Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Polyamines and WOX genes in the recalcitrance to plant conversion of somatic embryos of Habanero pepper (Capsicum chinense Jacq.)

In order to determine the role of polyamines in the formation and development of the somatic embryos of Capsicum chinense, the effect of different concentrations (0, 0.01, 0.1, and 1.0 mM) of Putrescine, Spermidine and Spermine on the efficiency and morphology of the embryos was evaluated. The results show that none of the three polyamines evaluated had a significant effect on the number of embryos formed, except Spermidine (1 mM), which caused a significant reduction in their numbers, in comparison with the control treatment. However, the most noteworthy result was observed in the treatment containing 0.1 mM of Spermine. The embryos developed in this treatment showed harmonic apex-radicle development, pale-green coloration and the formation of two tiny cotyledonary leaves. Real-time PCR was used to analyze the differential expression of the WUS, WOX1 and WOX3 genes in somatic embryos treated with Spermine and untreated, including the zygotic embryo. The transcript levels of the genes analyzed were found to differ significantly between both types of embryos (somatic and zygotic), with the zygotic embryos presenting a higher level of transcripts; however, compared to the untreated somatic embryos, the somatic embryos treated with Spermine showed an increase in the transcript levels of the three genes analyzed (WUS, WOX1 and WOX3); the WOX1 gene in particular presented an accumulation pattern similar to that of the zygotic embryo of the species. Keywords: Somatic embryos, zygotic embryos, polyamines, transcript patterns and morphology.