Strigolactones affect lateral root formation and root-hair elongation in Arabidopsis

Strigolactones (SLs) have been proposed as a new group of plant hormones, inhibiting shoot branching, and as signaling molecules for plant interactions. Here, we present evidence for effects of SLs on root development. The analysis of mutants flawed in SLs synthesis or signaling suggested that the absence of SLs enhances lateral root formation. In accordance, roots grown in the presence of GR24, a synthetic bioactive SL, showed reduced number of lateral roots in WT and in max3-11 and max4-1 mutants, deficient in SL synthesis. The GR24-induced reduction in lateral roots was not apparent in the SL signaling mutant max2-1. Moreover, GR24 led to increased root-hair length in WT and in max3-11 and max4-1 mutants, but not in max2-1. SLs effect on lateral root formation and root-hair elongation may suggest a role for SLs in the regulation of root development; perhaps, as a response to growth conditions.     

Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis

Plant hormone brassinosteroids (BRs) and auxin exert some similar physiological effects likely through their functional interaction, but the mechanism for this interaction is unknown. In this study, we show that BRs are required for lateral root development in Arabidopsis and that BRs act synergistically with auxin to promte lateral root formation. BR perception is required for the transgenic expression of the beta-glucuronidase gene fused to a synthetic auxin-inducible promoter (DR5::GUS) in root tips, while exogenous BR promotes DR5::GUS expression in the root tips and the stele region proximal to the root tip. BR induction of both lateral root formation and DR5::GUS expression is suppressed by the auxin transport inhibitor N-(1-naphthyl) phthalamic acid. Importantly, BRs promote acropetal auxin transport (from the base to the tip) in the root. Our observations indicate that BRs regulate auxin transport, providing a novel mechanism for hormonal interactions in plants and supporting the hypothesis that BRs promote lateral root development by increasing acropetal auxin transport.     

Role of auxin in regulating Arabidopsis flower development

To elucidate the role of auxin in flower morphogenesis, its distribution patterns were studied during flower development in Arabidopsis thaliana (L.) Heynh. Expression of DR5::GUS was regarded to reflect sites of free auxin, while immunolocalization with auxin polyclonal antibodies visualized conjugated auxin distribution. The youngest flower bud was loaded with conjugated auxin. During development, the apparent concentration of free auxin increased in gradual patterns starting at the floral-organ tip. Anthers are major sites of high concentrations of free auxin that retard the development of neighboring floral organs in both the acropetal and basipetal directions. The IAA-producing anthers synchronize flower development by retarding petal development and nectary gland activity almost up to anthesis. Tapetum cells of young anthers contain free IAA which accumulates in pollen grains, suggesting that auxin promotes pollen-tube growth towards the ovules. High amounts of free auxin in the stigma induce a wide xylem fan immediately beneath it. After fertilization, the developing embryos and seeds show elevated concentrations of auxin, which establish their axial polarity. This developmental pattern of auxin production during floral-bud development suggests that young organs which produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip. Electronic Supplementary Material Supplementary material is available for this article at This paper is dedicated to Orna Aloni for continuous support and management over many years.     

Multilevel interactions between ethylene and auxin in Arabidopsis roots

Hormones play a central role in the coordination of internal developmental processes with environmental signals. Herein, a combination of physiological, genetic, cellular, and whole-genome expression profiling approaches has been employed to investigate the mechanisms of interaction between two key plant hormones: ethylene and auxin. Quantification of the morphological effects of ethylene and auxin in a variety of mutant backgrounds indicates that auxin biosynthesis, transport, signaling, and response are required for the ethylene-induced growth inhibition in roots but not in hypocotyls of dark-grown seedlings. Analysis of the activation of early auxin and ethylene responses at the cellular level, as well as of global changes in gene expression in the wild type versus auxin and ethylene mutants, suggests a simple mechanistic model for the interaction between these two hormones in roots, according to which ethylene and auxin can reciprocally regulate each other's biosyntheses, influence each other's response pathways, and/or act independently on the same target genes. This model not only implies existence of several levels of interaction but also provides a likely explanation for the strong ethylene response defects observed in auxin mutants.     

A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis

Although auxin response factors (ARFs) are the first well-characterized proteins that bind to the auxin response elements, elucidation of the roles of each ARF gene in auxin responses and plant development has been challenging. Here we show that ARF19 and ARF7 not only participate in auxin signaling, but also play a critical role in ethylene responses in Arabidopsis (Arabidopsis thaliana) roots, indicating that the ARFs serve as a cross talk point between the two hormones. Both arf19 and arf7 mutants isolated from our forward genetic screens are auxin resistant and the arf19arf7 double mutant had stronger auxin resistance than the single mutants and displayed phenotypes not seen in the single mutants. Furthermore, we show that a genomic fragment of ARF19 not only complements arf19, but also rescues arf7. We conclude that ARF19 complements ARF7 at the protein level and that the ARF7 target sequences are also recognized by ARF19. Therefore, it is the differences in expression level/pattern and not the differences in protein sequences between the two ARFs that determines the relative contribution of the two ARFs in auxin signaling and plant development. In addition to being auxin resistant, arf19 has also ethylene-insensitive roots and ARF19 expression is induced by ethylene treatment. This work provides a sensitive genetic screen for uncovering auxin-resistant mutants including the described arf mutants. This study also provides a likely mechanism for coordination and integration of hormonal signals to regulate plant growth and development.     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

Inhibitory action of auxin on root elongation not mediated by ethylene

The inhibitory effects of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) on elongation growth of pea (Pisum sativum L.) seedling roots were investigated in relation to the effects of these compounds on ethylene production by the root tips. When added to the growth solution both compounds caused a progressively increasing inhibition of growth within the concentration range of 0.01 to 1 micromolar. However, only ACC increased ethylene production in root tips excised from the treated seedlings after 24 hours. High auxin concentrations caused a transitory increase of ethylene production during a few hours in the beginning of the treatment period, but even in 1 micromolar IAA this increase was too low to have any appreciable effect on growth. ACC, but not IAA, caused growth curvatures, typical of ethylene treatment, in the root tips. IAA caused conspicuous swelling of the root tips while ACC did not. Cobalt and silver ions reversed the growth inhibitory effects induced by ACC but did not counteract the inhibition of elongation or swelling caused by IAA. The growth effects caused by the ACC treatments were obviously due to ethylene production. We found no evidence to indicate that the growth inhibition or swelling caused by IAA is mediated by ethylene. It is concluded that the inhibitory action of IAA on root growth is caused by this auxin per se.     

Brassinolide-induced elongation and auxin

Segments from the hook and subhook zone of the stem of 6-day-old etiolated Pisum sativum L. cv. Victory Freezer seedlings were used to study the relationship between brassinolide and auxin in the promotion of elongation. Minor changes in exogenous indole-3-acetic acid or4-chloroindole-3-acetic acid concentration affected the kinetics markedly and the ethylene generator ethephon overcame brassinolide-induced elongation in an antagonistic interaction. Brassinolide-induced elongation was markedly inhibited by low concentrations of the cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile, and diagnostic concentrations of the antiauxin 2-( p -chlorophenoxy) isobutyric acid did not affect brassinolide-induced elongation. As the characteristics of auxin-induced growth are not displayed in brassinolide-induced elongation of the upper stem segment, it is proposed that brassinolide does not depend on auxin as a mediator in the promotion of elongation of younger tissues but that it can interact in a very complex manner with auxin. In the elongation of more mature tissues, and in bending responses, brassinolide probably accelerates auxin effects. When split, the upper stem segment was unusual in its lack of specific response to growth regulators, and the slight relief of epidermal tension.     

Strigolactones suppress adventitious rooting in Arabidopsis and pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.     

The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport

BACKGROUND: Plants achieve remarkable plasticity in shoot system architecture by regulating the activity of secondary shoot meristems, laid down in the axil of each leaf. Axillary meristem activity, and hence shoot branching, is regulated by a network of interacting hormonal signals that move through the plant. Among these, auxin, moving down the plant in the main stem, indirectly inhibits axillary bud outgrowth, and an as yet undefined hormone, the synthesis of which in Arabidopsis requires MAX1, MAX3, and MAX4, moves up the plant and also inhibits shoot branching. Since the axillary buds of max4 mutants are resistant to the inhibitory effects of apically supplied auxin, auxin and the MAX-dependent hormone must interact to inhibit branching. RESULTS: Here we show that the resistance of max mutant buds to apically supplied auxin is largely independent of the known, AXR1-mediated, auxin signal transduction pathway. Instead, it is caused by increased capacity for auxin transport in max primary stems, which show increased expression of PIN auxin efflux facilitators. The max phenotype is dependent on PIN1 activity, but it is independent of flavonoids, which are known regulators of PIN-dependent auxin transport. CONCLUSIONS: The MAX-dependent hormone is a novel regulator of auxin transport. Modulation of auxin transport in the stem is sufficient to regulate bud outgrowth, independent of AXR1-mediated auxin signaling. We therefore propose an additional mechanism for long-range signaling by auxin in which bud growth is regulated by competition between auxin sources for auxin transport capacity in the primary stem.     

hydra Mutants of Arabidopsis are defective in sterol profiles and auxin and ethylene signaling

The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Delta8-Delta7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file-specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function.     

Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis

Strigolactones (SL) fulfil important roles in plant development and stress tolerance. Here, we characterized the role of SL in the dark chilling tolerance of pea and Arabidopsis by analysis of mutants that are defective in either SL synthesis or signalling. Pea mutants (rms3, rms4, and rms5) had significantly greater shoot branching with higher leaf chlorophyll a/b ratios and carotenoid contents than the wild type. Exposure to dark chilling significantly decreased shoot fresh weights but increased leaf numbers in all lines. Moreover, dark chilling treatments decreased biomass (dry weight) accumulation only in rms3 and rms5 shoots. Unlike the wild type plants, chilling‐induced inhibition of photosynthetic carbon assimilation was observed in the rms lines and also in the Arabidopsis max3‐9, max4‐1, and max2‐1 mutants that are defective in SL synthesis or signalling. When grown on agar plates, the max mutant rosettes accumulated less biomass than the wild type. The synthetic SL, GR24, decreased leaf area in the wild type, max3‐9, and max4‐1 mutants but not in max2‐1 in the absence of stress. In addition, a chilling‐induced decrease in leaf area was observed in all the lines in the presence of GR24. We conclude that SL plays an important role in the control of dark chilling tolerance.     

Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation

Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis thaliana have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin biosynthesis in the root apex. We subsequently demonstrated that ethylene-regulated root growth is dependent on (1) the transport of auxin from the root apex via the lateral root cap and (2) auxin responses occurring in multiple elongation zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by upregulating auxin biosynthesis, ethylene facilitates its ability to inhibit root cell expansion.     

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.     

A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid

Summary We have screened a large population of M₂ seeds ofArabidopsis thaliana for plants which are resistant to exogenously applied indole-acetic acid (IAA). One of the resistant lines identified in this screen carries a dominant mutation which we have namedaxr2. Linkage analysis indicates that theaxr2 gene lies on chromosome 3. Plants carrying theaxr2 mutation are severe dwarfs and display defects in growth orientation of both the shoot and root suggesting that the mutation affects some aspect of gravitropic growth. In addition, the roots ofaxr2 plants lack root hairs. Growth inhibition experiments indicate that the roots ofaxr2 plants are resistant to ethylene and abscisic acid as well as auxin.     

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506–508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4–17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways. Experiment station: Plant Biotechnology Centre, Primary Industries Research Victoria, Department of Primary Industries, La Trobe University, Bundoora, Victoria 3086, and the Victorian Microarray Technology Consortium (VMTC).     

Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis

Cytokinins and auxins are major phytohormones involved in various aspects of plant growth and development. These phytohormones are also known to antagonize the effects of abscisic acid (ABA) on stomatal movement, and to affect ethylene biosynthesis. As ethylene has an antagonistic effect on ABA-induced stomatal closure, the possibility that the antagonistic effects of these phytohormones on ABA were mediated through ethylene biosynthesis was investigated. Both the cytokinin, 6-benzyladenine (BA), and the auxin, 1-naphthaleneacetic acid (NAA), antagonized ABA-induced stomatal closure in a manner similar to that following application of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). However, these effects were negated when ethylene signalling, perception, or biosynthesis were blocked. As stomatal aperture is regulated by changes in guard cell volume, ABA application was found to reduce the volume of the guard cell protoplasts (GCP). It was found that BA, NAA, or ACC application compensated perfectly for the reduction in GCP volume by ABA application in WT plants. The above observations suggest that cytokinins and auxins inhibit ABA-induced stomatal closure through the modulation of ethylene biosynthesis, and that ethylene inhibits the ABA-induced reduction of osmotic pressure in the guard cells.     

The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin

Ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) can stimulate hypocotyl elongation in light-grown Arabidopsis seedlings. A mutant, designated ACC-related long hypocotyl 1 (alh1), that displayed a long hypocotyl in the light in the absence of the hormone was characterized. Etiolated alh1 seedlings overproduced ethylene and had an exaggerated apical hook and a thicker hypocotyl, although no difference in hypocotyl length was observed when compared with wild type. Alh1 plants were less sensitive to ethylene, as reflected by reduction of ACC-mediated inhibition of hypocotyl growth in the dark and delay in flowering and leaf senescence. Alh1 also had an altered response to auxin, whereas auxin levels in whole alh1 seedlings remained unaffected. In contrast to wild type, alh1 seedlings showed a limited hypocotyl elongation when treated with indole-3-acetic acid. Alh1 roots had a faster response to gravity. Furthermore, the hypocotyl elongation of alh1 and of ACC-treated wild type was reverted by auxin transport inhibitors. In addition, auxin up-regulated genes were ectopically expressed in hypocotyls upon ACC treatment, suggesting that the ethylene response is mediated by auxins. Together, these data indicate that alh1 is altered in the cross talk between ethylene and auxins, probably at the level of auxin transport.