Developmental modulation of tubulin protein and mRNA levels during somatic embryogenesis in cultured carrot cells

Abstract

The number of cortical microtubules (MTs) increases considerably as cultured carrot (Daucus carota L.) cells initiate and progress through somatic embryogenesis. The basis for this increase in MT number was investigated. A radioimmune assay was used to show that tubulin-protein per cell first decreased as the undifferentiated cells initiated embryonic development, but subsequently increased approximately fivefold between the globular and torpedo/plantlet stages. The increase during the torpedo/plantlet stage was correlated with the increase in cell size that occurred during the latter stages of embryogenesis. The cellular levels of tubulin mRNA were determined by Northern blot analysis, using labeled probes derived from soybean α- and β-tubulin genomic sequences, cloned in the vectors pSP64 and pSP65. This analysis demonstrated that the levels of tubulin-gene transcripts varied with the tubulin-protein levels. Cell-free translation of polyadenylated RNA, followed by immunoprecipitation with an anti-tubulin antiserum, established that these transcripts represented functional tubulin mRNA. These results indicate that MT formation in early embryogenesis is controlled by factors other than the availability of tubulin, but that MT formation later in embryogenesis is coordinated with concomitant changes in tubulin-gene transcription and in the size of the total tubulin-heterodimer pool.