- Anthocyanins, one of the important classes of flavonoids are important pigmented compounds in plants which play protective role under different stress conditions.
- Pigmentation is the oldest trait employed for studies in genetics.
- Sir Gregor John Mendel, the founder of modern genetics, studied inheritance of flower and seed color in pea. Mendel’s work on flower color in peas was the initiative to study the genetics of anthocyanin synthesis.
- Barbara Mc Clintock, the discoverer of transposable elements and epigenetic gene regulation, used pigmentation pattern of maize kernels as marker in her research.
- Before the structures of anthocyanins and flavonoids were determined, the color changes were observed with respect to genetic loci. Before the elucidation of the structures, the particular structural alterations of anthocyanins were correlated with single genes in the presence or absence of particular flavonoid.
- Mutations in the anthocyanin genes are easily identified because they have no harmful effect on plant growth and development. More recently, genes involved in the biosynthesis of anthocyanin pigments have been isolated and characterized by using recombinant DNA technologies.
- For elucidating the anthocyanin biosynthetic biosynthetic pathway, two species are particularly studied Maize and Petunia.
- In this context, we are going to describe the genes controlling biosynthesis in Zea mays and Petunia hybrida. The biosynthetic pathways of both the plant species have some major common reactions, but there are some differences regarding the types of anthocyanins produced by each of the above two species.
- Maize can't produce delphinidin whereas Petunia is incapable of producing pelargonidin pigments.
- Genetic engineering of a secondary metabolic pathway aims to either increase or decrease the quantity of a certain compounds or a group of compounds. To increase the production of a group of compounds, two general approaches have been followed.
1. Firstly methods have been employed to change the expression of one or few genes, thereby overcoming specific rate limiting steps in the pathway to shut down competitive pathways and to decrease the catabolism of the product of interest.
2. Secondly attempts have been made to change the expression of regulatory genes that control multiple biosynthesis genes. Flavonoid and anthocyanin biosynthesis was the first target for genetic engineering, as the biosynthetic pathway was well known and the results could easily be observed by changes in flower colour.
- In the past few years several secondary metabolism genes has been over-expressed in the original plant or in other plant species.
- In some cases, over-expression resulted in an improved production of the desired production of the desired products, whereas in other cases only an increase in the level of the direct product of the over-expressed enzyme was achieved.
- The below table shows increased product accumulation due to gene expression of some of the enzymes involved in the metabolic pathway of the plants.
Maize Anthocyanin Pathway
- The anthocyanins generally accumulate in the vacuoles of the aleurone cells late during development of the kernel after abscisic acid; a plant hormone activates the biosynthetic pathway.
- The production of anthocyanin pigments in the aleurone layer of maize endosperm requires the products of both structural and regulatory genes.
- The anthocyanin pathway of maize includes eight known enzymatic genes(a1,a2,bz1,bz2,c2,chi, pr and whp) that catalyze the biosynthesis or transport of anthocyanin and five regulatory genes(b, cl ,pl, r and vp1) that govern the tissue-specific expression of anthocyanin synthesis.
- The over-expression of the maize transcriptions factors C1 and R in combination with the chalcone synthase gene resulted in activation of anthocyanin biosynthesis in rice, causing an increased resistance to fungi.
- The anthocyanin color in the aleurone layer of the endosperm requires dominant alleles at eight loci a1, a2, c1, c2, r, bz1, bz2, vp and r. The regulatory c1 locus is required for the synthesis of anthocyanins in the aleurone and scutellar tissues of maize kernels.
- The mutants can effect embryo and endosperm development (defective kernel, dek, mutants), endosperm development (defective endosperm, mutants) and embryo development (embryo specific, emb, mutants). These mutants affect embryo development at many different times and in different ways.
- The mutants affecting endosperm have also been shown to differentially affect development in other tissues. The genes C, C2, R, A, A2, Bz, Bz2 and Pr are required for the formation of purple anthocyanin in the aleurone tissue of maize, and the recessive gene(s) results in non-purple (red, bronze and colorless).
Transport of Anthocyanins in Zea mays
- In Zea mays, anthocyanins are synthesized in the cytoplasm and they are transported in the vacuole by Multidrug Resistance Transporter (MRP).
- There are two genes namely ZmMrp3 and ZmMrp4 involved in the expression of them in the pigmented aleurones of the maize kernels.
- Phytic acid is the major source of the phosphate content in maize kernels. Phytic acid generally accumulates in the scutellum as a mixture of phytae salts of several cations such as potassium, iron, zinc etc). The phytase enzyme results in degredation of the phytic acid during the seed germination.
- A novel gene ZmMrp4 coding multidrug resistance-associated protein is mainly responsible for lpa1 (low phytic acid) mutation. Lesions in the ZmMrp4 cause lpa1 mutations in maize.
- The mutation caused by this gene leads to the higher level of accumulation of anthocyanins. Depending on the pH of the surrounding where anthocyanins accumulate, their color varies. This lpa1 mutant may be responsible for the color variation of the maize kernels.
Maize genes studied by Barbara Mc Clintock
C’- Dominant allele on the short arm of chromosome 9 that prevents color from being expressed in the aleurone layer of maize kernels, causing a so called “colorless” phenotype (which is actually white or yellow in color). This is also known as inhibitor allele.
C- Recessive allele on the short arm of chromosome 9 that leads to color development.
Bz- Dominant allele on the short arm of chromosome 9 that leads to purple phenotype.
bz- Recessive allele on the short arm of chromosome 9 that leads to dark brown phenotype.
Genes involved in Maize anthocyanin pathway and their role
- Chalcone Synthase(CHS)- Two genes encode the chalcone synthase activity in Maize anthocyanin pathway. colorless2 (c2), is involved in the anthocyanin biosynthesis in seed and white pollen 1(whp1) controls CHS activity in pollen.
- Chalcone Isomerase (CHI) – (chi) encodes the enzyme chalcone isomerase in Maize. Expression of this gene is seen in light grown seedlings (pigmented) and pigmented pericarps but not expressed in unpigmented pericarps.
- Flavanone-3-hydroxylase (F3H) – In Maize, this enzyme is encoded by the gene (fht1). F3H expression directly correlates with the pigmentation levels in kernels and flavonol level in anthers.
- Flavonoid-3’-hydroxylase (F3’H) - This enzyme is encoded by the maize red aleurone1 (pr1) gene. Red Aleurone1 (pr1) describes the kernel color associated with the recessive phenotype phenotype and identifies a specific structure of the seed involved in its expression, the aleurone. This structural gene encoded for the protein flavonoid 3- hydroxylase, an enzyme that is responsible for the production of cyanidin-glucoside, the purple pigment produced in the anthocyanin pathway. The color of the aleurone pr plants is purple due to the accumulation of mostly cyanidin glucoside whereas aleurone of (pr) plants is red due to accumulation of mostly pelargonidin glucoside.
- Dihydroflavonol-4-reductase (DFR) – (a1) gene encodes the DFR enzyme in maize. Mutations at the a1 gene of maize leads to the production of colorless aleurone layer.
- Anthocyanidin synthase (ANS) – (a2) gene controls the enzymatic conversion of leucoanthocyanidins to anthocyanidins. Mutation of the (a2) gene blocks thee enzymatic conversion of leucoanthocyanidins to anthocyanidins. The (a2) genes codes for a protein that shares sequence similarity to the family of 2-oxo-glutarate-dependent-oxygenases like F3H.
- UDP-Glucose Flavonoid-3-Glucosyl transferase (UFGT) - The maize (bz1) gene encodes the activity of this enzyme.
- Glutathione-S-transferase (GST) - In Maize (bz2) gene encodes the activity of this enzyme. Recessive mutations of the bronze (bz2) gene of maize results in bronze pigmentation of the aleurone layer and modify purple plant color to reddish brown. Maize anthocyanin accumulates within the vacuole in the presence of (bz2) gene. Lack of this gene leads to the accumulation of anthocyanins in the cytosol.
Gene Gene product
c2 chalcone synthase
chi1 chalcone isomerase
pr1 flavonoid 3’-hydroxylase
fht1 flavanone 3-hydroxylase
a2 leucoanthocyanidin reductase
bz1 UDP glucose flavonol-3-O-glucosyl transferase
ZmMrp4 Multidrug resistance like transporter
Table 2 : Maize anthocyanin genes and their products:
Regulatory genes of the Maize anthocyanin pathway
- In Maize kernels, anthocyanin biosynthesis is regulated by a combination of two transcription factors R and Cl.
- The R protein shares homology with the basic helix-loop-helix protein encoded by the vertebrate proto-oncogene c-MYC, whereas the C1 protein has homology with the proto-oncogene c-MYB product. Induction of the complete flavonoid pathway has been achieved by the over-expression of the transcription factors R and C1 in undifferentiated maize cultures.
- The expression of the structural genes of the anthocyanin biosynthetic pathway is controlled by the regulatory genes. The intensity and pattern of anthocyanin biosynthesis are influenced by the regulatory genes.
- Each gene determines pigmentation of different parts of the plant. Accumulation of anthocyanins in competent tissues also requires the presence of either C1 (in the seed) or P1 (in the plant tissue).
- Viviparous-1(vp1) controls the anthocyanin pathway in the developing maize seed primarily through regulation of the C1 gene. It is one of the regulatory genes whose product appears to influence the coordinate regulation of the expression of at least two structural genes in the anthocyanin pathway.
- Kernels containing the dominant allele of C1 together, with dominant alleles of other genes in the anthocyanin pathway are deeply pigmented. A number of recessive c1 mutants have been identified which are colorless if homozygous and colored if heterozygous in the presence of c1 wild type allele.
- Aleurones from kernels that are homozygous for a recessive c2 allele have low CHS activity. This low CHS activity leads to the formation of pale or colorless kernels of maize.
- The (pl1) gene is a duplicate of (c1). It is generally described as the dominant gene responsible for the blotched aleurone pigmentation in kernels that were homozygous recessive for c1.
- The (r1/b1) gene family acts as regulatory partners of (c1/pl1) in activating anthocyanin synthesis. The (r1/b1) only differs in the tissue distribution of pigmentation under their control.
- Mutation in the recessive (in1) gene changes the pigment color of homozygous (pr1) aleurone from red to almost black. The mutation in the (in1) gene leads to increased levels of UDP-Glucose Flavonoid-3-Glucosyl transferase (UFGT) enzyme.
- The (pac1) gene is identified as mutational screen for new regulators of the maize anthocyanin pathway. The mutation in the (pac1) gene results in pale pigmentation of the aleurone and the seedling roots. The (pac1) mutants have pale rather than colorless phenotype. Anthocyanin pigment in the aleurone and the scutellum of the maize seed requires the (pac1) locus.
Locus Genes regulated
R CHS, DFR, 3GT
R(S) CHS, DFR, 3GT
R (Sn) CHS, DFR
R (Lc) CHS, DFR
B DFR, 3GT
c1 CHS, DFR, 3GT
P1 CHS, DFR, 3GT
Table 3: Regulatory genes that control the anthocyanin biosynthesis in Maize
Fig 1: Anthocyanin biosynthetic pathway in Maize
Anthocyanin Biosynthetic pathway in Petunia
- The anthocyanin biosynthetic genes can be divided into early and late genes in Petunia. The regulatory division between the early and late genes occurs before and after Flavanone-3-hydroxylase (F3H) in Petunia.
- Thus the regulations of the genes in the flavones and flavonol biosynthetic pathways in Petunia are different from those in their anthocyanin biosynthetic pathways. An1, An2 and An11 are known to control the anthocyanin synthesis. They also control the vacuolar pH in petal cells and the morphology of the seed coat epidermis.
- Loss of anthocyanin pigments is seen in An1, An2 and An11 petals of Petunia which is associated with increase in vacuolar pH.
- Two genetic loci, Ht1 and Ht2 control the flavonoid-3’-hydroxylase activity in the flowers of petunia. Ht1 acts in the limb and tube of the corolla, whereas Ht2 acts only in the corolla tube. The Ht1 and Ht2 genes control 3’-hydroxylation of anthocyanins and flavonols.
- Two genetic loci, Hf1 and Hf2 control F3’,5’H activity in Petunia. Hf1 acts in the corolla, stigma and pollen, whereas Hf2 acts only in the corolla limb.
- Petunia contains the three DFR genes (dfrA, dfrB and dfrC). Among the three dfrA gene is transcribed in floral tissue. The dfrA gene corresponds to the (An6) locus.
- UDP-rhamnose: anthocyanidin-3-glucoside rhamnosyltransferase (3RT)- (Rt) gene encodes the activity of this enzyme in Petunia flower.
- Anthocyanin acyltransferase (AAT)-The activity of this enzyme in Petunia flower is encoded by the gene (Gf).
- The (An13) gene encodes GST enzyme activity in Petunia. This gene also share homology with GSTs. The conjugation of the (An13) gene with Glutathione is required for the transport of anthocyanins into the vacuole.
Locus Gene regulated
An1 DFR, ANS, 3GT, 3RT, AMT, F3, 5’H
An2 DFR, ANS, 3GT, 3RT, AMT, GST
An4 DFR, ANS, 3GT, 3RT, AMT, GST
An11 DFR, ANS, 3RT, AMT, GST
Table 4: Regulatory genes that control the anthocyanin biosynthesis in Petunia
Fig 2: Anthocyanin biosynthetic pathway in Petunia