Arabidopsis ROOT UVB SENSITIVE2/WEAK AUXIN RESPONSE1 Is Required for Polar Auxin Transport

Auxin is an essential phytohormone that regulates many aspects of plant development. To identify new genes that function in auxin signaling, we performed a genetic screen for Arabidopsis thaliana mutants with an alteration in the expression of the auxin-responsive reporter DR5rev:GFP (for green fluorescent protein). One of the mutants recovered in this screen, called weak auxin response1 (wxr1), has a defect in auxin response and exhibits a variety of auxin-related growth defects in the root. Polar auxin transport is reduced in wxr1 seedlings, resulting in auxin accumulation in the hypocotyl and cotyledons and a reduction in auxin levels in the root apex. In addition, the levels of the PIN auxin transport proteins are reduced in the wxr1 root. We also show that WXR1 is ROOT UV-B SENSITIVE2 (RUS2), a member of the broadly conserved DUF647 domain protein family found in diverse eukaryotic organisms. Our data indicate that RUS2/WXR1 is required for auxin transport and to maintain the normal levels of PIN proteins in the root.     

SMALL ACIDIC PROTEIN1 Acts with RUB Modification Components, the COP9 Signalosome, and AXR1 to Regulate Growth and Development of Arabidopsis

Previously, a dysfunction of the SMALL ACIDIC PROTEIN1 (SMAP1) gene was identified as the cause of the anti-auxin resistant1 (aar1) mutant of Arabidopsis (Arabidopsis thaliana). SMAP1 is involved in the response pathway of synthetic auxin, 2,4-dichlorophenoxyacetic acid, and functions upstream of the auxin/indole-3-acetic acid protein degradation step in auxin signaling. However, the exact mechanism by which SMAP1 functions in auxin signaling remains unknown. Here, we demonstrate that SMAP1 is required for normal plant growth and development and the root response to indole-3-acetic acid or methyl jasmonate in the auxin resistant1 (axr1) mutation background. Deletion analysis and green fluorescent protein/glutathione S-transferase pull-down assays showed that SMAP1 physically interacts with the CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME (CSN) via the SMAP1 F/D region. The extremely dwarf phenotype of the aar1-1 csn5a-1 double mutant confirms the functional role of SMAP1 in plant growth and development under limiting CSN functionality. Our findings suggest that SMAP1 is involved in the auxin response and possibly in other cullin-RING ubiquitin ligase-regulated signaling processes via its interaction with components associated with RELATED TO UBIQUITIN modification.     

A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid

2,4-dichlorophenoxyacetic acid (2,4-D), a chemical analogue of indole-3-acetic acid (IAA), is widely used as a growth regulator and exogenous source of auxin. Because 2,4-D evokes physiological and molecular responses similar to those evoked by IAA, it is believed that they share a common response pathway. Here, we show that a mutant, antiauxin resistant1 (aar1), identified in a screen for resistance to the anti-auxin p-chlorophenoxy-isobutyric acid (PCIB), is resistant to 2,4-D, yet nevertheless responds like the wild-type to IAA and 1-napthaleneacetic acid in root elongation and lateral root induction assays. That the aar1 mutation alters 2,4-D responsiveness specifically was confirmed by analysis of GUS expression in the DR5:GUS and HS:AXR3NT-GUS backgrounds, as well as by real-time PCR quantification of IAA11 expression. The two characterized aar1 alleles both harbor multi-gene deletions; however, 2,4-D responsiveness was restored by transformation with one of the genes missing in both alleles, and the 2,4-D-resistant phenotype was reproduced by decreasing the expression of the same gene in the wild-type using an RNAi construct. The gene encodes a small, acidic protein (SMAP1) with unknown function and present in plants, animals and invertebrates but not in fungi or prokaryotes. Taken together, these results suggest that SMAP1 is a regulatory component that mediates responses to 2,4-D, and that responses to 2,4-D and IAA are partially distinct.     

Serotonin, a tryptophan-derived signal conserved in plants and animals, regulates root system architecture probably acting as a natural auxin inhibitor in Arabidopsis thaliana

Serotonin (5-hydroxytryptamine) is a well-known neurotransmitter in mammals and is widely distributed in plants. This compound is synthesized from tryptophan and shares structural similarity with IAA. To date, little is known about the morphological, physiological and molecular responses of plants to serotonin. In this study, we characterized the effects of serotonin on growth and development in Arabidopsis thaliana seedlings. Gas chromatography-mass spectrometry (GC-MS) analysis showed that plants are able to take up serotonin from the growth medium, which coincided with greatly stimulated lateral root development at concentrations from 10 to 160 μM. In contrast, higher doses of serotonin repressed lateral root growth, primary root growth and root hair development, but stimulated adventitious root formation. To investigate the role of serotonin in modulating auxin responses, we performed experiments using transgenic Arabidopsis lines expressing the auxin-responsive marker constructs DR5:uidA, BA3:uidA and HS::AXR3NT-GUS, as well as a variety of Arabidopsis mutants defective at the AUX1, AXR1, AXR2 and AXR4 auxin-related loci. We found that serotonin strongly inhibited both DR5:uidA and BA3:uidA gene expression in primary and adventitious roots and in lateral root primordia. This compound also abolished the effects of IAA or naphthaleneacetic acid on auxin-regulated developmental and genetic responses, indicating an anti-auxin activity in the plant. Mutant analysis further showed that lateral root induction elicited by serotonin was independent of the AUX1 and AXR4 loci but required AXR1 and AXR2. Our results show that serotonin regulates root development probably by acting as a natural auxin inhibitor.     

Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling in Arabidopsis

Arabidopsis (Arabidopsis thaliana) mutants lacking a functional ERA1 gene, which encodes the beta-subunit of protein farnesyltransferase (PFT), exhibit pleiotropic effects that establish roles for protein prenylation in abscisic acid (ABA) signaling and meristem development. Here, we report the effects of T-DNA insertion mutations in the Arabidopsis GGB gene, which encodes the beta-subunit of protein geranylgeranyltransferase type I (PGGT I). Stomatal apertures of ggb plants were smaller than those of wild-type plants at all concentrations of ABA tested, suggesting that PGGT I negatively regulates ABA signaling in guard cells. However, germination of ggb seeds in response to ABA was similar to the wild type. Lateral root formation in response to exogenous auxin was increased in ggb seedlings compared to the wild type, but no change in auxin inhibition of primary root growth was observed, suggesting that PGGT I is specifically involved in negative regulation of auxin-induced lateral root initiation. Unlike era1 mutants, ggb mutants exhibited no obvious developmental phenotypes. However, era1 ggb double mutants exhibited more severe developmental phenotypes than era1 mutants and were indistinguishable from plp mutants lacking the shared alpha-subunit of PFT and PGGT I. Furthermore, overexpression of GGB in transgenic era1 plants partially suppressed the era1 phenotype, suggesting that the relatively weak phenotype of era1 plants is due to partial redundancy between PFT and PGGT I. These results are discussed in the context of Arabidopsis proteins that are putative substrates of PGGT I.     

Mutation of E1-CONJUGATING ENZYME-RELATED1 decreases RELATED TO UBIQUITIN conjugation and alters auxin response and development

The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.     

The jasmonate receptor COI1 plays a role in jasmonate-induced lateral root formation and lateral root positioning in Arabidopsis thaliana

Jasmonic acid (JA) regulates a broad range of plant defense and developmental responses. COI1 has been recently found to act as JA receptor. In this report, we show that low micromolar concentrations of JA inhibited primary root (PR) growth and promoted lateral root (LR) formation in Arabidopsis wild-type (WT) seedlings. It was observed that the coi1-1 mutant was less sensitive to JA on pericycle cell activation to induce lateral root primordia (LRP) formation and presented alterations in lateral root positioning and lateral root emergence on bends. To investigate JA-auxin interactions important for remodeling of root system (RS) architecture, we tested the expression of auxin-inducible markers DR5:uidA and BA3:uidA in WT and coi1-1 seedlings in response to indole-3-acetic acid (IAA) and JA and analyzed the RS architecture of a suite of auxin-related mutants under JA treatments. We found that JA did not affect DR5:uidA and BA3:uidA expression in WT and coi1-1 seedlings. Our data also showed that PR growth inhibition in response to JA was likely independent of auxin signaling and that the induction of LRP required ARF7, ARF19, SLR, TIR1, AFB2, AFB3 and AXR1 loci. We conclude that JA regulation of postembryonic root development involves both auxin-dependent and independent mechanisms.     

Mutations in an auxin receptor homolog AFB5 and in SGT1b confer resistance to synthetic picolinate auxins and not to 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid in Arabidopsis

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.     

Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root.

The Arabidopsis root produces a position-dependent pattern of hair-bearing and hairless cell types during epidermis development. Five loci (TRANSPARENT TESTA GLABRA [TTG], GLABRA2 [GL2], ROOT HAIR DEFECTIVE6 [RHD6], CONSTITUTIVE TRIPLE RESPONSE1 [CTR1], and AUXIN RESISTANT2 [AXR2]) and the plant hormones ethylene and auxin have been reported to affect the production of root hair and hairless cells in the Arabidopsis root. In this study, genetic, molecular, and physiological tests were employed to define the roles of these loci and hormones. Epistasis tests and reporter gene studies indicated that the hairless cell-promoting genes TTG and GL2 are likely to act early to negatively regulate the ethylene and auxin pathways. Studies of the developmental timing of the hormone effects indicated that ethylene and auxin pathways promote root hair outgrowth after cell-type differentiation has been initiated. The genetic analysis of ethylene-and auxin-related mutations showed that root hair formation is influenced by a network of hormone pathways, including a partially redundant ethylene signaling pathway. A model is proposed in which the patterning of root epidermal cells in Arabidopsis is regulated by the cell position-dependent action of the TTG/GL2 pathway, and the ethylene and auxin hormone pathways act to promote root hair outgrowth at a relatively late stage of differentiation.     

yucca6, a dominant mutation in Arabidopsis, affects auxin accumulation and auxin-related phenotypes

Auxin plays critical roles in many aspects of plant growth and development. Although a number of auxin biosynthetic pathways have been identified, their overlapping nature has prevented a clear elucidation of auxin biosynthesis. Recently, Arabidopsis (Arabidopsis thaliana) mutants with supernormal auxin phenotypes have been reported. These mutants exhibit hyperactivation of genes belonging to the YUCCA family, encoding putative flavin monooxygenase enzymes that result in increased endogenous auxin levels. Here, we report the discovery of fertile dominant Arabidopsis hypertall1-1D and hypertall1-2D (yucca6-1D, -2D) mutants that exhibit typical auxin overproduction phenotypic alterations, such as epinastic cotyledons, increased apical dominance, and curled leaves. However, unlike other auxin overproduction mutants, yucca6 plants do not display short or hairy root phenotypes and lack morphological changes under dark conditions. In addition, yucca6-1D and yucca6-2D have extremely tall (>1 m) inflorescences with extreme apical dominance and twisted cauline leaves. Microarray analyses revealed that expression of several indole-3-acetic acid-inducible genes, including Aux/IAA, SMALL AUXIN-UP RNA, and GH3, is severalfold higher in yucca6 mutants than in the wild type. Tryptophan (Trp) analog feeding experiments and catalytic activity assays with recombinant YUCCA6 indicate that YUCCA6 is involved in a Trp-dependent auxin biosynthesis pathway. YUCCA6:GREEN FLUORESCENT PROTEIN fusion protein indicates YUCCA6 protein exhibits a nonplastidial subcellular localization in an unidentified intracellular compartment. Taken together, our results identify YUCCA6 as a functional member of the YUCCA family with unique roles in growth and development.     

Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis

Degradation of Aux/IAA proteins which are triggered by the ubiquitin ligase complex containing the auxin F-box receptors (AFBs), is thought to be the primary reaction of auxin signaling. Upon auxin perception, AFBs bind domain II of Aux/IAA proteins that is conserved in most of the 29 family members in Arabidopsis. However, IAA20 and IAA30 lack domain II. Furthermore, IAA31, which forms a single clade with IAA20 and IAA30 in Aux/IAA protein family, has a partially conserved domain II, which contains an amino acid substitution that would cause a dominant mutation of Aux/IAA genes. It has been shown that the half-lives of these proteins are much longer than those of the canonical Aux/IAA proteins. We generated overexpression lines (OXs) of IAA20 , IAA30 and IAA31 by the use of cauliflower mosaic virus 35S promoter to better understand the molecular function of atypical Aux/IAA proteins in Arabidopsis. OXs of the three genes exhibited similar auxin-related aberrant phenotypes, with IAA20 OX showing the most severe defects: Some of them showed a semi-dwarf phenotype; gravitropic growth orientation was often affected in hypocotyl and root; vasculature of cotyledons was malformed; the primary root stopped growing soon after germination because of collapse of root apical meristem. IAA 20 and IAA30 were early auxin inducible, but IAA31 was not. These results showed that the wild-type genes of the three Aux/IAAs could disturb auxin physiology when ectopically overexpressed.     

MM31/EIR1 promotes lateral root formation in Arabidopsis

Lateral root formation in Arabidopsis provides a model for the study of auxin function. Tryptophan (Trp) is a precursor of the auxin indoleacetic acid (IAA). To study the physiological function of Trp in auxin-related phenotypes, we examined the effect of Trp on lateral root formation. We found that Trp treatment enhanced lateral root formation and, by screening for mutants in which the effect of Trp on lateral root formation was enhanced, we isolated the mm31 mutant. Based on genetic and physiological analyses, we propose that MM31/EIR1 modulates lateral root formation by regulating the IAA polar transport system, and that auxin transport from the shoot to the root regulates lateral root formation.Key words: lateral root formation, Arabidopsis, EIR1, IAA, auxin     

Small acidic protein 1 and SCFTIR1 ubiquitin proteasome pathway act in concert to induce 2,4‐dichlorophenoxyacetic acid‐mediated alteration of actin in Arabidopsis roots

2,4‐Dichlorophenoxyacetic acid (2,4‐D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole‐3‐acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4‐D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4‐D‐specific mutants suggested that 2,4‐D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4‐D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4‐D but not IAA altered the actin structure in long‐term and short‐term assays. Analysis of the 2,4‐D‐specific mutant aar1‐1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4‐D‐induced depolymerization of actin. The ubiquitin proteasome mutants tir1‐1 and axr1‐12, which show enhanced resistance to 2,4‐D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4‐D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4‐D on the organization of actin filaments. Roots of the double mutant aar1‐1 tir1‐1 also showed enhanced resistance to 2,4‐D‐induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4‐D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCF^TIR1 ubiquitin proteasome components.