The biosynthetic pathway for indole-3-acetic acid changes during tomato fruit development

Phytohormone metabolism during fruit ripening is critical to the controlof this developmental process, yet we know little about pathways for theproduction of many of these signaling compounds. Using stable isotope labelingin both an in vitro aseptic tomato fruit culture systemanddetached greenhouse-grown tomato fruit, we have shown by mass spectral analysisthat tomato uses the tryptophan-independent pathway to produce IAA fromanthranilate or indole. We also show that there is a developmental switch fromtryptophan utilization to tryptophan-independent production that occurs betweenmature green and red-ripe stages of fruit development. Moreover, this pathwayswitch does not appear to be associated with ripening per se in that fruit fromneverripe tomato plants also utilize the tryptophanindependent pathway.     

Development of an abscisic acid biosynthesizing cell-free system from flavedo of Citrus sinensis fruit

Cell-free extracts prepared from an acetone powder derived fromthe flavedo of mature, coloured fruits of Citrus sinensis L.cv. Midknight transformed mevalonic acid, isopentenyl pyrophosphate,all-trans-ß-carotene and 1' ,4' -trans-ABAdiol intoABA. This is the first report of a cell-free system capableof producing ABA from both a terpenyl pyrophosphate and carotenoidorigin. ABA biosynthesizing activity was stimulated by reducednicotinamide nucleotides, molybdate, AM01618 and a cold-pooltrap of ()-ABA, but was inhibited by FAD. Addition of FAD causedaccumulation of label from isopentenyl pyrophosphate in a compoundwith similar chromatographic and spectrophotometric propertiesto those of ß-carotene. 1',4'-Trans-ABAdiol, ABA andPA were unequivocally characterized as products of ß-carotenemetabolism in this cell-free system, a process that was markedlyimproved by extraction of enzyme in the presence of detergents.Dithiothreitol enhanced ABA biosynthesis from all-trans-ß-carotene.Stigmasterol, and to a lesser extent cholesterol reduced conversionof ß-carotene to ABA, but did not influence transformationof 1',4'-trans-ABAdiol to ABA. AM01618 stimulated formationof ABA and appeared to exert its effect at the level of conversionof 1',4'-trans-ABAdiol to ABA. Ancymidol and zeatin reducedincorporation of label from all-trans-ß-carotene intoABA suggesting involvement of a mixed function oxidase in thisreaction sequence. Sodium dodecylsulphate polyacrylamide gelelectrophoresis of the enzyme extract derived from Citrus flavedorevealed the presence of a 53 kDa protein with peroxidase activitycharacteristic of a cytochrome P-450. Key words: Abscisic acid, biosynthesis, cell-free system Citrus sinensis, flavedo, Rutaceae     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Metabolism of indole-3-acetic acid in rice: identification and characterization of N-beta-D-glucopyranosyl indole-3-acetic acid and its conjugates

A search was made for conjugates of indole-3-acetic acid (IAA) in rice (Oryza sativa) using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in order to elucidate unknown metabolic pathways for IAA. N-beta-d-Glucopyranosyl indole-3-acetic acid (IAA-N-Glc) was found in an alkaline hydrolysate of rice extract. A quantitative analysis of 3-week-old rice demonstrated that the total amount of IAA-N-Glc was equal to that of IAA. A LC-ESI-MS/MS-based analysis established that the major part of IAA-N-Glc was present as bound forms with aspartate and glutamate. Their levels were in good agreement with the total amount of IAA-N-Glc during the vegetative growth of rice. Further detailed analysis showed that both conjugates highly accumulated in the root. The free form of IAA-N-Glc accounted for 60% of the total in seeds but could not be detected in the vegetative tissue. An incorporation study using deuterium-labeled compounds showed that the amino acid conjugates of IAA-N-Glc were biosynthesized from IAA-amino acids. IAA-N-Glc and/or its conjugates were also found in extracts of Arabidopsis, Lotus japonicus, and maize, suggesting that N-glucosylation of indole can be the common metabolic pathway of IAA in plants.     

Changes in indole-3-acetic acid and abscisic acid levels during tomato (Lycopersicon esculentum Mill.) fruit development and ripening

Changes in the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) in tomato (Lycopersicon esculentum Mill.) fruit pericarp tissue during development through ripening were measured by GC-SIM-MS using d₃-ABA and ¹³C₆-IAA internal standards. In the two cultivars of fieldgrown tomatoes analyzed, the highest IAA levels (8–24 ng/g fw) were found at the earliest stage of development (7 days after anthesis) followed by a rapid decline in levels of the hormone. ABA levels of 40–60 ng/g fw were found at the earliest stages of development followed by a decline in levels until ripening occurred when elevated ABA levels (125 ng/g fw) were measured.     

Dynamics of indole-3-acetic acid and indole-3-ethanol during development and germination of Pinus sylvestris seeds

Indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified in immature seeds of Pinus sylvestris L. by combined gas chromatography-mass spectrometry. Indole-3-methanol was tentatively identified using multiple ion monitoring. Anatomical investigations of seeds, as well as measurements of free and alkali-hydrolysable IAA and IEt, were made during seed development and germination. Levels of free IAA and IEt decreased during seed development. In the later stages of seed maturation most IAA and IEt were present in alkali-hydrolysable forms. Bound IAA and bound IEt rapidly decreased during germination, while levels of free IAA and IEt increased dramatically for a short period.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

Changes in indole-3-acetic acid levels during tomato (Lycopersicon esculentum Mill.) seed development

Summary The changes in the level of indole-3-acetic acid (IAA) were investigated in seeds and fruit tissues-placenta and mesocarp-during tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis, which was characterized through eight morphological embryo stages [from globular (stage 1) to mature embryo (stage 8)]. In whole seeds, IAA levels increased mainly at stage 3 (young torpedo) and at stage 5 (late torpedo stage). As the seed matured and dehydrated, IAA levels decreased and showed a new distribution pattern within seed structures, preferentially in endosperm tissue. IAA contents in fruit tissues were lower but followed the same pattern as those of seeds. These data support the hypothesis of IAA biosynthesis in seeds with a transient role of the endosperm at the end of embryo development and suggest a role of IAA in fruit and seed growth. Moreover a comparison of IAA and ABA changes suggests that IAA could be especially necessary for the beginning of embryo growth, whereas ABA could act mainly at the end of the growth phase.Abbreviations ABA abscisic acid - ABTS 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) - BHT butylhydroxytoluene - DW dry weight - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid. PGRs: plant growth regulators     

Levels of endogenous indole-3-acetic acid and indole-3-acetyl-aspartic acid during adventitious rooting in avocado microcuttings

Quantification of endogenous IAA and lAAsp was carried out duringadventitious root formation in avocado microcuttings. Both auxinand conjugate were monitored in control cuttings (rooted inthe absence of auxin) as well as in cuttings treated with arooting promotor (IBA) or an auxin transport inhibitor (TIBA).Additionally, a histological study to follow root differentiationwas carried out. In control cuttings IAA levels remained constantthroughout the rooting process, however, in IB A-treated cuttingsIAA levels increased 2-fold during the first 6 d. Addition of200 µM TIBA induced a slight decrease of IAA levels andinhibited root formation. As for IAAsp levels, both control and IBA-treated cuttings showeda big increase before root differentiation occurred and as theprocess went on, a progressive decrease took place. However,in TIBA-treated cuttings IAAsp levels not only did not increasebut diminished progressively during the process. The role ofauxin conjugates during the rooting process of avocado is discussed. Key words: Avocado, IAA, IAAsp, rooting     

An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.     

Magnesium influences on the fruit quality of sweet orange (Citrus sinensis L. osbeck)

Plant and Soil - The influence of magnesium nutrition on fruit quality of sweet orange was investigated in sand culture, and the relationships between Mg and fruit quality were explored in the...     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

Summary Nucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

The biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Destruction of indole-3-acetic acid during the aerobic oxidation of sulfite

Indole-3-acetic acid (IAA) was rapidly destroyed in the presence of Mn²⁺, oxygen and sulfite ion. The optimal pH for the reaction was between 5 and 6. The destruction was dependent on the aerobic oxidation of sulfite, but was not inhibited by superoxide dismutase. Tracer studies indicate that IAA was converted into at least 3 compounds. Decarboxylation of IAA was not involved in the destruction.     

Uptake and metabolism of indole-3-butyric acid and indole-3-acetic acid by Petunia cell suspension culture

The uptake and metabolism of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were studied in suspension cell cultures of Petunia hybrida. The initial uptake of ³H-IBA was much higher than that of ³H-IAA, and after 10 min of incubation with labeled IBA and IAA, 4.6 pM vs 0.35 (39% vs 12% of total applied radioactivity) respectively, were found in the cell extracts. The uptake of IBA reached a plateau of 6.0 pM (62%) after 2 h while that of IAA increased continuously up to 1.5 pM (46%) after 24 h. Following the addition of 40 µM of unlabeled auxin more IBA was taken in initially than IAA (39% vs 12%), but the level almost equalized after 24 h of incubation when IBA uptake reached 890 nM (55%) and IAA 840 nM (46%).IBA was metabolized very rapidly by Petunia cell suspension to new compounds. HPLC of the cell extracts demonstrated a new metabolite after only 2 min of incubation, and after 30 min 60% of the radioactivity was in the new metabolite vs 10% in the IBA. The new compound was resolved by autofluorography to two metabolites but after 24 h only one metabolite was present. The IBA metabolites were identified tentatively as IBA aspartic acid (IBAasp) and IBA glucose (IBAglu). In the medium IBA disappeared at a fast rate and after 24h most of the radioactivity was present in the new metabolite, probably IBAasp. IAA was also converted rapidly to two new metabolites and both were still present after 24 h. No attempt was made to identify the metabolites of IAA. IAA metabolism proceeded at a slower rate, and autofluorography showed that while free IBA disappeared after 0.5 h, free IAA was still present after 1 h of incubation. We postulate that Petunia cells conjugate IBA rapidly to IBAglu which in turn is converted to form IBAasp which probably acts as a slow release hormone. Only intact cells were able to metabolize IBA and the reaction was affected by low temperature and anaerobic conditions. The fast rate of IBA uptake, the need for whole cells for the metabolism to proceed, and the fast change of IBA to a new metabolite in the medium, all suggest that both uptake and metabolism of IBA in Petunia cells occur on the cell surface.