Molecular strategies for developing aluminium tolerance in plants
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. Aluminium (Al) toxicity is a major limitation to crop plant growth in acid soils. The work presented in this thesis is concerned with the identification and isolation of genes capable of conferring Al tolerance on plants. Two experimental approaches were undertaken to achieve this aim. The first strategy employed was to search for Al tolerant plants (Nicotiana plumbaginifolia and Arabidopsis thaliana) as donors of genes for use in a shotgun approach to cloning Al tolerance genes. No Al tolerant plants showing Mendelian inheritance of a single dominant gene were characterised during these studies. Hence, a second strategy was initiated. The approach taken in this case was to investigate whether Al binding proteins were able to protect plants from the effects of Al. In order to test this idea, Al binding proteins were identified, chimaeric genes encoding these proteins were introduced into plants and the Al tolerance of the transgenic plants was assessed. In the first step, a series of proteins and polypeptides were screened for their ability to bind Al using biochemical assays. Two out of the three assays were based on measurement of Al induced changes to calmodulin, an important Ca effector in eucaryotic cells. The test compounds that were most effective in relieving the interference by Al on calmodulin structure and function were transferrin and two acidic polypeptides, poly (L-glutamic) acid and poly (L-aspartic) acid. The results of equilibrium dialysis also indicated that transferrin, calmodulin and the acidic polypeptides bound Al. The acidic polypeptides subsequently were shown to protect tea pollen tubes from Al induced inhibition of growth. Experiments then were performed to introduce genes coding for some of these proteins and polypeptides into plants. Six chimaeric genes were constructed that were all designed to be transcribed under the control of the CaMV 35S promoter. Two of the genes contained cDNA copies of eucaryotic genes: conalbumin (chicken ovotransferrin) and Xenopus calmodulin and a third contained the yeast ubiquinol-cytochrome c reductase gene (an acidic protein with untested Al binding capacity). The other three genes were NH2 terminal fusions to ?-glucuronidase (GUS). These genes were constructed by inserting synthetic oligonucleotides encoding poly (L-aspartic acid) and a consensus Ca binding site sequence upstream of the GUS reporter gene. Transient expression assays in carrot cell protoplasts using the GUS gene constructions showed that GUS fusion proteins of the expected size were produced. All six genes then were inserted into binary vectors and the constructions were introduced into Nicotiana plumbaginifolia using Agrobacterium rhizogenenes mediated gene transfer. The resulting transgenic hairy roots were grown under kanamycin selection. Southern, Northern, Western and polymerase chain reaction (PCR) analyses were employed to determine integration and expression of the introduced genes. The results of these analyses showed that most of the transgenic hairy roots contained intact copies of the genes. Messenger RNA transcripts of the correct size were produced in some of the roots. The protein products of the chimaeric GUS genes were detected using fluorimetric and histochemical assays. Western analysis of protein extracts made from hairy roots indicated that the conalbumin protein was present at ~0.1%-0.7% of protein in total protein extracts. Unexpectedly, conalbumin was not detected in the soluble protein fraction, but was present in the culture medium of the hairy roots. These results suggested that the NH2 terminal signal sequence for secretion in animals, present upstream of the mature conalbumin protein, may be functional in plants. Progeny seedlings from plants regenerated from hairy roots were tested for kanamycin resistance. The results of this analysis showed that the kanamycin resistance gene in the transgenic hairy roots was heritable as a single dominant Mendelian gene in some cases. Crude purification of the GUS fusion proteins was achieved, but Al binding tests using these proteins have proven inconclusive. Transgenic hairy roots and plants were screened for Al tolerance, but did not exhibit improved tolerance to Al.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/1990