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