Flavonols modulate plant development, signaling, and stress responses

Abstract

Flavonols are plant-specialized metabolites with important functions in plant growth and development. Isolation and characterization of mutants with reduced flavonol levels, especially the transparent testa mutants in Arabidopsis thaliana, have contributed to our understanding of the flavonol biosynthetic pathway. These mutants have also uncovered the roles of flavonols in controlling development in above- and below-ground tissues, notably in the regulation of root architecture, guard cell signaling, and pollen development. In this review, we present recent progress made towards a mechanistic understanding of flavonol function in plant growth and development. Specifically, we highlight findings that flavonols act as reactive oxygen species (ROS) scavengers and inhibitors of auxin transport in diverse tissues and cell types to modulate plant growth and development and responses to abiotic stresses.

Keywords: Flavonoids; Flavonols; Pollen development; Reactive oxygen species; Root development; Stomatal closure.

Figure 1. The flavonoid biosynthetic pathway.

Enzymes are indicated next to arrows with mutant names in parentheses under the enzyme abbreviation. Arabidopsis mutants are in black and tomato mutants are in red. The flavonol biosynthesis branch is illustrated in the yellow dashed box. Chemical structure of specific flavonols are presented on the upper right. Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase; F3H, flavanone 3-hydroxylase; IFS, isoflavone synthase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; ANS/LDOX, anthocyanidin synthase/leucoanthocyanidin dioxygenase; RT, rhamnosyl transferase; UFGT, UDP-glucose flavonoid 3-O-glucotransferase; F3’5’H, flavonoid 3’5’-hydroxylase; F3’H, flavonoid 3’-hydroxylase; FLS, flavonol synthase; OMT-1, O-methyltransferase-1. Figure adapted from [64 & 66], B. The predicted mechanism by which the flavonol quercetin donates electrons to convert hydrogen peroxide to water is shown, along with a potential mechanism by which reduced flavonols are regenerated. The transfer of electrons and protons to hydrogen peroxide are indicated above the dashed arrow. Types of enzymes that may catalyze these chemical reactions are presented in parentheses. It is also possible that the quinone reductase may convert the quinone back to a semiquinone. C. This diagram summarizes environmental or hormonal factors that increase levels of reactive oxygen species (ROS) and the role of flavonols as modulators of ROS to control developmental and physiological responses. Factors affecting ROS levels are indicated in colored boxes and the resulting changes in ROS concentration are depicted by increased size of boxes. Hormonal and environment increases in ROS levels are indicated by arrows and the ability of flavonols to reduce ROS accumulation is indicated by a blunt-end arrow. Observed developmental or physiological changes linked to the different ROS levels are indicated below the ROS changes. Abbreviation: ABA, abscisic acid.

Figure 2.. The flavonol biosynthetic machinery is localized to the root epidermis to regulate root hair initiation.

(a) Fluorescence of transgenic Arabidopsis lines containing CHS-GFP and FLS1-GFP reporters (green) stained with the cell wall probe propidium iodide (PI) (magenta) show that flavonol biosynthetic enzymes localize to the root epidermis. Images without PI channel have epidermal tissues outlined by dashed lines. Scale bar =100 μm (b) LSCM images of WT Arabidopsis roots treated with either the flavonol-selective probe, DPBA, with yellow fluorescence of quercetin and green fluorescence of kaempferol (left image), or the fluorescence of the general ROS sensor, dichlorofluorescein (DCF) (green, right image), reveals ROS accumulate in higher levels in root epidermal tissues where flavonols are less abundant. (c) Representative images of root hair number in WT Arabidopsis and mutants with impaired flavonol accumulation, tt4-ll and fls1-3, and tt4-11 complemented with a CHS-GFP transgene (ttd-11 CHS-GFP) show increased root hair numbers in mutants with defects in flavonol synthesis. Scale bar = 200 μm (d) Confocal images of WT, tt4-2, and tt4-11 Arabidopsis lines stained with the H₂O₂ probe, Peroxy orange 1, display elevated H₂O₂ accumulation in root hair forming cells (denoted as 1, 3, and 5) relative to nonhair cells (2 and 4). H₂O₂ accumulation is increased in tt4-2 and tt4-11, though this phenotype can be restored to WT levels through treatment with the flavonol precursor, naringenin. Scale bar = 50 μm. Adapted from Ref. [64].

Figure 3.. Flavonols accumulate within guard cells to influence stomatal aperture.

(a) Confocal micrograph of a VF36 tomato leaf stained with the flavonol-selective probe DPBA (yellow) reveals flavonol accumulation in guard cells. Chlorophyll autofluorescence is visualized in magenta, (b) Fluorescence of DPBA (yellow) or the general ROS sensor dichlorofluorescein (DCF) (green) in guard cells of the are and aw tomato mutants and their respective wild-type parental lines, VF36 and AC, reveals an inverse relationship between flavonol levels and general ROS accumulation, (c) Representative images of stomatal aperture of guard cells treated with ABA show that the are (anthocyanin reduced) mutant with decreased flavonols and elevated ROS levels has enhanced rates of stomatal closure, while the anthocyanin without (aw) mutant with increased flavonols and decreased ROS accumulation has reduced rates of stomatal closure, (d) DPBA fluorescence is increased in guard cells following treatment with ethylene gas, while they remain unchanged in the ethylene insensitive Neverripe mutant. Scale bars = 5 μm. (Adapted from Ref. [78]. Copyright 2017 American Society of Plant Biologists.

Figure 4.. Flavonols promote pollen development and tube growth in tomato.

(a) Confocal images of VF36 and flavonol-deficient are mutant pollen grains stained with fluorescein diacetate (FDA, green; live grains) and propidium iodide (PI, magenta; dead grains) and scanning electron micrographs show that the are mutant produces less viable pollen. Scale bars = 200 μm for confocal images and 20 μm for electron micrographs, (b) The general ROS sensor, DCF, and the H₂O₂-selective chemical probe, Peroxy orange 1 (PO1) reveal increased ROS accumulation in pollen grains of the are mutant. Scale bar = 50 μm (c) Confocal micrographs display significantly impaired pollen tube growth as well as increased levels of ROS in the are mutant. Scale bar = 50 μm (top) and 100 μm (bottom). Adapted from Ref. [18]. Copyright 2018 Proceedings of the National Academy of Sciences.