Discussion:
Genotyping in the BC2F2 segregating population with 987 individuals with the marker bropsK18 was perfectly consistent with the phenotyping data. However, genotyping in the F2 segregating population derived from PS and GS with the marker bropsK18 was not completely consistent with the phenotyping. On the contrary, phenotyping concurred entirely with the genotyping of marker bropsK49, whose primers were designed according to the DNA sequences of the BolC9t59639H (F3’H ) gene on the target region between HX-11 and HX-16, where a 43-bp deletion in the second exon of BolC9t59639H has occurred. These results showed that the 43-bp deletion in BolC9t59639H (F3’H ) should be responsible for the buds keeping-green at low temperature (KGLT) in HX-16, the mutant GS and most broccoli materials with this trait.
The core structures of anthocyanins have only six types, cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin (Saigo et al. , 2020). In the biosynthetic pathway of anthocyanins, the substrate specificity of some key enzymes and the competition among them on the branch nodes results in the genus- and -species specificity of anthocyanins (Saigo et al ., 2020). In this study, no pelargonidin, peonidin or petunidin was detected, and only cyanidin, delphinidin and malvidin were detected in three broccoli materials. Cyanidin was absolutely dominant in the purple broccoli, had much the same content as delphinidin in PS, and had less content than delphinidin in GS. The result was almost consistent with the previous study (Moreno et al ., 2010) and demonstrated that species characteristics of anthocyanins were prominent in broccoli. It appeared that the vivid purple color of buds was due to the accumulation of cyanidins in purple broccoli, and low temperature mainly promoted the accumulation of cyanidins in PS, as the cyanidin content was decreased the most in GS compared to PS. InArabidopsis , many genes associated with anthocyanins biosynthesis were identified by corresponding mutants whose seed coat hastransparent testa (tt ) due to the lack of a particular step of anthocyanin biosynthesis (Li et al ., 2017). Arabidopsistt7 mutant, lacking a flavonoid 3’-hydroxylase lacks anthocyanins accumulation. Transgenic Arabidopsis tt7 seedlings expressing apple MdF3’H regained red color pigmentation in seed coats, and the seedlings increased the accumulation of both pelargonidin and cyanidin under nitrogen-deficient conditions (Han et al. , 2010). This illuminates that MdF3’H affect the accumulation of pelargonidin and cyanidin in Arabidopsis . According to the biosynthetic pathway of anthocyanins, F3’H , which encodes flavonoid 3’-hydroylase that catalyzes the conversion of kaempferol into quercetin and dihydrokaempferol into dihydroquercetin, is the key gene for cyanidins and delphinidins biosynthesis (Seitz et al. , 2007; Han et al. , 2010; ko00941 in https://www.kegg.jp). Therefore, in this study, KGLT in GS and HX-16 could result from the 43-bp deletion in the F3’H CDS, decreasing the accumulation of cyanidins and delphinidins.
In the mutant GS and wild line PS, there were only two genes relative to anthocyanins biosynthesis, which expressed differently in the buds; F3’H expressing lower and FLS expressing higher in the mutant. Although F3’H andFLS are involved in the biosynthesis pathway of anthocyanin, flavone and flavonol, respectively, a part of the substrates that F3’H catalyzes, i.e., dihydroflavonol is also the substrate for FLS(Wang et al ., 2021; Guo et al ., 2019). FLS could catalyze dihydroflavonol to flavonol, while F3’H could hydroxylate the 3’-position of B-ring in flavanone, flavonol and dihydroflavonol (Guo et al ., 2019; Jia et al ., 2019). Therefore, FLS could express higher if F3’H expresses lower due to the competitive relationship of the substrate. In the mutant, the expression profile of F3’H and FLS should result from their competitive relationship for the same substrate.
The accumulation of anthocyanins is affected by ambient temperature in plants, and usually, low temperature can stimulate the expression of the relevant genes. Low-temperature treatment would stimulate the gene expression of anthocyanin biosynthesis, such as PAL, C4H, 4CL, CHS, CHI, F3H, F3’H, DFR, ANS and UGT75C1 , and regulating factors MYB (Dai et al ., 2022). In this study, cold temperature also could promote the expression levels of PAL, C4H, FLS, F3H, F3’H, some of 4CL, CHS and MYB but would reduce the expression of ANS, CHI, and some of 4CL, CHS andMBY . The expression of ANS reduces at low temperature, which seems contradictory to the pigment accumulation because ANSis an important enzyme that can catalyze the production of anthocyanins monomers from leucoanthocyanidins (Saito et al ., 1999; Wanget al ., 2021). However, ANS not only catalyzes the synthesis of anthocyanins, but also has the same activity as FL S (Turnbull et al ., 2000; Wellmann et al ., 2006; Wilmouthet al ., 2002). Meanwhile, in Arabidopsis, AtFLS1and/or AtANS would have partial activity of the F3H enzyme (Owens et al. , 2008). Additionally, FLS could partially complement ANS in Arabidopsis tt6 mutants (Owens et al. , 2008). FLSand ANS shared highly similar polypeptides, and both could react with the leucoanthocyanidins (Turnbull et al ., 2004).
Consequently, due to the species-specificity and complexity of anthocyanins, low temperature primarily induces the accumulation of anthocyanins in most plants (Li et al ., 2017; Ubi et al ., 2006; Jiang et al .,2022; Zhang et al ., 2012), while in some plants, it would reduce the accumulation of anthocyanins (Zhuet al. , 2020; Mao et al ., 2022). Hence, although the biosynthesis pathway of anthocyanins in Arabidopsis has been well explained, the specificity of anthocyanins biosynthesis and regulation in broccoli is yet to be discovered. In the present investigation, the 43-bp deletion in the F3’H CDS decreased the expression of the gene and ultimately reduced the accumulation of cyanidin and delphinidins in some broccoli cultivars and accessions, which would keep green buds at cold temperature. However, the application accuracy of the marker bropsK49 in the germplasm accessions of Brassica was 96 percent. This showed that besides the 43-bp deletion in F3’H, there might be different mutations occurring in F3’H or other relative factors affecting the accumulation of anthocyanins in Brassica at low temperature. This has been illustrated by other reports, where a 68-bp deletion in the DNA sequences of F3’H and a 1-bp insert in the exon of DFR are responsible for green head and green leaves in broccoli and kale, respectively (Liu et al. , 2021; Tang et al. , 2017).
Data Availability: Raw sequence data were deposited under NCBI BioProject PRJNA872779.
Funding: Funding was provided by Zhejiang Provincial Natural Science Foundation (LGN20C150005, LD22C150002), the Science and Technology Department of Zhejiang Province (2021C02065-4, 2022C02051, 2021C02042), key projects of international scientific and technological innovation cooperation between governments (2017YFE0114500), and the European Union’s Horizon 2020 research and innovation program under Grant agreement No. 774244.
Conflicts of Interest: The authors declare no conflict of interest.