Abscisic acid (ABA) increases reactive oxygen species (ROS) in guard cells to close Arabidopsis (Arabidopsis thaliana) stomata. In tomato (Solanum lycopersicum), we find that ABA-increased ROS is followed by stomatal closure and that both responses are blocked by inhibitors of ROS-producing respiratory burst oxidase enzymes. ABA-induced ROS sensor fluorescence accumulates in the nucleus, chloroplasts, and endomembranes. The accumulation of flavonol antioxidants in guard cells, but not surrounding pavement cells, was visualized by confocal microscopy using a flavonol-specific fluorescent dye. Decreased flavonols in guard cells in the anthocyanin reduced (are) mutant and elevated levels in the anthocyanin without (aw) mutant were quantified by confocal microscopy and in leaf extracts by mass spectrometry. Consistent with flavonols acting as antioxidants, higher levels of ROS were detected in guard cells of the tomato are mutant and lower levels were detected in aw both at homeostasis and after treatment with ABA. These results demonstrate the inverse relationship between flavonols and ROS. Guard cells of are show greater ABA-induced closure than the wild type, reduced light-dependent guard cell opening, and reduced water loss, with aw having opposite responses. Ethylene treatment of wild-type tomato plants increased flavonol accumulation in guard cells; however, no flavonol increases were observed in Neverripe (Nr), an ethylene receptor mutant. Consistent with lower levels of ROS due to elevated flavonols, ethylene treatments decreased ABA-induced stomatal closure in the wild type, but not Nr, with ethylene responses attenuated in the are mutant. Together, these results are consistent with flavonols dampening the ABAdependent ROS burst that drives stomatal closure and facilitating stomatal opening to modulate leaf gas exchange.

Guard cell swelling controls the aperture of stomata, pores that facilitate gas exchange and water loss from leaves. The hormone abscisic acid (ABA) has a central role in regulation of stomatal closure through synthesis of second messengers, which include reactive oxygen species (ROS). ROS accumulation must be minimized by antioxidants to keep concentrations from reaching damaging levels within the cell. Flavonols are plant metabolites that have been implicated as antioxidants; however, their antioxidant activity in planta has been debated. Flavonols accumulate in guard cells of Arabidopsis thaliana, but not surrounding pavement cells, as visualized with a flavonolspecific dye. The expression of a reporter driven by the promoter of CHALCONE SYNTHASE, a gene encoding a flavonol biosynthetic enzyme, in guard cells, but not pavement cells, suggests guard cell-specific flavonoid synthesis. Increased levels of ROS were detected using a fluorescent ROS sensor in guard cells of transparent testa4-2, which has a null mutation in CHALCONE SYNTHASE and therefore synthesizes no flavonol antioxidants. Guard cells of transparent testa4-2 show more rapid ABA-induced closure than the wild type, suggesting that flavonols may dampen the ABA-dependent ROS burst that drives stomatal closing. The levels of flavonols are positively regulated in guard cells by ethylene treatment in the wild type, but not in the ethylene-insensitive2-5 mutant. In addition, in both ethyleneoverproducing1 and ethylene-treated wild-type plants, elevated flavonols lead to decreasing ROS and slower ABA-mediated stomatal closure. These results are consistent with flavonols suppressing ROS accumulation and decreasing the rate of ABA-dependent stomatal closure, with ethylene-induced increases in guard cell flavonols modulating these responses.

Transcriptomic analyses with high temporal resolution provide substantial new insight into hormonal response networks. This study identified the kinetics of genome-wide transcript abundance changes in response to elevated levels of the plant hormone ethylene in roots from light-grown Arabidopsis () seedlings, which were overlaid on time-matched developmental changes. Functional annotation of clusters of transcripts with similar temporal patterns revealed rapidly induced clusters with known ethylene function and more slowly regulated clusters with novel predicted functions linked to root development. In contrast to studies with dark-grown seedlings, where the canonical ethylene response transcription factor, EIN3, is central to ethylene-mediated development, the roots of and single and double mutants still respond to ethylene in light-grown seedlings. Additionally, a subset of these clusters of ethylene-responsive transcripts were enriched in targets of EIN3 and ERFs. These results are consistent with EIN3-independent developmental and transcriptional changes in light-grown roots. Examination of single and multiple gain-of-function and loss-of-function receptor mutants revealed that, of the five ethylene receptors, ETR1 controls lateral root and root hair initiation and elongation and the synthesis of other receptors. These results provide new insight into the transcriptional and developmental responses to ethylene in light-grown seedlings.

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This work describes the learning gains of undergraduates who participated in a service-learning program involving teaching genetics to middle and high school students. Results show increases in content knowledge of topics taught by undergraduates, compared with other course content, assessed by both an immediate posttest and a delayed course final exam.

In animals, endocytosis of a seven-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic G protein–mediated signaling, depending on the bias of the receptor or ligand, which determines how much one transduction pathway is used compared to another. In Arabidopsis thaliana, GPCRs are not required for G protein–coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the seven-transmembrane protein AtRGS1 modulates G protein signaling through ligand-dependent endocytosis, which initiates derepression of signaling without the involvement of canonical arrestins. Here, we found that endocytosis of AtRGS1 initiated from two separate pools of plasma membrane: sterol-dependent domains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and candidate arrestin-like adaptors. Ligand identity (either the pathogen-associated molecular pattern flg22 or the sugar glucose) determined the origin of AtRGS1 endocytosis. Different trafficking origins and trajectories led to different cellular outcomes. Thus, in this system, compartmentation with its associated signalosome architecture drives biased signaling.

flg22 is a 22 amino peptide released from bacterial flagellin a Microbe Associated Molecular 51 Pattern ( that is recognized by the plant cell as a signal indicating that bacteria are present. On its own, flg22 initiates a rapid increase in cytoplasmic calcium, extracellular reactive oxygen species, and activation of a Mitogen Activated Protein Kinase (cascade all of which are activated within 15 minutes after the cell perceives flg22. Here we show a massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome within this 15 minute duration in wildtype and a mutant deficient in G protein coupled signaling Integration of phosphoproteome with protein protein interactome data followed by network topology analyses discovered that many of the flg22 induced phosphoproteome changes fall on proteins that comprise the G protein interactome and on the most highly populated hubs of the immunity network approximately 95% of the phosphorylation changes in the G protein interactome depend on a functional heterotrimeric G protein complex some occur on proteins that interact directly with components of G coupled signal transduction. One of these is ATBα, a substrate recognition sub-unit of the PP2A Ser/Thr phosphatase and an interactor to Arabidopsis thaliana REGULATOR OF G SIGNALING 1 protein (a 7 transmembrane spanning modulator of the nucleotide binding state of the core G protein complex. AtRGS1 is phosphorylated by BAK1, a component of the flg22 receptor, to initiate AtRGS1 endocytosis. A null mutation of ATB α confer s high 67 basal endocytosis of AtRGS1 suggesting sustained phosphorylated status. Loss of ATB α confers 68 trait s associated with loss of AtRGS1. Because the basal level of AtRGS1 is lower in the atbα null mutant in a proteasome dependent manner we propose that phosphorylation dependent endocytosis of AtRGS1 is part of a mechanism to degrade AtRGS1 which then sustains activation of the 71 G protein complex Thus, the role of ATB α is now established as a central component of phosphorylation dependent regulation of system dynamics in innate immunity

Abbreviations: Arabidopsis thaliana Regulator of G Signaling (AtRGS1), Clathrin-Mediated 43 Endocytosis (CME), Sterol-Dependent Endocytosis (SDE), Total Internal Reflection 44 Fluorescence Microscopy (TIRF), Clathrin Light Chain (CLC), Flotilin 1 (FLOT1), 45 Vacuolar Protein Sorting 26 (VPS26) 46 SUMMARY 47 Biased signaling occurs when different ligands that are directed at the same receptor launch 48 different cellular outcomes. Because of their pharmacological importance, we know the most 49 about biased ligands and little is known about other mechanisms to achieve signaling bias. 50 In the canonical animal G protein system, endocytosis of a 7-transmembrane GPCR 51 desensitizes a cell from its extracellular signal. β-arrestins facilitate GPCR endocytosis but 52 also propagate cytoplasmic signaling depending on the bias. In Arabidopsis, GPCRs are not 53 required for G protein coupled signaling because the heterotrimeric G protein complex 54 spontaneously exchanges nucleotide. Instead, the prototype 7-transmembrane Regulator of 55 G Signaling 1 protein AtRGS1 modulates G signaling and through ligand-dependent 56 endocytosis, de-repression of signaling is initiated but canonical arrestins are not involved. 57 Endocytosis initiates from two separate pools of plasma membrane: microdomains and a 58 clathrin-accessible neighborhood, each with a select set of discriminators, activators, and 59 newly-discovered arrestin-like adaptors. Different trafficking origins and trajectories lead 60 to different cellular outcomes. Thus, compartmentation with its attendant signalosome 61