Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth

The distribution and biosynthesis of indole-3-acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg(-1) of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred-fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten-fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue-dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue-specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis.     

Indole-3-methylglucosinolate biosynthesis and metabolism in clubroot diseased plants

lndole-3-methylglucosinolate biosynthesis and metabolism in roots of Brassica napus (swede, cv. Danestone II) infected with Plasmodiophora brassicae Wor. were investigated with a pulse feeding technique developed to infiltrate intact tissue segments with labelled substrates. Infected root tissue metabolized [¹⁴C]-L-tryptophan to indole-3-methylglucosinolate, indole-3-acetonitrile, and some other lipophilic indole compounds. The incorporation of radioactivity into these compounds was significantly enhanced in infected tissue compared with control tissue. A time course study showed a high turnover of indole-3-methylglucosinolate and indole-3-acetonitrile in infected tissue. However, thioglucoside glucohydrolase activity was not changed in infected tissue compared with control tissue. Disc electrophoresis revealed the same isoenzyme in both tissues. Control and infected tissues both rapidly hydrolyzed [¹⁴C]-indole-3-acetonitrile in vivo. The possibility of a disease specific biosynthesis of indole-3-acetic acid from indole-3-methylglucosinolate as the result of a changed compartmentation is discussed.     

Many roads lead to "auxin": of nitrilases, synthases, and amidases

Recent progress in understanding the biosynthesis of the auxin, indole-3-acetic acid (IAA) in Arabidopsis thaliana is reviewed. The current situation is characterized by considerable progress in identifying, at the molecular level and in functional terms, individual reactions of several possible pathways. It is still too early to piece together a complete picture, but it becomes obvious that A. thaliana has multiple pathways of IAA biosynthesis, not all of which may operate at the same time and some only in particular physiological situations. There is growing evidence for the presence of an indoleacetamide pathway to IAA in A. thaliana, hitherto known only from certain plant-associated bacteria, among them the phytopathogen Agrobacterium tumefaciens.     

Action of Rice α-Glucosidase on Maltose and Starch

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3~2.6 times in both enzymes at the concentration of 50 mM of potassium chloride. Such activation was also caused by mono and di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-glucosidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.     

Arabidopsis Seedlings Over-Accumulated Indole-3-acetic Acid in Response to Aminooxyacetic Acid

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.     

Alternative splicing of the auxin biosynthesis gene YUCCA4 determines its subcellular compartmentation

Auxin is a major growth hormone in plants, and recent studies have elucidated many of the molecular mechanisms underlying its action, including transport, perception and signal transduction. However, major gaps remain in our knowledge of auxin biosynthetic control, partly due to the complexity and probable redundancy of multiple pathways that involve the YUCCA family of flavin-dependent mono-oxygenases. This study reveals the differential localization of YUCCA4 alternative splice variants to the endoplasmic reticulum and the cytosol, which depends on tissue-specific splicing. One isoform is restricted to flowers, and is anchored to the cytosolic face of the endoplasmic reticulum membrane via a hydrophobic C-terminal transmembrane domain. The other isoform is present in all tissues and is distributed throughout the cytosol. These findings are consistent with previous observations of yucca4 phenotypes in flowers, and suggest a role for intracellular compartmentation in auxin biosynthesis.     

The role of endogenous auxin in root initiation

This paper describes the process of the formation of adventitious roots. There appears to be good agreement that this consists of four stages, defifferentiation coupled with the formation of a meristematic locus, cell division to form a radially symmetrical cluster of cells, further divisions coupled with organisation into a bilaterally symmetrical meristem and finally growth of cells in the basal part of the meristem which causes its protursion through the epidermis. Evidence for the involvement of auxins in these various stages is reviewed and the extent to which rooting of easy- and hard-to-root species can be accounted for in terms of auxin content discussed. Peaks of IAA occur soon after excision of cuttings in some species and there is some evidence suggesting that this is correlated with changes in peroxidase activity. The possible involvement of cytokinins with auxins is briefly considered.     

Evolutionary patterns in auxin action

This review represents the first effort ever to survey the entire literature on auxin (indole-3-acetic acid, IAA) action in all plants, with special emphasis on the green plant lineage, including charophytes (the green alga group closest to the land plants), bryophytes (the most basal land plants), pteridophytes (vascular non-seed plants), and seed plants. What emerges from this survey is the surprising perspective that the physiological mechanisms for regulating IAA levels and many IAA-mediated responses found in seed plants are also present in charophytes and bryophytes, at least in nascent forms. For example, the available evidence suggests that the apical regions of both charophytes and liverworts synthesize IAA via a tryptophan-independent pathway, with IAA levels being regulated via the balance between the rates of IAA biosynthesis and IAA degradation. The apical regions of all the other land plants utilize the same class of biosynthetic pathway, but they have the potential to utilize IAA conjugation and conjugate hydrolysis reactions to achieve more precise spatial and temporal control of IAA levels. The thallus tips of charophytes exhibit saturable IAA influx and efflux carriers, which are apparently not sensitive to polar IAA transport inhibitors. By contrast, two divisions of bryophyte gametophytes and moss sporophytes are reported to carry out polar IAA transport, but these groups exhibit differing sensitivities to those inhibitors. Although the IAA regulation of charophyte development has received almost no research attention, the bryophytes manifest a wide range of developmental responses, including tropisms, apical dominance, and rhizoid initiation, which are subject to IAA regulation that resembles the hormonal control over corresponding responses in seed plants. In pteridophytes, IAA regulates root initiation and vascular tissue differentiation in a manner also very similar to its effects on those processes in seed plants. Thus, it is concluded that the seed plants did not evolve de novo mechanisms for mediating IAA responses, but have rather modified pre-existing mechanisms already operating in the early land plants. Finally, this paper discusses the encouraging prospects for investigating the molecular evolution of auxin action.     

Roles of YUCCAs in auxin biosynthesis and drought stress responses in plants

Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.¹ The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.     

Chlorophyll-protein levels and degree of thylakoid stacking in radish chloroplasts from high-light, low-light and bentazon-treated plants

Lemna gibba plants were incubated aseptically on medium containing labelled 10^-7 M indole-3-acetic acid (IAA-1-¹⁴C). Most of the radioactivity disappeared from the culture medium during a 24 h light period. A high percentage of the loss was due to photolysis and only a low percentage of the radioactivity was recovered in the plants. Uptake of ¹⁴C by the plants was strongly stimulated by light. The radioactivity taken up by the plants was the sum of photosynthetically taken up ¹⁴CO₂ and ¹⁴C taken up in IAA. Analyses with the indolo-α-pyrone fluorescence method revealed that the free IAA content was almost the same in plants grown in control and in IAA media for 5 h, whereas the amount of IAA which could be liberated by alkaline hydrolysis was doubled by the presence of IAA in the medium.     

Local auxin production: a small contribution to a big field

Auxin is a plant growth regulator involved in diverse fundamental developmental responses. Much is now known about auxin transport, via influx and efflux carriers, and about auxin perception and its role in gene regulation. Many developmental processes are dependent on peaks of auxin concentration and, to date, attention has been directed at the role of polar auxin transport in generating and maintaining auxin gradients. However, surprisingly little attention has focussed on the role and significance of auxin biosynthesis, which should be expected to contribute to active auxin pools. Recent reports on the function of the YUCCA flavin monooxygenases and a tryptophan aminotransferase in Arabidopsis have caused us to look again at the importance of local biosynthesis in developmental processes. Many alternative and redundant pathways of auxin synthesis exist in many plants and it is emerging that they may function in response to environmental cues.     

Effect of light in the control of growth by auxin and its inhibitor(s) in the sunflower

Diffusates from the hypocotyl and leaves of sunflower seedlings ( Helianthus annuus L. cv. Mammoth) contain both auxin and inhibitor(s) of auxin-induced growth. The auxin activity has been evaluated with the conventional Avena coleoptile bioassay employing negative curvature, The inhibitor activity has been determined with a newly developed bioassay, measuring the positive curvature response of the Avena coleoptile. This bioassay has been standardized by the response to 2,3,5-triiodo-benzoic acid. Diffusates from plants in darkness have higher auxin activity and lower inhibitor activity than diffusates from plants in light. Irradiation at 730 nm promotes auxin synthesis in leaves, and! irradiation at 660 nm promotes synthesis of the inhibitor.     

Structural characterization and auxin properties of dichlorinated indole-3-acetic acids

The dichlorinated indole-3-acetic acids: 4,5-Cl2-IAA, 4,6-Cl2-IAA, 4,7-Cl2-IAA, 5,6-Cl2-IAA, 5,7-Cl2-IAA and 6,7-Cl2-IAA were synthesized and characterized by X-ray structure analysis to unambiguously identify the substances for bioassays required to establish structure activity relationships of auxins and their analogues. Straight-growth tests were performed on Avena sativa coleoptiles to correlate their auxin activity with molecular properties which could reveal information on the topology of the auxin binding site. Structure/activity correlations revealed that the 5,6-Cl2-IAA molecule, by virtue of its size and shape, fits particularly well into the active site cavity of the receptor protein. The main contact of the substrate or inhibitor in the receptor active site via the carboxylic group determines their orientation in the active site cavity. As a consequence, the 5,6-substituted sites protrude into the widest part of the active site whereas the 7-, 4-, and 5-substituted sites are oriented towards the narrowest part of the active site. These topological parameters are in agreement with the high auxin activity of 5,6-Cl2-IAA and the low activity of 4,7-Cl2-IAA.     

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.     

Brassinolide-induced elongation and auxin

Segments from the hook and subhook zone of the stem of 6-day-old etiolated Pisum sativum L. cv. Victory Freezer seedlings were used to study the relationship between brassinolide and auxin in the promotion of elongation. Minor changes in exogenous indole-3-acetic acid or4-chloroindole-3-acetic acid concentration affected the kinetics markedly and the ethylene generator ethephon overcame brassinolide-induced elongation in an antagonistic interaction. Brassinolide-induced elongation was markedly inhibited by low concentrations of the cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile, and diagnostic concentrations of the antiauxin 2-( p -chlorophenoxy) isobutyric acid did not affect brassinolide-induced elongation. As the characteristics of auxin-induced growth are not displayed in brassinolide-induced elongation of the upper stem segment, it is proposed that brassinolide does not depend on auxin as a mediator in the promotion of elongation of younger tissues but that it can interact in a very complex manner with auxin. In the elongation of more mature tissues, and in bending responses, brassinolide probably accelerates auxin effects. When split, the upper stem segment was unusual in its lack of specific response to growth regulators, and the slight relief of epidermal tension.     

Chitosan and silver nanoparticles are attractive auxin carriers: A comparative study on the adventitious rooting of microcuttings in apple rootstocks

Nanocarriers for encapsulation and sustained release of agrochemicals such as auxins have emerged as an attractive strategy to provide enhanced bioavailability and efficacy for improved crop yields and nutrition quality. Here, a comparative study was conducted on the effectiveness of chitosan-as a biopolymeric nanocarrier- and silver-as a metallic nanocarrier- on in vitro adventitious rooting potential of microcuttings in apple rootstocks, for the first time. Auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) loaded silver (nAg) or chitosan nanoparticles (nChi) were synthesized. Scanning electron microscopy and transmission electron microscopy studies showed the spherical shape of the nanoparticles. The average particle size of IAA-nChi was 167.5 ± 0.1 nm while that of IBA-nChi was 123.2 ± 2.6 nm. The hydrodynamic diameter of the nAg-IAA and nAg-IBA particles were measured as 93.66 ± 5 nm and 71.41 ± 3 nm, respectively. Fourier transform infrared spectroscopy analyses confirmed the encapsulation of IAA or IBA in the chitosan nanoparticles. Meanwhile, the characteristic peaks of IAA or IBA were detected on silver nanoparticles. In-vitro adventitious rooting of microcuttings of Malling Merton 106 (MM 106) was significantly higher both in chitosan and silver nanoparticles loaded with IAA or IBA (91.7%–62.5%) compared to free IAA or IBA applications (50.0%–33.3%), except for 2.0 mg L^–1 IBA (66.7%). However, the application of 2 mg L^–1 IBA and IBA-nChi at all concentrations caused an undesirable large callus development.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.     

生长素合成途径的研究进展

生长素是一类含有一个不饱和芳香族环和一个乙酸侧链的内源激素, 参与植物生长发育的许多过程。植物和一些侵染植物的病原微生物都可以通过改变生长素的合成来调节植株的生长。吲哚-3-乙酸(IAA)是天然植物生长素的主要活性成分。近年来, 随着IAA生物合成过程中一些关键调控基因的克隆和功能分析, 人们对IAA的生物合成途径有了更加深入的认识。IAA的生物合成有依赖色氨酸和非依赖色氨酸两条途径。依据IAA合成的中间产物不同, 依赖色氨酸的生物合成过程通常又划分成4条支路: 吲哚乙醛肟途径、吲哚丙酮酸途径、色胺途径和吲哚乙酰胺途径。该文综述了近几年在IAA生物合成方面取得的新进展。