Monoclonal antibodies as developmental markers to characterize pea floral homeotic transformations

Abstract

Monoclonal antibodies (mAbs) specific for the different floral organs of the garden pea (Pisum sativum L.) were raised using two different types of immunization procedures. These antibodies were powerful tools to study the functionality of floral organ identity genes in pea homeotic mutants. The mAbs were used extensively as developmental markers for the immunohistochemical characterization of two pea floral homeotic mutants (stp-1 and stp-2) to elucidate the different degree of transformation present in the mutated organs. Mutations in the Stamina pistilloida (Stp) locus, the pea ortholog of Fim (Antirrhinum) and UFO (Arabidopsis), produce floral phenotypes resembling those of loss-of-B-function. The initial screening of the antibodies obtained against protein extracts from complete flower buds or individual floral organs allowed the isolation of a mAb (S1) that was used to identify sepal tissues in flower sections and to visualize the complete transformation of petals into sepals in stp-2 or to identify partial homeotic transformations (sepaloid sectors) in the keel of stp-1. A second mAb (P1), which recognized an antigen present in petals, was used to identify the abnormal sepaloid features of the two petals forming the keel in the stp-1 mutant. However, using this immunization procedure we did not obtain any antibody specific for carpel or stamen tissues. To overcome this problem we developed an improved immunosubtractive strategy and several mAbs specific for these floral organs were obtained. mAbs OV1, OV4, OV5 recognized antigens mainly present in different carpel tissues and mAb A1 a protein present only in anther tissues (epidermis, connective, endothecium). These mAbs were used as markers to identify carpelloid or stamenoid tissues present in the stp-1 and stp-2 floral homeotic mutants.