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.     

AXR2 encodes a member of the Aux/IAA protein family

The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.     

The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation

To understand the molecular mechanism of auxin action, mutants of Arabidopsis thaliana with altered responses to auxin have been identified and characterized. Here the isolation of two auxin-resistant mutants that define a new locus involved in auxin response, named AXR4, is reported. The axr4 mutations are recessive and map near the ch1 mutation on chromosome 1. Mutant plants are specifically resistant to auxin and defective in root gravitropism. Double mutants between axr4 and the recessive auxin-resistant mutants axr1-3 and aux1-7 were characterized to ascertain possible genetic interactions between the mutations. The roots of the axr4 axr1-3 double mutant plants are less sensitive to auxin, respond more slowly to gravity, and form fewer lateral roots than either parental single mutant. These results suggest that the two mutations have additive or even synergistic effects. The AXR1 and AXR4 gene products may therefore act in separate pathways of auxin response or perhaps perform partially redundant functions in a single pathway. The axr4 aux1-7 double mutant has the same sensitivity to auxin as the aux1-7 mutant but forms far fewer lateral roots than either parental single mutant. The aux1-7 mutation thus appears to be epistatic to axr4 with respect to auxin-resistant root elongation, whereas in lateral root formation, the effects of the two mutations are additive. The complexity of the genetic interactions indicated by these results may reflect differences in the mechanism of auxin action during root elongation and the formation of lateral roots. The AXR4 gene product, along with those of the AXR1 and AUX1 genes, is important for normal auxin sensitivity, gravitropic response in roots and lateral root formation.     

The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin response and early development

The indolic compound auxin regulates virtually every aspect of plant growth and development, but its role in embryogenesis and its molecular mechanism of action are not understood. We describe two mutants of Arabidopsis that define a novel gene called AUXIN-RESISTANT6 (AXR6) which maps to chromosome 4. Embryonic development of the homozygous axr6 mutants is disrupted by aberrant patterns of cell division, leading to defects in the cells of the suspensor, root and hypocotyl precursors, and provasculature. The homozygous axr6 mutants arrest growth soon after germination lacking a root and hypocotyl and with severe vascular pattern defects in their cotyledons. Whereas previously described mutants with similar developmental defects are completely recessive, axr6 heterozygotes display a variety of morphological and physiological alterations that are most consistent with a defect in auxin physiology or response. The AXR6 gene is likely to be important for auxin response throughout the plant, including early development.     

Growth and development of the axr1 mutants of Arabidopsis.

We have recovered eight new auxin-resistant lines of Arabidopsis that carry mutations in the AXR1 gene. These eight lines, together with the 12 lines described in a previous report, define at least five different axr1 alleles. All of the mutant lines have a similar phenotype. Defects include decreases in plant height, root gravitropism, hypocotyl elongation, and fertility. Mutant line axr1-3 is less resistant to auxin than the other mutant lines and has less severe morphological abnormalities. This correlation suggests that the morphological defects are a consequence of a defect in auxin action. To determine whether the altered morphology of mutant plants is associated with changes in cell size or tissue organization, tissue sections were examined using scanning electron microscopy. No clear differences in cell size were observed between wild-type and mutant tissues. However, the vascular bundles of mutant stems were found to be less well differentiated than those in wild-type stems. The auxin sensitivity of rosette-stage plants was determined by spraying plants with auxin solutions. Mutant rosettes were found to be significantly less sensitive to exogenously applied auxin than wild-type rosettes, indicating that the AXR1 gene functions in aerial portions of the plant. Our studies suggest that the AXR1 gene is required for auxin action in most, if not all, tissues of the plant and plays an important role in plant development. Linkage studies indicate that the gene is located on chromosome 1 approximately 2 centiMorgans from the closest restriction fragment length polymorphism.     

A principal role for AtXTH18 in Arabidopsis thaliana root growth: a functional analysis using RNAi plants

Rearrangement of cellulose microfibrils within cell-wall matrices is considered one of the most critical steps in the regulation of both the orientation and extent of cell expansion in plants. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of load-bearing cross links among cellulose microfibrils. The Arabidopsis thaliana XTH genes AtXTH17, 18, 19, and 20 are phylogenetically closely related to one another and are preferentially expressed in the roots. However, they exhibit different expression profiles within the root and respond to hormonal signals differently. To investigate their functions in root growth, we examined phenotypes of loss-of-function mutants for these genes using T-DNA insertion lines and RNAi plants. These functional analyses disclosed a principal role for the AtXTH18 gene in primary root elongation. Of the four XTH genes, AtXTH18 exhibits the highest level of mRNA expression. We also determined auxin-signaling pathways for these genes using a mutant with a defect in the AXR2/IAA7 gene and found that the expression of AtXTH19 in the elongation/maturation region of the root is under the control of the AXR2/IAA7 signaling pathway.     

AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis

Cullin-RING ubiquitin-protein ligases such as the Skp1, cullin, F-box protein (SCF) have been implicated in many growth and developmental processes in plants. Normal SCF function requires that the CUL1 subunit be post-translationally modified by related to ubiquitin (RUB), a protein related to ubiquitin. This process is mediated by two enzymes: the RUB-activating and RUB-conjugating enzymes. In Arabidopsis, the RUB-activating enzyme is a heterodimer consisting of AXR1 and ECR1. Mutations in the AXR1 gene result in a pleiotropic phenotype that includes resistance to the plant hormone auxin. Here we report that the AXL (AXR1-like) gene also functions in the RUB conjugation pathway. Overexpression of AXL in the axr1-3 background complements the axr1-3 phenotype. Biochemical analysis indicates that AXL overexpression restores CUL1 modification to the wild-type level, indicating that AXR1 and AXL have the same biochemical activity. Although the axl mutant resembles wild-type plants, the majority of axr1 axl-1 double mutants are embryo or seedling lethal. Furthermore, the axl-1 mutation reveals novel RUB-dependent processes in embryo development. We conclude that AXR1 and AXL function redundantly in the RUB conjugating pathway.     

AXR1-ECR1-dependent conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 is required for auxin response

Mutations in the AXR1 gene result in a reduction in auxin response and diverse defects in auxin-regulated growth and development. In a previous study, we showed that AXR1 forms a heterodimer with the ECR1 protein. This enzyme activates the ubiquitin-related protein RUB1 in vitro. Furthermore, we showed that the Skp1-Cul1/Cdc53-F-box (SCF) subunit AtCUL1 is modified by RUB1 in vivo. In this report, we demonstrate that the formation of RUB-AtCUL1 is dependent on AXR1 and ECR1 in vivo. The expression of AXR1 and ECR1 is restricted to zones of active cell division and cell elongation, consistent with their role in growth regulation. These results provide strong support for a model in which RUB conjugation of AtCUL1 affects the function of SCF E3s that are required for auxin response.     

The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways

The recessive mutations aux1 and axr1 of Arabidopsis confer resistance to the plant hormone auxin. The axr1 mutants display a variety of morphological defects. In contrast, the only morphological defect observed in aux1 mutants is a loss of root gravitropism. To learn more about the function of these genes in auxin response, the expression of the auxin-regulated gene SAUR-AC1 in mutant and wild-type plants has been examined. It has been found that axr1 plants display a pronounced deficiency in auxin-induced accumulation of SAUR-AC1 mRNA in seedlings as well as rosette leaves and mature roots. In contrast, the aux1 mutation has a modest effect on auxin induction of SAUR-AC1. To determine if the AUX1 and AXR1 genes interact to facilitate auxin response, plants which are homozygous for both aux1 and axr1 mutations have been constructed and characterized. The two mutations are additive in their effects on auxin response, suggesting that each mutation confers resistance by a different mechanism. However, the morphology of double mutant plants indicates that there is an inter-action between the AXR1 and AUX1 genes. In mature plants, the aux1-7 mutation acts to partially suppress the morphological defects conferred by the axr1-12 mutation. This suppression is not accompanied by an increase in auxin response, as measured by SAUR-AC1 expression, suggesting that the interaction between the AUX1 and AXR1 genes is indirect.     

The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF(COI1) complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component of SCF(COI1) complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modification of the AtCUL1 subunit of SCF(COI1) complexes is important for JA signaling.     

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCF(TIR1). Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCF(TIR1) substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.     

FRD3 controls iron localization in Arabidopsis

The frd3 mutant of Arabidopsis exhibits constitutive expression of its iron uptake responses and is chlorotic. These phenotypes are consistent with defects either in iron deficiency signaling or in iron translocation and localization. Here we present several experiments demonstrating that a functional FRD3 gene is necessary for correct iron localization in both the root and shoot of Arabidopsis plants. Reciprocal grafting experiments with frd3 and wild-type Arabidopsis plants reveal that the phenotype of a grafted plant is determined by the genotype of the root, not by the genotype of the shoot. This indicates that FRD3 function is root-specific and points to a role for FRD3 in delivering iron to the shoot in a usable form. When grown under certain conditions, frd3 mutant plants overaccumulate iron in their shoot tissues. However, we demonstrate by direct measurement of iron levels in shoot protoplasts that intracellular iron levels in frd3 are only about one-half the levels in wild type. Histochemical staining for iron reveals that frd3 mutants accumulate high levels of ferric iron in their root vascular cylinder, the same tissues in which the FRD3 gene is expressed. Taken together, these results clearly indicate a role for FRD3 in iron localization in Arabidopsis. Specifically, FRD3 is likely to function in root xylem loading of an iron chelator or other factor necessary for efficient iron uptake out of the xylem or apoplastic space and into leaf cells.