Molecular biology and physiology of isolated chloroplasts from the algae Vaucheria

Abstract

Sea slugs of the genus Elysia (e.g. E. chlorotica) are known for their ability to incorporate chloroplasts from the yellow-green alga Vaucheria litorea. These “kleptoplasts” stay active in the digestive tract of the sea slug for several months. Chloroplasts from Vaucheria litorea are also reported to be significantly more stable after in vitro isolation than chloroplasts of other algae or of higher plants. In organello assays with isolated chloroplasts are used in studies on photosynthetical and biochemical processes in these organelles, chloroplast-nucleus communication, plant development and plant responses to environmental changes. The major limiting factor of the chloroplast in organello systems is the rapid decline in function and intactness of the plastids. This study is investigating the stability and longevity of chloroplasts isolated from V. litorea and its relative V. bursata in comparison to an angiosperm Pisum sativum. The structural intactness of the chloroplasts was investigated by phase contrast microscopy and the overall transcriptional activities were analyzed by run-on transcription assays. The recently completed sequence of the plastid genome from V.litorea has made it possible to investigate also the specific changes in isolated chloroplasts on the transcriptional level. The expression patterns of chloroplast-encoded genes trnE, rrn23, rrn16, rbcL, psbD, psbA and psaA directly after isolation and 4 hours post-isolation time were analyzed using dot blot hybridization. The ability of isolated chloroplasts to incorporate 35S-methionine into de novo synthesized proteins at 0, 4 and 24 hours after isolation was tested by translational assays. The chloroplasts of Vaucheria litorea were both transcriptionally and translationally stable over an extended period of time. However, the culturing limitations and low plastid yields diminish the potential of this alga as a chloroplast donor for in organello assays. Chloroplasts of Pisum sativum, previously suspected to be more unstable, did not show a decline in transcription rate until 4 hours after isolation. The decrease in incorporation of 35S-methionine between time 0 and 4 hours after isolation was only minor, between 4 and 24 hours more significant but the radioactive signal was still readily detectable at 24 hours. Pisum sativum has simple growth requirements and offers high yields of isolated chloroplasts. Chloroplasts from this plant should therefore be in the centre of further investigations into their suitability to express foreign genes and their potential for in organello assays.