The role of flavonoids as the major red, blue, and purple pigments in plants has gained these secondary products a great deal of attention over the years. From the first description of acid and base effects on plant pigments by Robert Boyle in 1664 to the characterization of structural and

Flavonoid Biosynthesis. A Colorful Model for Genetics, Biochemistry, Cell Biology, and Biotechnology

The r2r3-myb gene family in arabidopsis thaliana

Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks.

Genetics and biochemistry of seed flavonoids

Seed dormancy and germination.

Comprehensive functional data on plant R2R3-MYB transcription factors is still scarce compared to the manifold of their occurrence. Here, we identified the Arabidopsis (Arabidopsis thaliana) R2R3-MYB transcription factor MYB12 as a flavonol-specific activator of flavonoid biosynthesis. Transient expression in Arabidopsis protoplasts revealed a high degree of functional similarity between MYB12 and the structurally closely related factor P from maize (Zea mays). Both displayed similar target gene specificity, and both activated target gene promoters only in the presence of a functional MYB recognition element. The genes encoding the flavonoid biosynthesis enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase, and flavonol synthase were identified as target genes. Hence, our observations further add to the general notion of a close relationship between structure and function of R2R3-MYB factors. High-performance liquid chromatography analyses of myb12 mutant plants and MYB12 overexpression plants demonstrate a tight linkage between the expression level of functional MYB12 and the flavonol content of young seedlings. Quantitative real time reverse transcription-PCR using these mutant plants showed MYB12 to be a transcriptional regulator of CHALCONE SYNTHASE and FLAVONOL SYNTHASE in planta, the gene products of which are indispensable for the biosynthesis of flavonols.

The Arabidopsis Transcription Factor MYB12 Is a Flavonol-Specific Regulator of Phenylpropanoid Biosynthesis

To investigate the nature of mutations induced by accelerated ions in higher plants, the effects of carbon-ion-irradiation were compared with those of electron-irradiation in Arabidopsis thaliana. Point-like mutations and rearrangements were induced at a similar frequency after carbon-ion-irradiation, whereas point-like mutations were more frequently induced after electron-irradiation. Sequence analysis revealed that carbon-ion-induced point-like mutations were mostly short deletions. In the case of rearrangements, deletions, inversions, insertions, and translocations were found. The estimated frequency of deletion induction was comparable to that of fast neutrons. Analysis of chromosome breakpoints revealed that carbon ions frequently deleted small regions around the breakpoints, whereas electron-irradiation often duplicated these regions. Moreover, for both types of radiation, broken ends with microhomologies were frequently rejoined. Results of the breakpoint and broken end analyses suggest that non-homologous end-joining (NHEJ) leads to the rejoining of double strand breaks (dsbs) after cells are exposed to both types of radiation, but the type of NHEJ that occurs as a result of damage is different. The results indicated that carbon-ion-induced mutations are most likely nulls and that the induced rearrangements may arise through a unique mechanism. These findings indicate that accelerated ions are a useful mutagen for both forward and reverse genetics for plants.

Analysis of mutations induced by carbon ions in Arabidopsis thaliana.

Irradiation of Arabidopsis thaliana by carbon ions was carried out to investigate the mutational effect of ion particles in higher plants. Frequencies of embryonic lethals and chlorophyll-deficient mutants were found to be significantly higher after carbon-ion irradiation than after electron irradiation (11-fold and 7.8-fold per unit dose, respectively). To estimate the mutation rate of carbon ions, mutants with no pigments on leaves and stems (tt) and no trichomes on leaves (gl) were isolated at the M2 generation and subjected to analysis. Averaged segregation rate of the backcrossed mutants was 0.25, which suggested that large deletions reducing the viability of the gametophytes were not transmitted, if generated, in most cases. During the isolation of mutants, two new classes of flavonoid mutants (tt18, tt19) were isolated from carbon-ion-mutagenized M2 plants. From PCR and sequence analysis, two of the three tt18 mutant alleles were found to have a small deletion within the LDOX gene and the other was revealed to contain a rearrangement. Using the segregation rates, the mutation rate of carbon ions was estimated to be 17-fold higher than that of electrons. The isolation of novel mutants and the high mutation rate suggest that ion particles can be used as a valuable mutagen for plant genetics.

https://www.genetics.org/content/genetics/163/4/1449.full.pdf

Mutation rate and novel tt mutants of Arabidopsis thaliana induced by carbon ions.

An ultraviolet-B (UV-B)-resistant mutant, uvi1 ( UV-B insensitive 1 ), of Arabidopsis was isolated from 1,280 M 1 seeds that had been exposed to ion beam irradiation. The fresh weight of uvi1 under high-UV-B exposure was more than twice that of the wild type. A root-bending assay indicated that root growth was less inhibited by UV-B exposure in uvi1 than in the wild type. When the seedlings were grown under white light, the UV-B dose required for 50% inhibition was about 6 kJ m −2 for the wild type and 9 kJ m −2 for uvi1 . When the seedlings were irradiated with UV-B in darkness, the dose required for 50% inhibition was about 1.5 kJ m −2 for the wild type and 4 kJ m −2 for uvi1 . An enzyme-linked immunosorbent assay showed that the reduction in levels of cyclobutane pyrimidine dimers (CPDs) under white light and of (6-4) photoproducts in darkness occurred faster in uvi1 than in the wild type. These results indicate that uvi1 had increased photoreactivation of CPDs and dark repair of (6-4) photoproducts, leading to strong UV-B resistance. Furthermore, the transcript levels of PHR1 (CPD photolyase gene) were much higher in uvi1 than in the wild type both under white light and after UV-B exposure. Placing the plants in the dark before UV-B exposure decreases the early reduction of CPDs in the wild type but not in uvi1 . Our results suggest that UVI1 is a negative regulator of two independent DNA repair systems.

An Ultraviolet-B-Resistant Mutant with Enhanced DNA Repair in Arabidopsis

A new stable mutant of Arabidopsis thaliana with a spotted pigment in the seed coat, named anthocyanin spotted testa (ast), was induced by carbon ion irradiation. The spotted pigmentation of ast mutant was observed in immature seeds from 1-2 days after flowering (DAF), at the integument of the ovule, and spread as the seed coat formed. Anthocyanin accumulation was about 6 times higher in ast mutant than in the wild-type at 6 DAF of the immature seeds, but was almost the same in mature dry seeds. A higher anthocyanin accumulation was not observed in the seedlings, leaves or floral buds of ast mutant compared with the wild-type, which suggests that a high accumulation of anthocyanins is specific to the seed coat of the immature ast seeds. Reciprocal crosses between ast mutant and the wild-type indicated that ast is a single recessive gene mutation and segregates as a delayed inheritance. The results of crossing with tt7 and ttg mutants also confirmed that the AST gene is probably a regulatory locus that controls flavonoid biosynthesis. A mapping analysis revealed that the gene is located on chromosome I and is closely linked to the SSLP DNA marker nga280 with a distance of 3.2 cM. AST has been registered as a new mutant of Arabidopsis.

A new Arabidopsis mutant induced by ion beams affects flavonoid synthesis with spotted pigmentation in testa.

To clarify the effect of heavy ions in plants, dry seeds of Arabidopsis were irradiated with carbon, neon, and argon ions with various linear energy transfer (LET) values. The relative biological effectiveness (RBE) for lethality peaked at LET values over 350 keV/µm for neon and argon ions. This LET giving the peak RBE was higher than the LET of 100–200 keV/µm which was reported to have a maximum RBE for other types of cells, such as mammalian cells. Furthermore, sterility showed a higher RBE at an LET of 354 keV/µm with neon ions than that at an LET of 113 keV/µm with carbon ions. Lethality and sterility are both considered to be caused by damage to DNA. The results indicate that the LET having a maximum of RBE for lethality is higher in Arabidopsis seeds than in other unicellular systems. The most likely explanation for this shift of LET is that the DNA in dry seeds has a different chemical environment and/or hydration state than the DNA in cells in culture.

Shigemitsu Tano

Papers

Analysis of mutations induced by carbon ions in Arabidopsis thaliana.

Mutation rate and novel tt mutants of Arabidopsis thaliana induced by carbon ions.

An Ultraviolet-B-Resistant Mutant with Enhanced DNA Repair in Arabidopsis

A new Arabidopsis mutant induced by ion beams affects flavonoid synthesis with spotted pigmentation in testa.

LET dependence of lethality in Arabidopsis thaliana irradiated by heavy ions.