Toyocamycin specifically inhibits auxin signaling mediated by SCF pathway

The auxins, plant hormones, play a crucial role in many aspects of plant development by regulating cell division, elongation and differentiation. Toyocamycin, a nucleoside-type antibiotic, was identified as auxin signaling inhibitor in a screen of microbial extracts for inhibition of the auxin-inducible reporter gene assay. Toyocamycin specifically inhibited auxin-responsive gene expression, but did not affect other hormone-inducible gene expression. Toyocamycin also blocked auxin-enhanced degradation of the Aux/IAA repressor modulated by the SCF(TIR1) ubiquitin-proteasome pathway without inhibiting proteolytic activity of proteasome. Furthermore, toyocamycin inhibited auxin-induced lateral root formation and epinastic growth of cotyledon in the Arabidopsis thaliana plant. This evidence suggested that toyocamycin would act on the ubiquitination process regulated by SCF(TIR1) machineries. To address the structural requirements for the specific activity of toyocamycin on auxin signaling, the structure-activity relationships of nine toyocamycin-related compounds, including sangivamycin and tubercidin, were investigated.     

Characterisation of root amino acid exudation in white clover (Trifolium repens L.)

Lateral roots are crucial for the plasticity of root responses to environmental conditions in soil. The bacterivorous microfauna has been shown to increase root branching and to foster auxin producing soil bacteria. However, information on modifications of plant internal auxin content by soil bacteria and bacterivores is missing. Therefore, the effects of a rhizosphere bacterial community and a common soil amoeba (Acanthamoeba castellanii) on root branching and on auxin (indole-3-acetic acid) metabolism in Lepidium sativum and Arabidopsis thaliana were investigated. In a first experimental series, bacteria increased conjugated auxin concentrations in L. sativum shoots, but did not alter free bioactive auxin content nor root branching. In contrast, in presence of soil bacteria plus amoebae free auxin concentrations in shoots and root branching increased, demonstrating that effects of bacteria on auxin metabolism in plants were strongly modified by the bacterivorous amoebae. In a second experiment, A. thaliana reporter plants for auxin (DR5) and cytokinin (ARR5) responded similarly with increased root branching in the presence of amoebae. Surprisingly, in reporter plants cytokinin but not auxin responses were detectable, accompanied by higher soil nitrate concentrations in the presence of amoebae. Likely, increased nitrate concentrations in the rhizosphere led to an accumulation of cytokinin and interactions with free auxin in plants and finally to increased root growth in the presence of amoebae. Altogether, the results show that mutual control mechanisms exist between plant hormone metabolism and microbial signalling, and that effects on hormonal concentrations of plants by free-living bacteria are strongly influenced by bacterial grazers like amoebae.     

Arabidopsis ROP9 and ROP10 GTPases differentially regulate auxin and ABA responses

Auxin and abscisic acid (ABA) are major plant hormones that act together to modulate numerous aspects of plant growth and development, including seed germination, primary root elongation, and lateral root formation. In this study, we analyzed the loss-of-function mutants of two closely related ROP (Rho of plants) GTPases, ROP9 and ROP10, and found that these ROP GTPases differentially regulate the auxin and ABA responses. rop9 and rop10 mutations enhanced the ABA-induced suppression of seed germination, primary root growth, and lateral root formation and the expression of ABA-responsive genes, whereas rop9 but not rop10 suppressed auxin-induced root phenotypes and auxin-responsive gene expression. These results suggest that both ROP9 and ROP10 function as negative regulators of ABA signaling, and that ROP9, but not ROP10, functions as a positive regulator of auxin signaling. Previously, ROPinteractive CRIB motif-containing protein 1 (RIC1) was reported to participate in auxin and ABA responses, and to have a similar effect as ROP9 and ROP10 on gene expression, root development, and seed germination. Because RIC proteins mediate ROP GTPase signaling, our results suggest that ROP9 and ROP10 GTPases function upstream of RIC1 in auxin- and ABA-regulated root development and seed germination.     

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild‐type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss‐of‐function mutations, or in a dominant (gain‐of‐function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.     

dhm1, an Arabidopsis mutant with increased sensitivity to alkamides shows tumorous shoot development and enhanced lateral root formation

The control of cell division by growth regulators is critical to proper shoot and root development. Alkamides belong to a class of small lipid amides involved in plant morphogenetic processes, from which N-isobutyl decanamide is one of the most active compounds identified. This work describes the isolation and characterization of an N-isobutyl decanamide-hypersensitive (dhm1) mutant of Arabidopsis (Arabidopsis thaliana). dhm1 seedlings grown in vitro develop disorganized tumorous tissue in petioles, leaves and stems. N-isobutyl decanamide treatment exacerbates the dhm1 phenotype resulting in widespread production of callus-like structures in the mutant. Together with these morphological alterations in shoot, dhm1 seedlings sustained increased lateral root formation and greater sensitivity to alkamides in the inhibition of primary root growth. The mutants also show reduced etiolation when grown in darkness. When grown in soil, adult dhm1 plants were characterized by reduced plant size, and decreased fertility. Genetic analysis indicated that the mutant phenotype segregates as a single recessive Mendelian trait. Developmental alterations in dhm1 were related to an enhanced expression of the cell division marker CycB1-uidA both in the shoot and root system, which correlated with altered expression of auxin and cytokinin responsive gene markers. Pharmacological inhibition of auxin transport decreased LR formation in WT and dhm1 seedlings in a similar manner, indicating that auxin transport is involved in the dhm1 root phenotype. These data show an important role of alkamide signaling in cell proliferation and plant architecture remodeling likely acting through the DHM1 protein.     

The TRANSPORT INHIBITOR RESPONSE2 Gene Is Required for Auxin Synthesis and Diverse Aspects of Plant Development

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.Auxin is known to play an important role in plant development (Davies, 1995). However, many aspects of auxin biology remain poorly understood. Auxin is synthesized primarily in young tissues, such as cotyledons, leaves, and roots (Ljung et al., 2001, 2005), and transported to other tissues where it is perceived by members of the TRANSPORT INHIBITOR RESPONSE1 (TIR1) auxin receptor family. Recent studies have dramatically increased our knowledge of auxin transport and signaling (Quint and Gray, 2006; Vieten et al., 2007). However, the pathways of auxin synthesis and their regulation are still relatively unclear.Several indole-3-acetic acid (IAA) biosynthetic pathways have been proposed in plants based on research in plant-associated bacteria (Patten and Glick, 1996; Woodward and Bartel, 2005; Spaepen et al., 2007). There are two major types of pathways: the Trp-dependent and Trp-independent pathways. It has been hypothesized that plants have four Trp-dependent pathways that are generally named after an intermediate. In bacteria, the indole-3-pyruvic acid (IPA) pathway, one of the Trp-dependent pathways, has been described in detail (Koga, 1995; Spaepen et al., 2007). The current model for the IPA pathway involves a Trp aminotransferase oxidatively transaminating Trp to IPA. Subsequently, an IPA decarboxylase converts IPA to indole-3-acetaldehyde, and indole-3-acetaldehyde is oxidized to IAA. The IPA pathway is considered a major IAA biosynthetic pathway in plants, since potential intermediates have been isolated from different species (Sheldrake, 1973; Cooney and Nonhebel, 1991; Koga, 1995; Tam and Normanly, 1998). In addition, Trp transamination activity has been found in many plants (Gamborg, 1965; Forest and Wightman, 1972; Truelsen, 1973). Recently, two groups reported the identification of a gene called TAA1. This gene encodes an aminotransferase that converts Trp to IPA and functions in IAA biosynthesis (Stepanova et al., 2008; Tao et al., 2008).To identify genes that are required for auxin synthesis, transport, and signaling, we previously screened for Arabidopsis (Arabidopsis thaliana) mutants that are resistant to auxin transport inhibitors, such as N-1-napthylpthalamic (NPA; Ruegger et al., 1997). The treatment of seedlings with NPA results in auxin accumulation in the root tip (Ljung et al., 2005). Thus, mutants that are resistant to NPA may have defects in synthesis, transport, or response because roots of these mutants are expected to have lower levels of IAA or reduced sensitivity to IAA. This screen succeeded in isolating mutations in seven genes with weak NPA-resistant phenotypes, including genes related to auxin signaling (TIR1), auxin transport (TIR3), and auxin synthesis (TIR7; Ruegger et al., 1997, 1998; Ljung et al., 2005).Here, we describe the characterization of TIR2, a gene whose function is required for auxin synthesis. Genetic and physiological analyses of the tir2 mutant suggest that TIR2 is required for the Trp-dependent auxin synthesis pathway and functions as a Trp aminotransferase. Molecular cloning of TIR2 reveals that the gene is identical to TAA1 (Stepanova et al., 2008; Tao et al., 2008). We show that auxin regulates expression of TIR2 in a tissue-specific manner. Furthermore, we show that TIR2 is required for temperature-dependent hypocotyl elongation and that high temperature positively regulates expression of the TIR2 gene, suggesting that temperature regulates hypocotyl elongation directly by stimulating auxin synthesis. Finally, we provide evidence that TIR2 functions in a positive regulatory loop required for root gravitropism.     

Aeromonas punctata PNS-1: a promising candidate to change the root morphogenesis of Arabidopsis thaliana in MS and sand system

A model system of sand, comprising Arabidopsis plants inoculated with Aeromonas punctata PNS-1 strain, was used to evaluate the bacterial effect in modulation of plant root structure at second-order lateral root level. In MS media, the root morphogenesis was changed only at first-order lateral root level when inoculated with PNS-1 strain. Inoculation with PNS-1 strain was subjected to significant (P < 0.01) increase in primary root length and lateral root density in both MS and sand system. However, this strain modulated the root structure in the sand environment in a complex manner that may be helpful for incitation of the plant–microbe interaction close to natural environment. In order to determine whether this change in root morphology was due to bacterial auxin, Arabidopsis transgenic line (DR5:GUS) was used to reveal the change in homeostasis of endogenous auxin. In PNS-1 inoculated transgenic seedlings of Arabidopsis plant (DR5:GUS), endogenous auxin in primary root apices and lateral roots was enhanced. For confirmation, PNS-1 was evaluated for auxin production in vitro, showed an increase in auxin production after supplementation of l-tryptophan. The presence of ACC deaminase activity in PNS-1 showed its possible involvement in primary root elongation. In the present study Aeromonas punctata PNS-1 is the potential candidate for triggering the change in root morphogenesis of Arabidopsis thaliana with the involvement of auxin and ACC deaminase production.     

Abscisic Acid Represses Growth of the Arabidopsis Embryonic Axis after Germination by Enhancing Auxin Signaling

Under unfavorable environmental conditions, the stress phytohormone ABA inhibits the developmental transition from an embryo in a dry seed into a young seedling. We developed a genetic screen to isolate Arabidopsis thaliana mutants whose early seedling development is resistant to ABA. Here, we report the identification of a recessive mutation in AUXIN RESISTANT1 (AUX1), encoding a cellular auxin influx carrier. Although auxin is a major morphogenesis hormone in plants, little is known about ABA–auxin interactions during early seedling growth. We show that aux1 and pin2 mutants are insensitive to ABA-dependent repression of embryonic axis (hypocotyl and radicle) elongation. Genetic and physiological experiments show that this involves auxin transport to the embryonic axis elongation zone, where ABA enhances the activity of an auxin-responsive promoter. We propose that ABA represses embryonic axis elongation by potentiating auxin signaling in its elongation zone. This involves repression of the AUXIN INDUCIBLE (Aux/IAA) gene AXR2/IAA7, encoding a key component of ABA- and auxin-dependent responses during postgerminative growth.     

Chloroplast protein PLGG1 is involved in abscisic acid-regulated lateral root development and stomatal movement in Arabidopsis

The plant hormone abscisic acid (ABA) plays a crucial role in root architecture; however, the molecular mechanism of ABA-regulated lateral root (LR) growth is not well known. We screened an Arabidopsis thaliana mutant with LR growth that was sensitive to ABA from a T-DNA insertion mutant library, which was an allelic mutant of plgg1-1, termed plgg1-2. PLGG1 encodes a chloroplast protein that transports plastidic glycolate and glycerate. The length and number of LRs at the root-hypocotyl junction of plgg1-1 and plgg1-2 were significantly impaired under exogenous ABA treatment, and the transgenic plant complementary lines of plgg1-2 restored LR growth in response to ABA. In addition, we found that PLGG1 is involved in other major ABA responses, including ABA-inhibited seed germination, ABA-mediated stomatal movement, and drought tolerance. These findings open new perspectives on elucidating the mechanism of ABA response, and provide clues for analysing the functions of chloroplast proteins in regulating root growth.     

The involvement of auxin in root architecture plasticity in Arabidopsis induced by heterogeneous phosphorus availability

Homogeneous low phosphorus availability was reported to regulate root architecture in Arabidopsis via auxin, but the roles of auxin in root architecture plasticity to heterogeneous P availability remain unclear. In this study, we employed auxin biosynthesis-, transport- and signalling-related mutants. Firstly, we found that in contrast to low P (LP) content in the whole medium, primary root (PR) growth of Arabidopsis was partially rescued in the medium divided into two parts: upper with LP and lower with high P (HP) content or in the reverse arrangement. The down part LP was more effective to arrest PR growth as well as to decrease density of lateral roots (DLR) than the upper LP, and effects were dependent on polar auxin transport. Secondly, we verified that auxin receptor TIR1 was involved in the responses of PR growth and lateral root (LR) development to P supply and loss of function of TIR1 inhibited LR development. Thirdly, effects of heterogeneous P on LRD in the upper part of PR was dependent on PIN2 and PIN4, and in the down part on PIN3 and PIN4, whereas density of total LRs was dependent on auxin transporters PIN2 and PIN7. Finally, heterogeneous P availability altered the accumulation of auxin in PR tip and the expression of auxin biosynthesisrelated genes TAA1, YUC1, YUC2, and YUC4. Taken together, we provided evidences for the involvement of auxin in root architecture plasticity in response to heterogeneous phosphorus availability in Arabidopsis.     

Abscisic Acid Elicits the Water-Stress Response in Root Hairs of Arabidopsis thaliana

Water stress has been shown to cause root hairs to become short and bulbous. Because abscisic acid (ABA) mediates a variety of water-stress responses, we investigated the response of Arabidopsis thaliana root hairs to ABA. When wild-type root hairs were treated with ABA, they exhibited the water-stress response. The Arabidopsis mutants abi1 and abi2, which are insensitive to ABA at the seedling stage, did not display the root hair response. These data suggest that ABA may mediate the response of root hairs to water stress. The drought response of root hairs resulting in an inhibition of tip growth will provide an easy screen to select mutations that are insensitive to ABA and/or involved in tip growth.     

The eta7/csn3-3 Auxin Response Mutant of Arabidopsis Defines a Novel Function for the CSN3 Subunit of the COP9 Signalosome

The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCF^TIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCF^TIR1-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.     

Genetic identification of a second site modifier of ctr1-1 that controls ethylene-responsive and gravitropic root growth in Arabidopsis thaliana

Ethylene controls myriad aspects of plant growth throughout developmental stages in higher plants. It has been well established that ethylene-responsive growth entails extensive crosstalk with other plant hormones, particularly auxin. Here, we report a genetic mutation, named 1-aminocyclopropane carboxylic acid (ACC) resistant root1-1 (are1-1) in Arabidopsis thaliana (L.) Heynh. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) encodes a Raf-related protein, functioning as an upstream negative regulator of ethylene signaling in Arabidopsis thaliana. We found that the ctr1-1, a kinase-inactive allele exhibited slightly, but significantly, longer root length, compared to ACC-treated wild-type or ctr1-3, a null allele. Our genetic studies unveiled the existence of are1-1 mutation in the ctr1-1 mutant, as a second-site modifier which confers root-specific ethylene-resistance. Based on well-characterized crosstalk between ethylene and auxin during ethylene-responsive root growth, we performed various physiological analyses. Whereas are1-1 displayed normal sensitivity to synthetic auxins, it showed modest resistance to an auxin transport inhibitor, 1-Nnaphthylphthalamic acid. In addition, are1-1 mutant exhibited ectopically altered DR5:GUS activity upon ethylenetreatment. The results implicated the involvement of are1-1 in auxin-distribution, but not in auxin-biosynthesis, -uptake, or -sensitivity. In agreement, are1-1 mutant exhibited reduced gravitropic root growth and defective redistribution of DR5:GUS activity upon gravi-stimulation. Taken together with genetic and molecular analysis, our results suggest that ARE1 defines a novel locus to control ethylene-responsive root growth as well as gravitropic root growth presumably through auxin distribution in Arabidopsis thaliana.