A carrot cell variant temperature sensitive for somatic embryogenesis reveals a defect in the glycosylation of extracellular proteins

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Abstract
The temperature-sensitive carrot cell variant ts11c, arrested in somatic embryogenesis after the globular stage, was characterized. The sensitivity to a shift from 24° C (permissive temperature) to 32° C (non-permissive temperature) is greatest at the globular stage of embryogenesis, while cells proliferating in unorganized fashion and plantlets are not affected. Embryogenesis in ts11c is also arrested at the permissive temperature by replacement of conditioned culture medium with fresh medium. The timing of sensitivity of ts11c to medium replacement coincides with the sensitivity to temperature shift. Both sensitivities are recessive in somatic hybrids between ts11c and wild-type cells. Extracellular glycoproteins synthesized by ts11c at the non-permissive temperature contain much less fucose than those synthesized by the wild type. The glycoproteins synthesized by the variant under non-permissive conditions do not accumulate at the periphery of the embryo, as their wildtype counterparts do, but instead show a diffuse distribution throughout the embryo. The defect in ts11c can be fully complemented by the addition of extracellular wild-type proteins. A revertant of ts11c was isolated that simultaneously reacquired temperature insensitivity and normal glycosylation ability. Collectively, these observations indicate that ts11c is not able to perform proper glycosylation at the non-permissive temperature and suggest that the activity of certain extracellular proteins, essential for the transition of globular to heart stage somatic embryos, depends on the correct modification of their oligosaccharide side-chains.