[Show abstract][Hide abstract] ABSTRACT: The initial reaction in the pathway leading to the production of indole-3-acetic acid (IAA) in plants is the reaction between chorismate and glutamine to produce anthranilate, catalysed by the enzyme anthranilate synthase (ASA; EC 22.214.171.124). Compared with non-transgenic controls, leaves of transgenic poplar with ectopic expression of the pine cytosolic glutamine synthetase (GS1a; EC 126.96.36.199) produced significantly greater glutamine and significantly enhanced ASA α-subunit (ASA1) transcript and protein (approximately 130% and 120% higher than in the untransformed controls, respectively). Similarly, tobacco leaves fed with 30 mM glutamine and 2 mM chorismate showed enhanced ASA1 transcript and protein (175% and 90% higher than controls, respectively). Furthermore, free IAA was significantly elevated both in leaves of GS1a transgenic poplar and in tobacco leaves fed with 30 mM glutamine and 2 mM chorismate. These results indicated that enhanced cellular glutamine may account for the enhanced growth in GS transgenic poplars through the regulation of auxin biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: This study examined the role of endogenous abscisic acid (ABA) and jasmonic acid (JA) in indirect somatic embryogenesis of
Medicago sativa L. A multiplex GC-MS/MS technique allowed quantitative single-run analyses of ABA, JA, 12-oxophytodienoic acid (OPDA) and
indole-3-acetic acid (IAA). The preparation of initial explants led to a strong accumulation of ABA, JA and OPDA but not of
IAA. Substantially higher levels of ABA, JA and OPDA were detected in developing somatic embryos than in callus or embryogenic
suspension. Fluridone (FLD) decreased ABA, JA and OPDA levels. Indoprofen (INP) appeared to be a specific inhibitor of octadecanoid
biosynthesis. Somatic embryo production and development were negatively affected by FLD or INP. Only INP (0.5μM) applied
during proliferation phase increased the number of cotyledonary embryos. The results strongly indicate the involvement of
ABA and JA in somatic embryogenesis of M. sativa. Surprisingly, low IAA contents in comparison to stress-related compounds (ABA, JA and OPDA) were detected in explants, embryogenic
tissues and somatic embryos.
[Show abstract][Hide abstract] ABSTRACT: Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.
[Show abstract][Hide abstract] ABSTRACT: The plant photoreceptor phytochrome is organised in a small gene family with phytochrome A (phyA) being unique, because it is specifically degraded upon activation by light. This so called photodestruction is thought to be important for dynamic aspects of sensing such as measuring day length or shading by competitors. Signal-triggered proteolytic degradation has emerged as central element of signal crosstalk in plants during recent years, but many of the molecular players are still unknown. We therefore analyzed a jasmonate (JA)-deficient rice mutant, hebiba, that in several aspects resembles a mutant affected in photomorphogenesis. In this mutant, the photodestruction of phyA is delayed as shown by in vivo spectroscopy and Western blot analysis. Application of methyl-JA (MeJA) can rescue the delayed phyA photodestruction in the mutant in a time- and dose-dependent manner. Light regulation of phyA transcripts thought to be under control of stable phytochrome B (phyB) is still functional. The delayed photodestruction is accompanied by an elevated sensitivity of phytochrome-dependent growth responses to red and far-red light.
[Show abstract][Hide abstract] ABSTRACT: Photosynthesis and biomass production of plants are controlled by the water status of the soil. Upon soil drying, plants can reduce water consumption by minimizing transpiration through stomata, the closable pores of the leaf. The phytohormone abscisic acid (ABA) mediates stomatal closure, and is the assigned signal for communicating water deficit from the root to the shoot. However, our study does not support ABA as the proposed long-distance signal. The shoot response to limited soil water supply is not affected by the capacity to generate ABA in the root; however, the response does require ABA biosynthesis and signalling in the shoot. Soil water stress elicits a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure. Attenuation of the hydraulic response in various plants prevented long-distance signalling of water stress, consistent with root-to-shoot communication by a hydraulic signal.
The Plant Journal 11/2007; 52(1):167-74. DOI:10.1111/j.1365-313X.2007.03234.x · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plants respond to situations requiring the initiation of inducible defence reactions with a complex array of signalling events that ultimately result in the activation of sets of defence genes. Among the chemical signals involved in the induction of defence reactions are cyclic oxylipins derived from C18- or C16-unsaturated fatty acids, the octadecanoids and the hexadecanoids. Key to understanding octadecanoid biology are the C18-metabolite 12-oxophytodienoic acid (OPDA) and the C12-compound jasmonic acid which is biosynthetically derived from 12-oxophytodienoic acid. Different octadecanoids likely have different biological functions. The bouquet of signalling compounds, rather than any single compound, is probably decisive for the biological response that results. This means that the processes regulating the pool sizes of different octadecanoids and their distribution within the plant are key to understanding octadecanoid biology. Recent results, including the cloning of several enzymes of the octadecanoid biosynthetic pathway, have provided first insights into these processes and into how the octadecanoid system is linked to other defence-related signalling pathways of the plant cell.
Novartis Foundation Symposium 223 - Insect-Plant Interactions and Induced Plant Defence, 09/2007: pages 191 - 204; , ISBN: 9780470515679
[Show abstract][Hide abstract] ABSTRACT: cyclo-Oxylipin-galactolipids (cGL) are mono- or digalactosyldiglycerides carrying a cyclo-oxylipin in the sn1- and/or sn2-position or esterified to the galactose moiety. These compounds were recently identified in Arabidopsis thaliana. We provide evidence that cGL are mainly, if not exclusively, part of the thylakoid and can be hydrolysed by lipolytic activities associated with photosynthesis-related protein complexes in vitro. Using HPLC/ESI-mass spectrometry, cGL are shown to be restricted in occurrence to the genus Arabidopsis, they do not occur in other plants tested. A. thaliana cGL are rapidly and transiently formed upon wounding with characteristic changes in composition of the cGL-fraction. While the biological role of cGL is not understood, the genus Arabidopsis may present a model-case of chemical evolution of a novel class of regulatory molecules.
[Show abstract][Hide abstract] ABSTRACT: Most studies on the function of tocopherols in plants have focused on their photo-protective and antioxidant properties, and it has been recently suggested, though not yet demonstrated, that they may also play a role in cellular signaling. By using vte1 mutants of Arabidopsis thaliana, with an insertion in the promoter region of the gene encoding tocopherol cyclase, we demonstrate here for the first time that tocopherol deficiency may alter endogenous phytohormone levels in plants, thereby reducing plant growth and triggering anthocyanin accumulation in leaves. In plants grown under a combination of high light and low temperature conditions to induce anthocyanin accumulation, we evaluated age-dependent changes in tocopherols, indicators of photo-oxidative stress, phytohormone levels, plant growth and anthocyanin levels in wild type and vte1 mutants. These mutants showed lower tocopherol levels, reduced growth and enhanced anthocyanin accumulation compared with the wild type, while both the maximum and relative efficiencies of PSII, chlorophylls, and carotenoids were not significantly altered. Analyses of phytohormone levels revealed that reduced growth and enhanced anthocyanin accumulation in tocopherol-deficient plants were preceded by increased jasmonic acid levels. This is the first study suggesting a direct effect of tocopherols on phytohormones levels in plants and will undoubtedly help us to better understand the multiple functions tocopherols play in plants, as well as the cellular signaling mechanisms responsible for the phenotypes thus far described in tocopherol-deficient plants.
[Show abstract][Hide abstract] ABSTRACT: The pathway of jasmonic acid (JA) biosynthesis was established in the 1980s by Vick and Zimmerman but, until now, the preparative biosynthesis of the jasmonic acid precursors 12-oxo-phytodienoic acid (OPDA) and 3-oxo-2-[2'-pentenyl]-cyclopentan-1-octanoic acid (OPC-8:0) in their endogenous and biologically relevant cis(+)-configuration was only possible in small amounts and had to put up with high costs. This was mainly due to the lack of high amounts of pure and enzymatically active allene oxide cyclase (AOC), which is a key enzyme in the biosynthesis of jasmonates in that it releases, in a coupled reaction with allene oxide synthase (AOS), the first cyclic and biological active metabolite - OPDA. We describe here the expression and purification of AOS and AOC and their subsequent coupling to solid matrices to produce an enantioselective, reusable bioreactor for octadecanoid production. With the method described here it is possible to produce optically pure enantiomers of octadecanoids in high amounts in a cost- and time-efficient manner. Furthermore, it could be demonstrated that a physical interaction of AOS and AOC, hitherto postulated to be required for substrate channeling from AOS to AOC, is not necessary for the in vitro cyclization of the unstable epoxide generated by the AOS reaction.
[Show abstract][Hide abstract] ABSTRACT: Amidase 1 (AMI1) from Arabidopsis thaliana converts indole-3-acetamide (IAM), into indole-3-acetic acid (IAA). AMI1 is part of a small isogene family comprising seven members in A. thaliana encoding proteins which share a conserved glycine- and serine-rich amidase-signature. One member of this family has been characterized as an N-acylethanolamine-cleaving fatty acid amidohydrolase (FAAH) and two other members are part of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) or mitochondria (Tom complex) and presumably lack enzymatic activity. Among the hitherto characterized proteins of this family, AMI1 is the only member with indole-3-acetamide hydrolase activity, and IAM is the preferred substrate while N-acylethanolamines and oleamide are not hydrolyzed significantly, thus suggesting a role of AMI1 in auxin biosynthesis. Whereas the enzymatic function of AMI1 has been determined in vitro, the subcellular localization of the enzyme remained unclear. By using different GFP-fusion constructs and an A. thaliana transient expression system, we show a cytoplasmic localization of AMI1. In addition, RT-PCR and anti-amidase antisera were used to examine tissue specific expression of AMI1 at the transcriptional and translational level, respectively. AMI1-expression is strongest in places of highest IAA content in the plant. Thus, it is concluded that AMI1 may be involved in de novo IAA synthesis in A. thaliana.
[Show abstract][Hide abstract] ABSTRACT: To investigate the physiological basis of salt adaptation in poplar, we compared the effect of salt stress on wood anatomy and auxin physiology of the salt-resistant Populus euphratica and salt-sensitive Populus x canescens. Both poplar species showed decreases in vessel lumina associated with increases in wall strength in response to salt, however, in P. euphratica at three-fold higher salt concentrations than in P. x canescens. The predicted hydraulic conductivity of the wood formed under salt stress decreased in P. x canescens, while in P. euphratica, no significant effects of salt on conductivity and transpiration were observed. The concentration of free indole-3-acetic acid (IAA) decreased under salt stress in the xylem of both poplar species, but to a larger extent in P. x canescens than in P. euphratica. Only salt-treated P. euphratica exhibited an increase in IAA-conjugates in the xylem. Genes homologous to the auxin-amidohydrolase ILL3 were isolated from the xylems of P. euphratica and P. x canescens. For functional analysis, the auxin-amidohydrolase from P. x canescens was overexpressed in Arabidopsis. Transgenic Arabidopsis plants were more resistant to salt stress than the wild-type plants. Increased sensitivity of the transgenic Arabidopsis to IAA-Leu showed that the encoded hydrolase used IAA-Leu as a substrate. These results suggest that poplar can use IAA-amidoconjugates in the stem as a source of auxin to balance the effects of salt stress on auxin physiology.
[Show abstract][Hide abstract] ABSTRACT: NtWIF is a transcription factor activated upon phosphorylation by wound-induced protein kinase (WIPK) in tobacco plants. Transgenic tobacco plants overexpressing NtWIF exhibited constitutive accumulation of transcripts for pathogenesis-related genes, PR-1a and PR-2. Salicylic acid levels were 50-fold higher than those in wild-type plants. The levels of jasmonic acid and IAA did not significantly differ, while an increase of ABA upon wounding was delayed by 3 h in the transgenics. When challenged with tobacco mosaic virus, lesions developed faster and were smaller in the transgenic plants. The results suggest that NtWIF is likely to influence salicylic acid biosynthesis, being located downstream of WIPK.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 2-Cis(-)xanthoxin (XA) was linked to bovine serum albumin through a Schiff's base and the adduct stabilized by sodium borohydride reduction. The conjugate (molar coupling ratio: 3 mol XA per mol protein) was highly immunogenic in rabbits. Antisera contained antibodies binding XA with high affinity (Ka= 1.8 × 108M−1). [3H]-XA (2.2 × 1014 Bq mol−1) was synthesized by oxidation of [3H]-XA alcohol with MnO2 and used to set up a radioimmunoassay [RIA, detection limit, 1 pmol; measuring range, 1 to 200 pmol (0.3 to 60 ng) XA]. The sera were also suitable for enzyme-linked-immunosorbent assay (ELISA) using XA-alkaline phosphatase conjugates. The technique was more sensitive [detection limit, 0.1 pmol; measuring range, 0.1 to 50 pmol (0.05 to 15 ng) XA] than the radioimmunoassay, but less precise.
[Show abstract][Hide abstract] ABSTRACT: Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K+ -stimulated, mg2+-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, Km for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). Vmax -480 nmol (mg protein)1 min1 (whole leaf) and 510 nmol (mg protein)1 min1 (epidermis), I50 (Na3,VO4) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO3(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca2+ inhibited the ATPase [I50, C2+: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca2+ inhibited the ATPase [I50, C2+ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.
[Show abstract][Hide abstract] ABSTRACT: A solid-phase enzyme-immunoassay for abscisic acid and abscisic acid conjugates in fmol amounts (detection limit ca 50–60 fmol) has been developed. Only little procedural effort is required for the assay which can be completed within 6 h. With this method, abscisic acid was detected in a range of heterotrophically growing plant suspension cultures. In addition, abscisic acid-glucosyl ester was identified in a Lonicera prolifera Rehd. tissue culture. In this tissue, the level of free abscisic acid reaches ca 2 × 10−6M in the late log-phase of growth.
[Show abstract][Hide abstract] ABSTRACT: Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U3+U3′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven 45Ca2+ transport [? 15 pkat (mg protein)−1]. Ca2+ uptake into the vesicles had a pH optimum of 7.2 and apparent Km values for Ca2+ of 4.4 μM and for Mg-ATP of 300 μM. Ca2+ uptake, K+, Mg2+-ATPase (EC 188.8.131.52) activity as well as glucan synthase II (EC 184.108.40.206) activity were all maximal at the same equilibrium density (1.17 g cm−3) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca2+ transport into the vesicles, excluding a Ca2+/H+ exchange driven by a proton gradient. ATP-dependent Ca2+ uptake was inhibited by erythrosin B (I50= 0.1 μM), ruthenium red (I50= 30 μM), La3+ (I50= 10 μM) and vanadate (I50= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I50= 70 μM) and W-7 (I50= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca2+ efflux from the vesicles rather than inhibit Ca2+ uptake. Our results demonstrate the presence of a Ca2+-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca2+ out of the cell. Its high affinity for Ca2+ makes it a likely component involved in adjusting low cytoplasmic Ca2+ levels. No indications for a secondary active Ca2+/H+ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca2+-ATPase from the H+-translocating K+. Mg2+-ATPase in C. communis plasma membranes.
[Show abstract][Hide abstract] ABSTRACT: The distribution of immunoassayable xanthoxin (XA), abscisic acid (ABA) and indole-3-acetic acid (IAA) in all parts of sunflower (Helianthus annuus L.) seedlings was determined. During the course of phototropic curvature, including the lag phase (5 min), the distribution of these growth regulators was analyzed in the illuminated and shaded side of the hypocotyl, as well as in the peripheral and central tissues. All three growth regulators showed no detectable asymmetries between the illuminated and shaded hypocotyl halves during the lag phase and early phototropic curvature. Also, no indication for an exchange of XA, ABA or IAA between the peripheral and central tissues was observed. Partial removal of the peripheral cell layers revealed that changes in the growth properties of this tissue, preferentially at the illuminated side of the hypocotyl, are required for the phototropic reaction. Complete removal of the peripheral cell layers abolishes the phototropic response. In dark-incubated, green sunflower seedlings, the loss of sensitivity to phototropic stimulation is correlated with decreasing levels of IAA immunoreactivity, whereas no changes in the levels of ABA- and XA immunoreactivity were recorded. The findings are discussed with respect to the involvement of ABA, XA and IAA in phototropic reactions of green dicotyledonous shoots.
[Show abstract][Hide abstract] ABSTRACT: Among the Agrobacterium T-DNA genes, rolB, rolC, orf13, orf8, lso, 6b and several other genes encode weakly homologous proteins with remarkable effects on plant growth. The 6b oncogene induces tumors and enations. In order to study its properties we have used transgenic tobacco plants that carry a dexamethasone-inducible 6b gene, dex-T-6b. Upon induction, dex-T-6b plants develop a large array of morphological modifications, some of which involve abnormal cell expansion. In the present investigation, dex-T-6b-induced expansion was studied in intact leaves and an in vitro leaf disc system. Although T-6b and indole-3-acetic acid (IAA) both induced expansion and were non-additive, T-6b expression did not increase IAA levels, nor did it induce an IAA-responsive gene. Fusicoccin (FC) is known to stimulate expansion by increasing cell wall plasticity. T-6b- and FC-induced expansion were additive at saturating FC concentrations, indicating that T-6b does not act by a similar mechanism to FC. T-6b expression led to higher leaf osmolality values, in contrast to FC, suggesting that the T-6b gene induces expansion by increasing osmolyte concentrations. Metabolite profiling showed that glucose and fructose played a major role in this increase. We infer that T-6b disrupts the osmoregulatory controls that govern cell expansion during development and wound healing.
The Plant Journal 04/2006; 45(6):1017-27. DOI:10.1111/j.1365-313X.2006.02663.x · 5.97 Impact Factor