Structural analysis of APOLLO, a candidate apomixis factor

Abstract

Apomixis is a naturally occurring phenomenon in which plants reproduce without fertilization, resulting in the asexual production of seed. This phenomenon has been well-documented in Boechera, a genus in the Brassicacea family that includes members that reproduce through diplospory (gametophytic apomixis). The occurrence of apomixis in Boechera is associated with expression of the APOLLO gene. Apomictically-reproducing Boechera species carry one copy of an apomictic APOLLO allele and one copy of a sexual APOLLO allele. There are twelve apomictic-specific non-synonymous polymorphisms in the APOLLO coding sequence, compared to the sex-allele. The structure and function of the APOLLO exonuclease in Boechera may be affected by these polymorphisms. APOLLO has predicted regions of structural disorder and potential sites of post-translational modifications. An expression system was created to obtain the sex- and apo-alleles in E. coli cells. Whole cell lysates were examined by SDS-PAGE using stain-free technology. The proteins present on the PAGE were transferred to a PVDF membrane using a wet membrane transfer. The histidine tag present on the apo- and sex- proteins were visualized by a western blot. The results indicate that a functional expression vector system, pMCSG7 in BL21 (DE3) E. coli cells grown in LB media was identified for the apo-allele and pMCSG7 in C41 E. coli cells grown in auto-induction media was identified for the sex-allele. The predicted intrinsic disorder profile established that near the middle of the APOLLO protein there is a region of disorder, that appears to be smaller in the apo-allele than the sex-allele. This region is potentially a binding site for interactions with other cellular components. The difference in size of the binding site between the alleles is the first evidence of a proteomic difference between the sex- and apo alleles of APOLLO.