Physiological, genetic and molecular analysis of two Arabidopsis mutants with defects in normal development

• Physiologische, genetische und molekulare Analyse von zwei Arabidopsis-Mutanten mit Wachstumsdefekt

Pooraiiouby, Rana; Slusarenko, Alan (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2008)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2008

Abstract

Unlike animal cells, plant cells have a rigid wall and hence cannot migrate. Rather, they are permanently fixed in their relative position to neighbouring cells after cell division. Therefore, for normal plant organ morphogenesis the orientation of the cell division plane is critical. Cortical microtubules (MTs) play an important role in this process by forming a structure called the preprophase band (PPB). After mitosis, a new cell wall is formed during cytokinesis in the plane between the two daughter cells. The phragmoplast, another structure containing MTs, forms in the plane between the two daughter nuclei and lays down the new cell wall. A further role for MTs in determining plant stature stems from the fact that MTs are important for the parallel deposition of cellulose fibres in the cell wall. Thus, the patterns formed by the MTs direct the patterning of cellulose microfibrils on the outer side of the plasma membrane. Cellulose microfibril orientation has consequences on cell wall flexibility and controls the direction of cell expansion-growth. Plant cells are cemented together by pectic polysaccharides containing polygalacturonic acid, and pectin is a component of the matrix surrounding the cellulose microfibrils. Thus, MTs and pectic polysaccharides play an active and important role in establishing cell and organ morphology. In the process of selecting Arabidopsis transgenic lines two mutants were noticed with interesting phenotypes and these were selected for further study. In one mutant a gene coding for an enzyme involved in pectic polysaccharide metabolism was affected ("pgase") and the other mutant ("bushy-ff") had T-DNA insertions in two genes: one in a gene known to affect MT organization and the other in a gene of unknown function. During my thesis I characterized those mutants. Specifically I addressed the following aspects: What are the morphological and anatomical defects in the "pgase" mutant? Using light and scanning electron microscopy, it was shown that the total size of the plant in diameter and height was reduced and the leaves were somewhat twisted. At the tissue level the most obvious defects were the ~30% decreased number of the trichomes and reduced number of the epidermal guard cells in comparison to wild-type plants. What was the molecular basis of this mutant? Thermal asymmetric interlaced (=TAIL) PCR showed a T-DNA insertion in the Arabidopsis polygalacturonase (PGase) family member, At3g07970. Semi-quantitative RT-PCR showed a ~3Xfold lower expression of At3g07970 in the "pgase" mutant compared to the wild-type. Complementation of the "pgase" mutant phenotypes by over expression of the PGase At3g07970 gene, and analysis of an independent T-DNA insertion in At3g07970 that displayed an indistinguishable morphological phenotype to the original mutant, showed that this mutation was responsible for the observed phenotypes. Cell walls are important barriers for biotic and abiotic stresses. Since a mutation in a PGase At3g07970 gene likely results in altered cell walls, the responses of the "pgase" mutant to biotic and abiotic stresses were studied. To this end, this mutant was exposed to infection with Hyaloperonospora arabidopsidis, cold, heat and drought stress. We found that this mutant was hypersusceptible to infection with H. arabidopsidis, cold and heat stress.For the "bushy-ff" mutant the following aspects were addressed: What are the morphological and anatomical defects in the "bushy-ff" mutant? Using light and scanning electron microscopy, we found all of the organs including roots, leaves and flowers were abnormal in this mutant. The "bushy-ff" plants were strongly compressed in all dimensions. At the tissue and cellular level, several defects were observed, e.g. the size of the root cells of "bushy-ff" was ~30% smaller than Col-0 root cells, the total number of the trichomes was half of that in wild-type plants, branching of the trichomes was altered and differentiation of mesophyll cells into palisade and spongy cells in the"bushy-ff" mutant was aberrant. What was the molecular basis of this mutant? Thermal asymmetric interlaced (=TAIL) PCR showed that two independent T-DNA insertions, one in the promoter of a so far uncharacterized F-box At1g77000 gene and another in the promoter of the previously characterized, cortical microtubule organizing FASS At5g18580 gene occurred in this mutant. Further molecular analysis indicated about 50% lowered expression of the FASS and F-box At1g77000 gene as compared to wild-type. By generating lines constitutively expressing either F-box At1g77000 or FASS in the "bushy-ff" mutant background, it was observed that the "bushy-ff" phenotype could be complemented in both cases. This suggested that concomitant mutations in both genes were required for the "bushy-ff" mutant phenotype. Genetic experiments involving crosses between "bushy-ff" and wild-type as well as crossing single f-box At1g77000 to single fass mutants further confirmed that both mutations occurring in the "bushy-ff" mutant were both necessary and responsible for the "bushy-ff" phenotype. The expression pattern and organ specificity analysis of the F-box At1g77000 and FASS showed that both genes were expressed at similar levels relative to each other in all organs tested. Does the genetic interaction between the F-box At1g77000 and FASS genes reflect a physical interaction? Using yeast two hybrid analysis we could in fact show that the F-box At1g77000 and FASS proteins can also interact physically with each other. Where is the F-box At1g77000 protein localized inside the plant cell? Transgenic lines constitutively expressing a fusion protein of F-box At1g77000 with green fluorescent protein (35S::F-box At1g77000-GFP) in the "bushy-ff" mutant background were generated and showed an intracellular distribution of F-box At1g77000 that was similar to that of a microtubule associated protein (GFP-TUA6). In summary our data suggest that FASS might organize cortical microtubules through its interaction with an F-box At1g77000 protein.