HIGH‐THROUGHPUT SCREENING TECHNOLOGY FOR MONITORING PHYTOHORMONE PRODUCTION IN MICROALGAE¹

Abstract

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.

Phytohormones of microalgae: biological role and involvement in the regulation of physiological processes. Pt II. Cytokinins and gibberellins

“…В последующие годы появились публикации, посвященные прямой идентификации цитокининов с использованием современных хроматографических методов, в частности ЖХ/МС Jirásková et al, 2009;Hussain et al, 2010;.…”

“…1), с преобладанием изопентениладенина и цис-зеатина, тогда как транс-зеатин, дегидрозеатин и их производные выявлены в незначительных количествах Jirásková et al, 2009;. Нуклеотиды и метилтиолы iP (изопентенильного) и Z (зеатинового) типов выявлены лишь у Euglena gracilis .…”

“…Нуклеотиды и метилтиолы iP (изопентенильного) и Z (зеатинового) типов выявлены лишь у Euglena gracilis . Ароматические ЦТК представлены бензиладенином, бензиладенозином, орто-, мета-и пара-тополинами, при этом их суммарное содержание всегда было значительно ниже, чем изопреноидных ЦТК Jirásková et al, 2009;. Бензиладенин, орто-и мета-тополины встречались в более высоких концентрациях, чем пара-изомеры .…”

A Novel Miniature Culture System to Screen CO2-Sequestering Microalgae

“…Such methods provide reliable screening results, but are time consuming and labor intensive [5]. Some miniature culture technologies have been used to increase experimental throughput in microalgae research [6][7][8]. Most of these are adaptations of Lukavský's [9] system.…”

Biorefineries – factories of the future

“…Algae based polyunsaturated fatty acids include: arachidonic acid (ARA, an important omega-6 fatty acid), docosahexaenoic acid (DHA, an omega-3 acid, is an essential part of the human brain, cerebral cortex, skin and retina), gamma linolenic acid (GLA, an organic compound from the group of omega-6, which is a precursor of prostaglandin PGE1 -hormonally active arachidonic acid derivative with many biological functions). Algae can produce also flavouring substances as: pyroles, cyclopentenones (Maksymiec et al, 2005), phenols (Li et al, 2007), furans, pyrrolidones, indoles and skatol (Jirásková et al, 2009). In the pharmaceutical industry, algae are useful for the preparation of biologically active compounds used for the production of antioxidants and various antibacterial, antifungal, antiviral, antiprotozoal, antineoplastic, anti-obesity, anti-diabetic and anti-inflammatory agents, and as a support for the immune, cardiovascular and nervous system, and vitamins (Borowitzka, 1986).…”