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     

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     

Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin

Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 μg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.     

A comparison of 1-aminocyclopropane-1-carboxylate synthase in vitro translation product and in-vivo-labeled protein in ripening tomatoes

We determined the time course of increases in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in ripening tomato (Lycopersicon esculentum (L.) Mill.) pericarp discs following wounding and treatment with 75 mM LiCl. Over the course of 24 h, we detected oscillations in the amount of enzyme activity from an initial peak at 6 h to a subsequent, even higher level at 18 h. In-vitro translation products derived from poly(A)⁺ RNAs isolated at various times of treatment and in-vivo-labeled proteins were immunoprecipitated using antibodies specific for ACC synthase. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that wounding and treatment with LiCl induced an accumulation of translatable ACC-synthase-specific mRNAs. In addition, single, prominent bands were apparent for both in-vivo and in-vitro samples but their molecular masses differed. It appears that the in-vitro translation product is a polypeptide of 56 kDa while the in-vivo-labeled enzyme has a molecular mass of 47 kDa. The authors greatly appreciate the skilled technical assistance of Renate deZacks and Gail Robinson. This research was supported by the National Science Foundation through Grant No. DCB-8718873 and by the Department of Energy through Contract No. DE-AC02-76ER-01338.     

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds

 A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein. Received: 15 March 1999 / Accepted: 12 July 1999     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)⁺ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3-end was intact, it lacked a portion of sequence at the 5-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.     

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase

 Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination. Received: 28 September 1999 / Accepted: 27 December 1999     

Identification of oxindole-3-acetic acid,and metabolic conversion of indole-3-acetic acid to oxindole-3-acetic acid in Pinus sylvestris seeds

Oxindole-3-acetic acid (OxIAA) has been identified in germinating seeds of Scots pine (Pinus sylvestris) using gas chromatography-mass spectrometry. Seeds germinated for 5 d contained 2.7 ng OxIAA·g^-1 (dry weight) whereas ungerminated seeds contained 0.2 ng·g^-1. Isotopically labelled OxIAA was formed in seeds incubated with [1-¹⁴C]-, [2-¹⁴C]- or [²H₅]indole-3-acetic acid.Abbreviations DDC sodium diethyldithiocarbamate - GC gas chromatography - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - MS mass spectrometry - OxIAA oxindole-3-acetic acid - PVP polyvinylpyrrolidone - TMS trimethylsilyl     

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a ²H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - FW fresh weight - GC/MS coupled gas chromatography/mass spectrometry - HPLC high-performance liquid chromatography     

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     

The onset of starch degradation during banana ripening is concomitant to changes in the content of free and conjugated forms of indole-3-acetic acid

Banana (Musa acuminata AAA cv. Nanicão) slices were infiltrated with mannitol (control) and mannitol plus indole-3-acetic acid (IAA); then, some important ripening parameters like starch degradation, synthesis of ethylene and respiration were monitored. The contents of free-IAA and conjugated forms of IAA (ester and amide) were analyzed, by GC-MS-SIM, throughout the ripening in both banana slices and whole bananas. The starch degradation of IAA-treated slices was delayed for several days, but there was no difference between control and IAA-treated slices in the ethylene and respiration profiles. On day zero after infiltration, free-IAA levels were 500-fold higher in IAA-treated slices than in the control slices, but within 72 hours they declined to values 15-fold higher than those in the control group, with concomitant increase in IAA-ester. Similar to the banana slices, the onset of starch degradation occurred in whole bananas only when the free-IAA concentration was about 4 ng/g FW. The results herein suggest that IAA levels play a role during banana ripening in events like starch degradation with the consequence of banana sweetening.