Overexpressing CAPRICE and GLABRA3 did not change the anthocyanin content of tomato (solanum lycopersicum) fruit peel

Abstract

In Arabidopsis thaliana, the R3-type MYB transcription factor CAPRICE (CPC) and bHLH transcription factor GLABRA3 (GL3) cooperatively regulate epidermal cell differentiation. CPC and GL3 are involved in root-hair differentiation, trichome initiation and anthocyanin biosynthesis in Arabidopsis epidermal cells. Previously, we showed that CPC and GL3 also influence anthocyanin accumulation in tomato. Introduction of 35S::CPC into tomato significantly inhibits anthocyanin accumulation in cotyledons, leaves and stems. In contrast, introduction of GL3::GL3 strongly enhances anthocyanin accumulation in cotyledons, leaves and stems of tomato. In this study, we investigated the effect of CPC and GL3 on anthocyanin accumulation in the epidermis of tomato fruit. Unlike the results with vegetative tissues, overexpression of CPC and GL3 did not influence anthocyanin biosynthesis in tomato fruit peel.

Anthocyanins belong to the group of secondary plant metabolites called flavonoids that are important polyphenolic pigments derived from the phenylpropanoid biosynthetic pathway.¹ Anthocyanins protect plants from high irradiance and low temperatures because they function as a light-screen and as scavengers for radicals.² Recently, anthocyanins have been reported to be beneficial biomolecules for human health, specifically as anticancer agents due to their antioxidant activity.³ Normally anthocyanins do not accumulate in tomato fruit. Tomato produces anthocyanins in cotyledons, leaves and stems; however, only a few other flavonoids that are not anthocyanins are found in tomato fruit.^4-6 Recently, Buetlli et al. produced transgenic tomato plants that contained significant levels of anthocyanin in the fruit.⁷ This transgenic tomato harbors 2 transcription factors, Delila (DEL) and Rosea1 (ROS1), that control anthocyanin biosynthesis in snapdragon.⁷ DEL encodes a bHLH transcription factor, and ROS1 encodes a MYB-related transcription factor.^8,9 Thus, on the basis of this finding the anthocyanin biosynthetic pathway is fully present and functional in the fruit of tomatoes.

Previously, we showed that the CAPRICE (CPC) and GLABRA3 (GL3) genes can affect anthocyanin biosynthesis in tomato.¹⁰ CPC encodes an R3-type MYB transcription factor that induces root-hair cell differentiation and inhibits trichome initiation in Arabidopsis thaliana.¹¹ GL3 encodes a bHLH-type transcription factor that inhibits root-hair cell differentiation and induces trichome formation in Arabidopsis.¹² In addition to these functions, CPC and GL3 are involved in anthocyanin biosynthesis in Arabidopsis.^13,14 35S::CPC transgenic tomato vegetative tissues contain reduced amounts of anthocyanins compared with control tomato tissues.¹⁵ Furthermore, GL3::GL3 transgenic tomato vegetative tissues contain increased amounts of anthocyanins compared with control tomato tissues.¹⁵ We previously isolated Solanum lycopersicum TRYPTICHON (SlTRY) and Solanum lycopersicum GLABRA3 (SlGL3) from tomato as homologous genes of Arabidopsis CPC and GL3, respectively.¹⁶ SlTRY had similar functions to CPC including root-hair induction, inhibition of trichome formation and anthocyanin accumulation in Arabidopsis.^16,17 On the other hand, SlGL3 did not show any observable GL3-like functions in Arabidopsis.^16,17

To evaluate evolutionary relationships among anthocyanin synthesis-related transcription factors, we performed phylogenetic analyses (Fig. 1). Snapdragon ROS1 and ROS2⁸ are more closely related to Arabidopsis PAP1 and PAP2¹⁸ functioning in anthocyanin pigmentation than Arabidopsis GLABRA1 (GL1),¹⁹ WEREWOLF (WER),²⁰ CPC, TRYPTICHON (TRY)²¹ and tomato SlTRY¹⁶ that are mainly involved in root-hair and trichome formation (Fig. 1A). The snapdragon bHLH transcription factor DEL is closely related to tomato SlGL3 and belongs to the same cluster (Fig. 1B). Arabidopsis GL3, ENHANCER OF GL3 (EGL3),²² TT8,²³ petunia AN1,²⁴ maize R(Lc)²⁵ and rice Ra/OSB1²⁶ form another cluster (Fig. 1B).

Figure 1.

Phylogenic tree based on the deduced amino acid sequences of MYB and bHLH transcription factors. (A) Phylogenic tree of ROS1, ROS2, PAP1, PAP2, GL1, WER, CPC, TRY and SlTRY. (B) Phylogenic tree of GL3, EGL3, TT8, AN1, R(Lc), Ra/OSB1, DEL and SlGL3. Numbers above or below branches are genetic distances based on 1,000 bootstrap replicates. The tree was obtained by the neighbor-joining method using Genetyx ver. 16.0.2 software (Genetyx, Tokyo, Japan).

In this study, we examined the functions of CPC and GL3 as they relate to anthocyanin accumulation in the epidermis of tomato fruit. Anthocyanin extraction from the skin of tomato fruit was performed as described by Solfanelli et al.²⁷ In contrast to the results from the vegetative tissues including the cotyledons, leaves and stems of tomato,¹⁰ no obvious color changes were observed in 35S::CPC and GL3::GL3 transgenic tomato fruit (Fig. 2A). CPC and GL3 did not affect anthocyanin accumulation in tomato fruit epidermis (Fig. 2B). Very little anthocyanin accumulation was observed in the peel of control tomato fruit compared with that in the leaves of control tomato plants, and hardly any anthocyanin accumulated in the peels of 35S::CPC and GL3::GL3 tomato fruit (Fig. 2B). Therefore, overexpression of CPC and GL3 did not change the levels of anthocyanin accumulation in tomato fruit peel.

Figure 2.

Anthocyanin accumulation in the peel of 35S::CPC and GL3::GL3 transgenic tomato fruit. (A) Mature red tomato fruit from the control, 35S::CPC and GL3::GL3 transgenic plants. Scale bar: 1 cm. (B) Anthocyanin content in the leaves of control tomato plants, peels of control tomato fruit, peels of 35S::CPC transgenic tomato fruit and peels of GL3::GL3 transgenic tomato fruit were measured. Error bars indicate the standard deviations. Data evaluated by Student's t-test determined that there were no significant differences.

Since the anthocyanin biosynthetic pathway is present and functional in the fruit of tomato,⁷ there may be 2 reasons why CPC and GL3 do not function in tomato fruit peel. The first possibility is that the 35S and GL3 promoters are not strong enough to express their respective genes in tomato fruit peel. Secondly, there may be other MYB or bHLH factors that function primarily in fruit tissues.

Our next task is to determine which MYB or bHLH mainly functions in the floral tissue of Arabidopsis and tomato. PAP1 is reported to induce anthocyanin pigmentation in Arabidopsis petals when this transcription factor is overexpressed.¹⁸ From this study, it is clear again that epidermal-specific MYB and bHLH seem to be divided into 2 categories.^28,29 The former is mainly functioning in the formation of root-hairs and trichomes. The latter functions in regulating the production of anthocyanin pigments in many plant species.

Acknowledgments

We thank Y. Nukumizu for technical support, and T. Ishida, R. Sano and T. Kurata for useful suggestions.

Funding

This work was financially supported by JSPS KAKENHI Grant numbers 24658032, 23570057 and 25114513.