Introduction
Anthocyanins, which endow plants with orange, red, purple, or blue, belong to a kind of second metabolites called flavonoids widely distributed in plants (Springob et al ., 2003; Lepiniec et al ., 2006; Saito et al ., 2013). Besides, anthocyanins are regarded as stress protectants protecting plants against abiotic/biotic stress such as cold, strong sunshine, and microbe infection (Tohge and Fernie, 2017). As anthocyanins are prevalent but not indispensable in plants, the biosynthesis of anthocyanins, which is a part of the flavonoid biosynthesis pathway, is studied widely by relevant mutants, especially in the model plant Arabidopsis thaliana(Winkel-Shirley, 2001; Grotewold, 2006; Saito et al ., 2013). InArabidopsis , many genes associated with anthocyanins biosynthesis were identified by the mutant lines whose seed coat hastransparent testa (tt ) (Li et al ., 2017). The biosynthesis pathway of anthocyanins starts with phenylalanine which converts into 4-coumaroyl CoA by a series of enzymes: phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase (4CL), that’s the general phenylpropanoid pathway (Winkel-Shirley, 2001; Zhang et al ., 2014). Malonyl-CoA and 4-coumaroyl CoA were catalyzed by a series of enzymes such as chalcone synthase (CHS), chalcone isomerase (CHI), and flavone 3-hydroxylase (F3H) to produce dihydroflavonols, which would then convert into anthocyanidins by another series of enzymes, such as F3’H (flavonoid 3’-hydroxylase), DFR (Dihydroflavonol 4-Reductase), ANS (Anthocyanidin Synthase)/LDOX (leucoanthocyanidin dioxygenase). Dihydroflavonols could also be oxidized to flavonols by FLS (flavonol synthase). The anthocyanidins are unstable and usually become stable by glycosylation, methylation and/or acylation (Winkel-Shirley, 2001; Zhang et al ., 2014). The biosynthesis of anthocyanins is also regulated by many factors, including transcription factors (R2R3MYB and bHLH), WD Repeat Proteins (WDR), epigenetic modification, and environmental factors (light, temperature, sugar, hormones, fertilizer, drought, infection of pathogens, etc.) (Espley et al ., 2007; Spelt et al. , 2000; Winkel-Shirley, 2001; Lorenc-Kukula et al. , 2005; Zhang et al ., 2014; Wang et al ., 2020; Yu et al ., 2022).
Environmental factors regulate anthocyanin biosynthesis by modifying the expression of its structural or regulatory genes. Low temperature and strong sunshine could together induce the accumulation of anthocyanins by promoting the expression of CHS, ANS and UFGT in apple skin (Ubi et al ., 2006), while light promotes anthocyanin accumulation in apple fruit skin by MdSIZ1 modifying MdMYB1 (Takoset al., 2006; Zhou et al. , 2017). Anthocyanin rhamnosyl transferases, UGT79B2 and UGT79B3, were regulated by CBF1 (CRT/DRE-binding factor1, also named DREB1B) and increase low temperatures tolerance via modulating anthocyanin accumulation inArabidopsis (Li et al. , 2017). Structure genes of synthesis of anthocyanins, such as CHS, F3’H , F3H , andUFGT , and transcript factor MYB , were regulated by low temperature (Zhang et al ., 2010 and 2012). Low temperature has different influences on anthocyanin biosynthesis in different species (Mao et al. , 2022; Jiang et al. , 2022). The low temperature usually induces the accumulation of anthocyanins in plants, such as Arabidopsis (Li et al ., 2017), apple (Ubi et al ., 2006; Jiang et al .,2022), and kale (Zhang et al ., 2012), while exerts converse function to accumulation of anthocyanins in peach and strawberry (Zhu et al. , 2020; Mao et al ., 2022). Anthocyanin decoration was also important in increasing environmental stress tolerance (Saigo et al ., 2020).
There are several cultivars or accessions with purple leaves, bulbs or curds at normal growth environment in Brassica (Chiu et al. , 2010; Yan et al ., 2019). A Harbinger DNA transposon insertion in the regulatory region of R2R3 MYB transcription factor activated the gene and some anthocyanins structural genes such asF3’H, DFR , and ANS to promote the accumulation of the pigments in the purple cauliflower (Chiu et al. , 2010). A 7600 bp CACTA-like transposon and point mutation and 1-bp insertion inBoMYB2 promoter region were found in purple kohlrabi and purple cabbage, respectively (Yan et al ., 2019). Most broccoli cultivars or accessions show no purple or red bud trait in optimum growth temperature but would turn red or purple in abiotic stressed environments, such as low temperature and strong sunshine. In contrast, some broccoli plants would keep green buds even at cold temperature. In this study, we have investigated the genetics of controlling the keeping-green trait at low temperature (KGLT) and the turning-purple at low temperature (TPLT) of the buds in broccoli (Brassicaoleracea L. var. italica ). Segregating population with about 1000 individuals was constructed, the KGLT trait was fine-mapped, and the candidate gene was cloned. Another segregating population with the same trait and other accessions in Brassica were investigated. This study provides some important information for the genetic breeding of B. oleracea and illuminates the genetic mechanism of the low-temperature effect on buds’ color in broccoli.