Dongxin Huai, Jie Wu, Xiaomeng Xue, Hao Liu, Nian Liu, Li Huang, Liying Yan, Yuning Chen, Xin Wang, Qianqian Wang, Yanping Kang, Zhihui Wang, Yanbin Hong, Huifang Jiang, Boshou Liao, Yong Lei
With the progress of urbanization, rural tourism has emerged as a popular leisure activity in China. The crop field with pattern art has been growing in popularity over the years, using strains with colorful leaves to create a variety of impressive designs (Xu 2024). A lot of successful cases in rice field has not only attracted tourists but also increased the income of farmers (Song et al. 2020). Plants with colorful leaves also have gained significant popularity in ornamental agriculture. Therefore, the development of plants with colorful leaves have gained significant popularity in ornamental agriculture.
Betalains are natural tyrosine-derived pigments in red-violet and yellow hues, which are exclusively found in plants of the Caryophyllales order (Azeredo 2009). In addition to their attractive colors, betalains have demonstrated robust antioxidant activity, thereby demonstrating potential health-promoting properties, including anticancer, hypolipidemic, hepatoprotective, anti-inflammatory and antidiabetic activities (Polturak and Aharoni 2018). Biosynthesis of betalains has been extensively investigated and only needs three enzymatic reactions to convert tyrosine into betalain. Tyrosine is initially hydroxylated on the benzene ring, resulting in the formation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by the P450 oxygenase CYP76AD1. Then, L-DOPA could be further oxidized into cyclo-DOPA by CYP76AD1, or undergoes catalysis by L-DOPA 4,5-dioxygenase (DODA) to yield betalamic acid. Subsequently, betalamic acid is condensed with cyclo-DOPA into betanidin in the absence of any enzymatic activity. Finally, a sugar moiety is added to betanidin by a glucosyltransferase to generate the betalain (Khan and Giridhar 2015). An artificial open reading frame named RUBY containing the three key genes (CYP76AD1, DODA and glucosylstransferase) has been engineered to enable betalain production in Arabidopsis, tomato, carrot and cotton (He et al. 2020; Grützner et al. 2021; Deng et al. 2023; Ge et al. 2023; Li et al. 2023). Therefore, there is an impetus to engineer colorful-leaved peanuts through expression of the RUBY gene.
Peanut (Arachis hypogaea L.) is an important oilseed crop worldwide, which grown on 30 million ha between latitudes 40°N and 40°S (Hariharan et al. 2023). In China, the area of peanut planting has exceeded 4.46 million ha in 2022 (FAO 2022), indicating great potential for ornamental agriculture. However, peanut leaves are predominantly green, which limits the development of sightseeing agriculture in peanut. Hence, the RUBY gene was employed to genetically modify peanuts with colorful leaves in this study.
To create peanut plants with colorful leaves, a transgenic construct named pBinBarRuby was developed, harboring the RUBY gene driven by 35S-promoter (Fig. 1-A). Then this construct was introduced into the peanut cultivar Zhonghua 12 (ZH12) through Agrobacterium tumefaciens-mediated transformation (Huai et al. 2023). The transformed callus turned red-violet, indicative of betalain biosynthesis, whereas the wild-type (WT) callus displayed a green coloration. Subsequently, the transgenic shoots also exhibited a red-purple color, while the WT shoots retained their green coloration. Upon the emergence of roots, the entire transgenic plantlets showed a vibrant purple hue in leaves, stems and roots (Fig. 1-B). Finally, a total of 18 independent positive T0 transgenic plants were successfully obtained. Each independent transgenic peanut plant accumulated betalain, but the accumulation patterns were different depending on the expression level of the RUBY gene. The transgenic plants exhibited purple leaves, orange flowers, purple pod shell, purple testa and purple embryo, whereas the WT control showed green leaves, yellow flowers, white pod shell, red testa and white embryo (Fig. 1-C). The T1 progeny of RUBY peanut exhibited the same morphological characteristics as T0 generation, indicating that the betalain can be inherited by subsequent generations.
Five transgenic lines displaying red-purple color were selected and were confirmed by PCR. The expression levels of RUBY were detected in leaves of the five transgenic lines by qRT-PCR. As expected, the expression of RUBY was not detectable in the WT, while was detected in transgenic lines (Fig. 1-D). The betalain contents in leaves of WT and the five transgenic lines were determined. In WT leaves, the betalain content was 0.57 mg g-1, while a significant increase ranging from 0.95 to 2.03 mg g-1 was observed in transgenic purple leaves. Compared to WT, the transgenic lines exhibited a substantial enhancement in betalain contents ranging from 0.66 to 2.55-fold (Fig. 1-E).
The betalains exhibit remarkable attractiveness as natural pigments due to their vibrant color and relatively simple biosynthesis pathway. The RUBY gene is a synthetic cassette consisting of the three key genes for involved in the betalain biosynthesis pathway (He et al. 2020), which has been used in cotton to produce pink fibers (Ge et al. 2023; Li et al. 2023). Due to the powerful antioxidant properties of betalain, the RUBY gene has been expressed in vegetable and fruits including tomatoes, cucumbers and carrots (Grützner et al. 2021; Deng et al. 2023; Liao et al. 2023). For ornamental purposes, the RUBY gene was applied used in Torenia fournieri, Gentiana scabra and Portulaca grandiflora to change the flower colors (Sakuta et al. 2021; Nishihara et al. 2023, 2024). In this study, the RUBY gene was used to modify the colors of both of leaves and flowers, and generated novel peanut germplasms which are not present in current varieties (Fig. 1-C). The distinctiveness of purple leaves compared to green leaves highlights the potential application of purple-leaved peanuts in field pattern artistry. This is an eco-friendly and health-conscious alternative to synthetic colorants for the cultivation of ornamental plants.
The RUBY gene has also been widely employed as a prominent reporter to visualize transgenic events in Arabidopsis, rice and soybean (He et al. 2020; Wang et al. 2023; Chen et al. 2024). In this study, the efficiency of identifying transgenic peanut plants using the RUBY reporter was investigated. The red-violet pigment could be observed at very early stage of callus formation (Fig. 1-B), indicating that the screening could be started very early as well. Introduction of RUBY facilitated the discrimination between transformed and untransformed callus. Compared to the DsRed2 reporter, RUBY is clearer and much more convenient to operate during tissue culture condition. Therefore, the utilization of RUBY as a visible reporter proved to be highly advantageous in the monitoring of transgenes within peanut plants.
In summary, we successfully created novel peanut germplasms with purple leaves by heterologous expression of the RUBY gene, thereby showcasing their potential application in field pattern artistry. Meanwhile, we also demonstrated that RUBY can serve as a visible reporter in peanut transformation without the need for any additional instrumentation. Additionally, we provided an eco-friendly and health-conscious alternative to synthetic colorants for the cultivation of ornamental plants.