Introduction

Anthocyanins, one of the important natural water-soluble pigments that determine the colors of flowers, fruits, seed coats, stems, and leaves, are widely found in plants. In addition to responding to biological and abiotic stresses to scavenge oxygen free radicals (Fan et al. 2016; Nakabayashi et al. 2014; Dong et al. 2020; Li et al. 2020), and protecting themselves from high-density light damage (Li et al. 2018; Hatier et al. 2013), anthocyanins also have nutritional and healthcare functions for humans in terms of anti-cancer, disease resistance, and anti-oxidation (Peiffer et al. 2016; Isaak et al. 2017; Mrkvicova et al. 2016). Many studies related to the metabolic pathways of anthocyanins and key genes involved in this pathway have been performed. Plant anthocyanidin biosynthesis belongs to the flavonoid biosynthesis pathway and the precursor of anthocyanin biosynthesis is phenylalanine which is catalyzed by a series of enzymes to finally generate anthocyanins. The main synthase enzymes in this pathway include phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavanone 3′-hydroxylase (F3′H), flavanone 3′5′-hydroxylase (F3′5′H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase/leucoanthocyanidin dioxygenase (ANS/LDOX), and uridine diphosphate glucose-flavonoid glucosyltransferase (UFGT), etc. (Martin and Gerats 1993; Mol et al. 1998; Himi et al. 2005). The expression of structural genes encoding these enzymes is mainly regulated by transcription factors (TFs). There are three main types of TFs involved in regulating anthocyanin biosynthesis, namely MYB, bHLH, and WD40 proteins. These TFs can be combined with corresponding cis-acting elements in structural gene promoters independently or synergistically to regulate the temporal and spatial expression of structural genes, thereby regulating the accumulation of anthocyanins in plants (Koes et al. 2005; Petroni and Tonelli 2011).

The accumulation of anthocyanins in the pericarp of purple wheat has been reported by the interaction of two complementary dominant genes. Previous research found that Pp1 and Pp3 were the genes that determined the purple pericarp in wheat, which were located on chromosomes 7D (Pp-D1) and 2A, respectively (Dobrovolskaya et al. 2006; Tereshchenko et al. 2012). Pp-1 is a homologous gene of ZmC1 in maize and OsC1 in rice. It encodes an MYB TF and activates the structural genes involved in anthocyanin biosynthesis (Reddy et al. 1998; Saitoh et al. 2004; Khlestkina 2013). Pp3 has been shown to be homologous to Lc/R encoding a bHLH TF in maize and Pb/Ra in rice (Khlestkina 2013; Ludwig et al. 1989; Hu et al. 1996). The latest studies identified TaPpm1 (encoding an MYB TF) as the candidate gene for Pp1, and TaPpb1/TaMYC1 (encoding a bHLH TF) for Pp3. There are four variants of TaPpm1(TaPpm1a/b/c/d) in the genic region, and two variants of TaPpb1(TaPpb1a/b) in the promoter region (Jiang et al. 2018). Co-expression of TaMYC1 with ZmC1 could induce the accumulation of anthocyanins in the coleoptile of white pericarp grains (Zong et al. 2017). The interaction of TaPpm1 and TaPpb1 could also co-regulate anthocyanin biosynthesis in the wheat pericarp (Jiang et al. 2018). However, the interaction of these two TFs was most performed in vitro, there was no reported study about the successful generation of progenies with purple grains by crossing with complementary alleles of TaPpm1 and TaPpb1.

Homozygous and heterozygous genotypes have different dosage effects with different numbers of genes, which affects the corresponding phenotypic changes. For example, the dosage of the tb1-ref mutant allele affected maize roots, and homozygous tb1-ref mutant plants showed a greater shoot and root biomass than heterozygotes, resulting in the similarity of the modern maize homozygous tb1-ref mutants to its ancestor Balsas teosinte (Gaudin et al. 2014). The common wheat AGL6 gene was involved in the development of the spikelet meristem, and its overexpression increased the spikelet number per spike. Heterozygous mutant progenies (aaBb or Aabb) obtained by crossing the tetraploid Kronos single mutant plants (aaBB and AAbb) were partially fertile, while the homozygous mutant plants (aabb) were completely sterile (Kong et al. 2022). Likewise, whether the expression of TaPpm1 and TaPpb1 genes had a dosage effect on anthocyanin biosynthesis in pericarp, and grain color was unknown. Additionally, it has been found that different regulatory genes act on different structural genes (Zimmermann et al. 2004; Grotewold et al. 1994; Mano et al. 2007; Yuan et al. 2009; Quattrocchio et al. 2006). In Arabidopsis, R2R3-MYB, bHLH, and WD40 form the MBW complex to activate the expression of late biosynthesis genes, including F3′H, DFR, ANS, and UFGT and ultimately lead to the accumulation of anthocyanins in the plant (Chen and Wang 2019). The MYB-bHLH-WD40 complex activates structural genes including OsCHS, OsCHI, OsF3'H, OsF3H, OsDFR, and OsANS, and thus regulates anthocyanin biosynthesis in rice leaves (Zheng et al. 2019). Nevertheless, it is still unclear which structural genes are regulated by the interaction of TaPpm1 and TaPpb1 in the anthocyanin biosynthesis pathway of the wheat pericarp.

In this study, several selected white pericarp lines in a DH population were constructed earlier by crossing purple-grained parent Nongda3753 (ND3753) with white-grained parent Liangxing99 (LX99) and an EMS-induced mutant of ND3753 was used to investigate the effects of TaPpm1 and TaPpb1 on anthocyanin biosynthesis. The anthocyanin content, the expression of TaPpm1 and TaPpb1 as well as the expression of key structural genes related to anthocyanin biosynthesis were measured and compared at various grain developmental stages. In this way, we can also verify there is a dosage effect in homozygous and heterozygous genotypes about TaPpm1 and TaPpb1, and speculate which structural genes are regulated by the interaction of these two TFs. The purpose of this study was to clarify the regulation mechanism of anthocyanin biosynthesis in the purple pericarp of wheat and to provide the molecular basis for cultivating purple wheat.

Materials and methods

Plant materials and experimental design

A DH population generated by crossing a purple pericarp variety ND3753 with a white pericarp variety LX99 (120 DH lines) was used in this study (Tian et al. 2021). Six white pericarp lines among the DH population with known alleles of TaPpm1 and TaPpb1, and an EMS-induced mutant from ND3753 named M564 with white pericarp were chosen and crossed with each other to generate different allele combinations. Two groups of crosses were made within DH lines (DH41/DH33, DH93/DH10, DH103/DH82) and between DH lines and M564 (DH41/M564, DH93/M564, DH103/M564) (Fig. S1). The developing grains of the progenies from the crossing were collected at 12, 17, 22, 27, and 32 days after pollination (DAP), and were stored in a refrigerator at  − 80 °C. The grains were taken from the middle part of frozen wheat spikes and used for observation, determination of anthocyanin content, and analysis of related gene expression.

Determination of anthocyanin content

The extraction of total anthocyanins used the colorimetric method of Ficco and Zhu (Ficco et al. 2014; Zhu et al. 2015) with some modifications. The grains were ground into a fine powder. A mixture of methanol acidified with 1 N HCl (85:15; v/v) was added to the samples and then stored at 4 °C for 24 h in dark. After centrifugation at 12,000 rpm for 30 s at 4 °C, the supernatants were stored at  − 20 °C, and re-extraction of the sediments was done iterative procedure. The mixed supernatants were filtered using 0.45 μm regenerated cellulose syringe filters. The absorbance values at wavelengths of 530 nm and 650 nm were measured. The anthocyanin content was quantified as (A530-0.25 × A650)/fresh weight.

DNA extraction and gene cloning

Previous studies have shown that the purple pericarp of wheat is co-regulated by two regulatory genes TaPpm1 (7D) and TaPpb1/TaMYC1 (2A) (Jiang et al. 2018). To identify the genotype, we used the published specific primers to amplify TaPpm1 and TaPpb1 in ND3753, LX99, M564, and the white pericarp DH lines respectively. Genomic DNA was extracted from the young leaves by the modified cetyltrimethylammonium bromide (CTAB) method (Devi et al. 2013). Then the extracted DNA was used to amplify the promoter and genic regions of TaPpm1 and TaPpb1 with Tks Gflex™ DNA Polymerase (TaKaRa). The specific primers previously reported (Table S1) were used to amplify the sequence (Jiang et al. 2018). The reaction system of PCR amplification comprised 10 μl 2 × Gflex PCR Buffer, 2.0 μl template DNA, 2.0 μl mixture of left and right primers (10 μM), 5.6 μl ddH2O and 0.4 μl Tks Gflex DNA Polymerase to the final volume of 20 μl. The PCR cycling conditions included 1 min at 95 °C; 34 cycles of 10 s at 98 °C, 15 s at 55–63 °C (according to the different Tm of primers), and 1–3 min at 68 °C; the final extension of 5 min at 68 °C. The PCR product was separated by 1% agarose gel electrophoresis.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

The total RNA was extracted from grains at different developmental stages using the MiniBEST Plant RNA Extraction Kit (TaKaRa), and the first-strand cDNA was obtained by using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). Quantitative real-time PCR (qRT-PCR) was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa). TaActin was used as an internal reference gene to measure the expression of related genes in the anthocyanin biosynthesis pathway. The reaction system was run in the Bio-Rad CFX96 Touch Real-Time PCR Detection System. The expression levels were calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001). All experiments were performed with three biological replicates. Previously reported primers were used with slight changes (Jiang et al. 2018) and the sequences were shown in Table S1.

Results

Gene cloning and genotype determination

The grain pericarp color of the DH population derived from the cross of purple pericarp ND3753 with white pericarp LX99 could be visually divided into purple and white groups (Fig. 1a), which contained 29 and 91 lines and fitted the separation ratio of 1:3 (χ2 = 0.044). These results correspond to the genetic mechanism of two complementary dominant genes. Parents' genotype detection by amplifying and sequencing TaPpm1 and TaPpb1 revealed that ND3753, LX99, and M564 carried alleles of TaPpm1a/TaPpb1a, TaPpm1d/TaPpb1b, and TaPpm1b/TaPpb1a, respectively (Fig. 2a). Same strategy was used to screen the white pericarp DH lines and found that there were three homozygous genotypes including TaPpm1d/TaPpb1a, TaPpm1a/TaPpb1b as well as TaPpm1d/TaPpb1b. The results of genotyping of some lines were shown in Fig. 2b.

Fig. 1
figure 1

Identification of wheat pericarp color. a Phenotypes of ND3753, M564, and LX99. b Phenotypes of the progenies of hybrid combinations by different white pericarp DH lines. c Phenotypes of the progenies of hybrid combinations by white pericarp DH lines and M564. Bars, 1 cm

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Fig. 2
figure 2

Cloning of the promoters and genic regions of TaPpm1 and TaPpb1 and genotyping of some white pericarp lines in the DH population. a Amplification results of promoters and genic regions of TaPpm1 and TaPpb1 in ND3753, M564, and LX99. 1, 2, 3, amplification results of promoters in ND3753, M564, and LX99; 4, 5, 6, amplification results of genic regions in ND3753, M564, and LX99. The agarose gel diagram at the top is the amplification results of TaPpm1, and at the bottom is the amplification results of TaPpb1. b Genotyping of TaPpm1 and TaPpb1 in some white pericarp lines in the DH population. 1, ND3753; 2, LX99; 3–8, DH lines with TaPpm1d/TaPpb1a; 9–14, DH lines with TaPpm1a/TaPpb1b; 15–20, DH lines with TaPpm1d/TaPpb1b. The agarose gel diagram at the top is the genotyping of TaPpm1, and at the bottom is the genotyping of TaPpb1

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Genetic complementation experiment of two transcription factors

The white pericarp DH lines DH41, DH93, and DH103 with TaPpm1a/TaPpb1b as well as DH33, DH10, and DH82 with TaPpm1d/TaPpb1a were chosen to cross each other for generating complementary genotype. Meanwhile, DH41, DH93, and DH103 were also used to make a cross with M564. So, two groups of complementary heterozygous genotypes were obtained, namely TaPpm1a/1d//TaPpb1a/1b and TaPpm1a/1b//TaPpb1a/1b including 3 hybrids separately (Fig. S1). Mature seeds of these 6 F1 plants all emerged purple color, though obviously lighter than that of ND3753 (Fig. 1b and c). This verified that the interaction of two transcription factors recovered anthocyanin biosynthesis but showed a certain dosage effect. Since the cross DH41/DH33 bared relatively deeper color seeds, it was chosen for further studies. Observation color change of seeds at 12, 17, 22, 27, and 32 DAP showed that DH41, DH33, and LX99 did not accumulate anthocyanins during the whole development of grains, whereas ND3753 began to emerge purple color at 17 DAP while DH41/DH33 at 22 DAP (Fig. 3). Correspondingly, measurement of grain anthocyanin content revealed that anthocyanin was not detected in DH41, DH33, and LX99, while its content reached 1.35 mg/100 g in ND3753 at 17 DAP and 0.25 mg/100 g in DH41/DH33 at 22 DAP. Moreover, grains of the final stage of ND3753 and DH41/DH33 contained anthocyanins of 29.74 mg/100 g and 22.26 mg/100 g, respectively (Table 1). In summary, heterozygous complementation of TaPpm1 and TaPpb1 could recover anthocyanin biosynthesis, however, its efficacy at this status was significantly lower than that of homozygous.

Fig. 3
figure 3

Phenotypic observation on pericarp color of different genotypes of regulatory genes at 12, 17, 22, 27, and 32 DAP (days after pollination) during grain development stages

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Table 1 Determination of anthocyanin content (mg/100 g) in grains of different genotypes of regulatory genes at five grain development stages
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Relative expression levels of TaPpm1 and TaPpb1 in different genotypes

To explore the reason why ND3753 accumulated anthocyanins earlier and quicker than DH41/DH33, the qRT-PCR was used to determine the expression levels of TaPpm1 and TaPpb1 in developing grains of different genotypes. LX99 carried double recessive alleles on both loci while ND3753 carried dominant ones, it isn’t unexpected that the two genes remained very low- or un-expression in LX99 while they were highly expressed in ND3753. TaPpm1 expressed highly in ND3753 at the middle grain development stages (17 and 22 DAP) and decrease rapidly at later stages (27 and 32 DAP), whereas TaPpb1 maintained high expression through middle and later stages (Fig. 4). It is also reasonable that TaPpm1 in DH33 and TaPpb1 in DH41 remained low- or un-expression because of corresponding double recessive alleles separately carried in them. What is interesting is that the expression patterns of these two genes in other statuses were very different. Dominant alleles of TaPpm1 exited all in DH41, DH41/DH33, and ND3753, but its expression levels in DH41 and DH41/DH33 were significantly lower than in ND3753 at 17 and 22 DAP. However, when its expression decreased at later stages in ND3753, its expression increased in DH41 and DH41/DH33 and reached a relatively higher level in DH41/DH33 at 32 DAP (Fig. 4a). On the contrary, TaPpb1 in DH41/DH33 and DH33, despite its status of single or double dominant alleles, expressed quite consistent with that of ND3753 at all stages (Fig. 4b). In other words, TaPpb1 can express persistently once its dominant allele exists, whereas expression of TaPpm1 is affected by its own and TaPpb1 dominant allele status. These results implied that TaPpm1 but not TaPpb1 should be the key factor that resulted in anthocyanin reduction in the heterozygous genotype of purple-grained wheat.

Fig. 4
figure 4

Relative expression levels of TaPpm1 and TaPpb1 in different combinations of genotypes on different days after pollination. a Relative expression levels of TaPpm1 at 12, 17, 22, 27, 32 DAP. b Relative expression levels of TaPpb1 at 12, 17, 22, 27, 32 DAP. Data are shown as means ± SD of three biological replicates

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Relative expression levels of structural genes of anthocyanin biosynthesis

TaPpm1 and TaPpb1 should be through regulating structural genes on the anthocyanin biosynthesis pathway to control anthocyanin accumulation in purple-grained wheat. So, expression of structural genes including CHS, CHI, F3H, F3'H, F3′5'H, DFR, and ANS was detected during grain development in different genotypes. Because anthocyanin accumulated only in grains of ND3753 and DH41/DH33, target genes should be those expressed higher at key grain development stages in these two genotypes than that in others without anthocyanin accumulation. Besides, it is also crucial that their expression patterns should correspond with that of TaPpm1 which was the key factor that caused the difference in anthocyanin accumulation. Upon these criteria, CHI and F3H could not be target genes, because their expression levels in some non-purple-grained genotypes were significantly higher than that in ND3753 and DH41/DH33 at 17 and/or 22 DAP stages (Fig. 5b and c). The expression level of CHS in ND3753 was ever-increasing through grain development stages but at 22 DAP was significantly lower than that in DH41/DH33 (Fig. 5a), whereas F3′5'H maintained lower expression during 12–27 DAP in all genotypes but simultaneously increased sharply at 32 DAP in ND3753 and DH41/DH33 (Fig. 5e). So, CHS and F3′5'H also should not be target genes according to their inconsistency expression patterns with that of TaPpm1. On the other hand, expression patterns of F3'H, DFR, and ANS met the criteria of target genes quite well. They all expressed significantly higher in purple-grained genotypes than in nonpurple ones at all grain development stages except ANS at a few stages, and between purple-grained genotypes their expression levels in heterozygous were significantly lower than in homozygous at most stages (Fig. 5d, f and g).

Fig. 5
figure 5

Relative expression levels of seven structural genes in different genotypes of regulatory genes on different days after pollination. Data are shown as means ± SD of three biological replicates

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Discussion

The formation of anthocyanins is regulated by a complex formed by MYB, bHLH, and WD40 transcription factors. In natural materials, WD40 transcription factors have complex functions and have not yet been studied in-depth, and they can all be expressed normally (Strygina and Khlestkina 2019). Therefore, there were two complementary dominant genes controlling the purple pericarp of wheat in previous mapping studies (Tereshchenko et al. 2012; Jiang et al. 2018). In our research, the anthocyanins could not be detected in the pericarp of genotypes with a single transcription factor across the five stages of grain development. The purple appeared only when two transcription factors were present at the same time (Fig. 1 and 3), which was consistent with previous studies. It is worth mentioning that for these two transcription factors, we used two white pericarp lines to obtain purple pericarp seeds of F1 plants through genetic complementarity (Fig. 1b and c), which was consistent with the principle of heterosis utilization. As an extremely important genetic improvement technique in current production practice, heterosis plays an important role in genetic breeding (Jiang et al. 2017). Previous research has shown that gene differential expression between the hybrids and parents is one of the molecular sources of plant heterosis. The differentially expressed genes between the parents and hybrid at the transcriptome level in early maize root tissue development mainly show a complementary expression pattern (Paschold et al. 2012). Similar results were reported in the current study, in which the expression of structural genes related to anthocyanin biosynthesis in the seeds of F1 plants was higher than that of the two parents, resulting in the pericarp of heterozygote DH41/DH33 appearing purple (Fig. 3 and 5, Table 1).

It is difficult for breeders to make the crossing between germplasms with white grains to get progenies with purple grains. A previous study has found that in 102 samples of common hexaploid wheat, 14 materials with purple pericarp contained TaMYC1p (TaPpb1a), and the remaining 88 materials with white pericarp all carried TaMYC1w (TaPpb1b) (Zong et al. 2017). Another study used the developed marker to detect the allelic variation of TaPpm1 and TaPpb1 in 34 varieties of wheat. The four purple-grain varieties all contained TaPpm1a/TaPpb1a, while the genotypes of other non-purple-grain varieties were TaPpm1a/TaPpb1b, TaPpm1b/TaPpb1b, TaPpm1c/TaPpb1b, and TaPpm1d/TaPpb1b respectively (Jiang et al. 2018). It seems that most varieties containing TaPpb1b had white pericarp and thus this allele had epistasis effects on grain color. Therefore, even if these white pericarp materials were crossed with white pericarp carrying the TaPpm1a, their progeny couldn’t produce purple pericarp either. In this study, we successfully obtained purple grains progeny by the selection of two white pericarp DH lines carrying TaPpm1a and TaPpb1a through genotyping (Fig. 1b and c, 2). At present, the use of assisted selection with markers closely linked to important agronomic traits to achieve multi-gene aggregation breeding has become the mainstream of breeding development. For example, molecular markers were used to aggregate two QTLs for resistance to Fusarium head blight (FHB) to accelerate the improvement of disease-resistant and high-yielding varieties (Salameh et al. 2011). In addition, the molecular marker-assisted selection was successfully used to pyramid excellent genes that control different traits in single plants with both disease resistance and high quality (Zheng et al. 2020). Results from this study were in line with these studies and confirmed the valuable roles of molecular markers in increasing the breeding efficiency of traits that was hard to select via conventional breeding.

Since the pericarp is developed from the ovary wall, the heterozygous material for regulatory genes used in this experiment was obtained from the seeds of F1 plants. In the current research, the DH population hybridization showed that the anthocyanin content in the purple pericarp of the heterozygous genotype of the two regulatory genes was lower than that of the homozygous genotype, and there was a certain color gradient (Fig. 1 and 3, Table 1). This may be due to differences in the genetic background of the DH lines, which showed lighter pericarp color than ND3753. Furthermore, the diversity in phenotype may be related to the difference in gene expression (Veitia et al. 2008; Schadt et al. 2003). For instance, the maximum transcriptional levels of the large and small alleles of fw2.2, which is associated with fruit weight, differ by about one week during tomato fruit size development, and changes in the duration of gene expression and differences in total transcriptional levels caused significant differences in fruit weight (Cong et al. 2002). In rice, GS5 expression levels in two near-isogenic lines (NILs) are measured at different times and in tissues. It is found that such expression differences are consistent with grain width and filling stage, and individual plants with more sustained high expression levels can produce wider grains (Li et al. 2011). The expression time and peak period of the cell cycle-related genes in cucumber affect the replication level and cell division of cucumber fruit, thereby affecting fruit development (Fu et al. 2010). Likewise, this study also found that there may be dosage effects of gene expression on anthocyanin biosynthesis. The expression time of the regulatory genes of the DH41/DH33 in the anthocyanin biosynthesis pathway was generally later than that of ND3753, and the overall expression was lower than ND3753 during grain development, as well as the same trend was observed among the structural genes (Fig. 4 and 5). As a result, the different expression amount of these genes may cause different levels of secondary metabolites involved in anthocyanin synthesis, which then eventually leads to less accumulation of anthocyanin content between grains of DH41/DH33 than those of ND3753.

A previous study has used NILs to analyze the specific expression of anthocyanin synthesis structural genes CHI and F3H in the pericarp by using different combinations of Pp alleles by qRT-PCR. The results showed that one of the genes was sufficient to activate the transcription of CHI, while the activation of F3H depended on the coexistence of Pp-D1 and Pp3 (Gordeeva et al. 2015). In addition, Ant1 encoding an R2R3-MYB TF and Ant2 encoding a bHLH TF form a complex to activate anthocyanin synthesis in barley lemma and pericarp by affecting the expression of F3'H and ANS (Gordeeva et al. 2019). Furthermore, A model of C-S-A gene regulation of rice hull pigmentation has been proposed, in which C1 (an R2R3-MYB TF) interacts with S1 (a bHLH TF) and activates the expression of A1 (OsDFR), giving rice hull purple color (Sun et al. 2018). By contrast, with the increased expression of TaPpm1 and TaPpb1 in the purple pericarp, all the structural genes in the anthocyanin biosynthesis pathway were expressed in the purple pericarp, and the expression levels of CHS, F3'H, F3′5'H, DFR, and ANS were significantly higher than those in white pericarp except for CHI and F3H. CHS, CHI, and F3H were expressed in the white pericarp, while F3'H, F3′5'H, DFR, and ANS were very low or almost not expressed in the white pericarp (Fig. 5). Among them, CHS and CHI are key enzymes in the metabolic pathway of flavonoids, in addition to anthocyanins, they are also involved in the biosynthesis of multiple pigment pathways such as proanthocyanidins (PA) and flavonols (Koes et al. 2005; Petroni and Tonelli 2011; Khlestkina et al. 2015). Nevertheless, the expression levels of TaPpm1 were highly correlated with the expression levels of F3'H, DFR, and ANS in grains (Fig. 5d, f and g). Therefore, we speculated that the co-expression of TaPpm1 and TaPpb1 in wheat purple pericarp mainly regulated F3'H, DFR, and ANS, whereas more methods are needed to verify their interaction with these regulatory genes.

Conclusion

The dosage effect of purple grain color in wheat should be due to the different expression of genes involving anthocyanin biosynthesis in different genotypes. This study demonstrated that out of two regulatory genes, only TaPpm1 was decreased expression at early grain developing stages, and consequently down regulated structural genes F3'H, DFR, and ANS and finally reduced anthocyanin accumulation in grains of the heterozygous genotype.