Targeted knockout of PpORS encoding an ancient type III polyketide synthase in the moss Physcomitrella patens

Li, Li
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Faculty of Graduate Studies and Research, University of Regina

The ubiquitous presence of type III polyketide synthases (PKS) in the plant kingdom suggests their important roles in plant evolution. Among plant type III PKSs, PpORS from the model moss, Physcomitrella patens, has previously been suggested to closely resemble the most recent common ancestor of plant type III PKSs. In vitro analysis of PpORS revealed its function as a 2'-oxoalkylresorcinol synthase. PpORS is highly expressed in gametophores, but not in protonema. This, together with previous phytochemical analysis, suggested that in planta PpORS products may be incorporated into the moss cuticular structure to confer protection from environmental stresses. In this study, to gain insight into the in planta function of PpORS, PpORS was targeted for knockout in Physcomitrella. Three stable transformants were confirmed by PCR, Southern blot and RT-PCR, and PpORS stable transformants (ors) were phenotypically analyzed. Ors plants were similar to the wild-type plant in developmental characteristics, including branch numbers and spore viability. Both the wild-type and ors plants responded similarly to UV-B irradiation, suggesting that UV protection is not a main function of PpORS. In contrast, abnormal leaves, including (i) curly leaves with a large number of protuberances, (ii) highly deformed leaves, (iii) irregularly shaped leaves, and (iv) leaves with cell outgrowths on the surface or margin, were frequently observed in ors. Abnormal leaves were heavily stained by a hydrophilic dye Toluidine Blue O, whereas both normal and abnormal leaves were stained by Sudan IV. Interestingly, the incidence of morphological abnormalities in ors leaves significantly decreased with water immersion treatment. These results suggest that ors shoots may be covered with a defective cuticle layer.

A Thesis Submitted to the Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Degree of Master of Science in Biochemistry, University of Regina. ix, 68 p.