Indole-3-butyric acid in Arabidopsis thaliana

Indole-3-butyric acid (IBA) was identified by HPLC and GC-MS as an endogenous compound in plantlets of the crucifer Arabidopsis thaliana (L.) Heynh. A. thaliana was cultivated under sterile conditions as shaking culture in different liquid media with and without supply of hormones. Free and total IBA and indole-3-acetic acid (IAA) were determined at different stages of development during the culture period as well as in culture media of different initial pH values. The results showed that IAA was present in higher concentrations than IBA, but both hormones seemed to show the same behaviour under the different experimental conditions. Differences were found in the mode of conjugation of the two hormones. While IAA was mostly conjugated via amide bonds, the main IBA conjugates were ester bound. The ethylene concentration derived from the seedlings, when they were grown in flasks of different size, seemed not to influence the auxin content in the same cultures.     

Indole-3-butyric acid in plant growth and development

Within the last ten years it has been established by GC-MS thatindole-3-butyric acid (IBA) is an endogenous compound in a variety ofplant species. When applied exogenously, IBA has a variety of differenteffects on plant growth and development, but the compound is stillmainly used for the induction of adventitious roots. Using moleculartechniques, several genes have been isolated that are induced duringadventitious root formation by IBA. The biosynthesis of IBA in maize(Zea mays L.) involves IAA as the direct precursor. Microsomalmembranes from maize are able to convert IAA to IBA using ATP andacetyl-CoA as cofactors. The enzyme catalyzing this reaction wascharacterized from maize seedlings and partially purified. The invitro biosynthesis of IBA seems to be regulated by several externaland internal factors: i) Microsomal membranes from light-grownmaize seedlings directly synthesize IBA, whereas microsomal membranesfrom dark-grown maize plants release an as yet unknown reaction product,which is converted to IBA in a second step. ii) Drought and osmoticstress increase the biosynthesis of IBA maybe via the increaseof endogenous ABA, because application of ABA also results in elevatedlevels of IBA. iii) IBA synthesis is specifically increased byherbicides of the sethoxydim group. iv) IBA and IBA synthesizingactivity are enhanced during the colonization of maize roots with themycorrhizal fungus Glomus intraradices. The role of IBA forcertain developmental processes in plants is discussed and somearguments presented that IBA is per se an auxin and does notact via the conversion to IAA.     

Indole-3-butyric acid synthesis in ecotypes and mutants of Arabidopsis thaliana under different growth conditions

Although IBA is a naturally occurring auxin, its role in plant development is still under debate. In this study a set of Arabidopsis mutants was used to analyze the biosynthesis of IBA in vitro. The mutants chosen for this study can be classified as: (1) involvement in auxin metabolism, transport or synthesis (amt1, aux1, ilr1, nit1, rib1, sur1, trp1-100); (2) other hormones possibly involved in the regulation of IBA synthesis (aba1, aba3, eto2, fae1, hls1, jar1); (3) photomorphogenesis (det1, det2, det3); and (4) root architecture (cob1, cob2, scr1). In addition, two transgenic lines overexpressing the IAA glucose synthase (iaglu) gene from maize were analyzed. The ecotypes No-0 and Wassilewskija showed the highest IBA synthetase activity under control conditions, followed by Columbia, Enkheim and Landsberg erecta. In the mutant lines IBA synthetase activity differed in most cases from the wild type, however no particular pattern of up- or down-regulation, which could be correlated to their possible function, was found. For rib1 mutant seedlings it was tested whether reduced IBA synthetase activity correlates with the endogenous IBA levels. Free IBA differed only depending on the culture conditions, but gave no clear correlation with IBA synthetase activity compared to the wild type. Since drought and osmotic stress as well as abscisic acid (ABA) application enhanced IBA synthesis in maize, it was tested whether IBA synthetase from Arabidopsis is also inducible by drought stress conditions. This was confirmed for the two ecotypes Col and Ler which showed different IBA synthetase activity when cultivated with various degrees of drought stress. IBA synthetase was also determined in photomorphogenic mutants under different light regimes. Induction of IBA synthetase in det1 and det3 plants was found under short day plus a red light pulse or in the dark, respectively. The results are discussed with respect to the functions of the mutated genes.     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.     

Effect of inhibitors of gibberellin biosynthesis on the induction of α-amylase in embryoless caryopses of Hordeum vulgare cv. Himalaya

The induction of -amylase by exogenously supplied gibberellin A₁ (GA₁) and GA₄ in embryoless caryopses of Hordeum vulgare (cv. Himalaya) was determined indirectly by measuring reducing sugars released from the endosperm. The presence of the inhibitors of GA biosynthesis, 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo 1618), Ancymidol, 2-chloroethyl trimethyl ammonium chloride (CCC) or (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl)pentan-3-ol (PP333) did not inhibit -amylase production by either GA₁ or GA₄.Abbreviations Amo-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - CCC 2-chloroethyl trimethyl ammonium chloride - cv. cultivar - GA gibberellin - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - PP333 (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl) pentan-3-01     

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC arginine decarboxylase - DFMA dl--dif-luoromethylarginine - DFMO dl--difluoromethylornithine - MPO N-methylputrescine oxidase - ODC ornithine decarboxylase - PMT putrescine N-methyltransferase We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion.     

Molecular cloning and expression analysis of the mevalonate kinase gene from Arabidopsis thaliana

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5 ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and inflorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5 region of the MVK gene to the -glucuronidase (GUS) reporter gene, indicated that the MVK 5-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions –295 and –194 of the MVK 5-flanking region are crucial for high-level MVK gene expression.     

Facile Preparation of Deuterium-Labeled Standards of Indole-3-Acetic Acid (IAA) and Its Metabolites to Quantitatively Analyze the Disposition of Exogenous IAA in Arabidopsis thaliana

[2′,2′-²H₂]-indole-3-acetic acid ([2′,2′-²H₂]IAA) was prepared in an easy and efficient manner involving base-catalyzed hydrogen/deuterium exchange. 1-O-([2′,2′-²H₂]-indole-3-acetyl)-β-D-glucopyranose, [2′,2′-²H₂]-2-oxoindole-3-acetic acid, and 1-O-([2′,2′-²H₂]-2-oxoindole-3-acetyl)-β-D-glucopyranose were also successfully synthesized from deuterated IAA, and effectively utilized as internal standards in the quantitative analysis of IAA and its metabolites in Arabidopsis thaliana by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The use of this technique shows that these metabolites were accumulated in the roots of Arabidopsis seedlings. Dynamic changes in the metabolites of IAA were observed in response to exogenous IAA, revealing that each metabolic action was regulated differently to contribute to the IAA homeostasis in Arabidopsis.     

Cloning and characterisation of an Arabidopsis thaliana cDNA clone encoding an organellar isoform of serine acetyltransferase

We have cloned an Arabidopsis thaliana cDNA encoding serine acetyltransferase (EC 2.3.1.30) by functional complementation of the Escherichia coli cysE mutant JM15. The cDNA clone Sat-1 conferred serine acetyltransferase activity (with apparent K _m for the two substrates acetyl CoA and L-serine of 0.043 and 3.47 mmol/dm³ respectively) on the cysE mutant. The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids which includes a putative chloroplastic targeting presequence. Northern analysis revealed a single message of 1.5 kb, while Southern hybridisation suggests a small multigene family of related sequences.     

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), -naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^–3 TDZ and either 0.25 or 0.50 mg dm^–3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range of media supplemented with 0.75 mg dm^–3 TDZ and 0.05 mg dm^–3 NAA or 2 mg dm^–3 KIN and 1 mg dm^–3 NAA. Regenerated shoots were excised and rooted in MS medium supplemented with 1 mg dm^–3 NAA. The rooted plantlets were finally transferred to pots.     

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the -glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO₄ were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.     

The Tom Thumb dwarfing gene,Rht3 in wheat,I. Reduced pre-harvest damage to breadmaking quality

Summary The effects of the Tom Thumb dwarfing gene, Rht3, on the quality and quantity of grain -amylase produced during germination and by induction with exogenous gibberellic acid are described. In a season conducive to high sprouting damage the gene reduced -amylase levels in the field by 77%. Selection among random Rht3 genotypes showed that other genetic factors can be combined with the dwarfing gene to further increase sprouting damage resistance.     

Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency.

Summary An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15–20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4–5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and -glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7–8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.     

Regulation of the lysine biosynthesis in Pichia guilliermondii

The regulatory properties of four enzymes (homocitrate synthase, -aminoadipate reductase, saccharopine reductase, saccharopine dehydrogenase) involved in the lysine biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory patterns found in other yeast species. The first enzyme of the pathway, homocitrate synthase, is feedback-inhibited by L-lysine. Some other amino acids (-aminoadipate, glutamate, tryptophan, leucine) and lysine analogues are also inhibitors of one or more enzymes. It is shown that only the synthesis of homocitrate synthase is weakly repressed by L-lysine.     

Molecular cloning,characterization and expression of an elongation factor 1α gene in maize

A cDNA (zmEF1A) and the corresponding genomic clone (zmgEF1A) of a member of the gene family encoding the subunit of translation elongation factor 1 (EF-1) have been isolated from maize. The deduced amino acid sequence is 447 residues long interrupted by one intron. Southern blot analysis reveals that the cloned EF-1 gene is one member out of a family consisting of at least six genes. As shown by northern hybridizations in leaves the mRNA level increases at low temperature whereas time-course experiments over 24 h at 5°C show that in roots the overall mRNA level of EF-1 is transiently decreased. These results indicate that the expression of EF-1 is differently regulated in leaves and roots under cold stress.     

Tissue-specific expression directed by an Arabidopsis thaliana pre-ferredoxin promoter in transgenic tobacco plants

We have isolated and analyzed a pre-ferredoxin gene from Arabidopsis thaliana. This gene encodes a 148 amino acid precursor protein including a chloroplast transit peptide of 52 residues. Southern analysis shows the presence of a single copy of this ferredoxin (Fd) gene in the A. thaliana genome. Its expression is tissue-specific and positively affected by light. Response times, both to dark and light conditions, are remarkably rapid.A chimeric gene consisting of a 1.2 kb Fd promoter fragment fused to the -glucuronidase reporter gene was transferred to tobacco. This fusion gene is expressed in a tissue-specific way; it shows high levels of expression in green leaves, as compared to root tissue.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Lateral root formation in rice (Oryza Sativa L.): differential effects of indole-3-acetic acid and indole-3-butyric acid

Comparative effects of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on lateral root (LR) formation were studied using 2-day-old seedlings of IR8 rice (Oryza sativa L.). Results showed that IBA at all concentrations (0.8–500 nmol/L) increased the number of LRs in the seminal root. However exogenous IAA, failed to increase the number of LRs. On the other hand, both IBA and IAA caused inhibition of seminal root elongation and promotion of LR elongation, but IAA can only reach to the same degree of that of IBA at a more than 20-fold concentration. Exogenous IBA had no effect on endogenous IAA content. We conclude from the results that IBA could act directly as a distinct auxin, promoting LR formation in rice, and that the signal transduction pathway for IBA is at least partially different from that for IAA.     

Purification and characterisation of soluble invertases from leaves of Arabidopsis thaliana

Multiple isoforms of -fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 sizeexclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a K _m for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 °C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.Abbreviations PEG polyethylene glycol - pI isoelectric point - PMSF phenylmethylsulphonyl fluoride We wish to acknowledge the support of the British/Swiss Joint Research Programme and the Sheffield University Research Support Fund. X.T. was in receipt of an Overseas Research Scholarship and a University of Sheffield Research Scholarship. We wish to thank Dr A. Moir for his help in N-terminal amino-acid sequencing.