Volker Magnus

Effect of halogen substitution of indole-3-acetic acid on biological activity in pea fruit

Abstract Auxins (a class of plant growth hormones naturally present in all plants) have been implicated in fruit growth of pea. Pea ( Pisum sativum L.) fruit contain the auxins indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-C1-IAA). Fruits grow poorly and subsequently abscise when seeds are removed two days after anthesis, but 4-C1-IAA can substitute for the seeds in maintaining growth of deseeded fruit (pericarp) in planta . Applications of 4-C1-IAA promoted pericarp growth, the effect increasing with concentration from 1 to 100 μM, but IAA was ineffective in stimulating growth when tested from 0.1 to 100 μM. The effect of the position of the halogen on pericarp growth was examined by assaying the activities of 4-, 5-, 6- and 7-chloro- and fluoro-substituted IAA. The position and type of halogen dramatically affected auxin activity, with the natural product 4-C1-IAA being most effective. Of the other compounds tested, only 5-C1-IAA stimulated pea pericarp elongation, and then only moderately. Fluoro-substituted IAAs did not stimulate pericarp growth, and 4-F-IAA was inhibitory. This study is unique in that it reports the biological activity of 4-C1-IAA and halogen-IAA analogues in tissues of intact plants known to contain 4-C1-IAA. The relative activity of the compounds is discussed in reference to previous reports of auxin activity in other systems, and 4-C1-IAAs possible importance in pea fruit growth.

4-chloroindole-3-acetic and indole-3-acetic acids in Pisum sativum

Abstract Endogenous IAA and 4-chloroindole-3-acetic acid (4-C1-IAA) were analysed in vegetative and reproductive tissues of the garden pea (Pisum sativum) using GC-MS selected ion monitoring in the presence of stable-isotope labelled internal standards. In fruit collected 3–8 days after anthesis (DAA) conjugates of both auxins were more abundant than the free hormones. Auxin levels (ng g−1 fr. wt) in the seeds were higher by 1–2 orders of magnitude than in the pericarps. However, as seeds are small at this stage, the pericarp nevertheless contains a substantial fraction of the overall quantity of IAA and 4-C1-IAA. While, in young fruit tissues, IAA was more abundant than 4-C1-IAA, the opposite was true for seeds at the ‘table-ready’ stage and for roots of nine-day-old seedlings. Our data suggest that both IAA and 4-C1-IAA are required to coordinate the vegetative and reproductive growth of pea plants.

Correlation of structural and physico-chemical parameters with the bioactivity of alkylated derivatives of indole-3-acetic acid, a phytohormone (auxin)

As part of molecular recognition studies on the phytohormone indole-3-acetic acid (IAA) a series of alkylated IAAs has been examined. Phenyl-ring substitution (alkyl = methyl and ethyl) at positions 4-, 6- or 7- as well as pyrrole substitution at the 2-site resulted in the six compounds which are analyzed: 2-Me-IAA, 4-Me-IAA, 6-Me-IAA, 7-Me-IAA, 4-Et-IAA and 6-Et-IAA. The structure-activity relationships investigated include those between the geometrical parameters of the molecular structures determined by X-ray analysis, the growth-promoting activities in the Avena coleoptile straight-growth bioassay and relative lipophilicities calculated from retention times on a reversed-phase HPLC column and from R(F) values in reversed-phase TLC. Lipophilicities are correlated with the moments of inertia, average polarizability, molecular mass, and the van der Waals radii of the ring substituents. The influence of substitution on the electronic properties of the indole ring and its geometry is discussed on the basis of the UV and 1H NMR spectra.

Auxin Amidohydrolases From Brassica rapa Cleave The Alanine Conjugate Of Indolepropionic Acid As A Preferable Substrate: A Biochemical And Modeling Approach

Two auxin amidohydrolases, BrIAR3 and BrILL2, from Chinese cabbage [Brassica rapa L. ssp. pekinensis (Lour.) Hanelt] were produced by heterologous expression in Escherichia coli, purified, and screened for activity towards N-(indol-3-ylacetyl)-L-alanine (IAA-Ala) and the long-chain auxin-amino acid conjugates, N-[3-(indol-3-yl)propionyl]-L-alanine (IPA-Ala) and N-[4-(indol-3-yl)butyryl]-L-alanine (IBA-Ala). IPA-Ala was shown to be the favored substrate of both enzymes, but BrILL2 was approximately 15 times more active than BrIAR3. Both enzymes cleaved IBA-Ala and IAA-Ala to a lesser extent. The enzyme kinetics were measured for BrILL2 and the obtained parameters suggested similar binding affinities for the long-chain auxin-amino acid conjugates (IPA-Ala and IBA-Ala). The velocity of the hydrolyzing reaction decreased in the order IPA-Ala > IBA-Ala > IAA-Ala. In a root growth bioassay, higher growth inhibition was caused by IPA-Ala and IBA-Ala in comparison with IAA-Ala. Neither these conjugates nor the corresponding free auxins affected the expression of the BrILL2 gene. A modeling study revealed several possible modes of IPA-Ala binding to BrILL2. Based on these results, two possible scenarios for substrate hydrolysis are proposed. In one the metal binding water is activated by the carboxyl group of the substrate itself, and in the other by a glutamate residue from the active site of the enzyme.

Molecular properties of 4-substituted indole-3-acetic acids affecting pea pericarp elongation

Pea (Pisum sativum L.) fruit naturally contain the auxins, indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA). However, only 4-Cl-IAA can substitute for the seeds in maintaining pea fruit growth in planta. The importance of the substituent at the 4-position of the indole ring was tested by comparing the molecular properties of 4-X-IAA (X = H, Me, Et, F, or Cl) and their effect on the elongation of pea pericarps in planta. Structure-activity is discussed in terms of structural data derived from X-ray analysis, computed conformations in solution, semiempirical shape and bulk parameters, and experimentally determined lipophilicities and NH-acidities. The size of the 4-substituent, and its lipophilicity are associated with growth promoting activity of pea pericarp, while there was no obvious relationship with electromeric effects.

N-(indol-3-ylacetyl)amino acids as sources of auxin in plant tissue culture

N-(Indol-3-ylacetyl) derivatives (IAA conjugates) of aliphatic amino acids with a two- to six-carbon backbone including α-l-amino acids, (ω-amino acids, and the α,ω-diamino acids ornithine and lysine were prepared, chemically characterized, and tested as sources of auxin in plant tissue culture. Stimulation of unorganized growth in Solanum nigrum L. callus and callus induction and developmental effects in tomato (Lycopersicon esculentum Mill. cv. Marglobe) hypocotyl explants were studied systematically. Relative auxin activities were estimated by comparing physiologically equivalent concentrations, in the optimal and suboptimal range, of the individual IAA conjugates. While the growth-promoting properties of some of the conjugates were species-dependent, those containing straight-chain two- to four-carbon α-l-amino acid moieties were generally up to 100 times more active than those of their five- to six-carbon homologues. Branching of the amino acid backbone at C-β (norvaline vs. valine and norleucine vs. isoleucine) and C-γ (norleucine vs. leucine) had a minor effect, but substitution of H-α by a methyl group (α-amino-l-butyric vs. α-aminoisobutyric acids) almost completely blocked growth-promoting activity. IAA conjugates of ω-amino acids were, in most cases, nearly as active as those of their α-amino-l-isomers. Among the conjugates of α,ω-diamino acids Nδ-(IAA) ornithine was less active than Nε-(IAA)lysine. The activity of Nα-(IAA)lysine was less than for the ε-(IAA) isomer, and that of Nα,Nε-(IAA)2-lysine was different in tomato and Solanum nigrum. The l-alanine and ε-lysine conjugates were also found to be useful for induction and development of Oenothera leaf callus and in tomato cell-suspension culture, two systems which require highly active sources of auxin.

Stable-isotope labeled metabolites of the phytohormone, indole-3-acetic acid

1,3-Dicyclohexylcarbodiimide-mediated condensation of [3a,4,5,6,7,7a-13C6]indole-3-acetic acid with the bis(tert-butyl) esters of L-aspartic or L-glutamic acids, followed by removal of the ester groups by dilute alkali, afforded N-([3a,4,5,6,7,7a-13C6]indol-3-ylacetyl)-L-aspartic and N-([3a,4,5,6,7,7a-13C6]indol-3-ylacetyl)-L-glutamic acids, labeled forms of compounds involved in the regulation of plant growth and development. The corresponding conjugates of (R,S)-2,3-dihydro-2-oxoindole-3-acetic acid, which are likewise of physiological significance, were labeled with 15N in the amino acid moieties and were synthesized via the N-hydroxysuccinimide ester.

Interaction of free indole-3-acetic acid and its amino acid conjugates in tomato hypocotyl cultures

The interaction of free IAA and its amino acid conjugates on growth and development of cultured tomato hypocotyl tissue (Lycopersicon esculentum Mill. cv. Marglobe) was studied. In a nutrient medium containing 10 μmol/L of benzyladenine, free IAA stimulated shoot and root development with little callus proliferation. In contrast, all IAA-amino acid conjugates tested supported mostly callus growth. Simultaneous application of free IAA and its conjugates resulted in the expression of mixed morphogenetic responses (i.e., both vigorous callus growth and organogenesis resulted). Growth kinetics and the effect of temporal exposure of the tissues to the bound and the free auxin suggest that some IAA-amino acid conjugates may specifically influence plant morphogenesis in ways that cannot be easily explained as simply a function of their slow hydrolysis to release free IAA.

Endogenous gibberellin profile during Christmas rose (Helleborus niger L.) flower and fruit development.

Gibberellins (GAs) were identified and quantified during flower and fruit development in the Christmas rose (Helleborus niger L.), a native of southeastern Europe with a long international horticultural tradition. Physiologically, the plant differs from popular model species in two major respects: (1) following anthesis, the initially white or rose perianth (formed in this species by the sepals) turns green and persists until fruit ripening, and (2) the seed is shed with an immature embryo, a miniature endosperm, and a prominent perisperm as the main storage tissue. GA1 and GA4 were identified by full-scan mass spectra as the major bioactive GAs in sepals and fruit. LC-MS/MS system in accord with previously verified protocols also afforded analytical data on 12 precursors and metabolites of GAs. In the fruit, GA4 peaked during rapid pericarp growth and embryo development and GA1 peaked during the subsequent period of rapid nutrient accumulation in the seeds and continued pericarp enlargement. In the sepals, the flux through the GA biosynthetic pathway was highest prior to the light green stage when the photosynthetic system was induced. Unfertilized, depistillated, and deseeded flowers became less green than the seed-bearing controls; chlorophyll accumulation could be restored by applying GA1, GA4, and, less efficiently, GA3 to the deseeded fruit. The sepals of unfertilized and depistillated flowers indeed contained very low levels of GA4 and gradually decreasing levels of GA1. However, the concentrations of their precursors and metabolites were less affected. These data suggest that a signal(s) from the fruit stimulates GA biosynthesis in the sepals resulting in greening. The fruit-derived GAs appear to be mainly involved in pericarp growth and seed development.

Endogenous Auxin Profile in the Christmas Rose (Helleborus niger L.) Flower and Fruit: Free and Amide Conjugated IAA

The reproductive development of the Christmas rose (Helleborus niger L.) is characterized by an uncommon feature in the world of flowering plants: after fertilization the white perianth becomes green and photosynthetically active and persists during fruit development. In the flowers in which fertilization was prevented by emasculation (unfertilized) or entire reproductive organs were removed (depistillated), the elongation of the peduncle was reduced by 20–30%, and vascular development, particularly lignin deposition in sclerenchyma, was arrested. Chlorophyll accumulation in sepals and their photosynthetic efficacy were up to 80% lower in comparison to fertilized flowers. Endogenous auxins were investigated in floral and fruit tissues and their potential roles in these processes are discussed. Analytical data of free indole-3-acetic acid, indole-3-ethanol (IEt), and seven amino acid conjugates were afforded by LC-MS/MS in floral tissues of fertilized as well as unfertilized and depistillated flowers. Among amino acid conjugates, novel ones with Val, Gly, and Phe were identified and quantified in the anthers, and in the fruit during development. Reproductive organs before fertilization followed by developing fruit at post-anthesis were the main source of auxin. Tissues of unfertilized and depistillated flowers accumulated significantly lower levels of auxin. Upon depistillation, auxin content in the peduncle and sepal was decreased to 4 and 45%, respectively, in comparison to fruit-bearing flowers. This study suggests that auxin arising in developing fruit may participate, in part, in the coordination of the Christmas rose peduncle elongation and its vascular development.