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. 2018 May 1;177(2):698–712. doi: 10.1104/pp.17.01826

FLOURY SHRUNKEN ENDOSPERM1 Connects Phospholipid Metabolism and Amyloplast Development in Rice1

Wuhua Long a,c,2, Yunlong Wang a,2, Susong Zhu c,2, Wen Jing d, Yihua Wang a, Yulong Ren b, Yunlu Tian a, Shijia Liu a, Xi Liu a, Liangming Chen a, Di Wang a, Mingsheng Zhong a, Yuanyan Zhang a, Tingting Hu a, Jianping Zhu a, Yuanyuan Hao a, Xiaopin Zhu a, Wenwei Zhang a, Chunming Wang a, Wenhua Zhang d,3, Jianmin Wan a,b,3
PMCID: PMC6001332  PMID: 29717019

The FLOURY SHRUNKEN ENDOSPERM1 gene, encoding a phospholipase-like protein, plays an important role in phospholipid metabolism and amyloplast development in rice endosperm.

Abstract

Starch synthesized and stored in amyloplasts serves as the major energy storage molecule in cereal endosperm. To elucidate the molecular mechanisms underlying amyloplast development and starch synthesis, we isolated a series of floury endosperm mutants in rice (Oryza sativa). We identified the rice mutant floury shrunken endosperm1 (fse1), which exhibited obvious defects in the development of compound starch grains, decreased starch content, and altered starch physicochemical features. Map-based cloning showed that FSE1 encodes a phospholipase-like protein homologous to phosphatidic acid-preferring phospholipase A1. FSE1 was expressed ubiquitously with abundant levels observed in developing seeds and roots. FSE1 was localized to both the cytosol and intracellular membranes. Lipid profiling indicated that total extra-plastidic lipids and phosphatidic acid were increased in fse1 plants, suggesting that FSE1 may exhibit in vivo phospholipase A1 activity on phosphatidylcholine, phosphatidylinositol, phosphatidyl-Ser, phosphatidylethanolamine, and, in particular, phosphatidic acid. Additionally, the total galactolipid content in developing fse1 endosperm was significantly reduced, which may cause abnormal amyloplast development. Our results identify FSE1 as a phospholipase-like protein that controls the synthesis of galactolipids in rice endosperm and provide a novel connection between lipid metabolism and starch synthesis in rice grains during endosperm development.

The endosperm of cereals accumulates large amounts of stored starch, which is the primary carbon source for humans and livestock (Burrell, 2003). Starch is mostly synthesized in the amyloplasts, which are specialized plastids in the endosperm cells (Martin and Smith, 1995). During rice (Oryza sativa) endosperm development, starch forms into insoluble particles in amyloplasts, referred to as starch grains (SGs). A compound SG can accumulate dozens of starch granules, which differs from single SGs in other crops (Jane et al., 1994; Yun and Kawagoe, 2010). When SGs are filled with starch, the amyloplast envelope begins to degrade, thus releasing SGs into the cytoplasm, which remain in situ and maintain the shape of the amyloplast (Wei et al., 2008). In mature endosperm, starch granules in a compound SG are polyhedral, sharp-edged, and easily separable (Jane et al., 1994; Yun and Kawagoe, 2010). In rice, starch synthesis in the cereal endosperm begins with ADP-Glc pyrophosphorylase (AGPase) catalyzing the reaction of Glc-1-phosphate (Glc-1-P) with ATP to produce ADP-Glc (ADPG). ADPG is used as a substrate for both amylose-specific granule-bound starch synthase (GBSS) and amylopectin unique starch biosynthetic enzymes, including soluble starch synthase (SS), starch branching enzyme, and starch debranching enzyme, to initiate starch synthesis and elongation (Tian et al., 2009). Mutations of key enzymes in the starch biosynthesis pathway generally lead to defective amyloplast development (Kubo et al., 1999; Nishi et al., 2001; Fujita et al., 2003, 2006, 2007, 2011; Satoh et al., 2003; Kawagoe et al., 2005; Ryoo et al., 2007; Tang et al., 2016; Toyosawa et al., 2016). For example, pyruvate orthophosphate dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and pyruvate to AMP, PPi, and PEP. The OsPPDKB gene encoding PPDK has been found to function in rice to modulate carbon metabolism and starch synthesis during grain filling (Kang et al., 2005). In addition, other factors involved in amyloplast development have also been reported, including Rice Starch Regulator 1 (RSR1; Fu and Xue, 2010), FLOURY ENDOSPERM2 (FLO2; She et al., 2010), FLO6 (Peng et al., 2014), FLO7 (Zhang et al., 2016), OsbZIP58 (Wang et al., 2013), Small kernel 1 (Li et al., 2014), SUBSTANDARD STARCH GRAIN4 (SSG4), and SSG6 (Matsushima et al., 2014, 2016).

Glycerolipids are the major constituents of all membranous structures in plants. Higher plants possess two distinct pathways for the synthesis of glycerolipids, i.e. the prokaryotic (plastidic) and eukaryotic (ER) pathways. In the prokaryotic pathway, phosphatidic acid (PA) has 16:0 fatty acids (FAs) at the sn-2 position and 18:1 FAs, generally, at the sn-1 position. PA is the starting molecule for other plastidic membrane lipids, including phosphatidylglycerol (PG) and diacylglycerol (DAG), which is converted to galactosyldiacylglycerol (GDG), including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG; Ohlrogge and Browse, 1995). In the eukaryotic pathway, PA has 18:1 or 16:0 FAs at the sn-1 position and 18:1 FAs at the sn-2 position. PA gives rise to phospholipids including phosphatidyl-Ser (PS), phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylethanolamine (PE), which are characteristic of the various extraplastidic membranes. In rice, the lack of PA dephosphorylation activity means that DAG cannot be formed via the plastidic pathway. The prokaryotic pathway contributes only PG in plastidic lipids, whereas the rest of the plastidic lipids are generated from DAG formed by the eukaryotic (ER) pathway and transported into plastids (Heinz and Roughan, 1983; Ohlrogge and Browse, 1995). MGDG and DGDG are the primary nonprotein components of plastid membranes, serving both structural and functional purposes. These galactolipids account for more than 40% of total polar lipids in maize (Zea mays) developing endosperm (Myers et al., 2011). However, only a minor portion of polar lipids exist in mature endosperm due to the degradation of the amyloplast envelope in rice (Wang et al., 2012; Toyosawa et al., 2016). Starch is the major component of rice grains, and starch and lipid synthesis compete for carbon flow during grain filling (Kang et al., 2005). The altered content and composition of galactolipids could in turn affect starch synthesis and the morphology of starch grains during endosperm development, although the molecular mechanisms are largely unknown (Myers et al., 2011; Toyosawa et al., 2016).

Phospholipases catalyze the initial step of phospholipid hydrolysis and play an important role in lipid signaling. Phospholipases have been broadly categorized as phospholipase A (PLA), phospholipase C (PLC), and phospholipase D (PLD), based on the action of respective enzymes at different sites on a glycerophospholipid molecule. The PLA superfamily has two subtypes, PLA1 and PLA2, which catalyze the hydrolysis of membrane glycerophospholipids at sn-1 and sn-2 positions, respectively, producing a free fatty acid and a lysophospholipid. PLAs play important roles in plants, in cell elongation, gravitropism, anther dehiscence, seed development, jasmonic acid biosynthesis, and defense signaling (Ishiguro et al., 2001; Kato et al., 2002; Viehweger et al., 2002; Lee et al., 2003; Froidure et al., 2010; Yang et al., 2012a; Liu et al., 2015). For example, a patatin-related phospholipase A (pPLA), AtpPLAIIα, could hydrolyze glycerophospholipids at the sn-1 and sn-2 positions in Arabidopsis (Arabidopsis thaliana; Yang et al., 2012), whereas a PLA2 from Eschscholzia californica (Papaveraceae) initiates a signal transduction pathway that is required for the expression of alkaloid biosynthesis (Heinze et al., 2015). In mammalian cells, phosphatidic acid-preferring phospholipase A1 (PA-PLA1), which preferentially hydrolyzes PA at the sn-1 position, was first identified from bovine testis (Higgs and Glomset, 1994). Its homolog (AtSGR2), identified in Arabidopsis, regulates gravitropism and seed development (Kato et al., 2002). Nevertheless, the functions and mechanisms of PLA1 have remained elusive in the plant kingdom.

In this study, we identified and characterized the rice mutant floury shrunken endosperm1 (fse1). FSE1 encodes a phospholipase-like protein homologous to PA-PLA1. Lipid profiling indicated that total extraplastidic lipids and PA are increased in fse1 plants, suggesting that FSE1 may exhibit PLA1 activity on PC, PI, PS, PE, and, in particular, PA. Additionally, total galactolipid abundance in developing fse1 endosperm is strongly reduced, which may cause abnormal amyloplast development.

RESULTS

Phenotypic Characterization of the fse1 Mutant

To identify new regulators of endosperm development, we isolated a set of rice floury endosperm mutants from a mutant pool induced by N-methyl-N-nitrosourea (in the japonica variety Dianjingyou 1 background). Under paddy field conditions, the mutant fse1 showed no significant differences from the wild-type plants during the seedling and tillering stages. However, fse1 displayed a remarkably slower grain filling rate during seed development (Supplemental Fig. S1A). In contrast to the transparent endosperm observed in wild-type seeds, mature seeds from the fse1 mutant showed floury and shrunken endosperm (Fig. 1, A and B). Notably, cross-section analysis showed that the central region of the fse1 mutant grain was floury white (Fig. 1C). Furthermore, scanning electron microscopy images indicated that the fse1 endosperm was filled with loosely packed, small, and spherical starch grains with large air spaces, whereas the wild-type endosperm consisted of densely packed, large, and irregularly polyhedral starch grains (Fig. 1D). Consistent with the above observations, the 1,000-grain weight of the fse1 mutant was reduced by 12% compared with that of the wild type (Supplemental Fig. S1B). These results indicated that the fse1 mutation affects starch accumulation during endosperm development.

Figure 1.

Figure 1.

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Phenotypic analyses of the fse1 mutant. A and B, Comparison of wild-type (left) and fse1 (right) seeds placed on a black surface (A) or a light box (B). Bars = 5 mm. C, Transverse sections of wild-type (left) and fse1 seeds (right). Bar = 0.5 mm. D, Scanning electron microscopy analyses of wild-type (left) and fse1 (right) endosperm. Bar = 50 μm. E, The starch, amylose, lipid, and crude protein contents of mature wild-type and fse1 seeds, calculated based on dry weight. Values are means ± sd from three biological replicates. The asterisks indicate statistical significance between wild type and fse1, as determined by a Student’s t test (**P < 0.01).

The total starch and amylose contents of fse1 grains were 6.5% and 12.1% lower than those of the wild type, respectively (Fig. 1E), and the structure of amylopectin was also changed, with both the short and long chains consisting of 6 to 8 degrees of polymerization (DP) and ≥16 DP, respectively, decreasing and the middle chains with 9 to 15 DP increasing (Supplemental Fig. S2A). Furthermore, the pasting properties of endosperm starch were analyzed using a rapid visco analyzer (Supplemental Fig. S2B). The rate of increase in the viscosity of fse1 starch was slower than that of wild-type starch as temperature increased. The peak and the final viscosity of fse1 starch were 60% and 61% that of wild-type starch, respectively. However, the crude protein content of fse1 seed was increased, and the total lipid content of fse1 seed was dramatically increased by 47.7%, compared to that of wild-type seeds (Fig. 1E). Taken together, these results indicate that the mutation in fse1 affects the accumulation of stored substances in the endosperm.

Amyloplast Development Is Arrested in fse1 Endosperm

During endosperm development, a large number of developed starch grains fill the endosperm (Toyosawa et al., 2016). To determine the cytological basis of abnormal starch accumulation in fse1, semithin sections of developing endosperm at 6, 9, and 12 d after flowering (DAF) were prepared to observe the structure of compound starch grains. Iodine staining of the sections showed that in the central region of wild-type endosperm, each amyloplast produced densely packed, polyhedral starch grains, indicating that the amyloplasts were well developed (Fig. 2A, iii). In contrast, the central region of fse1 mutant endosperm contained abnormal amyloplasts, with smaller, scattered, and weakly iodine-stained starch grains (Fig. 2A, vi). Even at early developmental stages at 6 and 9 DAF, the fse1 mutant produced aberrant starch grains compared to that in the wild type (Fig. 2A, i, ii, iv, and v). Furthermore, we performed transmission electron microscopy to observe the compound starch grains in developing endosperm at 9 DAF. The results showed that the central region of wild-type endosperm contained fully developed compound starch grains, whereas the fse1 endosperm formed lots of smaller and scattered starch grains (Fig. 2B), with large amounts of irregular, single starch grains. This result is consistent with the phenotype of mature seeds observed by scanning electron microscopy and iodine staining analyses (Fig. 1D). Collectively, these data suggest that the fse1 mutation affects compound starch grain formation during early development of endosperm.

Figure 2.

Figure 2.

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Abnormal compound starch grain formation in fse1 seeds. A, Semithin sections wild-type (i–iii) and fse1 (iv–vi) developing endosperm at 6, 9, and 12 DAF. Bars = 50 μm. B, Transmission electron microscopy analysis of the central region of wild-type (a) and fse1 (b) endosperm at 9 DAF. Bars = 5 μm.

Map-Based Cloning of FSE1 and Complementation of the fse1 Mutation

To elucidate the molecular mechanisms underlying the fse1 phenotypes, a total of 155 individuals showing the fse1 phenotype were selected from an F2 population for map-based cloning. The FSE1 locus was first mapped to the short arm of chromosome 8 between the simple sequence repeats marker RM408 and the InDel marker WH-1. The FSE1 locus was further delimited to a 190-kb region, which contains 25 open reading frames (http://rice.plantbiology.msu.edu/; Fig. 3A). DNA sequence analysis revealed a single nucleotide mutation in the sixth exon of gene LOC_Os08g01920, leading to a Gly-282-Glu substitution in the fse1 mutant (Fig. 3B).

Figure 3.

Figure 3.

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Positional cloning and complementation of the fse1 mutant. A, Fine mapping of the FSE1 locus. The FSE1 locus was mapped to a 190-kb region between markers WH-2 and WH-4 on chromosome 8 (Chr.8), which contained 25 predicted genes. The number of recombinants is indicated below the map. B, The FSE1 single nucleotide change that led to Gly-282-Glu substitution in fse1. Black box, exon; white box, noncoding region; line, intron. C, Whole-seed phenotype (bottom) and seed transverse sections (top) of wild type, fse1, and fse1 complemented lines (CL-1-3) expressing FSE1-GFP. Bars = 2 mm. D, Western-blot analyses of FSE1 and FSE1-GFP in mature seeds of wild-type and fse1 complemented lines using anti-FSE1 and anti-GFP antibodies. M, Protein marker.

To test whether LOC_Os08g01920 corresponded to the candidate FSE1 gene, both vectors bearing the fused FSE1-GFP driven by the 35S promoter and the FSE1 coding region driven by the UBIQUITIN1 promoter were introduced into the fse1 mutant. Positive transgenic lines were identified by quantitative real-time PCR (qRT-PCR; Supplemental Fig. S3A). Both constructs completely rescued the developmental defects of fse1 endosperm (Fig. 3C; Supplemental Fig. S3B). Therefore, LOC_Os08g01920 is indeed the gene responsible for the fse1 mutation and the FSE1-GFP fusion protein is biofunctional in vivo. Furthermore, specific polyclonal antibodies against FES1 were raised, which recognized the same band of about 160 kD recognized by the GFP monoclonal antibody when total seed proteins of FSE1-GFP transgenic lines were analyzed by immunoblotting (Fig. 3D), indicating the effectiveness and specificity of the FSE1 antibodies.

FSE1 Encodes a Ubiquitously Expressed Phospholipase-Like Protein Homologous to PA-PLA1

qRT-PCR analysis revealed that FSE1 was expressed in all the tested organs, including root, leaf, leaf sheath, stem, spikelet, and developing seed (Fig. 4A), with relatively higher abundance in developing seed, spikelet, and root. To better characterize the accumulation pattern of FSE1 protein during seed development, total seed protein was extracted from developing seeds for subsequent immunoblot analysis. The FSE1 antibodies recognized the band of 130 kD, which accumulated continuously as the seed developed (Fig. 4B). Unexpectedly, we found that FSE1 protein level in fse1 mutant seed was much lower than that in the wild type at 9 DAF (Fig. 4C), whereas FSE1 mRNA level did not significantly differ between the two genotypes (Fig. 4D). Therefore, the fse1 mutation might affect the stability of the FSE1 protein in the mutant.

Figure 4.

Figure 4.

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Spatial expression patterns, sequence and phylogenetic analyses of FSE1. A, Wild-type expression levels of FSE1 in various plant tissues and in the developing endosperm at 2, 6, 9, 12, 15, 18, 21, and 24 DAF. Actin1 was used as an internal control. Values are means ± sd from three biological replicates. The asterisks indicate statistically significant differences compared to root FSE1 expression level, as determined by a Student’s t test (*P < 0.05 and **P < 0.01). B and C, Western-blot analyses of FSE1 in wild-type and fse1 seeds (C), and during seed development (B). EF-1α antibodies were used as a loading control. D, FSE1 expression levels in wild-type and fse1 seeds 9 DAF. Values are means ± sd from three biological replicates. E, Schematic comparison of FSE1 (O. sativa), AtSGR2 (Arabidopsis), PA-PLA1 (Bos taurus), p125 (Homo sapiens), and KIAA0725p (H. sapiens). Various shaded boxes represent conserved domains defined on the right; aa, amino acids. F, Neighbor-joining tree of FSE1 and its homologs. The tree was constructed using MEGA5.0 and bootstrapped with 1000 replicates. The proteins are named according to their gene/EST names or NCBI accession numbers.

FSE1 was found to be a novel and unique gene in the rice genome (Singh et al., 2012). The FSE1 gene was predicted to encode a protein composed of 937 amino acids (Fig. 4E). Genes homologous to FSE1 were found in other eukaryotes (Fig. 4F), but not in prokaryotes. Therefore, FSE1 could belong to a eukaryote-specific protein family. The bovine testis PA-PLA1, which has the robust activity of phosphatidic acid-preferring phospholipase A1, was the first protein identified in this family (Higgs et al., 1998). Human p125 was shown to be a Sec23p-interacting protein (Tani et al., 1999). AtSGR2, the Arabidopsis ortholog of FSE1, is the only member of this protein family identified in plants to date, and its impaired function affects shoot gravitropism and causes shrunken seeds (Kato et al., 2002). Similarly, in fse1, we also observed inhibition of root growth (Supplemental Fig. S4, A–C) and enhanced root gravitropic response (Supplemental Fig. S4, D and E).

The FSE1 protein contains the DDHD domain that is known to be critical for PLA1 activity (Inoue et al., 2012), and the lipase motif (GXSXG) that is highly conserved in all members of the protein family except PA-PLA1 (SHSLG; Fig. 4E; Supplemental Fig. S5). In contrast to PA-PLA1 and KIAA0725p, the PLA1 activities of p125 and AtSGR2 have not been detected to date (Kato et al., 2002; Nakajima et al., 2002). FSE1 has a coiled-coil domain predicted by the COILS program (http://www.ch.embnet.org/software/COILS_form.html; Lupas et al., 1991). This structure was also found in PA-PLA1 and AtSGR2 (Fig. 4E). In addition, FSE1 was predicted to contain a transmembrane domain (https://embnet.vital-it.ch/software/TMPRED_form.html; Hofmann and Stoffel, 1993; Figure 4E). This region was also found in p125, an endoplasmic reticulum exit site-localized protein (Shimoi et al., 2005), and AtSGR2, which is localized to the vacuoles and small organelles (Morita et al., 2002). Therefore, we speculate that the members of the PA-PLA1 protein family might be membrane proteins.

FSE1 Is Localized in the Cytoplasm and Intracellular Membranes

To determine the localization of FSE1, we observed the fluorescence signal of FSE1-GFP in the protoplasts and root tip cells of fse1 complemented lines. The results showed that GFP fluorescence was mostly located in unknown small organelles that were obviously different from the prevacuolar compartment and vacuoles (Fig. 5, A and B). When proteins from FSE1-GFP transgenic rice leaves were fractionated into cytosolic and microsomal fractions, FSE1-GFP was detected in both the soluble and membrane-associated fractions (Fig. 5C). The microsomal proteins were further partitioned into the plasma membrane and intracellular membrane fractions. FSE1-GFP was detected in the intracellular membrane fractions, rather than the plasma membrane (Fig. 5C). Additionally, localization of FSE1 in the wild type is consistent with FSE1-GFP in the transgenic complemented lines (Fig. 5D). Taken together, the results showed that FSE1 is located in both cytosolic and intracellular membrane fractions.

Figure 5.

Figure 5.

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Subcellular Localization of FSE1. A and B, Confocal micrographs of protoplasts (A) and root tip cells (B) of complemented fse1 seedlings. VSR2, A prevacuolar compartment (PVC) marker. Bars = 5 μm. C and D, Immunoblot analysis of FSE1 following subcellular fractionation of 25 µg of soluble protein and membrane fractions of complemented fse1 seedlings (C) and the wild type (D). Cytosol, soluble fraction; MM, microsomal membrane fraction; PM, plasma membrane; IM, intracellular membrane.

Changes in Lipid Content and Composition in the fse1 Mutant

Since the homologous proteins PA-PLA1 and KIAA0725p showed robust phospholipase A1 activity (Higgs and Glomset, 1994; Nakajima et al., 2002), we examined the effect of FSE1 mutation on membrane lipids in rice. The total lipid extracts from wild-type and fse1 mutant seeds at 10 DAF were identified and quantified on the basis of head group and molecular mass through an automated electrospray ionization-tandem mass spectrometry (ESI-MS/MS) approach (Welti et al., 2002). The results showed that total glycerolipid content was comparable between the wild type and fse1 (complete data set in Supplemental Data Set 1). The amounts of MGDG and DGDG constitute ∼60% of total glycerolipids in developing rice seeds (Supplemental Data Set 1). Interestingly, the plastidic lipid (MGDG, DGDG, and PG) contents in the fse1 mutant were decreased, whereas the ER lipid (PC, PE, PI, and PS) and PA contents were increased compared to those in the wild type (Fig. 6A). It is noteworthy that the PA content was dramatically increased by about 3.5-fold of that in the wild type. However, the contents of lysophophatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylglycerol were comparable between the wild type and fse1. These results indicate that mutation of FSE1 changes the contents of membrane lipids in developing rice seeds.

Figure 6.

Figure 6.

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Changes in lipid contents and composition in fse1 mutant seeds. A and B, Changes in total lipids (A) and lipid molecular species (B) in the fse1 mutant seeds compared with those in the wild type. Lipids were extracted from the dehulled seeds at 10 DAF and analyzed by ESI-MS/MS. The value (peak intensity) of each lipid species was obtained by normalizing to the internal standards. Values are means ± sd from five biological replicates. Asterisks indicate statistically significant difference between the wild type and fse1 (*P < 0.05 and **P < 0.01; Student’s t test).

Furthermore, detailed lipid changes were obtained by lipid species profiling. The amounts of the MGDG species 36:3 and 36:4 and DGDG species 34:2, 36:3, and 36:4 were significantly decreased in the fse1 mutant, whereas the 36:5 and 36:6 MGDG, and 36:6 DGDG were increased compared to those in the wild type (Fig. 6B). These are the main components of amyloplast membranes in maize and rice endosperm (Myers et al., 2011). PG is present in both plastids and extraplastidic membranes. Major PG species in rice are 32:0, 34:1, and 34:2, and the decrease in total PG content in the fse1 mutant could be due to the reduced amounts of 34:1 and 34:2 species. Major PC species in rice are 34:1, 34:2, 34:3, 36:2, 36:3, and 36:4. PA, PI, and PE had similar patterns to PC with respect to major species, and the highest PS species were 34:2 and 42:2 (Supplemental Data Set 1). The pattern of major species in each extraplastidic lipid was consistent with that reported in a previous study (Wang et al., 2012). In the fse1 mutant, the increased amounts of these major species in PC, PE, PS, PI, and PA resulted in a higher level of each lipid compared to those in the wild type (Fig. 6B; Supplemental Data Set 1). Taken together, these results indicated that mutation of FSE1 alters lipid content and composition.

Mutation of FSE1 Affected Starch Biosynthesis during Endosperm Development

A previous study showed that the change in membrane lipid composition affected starch synthesis (Myers et al., 2011). We therefore used qRT-PCR analysis to examine the expression profiles of 14 starch synthesis-related genes, namely, those that were preferentially expressed in developing endosperm (Fu and Xue, 2010), in the fse1 mutant and the wild type from 6 to 15 DAF during the grain filling stage. The results indicated that expression levels of these genes were significantly decreased at the early developing stage of endosperm before 10 DAF, including those genes encoding AGPases (AGPS1, AGPS2b, AGPL1, and AGPL2), soluble SSs (SSI, SSIIa, SSIIIa, and SSIVb), GBSSI, BEI, BEIIb, isoamylases (ISA1 and ISA2), and pullulanase (PUL; Fig. 7). Furthermore, immunoblot analysis of total proteins in fse1 seeds 10 DAF showed that the protein levels of AGPL2, ISA1, ISA2, GBSSI, and SSIIa were obviously decreased, and the accumulation of BEI was slightly decreased, compared with that in the wild type. The amount of other proteins, such as AGPS2b, FLO4, Pho1, and BEIIb, was largely comparable between the wild type and fse1 (Supplemental Fig. S6A). In addition, the fse1 mutant endosperm retained only about 44% and 78% of the catalytic activities of the wild-type endosperm AGPase and Suc synthase, respectively (Supplemental Fig. S6B).

Figure 7.

Figure 7.

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Expression profiles of rice starch synthesis-related genes during seed development in the wild type and the fse1 mutant. Total RNA was extracted from seeds at 6, 10, and 15 DAF. Actin1 was used as an internal control. Values are means ± sd from three biological replicates. Asterisks indicate statistically significant difference between the wild type and fse1 (*P < 0.05 and **P < 0.01; Student’s t test).

Generally, defects in starch biosynthesis will result in higher amounts of soluble sugars (Bläsing et al., 2005; Tang et al., 2016). Thus, less starch accumulation in fse1 seeds might cause elevated sugar contents. To confirm this speculation, we measured the sugar contents with a Glc, Fru, and Suc assay kit. The results showed the fse1 mutant had a significantly increased level of soluble sugars, including Suc, Glc, and Fru at the early stage of seed development (Supplemental Fig. S6C). Taken together, these results show that the fse1 mutation affects the expression of most starch synthesis-related genes in rice seeds during the grain-filling stage and subsequently affects protein accumulation and corresponding enzymatic activities. These alterations ultimately lead to compromised starch biosynthesis.

DISCUSSION

FSE1 Is Important for Starch Synthesis and Amyloplast Development

In this study, we isolated and characterized the fse1 mutant, which has a floury and shrunken endosperm phenotype. Compared with that in the wild type, the fse1 mutant displayed decreased grain weight and starch content, and altered pasting properties and structure of amylopectin. Semithin sections of seeds 12 DAF showed that the fse1 mutant endosperm contained smaller, scattered, weakly iodine-stained starch granules (Fig. 2A, vi), whereas the wild type produced densely packed, polyhedral starch granules (Fig. 2A, iii). These results indicate that FSE1 plays a vital role in regulating starch synthesis and compound granule formation in rice endosperm.

Expression of many starch synthesis-related genes was reduced in the fse1 mutant (Fig. 7), which may affect starch metabolism and starch-related phenotypes during endosperm development. Phenotypes similar to those of the fse1 mutant are found in several mutants involved in starch biosynthesis, but many differences are evident in their features. The amylopectin composition of fse1 mutant seeds was similar to that of the flo7 and flo5/ss3a mutants, although flo7 exhibited a floury white endosperm only in the periphery and flo5/ss3a had a white-core floury endosperm (Ryoo et al., 2007; Fujita et al., 2011; Zhang et al., 2016). It has been shown that AGPase activity strongly affects the yield of grain starch (Kawagoe et al., 2005; Tang et al., 2016). Amylose is mainly synthesized by the activity of GBSSI/Wx (Sano et al., 1985), suggesting that a decrease in amylose content could be attributed to the reduced expression of GBSSI in fse1. Branching enzymes, debranching enzymes, and SSs cooperatively determine the fine structure of amylopectin in a highly complex manner. Mutants with compromised function of BEI, BEIIb, SSI, SSIIa, and ISA have major changes in amylopectin composition, but display amylopectin structure that differs greatly from that of fse1 (Kubo et al., 1999; Nishi et al., 2001; Satoh et al., 2003; Nakamura et al., 2005; Fujita et al., 2006). Surprisingly, the expression pattern of starch synthesis genes observed in fse1 was also found in RSR1-overexpressing lines, and the fse1 amylopectin structure is very similar to that of RSR1-overexpressing lines, which produced smaller seeds but did not have a floury appearance (Fu and Xue, 2010). Thus, the starch phenotype in fse1 may be ascribed to the combined effects of altered expression of multiple rice starch synthesis-associated genes.

FSE1 Encodes a Phospholipase-Like Protein Homologous to PA-PLA1

FSE1 encodes a phospholipase-like protein that is homologous to PA-PLA1. Genes that are homologous to PA-PLA1 are found widely throughout eukaryotes and form a gene family (Fig. 4F). Members of this gene family have mainly been studied in mammals (Higgs and Glomset, 1994; Tani et al., 1999; Nakajima et al., 2002; Shimoi et al., 2005; Sato et al., 2010). To date, only FSE1 in this study and AtSGR2 (Kato et al., 2002) have been identified in plants. PA-PLA1 and KIAA0725p were shown to have PLA1 activity toward PA and several other phospholipids, whereas the PLA1 activity of p125 and AtSGR2 has not been detected to date (Higgs and Glomset, 1994; Kato et al., 2002; Nakajima et al., 2002). In this study, we could not detect clear PLA1 activity in vitro with the purified recombinant MBP-FSE1 protein expressed in E. coli BL21 cells (Supplemental Fig. S7). This result might be attributed to aberrant protein folding or protein modification in the prokaryotic system or because of the assay conditions. A previous study demonstrated that PLA1 activity of KIAA0725p on PE depended on whether Triton X-100 was present (Nakajima et al., 2002). Another possibility is that FSE1 has no PLA1 activity at all. However, lipid profiling of 10-DAF seeds clearly demonstrated that the lipid contents of PC, PI, PS, PE, and PA (about 3.5-fold of that in the wild type) were increased in the fse1 mutant compared to those in the wild type (Fig. 6A). These results strongly imply that FSE1, similar to PA-PLA1 and KIAA0725p, may exhibit in vivo PLA1 activity on PC, PI, PS, PE, and, in particular, PA. Measurement of the enzyme activity of FSE1 in vitro is under way to examine this intriguing possibility.

Unique Effects of the fse1 Mutation on Endosperm Lipids

There were two major phenotypes in the fse1 mutant, i.e. compositional change of lipids and abnormality of starch synthesis. From the function of causative genes, compositional changes of lipids could be the first phenotype. In the developing endosperm of the fse1 mutant, the total membrane glycerolipids were comparable with that in the wild type, whereas the extraplastidic lipids were increased and the total plastidic lipids were decreased. Lipids act not only as the matrix of cellular membranes, but also as first and second messengers in signal transduction. For instance, PIs and PA have been regarded as lipid signaling molecules in plant cells (Zhang et al., 2014; Gerth et al., 2017). In Arabidopsis, PA is found to bind a MYB transcription factor and regulate its nuclear localization, which affects gene expression and root hair development (Yao et al., 2013). Mutation of the FSE1 gene did affect PIs and PA content, as well as gene expression patterns for starch synthesis in rice seeds (Fig. 7). However, whether the lipid changes directly or indirectly alter gene expression needs to be investigated in future work.

Changes in the composition of lipids in the fse1 mutant may cause abnormalities in seed development, which consequently causes abnormalities in starch synthesis. Lipid effects on starch synthesis have already been found in maize. Mutation of the opaque5 (o5) locus coding for a MGDG synthase causes decreases in both MGDG and DGDG, leading to abnormalities in endosperm development and starch production (Myers et al., 2011). These results reveal a connection between membrane structure and storage compound metabolism in nonphotosynthetic plastids. To some extent, this may be true for rice, a monocot like maize. Total DGDG content was significantly decreased in the fse1 mutant compared to that in wild type, whereas the total MGDG tended to decrease. Lipid species profiling demonstrated that the predominant GDG species were 36:4 MGDG and 36:4 DGDG, which were identical to those in maize endosperm (Myers et al., 2011; Fig. 6). The reduced amount of species 34:4 together with 36:2 and 36:3 GDGs contributed to the decreased total content of MGDG and DGDG in the fse1 mutant. Little is known about initiation of starch granules, particularly in endosperm; however, the thylakoid-like membrane within the amyloplast has been proposed as a functional aspect of starch polymer synthesis, and galactolipids have an important function in amyloplasts (Myers et al., 2011; Bahaji et al., 2014). Our results partially support this hypothesis because the alteration in galactolipids is tightly related to the development in endosperm and starch granules.

Finally, one possibility to consider is that lipid composition indirectly affects starch granule formation by the regulation of transporter activity for metabolite (e.g. Suc) transport. In cereal endosperm cells, brittle1 (BT1) proteins facilitate the transfer of cytosolic ADPG into the amyloplast for starch biosynthesis and are rate-limiting factors in this process (Bahaji et al., 2014). Although it is unclear whether and how lipids regulate proteins like BT, lipid regulation of transporter activity has been reported in animal cells (Habeck et al., 2017). Recently, PA has been determined to bind several proteins involved in galactolipid metabolism and transfer in Arabidopsis, including ABC lipid transporter and DGD1 (Kelly et al., 2016). Thus, the lipid-mediated metabolite transport could be an important pathway to decipher the mechanisms of starch accumulation in seeds.

In summary, we report a vital role of FSE1 involved in glycerolipid metabolism and amyloplast development. We demonstrate a functional connection between starch synthesis and membrane lipid synthesis in amyloplasts and will conduct more in-depth studies to elucidate the precise molecular mechanism underlying this connection.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The fse1 mutant was generated by N-methyl-N-nitrosourea treatment of cv Dianjingyou 1 (Oryza sativa, japonica). An F2 population was produced from a cross between the fse1 mutant and cv N22 (O. sativa, indica) for mapping. All plants were grown in a paddy field at Nanjing Agricultural University (118°46’E, 32°03’N) during the natural rice growing season. The developing seeds at 2 to 24DAF from three different plants of wild-type and fse1 mutant were used in biochemical and electron microscopy studies. The developing seeds at 6, 9, 12, 15, 18, 21, 24, 27, 30, and 33 DAF were harvested with three replicates for measuring grain filling; samples were treated at 105°C for 30 min, then transferred to 65°C and dried to a set weight. After dehulling, the weight of 100 seeds per sample was determined and converted to weight per seed.

Microscopy

Scanning electron microscopy was performed as described previously (Kang et al., 2005). Samples were examined with a Hitachi S-3400N scanning electron microscope. The procedure used for transmission electron microscopy followed Zhang et al. (2016). Samples were examined with a Hitachi H-7650 transmission electron microscope. Semithin sections were prepared according to a previously described process (Peng et al., 2014). Samples were examined under a Nikon ECLIPSE80i light microscope.

Determination of Seed Metabolites

Rice grains harvested from three different mature plants were processed using a dehuller and ground into fine flour with a miller. Amylose, starch, lipid, and protein contents as well as the chain length distributions of amylopectin were determined according to a previous report (Han et al., 2012). The pasting properties of endosperm starch were measured as described previously (Peng et al., 2014). The developing seeds at 6 to 24DAF from five different plants of the wild type and fse1 mutant were treated at 105°C for 30 min, then transferred to 65°C and dried to a set weight, and processed using a dehuller and ground into fine flour with a miller. Suc, Glc, and Fru contents were measured with a Glc, Fru, and saccharide assay kit (BioSenTec).

Enzyme Assays

One hundred milligrams of 10-DAF developing endosperm of each sample from one plant was homogenized on ice in 1 mL of 50 mm HEPES NaOH (pH 7.4), 2 mm MgCl2, and 12.5% (v/v) glycerol. The homogenate was centrifuged at 20,000g for 10 min at 4°C, and the resulting supernatant was used for all enzyme assays. AGPase activities were assayed in the pyrophosphorylase direction as described previously (Nishi et al., 2001). Suc synthase activities were measured according to methods in a previous report (Doehlert et al., 1988).

Map-Based Cloning of the FSE1 Gene

To map the FSE1 locus, a total 155 floury shrunken seeds from the F2 population were used. Both parents and 10 F2 recessive seeds were used for preliminary mapping based on more than 160 genome-wide polymorphic simple sequence repeats markers. To fine map the fse1 locus, new genetic markers were designed by comparing the sequences of 93-11 and Nipponbare (Supplemental Table S1).

Vector Construction and Rice Transformation

For complementation of the fse1 locus, the wild-type FSE1 cDNA sequence was cloned into the binary vector pCUbi1390 under the control of the maize ubiquitin promoter to generate the transformation cassette pUbi:FSE1. pd35S:FSE1-GFP was generated by inserting the wild-type FSE1 cDNA downstream of the double 35S promoter of Cauliflower mosaic virus and translationally fusing it with the GFP tag at its C terminus into vector pCAMBIA1305.1 (Supplemental Table S2). These plasmids were introduced into Agrobacterium tumefaciens strain EHA105, which was then used to transfect the fse1 mutant calli (Hiei et al., 1994). Hygromycin-resistant calli were regenerated and seedlings were grown in a greenhouse.

Sequence and Phylogenetic Analysis

The amino acid sequences of FSE1 homologous proteins were aligned using ClustalX (http://www.clustal.org). A neighbor-joining tree was constructed using MEGA 5.0 (http://www.megasoftware.net) by the bootstrap method with 1000 replicates (Tamura et al., 2011).

RNA Extraction and qRT-PCR Analysis

Total RNA was prepared from tissues (root, stem, leaf sheath, flag leaf, panicle, and developing seeds) of three different plants using an RNAprep pure Plant kit (Tiangen Biotech). A 1-μg portion of total RNA was reverse transcribed by priming with oligo(dT)18 in a 20-μL reaction using the PrimeScript Reverse Transcriptase kit (TaKaRa). The value of Actin1 mRNA was used as an internal control. Primers for Actin1 and 14 starch synthesis-related genes were newly designed based on Yang et al. (2012b) and Ohdan et al. (2005), respectively. The FSE1 gene was amplified with primers R1 (5′-ATGGAAAGGCTGACAGGTTC-3′) and R2 (5′-GAGATCCCAGGGCAGATA AG-3′).

Protein Extraction and Immunoblot Analysis

Total protein extraction and immunoblot assays were performed as described previously (Wang et al., 2010, 2016). Recombinant protein of FSE1 (480–680 amino acids) was bacterially produced in pET-32a and purified. The synthetic peptides of AGPS2b, AGPL2, FLO4, Pho1, SBEI, SBEIIb, SSIIa, ISA1, ISA2, GBSSI, or recombinant protein of FSE1 were injected into rabbits or mice to generate corresponding polyclonal antibodies at Yingji Biotech (http://www.immunogen.com.cn/). The anti-EF-1α antibodies and anti-GFP antibodies were purchased from Agrisera and Abmart, respectively.

Subcellular Localization and Fractionation

Rice protoplasts were extracted from FSE1-GFP transgenic complemented seedlings (Chen et al., 2006). GFP fluorescent signals were detected using a confocal laser scanning microscope (Zeiss LSM780). FSE1-GFP transgenic complemented seedlings and wild-type endosperm (10 DAF) were used for fractionation. Proteins were extracted using buffer A (100 mm HEPES-KOH, pH 7.5, 0.3 m Suc, 5 mm EGTA, 5 mm MgCl2, and protease inhibitor [complete cocktail tablets; Roche]), followed by centrifugation at 6,000g for 10 min. The supernatant was centrifuged at 100,000g for 60 min. The resultant supernatant is referred to as the soluble cytosol fraction, and the pellet is referred to as the microsomal fraction. Using two-phase partitioning as described previously (Fan et al., 1999; Hong et al., 2009), the microsomal fraction was separated further into plasma membrane and intracellular membrane fractions.

Lipid Extraction and Mass Spectrometry Analysis

Lipid extraction was performed as described previously with minor modifications (Welti et al., 2002). Briefly, 10-DAF seeds of the wild type and fse1 mutant were husked in liquid nitrogen. Fifteen grains of each sample from one plant were transferred into 3 mL of isopropanol with 0.01% butylated hydroxytoluene and crushed with a glass bar at 75°C. After 15 min, 1.5 mL of chloroform and 0.6 mL of water were added. The tubes were shaken for 1 h, followed by removal of the extract. The pellets were reextracted three times with 2:1 (v/v) chloroform/methanol with 0.01% butylated hydroxytoluene with 2 h of agitation each time. The combined extracts were washed once with 1 mL of 1 m KCl and once with 2 mL of water. The solvent was evaporated under nitrogen gas, and the lipid extract was finally dissolved in chloroform for the assay. At this time, equivalent amounts of seeds were heated overnight at 105°C and weighed. The weights of the dried seeds were described as their “dry weight.” Lipid samples were analyzed by ESI-MS/MS at the KS Lipidomics Research Center. Five replicates each of the wild type and fse1 mutant were analyzed.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers: FSE1 (Os08g0110700), AtSGR2 (At1g31480), FLO4 (Os05g0405000), PHO (Os03g0758100), AGPS1 (Os09g0298200), AGPS2b (Os08g0345800), AGPL1 (Os05g0580000), AGPL2 (Os01g0633100), SSI (Os06g0160700), SSIIa (Os06g0229800), SSIIIa (Os08g0191433), SSIVb (Os05g0533600), GBSSI (Os06g0133000), BEI (Os06g0726400), BEIIb (Os02g0528200), ISA1 (AB093426), ISA2 (AC132483), and PUL (AB012915). The accession numbers for proteins in the phylogenetic analysis are FSE1, XP_015650828.1; AtSGR2, NP_174433.2; p125, NP_009121.1; KIAA0725p, NP_056029.2; and PA-PLA1, NP_788816.1.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

This work was supported by the Key Laboratory of Biology, Genetics, and Breeding of Japonica Rice in Mid-lower Yangtze River, Ministry of Agriculture, P.R. China, and the Jiangsu Collaborative Innovation Center for Modern Crop Production. We thank Prof. Hongwei Xue and Dr. Hongyan Yao for their kind help in measuring glycerolipid contents. We also thank Kansas Lipodomics Research Center, Kansas State University, for the lipid analyses.

Footnotes

1

This work was supported by grants from The National Key R&D Program of China (2016YFD0100500), The National Natural Science Foundation of China (31330054), Jiangsu Science and Technology Development Program (BE2017368), Agricultural Science and Technology Innovation Fund project of Jiangsu Province [CX(16)1029], and the Fundamental Research Funds for the Central Universities (KYTZ201601).

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