Upon encountering oxidative stress, proteins are oxidized extensively by highly reactive and toxic reactive oxidative species, and these damaged, oxidized proteins need to be degraded rapidly and effectively. There are two major proteolytic systems for bulk degradation in eukaryotes, the proteasome and vacuolar autophagy. In mammalian cells, the 20S proteasome and a specific type of vacuolar autophagy, chaperone-mediated autophagy, are involved in the degradation of oxidized proteins in mild oxidative stress. However, little is known about how cells remove oxidized proteins when under severe oxidative stress. Using two macroautophagy markers, monodansylcadaverine and green fluorescent protein-AtATG8e, we here show that application of hydrogen peroxide or the reactive oxidative species inducer methyl viologen can induce macroautophagy in Arabidopsis (Arabidopsis thaliana) plants. Macroautophagy-defective RNAi-AtATG18a transgenic plants are more sensitive to methyl viologen treatment than wild-type plants and accumulate a higher level of oxidized proteins due to a lower degradation rate. In the presence of a vacuolar H 1 -ATPase inhibitor, concanamycin A, oxidized proteins were detected in the vacuole of wild-type root cells but not RNAi-AtATG18a root cells. Together, our results indicate that autophagy is involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis.

SummaryVacuolar autophagy is a major pathway by which eukaryotic cells degrade macromolecules, either to remove damaged or unnecessary proteins, or to produce respiratory substrates and raw materials to survive periods of nutrient deficiency. During autophagy, a double membrane forms around cytoplasmic components to generate an autophagosome, which is transported to the vacuole. The outer membrane fuses with the vacuole or lysosome, and the inner membrane and its contents are degraded by vacuolar or lysosomal hydrolases. We have identified a small gene family in Arabidopsis thaliana, members of which show sequence similarity to the yeast autophagy gene ATG18. Members of the AtATG18 gene family are differentially expressed in response to different growth conditions, and one member of this family, AtATG18a, is induced both during sucrose and nitrogen starvation and during senescence. RNA interference was used to generate transgenic lines with reduced AtATG18a expression. These lines show hypersensitivity to sucrose and nitrogen starvation and premature senescence, both during natural senescence of leaves and in a detached leaf assay. Staining with the autophagosome-specific fluorescent dye monodansylcadaverine revealed that, unlike wild-type plants, AtATG18a RNA interference plants are unable to produce autophagosomes in response to starvation or senescence conditions. We conclude that the AtATG18a protein is likely to be required for autophagosome formation in Arabidopsis.

Upon encountering nutrient stress conditions, plant cells undergo extensive metabolic changes and induce nutrient recycling pathways for their continued survival. The role of nutrient mobilization in the response of Arabidopsis suspension cells to Suc starvation was examined. Vacuolar autophagy was induced within 24 h of starvation, with increased expression of vacuolar proteases that are likely to be required for degradation of cytoplasmic components delivered to the vacuole, and thus for nutrient recycling. After 48 h of starvation, culture viability began to decrease, and substantial cell death was evident by 72 h. To provide further insight into the pathways required for survival during Suc deficit, transcriptional profiling during Suc starvation was performed using the ATH1 GeneChip array containing 22,810 probe sets. A significant increase in transcript levels was observed for 343 genes within 48 h of starvation, indicating a response to nutrient stress that utilizes the recycling of cellular components and nutrient scavenging for maintaining cell function, the protection of the cell from death through activation of various defense and stress response pathways, and regulation of these processes by specific protein kinases and transcription factors. These physiological and molecular data support a model in which plant cells initiate a coordinated response of nutrient mobilization at the onset of Suc depletion that is able to maintain cell viability for up to 48 h. After this point, genes potentially involved in cell death increase in expression, whereas those functioning in translation and replication decrease, leading to a decrease in culture viability and activation of cell death programs.

SummaryAutophagy is a process that is thought to occur in all eukaryotes in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon induction of autophagy, double membrane-bound structures called autophagosomes engulf portions of the cytoplasm and transfer them to the vacuole or lysosome for degradation. In this study, we have characterized two potential markers for autophagy in plants, the fluorescent dye monodansylcadaverine (MDC) and a green fluorescent protein (GFP)-AtATG8e fusion protein, and propose that they both label autophagosomes in Arabidopsis. Both markers label the same small, apparently membrane-bound structures found in cells under conditions that are known to induce autophagy such as starvation and senescence. They are usually seen in the cytoplasm, but occasionally can be observed within the vacuole, consistent with a function in the transfer of cytoplasmic material into the vacuole for degradation. MDC-staining and the GFP-AtATG8e fusion protein can now be used as very effective tools to complement biochemical and genetic approaches to the study of autophagy in plant systems.

RNase T2 enzymes are conserved in most eukaryotic genomes, and expression patterns and phylogenetic analyses suggest that they may carry out an important housekeeping role. However, the nature of this role has been elusive. Here we show that RNS2, an intracellular RNase T2 from Arabidopsis thaliana, is essential for normal ribosomal RNA recycling. This enzyme is the main endoribonuclease activity in plant cells and localizes to the endoplasmic reticulum (ER), ER-derived structures, and vacuoles. Mutants lacking RNS2 activity accumulate RNA intracellularly, and rRNA in these mutants has a longer half-life. Normal rRNA turnover seems essential to maintain cell homeostasis because rns2 mutants display constitutive autophagy. We propose that RNS2 is part of a process that degrades rRNA to recycle its components. This process appears to be conserved in all eukaryotes. ribophagy R ibonucleases (RNases) belonging to the RNase T2 family are acidic endonucleases without base specificity that are either extracellular or targeted to the secretory pathway (1). This family is conserved in the genome of almost all eukaryotic organisms so far analyzed, suggesting that it performs an important function that has been maintained throughout evolution (2, 3). Phylogenetic analyses have identified three subclasses present in plant genomes (3, 4). Class I proteins are highly diversified and show evidence of gene sorting, and their expression is commonly tissue-specific and/or regulated by biotic and abiotic stress (3). Class III includes mostly members of the S-RNases, enzymes involved in the process of gametophytic self-incompatibility in three plant families (5). Finally, class II includes proteins that are highly conserved in all plant genomes and are normally expressed at high levels in most plant tissues (3). On the basis of their conservation and gene expression characteristics, we proposed that class II RNases may have a housekeeping role in plant cells (3). Similar gene expression and phylogenetic studies in animals led us to the hypothesis that metazoan RNase T2 enzymes also have a housekeeping role and may be equivalent to class II enzymes from plants (2).RNS2, one of five RNase T2 genes in the Arabidopsis genome, encodes the only class II protein in this model organism (3,4). This RNase is present in all tissues at high levels (6, 7) and is localized in an intracellular compartment. RNS2 expression is increased even further during senescence and during inorganic phosphate (Pi) starvation (6, 8). Thus, it was hypothesized that RNS2 is part of a phosphate scavenging system that rescues plants that are under nutritional stress (9). However, because RNS2 and other class II RNase T2 proteins accumulate to high levels even under optimal growth conditions, this rescue function is unlikely to be the main role of these enzymes. Moreover, in vertebrates, in which RNase T2 enzymes are absolutely conserved (2), the mechanisms that control the response to phosphate starvation seem to be specific to the intestine and kidney (10), wherea...

Plant cells frequently encounter oxidative stress, leading to oxidative damage and inactivation of proteins. We have recently demonstrated that oxidative stress induces autophagy in Arabidopsis seedlings in an AtATG18a-dependent manner and that RNAi-AtATG18a transgenic lines, which are defective in autophagosome formation, are hypersensitive to reactive oxygen species. Analysis of protein oxidation indicated that oxidized proteins are degraded in the vacuole after uptake by autophagy, and this degradation is impaired in RNAi-AtATG18a lines. Our results also suggest that in the absence of a functional autophagy pathway, plants are under increased oxidative stress, even under normal growth conditions.

SummaryEndosomes are a heterogeneous collection of organelles that function in the sorting and delivery of internalized material from the cell surface and the transport of materials from the Golgi to the lysosome or vacuole. Plant endosomes have some unique features, with an organization distinct from that of yeast or animal cells. Two clearly defined endosomal compartments have been studied in plant cells, the trans-Golgi network (equivalent to the early endosome) and the multivesicular body (equivalent to the late endosome), with additional endosome types (recycling endosome, late prevacuolar compartment) also a possibility. A model has been proposed in which the trans-Golgi network matures into a multivesicular body, which then fuses with the vacuole to release its cargo. In addition to basic trafficking functions, endosomes in plant cells are known to function in maintenance of cell polarity by polar localization of hormone transporters and in signaling pathways after internalization of ligand-bound receptors. These signaling functions are exemplified by the BRI1 brassinosteroid hormone receptor and by receptors for pathogen elicitors that activate defense responses. After endocytosis of these receptors from the plasma membrane, endosomes act as a signaling platform, thus playing an essential role in plant growth, development and defense responses. Here we describe the key features of plant endosomes and their differences from those of other organisms and discuss the role of these organelles in cell polarity and signaling pathways.

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