Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Hüner, Norman P. A.

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.

Table S1.1.docx (13 kB)
Table S1.1. The enzymes of the Calvin-Benson-Bassham cycle

Table S1.2.docx (14 kB)
Table S1.2. The enzymes of the tetrapyrrole biosynthesis pathway

Table S2.1.docx (30 kB)
Table S2.1. Studies which have used the cell suspension Arabidopsis thaliana var Landsberg erecta that was isolated by May and Leaver in 1993

Table S2.2.docx (14 kB)
Table S2.2. Immunoblot quantification and normalization of the polypeptide levels to fresh weight. The immunoblots (Fig. 2.4) probed for polypeptides from the major photosynthetic proteins: D1, A core subunit of PSII, PsaA, A core subunit of photosystem I polypeptide, Lhcb2, A chlorophyll-a/b binding proteins of PSII, and RbcL, the large subunit of Rubisco

Figure S2.1.doc (127 kB)
Figure S2.1. Growth curves of the Arabidopsis cell suspension culture grown in MS media containing 3% sucrose at 30 μmol photons m-2 s-1 of continuous light

Figure S2.2.docx (160 kB)
Figure S2.2. 77K chlorophyll fluorescence emission spectra of the Arabidopsis cell suspension culture

Table S3.1.xlsx (7622 kB)
Table S3.1. Cuffdiff estimates of gene expression (FPKM) with statistical comparisons (q-values)

Table S3.2.xlsx (2238 kB)
Table S3.2. Annotated heatmap of transcript profiles from the cell suspension cultures grown at 0%, 3%, 6%, and 9% (w/v) sucrose, and at 9% (w/v) mannitol

Table S3.3.xlsx (147 kB)
Table S3.3. Genes differentially regulated by osmotic pressure in the cell suspension culture

Table S3.4.xlsx (135 kB)
Table S3.4. Genes differentially regulated by sucrose in the cell suspension culture which were NOT regulated by osmotic effects

Table S3.5.xlsx (92 kB)
Table S3.5. Genes differentially regulated by both sucrose and osmotic effects in the cell suspension culture

Table S3.6.xlsx (17 kB)
Table S3.6. Gene ontology analysis of genes differentially regulated by sucrose in the cell suspension culture

Table S4.1.docx (77 kB)
Table S4.1. Genetic variation in genes required for WT sugar signalling (Continuation of Table 4.1)

Table S4.2.docx (16 kB)
Table S4.2. Genetic variation in genes involved in ABA biosynthesis

Table S4.3.xlsx (13 kB)
Table S4.3. Genes upregulated by sucrose in WT leaves which were not expressed at all in the cell suspension culture. These genes were identified through meta-analysis comparing transcript profiles from WT seedlings from Müller et al. (2007), with the transcript profiles of the cell suspension cultures

Table S4.4.xlsx (23 kB)
Table S4.4. Genes upregulated by sucrose in WT leaves which were not expressed at all in the cell suspension culture. These genes were identified through meta-analysis comparing transcript profiles from WT seedlings from Han et al. (2015), with the transcript profiles of the cell suspension cultures

Table S4.5.xlsx (49608 kB)
Table S4.5. The VCF file listing all genetic variants called by GATK compared to the TAIR10 reference genome. The list has been filtered for read depth (DP > 10), quality by depth (QD > 2.0), mapping quality (MQ > 30.0), and strand bias (FS < 80.0)

Table S4.6.xlsx (16185 kB)
Table S4.6. The variants identified in the sugar insensitive cell culture genome (vs. TAIR10) compared to the natural variants that occur in the 27 WT strains of Arabidopsis with WT sugar insensitivity: RLD-1, Col-0, Cvi-0, Es-0, Kas-1, Ler-0, Mv-0, Tsu-0, Yo-0, Gr-1, Hn-0, Hs-0, Kil-0, Le-0, Lm-2, Mnz-0, Nc-1, NFA-8, Ob-0, Or-0, Phw-3, Phw-34, Pi-0, Pt-0, Sg-1, Si-0, and Zu-1

Table S4.7.xlsx (4439 kB)
Table S4.7. The list of cell culture genetic variants that that did not occur in the 27 WT genomes with WT sugar sensitivity. These unique genomic variants that were sorted by the regions where they occur (e.g exon, intron, promoter, 3’ UTR, 5’ UTR, upstream, downstream). Variants in the exon regions were further categorized based on the predicted impact on the protein product

Appendix 4.1.docx (1487 kB)
Appendix 4.1. Genome sequencing quality statistics, ecotype identification, and analysis of genetic variants identified in the cell suspension culture genome

Available for download on Thursday, August 01, 2019

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