Distribution of endogenous gibberellins in etiolated broad bean (Vicia faba) seedlings

Abstract

The work involved here was undertaken as an attempt to extract, purify, fractionate, separate, and identify the endogenous gibberellins in the etiolated broad bean (Vicia faba) seedling. Following a series of preliminary experiments, it was decided to extract the gibberellins in each organ of the seedling separately by ice-cold absolute methanol and fractionate this solvent extract into three fractions, for rough separation of the gibberellins, as follows: i. Free gibberellins or acidic ethyl acetate-soluble fraction. ii. Conjugated-gibberellins or acidic butanol-soluble fraction. iii. Bound-gibberellins or water-soluble fraction. Both the conjugated-gibberellins and the bound-gibberellins were released by acid hydrolysis.

The term bound-gibberellin is used here for those gibberellins which appear neither in the acidic ethyl acetate-soluble fraction nor in the acidic butanol-soluble fraction and which are extracted by ethyl acetate at low pH upon hydrolysis. The term does not imply that I have any proof of binding to specific molecules or cell particles.

The studies carried out in this thesis revealed that PVP slurries, PVP column chromatography followed by TLC of the gibberellins recovered from the columns effluents are satisfactory tools for the purification and separation of the endogenous gibberellins in the different organs of the etiolated Vicia seedling. During the study, use has been made of both bioassay and GLC following TLC.

Peaks with retention time corresponding to the methyl esters of gibberellins A₇, A₉, A₁₃, A₁₅, A₁₇, and A ₂₄ were detected in the acidic ethyl acetate-soluble fraction obtained from the radicle tissue extracts after raethylation; that obtained from the plumule tissue extracts gave peaks with retention time corresponding to the methyl esters of gibberellins A₁₂, A₁₃, iso-A ₁₃, A₂₄; while the acidicethyl acetate-soluble fraction of the cotyledons tissue extracts gave peaks with retention time corresponding to the methyl esters of gibberellins A₃, A₅, A ₆, A₇, A₁₂, A₁₃, iso-A₁₃, A₁₄, and A₁₅.

The free gibberellins released by acid hydrolysis of the conjugated-gibberellins in radicle extracts after methylation gave peaks with retention time corresponding to the methyl esters of gibberellins A₉, A₁₂, A ₁₃, A₁₄, A₂₀, and A₂₄; those released from the conjugated-gibberellins of the plumule gave peaks v/ith retention time corresponding to the methyl esters of gibberellins A₈ and A₁₅; while the free gibberellins released in the case of the cotyledons tissue extracts gave peaks with retention time corresponding to the methyl esters of gibberellins A₁₃ and A₁₄.

On the other hand, acid hydrolysis of the bound-gibberellins in the radicle tissue extracts released gibberellins which after methylation gave peaks with retention time corresponding to the methyl esters of gibberellins A₇ , A₉, A₁₇, and A₂₄ those released from extracts of the plumule tissue gave peaks with retention time corresponding to the methyl esters of gibberellins A₇, A₈, A ₉, A₁₂, and A₂₄ while the free gibberellins released by acid hydrolysis of the cotyledons bound-gibberellins gave peak with retention time corresponding to the methyl ester of gibberellin A₁₃.

The cotyledons which are the main storage organs in broad bean contain a number of gibberellins in a "bound"? and/or a conjugated form. These gibberellins appear to release free gibberellins on germination. The possible sites of gibberellin metabolism is discussed on the basis of the results obtained.