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.     

Auxin-Induced Epinasty of Tobacco Leaf Tissues (A Nonethylene-Mediated Response)

Interveinal strips (10 x 1.5 mm) excised from growing tobacco (Nicotiana tabacum L. cv Xanthi) leaves curled >300[deg] when incubated for 20 h in 5 to 500 [mu]M [alpha]-naphthalene acetic acid or 50 to 500 [mu]M indole-3-acetic acid. Epinasty was not induced without auxin or by the auxin analog [beta]-naphthalene acetic acid, and less substantial epinasty was induced in midrib and vein segments. Auxin treatment increased the length of both surfaces of strips. Curvature resulted from greater growth on the adaxial side. Epinastic sensitivity of strips to auxin appeared first in the distal third of young leaves (blade 4.5-6.0 cm). In older leaves (8-10 and 12-14 cm), the interveinal tissues throughout were sensitive, whereas in leaves 16- to 18-cm long, sensitivity was reduced in the distal two-thirds. Amino-oxyacetic acid (AOA), an ethylene biosynthesis inhibitor, partially inhibited epinasty at 100 [mu]M. However, a poor correlation between inhibition of ethylene biosynthesis by AOA and its inhibition of curvature and the inability of ethylene to produce epinasty or to reverse the effects of AOA suggests that auxin-induced epinasty is not caused by auxin-induced ethylene production.     

Inhibition of brassinosteroid-induced epinasty in tomato plants by aminooxyacetic acid and Co2+

The inhibitory effects of aminooxyacetic acid (AOA) and cobalt chloride (CoCl₂) on brassinosteroid (BR)-induced epinasty in tomato plants ( Lycopersicon esculentum Mill. cv. Heinz 1350) are evaluated. CoCl₂ dramatically decreases petiole bending and ethylene production as the concentration increases from 50 to 200 μ M. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petiole, instead of accumulating, is reduced and does not change over the concentration range tested. Inhibition of BR-induced epinasty by AOA results from inhibition of ACC synthesis. There are dramatic reductions in petiole bending, ethylene and ACC production as the concentration of AOA is increased from 50 to 200 μ M. Maximum inhibition occurs when the plants are pretreated with the inhibitors. The degree of inhibition increases as the length of pretreatment increases from 1 to 4 h. The response of BR-treated plants to AOA and CoCl₂ is similar to the effect of auxin, indicating the integral relationship between BR and auxin.     

Auxin abolishes inhibitory effects of methylcyclopropen and amino oxyacetic acid on pollen grain germination,pollen tube growth,and the synthesis of ACC in petunia

As established by us earlier, ethylene behaves as a regulator of germination, development, and growth of male gametophyte during the progamic phase of fertilization. However, the mechanisms of the regulation of these processes remain so far unstudied. It is believed that the main factor providing variety of the ethylene responses is its interaction with other phytohormones. According to our working hypothesis, ethylene controls germination of pollen grains (PGs) and growth of pollen tubes (PTs) by interacting with auxin, which, as the available data indicate, is likely a key regulator of plant cell polarization and morphogenesis and one of the factors modulating the biosynthesis of ethylene at the level of ACC-synthase gene expression. In the present work, on germinating in vitro male gametophyte and the pollen-stigma system for petunia (Petunia hybrida L.) effects of phytohormones (ethylene and IAA) and known blockers repressing ethylene reception (1-methylcyclopropene, 1-MCP), the synthesis of ACC (amino oxyacetic acid, AOA) and transport IAA (triyodbenzoynaya acid, TYBA) on PGs germination, PTs growth and the synthesis of ACC were investigated. According to the data obtained, exogenous ethylene and IAA stimulated both PGs germination and PTs growth. 1-MCP and TYBA completely inhibited the first process, whereas IAA abolished the inhibitory action of 1-MCP and AOA on both the above processes. Etrel only partially weakened the inhibitory effect of TYBA. Examination of ACC synthesis modulation with AOA showed that IAA does not affect the level of ACC in germinating in vitro male gametophyte and nonpollinated stigmas, while this phytohormone insignificantly raised the level of ACC and abolished the inhibitory effect of AOA on its synthesis in the pollenstigma system. Pollination of stigmas with the pollen preliminarily treated with 1-MCP led to 2.5-fold decline in both the rate of PT growth and the level of ACC. At the same time, IAA abolished the inhibitory action of 1-MCP recovering the synthesis of ACC and growth of PTs to the control values. All these results, taken together, provide evidence for the interaction of the signal transduction pathways of ethylene and auxin at the level of ACC biosynthesis in the course of germination and growth of petunia male gametophyte during the progamic phase of fertilization.     

Abscisic acid is required for somatic embryo initiation through mediating spatial auxin response in Arabidopsis

Abscisic acid (ABA) regulates many aspects of plant development, including somatic embryo (SE) initiation. However, mechanisms of ABA functions on SE initiation have remained to be investigated. In this study, we examined the endogenous ABA contents of calli in Arabidopsis during the SE inductive process. We further found that the capacity for SE initiation was strongly impaired by treatment of fluridone, a potent inhibitor of ABA biosynthesis, as well as by mutation of ABA biosynthetic gene ABA2, suggesting that ABA is required for SE initiation. Furthermore, treatment of fluridone inhibited local auxin biosynthesis and auxin polar transport in the embryonic calli, resulting in the disturbance of auxin response pattern and the decreased regeneration frequency of SEs. However, application of exogenous ABA in the medium almost recovered patterns of auxin response and SE initiation. Thus, the results suggest that ABA functions on SE initiation through mediating both auxin biosynthesis and polar transport for establishment of auxin response pattern in callus. Our study provides new information for understanding mechanisms of SE initiation.     

The effect of Aminooxyacetic acid on ethylene production induced by methyl jasmonate in tomatoes

Aminooxyacetic acid (AOA), an inhibitor of ACC biosynthesis, applied together with methyl jasmonate (JA-Me) to mature green and light-green tomatoes cv. Venture greatly inhibited ethylene production stimulated by JA-Me, when analyzed in ripe and overripe stages. AOA applied alone did not affect ethylene production in the same conditions of treatment and analysis. It is suggested that after JA-Me treatment of tomatoes the turnover rate of ACC is higher (JA-Me stimulates EFE activity) in comparison to control tissues, and, consequently, AOA inhibited ethylene production stimulated by methyl jasmonate.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.     

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。     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.     

Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.     

Ethylene‐triggered abscisic acid: A principle in plant growth regulation?

The application of auxins to sensitive plant species or their overproduction in transgenic plants stimulates ethylene biosynthesis via induction of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. Recent studies with auxin herbicides and indole-3-acetic acid (IAA) have revealed that auxin-stimulated ethylene triggers an increase in the biosynthesis of abscisic acid (ABA), which then functions as a second messenger, leading to growth inhibition and senescence. This raises the question of whether ethylene-triggered ABA is restricted to the action of auxin herbicides or whether it is a widespread phenomenon in the normal plant growth regulation. Our own results and a reappraisal of the literature indicate that ethylene-induced ABA may, indeed, play a role in natural physiological phenomena, such as root gravireaction and suppression of lateral bud growth in apical dominance. In addition, it would be worthwhile to investigate whether ethylene-triggered ABA is involved in other processes which coincide with a strong stimulation of ethylene biosynthesis, such as growth inhibition induced by cytokinins and senescence elicited under stress conditions.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.     

Regulation of genes associated with auxin, ethylene and ABA pathways by 2,4-dichlorophenoxyacetic acid in Arabidopsis

The chemical 2,4-dichlorophenoxyacetic acid (2,4-D) regulates plant growth and development and mimics auxins in exhibiting a biphasic mode of action. Although gene regulation in response to the natural auxin indole acetic acid (IAA) has been examined, the molecular mode of action of 2,4-D is poorly understood. Data from biochemical studies, (Grossmann (2000) Mode of action of auxin herbicides: a new ending to a long, drawn out story. Trends Plant Sci 5:506–508) proposed that at high concentrations, auxins and auxinic herbicides induced the plant hormones ethylene and abscisic acid (ABA), leading to inhibited plant growth and senescence. Further, in a recent gene expression study (Raghavan et al. (2005) Effect of herbicidal application of 2,4-dichlorophenoxyacetic acid in Arabidopsis. Funct Integr Genomics 5:4–17), we have confirmed that at high concentrations, 2,4-D induced the expression of the gene NCED1, which encodes 9-cis-epoxycarotenoid dioxygenase, a key regulatory enzyme of ABA biosynthesis. To understand the concentration-dependent mode of action of 2,4-D, we further examined the regulation of whole genome of Arabidopsis in response to a range of 2,4-D concentrations from 0.001 to 1.0 mM, using the ATH1-121501 Arabidopsis whole genome microarray developed by Affymetrix. Results of this study indicated that 2,4-D induced the expression of auxin-response genes (IAA1, IAA13, IAA19) at both auxinic and herbicidal levels of application, whereas the TIR1 and ASK1 genes, which are associated with ubiquitin-mediated auxin signalling, were down-regulated in response to low concentrations of 2,4-D application. It was also observed that in response to low concentrations of 2,4-D, ethylene biosynthesis was induced, as suggested by the up-regulation of genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Although genes involved in ethylene biosynthesis were not regulated in response to 0.1 and 1.0 mM 2,4-D, ethylene signalling was induced as indicated by the down-regulation of CTR1 and ERS, both of which play a key role in the ethylene signalling pathway. In response to 1.0 mM 2,4-D, both ABA biosynthesis and signalling were induced, in contrast to the response to lower concentrations of 2,4-D where ABA biosynthesis was suppressed. We present a comprehensive model indicating a molecular mode of action for 2,4-D in Arabidopsis and the effects of this growth regulator on the auxin, ethylene and abscisic acid pathways. Experiment station: Plant Biotechnology Centre, Primary Industries Research Victoria, Department of Primary Industries, La Trobe University, Bundoora, Victoria 3086, and the Victorian Microarray Technology Consortium (VMTC).     

Indole-acetic acid binding proteins in soybean cotyledon

Summary Using the charcoal assay to separate free from bound hormone, the results showed that there is a high binding of IAA to cytosol proteins. Competition experiments were carried out using compounds with different auxin activity (indole-acetic acid, alphanaphtalene acetic acid, indole-butyric acid and phenyl-acetic acid), revealing that specificity of binding exists for those compounds with a molecular configuration appropiate for auxin activity. The protein nature of the indoleacetic acid-binding molecule was demonstrated by the use of enzymes and by its thermolability.Abbreviations IAA indole-acetic acid - NAA alpha-naphtaleneacetic acid - IBA indole-butyric acid - PAA phenyl-acetic acid     

Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root

Cytokinin (CK) influences many aspects of plant growth and development, and its function often involves intricate interactions with other phytohormones such as auxin and ethylene. However, the molecular mechanisms underlying the role of CK and its interactions with other growth regulators are still poorly understood. Here we describe the isolation and characterization of the Arabidopsis CK-induced root curling 1 (ckrc1) mutant. CKRC1 encodes a previously identified tryptophan aminotransferase (TAA1) involved in the indole-3-pyruvic acid (IPA) pathway of indole-3-acetic acid (IAA) biosynthesis. The ckrc1 mutant exhibits a defective root gravitropic response (GR) and an increased resistance to CK in primary root growth. These defects can be rescued by exogenous auxin or IPA. Furthermore, we show that CK up-regulates CKRC1/TAA1 expression but inhibits polar auxin transport in roots in an AHK3/ARR1/12-dependent and ethylene-independent manner. Our results suggest that CK regulates root growth and development not only by down-regulating polar auxin transport, but also by stimulating local auxin biosynthesis.     

Inter-tissue 7

Shedding of oil palm fruit from the spike takes place in twostages. The first, cell separation at the junction of the fruitbase and the pedicel (position 1) is initiated by ethylene orits precursor (ACC) or by treatments that accelerate the production.ofethylene (ABA). Separation is delayed or suppressed by treatmentsthat block ethylene biosynthesis (AOA) or oppose ethylene action(auxin, 2, 4-D). Separation of cells at the fruit base fromthe rudimentary androecium or from the ring of tepals at thepedicel edge is a second stage event that depends upon the achievementof separation at position 1. Abscission cells differentiatedat these secondary positions do not separate in response toethylene or to ethylene enhancing compounds alone. It is concludedthat a chemical stimulus from the separated position 1 providesthe signal that induces the second cell separation process essentialto the completion of fruit shedding. Key words: Oil palm, Elaeis guineensis, fruit abscission, ethylene, cell separation, inter-tissue signalling     

An Integrative Analysis of the Effects of Auxin on Jasmonic Acid Biosynthesis in Arabidopsis thaliana

Auxin and jasmonic acid （JA） are two plant phytohormones that both participate in the regulation of many developmental processes. Jasmonic acid also plays important roles in plant stress response reactions. Although extensive investigations have been undertaken to study the biological functions of auxin and JA, little attention has been paid to the cross-talk between their regulated pathways. In the few available reports examining the effects of auxin on the expression of JA or JA-responsive genes, both synergetic and antagonistic results have been found. To further investigate the relationship between auxin and JA, we adopted an integrative method that combines microarray expression data with pathway information to study the behavior of the JA biosynthesis pathway under auxin treatment. Our results showed an overall downregulation of genes involved in JA biosynthesis, providing the first report of a relationship between auxin and the JA synthesis pathway in Arabidopsis seedlings.     

Morphological and anatomical changes in soybean roots subjected to indole-3-acetic acid and tryptophol: indole compounds present in plant auxin metabolism

The auxin metabolism is practically elucidated, and the compounds that are part of the biosynthesis are well characterized, but the indole-3-ethanol or tryptophol, a molecule that has a regulatory position in the indole-3-acetic acid biosynthesis, still represents a gap in the understanding of this pathway. We examined the hypothesis that tryptophol present the function of plant growth regulation on soybean root development. We evaluated two doses of auxin and two doses of tryptophol (100 e 200 mg L^??1), respectively, beside a control treatment (water), via leaf application, in soybean plants under V1–V2 phonological stages. After 18 days of application, the roots were collected for their volume and area measurement, thereafter small segments (0.5 cm of length), were collected at 1 cm below the root-collar, for anatomical analysis. We observed that the control showed greater area and root volume, but using 200 mg L^??1 auxin and 100 mg L^??1 tryptophol led to a radial increase of roots with significant increases in width radius vascular and cortical parenchyma. These results suggest that the application of both compounds had a potential of modify the vascular and ground tissues in soybean roots, which may be beneficial for the development of plants.