ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

Cryopreservation of Chrysanthemum morifolium (Dendranthema grandiflora Ramat.) using different approaches.

Chrysanthemum species are grown both as ornamentals and for the production of pyrethrum. Recent increased production and breeding efforts have raised the need for the conservation of valuable germplasm. Chrysanthemum has been cryopreserved by controlled-rate-freezing as early as 1990. We report here deep-freezing of shoot tips of C. morifolium var. Escort by different technical procedures: controlled-rate-freezing, encapsulation/dehydration, ultra-rapid-freezing by the droplet method and vitrification. While vitrification yielded the highest shoot regeneration rates, the very simple droplet method was also successful in this respect. Droplet freezing was successfully performed with nine cultivars. Our results open the door to the successful use of alternative methods if one method fails to cryopreserve a variety. Furthermore, it enables comparative investigations of genetic stability and cyro-injury to be carried out.

Recovery of potato apices after several years of storage in liquid nitrogen.

A method for the systematic cryopreservation of potato apices was developed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and the Institute for Crop and Grassland Science of the Federal Agricultural Research Centre (FAL, Braunschweig). Designed specifically for routine use in genebanks, this method uses a very simple ultra-rapid freezing approach and was applied to a wide range of varieties within the Federal Centre for Breeding Research on Cultivated Plants (BAZ, Quedlinburg) Potato Collection. After several years of storage in liquid nitrogen, shoot tips from a random sample of 51 varieties were thawed and the survival and shoot regeneration percentages compared to those measured immediately after freezing. There were no major changes in either survival or recovery of frozen apices. Data presented are not the outcome of a systematic experiment but from that accumulated during our work from 1992 to 1999.

Partial purification and characterization of dihydrobenzophenanthridine oxidase from Eschscholtzia californica cell suspension cultures.

The observation that upon elicitation cell suspension cultures of Eschscholtzia california showed a decrease of dihydromacarpine with a concomittant increase of macarpine led to the discovery of a novel enzyme which catalyzes the oxidation of dihydrobenzophenanthridines in the presence of oxygen. The enzyme was enriched approx. 70-fold. It has a pH-optimum of 7.0, an isoelectric point at pH 8.8, molecular weight of 56 kD and shows a high degree of substrate specificity. The enzyme obviously catalyzes the terminal step in the formation of benzophenanthridine alkaloids containing methylene dioxy substitutions in rings A and D.

Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures.

Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.

Partial Purification and Properties of (S)-Norlaudanosoline Synthase from Eschscholtzia tenuifolia Cell Cultures.

A new enzyme, (S)-norlaudanosoline synthase, which catalyses the synthesis of (S)-norlaudanosoline from dopamine and 3,4-dihydroxyphenylacetaldehyde was isolated from the soluble protein extract of ESCHSCHOLTZIA TENUIFOLIA cell suspension cultures and purified approximately 40-fold. The apparent molecular weight of the enzyme is 15 500 Dalton. The pH optimum is 7.8, temperature optimum 40 degrees C, apparent K (M) values for dopamine and dihydroxyphenyl-acetaldehyde are 1.5 mM and 0.7 mM respectively. The synthase shows high substrate specificity in that only the phenylacetaldehydes are transformed but not the phenylpyruvates. No apparent cofactor requirement could be demonstrated. By means of isoelectric focusing and disc-gel electrophoresis evidence was obtained for the existence of four norlaudanosoline synthase isoenzymes, none of which catalyses the reaction of dopamine with 3,4-dihydroxyphenylpyruvate. These enzymes are responsible for the synthesis of (S)-norlaudanosoline, the key intermediate in the formation of isoquinoline alkaloids occurring in the plant kingdom.