Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice (Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm “Purple Endosperm Rice” (called “Zijingmi” in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.

Engineering of complex biosynthetic pathways or multiple traits often requires the assembly of multi-gene expression cassettes into single transformation vectors; this has remained difficult due to the limited number of available cloning sites and the low ligation efficiency of large plasmids. Several transgene-stacking vector systems have been developed for assembly of limited numbers of target genes into single vectors (usually up to four) with different strategies, such as using homing endonuclease sites, Multisite Gateway, or a recent method using homologous recombination in yeast (). Stacking of transgenes in plants with multi-gene vector constructs by single plant transformation has many advantages compared with other transgene-stacking methods such as co-transformation of mixed plasmids, retransformation, and crossing between transgenic plants (), as well as the recombinase-mediated in planta transgene-stacking strategy that needs multiple rounds of plant transformation (). In particular, use of a single construct for transgene stacking allows all relevant genes to be delivered in a single event, thus saving time. We previously developed a transgene-stacking system using Cre recombinase/loxP-mediated recombination, which can assemble multiple genes into a transformation-competent artificial chromosome (TAC)-based acceptor vector that is capable of cloning and transferring large-size (>100 kb) DNA fragments into plants (). However, dealing with large numbers of target genes (more than five) with this vector system remained challenging, because it required a separate step to remove the donor plasmid backbone in the intermediate recombinant plasmids, using homing endonuclease digestion, and the gene assembly cycles involved an inefficient step to circularize the large plasmids by linker ligation. To expedite biosynthetic biology studies and metabolic engineering in crops, in this study we developed a new, high-efficiency multi-transgene stacking vector system, TransGene Stacking II (TGSII), which offers substantial improvements in ease of manipulation and efficiency of multi-gene assembly. Furthermore, we used the TGSII system to engineer the anthocyanin biosynthesis pathway in the rice endosperm, generating a novel biofortified rice germplasm, “Purple Endosperm Rice”, which shows enhanced phytonutrient contents of this important food crop.

The endosperm of cereal crops is ideal for engineered production of recombinant proteins (). Rice is a major staple food crop, and development of biofortified rice has attracted much attention from breeders and biotechnologists. However, previous attempts to produce anthocyanins in the rice endosperm have been unsuccessful. For example, transgenic expression of two regulatory genes ZmR-S and ZmC1 from maize (encoding the bHLH and the MYB-type transcription factor, respectively) in the rice endosperm produced no target anthocyanins but did produce intermediate flavonoid products (). In another study, introduction of several endosperm-expressed structural genes for anthocyanin biosynthesis in rice also produced only intermediate flavonoids ().

The sophisticated anthocyanin biosynthesis pathway in plants involves conserved proteins responsible for the formation of a series of metabolites and decoration and transport of the anthocyanin products, as well as several regulatory proteins that control the temporal and spatial expression of anthocyanin-related genes in various organs and tissues (). In purple-leaf rice, the regulatory proteins include OsC1 (the homolog of the MYB-R2R3-type transcription factor Colored 1 encoded by ZmC1 in maize), OsB1, and OsB2 (the homologs of the basic helix-loop-helix [bHLH]-type transcription factor Booster 1 of maize), and OsWD40 (a WD40-type transcription factor); these proteins interact to form the MYB–bHLH–WD40 (MBW) complex, which activates the genes for anthocyanin biosynthesis in leaves (). Kala4, a promoter-diverged allele of OsB2, controls anthocyanin production in the pericarp of black-grained rice (), and OsRc (Red pericarp, a homolog of OsB1/OsB2), regulates proanthocyanidin production in the pericarp of red-grained rice (). Supplemental Figure 1 summarizes the anthocyanin and proanthocyanidin biosynthetic pathways in rice.

Some biofortification approaches have targeted anthocyanins, a group of water-soluble pigments that have high antioxidant activities. Consumption of anthocyanins can benefit human health, decreasing the risk of certain cancers, cardiovascular disease, diabetes, and other chronic disorders (). Plants produce anthocyanins in leaves, stems, flowers, and fruits. In cereal grains the pericarp produces anthocyanins, but the endosperm lacks anthocyanins. For example, some black- and red-grained varieties of rice (Oryza sativa L.) produce anthocyanins and proanthocyanidins in the pericarp. However, brown (unpolished) rice grains have a poor taste due to the high fiber content of the pericarp, and polishing of rice grains removes the pericarp, along with its nutritional components, leaving only the endosperm.

Micronutrient and phytonutrient deficiencies affect more than 2 billion people globally and can have devastating effects, especially in poor populations (). Biofortification may offer an effective solution to mitigate this problem, using conventional breeding or transgenic methods to develop food crops with increased contents of specific micronutrients and phytonutrients (). Several conventionally bred biofortified crops (such as provitamin A-enriched maize, high-iron bean, high-iron pearl millet, and high-zinc rice) have been developed and released to farmers (). In addition, several crops genetically engineered for biofortification of phytonutrients, such as β-carotene-enriched “Golden Rice” (), anthocyanin-enriched “Purple Tomatoes” (), and folate-enriched rice (), have been developed via transformation with a few (two to four) regulatory or structural genes of the target metabolic biosynthesis pathways.

To examine the expression of the transgenes and how they affect the expression of related endogenous genes, we performed semi-quantitative RT–PCR on the endosperm of the developing seeds of a PER line (PER-Z#3). All the transgenes were expressed at high levels, similar to or somewhat lower than that of the highly expressed reference gene OsActin1 ( Figure 5 ). In the PER line, the expression of 13 endogenous genes related to anthocyanin biosynthesis, which were silent or expressed at low levels in the endosperm of ZH11, was activated or upregulated ( Figure 5 ). These genes include upstream phenylalanine pathway genes and structural, decorating, and transporter genes of the anthocyanin biosynthesis pathway. We also found that the endogenous WD40 gene OsWD40 was upregulated in the PER line, probably by the MBW complex. However, the endogenous MYB gene OsC1 was not activated in the PER line.

Semi-quantitative RT–PCR (22 cycles for all reactions) showed the expression of the transgenes (red) and the activated (or enhanced) endogenous genes (blue) for the anthocyanin biosynthesis pathway and its upstream phenylalanine pathway in developing endosperm (7 and 14 days after pollination) of ZH11 and PER-Z#3 (a homozygous T 3 line). Four functional endogenous structural genes, OsF3H, OsANS, OsF3′5′H, and OsLAR, were not activated by the regulatory genes in both ZH11 and PER-Z#3.

We measured the total antioxidant activity of four PER lines and ZH11 using the Trolox equivalent antioxidant capacity (TEAC) assay. As expected, the antioxidant activities of unpolished and polished grain samples of the PER lines were much higher than those of ZH11 ( Figure 4 F).

To examine the anthocyanin composition in the PER lines, we performed high-performance liquid chromatography (HPLC) and identified two kinds of anthocyanins, cyanidin 3-O-glucoside (C3G) and its derivative, peonidin 3-O-glucoside (P3G), in the endosperm of the PER lines. These anthocyanins are also found in the pericarp of the black-grained rice variety BGR1 ( Figure 4 D). Quantitative analysis showed that the PER lines had a much higher proportion of P3G compared with that in BGR1, and the total anthocyanin content in one line (PER-Z#3) reached ∼1 mg/g of dried grain ( Figure 4 E and Supplemental Figure 13 ). We detected no anthocyanin in the unpolished grains of the non-transformed parental varieties, nor in the polished grains of BGR1 ( Figure 4 D). In addition, we detected low levels of some flavonoids and proanthocyanidins in the endosperm of the PER lines but not in ZH11 ( Supplemental Figure 14 ).

We used Agrobacterium-mediated transformation to introduce pYLTAC380MF-10G into the japonica rice variety ZH11 and the indica variety HG1. We obtained 15 and 13 transgenic (T) plants, respectively, and found that 11 and 9 of the ZH11- and HG1-transgenic plants, respectively, produced purple seed grains ( Figure 4 A–4C and Supplemental Figure 8 ). We named these lines “Purple Endosperm Rice” (PER) (also called “Zijingmi” in Chinese).We used PCR to verify the presence of the transgenes in the transgenic plants (T, T, and T), and the results indicated stable inheritance of the integrated transgenes in the PER lines ( Supplemental Figures 9 and 10 ). The grains of three transgenic lines (PER-Z#1, PER-Z#2, and PER-H#11) were a much lighter purple than those of the other PER lines, and PCR showed that these lines lacked three of the structural genes, SsCHS, SsCHI, and SsF3′H ( Figure 4 A; Supplemental Figures 9 and 10 ). This suggests that these transgenic structural genes enhanced anthocyanin biosynthesis, even though the introduced regulatory genes activated the corresponding endogenous genes OsCHS, OsCHI, and OsF3′H (see below). PCR analysis also showed that the selectable marker/Cre gene cassette was excised as expected, producing homozygous marker-free PER lines in the Tgeneration ( Supplemental Figure 11 ). Most of the growth characteristics of the PER lines, such as plant height, tiller number, and seed-setting rate, were similar to those of the parental varieties; however, the grain weight of the lines was reduced by about 25% compared with ZH11 ( Supplemental Figure 12 ).

(F) TEAC analysis of the unpolished and polished rice grains of the PER lines and ZH11, showing different levels of antioxidant activity. **p < 0.01 and ***p < 0.001, significances between the PER and ZH11 line, respectively (n = 3), with Student's t-test.

(E) Quantitative analysis (n = 3) of the C3G and P3G contents in grains of the PER lines and the control materials. gdw, grams of dry weight; nd, not detectable.

Unpolished and polished grains from the black-grained rice (BGR1), ZH11, and the “Purple Endosperm Rice” (PER) or “Zijingmi” (ZJM) lines (T) of transgenic ZH11. In PER-Z#2 with lighter purple-colored grains, three transgenes (SsCHS, SsCHI, and SsF3′H) were absent ( Supplemental Figures 9 and 10 ).

We cloned the eight anthocyanin pathway genes into six intermediate donor vectors with the ESPs, giving four vectors that each contained one target gene and two that carried two target genes. Each of the genes was controlled by a distinct endosperm-specific promoter ( Supplemental Figure 7 ). After six rounds of target-gene assembly and the final integration of the selectable marker/marker-excision cassette, the multi-transgene stacking construct carrying all the target genes, pYLTAC380MF-10G (with a T-DNA of ca. 31 kb), was completed ( Figure 3 A). Restriction analysis with NotI verified the vector structure and further confirmed its structural stability in E. coli and A. tumefaciens ( Figure 3 B).

(B) Not I-restriction analysis of the acceptor constructs from different rounds of gene assembly with increasing numbers of target genes (1G to 10G). Arrows indicate the newly added gene(s) in each gene assembly cycle. For structural stability testing of pYLTAC380MF-10G in the A. tumefaciens strain EHA105, the plasmid was introduced into EHA105, isolated from colonies, transferred back into E. coli, and isolated for restriction analysis.

Therefore, we explored a new strategy involving transformation with two regulatory genes and a complete set of the six structural genes of the anthocyanin pathway, all controlled by endosperm-specific promoters. We selected maize ZmLc (Leaf color) and ZmPl (Purple leaf), which encode the bHLH-type and MYB-type transcription factors, respectively, to activate the anthocyanin biosynthesis genes; ZmPl is a homolog of ZmC1 but has stronger activity (). Based on the high sequence conservation of the anthocyanin pathway genes in plants, we used PCR to isolate cDNAs for the six structural genes for anthocyanin biosynthesis from Coleus (Solenostemon scutellarioides, known for its colorful foliage), including SsCHS (encoding chalcone synthase), SsCHI (encoding chalcone isomerase), SsF3H (encoding flavanone 3-hydroxylase), SsF3′H (encoding flavonoid 3′-hydroxylase), SsDFR (encoding dihydroflavonol 4-reductase), and SsANS (encoding anthocyanidin synthase) ( Supplemental Table 1 ). For the promoters that drive the expression of these genes, we PCR-amplified eight endosperm-specific promoters (ESPs) from rice (), and cloned them into pYL322d1 and pYL322d2 to generate a suite of 16 intermediate donor vectors ( Supplemental Figure 6 ), which can be used to engineer various metabolic pathways in the endosperm of cereal crops. We used these structural genes and two regulatory genes, under the control of the ESPs, to engineer the anthocyanin pathway in the rice endosperm.

To determine why many rice varieties do not produce anthocyanins and why previous attempts to bioengineer anthocyanin production in the rice endosperm did not work (), we first analyzed sequences of the anthocyanin pathway genes in different rice varieties. The sequence analysis showed that OsB1 and OsB2 and the structural gene OsDFR (encoding dihydroflavonol-4-reductase) are defective in rice varieties that do not produce anthocyanins, including the japonica variety Zhonghua 11 (ZH11) and the indica variety Huaguang 1 (HG1), which we used for transformation in this study ( Supplemental Figure 4 ). Furthermore, analysis of public transcriptome data showed that the regulatory gene OsC1 and most of the genes encoding proteins of the anthocyanin pathway are silent or expressed at low levels in the rice endosperm ( Supplemental Figure 5 ). Our RT–PCR analysis in ZH11 (see below) was consistent with the transcriptome data for these genes. These findings suggest that transformation with only regulatory genes or structural genes would not be effective for engineering anthocyanin production in the rice endosperm.

To use the TGSII system, we individually cloned the target genes into the pYL322d1- and pYL322d2-based donor vectors (each capable of holding one, two, or more target genes) by various cloning methods, including Gibson cloning and Ω-PCR cloning (). The two groups of donor vectors carrying the target gene(s) are used in rotation for recombination with the original and target-gene-carrying acceptor vectors, in an E. coli strain (NS3529) expressing Cre () ( Figure 2 A). In each gene assembly cycle, the donor vector backbone in the intermediate recombinant plasmid is automatically removed by irreversible recombination between the paired compatible mutant loxP sites (loxP1L/loxP1R for odd-numbered rounds or loxP2L/loxP2R for even-numbered rounds). After each round of recombination, the unrecombined plasmids and intermediate recombinant plasmid are removed by digestion with I-SceI (odd-numbered rounds) or PI-SceI (even-numbered rounds), recovering the uncut target recombinant construct that lacks the corresponding homing endonuclease site. With this iterative Cre/loxP recombination strategy and the large cloning capacity of TAC, many target genes can be assembled into the acceptor vector, independent of the limitation imposed by the number of available “cloning sites” in the vectors. Finally, the marker/marker-excision cassette is introduced into the target-gene-carrying binary vector by Gateway reaction ( Figure 2 B). In transgenic plants with the stacked transgenes, the marker gene/Cre sequence is excised from the T-DNA insertion by loxP recombination in certain tissue(s) where Cre is expressed from a tissue-specific or inducible promoter; in this study we used the pollen-specific promoter Pv4 from the rice gene OsVillin 4 ( Figure 2 B).

(B) When all target genes are assembled, the selectable marker/marker-excision cassette is recombined into the acceptor vector by Gateway BP reaction to generate the final binary construct. In transgenic plants, Cre expression driven by Pv4 (or another suitable promoter) mediates loxP recombination to excise the cassette.

Multiple assembly cycles for multi-gene stacking. One or more target genes (A–F) are cloned into the donor vectors ( Supplemental Figures 6 and 7 ). For Round I of gene assembly, the acceptor vector and pYL322d1-A (with gene A) are co-transferred into an E. coli strain (NS3529) expressing Cre. The in vivo reversible recombination (i) between the wild-type loxP sites produces an intermediate recombinant plasmid, then an irreversible recombination (ii) between the compatible mutant loxP sites loxP1L (1L) and loxP1R (1R) excises the donor vector backbone (containing two I-SceI sites, a loxP site, and the bacterial selection marker gene), generating the target plasmid Acceptor-A. The irreversible recombination generates an inactive recombinant site 1LR (and 2LR in the even-numbered assembly cycles). After the gene assembly, a digestion with homing endonuclease I-SceI (with PI-SceI for even-numbered cycles) is performed to remove the original acceptor, donor, and intermediate plasmid (which contain one or two of the homing endonuclease sites), recovering the uncut target plasmid that lacks the corresponding homing endonuclease site. For Round II gene assembly, Acceptor-A and pYL322d2-C/B (with genes B and C in this example) are co-transferred into NS3529. The recombination events produce the recombination plasmid Acceptor-C/B/A. A third (and other odd-numbered, with pYL322d1-based vectors) and fourth (and other even-numbered, with pYL322d2-based vectors) round of gene assembly can be conducted to stack additional genes like Round I and Round II, respectively.

The TAC-based acceptor vector, pYLTAC380GW, uses the P1 plasmid replicon and the pVS1 replicon for replication of large plasmids in Escherichia coli and Agrobacterium tumefaciens, respectively. This vector contains three key components within the transfer DNA (T-DNA) region for multi-gene assembly: a wild-type loxP, a recognition site for the homing endonuclease I-SceI, and a mutant loxP1R. The target-gene donor vectors, pYL322d1 and pYL322d2, carry a wild-type loxP, two mutant loxP sites, and homing endonuclease sites; these sites are located in different arrangements in the two donor vectors ( Figure 1 B). Each of the marker-free donor vectors has a cassette containing a plant-selectable marker (HPT, NPTII, or Bar) and Cre driven by a pollen-specific promoter (Pv4) isolated in this study, and flanked by two wild-type loxP sites and two sites (attB1, attB2) for Gateway recombination ( Figure 1 C). We also developed two sets of binary acceptor vectors for different applications based on the TAC backbone and the pCAMBIA backbone. These vectors also could be adapted for use in non-plant systems by modifying suitable acceptor vectors with the key components loxP/I-SceI/loxP1R ( Supplemental Figure 3 A–3C).

Compared with our previous transgene-stacking vector system (), the major improvement we incorporated in this new TGSII system is the ability to automatically remove the donor vector backbone from the intermediate recombinant plasmid without separate restriction and ligation steps. Cre catalyzes reversible recombination between wild-type loxP sites and can also catalyze recombination between mutant loxP sites with a few nucleotide variations (). To enable Cre to automatically remove the donor vector backbone in the TGSII system, we designed and synthesized two pairs of mutant loxP sites, loxP1L/loxP1R and loxP2L/loxP2R, which can mediate irreversible recombination ( Supplemental Figure 2 ), and used them in the TGSII vectors. The two mutant loxP sites of each set contain variant nucleotides in the left arm or right arm and the spacer region; they can be recognized by Cre to undergo an irreversible recombination, because Cre cannot recognize the resulting recombinant sites loxP1LR or loxP2LR, which contain variant nucleotides in the both arms ( Supplemental Figure 2 ). Recombination does not occur between the sites of the different mutant sets (nor between a mutant site and a wild-type loxP site), because they have different, incompatible spacer sequences; identical spacer sequences are required for pairing and exchange of the strands.

The basic TGSII system consists of a binary acceptor vector, two target-gene donor vectors, and an optionally used donor vector with an antibiotic selectable marker and a marker excision (marker-free) cassette ( Figure 1 A–1C ). The donor vectors containing target genes are used in alternation for Cre/loxP-mediated recombination with the acceptor vector; the Cre recombinase then removes the donor vector backbone in the intermediate recombinant plasmid via an irreversible recombination between paired compatible mutant loxP sites. In a final step, the chosen selectable marker cassette is incorporated by Gateway recombination reaction; this cassette also contains Cre under the control of a tissue-specific promoter, allowing the markers to be removed by recombination in planta, thus resulting in plants that contain a marker-free transgenic insertion of a stack of target genes.

(C) Structural features of the donor vectors for insertion and excision of selectable markers. The plant selectable marker gene/marker-excision cassette between two loxP sites includes a marker gene (HPT, NPTII, or Bar, for pYLMF-H, pYLMF-N, or pYLMF-B) and the Cre gene (with an intron from the catalase gene) driven by a pollen-specific promoter (Pv4); these components are located between the Gateway attB1/attB2 sites. Pv4 from Villin4 (Os04g0604000) is expressed specifically in pollen. This cassette can be recombined into the pYLTAC380GW-based constructs by Gateway BP reaction to form a structure that is competent for in planta excision to produce marker-free plants.

(B) Structural features of the basic target-gene donor vectors. The wild-type and mutant loxP sites, the homing endonuclease sites, and MCSs are located in different arrangements (MCSs are in different orientations) in the donor vectors. The replicon is from pBR322, and Cm r and Amp r denote the chloramphenicol- and ampicillin-resistance genes, respectively. LacZ is used to indicate blue bacterial colonies of the recombinant plasmid with unremoved donor vector backbone.

(A) Structural features and the key sequence of the basic acceptor vector. The vector backbone contains a bacterial kanamycin-resistance gene (Kan r ), a P1 plasmid replicon for replication in E. coli, a P1 lytic replicon for increasing the plasmid copy number induced by IPTG, and a pVS1 replicon for replication in A. tumefaciens. The loxP and loxP1R sites are located in the T-DNA region (from right border [RB] to left border [LB]) for recombination with a target-gene donor plasmid and deletion of the donor backbone sequence. PI-PspI, I-SceI, and I-Ppo are homing endonuclease sites. A Gateway recipient region containing the SacB gene (which causes lethality in E. coli in the presence of sucrose) flanked by the attP1 and attP2 sites for recombining the excisable marker cassette into the vector after assembling all target genes.

Discussion

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Takaiwa F. Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits. Genetic engineering of complex metabolic pathways requires: (1) an understanding of the underlying gene networks and the regulatory relationship of the gene expression in these networks; (2) identification of the set of target genes that are required to establish the complete pathway in the target tissues of the organisms; and (3) an effective vector system and transformation method, particularly if a relatively large number of transgenes must be introduced. In the past decades, many studies on genetic engineering of metabolic pathways and multi-gene traits have been reported, but most of these studies involved transformation with relatively few genes, mostly up to three or four (). For example, the anthocyanin-enriched “Purple Tomato” was engineered using two regulatory genes, Ros1 (encoding an MYB protein) and Del (encoding a bHLH protein), which could activate the necessary endogenous genes (present in functional but silent forms) of the anthocyanin and flavonoid pathways in tomato. The resulting “Purple Tomato” plants produced high levels (0.23–2.83 mg/g fresh weight) of complex anthocyanin and flavonoid products in the fruit (). However, previous studies by transformation of rice with regulatory genes or structural genes failed to produce anthocyanins in the endosperm (). Therefore, engineering complex metabolic pathways in many plants remains a challenge because of our limited understanding of the gene networks and the mechanisms that regulate the expression of many metabolic pathways in certain plants, as well as the technical difficulty in linking and transferring multiple target genes.

Figure 6 Engineering of the Anthocyanin Biosynthesis Pathway in the Rice Endosperm. Show full caption The genetically engineered anthocyanin biosynthesis pathway involves the metabolic enzymes expressed from the transgenes (red), the rebuilt MBW transcription factor complex, and some functional endogenous genes (blue) activated (or enhanced) by the MBW complex. The pathway synthesizes cyanidin as the major anthocyanidin, which is further decorated by one and two steps to produce the anthocyanin products C3G and P3G, respectively. The minor proanthocyanidin branch is not shown. In this study, we successfully genetically engineered the complex anthocyanin biosynthesis pathway in the rice endosperm using the TGSII system. This success could be attributed to several factors in our design ( Figure 6 ). First, we examined the genes for anthocyanin biosynthesis (whether they were functional or non-functional) and their expression (expressed or silent) in the endosperm of white-grained rice varieties. We found that OsB1, OsB2, and OsDFR are defective, and that OsC1 and most of the related genes are silent or expressed at low levels in the endosperm. Based on this analysis, we selected the appropriate target genes for genetic transformation to reconstitute anthocyanin biosynthesis in the rice endosperm. Second, we used both the transgenic and endogenous regulatory genes where possible. For example, the transgenic ZmLc and ZmPl and the endogenous OsWD40 rebuilt the MBW complex, which enhanced or activated the expression of some other endogenous genes for gene regulation (e.g., OsWD40) and for biosynthesis, decoration, and transport of the anthocyanin products. In fact, the two lines (Z#8 and H#9) that lacked only ZmLc produced white grains ( Supplemental Figure 9 ), confirming the requirement of ZmLc for this engineered anthocyanin biosynthesis. Third, we used homologous structural genes to complement the anthocyanin pathway. Because the necessary endogenous structural gene OsDFR is defective in the parental varieties, and two other genes, OsF3H and OsANS, are not expressed in the endosperm of the wild-type and PER plants, we transferred the corresponding homologous genes from Coleus (SsF3H, SsDFR, and SsANS), which enabled the completion of anthocyanin biosynthesis. Consistent with this notion, a line (H#12) that lacked SsDFR did not produce purple seeds ( Supplemental Figure 9 ). Fourth, we supplemented endogenous genes with transgenes. For example, although three endogenous genes (OsCHS, OsCHI, and OsF3′H) were activated by the rebuilt MBW complex, the absence of the corresponding transgenes in the lines (Z#1, Z#2, and H#11; Supplemental Figures 9 and 10 ) resulted in lower anthocyanin contents and lower antioxidant activity ( Figure 4 A and 4E; Supplemental Figure 13 ). Therefore, introduction of the two necessary regulatory genes together with the complete set of structural genes successfully produced and enhanced anthocyanin biosynthesis in the rice endosperm.

6 ) and expressed the expected anthocyanin products ( Our results showed that although several transgenic plants lacked one to three gene fragments, which probably were lost during T-DNA biogenesis in A. tumefaciens and/or T-DNA infection/integration in callus cells, most of the transgenic plants contained the complete set of transgenes, and these transgenes were stably inherited in the progenies (we observed inheritance at least to T) and expressed the expected anthocyanin products ( Supplemental Figures 8–10 ). Compared with the complex anthocyanin and flavonoid products in the “Purple Tomato,” our PER lines produced only two anthocyanins (P3G and C3G) in relatively high amounts (up to ca. 1 mg/g dry weight), and low amounts of some flavonoids and proanthocyanidins. This new biofortified germplasm may provide a source of health-promoting food, improving the hydrophilic antioxidant levels in human diets.