Identification of new components involved in shoot gravitropism in Arabidopsis thaliana
Type of DegreeDissertation
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Plants can reorient their growth according to environmental cues such as light, water, nutrients, gravity etc. Shoots (hypocotyls and inflorescence stems) of the plants exhibit negative gravitropism. Germinating seeds are usually not exposed to the light therefore in the dark the directional growth of stem and root is solely determined by the orientation of gravity vector. Therefore, gravitropism is essential for plant survival because if a seedling does not break the ground it will not be able to photosynthesize and it will not survive. SCARECROW (SCR) plays key roles as transcription factor in Arabidopsis thaliana development. Confirmed functions of SCR include the radial patterning of axial organs, the development of endodermis and normal shoot gravitropism. The scr mutants exhibit abnormal internal shoot and root architecture. Analysis of shoot internal morphology indicates that both the hypocotyl and the inflorescence stem have defective radial patterning (missing one ground tissue layer) that leads to shoots to agravitropism. Functional orthologs of Arabidopsis SCR have already been identified in various agriculturally important crops such as maize and rice. Furthermore, at least some of the SCR functions have been found to be conserved throughout the plant kingdom. The goal of this research is to discover novel components that function along with the SCR gene in the SCR; regulated gravitropic pathway. To achieve this goal, a detailed study of scr1 mutant suppressors has been performed. Ten suppressors with improved hypocotyl gravitropism and three suppressors with improved root length have been identified. These suppressors show improved gravitropic response and root length respectively over scr1 mutant but below that of the wild type (WT) level. Most of the suppressors were isolated from different seed pools, and they show various degrees of gravitropic response as compared to WS (wild type) and scr1. Therefore, it is reasonable to predict that each suppressor represents a different mutation. Each mutation should point to a specific point in the gravitropic pathway in which the corresponding gene is involved. In order to confirm the number of different loci affected by scr1 suppressors, I conducted a complementation test. The results demonstrated that six different genes are mutated in this suppressor collection. The strongest scr1 hypocotyl gravitropic suppressor (shs1) was selected for mapping analysis. The rough map positions have been determined using Simple Sequence Length Polymorphism (SSLP) markers. Pollen of shs1 was used to cross with ovaries of scr3 flowers (SCR mutant on Columbia background). F2 generation seedlings with hypocotyl gravitropic response were selected for DNA isolation and the DNAs were used for mapping. To map the position of gene two to six SSLP markers were used for each of the five chromosomes of Arabidopsis thaliana. Only chromosome 5 showed the linkage. The second site mutation that rescued the hypocotyl gravitropic phenotype of shs1 maps to the lower arm of chromosome 5 about 27.9cM away from the closest SSLP marker tested. It has been suggested that shoot agravitropism in scr1 mutant is due to the abnormal radial pattern and/or the decreased hypocotyl cell elongation in the dark. However, my results do not support either of these hypotheses. The cell elongation in suppressors and scr1 mutant dark grown hypocotyls is indistinguishable. Also, the hypocotyls of the all ten gravitropic suppressors have a scr1 radial pattern that is characterized by a ground tissue deletion. These data suggest that decreased elongation and abnormal radial pattern are not responsible for scr1 hypocotyl agravitropism. I propose SCR is more directly involved in gravitropism by regulating genes involved in early stages of gravitropism, probably the aspects of gravity sensing stage. In order to address the function of SCR in gravitropism we have generated WS and scr1 lines that carry 35S::SCR and 35S::GFP::SCR constructs. Phenotypic analysis of these transgenic plants yielded some very interesting results. Both 35S::GFP::SCR/scr1(st1) and 35S::SCR/scr1(D7) plant hypocotyls showed a gravitropic response similar to WS plants while one 35S::SCR/WS (35S) line of plant hypocotyls showed complete agravitropism. The cross section analysis showed that st1 and D7 have not rescued their normal radial pattern. Both of them contain only two ground tissue layers. On the other hand hypocotyl of the 35S seedlings continued to have a normal radial pattern with three ground tissue layers. The result of radial pattern analysis is consistent with my hypothesis that the reason of agravitropism is not the absence of a cell layer but the deregulation. This finding also suggests that the SCR gene plays a more important role in gravity perception than previously considered. Currently, the most favored hypothesis to describe this complex mechanism is the “Starch-statolith hypothesis” which postulates that gravity sensing involves sedimentation of amyloplasts (statoliths) in specific cells known as statocytes. I performed whole-mount amyloplast staining in order to identify the presence and location of amyloplasts in suppressors with improved gravitropic response. Staining results show that suppressors resemble scr1 in both presence and position of amyloplasts rather than WS plants. 35S, st1 and D7 were also stained to check amyloplast sedimentation. The results demonstrated that amyloplast sedimentation in 35S is similar to WS while st1 and D7 resemble scr1. Our results on amyloplast sedimentation do not correlate with gravitropic responses and indicate that an alternative mechanism for sensing gravity other than amyloplast sedimentation must be operating in Arabidopsis thaliana hypocotyls.