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.     

Phospholipid composition and fatty acid peroxidation during ageing of Acer platanoides seeds

Seeds of Norway maple ( Acer platanoides L.) differing in water content (10, 20 and 30%) were stored for 6 weeks at 20 to 30°C. During this period changes in phospholipids and fatty acids as well as in seed viability and germination capacity were studied. A considerable decrease in the phospholipid content was observed, which depended on the water content in the seeds and was related to the decrease of the seed germination capacity. The level of linoleic (18:2) and linolenic (18:3) acids in the phospholipid fraction decreased considerably in the course of the accelerated seed ageing. The results obtained suggest that phospholipid degradation and peroxidation of unsaturated fatty acids, followed by membrane destruction, play a considerable role in maple seed ageing.     

The influence of the extraction procedure on yield of indole-3-acetic acid in plant extracts

Different types of plant material, including both dry and swollen maize kernels, swollen bean seeds, bean seedlings and dry rose seeds, were extracted by different methods and the yield of IAA was determined with the indolo-α-pyrone method. Extraction of dry maize kernels during short time experiments, varying from 3 to 24 h, gave the highest IAA yield when methanol was the extractant and a significant lower yield when diethyl ether or dichloromethane were used. The duration of the extraction period increased the yield with all the extractants. Progressive extractions for several days or weeks had little effect on the yield when 100% acetone was used in contrast to methanol and ether as extractants, which increased the yield during prolonged extraction. Extractions of tissue treated to 100°C for 1 h contradicted the hypothesis that IAA is enzymatically liberated during ether extraction. Water in the extractant solvents increased the yields. This was most pronounced when aqueous acetone was used instead of 100% acetone. Increased extraction temperature augmented the IAA yields. The yield of IAA from other types of tissue extracted with methanol for periods of 3 or 24 h was, however, independent of the duration of the extraction time. This indicates that some tissues contain less not easily extractable IAA than dry maize kernels. The terms “free” and “bound” IAA are discussed; they should be replaced by “easily extractable” and “not easily extractable” IAA. The results also show that IPyA in vitro can partly be converted to IAA during extraction and fractionation.     

Sequential temperature control of multi-phasic dormancy release and germination of Paeonia corsica seeds

Aims The physiological responses during dormancy removal and multi-phasic germination were investigated in seeds of Paeonia corsica (Paeoniaceae).Methods Seeds of P. corsica were incubated in the light at a range of temperatures (10–25 and 25/10°C), without any pre-treatment, after W (3 months at 25°C), C (3 months at 5°C) and W + C (3 months at 25°C followed by 3 months at 5°C) stratification, and a GA 3 treatment (250 mg·l^-1 in the germination substrate). Embryo growth, time from testa to endosperm rupture and radicle emergence were assessed as separate phases. Epicotyl–plumule emergence was evaluated incubating the germinated seeds at 15°C for 2 weeks, at 5 and 25°C for 2 months on agar water before transplanting to the soil substrate at 10, 15 and 20°C and at 15°C for 2 months on the surface agar water with GA 3 .Important findings Embryo growth, testa rupture, endosperm rupture (radicle emergence) and growth of the epicotyl were identified as four sequential steps in seeds of P. corsica. Gibberellic acid alone and warm stratification followed by 15°C promoted embryo growth and subsequent seed germination. Cold stratification induced secondary dormancy, even when applied after warm stratification. After radicle emergence, epicotyl–plumule emergence was delayed for ca. 3 months. Mean time of epicotyl–plumule emergence was positively affected by cold stratification (2 months at 5°C) and GA 3. P. corsica seeds exhibited differential temperature sensitivity for the four sequential steps in the removal of dormancy and germination processes that resulted in the precise and optimal timing of seedling emergence.     

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     

Changes in protein synthesis during stratification and dormancy release in embryos of sugar maple (Acer saccharum)

Protein synthesis in dormant embryos of sugar maple ( Acer saccharum ) was investigated in seeds stratified at 4°C or incubated at 15°C. Seeds stratified at 4°C germinated after 27 days; seeds incubated at 15°C failed to germinate. Stratification increased the embryo's capacity for protein synthesis by day 11 as measured by in vivo incorporation of [³⁵S]-methionine into purified protein. At 4°C protein synthesis in the embryonic axis rose in a linear fashion prior to germination, whereas in cotyledons it increased until day 20 and then declined. Analysis of radiolabelled proteins by two-dimensional gel electrophoresis revealed that the levels of specific proteins were altered by temperature, primarily in the cotyledons. Several proteins were expressed in the cotyledons at 15°C but were absent in unstratified embryos and in embryos stratified at 4°C. That is, the expression of these proteins was repressed during stratification and release from dormancy. Levels of other proteins in the cotyledons declined at 4°C during stratification. We suggest that one or more of these proteins may be associated with the inhibition of growth of the embryonic axis imposed by the cotyledons.     

Seed dormancy in Acer: The role of abscisic acid in the regulation of seed development in Acer platanoides L.

Germinability of isolated embryos from developing fruits of Acer platanoides was high at the earliest developmental stage assessed (90 dpa), but fell subsequently and at seed maturity was very low. These observations showed an inverse correlation with changes in endogenous free abscisic acid (ABA) levels in the embryo, which were low during early ontogeny, but reached maximum levels late in development (150–160 dpa). These observations suggest the possibility that dormancy may be induced during development as a result of ABA accumulation in the embryo, an argument strengthened by the obvious inhibitory effect of added ABA on the germinability of isolated embryos. The cotyledons appear to exert an inhibitory influence on embryo germinability that may result from their free ABA content although the embryonic axis itself possesses an innate dormancy that may reflect its own free ABA content. The increased germinability of isolated embryos resulting form added kinetin serves only to emphasise the complexity of the system and the dangers of simplistic interpretation.The correlation between germinability and ABA content is not complete, however, since much of the reduction in germinability had occurred before any appreciable increase in free ABA levels in the embryo was observed. Indeed the failure of the intact seed to respond to endogenous changes in embryonal ABA levels suggests that even though free ABA in the embryo may influence embryo germinability, it has little effect in the intact seed, where the presence of an intact testa may be a more important factor.The absence of a desiccation phase in the embryo during the late stages of development suggests that the large increases in endogenous free ABA did not cause dormancy by inhibiting water uptake, nor did they result from water stress in the embryonal tissues.     

Precision and accuracy of indole-3-acetic acid analyses performed with the 2-methylindolo-α-pyrone fluorescence assay and with a high performance liquid chromatography technique with spectrofluorimetric detection, exemplified on pine tissue (Pinus sylvestris L.)

The precision and accuracy of two versions of the 2-methylindolo-α-pyrone (2-MIP) assay and of a high performance liquid chromatography (HPLC) technique was assessed when applied to analysis of indole-3-acetic acid (IAA) in pine ( Pinus sylvestris ) tissue. In one version of the 2-MIP assay total fluorescence at wavelengths > 450 nm was recorded, whereas in the other version fluorescence spectra were recorded and analyzed. Such analysis revealed the occurrence in some samples of fluorescent contaminants that impaired both precision and accuracy. The precision and accuracy of the 2-MIP assay could be improved by additional sample purification, and the over-all accuracy of the assay was finally verified, at a high level of sample purity, by successive approximation. The precision of the assay was then 14%. Successive approximation also verified that the HPLC technique was accurate at a less advanced stage of sample purity than the 2-MIP assay, and had a precision of 14%.     

Identification of 4-chloroindole-3-acetic acid and indole-3-aldehyde in seeds of Pinus sylvestris

4-Chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-aldehyde (IAId) have been characterized as endogenous constituents in seeds of Pinus sylvestris L. by gas chromatography-mass spectrometry. Quantitative estimates indicate that immature seeds contained 640 pg 4-Cl-IAA (g fresh weight)^-1 while mature seeds contained 340 pg (g dry weight)^-1. 4-Cl-IAA could not be detected in seeds five days after germination. The content of IAld increased from 127 pg (g dry weight)^-1 in mature seeds to 315 pg (g dry weight)^-1 after five days of germination.     

Indole-3-acetic acid and indole-3-acetylaspartic acid isolated from seeds of Heracleum laciniatum Horn

Seeds from mature flowers of Heracleum laciniatum were collected locally (Tromsø, Norway). Seed coats were removed and the seeds were analyzed for their content of free, free plus ester-conjugate, and total indole-3-acetic acid (IAA) by quantitative gas chromatography-mass spectrometry. Seeds contained high levels of free and amide-linked IAA relative to other dicotyledonous seeds for which values have been published. The major amide conjugate in this material was identified as indole-3-acetylaspartate by gas chromatography-mass spectrometry of its bis-methyl ester.     

Tryptophan and indole-3-acetic acid accumulation in Acer cell cultures and its relationship with cell autolysis

The accumulation and decline of free indole-3-acetic acid (IAA) and tryptophan has been monitored in cells of Acer pseudoplatanus L. grown in batch suspension cultures. The period of maximal IAA accumulation per cell or per unit dry weight of tissue was found to precede the peak of tryptophan accumulation by several days. A study of cell viability throughout a growth passage indicated the presence of a basal level of non-viable cells of 5–7%, with only minor increases occurring during the first week of the three-week growth passage. The results suggest that IAA biosynthesis is not regulated by substrate availability arising from proteolysis in dead cells.Abbreviation GC-MS Gas chromatography-mass spectrometry - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - TLC thin-layer chromatography     

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.     

Membrane phospholipid composition during maturation of seeds of Acer platanoides and Acer pseudoplatanus in relation to desiccation tolerance

Membrane phospholipid composition was investigated in seeds of two species from the genus Acer: Norway maple (Acer platanoides L.) — tolerant to desiccation, and sycamore (Acer pseudoplatanus L.) — intolerant to desiccation, during their maturation, from 1 August to 25 September 1995, at weekly intervals. Seeds of Norway maple acquire tolerance to desiccation at the end of August ie. about 125 days after flowering (DAF). Phospholipid composition during development revealed marked differences between studied seeds. Seeds of Norway maple after acquiring tolerance to desiccation contained much more phosphatidylcholine (PC) and phosphatidylethanolamine (PE), compared to sycamore. The ratio of PC/PE in mature Norway maple seeds was evidently higher than those in sycamore. The level of unsaturated fatty acids in the phospholipid fraction substantially increased in Norway maple seeds during development and the saturation of PC and PE was less than in sycamore. The results suggest that phospholipid composition may be involved in desiccation tolerance of Norway maple seeds.     

Comparison of movement and metabolism of indole-3-acetic acid and indole-3-butyric acid in mung bean cuttings

Indole-3-butyric acid (IBA) was much more effective than indole-3-acetic acid (IAA) in inducing adventitious root formation in mung bean ( Vigna radiata L.) cuttings. Prolonging the duration of treatment with both auxins from 24 to 96 h significantly increased the number of roots formed. Labelled IAA and IBA applied to the basal cut surface of the cuttings were transported acropetally. With both auxins, most radioactivity was detected in the hypocotyl, where roots were formed, but relatively more IBA was found in the upper sections of the cuttings. The rate of metabolism of IAA and IBA in these cuttings was similar. Both auxins were metabolized very rapidly and 24 h after application only a small fraction of the radioactivity corresponded to the free auxins. Hydrolysis with 7 M NaOH indicates that conjugation is the major pathway of IAA and IBA metabolism in mung bean tissues. The major conjugate of IAA was identified tentatively as indole-3-acetylaspartic acid, whereas IBA formed at least two major conjugates. The data indicate that the higher root-promoting activity of IBA was not due to a different transport pattern and/or a different rate of conjugation. It is suggested that the IBA conjugates may be a better source of free auxin than those of IAA and this may explain the higher activity of IBA.     

The bound auxins: Protection of indole-3-acetic acid from peroxidase-catalyzed oxidation

Indole-3-acetic acid (IAA) was oxidized by horseradish peroxidase, but ester and amide conjugates of IAA were not degraded. Addition of indoleacetyl-myo-inositol, indoleacetyl-L-aspartate, indoleacetylglycine, indoleacetyl-L-alanine, indoleacetyl-D-alanine, or indoleacetyl--alanine did not affect the rate of oxidation of IAA by horseradish peroxidase. Peroxidase preparations from Pisum sativum L. and Zea mays L. behaved similarly in that they rapidly oxidized IAA, but not conjugates found in the plant from which the peroxidase was prepared. These results indicate that conjugation could affect the stability of IAA in vivo.Abbreviation IAA Indole-3-acetic acid     

Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid,naphthalene-1-acetic acid,and indole-3-acetic acid in suspension-cultured tobacco cells

Accumulation of radiolabelled naphthalene-1-acetic acid (1-NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA) has been measured in suspension-cultured tobacco (Nicotiana tabacum) cells. In this paper is presented a simple methodology allowing activities of the auxin influx and efflux carriers to be monitored independently by measuring the cellular accumulation of [³H]NAA and [¹⁴C]2,4-D. We have shown that 1-NAA enters cells by passive diffusion and has its accumulation level controlled by the efflux carrier. By contrast, 2,4-D uptake is mostly ensured by the influx carrier and this auxin is not secreted by the efflux carrier. Both auxin carriers contribute to IAA accumulation. The kinetic parameters and specificity of each carrier have been determined and new information concerning interactions with naphthylphthalamic acid, pyrenoylbenzoic acid, and naphthalene-2-acetic acid are provided. The relative contributions of diffusion and carrier-mediated influx and efflux to the membrane transport of 2,4-D, 1-NAA, and IAA have been quantified, and the data indicate that plant cells are able to modulate over a large range their auxin content by modifying the activity of each carrier.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 1-NAA naphthalene-1-acetic acid - 2-NAA naphthalene-2-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - V_m maximum transport capacity of the carrier In honour of Professor Dieter Klämbt's 65th birthdayThe authors thank Drs. A.E. Geissler and G.F. Katekar (CSIRO, Canberra City, Australia) for providing auxin efflux carrier inhibitors CPD, CPP, and PBA, and Dr. H. Barbier-Brygoo (Institut des Sciences Végétales, CNRS, Gif-sur-Yvette, France) for helpful discussions. This work was supported by funds from the Centre National de la Recherche Scientifique (UPR0040).     

Changes in indole-3-acetic acid,indole-3-acetic acid oxidase,and peroxidase isoenzymes in the seeds of developing peach fruits

Changes in indole-3-acetic acid (IAA) content of peach (Prunus persica L. Batsch cv. Merry) seeds were followed during fruit development. The highest concentration of IAA, 2.7 g/g fresh weight, was found at the beginning of Stage III of fruit development, approximately 50–60 days after anthesis. The IAA-decarboxylating capacity of crude extracts of seeds was also greatest at 55–60 days after anthesis. Four soluble peroxidase isoenzymes were found on anionic electrophoresis. There were no marked changes in two isoenzymes (R _f 0.23 and 0.51), which were present in all three stages of fruit growth. There was a marked increase in a band atR _f 0.59 between Stages II and III, and a decrease in a band atR _f 0.68 from Stages II to III. Neither band (R _f 0.59 and 0.68) was present at Stage I.     

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     

Promotion of hypocotyl elongation in loblolly pine (Pinus taeda L.) by indole-3-acetic acid

Elongation of excised loblolly pine ( Pinus taeda L.) hypocotyls was promoted by indole-3-acetic acid and the fungal metabolite, fusicoccin. Gibberellic acid, kinetin, zeatin, or zeatin-riboside were either without effect or promoted elongation only slightly. The most auxin-responsive tissue was just below the cotyledonary node, and elongation was confined to sections excised from the upper 2 cm of the hypocotyl. Indole-3-acetic acid induced elongation rates in the hypocotyl sections equal to those of intact hypocotyls when the sections were excised from young seedlings. Elongation rates decreased in intact hypocotyls before the excised tissues lost responsiveness to the auxin. Hypocotyl elongation in loblolly pine is discussed in relation to hypocotyl elongation in angiosperm species.