Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the β-glucuronidase gene (uidA) drives β-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [²H]₅-L-tryptophan, they converted this precusor to [²H]₅-indole-3-acetonitrile and [²H]₅-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [²H]₅-indole-3-acetic acid extracted from roots. When [1-¹³C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-¹³C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan. Received: 3 February 1998 / Accepted: 17 April 1998     

The relative importance of tryptophan-dependent and tryptophan-independent biosynthesis of indole-3-acetic acid in tobacco during vegetative growth

A quantitative study of indole-3-acetic acid (IAA) turnover, and the contribution of tryptophan-dependent and tryptophan-independent IAA-biosynthesis pathways, was carried out using protoplast preparations and shoot apices obtained from wild-type and transgenic, IAA-overproducing tobacco (Nicotiana tabacum L.) plants, during a phase of growth when the level of endogenous IAA was stable. Based on the rate of disappearance of [¹³C₆]IAA, the half-life of the IAA pool was calculated to be 1.1 h in wild-type protoplasts and 0.8 h in protoplasts from the IAA-overproducing line, corresponding to metabolic rates of 59 and 160 pg IAA (μg Chl)⁻¹ h⁻¹, respectively. The rate of conversion of tryptophan to IAA was 15 pg IAA (μg Chl)⁻¹ h⁻¹ in wild-type protoplasts and 101 pg IAA (μg Chl)⁻¹ h⁻¹ in protoplasts from IAA-overproducing plants. In both instances, IAA was metabolised more rapidly than it was synthesised from tryptophan. As the endogenous IAA pools were in a steady state, these findings indicate that IAA biosynthesis via the tryptophan-independent pathway was 44 pg IAA (μg Chl)⁻¹ h⁻¹ and 59 pg IAA (μg Chl)⁻¹ h⁻¹, respectively, in the wild-type and transformed protoplast preparations. In a parallel study with apical shoot tissue, the presumed site of IAA biosynthesis, the rate of tryptophan-dependent IAA biosynthesis exceeded the rate of metabolism of [¹³C₆]IAA despite the steady state of the endogenous IAA pool. The most likely explanation for this anomaly is that, unlike the protoplast system, injection of substrates into the apical tissues did not result in uniform distribution of label, and that at least some of the [²H₅]tryptophan was metabolised in compartments not normally active in IAA biosynthesis. This demonstrates the importance of using experimental systems where labelling of the precursor pool can be strictly controlled. Received: 18 January 2000 / Accepted 24 February 2000     

Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide

Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Heteroblasty in Arabidopsis thaliana (L.) Heynh

 Heteroblasty in Arabidopsis thaliana was analyzed in a variety of plants with mutations in leaf morphology using a tissue-specific β-glucuronidase gene marker. Some mutants exhibited their mutant phenotypes specifically in foliage leaves. The phenotypes associated with the foliage-leaf-specific mutations were also found to be induced ectopically in cotyledons in the presence of the lec1 mutation. Moreover, the features of an emf1lec1 double mutant showed that cotyledons can be partially converted into carpelloids. When heteroblastic traits were examined in foliage leaves in the presence of certain mutations or natural deviations by histochemical analysis of the expression of the tissue-specific marker gene, it was found that ectopic expression of the developmental program for the first foliage leaves in lec1 cotyledons seemed to affect the heteroblastic features of the first set of foliage leaves, while foliage leaves beyond the third position appeared normal. Similarly, in wild-type plants, discrepancies in heteroblastic features, relative to standard features, of foliage leaves at early positions seemed to be eliminated in foliage leaves at later positions. These results suggest that heteroblasty in foliage leaves might be affected in part by the heteroblastic stage of the preceding foliage leaves but is finally controlled autonomously at each leaf position. Received: 9 July 1999 / Accepted: 17 August 1999     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry     

Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidopsis

The extension rate of the first inflorescence node of Arabidopsis was measured during light/dark or continuous light exposure and was found to exhibit oscillations which showed a circadian rhythmicity. Decapitation induced a strong inhibition of stem extension. Subsequent application of IAA restored growth and the associated extension–rate oscillations. In addition, IAA treatments, after decapitation, re-established the circadian rhythmicity visible in the intact plants during free run. This indicates that the upper zone of the inflorescence has a major influence on the extension rate of floral stems and implies a role for auxin. Application of N-(1-naphthyl)phthalamic acid, an IAA transport inhibitor, to an intact floral stem inhibited growth and the rhythmicity in the extension rate oscillations, indicating that IAA polar transport may play a role in the dynamics of stem elongation. Furthermore, IAA-aspartate application, after decapitation, did not restore growth and rhythmicity. Nevertheless, biochemical analysis of IAA and IAA-aspartate demonstrated circadian fluctuations of the endogenous levels of both compounds. These observations suggest that IAA metabolism is an essential factor in the regulation of the circadian growth rhythm of Arabidopsis floral stems. Received: 21 September 1998 / Accepted: 23 January 1999     

A nonphotochemical-quenching-deficient mutant of Arabidopsis thaliana possessing normal pigment composition and xanthophyll-cycle activity

Higher-plant chloroplasts alter the distribution of absorbed radiant energy between photosynthesis and heat formation in response to changing illumination level or environmental stress. Fluorescence imaging was used to screen 62 yellow-green T-DNA insertion mutant lines of Arabidopsis thaliana (L.) Heynh. for reduced photoprotective nonphotochemical quenching (NPQ) capacity. Pulse-modulation fluorometry was employed to characterize one line (denoted Lsr1⁻) that exhibited an approximately 50% reduction in NPQ compared to the wild type (WT). The loss in NPQ capacity was associated with the ΔpH-dependent phase of quenching (qE). Under the growth conditions employed, pigment composition and levels of the six photosystem-II light-harvesting chlorophyll a/b proteins were identical in mutant and WT. Changes in the in-vivo levels of the xanthophyll pigments violaxanthin, antheraxanthin, and zeaxanthin in excess light were the same for mutant and WT. However, use of the violaxanthin de-epoxidase inhibitor dithiothreitol indicated that a zeaxanthin-dependent component of NPQ was specifically reduced in the mutant. The mutant exhibited diminished suppression of minimum fluorescence yield (F _o ) in intense light suggesting an altered threshold in the mechanism of response to light stress in the mutant. The NPQ-deficient phenotype was meiotically transmissible as a semidominant trait and mapped near marker T27K12 on chromosome 1. The results suggest that the mutant is defective in sensing the transthylakoid ΔpH that reports exposure to excessive illumination. Received: 26 May 1999 / Accepted: 17 June 1999     

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants

When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids. Received: 10 February 2000 / Accepted: 11 April 2000     

Root hair elongation is inhibited by hypaphorine, the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by indole-3-acetic acid

Hypaphorine, the major indolic compound isolated from the ectomycorrhizal fungus Pisolithus tinctorius, controls the elongation rate of root hairs. At inhibitory concentrations (100 μM), hypaphorine induced a transitory swelling of root hair tips of Eucalyptus globulus Labill. ssp. bicostata. When the polar tip growth resumed, a characteristic deformation was still visible on elongating hairs. At higher hypaphorine concentrations (500 μM and greater), root hair elongation stopped, only 15 min after application. However, root hair initiation from trichoblasts was not affected by hypaphorine. Hypaphorine activity could not be mimicked by related molecules such as indole-3-acetic acid (IAA) or tryptophan. While IAA had no activity on root hair elongation, IAA was able to restore the tip growth of root hairs following inhibition by hypaphorine. These results suggest that hypaphorine and endogenous IAA counteract in controlling root hair elongation. During ectomycorrhiza development, the absence of root hairs might be due in part to fungal release of molecules, such as hypaphorine, that inhibit the elongation of root hairs. Received: 27 October 1999 / Accepted: 14 March 2000     

The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots

 Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions. Received: 30 July 1999 / Accepted: 22 September 1999     

Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana

The pho2 mutant of Arabidopsis thaliana (L.) Heynh. accumulates excessive Pi (inorganic phosphate) concentrations in shoots compared to wild-type plants (E. Delhaize and P. Randall, 1995, Plant Physiol. 107: 207–213). In this study, a series of experiments was conducted to compare the uptake and translocation of Pi by pho2 with that of wild-type plants. The pho2 mutants had about a twofold greater Pi uptake rate than wild-type plants under P-sufficient conditions and a greater proportion of the Pi taken up accumulated in shoots of pho2. When shoots were removed, the uptake rate by roots was found to be similar for both genotypes, suggesting that the greater Pi uptake by the intact pho2 mutant is due to a greater shoot sink for Pi. Although pho2 mutants could recycle ³²Pi from shoots to roots through phloem the proportion of ³²Pi translocated to roots was less than half of that found in wild-type plants. When transferred from P-sufficient to P-deficient solutions, Pi concentrations in pho2 roots had a similar depletion rate to wild-type roots despite pho2 shoots having a fourfold greater Pi concentration than wild-type shoots throughout the experiment. We suggest that the pho2 phenotype could result from a partial defect in Pi transport in the phloem between shoots and roots or from an inability of shoot cells to regulate internal Pi concentrations. Received: 20 August 1997 / Accepted: 4 October 1997     

Differential expression of triplicate phosphoribosylanthranilate isomerase isogenes in the tryptophan biosynthetic pathway of Arabidopsis thaliana (L.) Heynh.

Phosphoribosylanthranilate isomerases (PAI) in the tryptophan biosynthetic pathway of Arabidopsis thaliana are encoded by a gene family. Expression patterns of each individual PAI isogene were investigated by analyzing expression of translation-fusions of promoter-β-glucuronidase (GUS) chimeras in transgenic plants. Quantification and histochemical staining of GUS activities expressed in PAI transgenic plants demonstrated that, first, expression of the three PAI isogenes was differentially regulated under normal growth conditions. Both PAI1 and PAI3 showed approximately 10-fold stronger expression than PAI2. Second, PAI isogenes differentially responded to environmental stresses such as ultraviolet irradiation and the abiotic elicitor silver nitrate. PAI2 displayed a stronger response to stresses than the other two PAI isogenes. Third, each individual PAI isogene was differentially expressed in a tissue- and cell-type-specific manner. Fourth, expression of PAI isogenes was coordinated to meet the requirement for normal growth and development of A. thaliana. Deletion of PAI1 is partially responsible for abnormal growth and development in the PAI deletion mutant trp6 as well as strong blue fluorescence in young leaves under ultraviolet irradiation. Received: 15 June 2000 / Accepted: 16 August 2000     

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition. Received: 21 March 2000 / Accepted: 15 August 2000     

Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding

1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14) is the key regulatory enzyme of the ethylene biosynthetic pathway and is encoded by a multigene family in Arabidopsis thaliana, tomato, mung bean and other plants. Southern blot analysis revealed the existence of at least five ACS genes in white lupin (Lupinus albus L.) genome. Four complete and one partial sequences representing different ACS genes were cloned from the lupin genomic library. The levels of expression of two of the genes, LA-ACS1 and LA-ACS3, were found to increase after hypocotyl wounding. Apparently, these two genes were up-regulated by exogenous IAA treatment of seedlings. The LA-ACS3 mRNA levels were also elevated in the apical part of hypocotyl, which is reported to contain a high endogenous auxin concentration. This gene may be involved in the auxin- and ethylene-controlled apical hook formation. The expression of the LA-ACS4 gene was found to be almost undetectable. This gene may represent a “silent” twin of LA-ACS5 as these two genes share a considerable level of homology in coding and non-coding regions. The LA-ACS5 mRNA is strongly up-regulated in the embryonic axis of germinating seeds at the time of radicle emergence, and was also found in roots and hypocotyls of lupin seedlings. Received: 19 July 1999 / Accepted: 3 March 2000     

The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings ofDalbergia dolichopetala

Germinating seed ofDalbergia dolichopetala converted both [²H₅]l-tryptophan and [²H₅]indole-3-ethanol to [²H₅]indole-3-acetic acid (IAA). Metabolism of [2-¹⁴C]IAA resulted in the production of indole-3-acetylaspartic acid (IAAsp), as well as several unidentified components, referred to as metabolites I, II, IV and V. Re-application of [¹⁴C]IAAsp to the germinating seed led to the accumulation of the polar, water-soluble compound, metabolite V, as the major metabolite, together with a small amount of IAA. Metabolites I, II and IV were not detected, nor were these compounds associated with the metabolism of [2-¹⁴C]IAA by shoots and excised cotyledons and roots from 26-d-oldD. dolichopetala seedlings. Both shoots and cotyledons converted IAA to IAAsp and metabolite V, while IAAsp was the only metabolite detected in extracts from excised roots. The available evidence indicates that inDalbergia, and other species, IAAsp may not act as a storage product that can be hydrolysed to provide the plant with a ready supply of IAA.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting - IAA indole-3-acetic acid - IAAsp indole-3-acetylaspartic acid - IAlnos 2-O-indole-3-acetyl-myo-inositol - IEt indole-3-ethanol     

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone,indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.     

Indole acetonitrile-sensitivity of transgenic tobacco containing Arabidopsis thaliana nitrilase genes

 The Arabidopsis thaliana genome has four nitrilase (nitrile aminohydrolase, EC 3.5.5.1) genes (NIT1 to NIT4). These nitrilases catalyze hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Growth of A. thaliana is inhibited by IAN probably due to hydrolysis of IAN to IAA, while the tobacco (Nicotiana tabacum) genome has only NIT4 homologs and is resistant to IAN. In this study, we introduced A. thaliana NIT1 to NIT4 into tobacco. Introduction of NIT1, NIT2 or NIT3 into tobacco conferred growth inhibition by IAN. NIT2 transgenic plants were highly sensitive to IAN, and NIT1 and NIT3 transgenic plants were moderately sensitive. On the other hand, NIT4 transgenic plants were less sensitive to IAN, although some morphological changes in the roots were observed as the wild-type tobacco. These findings suggest that the ability of transgenic tobacco to convert IAN to IAA in vivo is markedly different among transgenes of NIT1 to NIT4. Received: 22 November 1999 / Revision received: 28 January 2000 / Accepted: 4 February 2000     

A freezing-sensitive mutant of Arabidopsis, frs1, is a new aba3 allele

To investigate the molecular mechanisms controlling the process of cold acclimation and to identify genes involved in plant freezing tolerance, mutations that impaired the cold acclimation capability of Arabidopsis thaliana (L.) Heynh. were screened for. A new mutation, frs1 (freezing sensitive 1), that reduced both the constitutive freezing tolerance as well as the freezing tolerance of Arabidopsis after cold acclimation was characterized. This mutation also produced a wilty phenotype and excessive water loss. Plants with the frs1 mutation recovered their wild-type phenotype, their capability to tolerate freezing temperatures and their capability to retain water after an exogenous abscisic acid (ABA) treatment. Measurements of ABA revealed that frs1 mutants were ABA deficient, and complementation tests indicated that frs1 mutation was a new allele of the ABA3 locus showing that a mutation in this locus leads to an impairment of freezing tolerance. These results constitute the first report showing that a mutation in ABA3 leads to an impairment of freezing tolerance, and not only strengthen the conclusion that ABA is required for full development of freezing tolerance in cold-acclimated plants, but also demonstrate that ABA mediates the constitutive freezing tolerance of Arabidopsis. Gene expression in frs1 mutants was altered in response to dehydration, suggesting that freezing tolerance in Arabidopsis depends on ABA-regulated proteins that allow plants to survive the challenges imposed by subzero temperatures, mainly freeze-induced cellular dehydration. Received: 16 December 1999 / Accepted: 31 March 2000     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000