Nuclear export of replication protein A in the nonreplicative infective forms of Trypanosoma cruzi.

Replication protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.

Identification and expression analysis of ethylene biosynthesis and signaling genes provides insights into the early and late coffee cultivars ripening pathway.

The plant hormone ethylene is involved in the regulation of a multitude of plant processes, ranging from seed germination to organ senescence. Ethylene induces fruit ripening in climacteric fruits, such as coffee, being directly involved in fruit ripening time and synchronization. Coffee early cultivars usually show a more uniform ripening process although little is known about the genetic factors that promote the earliness of ripening. Thus, this work aimed to characterize the putative members of the coffee (Coffea arabica) ethylene biosynthesis and signaling pathways, as well as to analyze the expression patterns of these members during fruit ripening of early (Catucaí 785-15) and late (Acauã) coffee cultivars. Reverse Transcription-qPCR analysis of the four biosynthesis genes (CaACS1-like; CaACO1-like; CaACO4-like e CaACO5-like) analyzed in this study showed that CaACO1-like and CaACO4-like displayed an expression pattern typically observed in climacteric fruits, being up-regulated during ripening. CaACS1-like gene expression was also up-regulated during fruit ripening of both cultivars, although in a much lesser extent when compared to the changes in CaACO1-like and CaACO4-like gene expression. CaACO5-like was only induced in raisin fruit and may be related to senescence processes. On the other hand, members of the ethylene signaling pathway (CaETR1-like, CaETR4-like, CaCTR2-like, CaEIN2-like, CaEIN3-like, CaERF1) showed slightly higher expression levels during the initial stages of development (green and yellow-green fruits), except for the ethylene receptors CaETR1-like and CaETR4-like, which were constitutively expressed and induced in cherry fruits, respectively. The higher ethylene production levels in Catucaí 785-15 fruits, indicated by the expression analysis of CaACO1-like and CaACO4-like, suggest that it promotes an enhanced CaETR4-like degradation, leading to an increase in ethylene sensitivity and consequently to an earliness in the ripening process of this cultivar. Ethylene production in Acauã fruits may not be sufficient to inactivate the CaETR4-like levels and thus ripening changes occur in a slower pace. Thus, the expression analysis of the ethylene biosynthesis and signaling genes suggests that ethylene is directly involved in the determination of the ripening time of coffee fruits, and CaACO1-like, CaACO4-like and CaETR4-like may display essential roles during coffee fruit ripening.