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Title: Molecular and physiological investigations of arabidopsis insertional mutant lines under abiotic stresses
Author: Elhaj, Abobakir Ali
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2009
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Salt, cold, and drought are major abiotic stresses that impact plants and agricultural crops. Many plants and crops are able to increase their ability to survive these and other stresses by altering gene expression. Using the Arabidopsis model plant in studying gene function and regulation is of crucial importance to plant genetics and biotechnology. Our objective was to study plant survival and stress tolerance in Arabidopsis T-DNA lines and, also chemically mutagenized and RNAi lines, for specific genes that are hypothesised or expected to have a role in at least one of the known abiotic stresses. The selection of genes was based on some previous research including QTL analysis. I included in my investigations OSMl, CHXl7, KUPl, bHLH033, CBF4, dreb2a and the NW20 and N163 lines of ERECTA. Other non-mutated genes such as COR 78, DREB2A, CBFl, CBF3, CBF4, and OSMl were used to study the specificity in gene expression in Arabidopsis leaf and epidermal tissues under a combination of cold and osmolytes. The insertional mutant analysis showed that T-DNA can be located in a different site than the one shown in database and sometimes parts of the T-DNA left-border-sequence were absent. Most of the targeted Arabidopsis mutants showed a normal Mendelian pattern of segregation with the one exception of the CBF4 insertional mutant population. RT-PCR transcript detection of bHLH and DREB2A indicated that wild-type Arabidopsis in our growth environment expressed only one splice form of each of these genes rather than the two described by the TAIR and MIPS databases. Stress results indicated that inserting T-DNA within the Arabidopsis genome for a specific gene can lead to negative or positive effects on the gene's expression and plant survival. Investigation of the role of OSMl in cold tolerance indicated that the mutant plants were more tolerant to cold as measured by plant survival and electrolyte leakage. The higher expression of OSMl gene in the mutant plants has driven me to search in the promoter region of OSMl at OSM1::T-DNA junction. The results revealed the presence of different known eis-acting elements which might have affected plant survival. Using RNAi lines for OSMl (gene expression was not tested) is also consistent with the idea that this gene has a role in cold stress. Even though the high transcripts accumulation of bHLH (lCE2) in the mutant line was discovered, there was only slight difference between homozygous and wild type plants in response to cold. DREB2A, which is well known for its dehydration involvement, was tested under cold. Using dreb2a mutant for cold stress revealed that Salt stress tolerance was obtained with osml, ehx17, and kupl mutants as measured by the high average of plant survival. Mineral content was measured in leaves and roots of CHX17 and KUP 1 mutant and wild type plants under salt conditions. The mutant plants accumulated less sodium and higher potassium especially in roots compared to wild type. Also, roots accumulated more calcium and magnesium than the wild type. These results are clearly consistent with the higher survival in kupl mutant and may indicate a correlation with the salt tolerance in ehx17. Also, the high sensitivity to salt stress was strongly linked to some phenotypic and developmental changes, such as leaf wilting, rolling, chlorosis, and desiccation. Mutant plants that had higher survival developed dark leaf colour which probably indicates anthocyanin accumulation. A higher level of expression of genes in some mutant lines than in the wild type lines, linked with higher plant survival indicated there might be some eis-acting elements in the inserted T-DNA that were influencing stress tolerance. Compared to NW20 Landsberg ereeta line, carrying the chemically produced mutant, N163 plants, which carry wild-type ERECT A gene, exhibited more plant survival under salt stress especially at an older growth stage. Abstract IV covering plants during cold acclimation increased plant's sensitivity to cold compared to uncovered plants. This suggests that the role of DREB2A in cold acclimation probably is not through water stress signalling per se but other signalling must be involved. Also, drought stress was studied and was limited to CBF4 gene. The gene expression of CBF4 in the mutant was greatly reduced. However, plant survival under drought was not affected by the mutation. Gene expression-specificity of some cold regulated genes in epidermis and leaves of Arabidopsis wild type was studied under sugar and sorbitol with or without minerals. COR 78, DREB2A, CBF1, Eli-l-o; ACT2, and ACT8 were more highly expressed under cold in epidermis than in leaves. This expression was often several-fold higher when sucrose at 90 mM with mineral salts was supplied. CBF3 also showed a higher level of transcript accumulation by cold in epidermis than in leaves but both tissues exhibited similar and higher expression with sucrose at 40mM than the control and 90mM of sucrose with minerals. It seemed that removing minerals from sugar solution reduced the gene expression but epidermal tissues conserved the preference of most genes to be expressed in higher level (relative to total RNA) than leaves. Sorbitol at 90mM with minerals highly affected the expression of CBFl and CBF3 apart from type of tissue and reduced the expression of DREB2A, CBF4, and ACT8 in both tissues. OSMl had differential gene expression by different treatments. It showed slightly higher expression in epidermis with cold treatment and its expression was completely diminished in leaves at 90mM of sorbitol with minerals. These results indicate the regulatory role of sugar in cold- induced gene expression of several genes in epidermis which reflects its importance in stress tolerance. Abstract v All in all, using T-DNA can affect plant survival and alter gene expression in ways which can be interpreted as a direct or non-direct link in the overall stress tolerance. These investigations have added new facts to understanding stress tolerance in plants but others remain to be more deeply tested. A novel observation was that the T-DNA in the promoter can increase the regulation of gene expression and stress tolerance and this may be due to stress response elements in the T-DNA. Abstract
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available