Biofortified Rice Provides Rich Sakuranetin in Endosperm

Abstract

Sakuranetin plays a key role as a phytoalexin in plant resistance to biotic and abiotic stresses, and possesses diverse health-promoting benefits. However, mature rice seeds do not contain detectable levels of sakuranetin. In the present study, a transgenic rice plant was developed in which the promoter of an endosperm-specific glutelin gene OsGluD-1 drives the expression of a specific enzyme naringenin 7-O-methyltransferase (NOMT) for sakuranetin biosynthesis. The presence of naringenin, which serves as the biosynthetic precursor of sakuranetin made this modification feasible in theory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) validated that the seeds of transgenic rice accumulated remarkable sakuranetin at the mature stage, and higher at the filling stage. In addition, the panicle blast resistance of transgenic rice was significantly higher than that of the wild type. Specially, the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging was performed to detect the content and spatial distribution of sakuranetin and other nutritional metabolites in transgenic rice seeds. Notably, this genetic modification also did not change the nutritional and quality indicators such as soluble sugars, total amino acids, total flavonoids, amylose, total protein, and free amino acid content in rice. Meanwhile, the phenotypes of the transgenic plant during the whole growth and developmental periods and agricultural traits such as grain width, grain length, and 1000-grain weight exhibited no significant differences from the wild type. Collectively, the study provides a conceptual advance on cultivating sakuranetin-rich biofortified rice by metabolic engineering. This new breeding idea may not only enhance the disease resistance of cereal crop seeds but also improve the nutritional value of grains for human health benefits.

Keywords: Biofortified rice; MALDI-MS imaging; Metabolic engineering; Rice endosperm; Sakuranetin biosynthesis.

Fig. 1

The pathways of sakuranetin biosynthesis in rice. Metabolites are indicated in black and different enzymes are indicated in color. PAL, phenylalanine ammonialyase; C4H, cinnamic acid 4-hydrolase; 4CL, coumarin-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase; F3'H, flavonoid-3'-hydroxylase; F3H, flavanone-3-hydroxylase; NOMT, Naringenin 7-O-methyltransferase

Fig. 2

The expression pattern of OsNOMT and sakuranetin content in different rice tissues. A GUS staining of 7-day-old seedlings of pOsNOMT::GUS. Scale bar = 1 cm. B GUS staining of 20-day-old leaf (top) and leaf sheath (bottom) of pOsNOMT::GUS. Scale bar = 1 mm. C GUS staining of pOsNOMT::GUS seeds at 15 DAF. The top is the intact husked seeds, and the bottom is the cross section of the seeds (the left half contains the ridge of the seed, the right half does not). Scale bar = 0.5 mm. D qRT-PCR analysis of the expression levels of OsNOMT in different tissues of ZH11. E LC–MS/MS analysis of the sakuranetin content in different tissues of ZH11. Different letters indicate significant differences at P < 0.05 as determined by one-way ANOVA with Tukey’s test (mean ± s.d., n = 3, individual values and means are shown, biologically independent samples) (D, E). n.d., not detected

Fig. 3

The specific expression of OsNOMT in endosperm resulted in the accumulation of sakuranetin and increased the blast resistance in rice seeds. A Schematic diagram of the pOsGluD-1::OsNOMT construct with the OsNOMT CDS, driven by the OsGluD-1 promoter. B LC–MS/MS analysis of the sakuranetin content in ZH11 and pOsGluD-1::OsNOMT seeds at 15 DAF. #6, #7, and #17 represent three different transgenic lines. P-values were determined using two-tailed Student’s t-tests, ***P < 0.001 (mean ± s.d., n = 3, individual values and means are shown, biologically independent samples). FW, fresh weight. C The MALDI-MS images of metabolites in the sakuranetin biosynthesis pathway in ZH11 and pOsGluD-1::OsNOMT seeds at 25 DAF. The colors in the spatial distribution images represent the relative content of metabolites, and 0 to 1 indicates sequentially increasing metabolite content. Scale bar = 1 mm. D LC–MS/MS analysis of the sakuranetin content in ZH11 and pOsGluD-1::OsNOMT seeds at the mature stage. DW, dry weight; n.d., not detected. E The phenotype of pOsGluD-1::OsNOMT panicle inoculated with rice blast. The red triangles indicate M. oryzae-infected rice seeds. Scale bar = 2 cm. F The relative fungal growth in E. Relative fungal growth indicates the relative fungal biomass in M. oryzae-inoculated panicles and was determined with fungal 28S rDNA normalized to rice genomic ACTIN1 DNA by DNA-based qPCR. P-values were determined using two-tailed Student’s t-tests, ***P < 0.001 (mean ± s.d., n = 3, individual values and means are shown, biologically independent samples)

Fig. 4

Comparison of nutrition and quality between pOsGluD-1::OsNOMT and wild type seeds. A The MALDI-MS images of the essential amino acids and amino acids that determine the eating quality in ZH11 and pOsGluD-1::OsNOMT seeds at 25 DAF. Scale bar = 1 mm. B The MALDI-MS images of of targeted free soluble sugars in ZH11 and pOsGluD-1::OsNOMT seeds at 25 DAF. Scale bar = 1 mm. C–H Comparison of total amino acid, soluble sugar, total flavonoid, amylose, total protein and free fatty acid content between ZH11 and pOsGluD-1::OsNOMT seeds at the mature stage. P-values were determined using two-tailed Student’s t-tests (mean ± s.d., n = 3, individual values and means are shown, biologically independent samples)

Fig. 5

The phenotype of pOsGluD-1::OsNOMT. A Phenotype of 14-day-old ZH11 and pOsGluD-1::OsNOMT seedlings. Scale bar = 5 cm. B Phenotypes of ZH11 and pOsGluD-1::OsNOMT at the reproductive stage. Scale bar = 15 cm. C The phenotypes of panicles at the grain maturation stage detached from the ZH11 and pOsGluD-1::OsNOMT. Scale bar = 3 cm. D, E Mature grains of ZH11 and pOsGluD-1::OsNOMT. Scale bar = 0.5 cm. F Husked grains of ZH11 and pOsGluD-1::OsNOMT. Scale bar = 0.8 cm. G–I Comparison of grain width (n = 10), grain length (n = 10), and 1000-grain weight (n = 3) between ZH11 and pOsGluD-1::OsNOMT. P values were determined using two-tailed Student’s t-tests (mean ± s.d., individual values and means are shown, biologically independent samples)

Fig. 6

The proposed work model illustrating how endosperm-specific expression of OsNOMT leads to the accumulation of sakuranetin in the rice seeds. The expression of the sakuranetin biosynthesis gene OsNOMT was driven by the promoter of the endosperm-specific glutelin gene OsGluD-1, and OsNOMT successfully catalyzes naringenin synthesis of sakuranetin in the rice seeds. Sakuranetin content was highest at the filling stage and decreased with seed maturation. During the filling stage, the rapid and massive accumulation of sakuranetin is proposed to improve disease resistance in crop panicles by acting as a phytoalexin. At the mature stage, the lower abundance of sakuranetin confer potential nutritional and health benefits to humans. The dark brown color in the proposed model represents the rice seed embryo, the yellow color represents the cortex (rice bran layer), the beige color represents the endosperm, and the small red dots represent sakuranetin