Investigations on the Mechanism of the Brassinosteroid Response: VI. Effect of Brassinolide on Gravitropism of Bean Hypocotyls.

Brassinosteroids are steroidal lactones of plant origin that promote growth of a number of plant systems, and particularly the growth induced by auxins. Biologically active brassinosteroids (BR) also promote the growth of gravisensitive hypocotyls of 7-day-old light grown Phaseolus vulgaris when gravistimulated. Brassinolide-mediated promotion of curvature of gravistimulated internodes occurs in the absence of exogenously supplied indole-3-acetic acid (IAA). This is in contrast to the BR-promoted bending of vertically positioned bean hypocotyls, which is dependent upon exogenous IAA. Brassinosteroid treatment increased the graviperception of young internode tissues and the bending of the gravistimulated sections as well as the subsequent reversal of bending after the sections were placed vertically. These results indicate that BR sensitizes bean hypocotyls to gravistimulation and potentiates the action of a growth factor that induces gravitropic growth.

Indole-3-acetic Acid (IAA) and IAA Conjugates Applied to Bean Stem Sections: IAA Content and the Growth Response.

High resolution growth recording techniques and reverse isotope dilution analysis were used to study the relationship between indole-3-acetic acid (IAA) concentration and curvature of excised bean (Phaseolus vulgaris L. cv Bush Burpee Stringless) first internode sections unilaterally treated with hormone. The maximum rate of curvature occurred rapidly (within 25 minutes) and was proportional to the log of the amount of applied IAA recovered in the tissue. The rate of curvature decreased after 30 minutes although little or no lateral migration of applied IAA occurred and tissue levels of IAA increased. The biologic activity of IAA-amino acid conjugates was found to be directly related to the amount of free IAA, resulting from their hydrolysis, which could be recovered from the tissue.

Investigations on the Mechanism of the Brassinosteroid Response: I. Indole-3-acetic Acid Metabolism and Transport.

A brassinosteroid treatment of light-grown first internode sections of Phaseolus vulgaris results in an increased bending response following unilateral indole-3-acetic acid (IAA) application. Reverse isotope dilution analysis shows that this increased response is not due to an increase in the concentration of applied IAA in the tissue or a change in the amount of IAA conjugated. Treatment with the brassinosteroid also does not affect the rate of IAA transport as measured using the agar block method. These results indicate that even though brassinosteroid potentiates auxin action, it does not have a direct effect on IAA uptake, metabolism, or cell to cell transport.

Synthesis of brassinosteroids and relationship of structure to plant growth-promoting effects.

A number of brassinosteroids with and without hydroxyl groups or an alkyl substituent in their side chain were synthesized. The alkyl substituent at C-24 highly influenced the oxidation of the C-22 double bond with osmium tetroxide and, hence the ratio of the 22 beta,23 beta- and 22 alpha,23 alpha-glycolic isomers obtained. Two different bean bioassays used to compare the plant growth-promoting capabilities of these compounds and of brassinolide and its three side chain 22,23-cis-glycolic isomers showed that brassinolide was the most active. The next most active brassinosteroids were generally those with 22 alpha-OH, 23 alpha-OH orientation and a beta-methyl or alpha-ethyl substituent at C-24. Similarly, of the synthetic precursor tetrahydroxy ketones of the brassinosteroids, those with 22 alpha-OH, 23 alpha-OH orientation (like brassinolide) and an alkyl group at C-24 were also the most active in both bioassays. The results indicate stringent structural features are required for a steroid to induce brassin activity. The structural requirements are: a trans A/B ring system (5alpha-hydrogen), a 6-ketone or a 7-oxa-6-ketone system in ring B, cis alpha-oriented hydroxyl groups at C-2 and C-3, cis hydroxy groups at C-22 and C-23 as well as a methyl or ethyl substituent at C-24.

Synthesis and biological activity of brassinolide and its 22 beta, 23 beta-isomer: novel plant growth-promoting steroids.

Brassinolide (2 alpha, 3 alpha, 22 alpha, 23 alpha-tetrahydroxy-24 alpha-methyl -B-homo-7-oxa-5 alpha-cholestan-6-one), a novel plant growth-promoting steroid isolated from rape pollen, and its hitherto unknown 22 beta, 23 beta-isomer were synthesized from a C-24 epimeric 60:40 mixture of 22-dehydroxampesterol (24 alpha-methyl) and brassicasterol (24 beta-methyl) from oysters. The method of synthesis favored the formation of the 22 beta, 23 beta-isomer by better than 4:1. Comparative plant growth-promoting capabilities of brassinolide, both natural and synthetic, and its three side chain cis-glycolic isomers in the bean second internode bioassay showed that the natural and synthetic brassinolides were equally active and caused splitting of the internode at the 0.1 microgram level. The least active was the 22 beta, 23 beta-isomer of brassinolide. The isomers with the 22 alpha, 23 alpha and 24 alpha, and the 22 beta, 23 beta and 24 beta configurations were highly active and were required at about 10 times the concentration of brassinolide to cause the same physiological response. In the bean first internode bioassay, an auxin-induced growth test system which employs isolated bean plant segments, the isomer with 22 beta, 23 beta and 24 beta configuration caused a greater response than brassinolide. Two of the four tetrahydroxy ketones obtained in the synthesis of the isomers were also active in both assays.

DPX 1840-induced Organogenesis in Xanthi-nc Tobacco Plants.

The synthetic growth regulant DPX 1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5, 1-a]isoindol-8-one) induced callus growth and subsequent tissue differentiation on cut surfaces of decapitated Xanthi-nc tobacco plants (Nicotiana tabacum). Callus formation and organogenesis induced by DPX 1840 depended on the presence of leaves. The adventitious meristems developed into either vegetative or flowering shoots. Pedicels that bore single flower buds developed two abscission zones that caused the buds to abscise before anthesis. The various morphological and physiological processes affected by DPX 1840 suggests that this growth regulator affects the endogenous hormonal distribution and/or activity.

Promotion of peroxidase activity in the cell wall of Nicotiana.

Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5-triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin.

Studies on the oxidation of indole-3-acetic Acid by peroxidase enzymes. I. Colorimetric determination of indole-3-acetic Acid oxidation products.

The method described here is based on a brief report by Harley-Mason and Archer. It involves the use of p-dimethylaminocinnamaldehyde (DMACA), a vinylogue of Ehrlich's reagent, as a color reagent for indoles. Colorimetric analyses of indoleacetic acid (IAA) oxidation reaction mixtures were made with the DMACA reagent as a solution rather than a spray. DMACA reagent will yield a wine-red color with IAA oxidation products in solution. Under similar conditions DMACA reacts with authentic IAA to yield only slight coloration at best. In comparison with other indoles, DMACA is more relative with IAA oxidation reaction products than either Salkowski or Ehrlich's reagents. Data discussed support a concept that the color produced with DMACA is due to the presence of tautomeric oxidation product(s) of IAA.