Comparisons of Metabolic Profiles for Carbohydrates, Amino Acids, Lipids, Fragrance and Flavones During Grain Development in indica Rice Cultivars

Chen Yibo, Wang Zhidong,Wang Chongrong, Li Hong, Huang Daoqiang, Zhou Degui, Zhao Lei, Pan Yangyang, Gong Rong, Zhou Shaochuan

Research Paper

Comparisons of Metabolic Profiles for Carbohydrates, Amino Acids, Lipids, Fragrance and Flavones During Grain Development inRice Cultivars

Chen Yibo, Wang Zhidong,Wang Chongrong, Li Hong, Huang Daoqiang, Zhou Degui, Zhao Lei, Pan Yangyang, Gong Rong, Zhou Shaochuan

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We used a widely targeted metabolomics approach to examine the metabolic changes in three pedigreedcultivars (Meixiangzhan 2, Meisizhan and Qixinzhan), with different eating qualities, at 8, 15 and 30 d after flowering (DAF) to explore the formation mechanism of rice eating and nutritional qualities at a global metabolic level. A total of 623 metabolites were identified, and results showed that metabolic variations among rice cultivars decreased with grain developmental stage, suggesting that sufficient carbohydrate and amino acid supply during grain development may contribute to excellent rice eating and nutritional quality formation. Lysophosphatidylcholines 19:0 and 16:1 were beneficial for excellent eating quality formation during grain development. Rice fragrance was attributed mainly to spermidine and γ-aminobutyric acid. Rice cultivars with excellent eating quality at 15–30 DAF had relatively higher flavone content, possibly because they had adequate carbohydrate and amino acid contents during grain development. These results provided insight into the mechanisms for establishing rice eating and nutritional qualities during grain formation at a global metabolic level, and can be applied towards improving rice quality.

eating quality; metabolomics; nutritional quality; high-quality rice; grain development

Rice has been cultivated and consumed by humans for nearly 5 000 years. At present, rice feeds almost half of the global human population (Liu et al, 2013). As standards of living improve, rice eating and nutritional qualities have become top priorities for breeders and consumers (Hori, 2018). Various rice cultivars are also used for medical, ceremonial or other special purposes (Tian et al, 2009).

Rice eating and nutritional qualities are closely related to amylose, amylopectin, protein and lipid compositions and contents of the grains. These metabolites are synthesized and genetically regulated during rice grain development. The three major stages of rice grain development are embryonic development, cell division and morphogenesis; grain maturation (when large amounts of storage reserves accumulate); and grain drying and dormancy (Deng et al, 2013; Sreenivasulu et al, 2015). All the three stages are associated with substantial spatiotemporal metabolic rearrangements regulated by global gene expression programs. Prior research mainly focuses on the mechanisms by which genes control rice quality formation. Zhao et al (2018) found thatis a transcriptional activator regulating rice grain shape and appearance. Natural variation incontrols rice grain protein content (Yang Y H et al, 2019). Wang et al (2015) reported that the lipid transfer protein OsLTPL36 is vital to rice grain development and quality. However, few studies have explored the mechanisms of rice eating and nutritional quality formations at a global metabolic level.

Rice grains are rich in a large number of primary and secondary metabolites. Primary metabolites are conducive to the synthesis of yield- and quality-related macromolecules, such as starch. Moreover, some secondary metabolites have nutritional functions. For example, the outer covering of a rice grain contains a unique profile of phytochemicals with medicinal and nutritional properties that are beneficial to human health. Some of these secondary metabolites have been targeted for nutraceutical development for managing cancer (Verschoyle et al, 2007; Henderson et al, 2012). However, few studies have been conducted on how metabolites affect the eating and nutritional qualities of rice in the process of grain formation.

In this study, we used widely targeted metabolomics, which is efficient at a large scale and complements traditional and genomic approaches (Chen et al, 2016; Zhu et al, 2018; Gang et al, 2019; Sulpice, 2019), to elucidate the metabolic changes that occur during grain development in three pedigreedcultivars with different eating qualities. We investigated the formation mechanism of rice eating and nutritional qualities by analyzing the changes in the metabolic levels of carbohydrates, amino acids and their derivatives, lipids, fragrance and flavones. This study provided insight into the formation mechanism of rice eating and nutritional qualities at a global metabolic level, and can be applied as an important reference towards improving rice quality.

RESULTS

Eating qualities of three indica cultivars

To investigate the changes in metabolites during rice grain development, we selected three pedigreedcultivars with different eating qualities, namely, Meixiangzhan 2 (MXZ), Meisizhan (MSZ) and Qixinzhan (QXZ) (Figs. 1-A and S1). The eating quality values for MXZ, MSZ and QXZ were 88.0, 84.2 and 64.7, respectively. MXZ is the most widely cultivated conventional rice in Guangdong Province, China, and QXZ is a negative control (Fig. 1-A).

Fig. 1. Eating quality values and metabolic profiles of three rice samples.

A, Eating quality values of MXZ, MSZ and QXZ.

B, Metabolite classes and numbers detected in samples.

C, Principal component analysis of metabolomes during grain development.

D, Venn diagram of results of two-way analysis of variance.

MXZ, Meixiangzhan 2; MSZ, Meisizhan; QXZ, Qixinzhan; DAF, Days after flowering.

Metabolic profiles of all rice samples

To investigate the changes in metabolites during rice grain development, we collected grain samples at 8, 15 and 30 d after flowering (DAF) and subjected them to metabolic profiling analysis using a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Totally, 623 metabolites were identified, including 95 amino acids and their derivatives, 85 organic acids and their derivatives, 67 lipids and 62 flavones (Fig. 1-B and Table S1).

Principal component analysis (PCA) was performed on these 623 metabolites to visualize kinetic metabolome patterns in the developing rice grains. All the three cultivars presented with similar dynamic patterns in the changes of their developing rice grain metabolomes (Fig. 1-C). During grain filling at 8 DAF, the metabolite patterns were distinct and widely separated among cultivars. During grain desiccation, the 15 and 30 DAF samples were virtually indistinguishable. Hence, metabolic variation among cultivars decreased with advancing grain development.

As PCA cannot resolve the exact contribution of each variable, two-way analysis of variance (ANOVA) and ANOVA-simultaneous component analysis (ASCA) were performed to deconstruct the contributions of stage, cultivar and their interaction to the metabolic variations. The abundances of 371, 531 and 254 metabolites were significantly influenced by stage, cultivar and their interaction, respectively (Figs. 1-D, S2 and S3). The abundances of 187 metabolites were simultaneously affected by stage, cultivar and their interaction. ASCA revealed that 66.21%, 89.25% and 46.43% of the observed metabolic variations were explained by developmental stage, cultivar and their interaction, respectively (Figs. S2 and S3). Developmental stage score plots based on submodel PC1 showed that the scores decreased from 8 to 30 DAF. This finding was consistent with the PCA results shown in Fig. 1. Over time, the metabolomes of the three cultivars shifted in the same direction, therefore, the metabolic variation among cultivars was distinct and wide at 8 DAF, but relatively narrower from 15?30 DAF. Moreover, the metabolites were affected by developmental stage and cultivar.

Carbohydrate metabolism during rice grain development

Starch is a photosynthetic end product stored in source tissues, and it is also an energy reserve in the rice grain and consists of amylose and amylopectin (Wang et al, 2013). We identified six of the eight metabolites involved in starch biosynthesis (Fig. 2-A and Table S2). The levels of the six carbohydrates were significantly higher in MXZ than in the other cultivars at 8 DAF (Fig. 2-A). Sucrose and uridine diphosphate glucose (UDPG) levels were significantly decreased in all the cultivars between 8 and 15 DAF (grain-filling). In both QXZ and MSZ, glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P) and glucose-1-phosphate (G-1-P) initially increased and then decreased between 8 and 30 DAF. In contrast, the levels of all the three carbohydrates were significantly higher in MXZ than in the other cultivars at 30 DAF. Hence, the sufficient foliar carbon supply for starch biosynthesis during grain development in MXZ may have contributed to the excellent eating quality of this cultivar.

Fig. 2.Changes of carbohydrate levels inthree rice cultivars at 8, 15 and 30 d after flowering (DAF).

A, Changes ofmetabolites mapped to starch biosynthesis pathway in three rice cultivars at 8, 15 and 30 DAF.

B, Heat map of carbohydrate metabolites that significantly changed in rice grains among three rice cultivars at 8, 15 and 30 DAF. Fold change ratios are indicated by red or blue shading according to the scale bar. Data are means of three biological replicates per cultivar and time point.Full metabolite names are listed in Tables S1 and S2.

MXZ, Meixiangzhan 2; MSZ, Meisizhan; QXZ, Qixinzhan; UPDG, Uridine diphosphate glucose; ADPG, Adenosine diphosphate glucose; G-6-P, Glucose-6-phosphate; F-6-P, Fructose-6-phosphate; G-1-P, Glucose-1-phosphate.

We compared other eight significantly differentially expressed carbohydrates during grain development of the three cultivars (Fig. 2-B and Table S2). The levels of all eight carbohydrates were significantly higher in MXZ than in QXZ at 8 DAF. However, only trehalose-6-phosphate significantly differed between these cultivars at 15 DAF (Table S2). The levels of-(?)-threose, ribulose-5-phosphate and-F-6-P were significantly higher in MXZ than in QXZ at 30 DAF (Table S2). Therefore, MXZ and QXZ significantly differed in terms of carbohydrate metabolite levels. There was no significant difference in carbohydrate metabolism between MSZ and QXZ from 8 to 15 DAF. At 30 DAF, however, these cultivars significantly differed in terms of-(+)-melezitose--rhamnoside. Thus, the superior eating quality of MXZ might be explained by the significant advantages that it had over the other cultivars in terms of starch synthesis and carbohydrate metabolism.

Amino acid and derivative metabolism during rice grain development

Amino acids are primarily utilized in the synthesis of grain storage proteins (Amir et al, 2018). Proteins influence rice grain eating and nutritional qualities and are precursors for secondary metabolite biosynthesis and energy sources (Amir et al, 2018). We identified 95 amino acids and their derivatives. They were the most abundant components in the metabolic profiles (Figs. 1-B and 3). We compared 66 significantly different amino acids and their derivatives among the three cultivars during grain development (Fig. 3 and Table S3).

There were significantly higher levels of various amino acids in MXZ than in QXZ during grain development. At 8 DAF, there were 16 significantly different amino acids between MXZ and QXZ. Of these, 14 were significantly upregulated in MXZ compared with QXZ. The level of glutamine, which is a main raw material in protein synthesis, was 3.7-fold higher in MXZ than in QXZ. At 15 DAF, there were 26 significantly different amino acids between MXZ and QXZ. Of these, 23 were significantly upregulated in MXZ relative to QXZ. At 30 DAF, there were 16 significantly different amino acids between MXZ and QXZ. Of these, 15 were significantly upregulated in MXZ compared with QXZ. During grain development, however, few amino acids significantly differed between MSZ and QXZ. At 8, 15 and 30 DAF, only nine metabolites differed between the two cultivars. Therefore, compared with QXZ and MSZ, MXZ had the most abundance in amino acids, especially at 15 DAF during grain development.

Fig. 3. Heat map of changes in amino acids and their derivativesin rice grains at 8, 15 and 30 d after flowering (DAF) among three cultivars.

Fold change ratios are indicated by red or blue shading according to the scale bar. Data are means of three biological replicates per cultivar and time point. Full metabolite names are listed in Tables S1 and S3.

MXZ, Meixiangzhan 2; MSZ, Meisizhan; QXZ, Qixinzhan.

We also investigated eight different essential amino acids in the three cultivars (Fig. S4). The levels of all these essential amino acids, except-threonine and-tryptophan, exhibited the same downward trend. Between 8 and 30 DAF, amino acid levels were always higher in MXZ than in the other two cultivars.

Lipid metabolism during grain development

Lipids maintain the structure of grain storage substances and contribute to rice eating quality and texture. However, they comprise a far smaller proportion of the total rice grain nutrient profile in comparison to starch and protein (Wang et al, 2015). We identified 66 lipids, of which, 23 (34.8%) were lysophospha- tidylcholines (LysoPCs), 8 (12.1%) were lysophospha- tidylethanolamines, 9 (13.6%) were monoacylglycerols (MAGs) and 26 (39.4%) were unnamed (Table S1). We compared 38 significantly different lipids among the three cultivars during grain development (Table S1).

The number of significantly different lipids between MXZ and QXZ decreased with grain development (Fig. 4 and Table S4). At 8 DAF, there were 15 significantly different lipids between them. Of these, 11 (mainly MAGs) were significantly upregulated in MXZ compared with QXZ. At 15 DAF, there were eight significantly different lipids between MXZ and QXZ, but only LysoPC 19:0 was significantly upregulated in MXZ relative to QXZ. At 30 DAF, there were five significantly different lipids between MXZ and QXZ, of which, four LysoPCs were significantly upregulated in MXZ compared with QXZ. The numbers of significantly different lipids were similar between MSZ and QXZ (Fig. 4 and Table S4). At 8 DAF, there were 22 significantly different lipids between QXZ and MSZ, of which, 19 (mainly LysoPCs) were significantly upregulated in MSZ relative to QXZ. At 15 and 30 DAF, there were seven and five significantly different lipids, respectively, between MSZ and QXZ. All of them were LysoPCs and were significantly upregulated in MSZ compared with QXZ. At 30 DAF, the levels of LysoPC 19:0 and LysoPC 16:1 were significantly higher in MXZ and MSZ than in QXZ. These results suggested that LysoPCs, particularly LysoPC 19:0 and LysoPC 16:1, are beneficial to the formation of excellent eating quality during rice grain development.

Fig. 4. Heat map of lipid metabolite changes in rice grains among three cultivars at 8, 15 and 30 d after flowering (DAF).

Fold change ratios are indicated by red or blue shading according to the scale bar. Data are means of three biological replicates per cultivar and time point. Full metabolite names are listed in Tables S1 and S4.

MXZ, Meixiangzhan 2; MSZ, Meisizhan; QXZ, Qixinzhan; LysoPC, Lysophosphatidylcholine; LysoPE, Lysophosphatidylethanolamine; MAG, Monoacylglycerol; 9-HOTrE, 9-hydroxy-(6Z,9Z,11E)-octadecatrienoic acid; 13-HPODE, 13-hydroperoxyoctadecadienoic acid; 9-HOA, 9- hydroxy-(10E,12Z,15Z)-octadecatrienoic acid; 12,13-EODE, 12,13-E- octadecadienoic acid; MGDG,Monogalatosyl diglyceride.

Changes in fragrance metabolism during grain development

An important fragrance constituent contributing to rice eating quality is 2-acetyl-1-pyrroline (2-AP) (Chen et al, 2008). Here, we identified nine metabolites in the 2-AP biosynthesis pathway (Pan et al, 2021) (Fig. 5-A). Only MXZ had fragrance and it also had the best eating quality. In contrast, neither MSZ nor QXZ displayed any fragrance. We analyzed nine substances participating in fragrance metabolism. For all the three cultivars, the levels of-glutamic acid, succinic acid,-proline, putrescine,-arginine and spermine all significantly declined with grain development (Fig. 5-B). The change patterns for α-ketoglutaric acid were the same for MXZ and QXZ (Fig. 5-B). Therefore, the seven aforementioned substances might not, in fact, be the key determinants of fragrance formation. Fragrant (MXZ) and non-fragrant (MSZ and QXZ) rice presented opposite change trends in spermidine and γ-aminobutyric acid (GABA) contents during grain development, suggesting that rice grain fragrance is attributed mainly to spermidine and GABA.

Fig. 5. Changes in levels of metabolites mapped to 2-AP biosynthesis pathway in three rice cultivars at 8, 15 and 30 d after flowering (DAF).

A, 2-AP biosynthesis pathway and its regulation in plants.

B, Relative changes in metabolites mapped to 2-AP biosynthesis pathway during grain development.

MXZ, Meixiangzhan 2; MSZ, Meisizhan; QXZ, Qixinzhan; 2-AP, 2-acetyl-1-pyrroline; BADH2, Betaine aldehyde dehydrogenase 2; DAO, Diamine oxidase; GABA, γ-aminobutyric acid; GAD, Glutamate decarboxylase; PAO, Polyamine oxidase; SpmS, Spermine synthase; TCA, Tricarboxylic acid cycle.

Flavone metabolites during grain development

We identified numerous secondary metabolites such as flavones that have an impact on human health. Flavones are nutraceuticals and radical scavengers and have other pharmacological properties (Tapas et al, 2008). We identified 62 flavones (Fig. 1-B) and compared 57 of them that were significantly different among the three cultivars during grain development (Fig. 6 and Table S5).

There were significantly higher levels of various flavones in MXZ than in QXZ at 15 and 30 DAF. At 8 DAF, there were 18 significantly different flavones between MXZ and QXZ. However, only luteolin--hexosyl--hexosyl--hexoside was significantly upregulated in MXZ relative to QXZ. At 15 DAF, there were 23 significantly different flavones (mainly tricin and its derivatives) between MXZ and QXZ. All of them were significantly upregulated in MXZ compared with QXZ. At 30 DAF, there were six significantly different flavones between MXZ and QXZ, of which four were significantly upregulated in MXZ relative to QXZ.

The numbers of flavones also significantly differed between MSZ and QXZ during grain development. At 8 DAF, there were 30 significantly different flavones, of which, only luteolin--hexosyl--hexosyl--hexosideand apigenin-5--glucoside were significantly upregulated in MSZ compared with QXZ. At 15 DAF, there were only 15 significantly different flavones between MSZ and QXZ, of which, 11 were significantly upregulated in MSZ relative to QXZ. At 30 DAF, there were 17 significantly different flavones between MSZ and QXZ, of which, 12 were significantly upregulated in MSZ compared with QXZ. At 30 DAF, the levels of luteolin 3′,7-di--glucoside, chrysoeriol--hexosyl-- rhamnoside, chrysoeriol-7--rutinoside and tricin-5--rutinoside were significantly higher in MXZ and MSZ than in QXZ. Therefore, compared with QXZ, similar changes occurred in the significantly different flavones of MXZ and MSZ during grain development. At 8 DAF, the levels of various flavones were significantly lower in MXZ and QXZ than in QXZ. However, the opposite was true at 15 and 30 DAF. Thus, rice cultivars with excellent eating quality might have relatively higher flavone content.

Fig. 6. Heat map of flavone changes in rice grains among three cultivars at 8, 15, and 30 d after flowering (DAF).

MXZ, Meixiangzhan 2; MSZ, Meisizhan; QXZ, Qixinzhan. Fold change ratios are indicated by red or blue shading according to the scale bar. Data are means of three biological replicates per cultivar and time point. Full metabolite names are listed in Tables S1 and S5.

DISCUSSION

As the final products of cellular biochemical activity, metabolites directly reflect rice eating and nutritional qualities. As profound changes in metabolites occur during grain development, understanding of these changes may help clarify rice eating and nutritional quality formation and facilitate rice breeding. In the present study, we determined changes in the metabolic profile of carbohydrates, amino acids and their derivatives, lipids, fragrance and flavones, and their roles in the formation of eating and nutritional qualities.

Rice grain development involves the synthesis, interconversion and accumulation of several metabolites, and the formation and concentration of various macromolecules that directly influence rice eating and nutritional qualities. PCA showed that metabolic variations among the three cultivars decreased with the advancement in grain development (Fig. 1-C). This discovery was consistent with a previous report (Hu et al, 2016). The widest separation among cultivars was observed between 8 and 15 DAF. The time interval corresponds to grain filling and storage compound accumulation, and a large number of metabolites are involved in the development of the rice grain. Therefore, we focused on the drastic changes in the metabolic type and the contents of carbohydrates, amino acids and their derivatives, lipids, fragrance and flavones, which are closely related to the formation of eating and nutritional qualities during the process.

Starch, especially amylose, is the most important factor affecting rice eating quality (Tian et al, 2009). At 8 DAF, the levels of six carbohydrates involved in starch biosynthesis were significantly higher in MXZ than in the other cultivars (Fig. 2 and Table S2). The level of sucrose was 4-fold higher in MXZ than in QXZ. Only trehalose-6-phosphate levels significantly differed between MXZ and QXZ at 15 DAF, while the levels of four carbohydrates (threose, ribulose-5- phosphate, G-1-P and F-6-P) were significantly higher in MXZ than in QXZ at 30 DAF. Only one carbohydrate (-(+)-melezitose--rhamnoside) was different between MSZ and QXZ at 30 DAF during grain development, indicating that the two cultivars have similar carbohydrate metabolism pathways. Therefore, sufficient carbon supply for starch biosynthesis at rice grain filling is vital to formation of starch and other carbohydrates, and might account for the excellent eating quality of MXZ.

Amino acids and their derivatives also affect rice eating and nutritional qualities (Amir et al, 2018). There were significantly higher levels of different amino acids in MXZ than in QXZ during grain development (Fig. 3 and Table S3). For instance, the level of glutamine was 2.7-fold higher in MXZ than in QXZ at 8 DAF. A total of 23/26 amino acids and their derivatives were significantly upregulated in MXZ compared with QXZ, while only 7/9 were significantly upregulated in MSZ compared with QXZ at 15 DAF. We further found that 15/16 and 2/9 amino acids in MXZ and QXZ, respectively, were significantly upregulated compared with QXZ at 30 DAF when the rice seeds were fully mature. In addition, three dipeptides (γ-Glu-Cys,-alanyl--alanine and-glycyl--leucine), which are closely related to human health (Wu, 2016), were rich in the mature grains (30 DAF) of MXZ. Hence, MXZ had a sufficient and continuous supply of nitrogen during grain development and may therefore synthesize relatively more proteins and secondary metabolites (fragrance and flavones, etc). Protein content is negatively correlated with rice eating quality (Yang Y H et al, 2019). However, continuous nitrogen supply late in grain maturity promotes the formation of orderly packed starch granules and protein bodies, which contribute to excellent rice grain appearance and eating qualities (An et al, 2020). Moreover, protein is an important nutrient in rice grains. The elevated protein, essential amino acid and dipeptide contents in MXZ demonstrated that sufficient nitrogen supply during grain development is conducive to excellent rice eating and nutritional qualities.

Rice grains contain a far smaller proportion of lipids than starch, however, lipids significantly contribute to rice eating and nutritional qualities (Ying et al, 2012). Here, LysoPCs comprised ≤ 36% of the total lipid content in rice grains (Table S1). We found that though MSZ and QXZ were similar in terms of carbohydrate metabolism, they showed significant difference in terms of lipid (especially LysoPCs) metabolism. For instance, 19/22 lipids (mostly LysoPCs) were significantly upregulated in MSZ compared with QXZ at 8 DAF. At 30 DAF, only five lipids in MSZ had significantly higher levels than QXZ, all of which were LysoPCs (LysoPCs 16:1, 18:1, 12:1, 19:0 and 14:0). This distinction can contribute to the excellent eating quality of MSZ. In addition, at 30 DAF, four of the five different lipids in MXZ were LysoPCs [LysoPCs 16:1, 16:1 (2n isomer), 10:0 and 19:0], and their levels were all higher than in QXZ. These results indicated that LysoPCs 19:0 and 16:1 may play an important role in excellent eating quality. LysoPC is closely related to human health, and can induce the proliferation of smooth muscle cell through a variety of ways (Balakrishnan et al, 2021). Therefore, LysoPCs, especially LysoPCs 19:0 and 16:1, might account for the excellent eating and nutritional qualities of MXZ and MSZ.

A major component imparting fragrance to rice grains is 2-AP (Chen et al, 2008). Here, we detected nine metabolites in the 2-AP biosynthesis pathway in fragrant (MXZ) and non-fragrant (MSZ and QXZ) rice (Fig. 5). We discovered that the rice cultivars were opposite in terms of changes in their GABA and spermidine contents. Therefore, these substances were vital to 2-AP synthesis. GABA is closely related to the synthesis of 2-AP (Bouché and Fromm, 2004). It is an important nutrient metabolite, which inhibits neuro- transmission, regulates blood pressure, improves immunity and prevents certain cancers in mammals (Zhang and Jackson, 1993; Inoue et al, 2003; Hayakawa et al, 2004). We found that GABA content was lower in fragrant MXZ than in non-fragrant MSZ and QXZ at 8 DAF. The activity of betaine aldehyde dehydrogenase 2 might have been inhibited and the synthesis of 2-AP may have been promoted during rice grain development. Spermidine has prominent cardioprotective and neuroprotective effects and stimulates anticancer immunosurveillance (Madeo et al,2018). We found that the spermidine content in fragrant rice was significantly higher than in non-fragrant rice at 8 DAF. Hence, it is possible that GABA and spermidine can be conducive to 2-AP production at 8 DAF which, in turn, enhance rice eating and nutritional qualities.

Flavones are the fourth most abundant metabolites in rice grains. They are crucial in the plant life cycle and beneficial to human health (Zhao et al, 2020). Flavone levels were significantly lower in MSZ and MXZ than in QXZ at 8 DAF (Fig. 6 and Table S5). However, the opposite was true at 15–30 DAF. By 30 DAF, four co-existing flavones were detected in MXZ and MSZ (Fig. 6 and Table S5). Thus, flavones were formed mainly at the later stages of grain development in rice cultivars with excellent nutritional quality. Flavones are secondary metabolites that may be converted from primary metabolites such as sugars and amino acids. Results showed that rice cultivars with excellent eating quality such as MXZ and MSZ received sufficient sugars and amino acids during grain development. We also discovered that adequate carbohydrate and amino acid supply during grain development is also conducive towards excellent nutritional quality.

In addition, the PCA, two-way ANOVA and ASCA results revealed that the changes in metabolite levels were also affected by the developmental stage (Figs. 1-C, S2 and S3). The cultivars were most widely separated at 8–15 DAF. We further found that changes in the levels of lipids and flavones at 15–30 DAF significantly influenced rice eating and nutritional qualities (Figs. 4 and 6). At 8 DAF, MXZ and MSZ differed from QXZ in their MAG and LysoPC contents. In contrast, they significantly differed only in LysoPC content at 30 DAF. Therefore, attention may be paid to the later stages of grain development during future investigation on the function of genes related to rice eating and nutritional quality formation.

In this study, we investigated the formation mechanism of rice eating and nutritional qualities at the global metabolic level. A total of 623 metabolites were identified in three pedigreedcultivars with different eating and nutritional qualities. There were metabolic variations among cultivars and grain developmental stages, but the variations decreased with grain maturation. Sufficient carbohydrate and amino acid supply during grain development may contribute to excellent rice eating and nutritional qualities. LysoPCs 19:0 and 16:1 were beneficial for the formation of excellent eating quality during grain development. GABA and spermidine played the most important roles in fragrance formation. Rice cultivars may simultaneously have superior eating and nutritional qualities based on the changes in flavones. These findings lay an empirical foundation for further investigation into the mechanisms of rice eating and nutritional quality formation. Moreover, they could facilitate the genetic breeding and improvement of novel rice cultivars with excellent eating and nutritional qualities.

METHODS

Rice materials and growth conditions

Rice plants were sown in a paddy field in Tianhe (23.2o N, 113.4o E), Guangzhou Province, China, late in 2018. Three biological replicates of rice grains at 8, 15 and 30 DAF were collected, frozen and shelled in liquid nitrogen, and stored at -80 oC.

Rice eating quality

Milled rice (17 g) was washed thrice with water, placed in a cup containing water (19.04 g), set in a rice cooker at 100 oC, and cooked for 20 min. Rice eating quality was evaluated with a rice taste meter (SATA1B; Japan Satake Corp., Hiroshima, Japan). Each experiment was repeated three times, and data were represented as Mean ± SD of three independent experiments.

Sample preparation and extraction

Freeze-dried grains were crushed in a mixer mill (MM 400; Retsch GmbH, Haan, Germany) with zirconia beads at 30 Hz for 1.5 min. The powder (100 mg) was weighed and extracted with 1.0 mL of 70% methanol at 4 oC overnight. The extract was centrifuged at 10 000 ×for 10 min, absorbed (CNWBOND Carbon-GCB SPE Cartridge; 250 mg; 3 mL; ANPEL, Shanghai, China; www.anpel.com.cn/cnw), and filtered (SCAA-104; 0.22 μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before liquid chromatography- mass spectrometry (LC-MS).

High-performance liquid chromatography (HPLC) conditions

Extracts were analyzed in the LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system; www. shimadzu.com.cn/; MS, Applied Biosystems 6500 Q TRAP; www.appliedbiosystems.com.cn/). The analytical conditions were as follows. HPLC: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm; 2.1 mm × 100 mm); solvent system, water-0.04% acetic acid: acetonitrile-0.04% acetic acid; gradient program, 95:5 at 0 min, 5:95 at 11.0 min, 5:95 at 12.0 min, 95:5 at 12.1 min and 95:5 at 15.0 min; flow rate, 0.40 mL/min; temperature, 40 oC; and injection volume, 2 μL. The effluent was connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS.

ESI-Q TRAP-MS/MS

Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple spectrometer Q TRAP API 6500 Q TRAP LC/MS/MS system fitted with an ESI Turbo Ion-Spray interface operating in positive ion mode and controlled by the Analyst v. 1.6.3 software (AB Sciex, Framingham, MA, USA). The ESI source operating parameters were as follows: ion source, turbo spray; source temperature, 500 oC; ion spray voltage, 5 500 V; ion source gas I, gas II and curtain gas were set to 55, 60 and 25 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed using 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as multiple reaction monitoring (MRM) experiments with the collision gas (nitrogen) set to 5 psi. The declustering potential and collision energy for the individual MRM transitions were optimized. A specific MRM transition set was monitored according to the metabolites eluted during each period.

Qualitative and quantitative metabolite determinations

Using in-house (MWDB) and public metabolite databases, primary and secondary mass spectrometry data were subjected to qualitative analyses. Interference from isotope signals and duplicate signals derived from K+, Na+and NH4+, and fragment ions from larger molecules were excluded (Fraga et al, 2010; Yang M et al, 2019).

Statistical analysis

PCA, two-way ANOVA, ASCA and heat maps were generated in R v. 3.5.0 (R Core Team, 439 Vienna, Austria). Data were normalized before analysis. Screening criteria for the significant differential metabolites were as follows: fold change ≥ 2 or ≤ 0.5 and< 0.05 (Student’stest).

ACKNOWLEDGEMENTS

This study was supported by the Natural Science Foundation of Guangdong Province of China (Grant Nos. 2018A030313465 and 2015B020231001), the National High-Tech Research and Development Program of China (Grant No. 2014AA10A604-19), the National Natural Science Foundation of China (Grant No. 31801448), and the Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science (Grant No. R2018PY-QF003).We thank Wuhan Metware and Guangzhou Gene Denovo Biotechnology Co. Ltd. for assisting in the metabolite and bioinformatics analyses.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Rice pedigree analysis based on information from Ricedata.

Fig. S2. Major patterns associated with developmental stage and cultivar.

Fig. S3. ANOVA-simultaneous component analysis.

Fig. S4. Relative changes in essential amino acids during grain development.

Table S1. List of 623 metabolites detected in this study.

Table S2. Carbohydrate with significant changes at 8, 15 and 30 d after flowering (DAF).

Table S3. Amino acid and derivatives with significant changes at 8, 15 and 30 d after flowering (DAF).

Table S4. Lipid with significant changes at 8, 15 and 30 d after flowering (DAF).

Table S5. Flavone with significant changes at 8, 15 and 30 d after flowering (DAF).

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22 April 2021;

28 July2021

Zhou Shaochuan (xxs123@163.com)

Copyright ? 2022, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/10.1016/j.rsci.2022.01.004.

(Managing Editor: Wu Yawen)