Indole-3-acetic Acid Distribution Research Articles

A high concentration of abscisic acid inhibits hypocotyl phototropism in Gossypium arboreum by reducing accumulation and asymmetric distribution of auxin.

Hypocotyl phototropism is mediated by the phototropins and plays a critical role in seedling morphogenesis by optimizing growth orientation. However, the mechanisms by which phototropism influences morphogenesis require additional study, especially for polyploid crops such as cotton. Here, we found that hypocotyl phototropism was weaker in Gossypium arboreum than in G. raimondii (two diploid cotton species), and LC-MS analysis indicated that G. arboreum hypocotyls had a higher content of abscisic acid (ABA) and a lower content of indole-3-acetic acid (IAA) and bioactive gibberellins (GAs). Consistently, the expression of ABA2, AAO3, and GA2OX1 was higher in G. arboreum than in G. raimondii, and that of GA3OX was lower; these changes promoted ABA synthesis and the transformation of active GA to inactive GA. Higher concentrations of ABA inhibited the asymmetric distribution of IAA across the hypocotyl and blocked the phototropic curvature of G. raimondii. Application of IAA or GA3 to the shaded and illuminated sides of the hypocotyl enhanced and inhibited phototropic curvature, respectively, in G. arboreum. The application of IAA, but not GA, to one side of the hypocotyl caused hypocotyl curvature in the dark. These results indicate that the asymmetric distribution of IAA promotes phototropic growth, and the weakened phototropic curvature of G. arboreum may be attributed to its higher ABA concentrations that inhibit the action of auxin, which is regulated by GA signaling.

Small Auxin Up RNAs influence the distribution of indole-3-acetic acid and play a potential role in increasing seed size in Euryale ferox Salisb

BackgroundAquatic Euryale ferox Salisb. is an economically important crop in China and India. Unfortunately, low yield limitations seriously hinder market growth. Unveiling the control of seed size is of remarkable importance in improvement of crops. Here, we generated a new hybrid line (HL) with larger seeds by crossing South Gordon Euryale and North Gordon Euryale (WT) which hasn’t been reported before. However, the functional genes and molecular mechanisms controlling the seed size in Euryale ferox Salisb. remain unclear. In this study, we focused on the differentially expressed genes in the auxin signal transduction pathway during fruit development between HL and WT to explore candidate regulatory genes participated in regulating seed size.ResultsBoth concentration and localization of indole-3-acetic acid (IAA) at two growth stages of fruits of WT and HL were detected by LC-MS and immunofluorescence. Although IAA content between the two lines did not differ, IAA distribution was significantly different. To elucidate the mechanism and to seek the key genes underlying this difference, RNA-seq was performed on young fruits at the two selected growth stages, and differentially expressed genes related to the auxin transduction pathway were selected for further analysis.ConclusionHybrid Euryale ferox Salisb. expressed significant heterosis, resulting in non-prickly, thin-coated, large seeds, which accounted for the significantly larger yield of HL than that of WT. Our study indicated that Small Auxin Up RNAs (SAURs) -mediated localization of IAA regulates seed size in Euryale ferox Salisb. We found that some SAURs may act as a positive mediator of the auxin transduction pathway, thereby contributing to the observed heterosis.

Electrochemical mapping of indole-3-acetic acid and salicylic acid in whole pea seedlings under normal conditions and salinity

Indole-3-acetic acid (IAA) and salicylic acid (SA) are two essential phytohormones for plants. Their regulating functions depend on their levels in different locations of plants. However, currently there is no satisfactory method for visual analysis of such small molecules in a whole plant. Herein the levels of IAA and SA in continuous parts of whole pea seedlings were analysed with paper-based electroanalytical devices coupled with a multichannel electrochemical station. The parts of pea seedlings were cut and put on the working electrodes for quantification. The contents of IAA and SA in continuous parts could be presented in the form of heatmaps, illustrating polar distribution of IAA levels and gradual changes of SA levels in whole pea seedlings. The contents distribution of IAA and SA further revealed variation of IAA and SA in different locations of pea seedlings. More importantly, the contents of IAA and SA in pea seedlings under normal conditions and salinity could also be differentiated with heatmaps and the contents distribution. Such results implied that our approach might be able to be extended for the study of mechanisms of plant hormones under various stresses. This study provided a possible route for visualization of small biochemical molecules in vivo with paper-based electrochemical devices.

옥수수 일차뿌리에서 oryzalin이 굴중성 반응과 에틸렌 생성에 미치는 효과

Oryzalin은 미세소관을 분열시키는 dinitroaniline계의 제초제이다. 미세소관과 미세섬유는 평형석 침강과 세포벽을 구성하는 세포골격들이다. 평형석은 뿌리 끝에 있는 columella 세포에서 중력 인지 조절을 한다. 본 연구는 oryzalin이 옥수수 일차 뿌리에서 ethylene 생성을 통하여 굴중성 반응에 미치는 영향을 연구하였다. 뿌리 끝 부분에 10^-4 M oryzalin의 처리는 뿌리 성장과 굴중성 반응을 저해하였으나, 신장대에 처리하게 되면 저해현상은 관찰되지 않았다. 10^-4 M oryzalin을 뿌리 끝에 15시간 처리하면 뿌리 끝의 생장이 억제되고 둥근 형태로 부풀었다. 에틸렌의 전구물질인 ACC를 뿌리 끝에 처리하여도 굴중성 반응이 억제되었다. Oryzalin의 작용과 에틸렌 생성에 대한 관련성을 연구하기 위하여 oryzalin 처리 후 에틸렌 생성을 측정하였다. Oryzalin 처리에 의해 ACC oxidase와 ACC synthase의 활성이 증가되어 에틸렌 생성이 촉진되었다. Oryzalin은 ACO와 ACS의 유전자의 발현도 증가 시켰다. Indole-3-acetic acid (IAA)는 굴중성 반응 동안 관찰되는 비 대칭적 신장에 중요한 역할을 한다. 이러한 연구 결과는 oryzalin이 뿌리 끝에서 IAA transport를 억제하여 뿌리 신장대의 윗면과 아랫면의 IAA 양의 차이를 감소시키고, 또한 에틸렌 생성을 촉진하며 미세소관의 배열을 방해하여 뿌리 글중성과 생장을 억제할 가능성을 제시하고 있다. Oryzalin is a dinitroaniline herbicide, which disrupts the arrangement of microtubules. Microtubules and microfilaments are cytoskeletal components that are thought to play a role in the sedimentation of statoliths and the formation of cell walls. Statoliths regulate the perception of gravity by columella cells in the root tip. To determine the effect of oryzalin on the gravitropic response, ethylene production in primary roots of maize was investigated. Treatment with 10^-4 M oryzalin to the root tip inhibited the growth and gravitropic response of the roots. However, the treatment had no effect on the elongation zone of the roots. An application of 10^-4 M oryzalin for 15 hr to the root tip caused root tip swelling. The application of 1-aminocycopropane-1-carboxylic acid (ACC), a precursor of ethylene, to the root tip also inhibited the gravitropic response. To understand the role of oryzalin in the regulation of the growth and gravitropic response of roots, ethylene production in the primary roots of maize was measured following treatment with oryzalin. Oryzalin stimulated ethylene production via the activation of ACC oxidase (ACO) and ACC synthase (ACS), and it increased the expression of ACO and ACS genes. Indole-3-acetic acid (IAA) played a key role in the asymmetric elongation rates observed during gravitropism. The results suggest that oryzalin alters the gravitropic response of maize roots through modification of the arrangement of microtubules. This might reduce the distribution of IAA in the upper and lower sides of the elongation zone and increase ethylene production, thereby inhibiting growth and gravitropic responses.

Differential expression of a novel gene EaF82a in green and yellow sectors of variegated Epipremnum aureum leaves is related to uneven distribution of auxin.

EaF82, a gene identified in previous studies of the variegated plant Epipremnum aureum, exhibited a unique expression pattern with greater transcript abundance in yellow sectors than green sectors of variegated leaves, but lower abundance in regenerated pale yellow plants than in green plants derived from leaf tissue culture. Studies of its full-length cDNA and promoter region revealed two members with only the EaF82a expressed. Immunoblotting confirmed that EaF82a encodes a 12 kDa protein and its accumulation consistent with its gene expression patterns in different color tissues. Transient expression of EaF82a-sGFP fusion proteins in protoplasts showed that EaF82a seems to be present in the cytosol as unidentified spots. Sequence motif search reveals a potential auxin responsive element in promoter region. Using transgenic Arabidopsis seedlings carrying EaF82a promoter driving the bacterial uidA (GUS) gene, an increased GUS activity was observed when IAA (indole-3-acetic acid) concentration was elevated. In E. aureum, EaF82a is more abundant at the site where axillary buds emerge and at the lower side of bending nodes where more IAA accumulates relative to the upper side. The measurement of endogenous IAA levels in different color tissues revealed the same pattern of IAA distribution as that of EaF82a expression, further supporting that EaF82a is an IAA responsive gene. EaF82a expression in etiolated transgenic Arabidopsis seedlings responded to IAA under the influence of light suggesting a microenvironment of uneven light condition affects the EaF82a transcript levels and protein accumulation in variegated leaves.

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-013-1907-z) contains supplementary material, which is available to authorized users.

Immunolocalization of IAA and ABA in roots and needles of radiata pine (Pinus radiata) during drought and rewatering

Anatomical, physiological and phytohormonal changes involved in drought tolerance were examined in different Pinus radiata D. Don breeds subjected to soil drying and rewatering. Breeds with the smallest stomatal chamber size had the lowest transpiration rate and the highest intrinsic water-use efficiency. Xylem cell size was positively correlated with leaf hydraulic conductance and needle indole-3-acetic acid (IAA) concentrations, whereas transpiration rate was negatively correlated with needle abscisic acid (ABA) levels. Since these two phytohormones seem important in regulating the P. radiata drought response, they were simultaneously immunolocalized in roots and needles of the most tolerant breed (P. radiata var. radiata × var. cedrosensis) during two sequential drought cycles and after rewatering. During drought, IAA was unequally distributed into the pointed area of the needle cross-section and mainly located in mesophyll and vascular tissue cells of needles, possibly inducing needle epinasty, whereas ABA was principally located in guard cells, presumably to elicit stomata closure. In the roots, at the end of the first drought cycle, while strong IAA accumulation was observed in the cortex, ABA levels decreased probably due to translocation to the leaves. Rewatering modified the distribution of both IAA and ABA in the needles, causing an accumulation principally in vascular tissue, with residual concentrations in mesophyll, likely favouring the acclimatization of the plants for further drought cycles. Contrarily, in the roots IAA and ABA were located in the exodermis, a natural barrier that regulates the phytohormone translocation to other plant tissues and hormone losses to the soil solution after rewatering. These results confirm that immunolocalization is an efficient tool to understand the translocation of IAA and ABA in plants subjected to different water stress situations, and clarify their role in regulating physiological responses such as stomata closure and epinasty in needles and root development.

Indole-3-acetic acid accumulation during poplar rhizogenesis revealed by immunohistochemistry

Poplar hybrid 741 [Populus alba × (P. davidiana + P. simonii) × P. tomentosa] leaves were rooted within 8 d when cultured in vitro on 1/2 Murashige and Skoog (MS) medium. The spatial distribution of endogenous indole-3-acetic acid (IAA) in the rhizogenesis was investigated, using an immunohistochemical approach. In addition, the effect of 2,3,5-triiodobenzoic acid (TIBA) on IAA distribution was also analyzed. The results showed that a strong IAA signal was detected in the vascular bundles of the basal regions of the petioles 3 d after root induction. Furthermore, the signal in vascular bundles of the basal regions of the petioles was stronger than that of the middle regions of the petioles. Application of TIBA on lamina delayed both the accumulation of IAA in the vascular bundles and rhizogenesis. These data indicate that an endogenous IAA rise in vascular bundles is among the first signals leading to the rhizogenesis, and that it results from transportation of the hormone from the lamina of the leaf to the base of the petiole, rather than by in situ IAA generation.

Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips

To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.

Feeding by a gall-inducing caterpillar species alters levels of indole-3-acetic and abscisic acid in Solidago altissima (Asteraceae) stems

Various plant antagonists appear to alter phytohormone levels for their own benefit. Among insects, gall-inducing species appear to influence phytohormones and it is widely believed that they alter levels of indole-3-acetic acid (IAA) to help produce their galls, but evidence exists for only a limited number of species. To further explore the role of phytohormones in gall formation, we measured levels of IAA and abscisic acid (ABA), a hormone involved in plant defenses and that can influence IAA, in tissues of control stems of Solidagoaltissima (Asteraceae) and those galled by Gnorimoschema gallaesolidaginis (Gelechiidae). This gall-inducing caterpillar species significantly altered the distribution of IAA in galls and the larvae themselves contained high concentrations of IAA. In contrast, the generalist caterpillar Heliothis virescens (Noctuidae) neither altered IAA nor accumulated significant concentrations of IAA, suggesting that G. gallaesolidaginis may have a distinctive influence over IAA. The gall-inducing caterpillars, particularly younger larvae, also contained high levels of ABA but did not increase levels of ABA, which is induced by herbivory of H. virescens. Because G. gallaesolidaginis also does not increase levels of other defense-related hormones, avoiding generalized plant defenses may facilitate gall induction and formation.

The responsiveness of the IAA2 promoter to IAA and IBA is differentially affected in Arabidopsis roots and shoots by flavonoids

The structural features of flavonoids which are involved in the modulation of auxin distribution in Arabidopsis thaliana were evaluated. An auxin-inducible promoter IAA2 fused to a reporter gene (GUS) was used to monitor the tissue responsiveness to auxins. The following aspects were investigated: 1) the influence of flavonoids (quercetin, naringenin, kaempferol, myricetin and isorhamnetin) on the distribution of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in roots and leaves, 2) differences in flavonoid uptake into roots and shoots depending on flavonoid concentration in the medium, and 3) influence of structurally different flavonoids on the gravitropic response and growth of roots. The same flavonoids differently affected IAA and IBA distribution in leaves and roots. There were several structural requirements for the flavonoids which resulted in the changes of auxin response/distribution. Great differences between the ability of shoots and roots to take up quercetin were showed. Also, flavonoids influenced gravitropism and root growth of Arabidopsis seedlings in a structure-dependent manner.

Auxin polar transport is essential for the development of zygote and embryo in Nicotiana tabacum L. and correlated with ABP1 and PM H+-ATPase activities.

Auxin is an important plant growth regulator, and plays a key role in apical–basal axis formation and embryo differentiation, but the mechanism remains unclear. The level of indole-3-acetic acid (IAA) during zygote and embryo development of Nicotiana tabacum L. is investigated here using the techniques of GC-SIM-MS analysis, immunolocalization, and the GUS activity assay of DR5::GUS transgenic plants. The distribution of ABP1 and PM H+-ATPase was also detected by immunolocalization, and this is the first time that integral information has been obtained about their distribution in the zygote and in embryo development. The results showed an increase in IAA content in ovules and the polar distribution of IAA, ABP1, and PM H+-ATPase in the zygote and embryo, specifically in the top and basal parts of the embryo proper (EP) during proembryo development. For information about the regulation mechanism of auxin, an auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid) and exogenous IAA were, respectively, added to the medium for the culture of ovules at the zygote and early proembryo stages. Treatment with a suitable IAA concentration promoted zygote division and embryo differentiation, while TIBA treatment obviously suppressed these processes and caused the formation of abnormal embryos. The distribution patterns of IAA, ABP1, and PM H+-ATPase were also disturbed in the abnormal embryos. These results indicate that the polar distribution and transport of IAA begins at the zygote stage, and affects zygote division and embryo differentiation in tobacco. Moreover, ABP1 and PM H+-ATPase may play roles in zygote and embryo development and may also be involved in IAA signalling transduction.

Asymmetric distribution of auxin correlates with gravitropism and phototropism but not with autostraightening (autotropism) in pea epicotyls.

The relationships between the distribution of the native auxin indole-3-acetic acid (IAA) and tropisms in the epicotyl of red light-grown pea (Pisum sativum L.) seedlings have been investigated. The distribution measurement was made in a defined zone of the third internode, using (3)H-IAA applied from the plumule as a tracer. The tropisms investigated were gravitropism, pulse-induced phototropism, and time-dependent phototropism. The investigation was extended to the phase of autostraightening (autotropism) that followed gravitropic curvature. It was found that IAA is asymmetrically distributed between the two halves of the zone, with a greater IAA level occurring on the convex side, at early stages of gravitropic and phototropic curvatures. This asymmetry was found in epidermal peels and, except for one case (pulse-induced phototropism), no asymmetry was detected in whole tissues. It was concluded, in support of earlier results, that auxin asymmetry mediates gravitropism and phototropism and that the epidermis or peripheral cell layers play an important role in the establishment of auxin asymmetry in pea epicotyls. During autostraightening, which results from a reversal of growth asymmetry, the extent of IAA asymmetry was reduced, but its direction was not reversed. This result demonstrated that autostraightening is not regulated through auxin distribution. In this study, the growth on either side of the investigated zone was also measured. In some cases, the measured IAA distribution could not adequately explain the local growth rate, necessitating further detailed investigation.

Defects in root development and gravity response in the aem1 mutant of rice are associated with reduced auxin efflux

The phytohormone auxin is involved in the regulation of a variety of developmental processes. In this report, we describe how the processes of lateral root and root hair formations and root gravity response in rice are controlled by auxin. We use a rice mutant aem1 (auxin efflux mutant) because the mutant is defective in these characters. The aem1 line was originally isolated as a short lateral root mutant, but we found that the mutant has a defect in auxin efflux in roots. The acropetal and basipetal indole-3-acetic acid (IAA) transports were reduced in aem1 roots compared to wild type (WT). Furthermore, gravitropic bending as well as efflux of radioactive IAA was impaired in the mutant roots. We also propose a unique distribution of endogenous IAA in aem1 roots. An immunoassay revealed a 4-fold-endogenous IAA content in the aem1 roots compared to WT, and the application of IAA to the shoot of WT seedlings mimicked the short lateral root phenotype of aem1, suggesting that the high content of IAA in aem1 roots impaired the elongation of lateral roots. However, the high level of IAA in aem1 roots contradicts the auxin requirement for root hair formation in the epidermis of mutant roots. Since the reduced development in root hairs of aem1 roots was rescued by exogenous auxin, the auxin level in the epidermis is likely to be sub-optimum in aem1 roots. This discrepancy can be solved by the ideas that IAA level is higher in the stele and lower in the epidermis of aem1 roots compared to WT and that the unique distribution of IAA in aem1 roots is induced by the defect in auxin efflux. All these results suggest that AEM1 may encode a component of auxin efflux carrier in rice and that the defects in lateral roots, root hair formation and root gravity response in aem1 mutant are due to the altered auxin efflux in roots.

The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light.

We examined whether flavonoids act as endogenous auxin transport regulators during gravity vector and light intensity changes in Arabidopsis thaliana roots. Flavonoid deficient transparent testa4 [tt4(2YY6)] seedlings had elevated root basipetal auxin transport compared with the wild type, consistent with the absence of a negative auxin transport regulator. The tt4(2YY6) roots had delayed gravitropism that was chemically complemented with a flavonoid intermediate. Flavonoid accumulation was found in wild-type columella cells, the site of gravity perception, and in epidermal and cortical cells, the site of differential growth, but flavonoid accumulation was absent in tt4(2YY6) roots. Flavonoid accumulation was higher in gravity-stimulated root tips as compared with vertical controls, with maximum differences coinciding with the timing of gravitropic bending, and was located in epidermal cells. Exogenous indole-3-acetic acid (IAA) also elevated flavonoid accumulation, suggesting that flavonoid changes in response to gravity might be partly as a result of changing IAA distribution. Acropetal IAA transport was also elevated in roots of tt4(2YY6). Flavonoid synthesis was repressed in the dark, as were differences in root acropetal transport in tt4(2YY6). These results are consistent with light- and gravity-induced flavonoid stimulation that alters auxin transport in roots and dependent physiological processes, including gravitropic bending and root development.

Patterns of auxin distribution during gravitational induction of reaction wood in poplar and pine.

Gravistimulation of tree stems affects wood development by unilaterally inducing wood with modified properties, called reaction wood. Commonly, it also stimulates cambial growth on the reaction wood side. Numerous experiments involving applications of indole-3-acetic acid (IAA) or IAA-transport inhibitors have suggested that reaction wood is induced by a redistribution of IAA around the stem. However, in planta proof for this model is lacking. Therefore, we have mapped endogenous IAA distribution across the cambial region tissues in both aspen (Populus tremula, denoted poplar) and Scots pine (Pinus sylvestris) trees forming reaction wood, using tangential cryosectioning combined with sensitive gas chromatography-mass spectrometry analysis. Moreover, we have documented the kinetics of IAA during reaction wood induction in these species. Our analysis of endogenous IAA demonstrates that reaction wood is formed without any obvious alterations in IAA balance. This is in contrast to gravitropic responses in roots and shoots where a redistribution of IAA has been documented. It is also of interest that cambial growth on the tension wood side was stimulated without an increase in IAA. Taken together, our results suggest a role for signals other than IAA in the reaction wood response, or that the gravitational stimulus interacts with the IAA signal transduction pathway.

Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid.

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.

Immunohistochemical localization of IAA in developing strawberry fruit

SummaryThe immunohistochemical localization of indole-3-acetic acid (IAA) was carried out by using an anti-indole-3-acetic acid (IAA) monoclonal antibody in the achenes and the receptacles of developing field-grown strawberries in May. The distribution of free-IAA in the achenes and the receptacles and their dynamic variations were established.A small IAA signal was detected in the achenes and the receptacles of strawberry at anthesis. In the receptacles, the IAA signal gradually increased at the immature green (IG) stage. From day 7 after bloom to the inception (IC) stage, the IAA signal was localized throughout the vascular bundles of the receptacle tissues, and the signal strengthened at the early phase of the mature green (MG) stage and at the inception (IC) stage. However, at the firm ripe (FR) stage, the signal was hardly detected in the receptacles. In the achenes, the IAA signal gradually increased from the late phase of the immature green (IG) stage to the late phase of the mature green (MG) stage and was mainly localized in developing embryos. Little signal could be detected after inception (IC) stage in the achenes. The results showed that IAA was especially localized in the phloem of the receptacles and was mainly localized in developing embryos in the achenes, which gave apparent evidence for IAA distribution in fleshy fruits. Moreover, in our research, there was still a strong IAA signal in the receptacles of strawberry at the IC stage, which differed significantly from data in some other reports.

Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos

Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are known to have a crucial role in the induction of somatic embryogenesis in many other culture and regeneration systems. The fact that in the sunflower system no exogenous auxin is required for the induction of somatic embryos makes this system very suitable for the study of the internal dynamics of IAA. We used an immuno-cytochemical approach to visualize IAA distribution within the explants before, during and after the induction phase. IAA accumulated transiently throughout cultured embryos during the induction phase. The detected signal was not uniform but certain tissues, such as the root cap and the root meristem, accumulated IAA in a more pronounced manner. IAA accumulation was not restricted to the reactive zone but the kinetics of endogenous variations strikingly mimic the pulse of IAA that is usually provoked by exogenous IAA application. The direct evidence presented here indicates that an endogenous auxin pulse is indeed among the first signals leading to the induction of somatic embryogenesis.

AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling.

Arabidopsis root architecture is regulated by shoot-derived signals such as nitrate and auxin. We report that mutations in the putative auxin influx carrier AUX1 modify root architecture as a result of the disruption in hormone transport between indole-3-acetic acid (IAA) source and sink tissues. Gas chromatography-selected reaction monitoring-mass spectrometry measurements revealed that the aux1 mutant exhibited altered IAA distribution in young leaf and root tissues, the major IAA source and sink organs, respectively, in the developing seedling. Expression studies using the auxin-inducible reporter IAA2::uidA revealed that AUX1 facilitates IAA loading into the leaf vascular transport system. AUX1 also facilitates IAA unloading in the primary root apex and developing lateral root primordium. Exogenous application of the synthetic auxin 1-naphthylacetic acid is able to rescue the aux1 lateral root phenotype, implying that root auxin levels are suboptimal for lateral root primordium initiation in the mutant.