Development of “Tea Rice” by engineering catechin biosynthesis in rice endosperm

Catechins, a subclass of flavan-3-ols, constitute the primary constituents of tea plant (Camellia sinensis), representing approximately 15–30% of its dry weight (Wei et al., 2018). These essential flavonoid phytonutrients are renowned for their pivotal role in bestowing the unique health benefits associated with tea plant consumption. Moreover, catechins exhibit significant bioactivity, contributing to human health through their anti-inflammatory, antibacterial, anticancer and cardioprotective properties (Isemura, 2019). In most cultivated rice varieties, low expression or non-functionality of flavonoid pathway genes results in scarce flavonoid content in the endosperm. Even in pigmented rice, flavonoids are mainly in the pericarp. The lack of catechins in polished rice, given their robust bioactivity, positions them as an optimal candidate for biofortification efforts. Therefore, there is an impetus to develop a “Tea Rice” with a high content of catechins in the endosperm by harnessing tissue-specific metabolic engineering of enzymes involved in catechin biosynthetic pathways.

The catechin biosynthesis pathway is well understood at the molecular genetics and enzymatic levels (Figure 1b) (Zhao et al., 2022). The tea plant is a rich source of catechins; to generate “Tea Rice”, five genes from the tea plant, including [CsCHI (KY615684.1), CsCHS (NM_001426674.1), CsF3H (XM_028251604.1), CsDFR (KY615690.1) and CsLAR (KY615699.1)], along with one gene from the Crocosmia x crocosmiiflora [CcF3′5′H (MK562521)], have been obtained through PCR. In addition, we have selected the transcription factors OsMYBP1 (XM_015773843.3) and OsRc (DQ204737.1) to activate the endogenous catechin synthesis genes in rice (Zheng et al., 2019). These genes were chosen as metabolic genes for catechin biofortification in this study.

To rapidly and efficiently assess the potential of these gene combinations in synthesizing catechins, we initially cloned each target gene into a tobacco overexpression vector pEAQ-HT-DEST2 and infiltrated the mixed Agrobacterium suspension into the leaves of 4-week-old Nicotiana benthamiana. Consistent with our expected function, liquid chromatography-mass spectrometry (LC–MS) analysis confirmed the production of catechins (Figure 1c).

The main objective of this study was to fortify rice endosperm to improve the catechin content via genetic engineering. Thus, employing the GoldenBraid 2.0 method (Kundert et al., 2020), we constructed two gene expression cassettes comprising (OsMYBP1, OsRc) and an additional set of eight gene expression cassettes including (OsMYBP1, OsRc, CsCHI, CsCHS, CsF3H, CcF3′5′H, CsDFR, and CsLAR). People prefer to consume polished rice that has a better taste and longer shelf life, with all metabolic genes controlled by endosperm-specific expression promoters. These cassettes have been designated as Tea Rice 1 (TR1) and Tea Rice 2 (TR2), respectively (Figure 1a), and they were introduced into 7-day-old zygotic embryos of rice cultivar Zhonghua 11 via Agrobacterium-mediated T-DNA transfer. T0 plants were identified by PCR for the target genes (Figure S1). qRT-PCR analysis was conducted on two developing T4 generation seeds for each construct to assess the stable expression of exogenous inserted genes. The results showed all transgenes were overexpressed in the endosperm, with their expression levels being approximately 0.5 to 80 times that of the OsUBC13 expression level, except for CcF3′5′H in TR2-3 (Figure 1d,e).

Vanillin-HCl and dimethylaminocinnamaldehyde (DMACA) staining revealed characteristic red (vs. ZH11's colourless) and deep blue (vs. ZH11's light pink) coloration in TR2 extracts, respectively (Figure 1g). Together, these staining techniques confirm the presence of catechin-like substances in the Tea Rice cultivar we have developed. LC–MS analysis revealed the presence of low levels of flavonoids in polished rice of the variety ZH11 (Figure 1f); In TR1, there was a significant accumulation of over 20 types of flavonoid compounds, but no catechins (Figure 1f). In the TR2, more than 12 types of increased flavonoid compounds were detected, among which three catechins [(+)-catechin, (+)-afzelechin and (+)-gallocatechin] accumulated in large amounts (Figure 1f). This suggests that the metabolic flux of flavonoid synthesis in TR2 was redirected towards the catechin pathway under the influence of key structural genes such as CsDFR, CcF3′5′H and CsLAR, aligning with the expected outcomes. Quantitative analysis of catechins in Tea Rice was conducted. In TR2-3, the levels of (+)-afzelechin, (+)-catechin and (+)-gallocatechin reached 339.5, 61.2 and 1.1 μg/g, respectively, totalling 401.8 μg/g. In TR2-4, these levels were 139.1, 107.5, and 285.6 μg/g, respectively, totalling 532.2 μg/g (Figure 1h). Compared to wild-type plants, TRs (TR1 and TR2) transgenic plants showed no significant differences, but their grains were smaller than ZH11, leading to reduced yield (Figure S2). Additionally, TR2 grains exhibited a darker colour compared to both ZH11 and TR1 (Figure 1i).

Due to the strong antioxidant capacity of catechins, the antioxidant activity of extracts from polished TRs were measured. In the ABTS⁺ assay, TR1 and TR2 showed 3.6 and 4.4 times higher antioxidant capacities than the control, respectively (Figure 1j). In the DPPH assay, their radical scavenging rates were 1.7 and 2.3 times higher than the control (Figure 1k). The results indicate that both TR1 and TR2 have significantly improved antioxidant capabilities relative to ZH11, with TR2 showing notably higher antioxidant activity than TR1 (Figure 1k). Broad-target metabolomics showed elevated levels of various metabolites in TRs transgenic rice, likely due to shared precursors/cofactors, with flavonoid synthesis potentially activating key metabolic pathways and providing an antioxidant environment that promotes B-vitamin synthesis and retention (Figure S3; Table S1).

Catechins, as key antioxidant components in tea plants with high content, offer certain benefits to human health. In this study, we create a metabolic sink towards de novo catechin biosynthesis in rice endosperm by introducing structural genes involved in catechin synthesis and transcription factors that activate the catechin pathway, and successfully create “Tea Rice” with a high content of catechins in the endosperm. The catechin-fortified rice in this study can be used as a functional grain to promote health.

The authors declare no competing interests.

Luo J and Shen SQ designed this research. Zhu JJ, Zhou S and Wang QM performed these experiments. Wang WZ, Lv YY, Yang CK and Zhang R analysed these data. Zhu JJ, Zhou S, Shen SQ and Luo J wrote the manuscript.