The anthocyanin reduced tomato mutant demonstrates the role of flavonols in tomato lateral root and root hair development

Abstract

This study utilized tomato (Solanum lycopersicum) mutants with altered flavonoid biosynthesis to understand the impact of these metabolites on root development. The mutant anthocyanin reduced (are) has a mutation in the gene encoding FLAVONOID 3-HYDROXYLASE (F3H), the first step in flavonol synthesis, and accumulates higher concentrations of the F3H substrate, naringenin, and lower levels of the downstream products kaempferol, quercetin, myricetin, and anthocyanins, than the wild type. Complementation of are with the p35S:F3H transgene reduced naringenin and increased flavonols to wild-type levels. The initiation of lateral roots is reduced in are, and p35S:F3H complementation restores wild-type root formation. The flavonoid mutant anthocyanin without has a defect in the gene encoding DIHYDROFLAVONOL REDUCTASE, resulting in elevated flavonols and the absence of anthocyanins and displays increased lateral root formation. These results are consistent with a positive role of flavonols in lateral root formation. The are mutant has increased indole-3-acetic acid transport and greater sensitivity to the inhibitory effect of the auxin transport inhibitor naphthylphthalamic acid on lateral root formation. Expression of the auxin-induced reporter (DR5-β-glucuronidase) is reduced in initiating lateral roots and increased in primary root tips of are. Levels of reactive oxygen species are elevated in are root epidermal tissues and root hairs, and are forms more root hairs, consistent with a role of flavonols as antioxidants that modulate root hair formation. Together, these experiments identify positive roles of flavonols in the formation of lateral roots and negative roles in the formation of root hairs through the modulation of auxin transport and reactive oxygen species, respectively.

Figure 1.

The biosynthetic pathway leading to flavonols and anthocyanidins, showing enzymatic steps. Gray numbers indicate gene copies in tomato. Greater than/less than symbols describe relative levels of free quercetin, kaempferol, and myricetin in tomato hypocotyl and root tissues.

Figure 2.

Transcript abundance of flavonoid biosynthetic genes in hypocotyls and roots of VF36. Quantities are reported normalized to F3′5′H in roots. Scales differ above and below the y axis break, to clearly show levels of low-abundance transcripts. Averages and se are reported for nine biological replicates, each with three technical replicates. Asterisks indicate differences between tissue types. Significance was determined by Student’s t test with P ≤ 0.05. a, Transcript level was below the threshold of detection.

Figure 3.

Overexpression of a wild-type F3H gene increases anthocyanin production in wild-type plants and restores anthocyanin production in the are mutant. A, Images show the apical regions of untransformed parents and representative plants from the T0 generation. B, Anthocyanin content from hypocotyls was measured by spectrophotometry. Averages and se are presented. Asterisks indicate significant differences from untransformed VF36 with P ≤ 0.05 (n = 3–9), determined by Student’s t test. White and black bars signify lines in the VF36 and are backgrounds, respectively.

Figure 4.

Lateral root numbers are reduced in the are mutant. A, Image showing 6-d-old seedlings of the wild type and are displaying differences in lateral rooting and hypocotyl pigmentation. B, Numbers of emerged lateral roots, unemerged lateral root primordia, and combined totals of emerged and primordial roots. Averages and se are presented. Asterisks indicate differences between genotypes with P ≤ 0.05 (n = 15). C, Lateral root numbers are reported for untransformed VF36, are, and three F3H overexpression lines in each background genotype. Asterisks indicate differences from VF36, and number symbols indicate differences from are with P ≤ 0.05 (n = 16). Black bars indicate lines in the are background.

Figure 5.

Auxin transport and the response to an auxin transport inhibitor are altered in are. A and B, [³H]IAA pulse-chase transport in wild-type and are mutant seedlings. A, [³H]IAA rootward transport through hypocotyls from the shoot apex in are compared with the wild type. B, [³H]IAA rootward transport through roots from the root-shoot junction in are compared with the wild type. Averages and se are reported for n = 16. For A and B, asterisks indicate differences between are and the wild type with P ≤ 0.05. C, NPA or control droplets were placed 2 cm below the root-shoot junction, and roots were quantified below the application site after 3 d. Averages and se are reported for 15 seedlings. Asterisks indicate significant differences from the control treatment within genotypes with P ≤ 0.05, and number symbols indicate significant differences within treatments with P ≤ 0.05. WT, Wild type.

Figure 6.

The DR5:GUS auxin reporter indicates that more auxin accumulates in the root tip of the are mutant than in the wild type. A, GUS-stained primary root tips of wild-type or are seedlings containing the pDR5:GUS transgene. Bar = 200 µm. B, Lateral root primordia with GUS staining in wild-type or are seedlings. Bar = 100 µm. Seedlings were 3 d old, the stage at which lateral root primordia start forming and before the emergence of lateral roots.

Figure 7.

ROS levels and root hair numbers are greater in roots of the are mutant compared with the wild type. A, Tile scan confocal images of mutant and wild-type roots showing the distribution of DCF staining along the root. Bar = 300 µm. B, Bright-field images of primary roots showing root hair density in mutant and the wild type. Bar = 200 µm. C, Bright-field images of mature regions of primary roots grown on control medium or medium containing H₂O₂ or ascorbic acid. Bar = 150 µm. D, Quantification of root hair numbers in mature regions of primary roots grown on control medium or medium containing H₂O₂ or ascorbic acid. Asterisks indicate significant differences from the control treatment within genotypes with P < 0.05. E, Confocal images of mature regions of 3-d-old roots treated with water, H₂O₂, or ascorbic acid and stained with DCF to observe ROS levels. Fluorescence was quantified, and numbers in each image represent percentages of wild-type fluorescence and se. Asterisks indicate significant differences from the wild type within treatments with P < 0.05. Bar = 200 µm.