The growth and gravitropic responses of wild-type and auxinresistant mutants of Arabidopsis thaliana

Growth and gravitropism have been studied in three mutant strains of Arabidopsis thaliana L, that are resistant to auxin-herbicide. Two of the mutations are allelic and recessive ( aux-1 and aux-2 ) and are unlinked to a dominant mutation, Dwf , which confers a very high level of auxin-resistance and is apparently lethal when homozygous. The aux-1 and Dwf strains have altered response to gravity whereas aux-2 appears to be gravitropically normal.
After 96 h in the normal, vertical position only minor differences in elongation were observed between roots of wild-type, aux-1 and aux-2 , but the hypocotyls of aux-1 were significantly retarded compared with the gravitropically normal aux-2 and wild-type. In the progeny of selfed Dwf plants, where both normal ( dwf ) and agravitropic ( Dwf ) seedlings are present, the Dwf seedlings had much longer roots and shorter hypocotyls than dwf ⁺. During 22 h of continuous stimulation the optimum angle for gravitropism in wild-type roots and hypocotyls was 135° (i.e. the organ points obliquely upwards), with decreasing responses in the order 90° and 45°. The agravitropic nature of the roots of aux-1 was confirmed as no significant response was obtained at any of the stimulation angles. In marked contrast, the negative gravitropic response of aux-1 hypocotyls was greater than the wild-type response in terms of the final angle attained at 22 h, but between 6 and 22 h the elongation rate was lower in aux-1 . After varying stimulation periods in the horizontal position, the curvature which had developed, decreased rapidly and almost disappeared during ensuing rotation on clinostats (2 and 4 rpm). Rotation on the clinostats had no effect on the agravitropic behaviour of aux-1 .     

The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana

Abscisic acid (ABA) insensitive mutants of Arabidopsis thaliana (L.) Heynh. were isolated by selecting plants which grew well on a medium containing 10 μ M ABA. From the progeny of approximately 3500 mutagen-treated seeds, five mutants of at least three different loci were isolated. Three mutants were characterized, moreover, by a reduced seed dormancy and by symptoms of withering, indicating disturbed water relations and, therefore, resembled phenotypically the ABA-deficient mutants we described earlier in this species. Two mutants showed in addition only a reduction of seed dormancy. Compared to wild type, all mutants showed similar or increased levels of endogenous ABA in developing seeds and fruits (siliquae). The role of the different genes involved is discussed in relation to the mechanism of ABA action.     

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2 , display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis . The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 m M . Stomatal apertures were increased by lowering external Ca²⁺ to 0.05 m M . The apertures of stomates incubated with 10 μ M ABA were not altered by changes in Ca²⁺ from 0.05 to 1.0 m M .
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.     

Early development and gravitropic response of lateral roots in Arabidopsis thaliana

Root system architecture plays an important role in determining nutrient and water acquisition and is modulated by endogenous and environmental factors, resulting in considerable developmental plasticity. The orientation of primary root growth in response to gravity (gravitropism) has been studied extensively, but little is known about the behaviour of lateral roots in response to this signal. Here, we analysed the response of lateral roots to gravity and, consistently with previous observations, we showed that gravitropism was acquired slowly after emergence. Using a lateral root induction system, we studied the kinetics for the appearance of statoliths, phloem connections and auxin transporter gene expression patterns. We found that statoliths could not be detected until 1 day after emergence, whereas the gravitropic curvature of the lateral root started earlier. Auxin transporters modulate auxin distribution in primary root gravitropism. We found differences regarding PIN3 and AUX1 expression patterns between the lateral root and the primary root apices. Especially PIN3, which is involved in primary root gravitropism, was not expressed in the lateral root columella. Our work revealed new developmental transitions occurring in lateral roots after emergence, and auxin transporter expression patterns that might explain the specific response of lateral roots to gravity.     

Embryonic mutants of Arabidopsis thaliana

Genetic analysis of plant em-bryogenesis has been approached in part through the isolation and characterization of recessive embryonic mutants. The most extensive studies have dealt with maize and Arabidopsis. The high frequency of mutants defective in plant embryogenesis is consistent with the presence of many target genes with essential functions at this stage of the life cycle. Some mutants are likely to be defective in genes with general housekeeping functions. Others should facilitate the identification of genes with a more direct role in the regulation of morphogesis. Over 300 embryonic mutants of Arabidopsis isolated following chemical mutagenesis and T-DNA insertional mutagenesis are currently being analyzed. This collection includes embryonic le-thals, defectives, and pattern mutants. Developmental abnormalities include the presence of fused cotyledons, twin embryos, abnormally large suspensors, distorted epidermal layers, single cotyledons, enlarged shoot apices, pattern deletions and duplications, embryos with altered patterns of symmetry, bloated embryos with giant vacuolated cells, reduced hypocotyls that fail to produce roots, and embryos that protrude through the seed coat late in maturation. This review describes the isolation and characterization of embryonic mutants of Arabidopsis and their potential application to plant biology. © 1992 Wiley-Liss, Inc.     

Cesium-insensitive mutants of Arabidopsis thaliana

The molecular analysis of solute transport across the plasma membrane in animals and microorganisms has been aided by the analysis of well-defined transport mutants. To obtain mutant plants with genetic defects in cation transport, the inhibitory effect of monovalent cations (Li⁺, Na⁺, Rb⁺, and Cs⁺) on Arabidopsis thaliana seed germination was tested. Cesium was unique in that at low concentration it strongly inhibited seedling development. In this report it is demonstrated that cesium is a competitive inhibitor for potassium transport in A. thaliana and its toxicity is closely tied to the level of potassium supplied. Conditions were obtained to maximize the cesium-sensitivity for seed germination in a large population, and selection for resistance using M₂ seeds derived from ethyl methane sulfonate (EMS)-treated plants yielded several dozen resistant plants. Seeds derived from these plants yielded cesium-insensitive mutant lines with heritable changes in energy-dependent potassium uptake. In progeny from a backcross to wild-type plants, at least one of the lines showed the segregation ratio expected for a single-gene recessive mutation and an RFLP analysis mapped the mutant locus to the top of chromosome 4.     

Grasshopper oral secretions increase salicylic acid and abscisic acid levels in wounded leaves of Arabidopsis thaliana

Recent investigations showed that the model plant Arabidopsis thaliana specifically responds to herbivory-associated molecular patterns by activating a sophisticated signaling network. The lipase activity of insect oral secretions was shown to elevate oxylipin levels when applied to puncture wounds in leaves. The results also demonstrated that the oral secretions of the generalist Schistocerca gregaria contained other, probably non-proteinous, elicitors of plant defense responses which induced mitogen-activated protein kinases, calcium signaling and ethylene levels.¹ This addendum presents data on the levels of additional phytohormones that are elevated after application of S. gregaria oral secretion to wounded leaves. Abscisic acid and salicylic acid levels are significantly elevated after elicitation with S. gregaria oral secretions, adding another layer of complexity to the herbivory-induced response of A. thaliana.Key words: abscisic acid, Arabidopsis, herbivory, salicylic acid, Schistocerca gregaria     

Restoration of gravitropic sensitivity in starch-deficient mutants of Arabidopsis by hypergravity

Despite the extensive study of plant gravitropism, there have been few experiments which have utilized hypergravity as a tool to investigate gravisensitivity in flowering plants. Previous studies have shown that starch-deficient mutants of Arabidopsis are less sensitive to gravity compared to the wild-type (WT). In this report, the question addressed was whether hypergravity could restore the sensitivity of starch-deficient mutants of Arabidopsis. The strains examined include a WT, a starchless mutant and a reduced-starch mutant. Vertical orientation studies with dark-grown seedlings indicate that increased centrifugal acceleration improves orientation relative to the acceleration vector for all strains, even the WT. For starchless roots, growth of seedlings under constant 5 g acceleration was required to restore orientation to the level of the WT at 1 g. In contrast, approximately 10 g was required to restore the orientation of the starchless mutant hypocotyls to a WT level at 1 g. Examination of plastid position in root cap columella cells of the starchless mutant revealed that the restoration of gravitropic sensitivity was correlated with the sedimentation of plastids toward the distal cell wall. Even in WT plants, hypergravity caused greater sedimentation of plastids and improved gravitropic capability. Collectively, these experiments support the hypothesis of a statolith-based system of gravity perception in plants. As far as is known, this is the first report to use hypergravity to study the mechanisms of gravitropism in Arabidopsis.     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

Morphogenesis in pinoid mutants of Arabidopsis thaliana

A series of mutants of Arabidopsis thaliana was selected in which the inflorescence stem elongates but loses the ability to produce flower primordia on its flanks. Mutants fell into two classes, further occurrences of pin-formed mutants and mutations at a new locus named pinoid. As well as causing inflorescence defects, pinoid mutations result in pleiotropic defects in the development of floral organs, cotyledons and leaves. Most changes involve the number of organs produced rather than their differentiation suggesting that PINOID controls an early general step in meristem development. pinoid mutant defects are similar to those seen in pin-formed mutants for inflorescences and flowers, but different for cotyledons and leaves indicating that the two genes have separate but overlapping functions. A defect in polar auxin transport is implicated in the pin-formed mutant phenotype, but in young inflorescence stems of even the strongest pinoid mutants it occurs at close to wild-type levels. It is markedly reduced only after stems have ceased elongating. Thus, it is likely that polar auxin transport is secondarily affected in pinoid mutants rather than being directly controlled by the PINOID gene product. Even so, double mutant studies indicate that the process controlled by PINOID overlaps with that specified by the AUXIN RESISTANT1 gene, suggesting that PINOID plays some role in an auxin-related process.     

Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants

The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.     

Arabidopsis thaliana mutants disrupted in lipid mobilization

To isolate mutants in the process of lipid mobilization during post-germinative growth we employed a screen using the pro-herbicide 2,4-dichlorophenoxybutyric acid (2,4-DB). The phenotypes of a number of 2,4-DB-resistant mutants are compared with previously characterized mutants disrupted in beta-oxidation or the glyoxylate cycle. We conclude that the strength of 2,4-DB resistance and the ability of the seedlings to grow in the absence of exogenous sugar are inversely correlated. Sugar dependence of 2,4-DB-resistant seedlings is a consequence of impaired storage-lipid mobilization.     

Genetic analyses of interactions among gibberellin, abscisic acid, and brassinosteroids in the control of flowering time in Arabidopsis thaliana

Background

Genetic interactions between phytohormones in the control of flowering time in Arabidopsis thaliana have not been extensively studied. Three phytohormones have been individually connected to the floral-timing program. The inductive function of gibberellins (GAs) is the most documented. Abscisic acid (ABA) has been demonstrated to delay flowering. Finally, the promotive role of brassinosteroids (BRs) has been established. It has been reported that for many physiological processes, hormone pathways interact to ensure an appropriate biological response.

Methodology

We tested possible genetic interactions between GA-, ABA-, and BR-dependent pathways in the control of the transition to flowering. For this, single and double mutants deficient in the biosynthesis of GAs, ABA, and BRs were used to assess the effect of hormone deficiency on the timing of floral transition. Also, plants that over-express genes encoding rate-limiting enzymes in each biosynthetic pathway were generated and the flowering time of these lines was investigated.

Conclusions

Loss-of-function studies revealed a complex relationship between GAs and ABA, and between ABA and BRs, and suggested a cross-regulatory relation between GAs to BRs. Gain-of-function studies revealed that GAs were clearly limiting in their sufficiency of action, whereas increases in BRs and ABA led to a more modest phenotypic effect on floral timing. We conclude from our genetic tests that the effects of GA, ABA, and BR on timing of floral induction are only in partially coordinated action.     

Genetic analysis of salt-tolerant mutants in Arabidopsis thaliana

Stress caused by the increased salinity of irrigated fields impairs plant growth and is one of the major constraints that limits crop productivity in many important agricultural areas. As a contribution to solving such agronomic problems, we have carried out a large-scale screening for Arabidopsis thaliana mutants induced on different genetic backgrounds by EMS treatment, fast neutron bombardment, or T-DNA insertions. From the 675,500 seeds we screened, 17 mutant lines were isolated, all but one of which yielded 25-70% germination levels on 250 mm NaCl medium, a condition in which their ancestor ecotypes are unable to germinate. Monogenic recessive inheritance of NaCl-tolerant germination was displayed with incomplete penetrance by all the selected mutants, which fell into five complementation groups. These were named SALOBRENO (SAN) and mapped relative to polymorphic microsatellites, the map positions of three of them suggesting that they are novel genes. Strains carrying mutations in the SAN1-SAN4 genes display similar responses to both ionic effects and osmotic pressure, their germination being NaCl and mannitol tolerant but KCl and Na(2)SO(4) sensitive. In addition, NaCl-, KCl-, and mannitol-tolerant as well as abscisic-acid-insensitive germination was displayed by sañ5, whose genetic and molecular characterization indicates that it carries an extremely hypomorphic or null allele of the ABI4 gene, its deduced protein product lacking the APETALA2 DNA binding domain.     

Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants. Received: 23 September 1999 / Accepted: 10 November 1999     

Anther developmental defects in Arabidopsis thaliana male-sterile mutants

 We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsis thaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsis thaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis. Received: 15 October 1998 / Revision accepted: 19 November 1998