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.     

Differential IAA dose response relations of the axr1 and axr2 mutants of Arabidopsis

In comparison to wild type Arabidopsis thaliana, the auxin resistant mutants axr1 and axr2 exhibit reduced inhibition of root elongation in response to auxins. Several auxin-regulated physiological processes are also altered in the mutant plants. When wild-type, axr1 and axr2 seedlings were grown in darkness on media containing indoleacetic acid (IAA), promotion of root growth was observed at low concentrations of IAA (10^?11 to 10^?7M) in 5-day-old axr2 seedlings, but not in axr1 or wild-type seedlings. In axr1 there was little or no measurable root growth response over the same concentration range. In wild type, root growth was inhibited at concentrations greater than 10^?10M and no detectable root growth response was observed at lower concentrations. In addition, production of lateral roots in response to IAA increased in axr2 seedlings and decreased in axr1 seedlings relative to wild type. Promotion of root elongation and initiation of lateral roots in axr2 seedlings in response to auxin indicate that axr2 seedlings are able to perceive and respond to IAA.     

The pef mutants of Arabidopsis thaliana define lesions early in the phytochrome signaling pathway

In a screen for early-flowering mutants, a number of mutants that were early flowering under both short and long days were isolated. One such mutant, pef1, was selectively insensitive to both red and far-red light in the inhibition of hypocotyl elongation response; a classic phytochrome phenotype mediated by both PHYA and PHYB. The pef1 mutant seedlings could not be phenotypically rescued by biliverdin, a precursor of the phytochrome chromophore, nor did they fail to complement any previously identified elongated hypocotyl (hy) mutants. Difference spectra and Western blot analysis showed normal concentrations of PHYA photoreceptor apoprotein, which appeared photochemically active. It was concluded that the pef1 mutant is defective in both PHYA- and PHYB- mediated signaling pathways, and may represent a lesion in an early step of the phytochrome signal transduction pathway. Additional pef mutants deficient specifically in PHYB-mediated responses were also identified by this screen.     

Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis

A new auxin homeostasis gene in Arabidopsis called SUR2 has been identified. This gene, mapped to the bottom of chromosome 4, is defined by two recessive nuclear mutants designated superroot2 (sur2), which display several abnormalities reminiscent of auxin effects. A number of these characteristics are similar to the phenotype of the previously described auxin-overproducing mutant superroot1 (sur1); however, several lines of evidences reveal that the SUR2 gene defines a new key point in the regulation of endogenous auxin concentrations. The phenotype of the sur1 sur2 double mutant is additive. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of free indole-3-acetic acid correlated with a decreased level of bound auxin in the sur2 mutant. These results suggest that SUR2 may be involved in the control of auxin conjugation.     

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.     

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.     

Protophloem differentiation in early Arabidopsis thaliana development

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.     

Arabidopsis mutants reveal multiple singlet oxygen signaling pathways involved in stress response and development

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a ¹O₂-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by ¹O₂ but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether ¹O₂ was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the ¹O₂-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that ¹O₂-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Aiswarya Baruah and Klára Šimková contributed equally to the article.     

Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana (L.) Heynh. glucosinolate mutants and the development of clubroot disease

Mutants and wild type plants of Arabidopsis thaliana were analysed for differences in glucosinolate accumulation patterns, indole-3-acetic acid (IAA) biosynthesis and phenotype. A previously identified series of mutants, termed TU, with altered glucosinolate patterns was used in this study. Only the line TU8 was affected in shoot phenotype (shorter stems, altered branching pattern). Synthesis of IAA and metabolism were not much affected in the TU8 mutant during seedling development, although the content of free IAA peaked earlier in TU8 during plant development than in the wild type. Indole glucosinolates and IAA may, however, be involved in the development of clubroot disease caused by the obligate biotrophic fungus Plasmodiophora brassicae since the TU3 line had a lower infection rate than the wild type, and lines TU3 and TU8 showed decreased symptom development. The decline in clubroot formation was accompanied by a reduced number of fungal structures within the root cortex and slower development of the fungus. Indole glucosinolates were lower in infected roots of TU3 and TU8 than in control roots of these lines, whereas in wild-type plants the differences were not as prominent. Free IAA and indole-3-acetonitrile (IAN) were increased in infected roots of the wild type and mutants with normal clubroot symptoms, whereas they were reduced in infected roots of mutants TU3 and TU8. These results indicate a role for indole glucosinolates and IAN/IAA in relation to symptom development in clubroot disease. Received: 23 July 1998 / Accepted: 12 January 1999     

The isolation of apparently homoplastidic mutants induced by a nuclear recessive gene in Arabidopsis thaliana

Summary The nuclear recessive gene, chm1, of Arabidopsis thaliana is a imitator that induces a variety of plastid alterations giving rise to mixed cells and variegated leaves. The variegation is maternally transmitted but chm1 is transmitted in a Mendelian fashion (Rédei 1973; Rédei and Plurad 1973). In order to characterize the different types of plastid alterations induced by chm1, isolating homoplastidic lines, each apparently containing one type of mutant plastid in its cells, was essential since such characterization cannot be carried out on mixed cells. We have used two genetic approaches to isolate several apparently homoplastidic mutant lines by the removal of the mutator from the genetic background, and the maternal transmission of the mutant plastids. The rapidity of obtaining homoplastidic lines in the absence of chm1 indicated a non-stochastic sorting-out of plastids in mixed cells. That each of the chm1-free homoplastidic mutant lines was apparently homoplastidic for one type of mutant plastids was confirmed by electron microscopic observations. Here we report, for the first time, the production of different homoplastidic lines in the absence of the nuclear-mutator gene. Such genetically-stable homogeneous material should be a useful tool for studying the molecular mechanism(s) by which chm1 induces a variety of heritable plastid alterations.     

Effects of natural and synthetic auxins on the gravitropic growth habit of roots in two auxin-resistant mutants of Arabidopsis, axr1 and axr4: evidence for defects in the auxin influx mechanism of axr4.

The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium. Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect. Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA. The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells. The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins. These results suggest that the lesion of axrl mutants is different from that of axr4.     

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg erecta) and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.     

The localization of auxin transporters PIN3 and LAX3 during lateral root development in Arabidopsis thaliana

Fluorophore tagged proteins are used in Arabidopsis thaliana to understand their functional role in plant development. This requires the analysis of their spatial localization in planta. However, the localization analysis is often perturbed by a significant overlap of the fluorophores used to label proteins of interest and the optical filtering methods available on the confocal microscope. This problem can be addressed by the use of spectral imaging with linear unmixing the image data. We applied this method to help us identify double transgenic A. thaliana lines which expressed two fluorescently tagged auxin transporter proteins: the auxin efflux protein PIN-FORMED-3 (PIN3), tagged with green fluorescent protein (GFP), and the auxin influx protein LIKE-AUX1-3 (LAX3), tagged with yellow fluorescent protein (YFP). This method allows the reliable separation of overlapping GFP and YFP fluorescence signals and subsequent localization analysis highlighting the potential benefit of this methodology in studies of lateral root development.     

A proteomics study of auxin effects in Arabidopsis thaliana

Many phytohormones regulate plant growth and development through modulating protein degradation. In this study, a proteome study based on multidimensional non-gel shotgun approach was performed to analyze the auxin-induced protein degradation via ubiquitin-proteasome pathway of Arabidopsis thaliana, with the emphasis to study the overall protein changes after auxin treatment (1 nM or 1 μM indole-3-acetic acid for 6, 12, or 24 h). More than a thousand proteins were detected by using label-free shotgun method, and 386 increased proteins and 370 decreased ones were identified after indole-3-acetic acid treatment. By using the auxin receptor-deficient mutant, tir1-1, as control, comparative analysis revealed that 69 and 79 proteins were significantly decreased and increased, respectively. Detailed analysis showed that among the altered proteins, some were previously reported to be associated with auxin regulation and others are potentially involved in mediating the auxin effects on specific cellular and physiological processes by regulating photosynthesis, chloroplast development, cytoskeleton, and intracellular signaling. Our results demonstrated that label-free shotgun proteomics is a powerful tool for large-scale protein identification and the analysis of the proteomic profiling of auxin-regulated biological processes will provide informative clues of underlying mechanisms of auxin effects. These results will help to expand the understanding of how auxin regulates plant growth and development via protein degradation.     

Female gametophytic mutants of Arabidopsis thaliana identified in a gene trap insertional mutagenesis screen

In plants, the male and female gametophytes represent the haploid generation that alternates with the diploid sporophytic generation. Male and female gametophytes develop from haploid micro- and megaspores, respectively. In flowering plants (angiosperms), the spores themselves arise from the sporophyte through meiotic divisions of sporogenous cells in the reproductive organs of the flower. Male and female gametophytes contain two pairs of gametes that participate in double fertilization, a distinctive feature of angiosperms. In this paper, we describe the employment of a transposon-based gene trap system to identify mutations affecting the gametophytic phase of the plant life cycle. Mutants affecting female gametogenesis were identified in a two-step screen for (i) reduced fertility (seed abortion or undeveloped ovules) and (ii) segregation ratio distortion. Non-functional female gametophytes do not initiate seed development, leading to semi-sterility such that causal or linked alleles are transmitted at reduced frequency to the progeny (non-Mendelian segregation). From a population of 2,511 transposants, we identified 54 lines with reduced seed set (2%). Examination of their distorted segregation ratios and seed phenotypes led to the isolation of 12 gametophytic mutants, six of which are described herein. Chromosomal sequences flanking the transposon insertions were identified and physically mapped onto the genome sequence of Arabidopsis thaliana. Surprisingly, the insertion sites were often associated with chromosomal rearrangements, making it difficult to assign the mutant phenotypes to a specific gene. The mutants were classified according to the process affected at the time of arrest, i.e. showing mitotic, karyogamic, maternal or degenerative phenotypes.