Ethylene signaling increases reactive oxygen species accumulation to drive root hair initiation in Arabidopsis.

Root hair initiation is a highly regulated aspect of root development. The plant hormone ethylene and its precursor, 1-amino-cyclopropane-1-carboxylic acid, induce formation and elongation of root hairs. Using confocal microscopy paired with redox biosensors and dyes, we demonstrated that treatments that elevate ethylene levels lead to increased hydrogen peroxide accumulation in hair cells prior to root hair formation. In the ethylene-insensitive receptor mutant, etr1-3, and the signaling double mutant, ein3eil1, the increase in root hair number or reactive oxygen species (ROS) accumulation after ACC and ethylene treatment was lost. Conversely, etr1-7, a constitutive ethylene signaling receptor mutant, has increased root hair formation and ROS accumulation, similar to ethylene-treated Col-0 seedlings. The caprice and werewolf transcription factor mutants have decreased and elevated ROS levels, respectively, which are correlated with levels of root hair initiation. The rhd2-6 mutant, with a defect in the gene encoding the ROS-synthesizing RESPIRATORY BURST OXIDASE HOMOLOG C (RBOHC), and the prx44-2 mutant, which is defective in a class III peroxidase, showed impaired ethylene-dependent ROS synthesis and root hair formation via EIN3EIL1-dependent transcriptional regulation. Together, these results indicate that ethylene increases ROS accumulation through RBOHC and PRX44 to drive root hair formation.

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 are signaling molecules that modulate plant reproduction.

Reactive oxygen species (ROS) can serve as signaling molecules that are essential for plant growth and development but abiotic stress can lead to ROS increases to supraoptimal levels resulting in cellular damage. To ensure efficient ROS signaling, cells have machinery to locally synthesize ROS to initiate cellular responses and to scavenge ROS to prevent it from reaching damaging levels. This review summarizes experimental evidence revealing the role of ROS during multiple stages of plant reproduction. Localized ROS synthesis controls the formation of pollen grains, pollen-stigma interactions, pollen tube growth, ovule development, and fertilization. Plants utilize ROS-producing enzymes such as respiratory burst oxidase homologs and organelle metabolic pathways to generate ROS, while the presence of scavenging mechanisms, including synthesis of antioxidant proteins and small molecules, serves to prevent its escalation to harmful levels. In this review, we summarized the function of ROS and its synthesis and scavenging mechanisms in all reproductive stages from gametophyte development until completion of fertilization. Additionally, we further address the impact of elevated temperatures induced ROS on impairing these reproductive processes and of flavonol antioxidants in maintaining ROS homeostasis to minimize temperature stress to combat the impact of global climate change on agriculture.

Flavonols regulate root hair development by modulating accumulation of reactive oxygen species in the root epidermis.

Reactive oxygen species (ROS) are signaling molecules produced by tissue-specific respiratory burst oxidase homolog (RBOH) enzymes to drive development. In Arabidopsis thaliana, ROS produced by RBOHC was previously reported to drive root hair elongation. We identified a specific role for one ROS, H2O2, in driving root hair initiation and demonstrated that localized synthesis of flavonol antioxidants control the level of H2O2 and root hair formation. Root hairs form from trichoblast cells that express RBOHC and have elevated H2O2 compared with adjacent atrichoblast cells that do not form root hairs. The flavonol-deficient tt4 mutant has elevated ROS in trichoblasts and elevated frequency of root hair formation compared with the wild type. The increases in ROS and root hairs in tt4 are reversed by genetic or chemical complementation. Auxin-induced root hair initiation and ROS accumulation were reduced in an rbohc mutant and increased in tt4, consistent with flavonols modulating ROS and auxin transport. These results support a model in which localized synthesis of RBOHC and flavonol antioxidants establish patterns of ROS accumulation that drive root hair formation.

Flavonols modulate lateral root emergence by scavenging reactive oxygen species in Arabidopsis thaliana.

Flavonoids are a class of specialized metabolites with subclasses including flavonols and anthocyanins, which have unique properties as antioxidants. Flavonoids modulate plant development, but whether and how they impact lateral root development is unclear. We examined potential roles for flavonols in this process using Arabidopsis thaliana mutants with defects in genes encoding key enzymes in flavonoid biosynthesis. We observed the tt4 and fls1 mutants, which produce no flavonols, have increased lateral root emergence. The tt4 root phenotype was reversed by genetic and chemical complementation. To more specifically define the flavonoids involved, we tested an array of flavonoid biosynthetic mutants, eliminating roles for anthocyanins and the flavonols quercetin and isorhamnetin in modulating lateral root development. Instead, two tt7 mutant alleles, with defects in a branchpoint enzyme blocking quercetin biosynthesis, formed reduced numbers of lateral roots and tt7-2 had elevated levels of kaempferol. Using a flavonol-specific dye, we observed that in the tt7-2 mutant, kaempferol accumulated within lateral root primordia at higher levels than wild-type. These data are consistent with kaempferol, or downstream derivatives, acting as a negative regulator of lateral root emergence. We examined ROS accumulation using ROS-responsive probes and found reduced fluorescence of a superoxide-selective probe within the primordia of tt7-2 compared with wild-type, but not in the tt4 mutant, consistent with opposite effects of these mutants on lateral root emergence. These results support a model in which increased level of kaempferol in the lateral root primordia of tt7-2 reduces superoxide concentration and ROS-stimulated lateral root emergence.

Flavonols control pollen tube growth and integrity by regulating ROS homeostasis during high-temperature stress.

Plant reproduction requires long-distance growth of a pollen tube to fertilize the female gametophyte. Prior reports suggested that mutations altering synthesis of flavonoids, plant specialized metabolites that include flavonols and anthocyanins, impair pollen development in several species, but the mechanism by which flavonols enhanced fertility was not defined. Here, we used genetic approaches to demonstrate that flavonols enhanced pollen development by reducing the abundance of reactive oxygen species (ROS). We further showed that flavonols reduced high-temperature stress-induced ROS accumulation and inhibition of pollen tube growth. The anthocyanin reduced (are) tomato mutant had reduced flavonol accumulation in pollen grains and tubes. This mutant produced fewer pollen grains and had impaired pollen viability, germination, tube growth, and tube integrity, resulting in reduced seed set. Consistent with flavonols acting as ROS scavengers, are had elevated levels of ROS. The pollen viability, tube growth and integrity defects, and ROS accumulation in are were reversed by genetic complementation. Inhibition of ROS synthesis or scavenging of excess ROS with an exogenous antioxidant treatment also reversed the are phenotypes, indicating that flavonols function by reducing ROS levels. Heat stress resulted in increased ROS in pollen tubes and inhibited tube growth, with more pronounced effects in the are mutant that could be rescued by antioxidant treatment. These results are consistent with increased ROS inhibiting pollen tube growth and with flavonols preventing ROS from reaching damaging levels. These results reveal that flavonol metabolites regulate plant sexual reproduction at both normal and elevated temperatures by maintaining ROS homeostasis.

Identification of Transcriptional and Receptor Networks That Control Root Responses to Ethylene.

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 (Arabidopsis thaliana) 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 ein3 and eil1 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.

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

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.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

Abscisic Acid-Induced Reactive Oxygen Species Are Modulated by Flavonols to Control Stomata Aperture.

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 ABA-dependent ROS burst that drives stomatal closure and facilitating stomatal opening to modulate leaf gas exchange.

Transcriptional and Hormonal Regulation of Gravitropism of Woody Stems in Populus.

Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation.

Transcriptional sequencing and analysis of major genes involved in the adventitious root formation of mango cotyledon segments.

MAIN CONCLUSION: A total of 74,745 unigenes were generated and 1975 DEGs were identified. Candidate genes that may be involved in the adventitious root formation of mango cotyledon segment were revealed. Adventitious root formation is a crucial step in plant vegetative propagation, but the molecular mechanism of adventitious root formation remains unclear. Adventitious roots formed only at the proximal cut surface (PCS) of mango cotyledon segments, whereas no roots were formed on the opposite, distal cut surface (DCS). To identify the transcript abundance changes linked to adventitious root development, RNA was isolated from PCS and DCS at 0, 4 and 7 days after culture, respectively. Illumina sequencing of libraries generated from these samples yielded 62.36 Gb high-quality reads that were assembled into 74,745 unigenes with an average sequence length of 807 base pairs, and 33,252 of the assembled unigenes at least had homologs in one of the public databases. Comparative analysis of these transcriptome databases revealed that between the different time points at PCS there were 1966 differentially expressed genes (DEGs), while there were only 51 DEGs for the PCS vs. DCS when time-matched samples were compared. Of these DEGs, 1636 were assigned to gene ontology (GO) classes, the majority of that was involved in cellular processes, metabolic processes and single-organism processes. Candidate genes that may be involved in the adventitious root formation of mango cotyledon segment are predicted to encode polar auxin transport carriers, auxin-regulated proteins, cell wall remodeling enzymes and ethylene-related proteins. In order to validate RNA-sequencing results, we further analyzed the expression profiles of 20 genes by quantitative real-time PCR. This study expands the transcriptome information for Mangifera indica and identifies candidate genes involved in adventitious root formation in cotyledon segments of mango.

A kinetic analysis of the auxin transcriptome reveals cell wall remodeling proteins that modulate lateral root development in Arabidopsis.

To identify gene products that participate in auxin-dependent lateral root formation, a high temporal resolution, genome-wide transcript abundance analysis was performed with auxin-treated Arabidopsis thaliana roots. Data analysis identified 1246 transcripts that were consistently regulated by indole-3-acetic acid (IAA), partitioning into 60 clusters with distinct response kinetics. We identified rapidly induced clusters containing auxin-response functional annotations and clusters exhibiting delayed induction linked to cell division temporally correlated with lateral root induction. Several clusters were enriched with genes encoding proteins involved in cell wall modification, opening the possibility for understanding mechanistic details of cell structural changes that result in root formation following auxin treatment. Mutants with insertions in 72 genes annotated with a cell wall remodeling function were examined for alterations in IAA-regulated root growth and development. This reverse-genetic screen yielded eight mutants with root phenotypes. Detailed characterization of seedlings with mutations in cellulase3/glycosylhydrolase9b3 and leucine rich extensin2, genes not normally linked to auxin response, revealed defects in the early and late stages of lateral root development, respectively. The genes identified here using kinetic insight into expression changes lay the foundation for mechanistic understanding of auxin-mediated cell wall remodeling as an essential feature of lateral root development.

Ethylene-induced flavonol accumulation in guard cells suppresses reactive oxygen species and moderates stomatal aperture.

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 flavonol-specific 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 ethylene-overproducing1 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.

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

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-ß-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.

Block of ATP-binding cassette B19 ion channel activity by 5-nitro-2-(3-phenylpropylamino)-benzoic acid impairs polar auxin transport and root gravitropism.

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target.

Nitric oxide plays a role in stem cell niche homeostasis through its interaction with auxin.

Nitric oxide (NO) is a unique reactive nitrogen molecule with an array of signaling functions that modulates plant developmental processes and stress responses. To explore the mechanisms by which NO modulates root development, we used a pharmacological approach and NO-deficient mutants to unravel the role of NO in establishing auxin distribution patterns necessary for stem cell niche homeostasis. Using the NO synthase inhibitor and Arabidopsis (Arabidopsis thaliana) NO biosynthesis mutants (nitric oxide-associated1 [noa1], nitrate reductase1 [nia1] and nia2, and nia1 nia2 noa1), we show that depletion of NO in noa1 reduces primary root elongation and increases flavonol accumulation consistent with elevated reactive oxygen species levels. The elevated flavonols are required for the growth effect, because the transparent testa4 mutation reverses the noa1 mutant root elongation phenotype. In addition, noa1 and nia1 nia2 noa1 NO-deficient mutant roots display small root meristems with abnormal divisions. Concomitantly, auxin biosynthesis, transport, and signaling are perturbed. We further show that NO accumulates in cortex/endodermis stem cells and their precursor cells. In endodermal and cortical cells, the noa1 mutant acts synergistically to the effect of the wuschel-related homeobox5 mutation on the proximal meristem, suggesting that NO could play an important role in regulating stem cell decisions, which has been reported in animals.

Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis.

Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds known as flavonols have been shown to act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, we demonstrate that the WRKY23 transcription factor is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway. Our results suggest a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis.

Ethylene inhibits lateral root development, increases IAA transport and expression of PIN3 and PIN7 auxin efflux carriers.

We used genetic and molecular approaches to identify mechanisms by which the gaseous plant hormone ethylene reduces lateral root formation and enhances polar transport of the hormone auxin. Arabidopsis thaliana mutants, aux1, lax3, pin3 and pin7, which are defective in auxin influx and efflux proteins, were less sensitive to the inhibition of lateral root formation and stimulation of auxin transport following treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). By contrast, pin2 and abcb19 mutants exhibited wild-type ACC responses. ACC and indole-3-acetic acid (IAA) increased the abundance of transcripts encoding auxin transport proteins in an ETR1 and EIN2 (ethylene signaling)-dependent and TIR1 (auxin receptor)-dependent fashion, respectively. The effects of ACC on these transcripts and on lateral root development were still present in the tir1 mutant, suggesting independent signaling networks. ACC increased auxin-induced gene expression in the root apex, but decreased expression in regions where lateral roots form and reduced free IAA in whole roots. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) had opposite effects on auxin-dependent gene expression. These results suggest that ACC affects root development by altering auxin distribution. PIN3- and PIN7-GFP fluorescence was increased or decreased after ACC or AVG treatment, respectively, consistent with the role of PIN3 and PIN7 in ACC-elevated transport. ACC treatment abolished a localized depletion of fluorescence of PIN3- and PIN7-GFP, normally found below the site of primordia formation. These results suggest that ACC treatment increased PIN3 and PIN7 expression, resulting in elevated auxin transport, which prevented the localized accumulation of auxin needed to drive lateral root formation.

Localized induction of the ATP-binding cassette B19 auxin transporter enhances adventitious root formation in Arabidopsis.

Adventitious roots emerge from aerial plant tissues, and the induction of these roots is essential for clonal propagation of agriculturally important plant species. This process has received extensive study in horticultural species but much less focus in genetically tractable model species. We have explored the role of auxin transport in this process in Arabidopsis (Arabidopsis thaliana) seedlings in which adventitious root initiation was induced by excising roots from low-light-grown hypocotyls. Inhibition of auxin transport from the shoot apex abolishes adventitious root formation under these conditions. Root excision was accompanied by a rapid increase in radioactive indole-3-acetic acid (IAA) transport and its accumulation in the hypocotyl above the point of excision where adventitious roots emerge. Local increases in auxin-responsive gene expression were also observed above the site of excision using three auxin-responsive reporters. These changes in auxin accumulation preceded cell division events, monitored by a cyclin B1 reporter (pCYCB1;1:GUS), and adventitious root initiation. We examined excision-induced adventitious root formation in auxin influx and efflux mutants, including auxin insensitive1, pin-formed1 (pin1), pin2, pin3, and pin7, with the most profound reductions observed in ATP-binding cassette B19 (ABCB19). An ABCB19 overexpression line forms more adventitious roots than the wild type in intact seedlings. Examination of transcriptional and translational fusions between ABCB19 and green fluorescent protein indicates that excision locally induced the accumulation of ABCB19 transcript and protein that is temporally and spatially linked to local IAA accumulation leading to adventitious root formation. These experiments are consistent with localized synthesis of ABCB19 protein after hypocotyl excision leads to enhanced IAA transport and local IAA accumulation driving adventitious root formation.