Hadas Peled-Zehavi

Arabidopsis ATG8-INTERACTING PROTEIN1 Is Involved in Autophagy-Dependent Vesicular Trafficking of Plastid Proteins to the Vacuole

A previously unrecognized plastid-to-vacuole protein trafficking pathway that is stress induced and autophagy dependent appears to involve ATI1 interaction with plastid proteins and ATG8 of the core autophagy machinery. Selective autophagy has been extensively studied in various organisms, but knowledge regarding its functions in plants, particularly in organelle turnover, is limited. We have recently discovered ATG8-INTERACTING PROTEIN1 (ATI1) from Arabidopsis thaliana and showed that following carbon starvation it is localized on endoplasmic reticulum (ER)-associated bodies that are subsequently transported to the vacuole. Here, we show that following carbon starvation ATI1 is also located on bodies associating with plastids, which are distinct from the ER ATI bodies and are detected mainly in senescing cells that exhibit plastid degradation. Additionally, these plastid-localized bodies contain a stroma protein marker as cargo and were observed budding and detaching from plastids. ATI1 interacts with plastid-localized proteins and was further shown to be required for the turnover of one of them, as a representative. ATI1 on the plastid bodies also interacts with ATG8f, which apparently leads to the targeting of the plastid bodies to the vacuole by a process that requires functional autophagy. Finally, we show that ATI1 is involved in Arabidopsis salt stress tolerance. Taken together, our results implicate ATI1 in autophagic plastid-to-vacuole trafficking through its ability to interact with both plastid proteins and ATG8 of the core autophagy machinery.

Translation and translational regulation in chloroplasts

The translation mechanism of chloroplast mRNAs originated as prokaryotic-type, but has sinceevolved considerably. Chloroplast translation became, in large part, uncoupled from transcription,and turned into a highly regulated process. Concomitantly, chloroplast ribosomes, general translationfactors, and transcripts changed substantially from their prokaryotic counterparts. A multitudeof nucleus encoded regulatory proteins evolved that interact in a specific manner with elementsin mRNAs to allow translation regulation in response to environmental and developmental cues. Inthis chapter, we sum up the current knowledge regarding the translation machinery in the chloroplastusing examples of mechanisms utilized for chloroplast translation regulation.

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

This study identifies a regulatory oxidative pathway, comprised of thioredoxin and peroxiredoxin, in Arabidopsis thaliana chloroplasts. It shows that the pathway is used to sense photosynthetic peroxide formation under low to moderate light intensity and proposes that the oxidative signal adjusts the photosynthetic linear electron flow to fluctuating environmental conditions. The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.

Chloroplast degradation: one organelle, multiple degradation pathways.

Degradation of chloroplasts is a hallmark of both natural and stress-induced plant senescence. Autophagy and senescence-associated vacuoles are two established cellular pathways for chloroplast degradation. Recently, a third independent pathway for chloroplast degradation was reported. Here we will discuss this new discovery in relation to the other known pathways.

A small family of chloroplast atypical thioredoxins.

The reduction and the formation of regulatory disulfide bonds serve as a key signaling element in chloroplasts. Members of the thioredoxin (Trx) superfamily of oxidoreductases play a major role in these processes. We have characterized a small family of plant-specific Trxs in Arabidopsis (Arabidopsis thaliana) that are rich in cysteine and histidine residues and are typified by a variable noncanonical redox active site. We found that the redox midpoint potential of three selected family members is significantly less reducing than that of the classic Trxs. Assays of subcellular localization demonstrated that all proteins are localized to the chloroplast. Selected members showed high activity, contingent on a dithiol electron donor, toward the chloroplast 2-cysteine peroxiredoxin A and poor activity toward the chloroplast NADP-malate dehydrogenase. The expression profile of the family members suggests that they have distinct roles. The intermediate redox midpoint potential value of the atypical Trxs might imply adaptability to function in modulating the redox state of chloroplast proteins with regulatory disulfides.

hfAIM: A reliable bioinformatics approach for in silico genome-wide identification of autophagy-associated Atg8-interacting motifs in various organisms

ABSTRACT Most of the proteins that are specifically turned over by selective autophagy are recognized by the presence of short Atg8 interacting motifs (AIMs) that facilitate their association with the autophagy apparatus. Such AIMs can be identified by bioinformatics methods based on their defined degenerate consensus F/W/Y-X-X-L/I/V sequences in which X represents any amino acid. Achieving reliability and/or fidelity of the prediction of such AIMs on a genome-wide scale represents a major challenge. Here, we present a bioinformatics approach, high fidelity AIM (hfAIM), which uses additional sequence requirements—the presence of acidic amino acids and the absence of positively charged amino acids in certain positions—to reliably identify AIMs in proteins. We demonstrate that the use of the hfAIM method allows for in silico high fidelity prediction of AIMs in AIM-containing proteins (ACPs) on a genome-wide scale in various organisms. Furthermore, by using hfAIM to identify putative AIMs in the Arabidopsis proteome, we illustrate a potential contribution of selective autophagy to various biological processes. More specifically, we identified 9 peroxisomal PEX proteins that contain hfAIM motifs, among which AtPEX1, AtPEX6 and AtPEX10 possess evolutionary-conserved AIMs. Bimolecular fluorescence complementation (BiFC) results verified that AtPEX6 and AtPEX10 indeed interact with Atg8 in planta. In addition, we show that mutations occurring within or nearby hfAIMs in PEX1, PEX6 and PEX10 caused defects in the growth and development of various organisms. Taken together, the above results suggest that the hfAIM tool can be used to effectively perform genome-wide in silico screens of proteins that are potentially regulated by selective autophagy. The hfAIM system is a web tool that can be accessed at link: http://bioinformatics.psb.ugent.be/hfAIM/.

Autophagy-related approaches for improving nutrient use efficiency and crop yield protection

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

Metabolic Engineering of the Phenylpropanoid and Its Primary, Precursor Pathway to Enhance the Flavor of Fruits and the Aroma of Flowers

Plants produce a diverse repertoire of specialized metabolites that have multiple roles throughout their life cycle. Some of these metabolites are essential components of the aroma and flavor of flowers and fruits. Unfortunately, attempts to increase the yield and prolong the shelf life of crops have generally been associated with reduced levels of volatile specialized metabolites and hence with decreased aroma and flavor. Thus, there is a need for the development of new varieties that will retain their desired traits while gaining enhanced scent and flavor. Metabolic engineering holds great promise as a tool for improving the profile of emitted volatiles of domesticated crops. This mini review discusses recent attempts to utilize metabolic engineering of the phenylpropanoid and its primary precursor pathway to enhance the aroma and flavor of flowers and fruits.

Fluorescence Imaging of Autophagy-Mediated ER-to-Vacuole Trafficking in Plants

Macroautophagy (hereafter referred to as autophagy) is a conserved mechanism in eukaryotic cells that delivers unneeded cellular components for degradation in the lytic organelle. In plants, as in other eukaryotes, autophagy begins in the formation of cup-shaped double membranes that engulf cytosolic material. The double membrane closes to form autophagosomes that are then transported to the vacuole for degradation. Autophagy can function as a bulk nonselective process or as a selective process targeting specific proteins, protein aggregates, organelles, or other cellular components for degradation. The endoplasmic reticulum (ER) is linked to autophagy-related processes in multiple ways. The ER was suggested as a possible site for the nucleation of autophagosomes, and as a source for autophagosomal membranes. Furthermore, autophagy has an important role in ER homeostasis, and the ER is a target for a selective type of autophagy, ER-phagy, in response to ER stress. However, the detailed molecular mechanisms, especially in plants, are only now starting to be revealed.In this chapter, we describe the use of confocal imaging to follow the delivery of fluorescently tagged ER-associated proteins to the vacuole. We also describe the utilization of fluorescent protein fusions to look at the co-localization of a protein of interest with the autophagosome marker protein ATG8, a core autophagy machinery protein that is essential for selective autophagy processes.

Corrigendum: Autophagy-related approaches for improving nutrient use efficiency and crop yield protection

Tamar Avin-Wittenberg, Frantisek Baluška, Peter V. Bozhkov, Pernilla H. Elander, Alisdair R. Fernie, Gad Galili, Ammar Hassan, Daniel Hofius, Erika Isono, Romain Le Bars, Céline Masclaux-Daubresse, Elena A. Minina, Hadas Peled-Zehavi, Núria S. Coll, Luisa M. Sandalio, Béatrice Satiat-Jeunemaitre, Agnieszka Sirko, Pilar S. Testillano and Henri Batoko* 1 Department of Plant and Environmental Sciences, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram, Jerusalem 9190401, Israel 2 Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany 3 Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7015, SE-75007 Uppsala, Sweden 4 Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany 5 Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot 76100 Israel 6 Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center of Plant Biology, SE75007 Uppsala, Sweden 7 Department of Biology, University of Konstanz, Universitätsstrasse 10, D-78464 Konstanz, Germany 8 Cell Biology Pôle Imagerie-Gif, Institute of Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91 198, Gif-sur-Yvette, France 9 INRA-AgroParisTech, Institut Jean-Pierre Bourgin, UMR1318, ERL CNRS 3559, Saclay Plant Sciences, Versailles, France 10 Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra-Cerdanyola del Valles 08193, Catalonia, Spain 11 Departmento de Bioquímica, Biología Celular y Molecular de Plantas Experimental del Zaidín, CSIC, 18008 Granada, Spain 12 Institute of Biochemistry and Biophysics Polish Academy of Sciences, ul. Pawinskiego 5A, 02-106 Warsaw, Poland 13 Pollen Biotechnology of Crop Plants group, Centro de Investigaciones Biológicas, Biological Research Centre (CIB), CSIC, Ramiro de Maeztu, 9, 28040 Madrid, Spain 14 Université Catholique de Louvain, Institute of Life Sciences, Croix du Sud 4, L7.07.14, 1348 Louvain-la-Neuve, Belgium