Involvement of autophagy in the direct ER to vacuole protein trafficking route in plants

Trafficking of proteins from the endoplasmic reticulum (ER) to the vacuole is a fundamental process in plants, being involved both in vacuole biogenesis as well as with plant growth and response to environmental stresses. Although the canonical transport of cellular components from the ER to the vacuole includes the Golgi apparatus as an intermediate compartment, there are multiple lines of evidence that support the existence of a direct ER-to-vacuole, Golgi-independent, trafficking route in plants that uses the autophagy machinery. Plant autophagy was initially described by electron microscopy, visualizing cellular structures that are morphologically reminiscent of autophagosomes. In some of these reports these structures were shown to transport vacuole residing proteins, particularly seed storage proteins, directly from the ER to the vacuole. More recently, following the discovery of the proteins of the core autophagy machinery, molecular tools were implemented in deciphering the involvement of autophagy in this special trafficking route. Here we review the relatively older and more recent scientific observations, supporting the involvement of autophagy in the special cellular trafficking pathways of plants

Transcriptome Responses to Combinations of Stresses in Arabidopsis

Biotic and abiotic stresses limit agricultural yields, and plants are often simultaneously exposed to multiple stresses. Combinations of stresses such as heat and drought or cold and high light intensity have profound effects on crop performance and yields. Thus, delineation of the regulatory networks and metabolic pathways responding to single and multiple concurrent stresses is required for breeding and engineering crop stress tolerance. Many studies have described transcriptome changes in response to single stresses. However, exposure of plants to a combination of stress factors may require agonistic or antagonistic responses or responses potentially unrelated to responses to the corresponding single stresses. To analyze such responses, we initially compared transcriptome changes in 10 Arabidopsis (Arabidopsis thaliana) ecotypes using cold, heat, high-light, salt, and flagellin treatments as single stress factors as well as their double combinations. This revealed that some 61% of the transcriptome changes in response to double stresses were not predictable from the responses to single stress treatments. It also showed that plants prioritized between potentially antagonistic responses for only 5% to 10% of the responding transcripts. This indicates that plants have evolved to cope with combinations of stresses and, therefore, may be bred to endure them. In addition, using a subset of this data from the Columbia and Landsberg erecta ecotypes, we have delineated coexpression network modules responding to single and combined stresses. Plants are often simultaneousl

Integrative analysis of Paneth cell proteomic and transcriptomic data from intestinal organoids reveals functional processes dependent on autophagy

Paneth cells are key epithelial cells providing an antimicrobial barrier and maintaining integrity of the small intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process which recycles cellular components. The direct effect of autophagy-impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells making these cells impaired in autophagy. Using 3D intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type (WT) and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy targeted proteins and functional information to identify the mechanistic link between autophagy-impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%), predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms degrading proteins key to Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration affect protein level and not gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred down-regulation of exocytosis. Our observations could explain how protein level alterations affect Paneth cell homeostatic functions upon autophagy impairment

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

Relative dependence: Autophagy in the mother plant and the embryo contributes to Arabidopsis seed development

ABSTRACTOur study focused on the role of autophagy in seed development and its impact on nutrient remobilization from the mother plant and seed physiology. By conducting reciprocal crosses between wild-type (WT) and autophagy-deficient (atg mutant) Arabidopsis thaliana (Arabidopsis) plants, we differentiated between autophagy in the maternal tissue and the embryo. We found that autophagy in the maternal tissues did not affect embryo development, yet led to reduced growth of etiolated F1 atg maternal plants, possibly resulting from altered protein accumulation in the seeds. Surprisingly, F1 seeds from maternal atg mutants showed faster germination due to altered seed coat structure, which probably reduced seed longevity. Our results highlight the tissue-specific functions of autophagy, providing insight into the various roles of autophagy in seed development.Abbreviations: Arabidopsis: Arabidopsis thaliana; ATG: autophagy-related; WT: wild-type

Exploring the Contribution of Autophagy to the Excess-Sucrose Response in Arabidopsis thaliana

Autophagy is an essential intracellular eukaryotic recycling mechanism, functioning in, among others, carbon starvation. Surprisingly, although autophagy-deficient plants (atg mutants) are hypersensitive to carbon starvation, metabolic analysis revealed that they accumulate sugars under such conditions. In plants, sugars serve as both an energy source and as signaling molecules, affecting many developmental processes, including root and shoot formation. We thus set out to understand the interplay between autophagy and sucrose excess, comparing wild-type and atg mutant seedlings. The presented work showed that autophagy contributes to primary root elongation arrest under conditions of exogenous sucrose and glucose excess but not during fructose or mannitol treatment. Minor or no alterations in starch and primary metabolites were observed between atg mutants and wild-type plants, indicating that the sucrose response relates to its signaling and not its metabolic role. Extensive proteomic analysis of roots performed to further understand the mechanism found an accumulation of proteins essential for ROS reduction and auxin maintenance, which are necessary for root elongation, in atg plants under sucrose excess. The analysis also suggested mitochondrial and peroxisomal involvement in the autophagy-mediated sucrose response. This research increases our knowledge of the complex interplay between autophagy and sugar signaling in plants