RBOH-Dependent ROS Synthesis and ROS Scavenging by Plant Specialized Metabolites To Modulate Plant Development and Stress Responses

Abstract

Reactive oxygen species (ROS) regulate plant growth and development. ROS are kept at low levels in cells to prevent oxidative damage, allowing them to be effective signaling molecules upon increased synthesis. In plants and animals, NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins provide localized ROS bursts to regulate growth, developmental processes, and stress responses. This review details ROS production via RBOH enzymes in the context of plant development and stress responses and defines the locations and tissues in which members of this family function in the model plant Arabidopsis thaliana. To ensure that these ROS signals do not reach damaging levels, plants use an array of antioxidant strategies. In addition to antioxidant machineries similar to those found in animals, plants also have a variety of specialized metabolites that scavenge ROS. These plant specialized metabolites exhibit immense structural diversity and have highly localized accumulation. This makes them important players in plant developmental processes and stress responses that use ROS-dependent signaling mechanisms. This review summarizes the unique properties of plant specialized metabolites, including carotenoids, ascorbate, tocochromanols (vitamin E), and flavonoids, in modulating ROS homeostasis. Flavonols, a subclass of flavonoids with potent antioxidant activity, are induced during stress and development, suggesting that they have a role in maintaining ROS homeostasis. Recent results using genetic approaches have shown how flavonols regulate development and stress responses through their action as antioxidants.

Figure 1.

Respiratory burst oxidase homologues (RBOHs) have developmental roles in distinct plant organs and tissues in Arabidopsis thaliana. This figure illustrates the plant model species Arabidopsis thaliana (left) and highlights plant tissues and developmental responses, in which the different RBOHs are implicated in controlling signaling or development (right). The image of the Arabidopsis plant was reprinted with permission from ref 311.

Figure 2.

Tissue and developmental patterns of Arabidopsis RBOH transcript abundance. The abundance of the transcripts encoding all the RBOH enzymes were quantified by microarray and were extracted from the eFP Browser using the developmental map and tissue-specific data sets. The values were normalized relative to the median transcript abundance within the eFP Browser, and the fold changes from that value are reported. Conditional formatting was used within each transcript column to highlight their abundances across tissues and cell types, with values in the lowest 10% shown in dark blue, those in the top 10% in red, and those at the 50th percentile in white. The font color was changed to white for the values in the highest 20%. The locus identifiers for the individual genes are listed at the bottom of the figure.

Figure 3.

The rhd2–6/rbohc mutant has impaired root hair elongation compared with the wild-type (Col-0). (A) An Arabidopsis root stained with propidium iodide, showing cell outlines found in the meristematic, transition, and elongation zones. Scale bar =100 μm. (B) Comparison of young Arabidopsis root tips for the wild-type (Col-0) and the root-hair-defective (rhd2) mutant, which has a mutation in the RBOHC gene. The area above the dashed lines indicates the maturation zone, where root hairs form. Scale bar = 200 μm.

Figure 4.

ABA-induced ROS burst in guard cells precedes stomatal closure in wild-type tomato leaves and requires RBOH activity. (A) Increases in DCF fluorescence were visualized in guard cells across a 45 min time course of ABA treatment in wild-type tomato leaves. Scale bar = 5 μm. (B) DCF fluorescence in entire guard cells and stomatal aperture shown as functions of time after treatment with 20 μM ABA. Asterisk and number signs represent significant differences in DCF fluorescence (P < 0.001) and stomatal aperture (P < 0.009) between time 0 and indicated times or treatment. (C) DCF fluorescence were quantified with and without 100 μM DPI at 0 and 45 min of treatment with ABA. Asterisks and number signs represents significant differences (P < 0.001) in DCF fluorescence between time 0 and the indicated times or between untreated and treated, respectively. Statistics were determined using two-way analysis of variance followed by Tukey’s posthoc test with N = 70. Reproduced with permission from ref 76. Copyright 2017 American Society of Plant Biologists.

Figure 5.

Flavonoid biosynthesis pathway highlighting the six major subclasses of flavonoids. Mutants are noted in parentheses under the enzyme abbreviation, with Arabidopsis mutants in italics and tomato mutants in bold italics. The flavonol biosynthesis branch is further detailed in the red box. General flavonoid structures are presented for the six subclasses of flavonoids. The chemical bonds and/or substituents highlighted in red indicate sites of structural variation within each subclass. 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; OMT, O-methyltransferase; (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 6.

ROS levels and root hair numbers are greater in roots of the are mutant, which is impaired in flavonol synthesis, compared with the wild-type (VF36). (A) ROS levels are concentrated in the epidermal cells and root hairs of the maturation zone of roots, where root hairs initiate. ROS is elevated in the mutant, as shown in tile-scan confocal images of the are mutant and wild-type (VF36) roots stained with CM-H₂DCFDA. Scale bar = 300 μm. (B) The higher number of root hairs in the are mutant than the wild-type is evident in bright-field images of primary roots. Scale bars =150 μm. (C) Quantification of root hair numbers in mature regions of primary roots grown on control media. Reproduced with permission from ref 272. Copyright 2014 American Society of Plant Biologists.

Figure 7.

Levels of flavonols are inversely proportional to the levels of ROS in tomato guard cells, with more rapid closure and higher ROS. (A) Confocal micrographs show DPBA-bound flavonols in yellow and chlorophyll autofluorescence in magenta in the are mutant with reduced flavonols and the aw mutant with increased flavonols compared with the parental lines (VF36 and AC, respectively). Scale bar = 5 μm. (B) DCF fluorescence is shown in green, and the levels are inversely proportional to flavonol levels. (C) ABA closes guard cells with closure proportional to levels of ROS. (D) Changes in DCF fluorescence upon ABA addition were quantified and are greater in the are mutant with low flavonols than the wild-type, while elevated flavonols in the aw mutant show the lowest rate of DCF changes. Reproduced with permission from ref 76. Copyright 2017 American Society of Plant Biologists.

Figure 8.

Flavonols regulate pollen development by scavenging ROS. (A) Confocal micrographs of the wild-type tomato (VF36) and the flavonol-deficient anthocyanin reduced (are) mutant show reduced pollen viability in the mutant. Pollen grains were stained with FDA (live pollen grains, shown in green) and PI (dead pollen grains, shown in magenta). Scale bar = 200 μm. (B) Bright-field images of pollen tube tips of VF36 and the are mutant show that flavonols reduce swelling of the pollen tube tip. Scale bar = 10 μm. (C) Confocal micrographs of VF36 and are mutant pollen grains and tubes stained with the general ROS sensor CM-H₂DCFDA show that flavonols reduce ROS levels (as evidenced by DCF fluorescence) in pollen grains and tubes. Scale bars = 50 μm. (D) Confocal micrographs of VF36 and are mutant pollen grains stained with the hydrogen peroxide-specific sensor peroxy orange 1 (PO1) show elevated hydrogen peroxide in the flavonol-deficient are mutant. Scale bar = 50 μm. These images were reprinted from ref 96.