Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Mapping of quantitative trait loci for floral scent compounds in cowpea (Vigna unguiculata L.)

Floral scent is a very important trait in plant evolution. Currently, little is known about the inheritance of floral scent in cowpea (Vigna unguiculata L.) or changes that might have occurred during its domestication. Therefore, we analysed scent volatiles and molecular markers in a population of 159 F7 recombinant inbred lines derived from a cross of a domesticated blackeye cowpea cultivar, ‘524B’ and a wild accession ‘219-01’. Using gas chromatography-mass spectrometry (GC–MS) 23 volatile compounds were identified that fall into five general functional categories. Twenty-two of the compounds displayed quantitative variation in the progeny, and a total of 63 QTLs influencing the amounts of these volatiles were mapped onto the cowpea genetic marker map. Although QTLs for volatile compounds putatively involved in cowpea flower scent were found on 9 of the 11 cowpea chromosomes, they were not evenly distributed with QTLs mainly clustered on LGs 1, LGs 2 and LG 4. Our results serve as a starting point for both more detailed analyses of complex scent biosynthetic pathways and the development of markers for marker-assisted breeding of scented rose varieties

Agro-ecological distribution of the phenotypic diversity of aerial yam (Dioscorea bulbifera L.) in Cameroon using multivariate analysis: prospect for germplasm conservation and improvement

Aerial yam (Dioscorea bulbifera L.) is a crop of great economic importance and an excellent candidate for improving food security in developing countries. Understanding the genetic variability of any crop species is a decisive step for its improvement and requires characterization and evaluation of available germplasm. The objectives of this study were to determine the extent of genetic variability, estimate the association between agromorphological traits and clustering among 57 genotypes of aerial yam from three distinct agro-ecological zones in Cameroon using multivariate analysis. Thirty nine characters (23 qualitative and 16 quantitative) were used for the study. Significant differences in genetic diversity indices were found. Accessions from the bimodal humid forest zone (Na = 2.08, He = 0.27) showed significantly lower diversity compared to both western highland (Na = 2.30, He = 0.34) and humid monomodal forest zones (Na = 2.57, He = 0.32). Means values of most quantitative traits also showed significant differences between agro-ecological zones. Batingla-3 and Bawouwoua-1 had important bulbil yield, reaching 3500 g / plant. Significant associations were found between many traits. The use of the Unweighted Pair Group Method with Arithmetic Mean allowed the distribution of the 57 genotypes into six distinct clusters with the clustering pattern not showing any parallelism with location sites or agro-ecological zones. Mahalanobis D2 statistics revealed the highest inter-cluster distance between cluster II and VI. Accessions of these clusters are potential parents for future breeding programs. This study showed that aerial yam from Cameroon has an enormous wealth of traits variation, indicating huge potential for its genetic improvement through selection and hybridization

Genetic structure and mating system of wild cowpea populations in West Africa

Abstract Background Cowpea is a highly inbred crop. It is part of a crop-weed complex, whose origin and dynamics is unknown, which is distributed across the African continent. This study examined outcrossing rates and genetic structures in 35 wild cowpea (Vigna unguiculata ssp. unguiculata var. spontanea) populations from West Africa, using 21 isozyme loci, 9 of them showing polymorphism. Results Outcrossing rates ranged from 1% to 9.5% (mean 3.4%), which classifies the wild cowpea breeding system as primarily selfing, though rare outcrossing events were detected in each population studied. Furthermore, the analyses of both the genetic structure of populations and the relationships between the wild and domesticated groups suggest possibilities of gene flow that are corroborated by field observations. Conclusions As expected in a predominantly inbred breeding system, wild cowpea shows high levels of genetic differentiation and low levels of genetic diversity within populations. Gene flow from domesticated to wild cowpea does occur, although the lack of strong genetic swamping and modified seed morphology in the wild populations suggest that these introgressions should be rare.</p

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

No abstract available

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present