Development-related changes of protein ubiquitination in pollen from male and female kiwifruit (Actinidia deliciosa)

Kiwifruit (Actinidia deliciosa) is a dioecious vine whose staminate and pis tillate flowers nonetheless develop non-functional reproductive structures of the opposite sex. Ubiquitin is a small, highly conserved protein found i n all eucaryotes: a covalent ATP-dependent attachment of ubiquitin marks pr oteins for degradation. In the present paper, we used immunoblotting to inv estigate the presence of free ubiquitin and ubiquitin conjugates during pol len development in male (androfertile) and in female (androsterile) genotyp es of kiwifruit. In the male, several high molecular mass protein conjugate s were present throughout development. On the contrary, such a pattern char acterized only early stages of pollen from the female genotype, where conju gates progressively disappeared, until they were detectable only in trace a mounts at anthesis. The highest content of conjugates in the male genotype was observed when microspores were approaching the first mitosis, Free ubiq uitin increased continuously during development of the male microgametophyt e so that mature pollen contained considerable amounts of the ubiquitin mon omer at the time of its release from the anther, By contrast, only low leve ls were detectable in the degenerating microspores in the pistillate flower s. In vitro experiments using labeled ubiquitin indicated that early-uninuc leate microspores of the female genotype had a much higher conjugation rate Ban those of the male genotype at the same stage. However, after feeding a lpha-lactalbumin as exogenous substrate, the rate of ubiquitin conjugation strongly increased and was quite similar in both sexes. Nuclear features of pollen development in both genotypes are also described. The nucleus progr essively degenerated in the microspores of the pistillate flowers starting from the early-uninucleate stage, in parallel with the progressive decrease in ubiquitin content and activity, At anthesis, the microspores in the pis tillate flowers either had no nucleus or showed only traces of chromatin, T hus, the ubiquitin system seems to play an important role in protein turnov er occurring during the normal developmental pathway of the kiwifruit micro gametophyte, while it was mainly involved in regressive events related to m icrospore degeneration in the female genotype.