Hormones are present in seaweeds, but little is known about their biosynthesis and regulation. The aim of this study was to investigate variation in concentration and composition of endogenous cytokinins, auxins and abscisic acid in two seaweeds collected over a 1 year period to gain a more complete picture of the types of hormones present during a range of environmental and developmental conditions. Ulva fasciata (Ulvales, Chlorophyta) and Dictyota humifusa (Dictyotales, Phaeophyta) were collected bimonthly from the inter-tidal zone at Rocky Bay, South Africa. Ethanol extracts of the samples containing a mixture of internal standards were purified using a combined DEAE-Sephadex-octadecylsilica column, followed by immuno-affinity chromatography and analysed by high-performance liquid chromatography -mass spectrometry to quantify different cytokinins, auxins and abscisic acid. cis-Zeatin, isopentenyladenine and their ribotide and riboside conjugates were the main cytokinins present in both U. fasciata and D. humifusa with low concentrations of trans-zeatin, dihydrozeatin and aromatic cytokinins. Very low concentrations of O-glucosides and riboside-O-glucosides and no N-glucosides were detected in any of the samples. Based on the cytokinins detected, we propose that as in higher plants, the ribotides are also the first intermediates formed during de novo biosynthesis in seaweeds and are subsequently converted to free-bases and ribosides. The only indole compounds detected in both species were free indole-3-acetic acid and indole-3-acetamide (IAM), suggesting that IAM is the intermediate in auxin biosynthesis. Endogenous abscisic acid was detected in most samples with levels in U. fasciata generally being higher than those measured in D. humifusa.

New miniaturized techniques for multiplying microalgae and estimating their phytohormone production were developed; in these methods, the strains to be tested are cultivated in microtitre plates, and the phytohormones in suspensions of the cultures are measured by direct ELISAs. Specific and sensitive ELISAs for determining abscisic acid (ABA), indole-3-acetic acid (IAA), cis-and trans-zeatin riboside, isopentenyladenosine (iPR), and other less common cytokinins were developed for this purpose. Polyclonal antibodies used in the ABA and IAA assays were raised against C1-and C1¢-conjugates of the compounds with BSA, respectively, and thus were specific for the free acids and their respective C1-derivatives. The use of cytokinin ribosides coupled via their sugar residues to BSA as haptens generally led to antibodies that bound free bases, 9-glycosides and nucleotides, but with high specificity for the corresponding N 6 -side chains. Using internal standards, dilution assays, and authentic [2 H] and [ 3 H] recovery markers, it was shown that the ELISAs could be used to estimate contents of the selected phytohormones in the cultures. The ELISAs provided reliable and very fast estimates of the selected phytohormones, at concentrations ranging from 0.01 to 10 pmol AE mL )1 in various microalgal strains. In addition, a recently developed HPLC selected ion monitoring mass spectrometry (HPLC-SIM-MS) method was used to calibrate and validate the ELISA results and confirm the presence of the detected phytohormones in immunoaffinity-purified extracts. Where independent validation of results is deemed necessary, the use of quantitative HPLC-MS is recommended for each new microalgal strain to be tested.

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