Transcription factors TgbHLH42‐1 and TgbHLH42‐2 positively regulate anthocyanin biosynthesis in Tulip (Tulipa gesneriana L.)

Abstract

The floral coloration of tulip flowers is one of the most prominent traits contributing to its high ornamental value. The molecular mechanisms of petal coloration remain elusive in tulip species. In this study, we performed comparative metabolome and transcriptome analyses using four tulip cultivars with distinguished petal colors. Four types of anthocyanins were identified, including cyanidin derivatives and pelargonidin derivatives. Comparative transcriptome analysis identified 22,303 differential expressed genes (DEGs) from the four cultivars, and 2589 DEGs were commonly regulated in three comparison groups (colored vs. white cultivar), including anthocyanins biosynthesis-related genes and regulatory transcription factors. Two basic helixloop-helix (bHLH) transcription factors, TgbHLH42-1 and TgbHLH42-2, with differential expression levels among cultivars and petal developmental stages, have high homology to TRANSPARENT TESTA 8 (AtTT8) of Arabidopsis. The anthocyanins accumulation in TgbHLH42-1 overexpressing (OE) seedlings was markedly greater than that in wild-type seedlings in the presence of methyl jasmonate (MeJA), but not for TgbHLH42-2 OE seedlings. Both TgbHLH42-1 and TgbHLH42-2 restored pigmentation defects in tt8 mutant seeds after complementation assay. TgbHLH42-1 could interact with MYB protein AtPAP1 to synergistically activate the transcription of AtDFR, whereas TgbHLH42-2 failed to. Silencing TgbHLH42-1 or TgbHLH42-2 individually could not, but simultaneously silencing both TgbHLH42 could reduce the anthocyanin in tulip petals. These results indicate that TgbHLH42-1 and TgbHLH42-2 function partially redundantly to positively regulate anthocyanin biosynthesis during tulip petal coloration. | INTRODUCTIONTulip (Tulipa gesneriana L.) is a bulbous plant belonging to the genus Tulipa in the Liliaceae family. It is distributed throughout the Mediterranean, Central and East Asia, Europe and Northern Africa and is widely grown around the world (Eker et al., 2014). Floral coloration is an important trait of tulips that determines their commercial value. Detailed color composition analyses in tulip have been performed since the 1950s, and the results indicated that tulip petal pigmentation is mainly due to the accumulation of carotenoids and anthocyanins. Carotenoids impart yellow color, while anthocyanins help produce various flower colors ranging from red, orange, pink, and purple (Nieuwhof et al., 1990).Eleven anthocyanin compounds have been identified in tulips, including pelargonidin, cyanidin, delphinidin and derivatives (Nakayama

The R2R3-MYB Transcriptional Repressor TgMYB4 Negatively Regulates Anthocyanin Biosynthesis in Tulips (Tulipa gesneriana L.)

“…TgMYB4 contains a bHLH-interacting motif ( Figure 1 B), which was identified as a conserved domain of subgroup 4 R2R3-MYBs and was shown to interact with the bHLH component of the MBW complex. TgbHLH42-1 plays a positive role in the regulation of anthocyanin in tulips, and has a high homology to AtTT8 [ 44 ]. In order to dissect the working mechanism of TgMYB4 , we explored the relationship between TgMYB4 and TgbHLH42-1.…”

“…We previously demonstrated that the AtPAP1-TgbHLH42-1 complex could activate the transcription of AtDFR through dual luciferase assays [ 44 ]. In this study, we investigated whether TgMYB4 could attenuate the ability of the MYB-bHLH complex to activate AtDFR .…”

“…The petals of the red cultivar ‘SF’ were used for the assay before coloration. The specific steps of the assay were conducted following a previously described protocol [ 44 , 65 ]. Firstly, the petals were immersed in a bacterial suspension containing pTRV1 and pTRV2 or pTRV2- TgMYB4 (OD600 = 1.5, Ratio = 1:1 v / v ) and vacuum-infiltrated at 0.7 Mpa for 5 min.…”

“…A subcellular localization assay was performed following the established protocol outlined in our previous work [ 44 , 65 ]. The ORF of TgMYB4 without a stop codon was amplified and inserted into the L101YFP vector to generate the 35S::TgMYB4 -YFP construct.…”

“…The TfANS1 (GenBank: KC261507) and AtDFR promoter fragments were ligated into the pGreen II 0800-LUC vector as reporters. The vectors were introduced into A. tumefaciens strain GV3101 and transiently expressed in 4-week-old tobacco leaves as previously described by Hu et al [ 44 ].…”