositions, i.e., galactolipid to phospholipid ratio, differ significantly. Indeed, galactolipids represent 30% of membrane lipids in the floury region, whereas they represent 20% in the vitreous region (Figure 4a). Similarly, vitreous endosperm contains more phospholipids than the floury one, with 80% of phospholipids in vitreous endosperm and 70% in floury endosperm. These tendencies are observed in most lines except for L05 and L06 where the proportions of galactolipids and phospholipids in their floury and vitreous regions were similar (data not shown). We do not observe significant differences in the galactolipid composition of the vitreous and floury endosperm (Figure 4b). MGDG and DGDG are the major galactolipids, and in most maize lines, except for L02 and L13 (data not shown), we observe the same MGDG/DGDG ratio in the vitreous and floury endosperm (Figure 4b). Similarly, we do not observe significant differences in PE, PA, and lysoPC proportions between the floury and vitreous regions (Figure 4c). Conversely, a higher proportion of PC is observed in the vitreous endosperm, where it represents about 59% of phospholipids versus 54% in the floury part. This difference is partially balanced by a significant increase of PI level in floury areas, i.e., 12.6% of phospholipids vs 10.6% in vitreous endosperm. Lipids of Vitreous Endosperm Display a Higher Level of PUFA than the Floury Endosperm. The fatty acid composition of vitreous and floury endosperm lipids display systematic differences. Acyl lipids of vitreous endosperm contained higher proportions of polyunsaturated fatty acid (PUFA) and lower levels of saturated and monounsaturated fatty acids (Figure 5). The proportions of linoleic acid and linolenic acid are significantly higher in the vitreous region. Nonstarch lipids consist of about 64% of PUFA in the vitreous region, whereas they represent at most 58% in the floury endosperm. In regard to the vitreous endosperm, the decrease of PUFA proportion in the floury endosperm is balanced by an increase of saturated and monounsaturated fatty acids, namely, palmitic, stearic, and oleic acids. We observed the same shift in fatty acid saturation level in both storage and membrane lipid classes. This shift was observed in starch lipid too, although it is less pronounced (data not shown). Therefore, a higher PUFA content in the lipid of the vitreous region than in the floury one is systematically observed in both starch and nonstarch lipids.

Figure 4. (a) Galactolipid and phospholipid levels in vitreous and floury endosperm of the 13 individual inbred lines (markers). The black line shows average lipid evolution. Lipids were quantified as FAME and expressed as percent of total polar lipids. Values are the average of two independent biological replicates (asterisks indicate significant difference; Student’s t test; ***P < 0.001). (b) Vitreous and floury endosperm galactolipid composition expressed as a percent of total galactolipids. MGDG, monogalactosyldiacylglycerol; DGDG, digalactosydiacylglycerol. Values are the average of three independent biological replicates for the 13 lines (error bar, SD; n = 39). (c) Vitreous and floury endosperm phospholipid composition expressed as percent of total phospholipids. PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; PC, phosphatidylcholine; LysoPC, lysophosphatidylcholine. Values are the average of three independent biological replicates for the 13 lines (error bar: SD; n = 39) (asterisks indicate significant difference; Student’s t test; **P < 0.01).

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DISCUSSION Although lipids are minor compounds of the cereal endosperm, they play a major role in the assembly of the starch−protein matrix of mature kernels and therefore in grain texture (hardness). In this context, numerous research works were conducted on the lipids of wheat endosperms.33 Surprisingly, only few research works were devoted to maize endosperm lipids and especially, no work had been realized on the relationships between endosperm lipids and vitreousness of maize seeds. Endosperm lipids can be classified in starch and nonstarch lipids. Starch lipids are embedded in the starch granules and are extracted only with hot alcohol−water solvents or after starch hydrolysis.27 They account for about 30% of total lipids and are characterized by a high palmitic acid content in regard to nonstarch lipids in agreement with previous results.28 These starch lipids are almost exclusively composed of monoacyllipids, i.e., FFA and lysophospholipids, mainly lysoPC. In this inbred line population, no correlation could be found between

these lipids and maize vitreousness or milling properties (semolina yield). Indeed, any significant correlation could be also observed between these physical properties and protein and starch contents as well. However, by considering separately the floury and vitreous regions obtained by hand dissection, 3555

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regard, it was shown that phospholipids could inhibit starch branching enzymes.44 Actually, differences in the biosynthesis of nonstarch lipids also exist between floury and vitreous endosperms. Indeed, nonstarch lipids are mainly composed by TAG and FFA for all inbred lines, and a higher accumulation of these lipids is observed in the floury than in the vitreous region of the maize endosperm. Concerning membrane lipids, galactolipids, and phospholipids, it was surprising to observe such low contents in all maize lines. Indeed, in wheat endosperm, with similar protein and starch content, the membrane lipid content29,45is more than 10 times the content found in maize endosperm (this work and ref 28). This low membrane lipid content in all inbred lines of our maize population seems to confirm and generalize the significant degradation of endosperm membrane lipids observed during endosperm development of a maize hybrid.46 This specificity of endosperm maize development could be related to autophagy. Actually, autophagy and especially macroautophagy is a ubiquitous cellular mechanism that is involved in the degradation of damaged organelles and their membrane components.47 Autophagy is operative in plants.48,49 It is worthy to note that in maize endosperm the lipidation of an autophagy protein, i.e., Atg8, a marker of the formation of functional autophagosomes, starts almost at the same time50 as membrane lipid degradation.46 Furthermore, it is interesting to note that the decrease of TAG contents and the increase of FFA contents from mid to the end of endosperm development46 occurs also at the onset of the expression of Atg8. Indeed, in animals as in plants, autophagy could be involved in TAG degradation.51,52 Whatever the origin and extent of the degradation of membrane lipids, it is worthwhile noting that there is a tendency toward higher galactolipid contents in the floury endosperm than in the vitreous one. This is in agreement with our observation that the floury endosperm has higher starch content than the vitreous one. Actually, galactolipids are specific compounds of the amyloplast membranes.18 However, the higher phospholipid contents in the vitreous endosperm can be related to its higher protein content. Indeed, protein bodies, where protein accumulates, emerge from the ER, and their membrane lipids are composed by phospholipids. Finally, the lipid composition of the floury and vitreous endosperm could reflect some specificity in the regulation of their metabolism, which is essentially directed toward carbon and nitrogen storage. In this regard, it is worthy to note that phospholipids of the floury endosperm display significantly higher PI level. Shank et al.19 reported an increase of PI content in the Fl2 mutant that was also paralleled with an augmentation of TAG content. These relationships are not surprising since phospholipid and TAG biosynthetic pathways share common steps.43 Shank et al.19 showed that, in the Fl2 mutant, these lipid changes were related to UPR. UPR is a ubiquitous mechanism that controls the ER production of correctly folded protein and ER homeostasis.53 Therefore, the PI and TAG increase observed in the floury endosperm in regard to the vitreous endosperm could suggest differences in the UPR responses between these endosperm regions. The lower proportion of PC in the floury endosperm also supports this. Indeed, studies on C. elegans report that PC decrease leads to UPR and is associated with an increase in saturated fatty acid.54 Moreover, we observed that the lower proportion of PUFA, i.e., both linoleic and linolenic acids, in the floury endosperm than in the vitreous one is

Figure 5. Fatty acid composition of nonstarch lipids quantified as FAME and expressed as percent of total fatty acid. Values are the average of two independent biological replicates for the 13 lines (error bar, SD; n = 26) (asterisks indicate significant difference; Student’s t test; **P < 0.01; ***P < 0.001).

significant differences were found. Starch lipid content was higher in the starch of vitreous endosperm than in the starch of floury endosperm. Similarly, the amylose content of starch is systemically higher in the vitreous than in the floury endosperm. This is in agreement with previous works reporting a relationship between the amylose and starch lipid contents when considering different groups, i.e., low-amylose (waxy), normal-amylose, and high-amylose maize starches. Indeed, higher amylose content is associated with higher starch lipid content.34,35 Furthermore, our work shows that the higher lipid content of starches from vitreous endosperm than that of the starches of floury endosperm was mainly due to a shift in the FFA to lysoPC ratio. On the contrary, both FFA and lysophospholipid contents increased when comparing normal and high-amylose maize mutants emphasizing the difference between normal maize and mutants.34,35 Starch lipids can form complexes with amylose in native starch granules36,37 and impact amylose chain assemblies, starch crystallinity, and obviously play a role in the architecture of starch granules.38 These changes of starch lipid content and composition may be compared with changes of the crystallinity and morphology of starch granules with an increase of endosperm vitreousness in QPM.4,39 This also has to be compared with differences observed for the surface structure of starch granules isolated from the vitreous and floury endosperm regions of maize hybrids and the QPM population.31 Therefore, the biosynthesis of starch lipids and its regulation can also be regarded as one of the significant molecular and cellular mechanisms determining maize endosperm vitreousness. Actually, the origin and the biological function of the monoacyl lipids entrapped in the starch granules are still unknown. Regarding lysoPC, it is the form used to import lipids from the ER into plastids for the synthesis of some plastid lipids.40 Although it is not known if this way is operative in amyloplasts, it is interesting to note that the highest lysoPC level is observed in the vitreous endosperm where the synthesis of storage proteins, i.e., zeins, which occurs in the ER, is particularly more intense than in the floury region. Indeed, it is also in the vitreous endosperm where the highest level of PC, the precursor of lysoPC, is observed. Beside a role of these lipids in the synthesis of amyloplast membrane lipids as in plastids,41−43 starch lipids could regulate the activity of enzymes involved in amylose and amylopectin synthesis. In this 3556

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Journal of Agricultural and Food Chemistry

quality protein maize; LysoPC, lysophosphatidylcholine; LysoPE, lysophosphatidylethanolamine; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PCD, programmed cell death; PI, phosphatidylinositol; PUFA, polyunsaturated fatty acid; quality protein maize; TAG, triacylglycerol; UPR, unfolded protein response

systematically observed and is balanced by a significant increase of the proportion of both saturated and monounsaturated fatty acids. These differences could be related to differences of Δ12desaturase activity. Actually, fatty acid saturation level and desaturase activity are good markers of cell stress in plants.55,56 Therefore, the nonstarch lipid contents and compositions of the floury vs those of the vitreous endosperm can be linked to the stress occurring during endosperm development. Indeed, PCD starts early in the central region, which almost will correspond to the floury endosperm of the mature grain.20,57 However, as outlined above for the membrane lipid and lysophospholipid partition, it could not be excluded that these differences in the fatty acid unsaturation level are also due to the metabolic specificities of the different endosperm regions. Indeed, the Δ12-desaturase activity seems centered in the endoplasmic reticulum of maize endosperm,45 a compartment highly solicited for zein synthesis in the vitreous endosperm. At this stage of the investigation, the role of nonstarch lipids of endosperm vitreousness cannot be clearly determined. It is, however, important to emphasize that lipids, especially membrane lipids, have to be considered in terms of surface potential in contrast with starch and proteins that have to be regarded in terms of volume occupancy. Actually, in dry mature wheat endosperm membrane lipids are mainly localized at the interface between the starch granule and the protein matrix,58 and correlations have been found between the extractability of membrane lipids and wheat hardness.59 Although lipids are minor components of maize endosperm, this first analytical approach reveals that these compounds could be good markers of endosperm development. Indeed, the significant changes impacting the content and composition of lipids in relation to endosperm vitreousness are related to differences in the metabolism of cells that will lead to the vitreous and floury regions of the endosperm. The most important feature of this study is the probable important role of starch lipids, especially lysoPC, in determining with amylose the starch architecture and finally the starch−protein interactions.

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ASSOCIATED CONTENT

S Supporting Information *

Vitreousness, semolina yield, and protein and starch content of maize endosperm; endosperm lipid composition; protein, starch, and amylose contents of vitreous and floury endosperm; principal component analysis (PCA) plots using semolina yield and vitreousness measurements (weight and surface, see Materials and Methods); and chromatogram of starch and nonstarch lipids from vitreous and floury endosperm. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by grants from the Fond Unique Interministériel (FUI GranoFlakes, F1204004C). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED DGDG, digalactosydiacylglycerol; ER, endoplasmic reticulum; FAME, fatty acid methyl ester; FFA, free fatty acids; QPM, 3557

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