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.