Flavonols improve thermotolerance in tomato pollen during germination and tube elongation by maintaining ROS homeostasis.

Elevated temperatures impair pollen performance and reproductive success, resulting in lower crop yields. The Solanum lycopersicum anthocyanin reduced ( are ) mutant has a FLAVANONE 3 HYDROXYLASE ( F3H ) gene mutation resulting in impaired synthesis of flavonol antioxidants. The are mutant has reduced pollen performance and seed set relative to the VF36 parental line, which is accentuated at elevated temperatures. Transformation of are with the wild-type F3H gene, or chemical complementation with flavonols, prevented temperature-dependent ROS accumulation in pollen and reversed are's reduced viability, germination, and tube elongation to VF36 levels. VF36 transformed with an F3H overexpression construct prevented temperature driven ROS increases and impaired pollen performance, revealing thermotolerance results from elevated flavonol synthesis. Although stigmas of are had reduced flavonols and elevated ROS, the growth of are pollen tubes were similarly impaired in both are and VF36 pistils. RNA-Seq was performed at optimal and stress temperatures in are , VF36, and the VF36 F3H overexpression line at multiple timepoints across pollen tube elongation. Differentially expressed gene numbers increased with duration of elevated temperature in all genotypes, with the largest number in are . These findings suggest potential agricultural interventions to combat the negative effects of heat-induced ROS in pollen that leads to reproductive failure. One sentence summary: Flavonol antioxidants reduce the negative impacts of elevated temperatures on pollen performance by reducing levels of heat induced reactive oxygen species and modulation of heat-induced changes in the pollen transcriptome.

Hydrogen peroxide-dependent oxidation of ERK2 within its D-recruitment site alters its substrate selection.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are dysregulated in many pervasive diseases. Recently, we discovered that ERK1/2 is oxidized by signal-generated hydrogen peroxide in various cell types. Since the putative sites of oxidation lie within or near ERK1/2's ligand-binding surfaces, we investigated how oxidation of ERK2 regulates interactions with the model substrates Sub-D and Sub-F. These studies revealed that ERK2 undergoes sulfenylation at C159 on its D-recruitment site surface and that this modification modulates ERK2 activity differentially between substrates. Integrated biochemical, computational, and mutational analyses suggest a plausible mechanism for peroxide-dependent changes in ERK2-substrate interactions. Interestingly, oxidation decreased ERK2's affinity for some D-site ligands while increasing its affinity for others. Finally, oxidation by signal-generated peroxide enhanced ERK1/2's ability to phosphorylate ribosomal S6 kinase A1 (RSK1) in HeLa cells. Together, these studies lay the foundation for examining crosstalk between redox- and phosphorylation-dependent signaling at the level of kinase-substrate selection.

Flavonols modulate plant development, signaling, and stress responses.

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.

Abscisic acid increases hydrogen peroxide in mitochondria to facilitate stomatal closure.

Abscisic acid (ABA) drives stomatal closure to minimize water loss due to transpiration in response to drought. We examined the subcellular location of ABA-increased accumulation of reactive oxygen species (ROS) in guard cells, which drive stomatal closure, in Arabidopsis (Arabidopsis thaliana). ABA-dependent increases in fluorescence of the generic ROS sensor, dichlorofluorescein (DCF), were observed in mitochondria, chloroplasts, cytosol, and nuclei. The ABA response in all these locations was lost in an ABA-insensitive quintuple receptor mutant. The ABA-increased fluorescence in mitochondria of both DCF- and an H2O2-selective probe, Peroxy Orange 1, colocalized with Mitotracker Red. ABA treatment of guard cells transformed with the genetically encoded H2O2 reporter targeted to the cytoplasm (roGFP2-Orp1), or mitochondria (mt-roGFP2-Orp1), revealed H2O2 increases. Consistent with mitochondrial ROS changes functioning in stomatal closure, we found that guard cells of a mutant with mitochondrial defects, ABA overly sensitive 6 (abo6), have elevated ABA-induced ROS in mitochondria and enhanced stomatal closure. These effects were phenocopied with rotenone, which increased mitochondrial ROS. In contrast, the mitochondrially targeted antioxidant, MitoQ, dampened ABA effects on mitochondrial ROS accumulation and stomatal closure in Col-0 and reversed the guard cell closure phenotype of the abo6 mutant. ABA-induced ROS accumulation in guard cell mitochondria was lost in mutants in genes encoding respiratory burst oxidase homolog (RBOH) enzymes and reduced by treatment with the RBOH inhibitor, VAS2870, consistent with RBOH machinery acting in ABA-increased ROS in guard cell mitochondria. These results demonstrate that ABA elevates H2O2 accumulation in guard cell mitochondria to promote stomatal closure.

Reactive oxygen species function as signaling molecules in controlling plant development and hormonal responses.

Reactive oxygen species (ROS) serve as second messengers in plant signaling pathways to remodel plant growth and development. New insights into how enzymatic ROS-producing machinery is regulated by hormones or localized during development have provided a framework for understanding the mechanisms that control ROS accumulation patterns. Signaling-mediated increases in ROS can then modulate the activity of proteins through reversible oxidative modification of specific cysteine residues. Plants also control the synthesis of antioxidants, including plant-specialized metabolites, to further define when, where, and how much ROS accumulate. The availability of sophisticated imaging capabilities, combined with a growing tool kit of ROS detection technologies, particularly genetically encoded biosensors, sets the stage for improved understanding of ROS as signaling molecules.

The Role of ROS Homeostasis in ABA-Induced Guard Cell Signaling.

The hormonal and environmental regulation of stomatal aperture is mediated by a complex signaling pathway found within the guard cells that surround stomata. Abscisic acid (ABA) induces stomatal closure in response to drought stress by binding to its guard cell localized receptor, initiating a signaling cascade that includes synthesis of reactive oxygen species (ROS). Genetic evidence in Arabidopsis indicates that ROS produced by plasma membrane respiratory burst oxidase homolog (RBOH) enzymes RBOHD and RBOHF modulate guard cell signaling and stomatal closure. However, ABA-induced ROS accumulates in many locations such as the cytoplasm, chloroplasts, nucleus, and endomembranes, some of which do not coincide with plasma membrane localized RBOHs. ABA-induced guard cell ROS accumulation has distinct spatial and temporal patterns that drive stomatal closure. Productive ROS signaling requires both rapid increases in ROS, as well as the ability of cells to prevent ROS from reaching damaging levels through synthesis of antioxidants, including flavonols. The relationship between locations of ROS accumulation and ABA signaling and the role of enzymatic and small molecule ROS scavengers in maintaining ROS homeostasis in guard cells are summarized in this review. Understanding the mechanisms of ROS production and homeostasis and the role of ROS in guard cell signaling can provide a better understanding of plant response to stress and could provide an avenue for the development of crop plants with increased stress tolerance.