Analysis of T-DNA alleles of flavonoid biosynthesis genes in Arabidopsis ecotype Columbia

Abstract

Background: The flavonoid pathway is a long-standing and important tool for plant genetics, biochemistry, and molecular biology. Numerous flavonoid mutants have been identified in Arabidopsis over the past several decades in a variety of ecotypes. Here we present an analysis of Arabidopsis lines of ecotype Columbia carrying T-DNA insertions in genes encoding enzymes of the central flavonoid pathway. We also provide a comprehensive summary of various mutant alleles for these structural genes that have been described in the literature to date in a wide variety of ecotypes.

Findings: The confirmed knockout lines present easily-scorable phenotypes due to altered pigmentation of the seed coat (or testa). Knockouts for seven alleles for six flavonoid biosynthetic genes were confirmed by PCR and characterized by UPLC for altered flavonol content.

Conclusion: Seven mutant lines for six genes of the central flavonoid pathway were characterized in ecotype, Columbia. These lines represent a useful resource for integrating biochemical and physiological studies with genomic, transcriptomic, and proteomic data, much of which has been, and continues to be, generated in the Columbia background.

Figure 1

Seed coat color phenotype of confirmed homozygous T-DNA lines with insertions disrupting genes involved in flavonoid biosynthesis. From top center, clockwise seeds are: Col-0 WT, tt4-13, tt5-3, tt5-2, tt6-3, tt7-5, tt11-11, and ban-4.

Figure 2

Schematic of homozygous T-DNA insertion lines. Boxes indicate exons, solid lines indicate introns and 5^′ leader sequence, and dashed lines indicate genomic sequence. Insertion sites are indicated by black triangles. The arrows above the insertion indicate the direction of the T-DNA left-border primer sequence used for mapping the insertion sites. The fls1 line is described in Owens et al. [45]. Genes are chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3 hydroxylase (F3H), flavonoid 3^′ hydroxylase (F3′H), flavonol synthase (FLS1), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR).

Figure 3

PCR confirmation of homozygous insertion lines for described tt alleles. T-DNA insertions were confirmed using T-DNA and gene-specific primers, while intact genes were confirmed using two gene-specific primers spanning the mapped T-DNA insertion site. Homozygous lines are indicated by the presence of a T-DNA insertion but not an intact gene.

Figure 4

UPLC analysis of flavonol aglycone profiles in T-DNA insertion lines.A) UPLC traces of hydrolyzed extracts prepared from 5-day-old seedlings, with arrows indicating the retention times of the flavonols, quercetin (Q) and kaempferol (K). B) Comparison of kaempferol and quercetin levels determined from integrated peak areas.