BACKGROUND
anthocyanins are widely distributed water-soluble pigments belonging to the flavanoid group of phytochemicals.
over  <dig> types of anthocyanins have been identified  <cit> .
these mainly possess six common aglycones  and various types of glycosylated and acylated compounds  <cit> .
anthocyanins protect plants from strong light, heavy metals, and pathogens, and play an important role in flowers  <cit> .
as they have low toxicity and vary in color they are often used as healthier alternatives to synthetic colorants  <cit> .
previous studies have confirmed that anthocyanins provide antioxidants for human health that protect against a broad range of diseases, including a high blood cholesterol level, cardiovascular disease, and ultraviolet radiation damage  <cit> .
carrots  are among the  <dig> most economically important vegetable crops grown worldwide  <cit> .
carrot cultivars appear in five taproot color types: purple, orange, yellow, red, and white.
although orange carrot cultivars  account for the majority of production, purple carrots  are enjoying increased popularity, largely because they contain high amounts of anthocyanin in their flesh taproots.
purple carrot cultivars have existed for over 3000 years, and are much older than orange carrot cultivars  <cit> .
anthocyanins from purple carrots are commonly used as natural food colorants in candies, ice cream, and beverages, this is because they remain stable when exposed to heat and light, and have increased ph values  <cit> .
purple carrots mainly contain cyanidin-based anthocyanins; some cultivars also contain trace amounts of peonidin- or pelargonidin-based anthocyanins in their taproots  <cit> .
the anthocyanin biosynthesis pathway has been extensively studied in numerous plant species, including bilberry , grape , apple , arabidopsis , mitchell petunia , and sweet potato   <cit> .
two classes of genes participate in the anthocyanin biosynthesis pathway: structural genes and regulatory genes.
structural genes encode enzymes that directly catalyze reaction steps leading to the formation of anthocyanins; the transcription of these genes is controlled by regulatory genes, such as myb, bhlh, and wd <dig> genes  <cit> .
structural genes that participate in anthocyanin biosynthesis have been identified in mumerous plant species  <cit> .
some functional genes participating in this pathway have also been identified in carrots; these include the phenylalanine ammonia-lyase , chalcone synthase , flavanone 3-hydroxylase , dihydroflavonol 4-reductase , and leucoanthocyanidin dioxygenase  genes  <cit> .
we described the presence of the udp-galactose: cyanidin 3-o-galactosyltransferase  in the purple carrot cultivars in our previous study  <cit> .
however, cinnamate 4-hydroxylase , 4-coumaroyl-coenzyme a ligase , chalcone-flavonone isomerase , and flavonoid 3'-hydroxylase  genes have not been identified in carrots.
to obtain insights into differences in anthocyanin biosynthesis between purple and non-purple carrot cultivars, we cloned six novel structural genes involved in anthocyanin biosyntheses from taproot-derived cdna.
the expression patterns of  <dig> structural genes in the taproots of three purple and six non-purple carrot cultivars were analyzed at the transcriptional level.
the accumulation of anthocyanins was determined in parallel.
the aim of this work was to determine the stage at which the anthocyanin biosynthesis pathway switches off, thus leading to loss of anthocyanins in non-purple carrot cultivars.
RESULTS
taproot color of nine carrot cultivars at different development stages
anthocyanins accumulate in different parts of carrot taproots at different development stages; this leads to taproots displaying a purple or dark color.
in 60-day-old carrots, anthocyanins accumulated in the cortex and xylem of ‘deep purple’ and ‘purple 68’ cultivar taproots, but only in the cortex of ‘tianzi2hao’ .
in the six other carrot cultivars, no purple or dark coloring was detected in the taproot.
in 90- and 120-day-old carrots, anthocyanins accumulated in the cortex, phloem, and xylem of ‘deep purple’, ‘purple 68’, and ‘tianzi2hao’ taproots.
in the six other carrot cultivars, no purple or dark coloring was detected in these taproot parts, with the exception of the hypocotyl-derived root part of 90- and 120-day-old ‘kuroda’, 90-day-old ‘sanhongliucun’, and 120-day-old ‘junchuanhong’; these displayed dark color in the epidermis when exposed to light .
as hypocotyl-derived parts of the taproots of 120-day-old ‘sanhongliucun’ and 90-day-old ‘junchuanhong’ cultivars were not exposed to light, purple or dark coloring was not detectable in their epidermis.figure  <dig>
colors of the cross-sections of various carrot taproots at three different stages.
cultivar abbreviations: dpp, deep purple; pp <dig>  purple 68; tz2h, tianzi2hao; krd, kuroda; shlc, sanhongliucun; jch, junchuanhong; bj, bejo1719; qth, qitouhuang; by, baiyu.
epidermis color of the taproots of nine carrots cultivars at three different stages.
anthocyanin content in the taproots of nine carrot cultivars at different developmental stages
total anthocyanin content in the taproots of the three purple carrot cultivars  increased significantly during development .
these cultivars accumulated anthocyanins more efficiently in their taproots between the 60- and 90-day-old stages than between the 90- and 120-day-old stages.
of these three purple carrot cultivars, ‘purple 68’ showed the highest anthocyanin content in the taproots at all three stages.
in the taproots of the three orange carrot cultivars , anthocyanin was not detected at the 60-day-old stage.
at the 90-day-old stage, the anthocyanin content in ‘kuroda’ and ‘sanhongliucun’ taproots was  <dig>  mg/100 g fresh weight  and  <dig>  mg/100 g fw, respectively; anthocyanin was not detected in ‘junchuanhong’ taproots at this stage.
the taproots of 120-day-old ‘kuroda’, ‘sanhongliucun’, and ‘junchuanhong’ contained  <dig> ,  <dig> , and  <dig>  mg/100 g fw anthocyanins, respectively.
anthocyanins did not accumulate in the taproots of the three yellow carrot cultivars in any of the three stages.figure  <dig>
total anthocyanin content in the taproot of various carrot cultivars at three different stages.
values are means of three independent experiments and are calculated as cyanidin 3-o-galactoside equivalents.
analysis of carrot structural genes for cyanidin-based anthocyanin biosynthesis
as cyanidin-based anthocyanins represent almost all anthocyanin content in carrots, we analyzed structural genes for cyanidin-based anthocyanin biosynthesis.
we propose the following cyanidin-based anthocyanin biosynthesis pathway in purple carrots .
pal, ca4h, and 4cl code enzymes implicated in the general phenylpropanoid pathway of anthocyanin biosynthesis in carrots.
chs, chi, f3h, f3′h, dfr, ldox, and ucgt code enzymes involved in the anthocyanin pathway of anthocyanin biosynthesis in carrots.
the full names of these genes and their corresponding accession numbers in genbank and carrotdb are listed in table  <dig>  <cit> .figure  <dig>
schematic of the proposed cyanidin-based anthocyanin biosynthetic pathway.
enzymes not identified in carrots are marked in red.
cyanidin-based anthocyanin biosynthetic genes annotation and accession numbers in genbank or carrotdb
gene family
annotation
genbank id
scaffolds id in carrotdb
predict genes id in carrotdb
pal
pal1
pal3/pal4
ca4h
ca4h1
ca4h2
4cl
4cl1
4cl2
chs
chs1
chs2/ chs9
chi
chi1
f3h
f3h1
f3'h
f3'h1
dfr
dfr1
ldox
ldox1/ ldox2
gene abbreviations: pal, phenylalanine ammonia-lyase; ca4h, cinnamate 4-hydroxylase; 4cl, 4-coumaroyl-coenzyme a ligase; chs, chalcone synthase; chi, chalcone–flavonone isomerase; f3h, flavanone 3-hydroxylase; f3′h, flavonoid 3′- hydroxylase; dfr, dihydroflavonol 4-reductase; ldox, leucoanthocyanidin dioxygenase.
pal <dig> , pal <dig> , pal <dig> , chs <dig> , chs <dig> , chs <dig> , f3h <dig> , dfr <dig> , ldox <dig> , and ldox <dig> genes  were present in genbank.
the ca4h <dig>  ca4h <dig>  4cl <dig>  4cl <dig>  chi <dig>  and f3′h <dig> genes were identified in carrotdb using a blast-based search tool; these genes were further identified by cloning and sequencing genes from the ‘deep purple’ cultivar.
the nucleotide and deduced amino acid sequences of these genes were deposited at the national center for biotechnology information .
the ncbi accession numbers of ca4h <dig>  ca4h <dig>  4cl <dig>  4cl <dig>  chi <dig>  and f3′h <dig> are listed in table  <dig>
the pal <dig> and pal <dig>  chs <dig> and chs <dig>  and ldox <dig> and ldox <dig> genes were considered as allelic genes because they share very high identity in their nucleotide acid sequences ; furthermore, only one gene corresponding to each pair of genes was found in the carrotdb.
therefore, only one pair of primers, specific to each pair of genes, was used for quantitative real-time polymerase chain reaction .
expression of cyanidin-based anthocyanin biosynthetic genes in the taproot at the 60-day-old stage
purple carrot cultivars had accumulated anthocyanins in taproots by the 60-day-old stage.
nucleotide sequences of primer pairs used for qrt-pcr specific to each anthocyanin biosynthetic genes are given in table  <dig>  pal3/pal <dig>  ca4h <dig>  4cl <dig>  chs <dig>  chi <dig>  f3h <dig>  f3′h <dig>  dfr <dig>  and ldox1/ldox <dig> genes showed significantly higher transcript abundance in the taproots of ‘deep purple’, ‘purple 68’, and ‘tianzi2hao’ than in the taproots of ‘kuroda’, ‘sanhongliucun’, ‘junchuanhong’, ‘bejo1719’, ‘qitouhuang’, and ‘baiyu’ .
correlation analysis results revealed that chs <dig>  chi <dig>  f3h <dig>  f3′h <dig>  dfr <dig>  and ldox1/ldox <dig> were highly correlated with anthocyanin presence among the genes encoding enzymes implicated in the anthocyanin pathway in anthocyanin biosynthesis .table  <dig>
nucleotide sequences of primers specific to cyanidin-based anthocyanin biosynthetic genes and
actin1
gene used for qrt-pcr
gene
forward primer 5′-3′
reverse primer 5′-3′
pal1
pal3/pal4
ca4h1
ca4h2
4cl1
4cl2
chs1
chs2/chs9
chi1
f3h1
f3′h1
dfr1
ldox1/ldox2
dcactin1expression of cyanidin-based anthocyanin biosynthetic pathway genes in 60-day-old stage carrot taproots.
the mrna level of actin <dig> was defined as  <dig>  data represents means of biological triplicate qrt-pcrs ± sd.
statistical analysis of differences was performed using duncan’s multiple range test.
significant differences are indicated by different letters at the p <  <dig>  level.
among the cultivars, ‘deep purple’ showed the highest taproot mrna levels of pal3/pal <dig>  ca4h <dig>  4cl <dig>  chs <dig>  f3h <dig>  f3′h <dig>  and ldox1/ldox <dig>  ‘purple 68’ had the highest taproot mrna levels of pal <dig>  4cl <dig>  and dfr <dig>  ‘tianzi2hao’ had the highest taproot mrna levels of ca4h <dig> and chi <dig>  and ‘kuroda’ showed the highest mrna levels of chs2/chs <dig> in the taproots.
transcript levels of pal <dig> and ca4h <dig> were lower than pal3/pal <dig> and ca4h <dig>  respectively, in the taproots of all carrot cultivars.
transcript levels of 4cl <dig> in the taproots of ‘kuroda’, ‘junchuanhong’, ‘bejo1719’, ‘qitouhuang’, and ‘baiyu’ were higher than those of 4cl <dig>  but lower than observed in the taproots of ‘deep purple’, ‘purple 68’, ‘tianzi2hao’, and ‘sanhongliucun’.
in the three purple carrot cultivars, mrna abundance of chs2/chs <dig> in the taproot was significantly lower than that of chs <dig>  in the six other carrot cultivars, chs2/chs <dig> showed similar mrna expression levels as chs <dig> in the taproots .
discussion
in many plants, anthocyanin biosynthesis can be light independent or light induced.
the light-independent anthocyanin biosynthesis pathway has been investigated in other plant species, including apple and sweet potato  <cit> .
light-induced anthocyanin biosynthesis has been observed in several plant species, including apple, grape, and mitchell petunia  <cit> .
in this work, purple carrot cultivars could produce rich anthocyanins in taproots light independently.
in contrast, yellow carrot cultivars failed to produce anthocyanins in the taproots, while orange carrots could only produce small amounts of anthocyanins in the epidermis of hypocotyl-derived root parts light dependently.
to determine genes involved in light-independent anthocyanin biosynthesis in purple carrots and those responsible for losses of light-independent anthocyanin production in non-purple carrots, we analyzed anthocyanin pathway structural genes in three purple and six non-purple cultivars.
some structural genes  involved in anthocyanin biosynthesis have been previously cloned, and their expression profiles analyzed under ultraviolet light  <cit> .
our previous study identified that ucgt <dig> expressed in purple carrot cultivars  <cit> .
in this study, six structural genes  present in purple  and non-purple  carrot cultivars were cloned and sequenced.
pal3/pal <dig>  ca4h <dig>  4cl <dig>  chs <dig>  f3h <dig>  f3′h <dig>  dfr <dig>  and ldox1/ldox <dig> genes may be involved in the anthocyanin biosynthesis in purple carrots as these genes showed higher expression levels in purple carrots than non-purple carrots.
chs <dig>  chi <dig>  f3h <dig>  f3′h <dig>  dfr <dig>  and ldox1/ldox <dig> genes were strongly correlated with the presence of anthocyanins, as indicated by high gene expression levels in the taproots of purple carrot cultivars but no or scarce expression in the taproots of non-purple carrot cultivars.
this suggests that these genes predominantly lead to loss of anthocyanin production in non-purple carrot cultivars.
a similar result was observed in sweet potatoes  <cit> .
loss of anthocyanins in the taproots of non-purple carrots is possibly caused by inactivation of regulator genes such as myb, bhlh, and wd <dig> genes.
future investigation will focus on transcription factors controlling expression of these structural genes to identify the key gene involved in anthocyanin production in purple carrot cultivars and responsible for anthocyanin loss in non-purple carrot cultivars.
the taproots of purple carrots are rich in anthocyanins, reaching a maximum of 175 mg/100 g fw in some cultivars  <cit> .
in this study, anthocyanin content varied significantly in the taproots of the three purple carrot cultivars at the three different stages, as visually indicated by the degree of root coloring.
anthocyanin content in 60-day-old stage taproots of the three purple carrot cultivars was comparable to previously reported for several genotypes of purple carrots  <cit> .
in 90- and 120-day-old purple carrots, anthocyanin content was higher than previously reported, this may be because of the different growth conditions and harvest time of the carrots  <cit> ; anthocyanin accumulation in carrots is sensitive to variations in growth conditions, such as temperature, light, and nutrients  <cit> .
anthocyanin contents in taproots of the three purple carrot cultivars at the 120-day-old stage were higher than those in strawberries, red onion, and red grapes but lower than that observed in blueberries  <cit> .
anthocyanins accumulated in the epidermis of the hypocotyl-derived root part of the three orange carrot cultivars after they were exposed to light.
this suggested that a light-induced anthocyanin biosynthesis pathway is found in these orange carrot cultivars.
CONCLUSIONS
purple carrot cultivars produced rich amounts of anthocyanins in the taproots light independently, whereas non-purple cultivars did not.
the anthocyanin content in purple carrot cultivars increased as root growth occurred.
six novel candidate structural genes that existed in both purple and non-purple carrots were successfully cloned and sequenced.
pal3/pal <dig>  ca4h <dig>  4cl <dig>  chs <dig>  chi <dig>  f3h <dig>  f3′h <dig>  dfr <dig>  and ldox1/ldox <dig> may participate in anthocyanin biosynthesis in the taproots of purple carrot cultivars.
chs <dig>  chi <dig>  f3h <dig>  f3′h <dig>  dfr <dig>  and ldox1/ldox <dig> were unexpressed or scarcely expressed in non-purple carrots, thus may lead to the loss of light-independent anthocyanins production in orange and yellow carrots.
our results provide new insights into anthocyanin biosynthesis in carrots at the molecular level and are of importance for other root vegetables.

 anthocyanins are widely distributed water-soluble pigments belonging to the flavanoid group of phytochemicals. over types of anthocyanins have been identified. these mainly possess six common aglycones and various types of glycosylated and acylated compounds. anthocyanins protect plants from strong light, heavy metals, and pathogens, and play an important role in flowers. as they have low toxicity and vary in color they are often used as healthier alternatives to synthetic colorants. previous studies have confirmed that anthocyanins provide antioxidants for human health that protect against a broad range of diseases, including a high blood cholesterol level, cardiovascular disease, and ultraviolet radiation damage. carrots are among the most economically important vegetable crops grown worldwide. carrot cultivars appear in five taproot color types: purple, orange, yellow, red, and white. although orange carrot cultivars account for the majority of production, purple carrots are enjoying increased popularity, largely because they contain high amounts of anthocyanin in their flesh taproots. purple carrot cultivars have existed for over 3000 years, and are much older than orange carrot cultivars. anthocyanins from purple carrots are commonly used as natural food colorants in candies, ice cream, and beverages, this is because they remain stable when exposed to heat and light, and have increased ph values. purple carrots mainly contain cyanidin-based anthocyanins; some cultivars also contain trace amounts of peonidin- or pelargonidin-based anthocyanins in their taproots. the anthocyanin biosynthesis pathway has been extensively studied in numerous plant species, including bilberry, grape, apple, arabidopsis, mitchell petunia, and sweet potato. two classes of genes participate in the anthocyanin biosynthesis pathway: structural genes and regulatory genes. structural genes encode enzymes that directly catalyze reaction steps leading to the formation of anthocyanins; the transcription of these genes is controlled by regulatory genes, such as myb, bhlh, and wd genes. structural genes that participate in anthocyanin biosynthesis have been identified in mumerous plant species. some functional genes participating in this pathway have also been identified in carrots; these include the phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase genes. we described the presence of the udp-galactose: cyanidin 3-o-galactosyltransferase in the purple carrot cultivars in our previous study. however, cinnamate 4-hydroxylase, 4-coumaroyl-coenzyme a ligase, chalcone-flavonone isomerase, and flavonoid 3'-hydroxylase genes have not been identified in carrots. to obtain insights into differences in anthocyanin biosynthesis between purple and non-purple carrot cultivars, we cloned six novel structural genes involved in anthocyanin biosyntheses from taproot-derived cdna. the expression patterns of structural genes in the taproots of three purple and six non-purple carrot cultivars were analyzed at the transcriptional level. the accumulation of anthocyanins was determined in parallel. the aim of this work was to determine the stage at which the anthocyanin biosynthesis pathway switches off, thus leading to loss of anthocyanins in non-purple carrot cultivars.  taproot color of nine carrot cultivars at different development stages anthocyanins accumulate in different parts of carrot taproots at different development stages; this leads to taproots displaying a purple or dark color. in 60-day-old carrots, anthocyanins accumulated in the cortex and xylem of ‘deep purple’ and ‘purple 68’ cultivar taproots, but only in the cortex of ‘tianzi2hao’. in the six other carrot cultivars, no purple or dark coloring was detected in the taproot. in 90- and 120-day-old carrots, anthocyanins accumulated in the cortex, phloem, and xylem of ‘deep purple’, ‘purple 68’, and ‘tianzi2hao’ taproots. in the six other carrot cultivars, no purple or dark coloring was detected in these taproot parts, with the exception of the hypocotyl-derived root part of 90- and 120-day-old ‘kuroda’, 90-day-old ‘sanhongliucun’, and 120-day-old ‘junchuanhong’; these displayed dark color in the epidermis when exposed to light. as hypocotyl-derived parts of the taproots of 120-day-old ‘sanhongliucun’ and 90-day-old ‘junchuanhong’ cultivars were not exposed to light, purple or dark coloring was not detectable in their epidermis.figure colors of the cross-sections of various carrot taproots at three different stages. cultivar abbreviations: dpp, deep purple; pp purple 68; tz2h, tianzi2hao; krd, kuroda; shlc, sanhongliucun; jch, junchuanhong; bj, bejo1719; qth, qitouhuang; by, baiyu. epidermis color of the taproots of nine carrots cultivars at three different stages. anthocyanin content in the taproots of nine carrot cultivars at different developmental stages total anthocyanin content in the taproots of the three purple carrot cultivars increased significantly during development. these cultivars accumulated anthocyanins more efficiently in their taproots between the 60- and 90-day-old stages than between the 90- and 120-day-old stages. of these three purple carrot cultivars, ‘purple 68’ showed the highest anthocyanin content in the taproots at all three stages. in the taproots of the three orange carrot cultivars, anthocyanin was not detected at the 60-day-old stage. at the 90-day-old stage, the anthocyanin content in ‘kuroda’ and ‘sanhongliucun’ taproots was  mg/100 g fresh weight and  mg/100 g fw, respectively; anthocyanin was not detected in ‘junchuanhong’ taproots at this stage. the taproots of 120-day-old ‘kuroda’, ‘sanhongliucun’, and ‘junchuanhong’ contained,, and  mg/100 g fw anthocyanins, respectively. anthocyanins did not accumulate in the taproots of the three yellow carrot cultivars in any of the three stages.figure total anthocyanin content in the taproot of various carrot cultivars at three different stages. values are means of three independent experiments and are calculated as cyanidin 3-o-galactoside equivalents. analysis of carrot structural genes for cyanidin-based anthocyanin biosynthesis as cyanidin-based anthocyanins represent almost all anthocyanin content in carrots, we analyzed structural genes for cyanidin-based anthocyanin biosynthesis. we propose the following cyanidin-based anthocyanin biosynthesis pathway in purple carrots. pal, ca4h, and 4cl code enzymes implicated in the general phenylpropanoid pathway of anthocyanin biosynthesis in carrots. chs, chi, f3h, f3′h, dfr, ldox, and ucgt code enzymes involved in the anthocyanin pathway of anthocyanin biosynthesis in carrots. the full names of these genes and their corresponding accession numbers in genbank and carrotdb are listed in table .figure schematic of the proposed cyanidin-based anthocyanin biosynthetic pathway. enzymes not identified in carrots are marked in red. cyanidin-based anthocyanin biosynthetic genes annotation and accession numbers in genbank or carrotdb gene family annotation genbank id scaffolds id in carrotdb predict genes id in carrotdb pal pal1 pal3/pal4 ca4h ca4h1 ca4h2 4cl 4cl1 4cl2 chs chs1 chs2/ chs9 chi chi1 f3h f3h1 f3'h f3'h1 dfr dfr1 ldox ldox1/ ldox2 gene abbreviations: pal, phenylalanine ammonia-lyase; ca4h, cinnamate 4-hydroxylase; 4cl, 4-coumaroyl-coenzyme a ligase; chs, chalcone synthase; chi, chalcone–flavonone isomerase; f3h, flavanone 3-hydroxylase; f3′h, flavonoid 3′- hydroxylase; dfr, dihydroflavonol 4-reductase; ldox, leucoanthocyanidin dioxygenase. pal, pal, pal, chs, chs, chs, f3h, dfr, ldox, and ldox genes were present in genbank. the ca4h ca4h 4cl 4cl chi and f3′h genes were identified in carrotdb using a blast-based search tool; these genes were further identified by cloning and sequencing genes from the ‘deep purple’ cultivar. the nucleotide and deduced amino acid sequences of these genes were deposited at the national center for biotechnology information. the ncbi accession numbers of ca4h ca4h 4cl 4cl chi and f3′h are listed in table  the pal and pal chs and chs and ldox and ldox genes were considered as allelic genes because they share very high identity in their nucleotide acid sequences; furthermore, only one gene corresponding to each pair of genes was found in the carrotdb. therefore, only one pair of primers, specific to each pair of genes, was used for quantitative real-time polymerase chain reaction. expression of cyanidin-based anthocyanin biosynthetic genes in the taproot at the 60-day-old stage purple carrot cultivars had accumulated anthocyanins in taproots by the 60-day-old stage. nucleotide sequences of primer pairs used for qrt-pcr specific to each anthocyanin biosynthetic genes are given in table  pal3/pal ca4h 4cl chs chi f3h f3′h dfr and ldox1/ldox genes showed significantly higher transcript abundance in the taproots of ‘deep purple’, ‘purple 68’, and ‘tianzi2hao’ than in the taproots of ‘kuroda’, ‘sanhongliucun’, ‘junchuanhong’, ‘bejo1719’, ‘qitouhuang’, and ‘baiyu’. correlation analysis results revealed that chs chi f3h f3′h dfr and ldox1/ldox were highly correlated with anthocyanin presence among the genes encoding enzymes implicated in the anthocyanin pathway in anthocyanin biosynthesis.table nucleotide sequences of primers specific to cyanidin-based anthocyanin biosynthetic genes and actin1 gene used for qrt-pcr gene forward primer 5′-3′ reverse primer 5′-3′ pal1 pal3/pal4 ca4h1 ca4h2 4cl1 4cl2 chs1 chs2/chs9 chi1 f3h1 f3′h1 dfr1 ldox1/ldox2 dcactin1expression of cyanidin-based anthocyanin biosynthetic pathway genes in 60-day-old stage carrot taproots. the mrna level of actin was defined as data represents means of biological triplicate qrt-pcrs ± sd. statistical analysis of differences was performed using duncan’s multiple range test. significant differences are indicated by different letters at the p <  level. among the cultivars, ‘deep purple’ showed the highest taproot mrna levels of pal3/pal ca4h 4cl chs f3h f3′h and ldox1/ldox ‘purple 68’ had the highest taproot mrna levels of pal 4cl and dfr ‘tianzi2hao’ had the highest taproot mrna levels of ca4h and chi and ‘kuroda’ showed the highest mrna levels of chs2/chs in the taproots. transcript levels of pal and ca4h were lower than pal3/pal and ca4h respectively, in the taproots of all carrot cultivars. transcript levels of 4cl in the taproots of ‘kuroda’, ‘junchuanhong’, ‘bejo1719’, ‘qitouhuang’, and ‘baiyu’ were higher than those of 4cl but lower than observed in the taproots of ‘deep purple’, ‘purple 68’, ‘tianzi2hao’, and ‘sanhongliucun’. in the three purple carrot cultivars, mrna abundance of chs2/chs in the taproot was significantly lower than that of chs in the six other carrot cultivars, chs2/chs showed similar mrna expression levels as chs in the taproots. discussion in many plants, anthocyanin biosynthesis can be light independent or light induced. the light-independent anthocyanin biosynthesis pathway has been investigated in other plant species, including apple and sweet potato. light-induced anthocyanin biosynthesis has been observed in several plant species, including apple, grape, and mitchell petunia. in this work, purple carrot cultivars could produce rich anthocyanins in taproots light independently. in contrast, yellow carrot cultivars failed to produce anthocyanins in the taproots, while orange carrots could only produce small amounts of anthocyanins in the epidermis of hypocotyl-derived root parts light dependently. to determine genes involved in light-independent anthocyanin biosynthesis in purple carrots and those responsible for losses of light-independent anthocyanin production in non-purple carrots, we analyzed anthocyanin pathway structural genes in three purple and six non-purple cultivars. some structural genes involved in anthocyanin biosynthesis have been previously cloned, and their expression profiles analyzed under ultraviolet light. our previous study identified that ucgt expressed in purple carrot cultivars. in this study, six structural genes present in purple and non-purple carrot cultivars were cloned and sequenced. pal3/pal ca4h 4cl chs f3h f3′h dfr and ldox1/ldox genes may be involved in the anthocyanin biosynthesis in purple carrots as these genes showed higher expression levels in purple carrots than non-purple carrots. chs chi f3h f3′h dfr and ldox1/ldox genes were strongly correlated with the presence of anthocyanins, as indicated by high gene expression levels in the taproots of purple carrot cultivars but no or scarce expression in the taproots of non-purple carrot cultivars. this suggests that these genes predominantly lead to loss of anthocyanin production in non-purple carrot cultivars. a similar result was observed in sweet potatoes. loss of anthocyanins in the taproots of non-purple carrots is possibly caused by inactivation of regulator genes such as myb, bhlh, and wd genes. future investigation will focus on transcription factors controlling expression of these structural genes to identify the key gene involved in anthocyanin production in purple carrot cultivars and responsible for anthocyanin loss in non-purple carrot cultivars. the taproots of purple carrots are rich in anthocyanins, reaching a maximum of 175 mg/100 g fw in some cultivars. in this study, anthocyanin content varied significantly in the taproots of the three purple carrot cultivars at the three different stages, as visually indicated by the degree of root coloring. anthocyanin content in 60-day-old stage taproots of the three purple carrot cultivars was comparable to previously reported for several genotypes of purple carrots. in 90- and 120-day-old purple carrots, anthocyanin content was higher than previously reported, this may be because of the different growth conditions and harvest time of the carrots; anthocyanin accumulation in carrots is sensitive to variations in growth conditions, such as temperature, light, and nutrients. anthocyanin contents in taproots of the three purple carrot cultivars at the 120-day-old stage were higher than those in strawberries, red onion, and red grapes but lower than that observed in blueberries. anthocyanins accumulated in the epidermis of the hypocotyl-derived root part of the three orange carrot cultivars after they were exposed to light. this suggested that a light-induced anthocyanin biosynthesis pathway is found in these orange carrot cultivars.  purple carrot cultivars produced rich amounts of anthocyanins in the taproots light independently, whereas non-purple cultivars did not. the anthocyanin content in purple carrot cultivars increased as root growth occurred. six novel candidate structural genes that existed in both purple and non-purple carrots were successfully cloned and sequenced. pal3/pal ca4h 4cl chs chi f3h f3′h dfr and ldox1/ldox may participate in anthocyanin biosynthesis in the taproots of purple carrot cultivars. chs chi f3h f3′h dfr and ldox1/ldox were unexpressed or scarcely expressed in non-purple carrots, thus may lead to the loss of light-independent anthocyanins production in orange and yellow carrots. our results provide new insights into anthocyanin biosynthesis in carrots at the molecular level and are of importance for other root vegetables. 
anthocyanins are important secondary metabolites in plants, protecting them from damage caused by strong light, heavy metals, and pathogens. six novel structural genes, ca4h ca4h 4cl 4cl chi and f3′h were isolated from purple carrots. this study isolated six novel structural genes involved in anthocyanin biosynthesis in carrots. these results suggest that numerous structural genes are involved in anthocyanin production in the taproots of purple carrot cultivars and in the loss of anthocyanin production in non-purple carrots. our results provide new insights into anthocyanin biosynthesis at the molecular level in carrots and for other root vegetables. purple carrot cultivars accumulate rich cyanidin-based anthocyanins in a light-independent manner in their taproots whereas other carrot color types do not. molecular mechanisms underlying anthocyanin accumulation in purple carrot cultivars and loss of anthocyanin production in non-purple carrot cultivars remain unknown. the taproots of the three purple carrot cultivars were rich in anthocyanin, and levels increased during development. conversely, the six non-purple carrot cultivars failed to accumulate anthocyanins in the underground part of taproots. the expression profiles of these genes, together with other structural genes known to be involved in anthocyanin biosynthesis, were analyzed in three purple and six non-purple carrot cultivars at the 60-day-old stage. pal3/pal ca4h and 4cl expression levels were higher in purple than in non-purple carrot cultivars. among the analyzed structural genes, pal3/pal ca4h 4cl chs chi f3h f3′h dfr and ldox1/ldox may participate in anthocyanin biosynthesis in the taproots of purple carrot cultivars. unexpressed or scarcely expressed genes in the taproots of non-purple carrot cultivars may be caused by the inactivation of regulator genes. chs chi f3h f3′h dfr and ldox1/ldox may lead to loss of light-independent anthocyanin production in orange and yellow carrots. furthermore, they are important nutrients for human health. carrots are among the most economically important vegetable crops grown worldwide.
 carrots are among the most economically important vegetable crops grown worldwide. purple carrot cultivars accumulate rich cyanidin-based anthocyanins in a light-independent manner in their taproots whereas other carrot color types do not. anthocyanins are important secondary metabolites in plants, protecting them from damage caused by strong light, heavy metals, and pathogens. furthermore, they are important nutrients for human health. molecular mechanisms underlying anthocyanin accumulation in purple carrot cultivars and loss of anthocyanin production in non-purple carrot cultivars remain unknown.  the taproots of the three purple carrot cultivars were rich in anthocyanin, and levels increased during development. conversely, the six non-purple carrot cultivars failed to accumulate anthocyanins in the underground part of taproots. six novel structural genes, ca4h ca4h 4cl 4cl chi and f3′h were isolated from purple carrots. the expression profiles of these genes, together with other structural genes known to be involved in anthocyanin biosynthesis, were analyzed in three purple and six non-purple carrot cultivars at the 60-day-old stage. pal3/pal ca4h and 4cl expression levels were higher in purple than in non-purple carrot cultivars. expression of chs chi f3h f3′h dfr and ldox1/ldox was highly correlated with the presence of anthocyanin as these genes were highly expressed in purple carrot taproots but not or scarcely expressed in non-purple carrot taproots.  this study isolated six novel structural genes involved in anthocyanin biosynthesis in carrots. among the analyzed structural genes, pal3/pal ca4h 4cl chs chi f3h f3′h dfr and ldox1/ldox may participate in anthocyanin biosynthesis in the taproots of purple carrot cultivars. chs chi f3h f3′h dfr and ldox1/ldox may lead to loss of light-independent anthocyanin production in orange and yellow carrots. these results suggest that numerous structural genes are involved in anthocyanin production in the taproots of purple carrot cultivars and in the loss of anthocyanin production in non-purple carrots. unexpressed or scarcely expressed genes in the taproots of non-purple carrot cultivars may be caused by the inactivation of regulator genes. our results provide new insights into anthocyanin biosynthesis at the molecular level in carrots and for other root vegetables. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords anthocyanin pathwayroot developmentpurple carrotcyanidindaucus carota lgene expressionissue-copyright-statement© the author 2014 