Defective ABA-mediated sugar signalling pathway in an established Arabidopsis thaliana cell suspension culture explains its stay-green phenotype at high sugar concentrations
Abstract
An unusual sugar insensitive phenotype was identified in an established cell suspension culture of Arabidopsis thaliana. We characterized the physiology, biochemistry and genetics of the sugar insensitive cell culture, in order to identify factors contributing to the phenotype. Chlorophyll levels of the cell suspension culture were insensitive to high sucrose (6-15% w/v) and maintained a green phenotype. Immunoblotting indicated that levels of key photosynthetic proteins (PsaA, Lhcb2 and Rubisco) increased as a function of external sucrose concentration. The green cell culture was photosynthetically competent based on light-dependent, CO2-saturated rates of O2 evolution as well as Fv/Fm and P700 oxidation. Transcript profiling indicated that key sugar signalling transcripts ABI3 and ABI4 were not detectable in the cell suspension culture, and this was confirmed by qPCR. Because the transcription factors ABI3 and ABI4 are essential for WT sugar signalling, the lack of these transcripts resulted in a sugar insensitive phenotype that mimicked the phenotype of abi3 and abi4 mutant seedlings. Addition of ABA to the external medium failed to rescue the suspension cells from its sucrose insensitivity. We sequenced the genome at over 300-fold coverage, and identified three hundred thousand genetic variants unique to the cell culture, not present in the genomes of twenty-seven Arabidopsis thaliana ecotypes with WT sugar sensitivity. Despite widespread genetic changes in the genome, there were no variations in the promoter regions or regulatory regions of ABI3 or ABI4 that could explain the lack of expression of these transcription factors. We conclude that the genetic change has impacted an unknown regulator of ABI3 or ABI4 impairing their expression resulting in a sugar insensitive phenotype. Because of the genetic variation that has occurred, and because of the unusual sugar and photosynthetic responses, caution must be exercised in the interpretation of physiological and biochemical data obtained from experimental use of this culture in any comparison with wild-type Arabidopsis seedlings.