Cell growth and homeostasis are disrupted in arabidopsis rns2-2 mutants missing the main vacuolar RNase activity.

BACKGROUND AND AIMS: Enzymes belonging to the RNase T2 family are essential for normal rRNA turnover in eukaryotes. In Arabidopsis thaliana, this function is performed by RNS2. The null mutant rns2-2 has increased rRNA half-life and constitutive autophagy. The aim of this work was to determine the molecular changes that take place in the rns2-2 mutant that may lead to altered cellular homeostasis, manifested by the observed cellular phenotype. METHODS: To determine the effect of defective rRNA turnover on cellular homeostasis, comparative transcriptome and metabolome analyses of 10-day-old wild-type and rns2-2 seedlings were used to identify molecular processes affected in the mutant. Bioinformatics analyses suggested additional phenotypes that were confirmed through direct plant size measurements and microscopy. KEY RESULTS: Few genes were differentially expressed in the rns2-2 mutant, indicating that control of autophagy in this genotype is mainly achieved at the post-transcriptional level. Among differentially expressed genes, transcripts related to carbon flux processes, particularly the pentose phosphate pathway (PPP), were identified. Metabolite analyses confirmed changes in the levels of PPP intermediates. Genes related to cell wall loosening were also differentially expressed in the mutant, and a decrease in monosaccharide components of cell wall hemicellulose were found. As a potential effect of weaker cell walls, rns2-2 plants are larger than wild-type controls, due to larger cells and increased water content. Elevated levels of reactive oxygen species (ROS) were also measured in rns2-2, and the constitutive autophagy phenotype was blocked by preventing ROS production via NADPH oxidase. CONCLUSIONS: Lack of rRNA recycling in rns2-2 cells triggers a change in carbon flux, which is redirected through the PPP to produce ribose-5-phosphate for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential for cellular homeostasis, probably through maintenance of nucleoside levels as part of the salvage pathway.

Validation of a hairy roots system to study soybean-soybean aphid interactions.

The soybean aphid (Aphis glycines) is one of the main insect pests of soybean (Glycine max) worldwide. Genomics approaches have provided important data on transcriptome changes, both in the insect and in the plant, in response to the plant-aphid interaction. However, the difficulties to transform soybean and to rear soybean aphid on artificial media have hindered our ability to systematically test the function of genes identified by those analyses as mediators of plant resistance to the insect. An efficient approach to produce transgenic soybean material is the production of transformed hairy roots using Agrobacterium rhizogenes; however, soybean aphids colonize leaves or stems and thus this approach has not been utilized. Here, we developed a hairy root system that allowed effective aphid feeding. We show that this system supports aphid performance similar to that observed in leaves. The use of hairy roots to study plant resistance is validated by experiments showing that roots generated from cotyledons of resistant lines carrying the Rag1 or Rag2 resistance genes are also resistant to aphid feeding, while related susceptible lines are not. Our results demonstrate that hairy roots are a good system to study soybean aphid-soybean interactions, providing a quick and effective method that could be used for functional analysis of the resistance response to this insect.

Localization of RNS2 ribonuclease to the vacuole is required for its role in cellular homeostasis.

MAIN CONCLUSION: Localization of the RNase RNS2 to the vacuole via a C-terminal targeting signal is essential for its function in rRNA degradation and homeostasis. RNase T2 ribonucleases are highly conserved enzymes present in the genomes of nearly all eukaryotes and many microorganisms. Their constitutive expression in different tissues and cell types of many organisms suggests a housekeeping role in RNA homeostasis. The Arabidopsis thaliana class II RNase T2, RNS2, is encoded by a single gene and functions in rRNA degradation. Loss of RNS2 results in RNA accumulation and constitutive activation of autophagy, possibly as a compensatory mechanism. While the majority of RNase T2 enzymes is secreted, RNS2 is located within the vacuole and in the endoplasmic reticulum (ER), possibly within ER bodies. As RNS2 has a neutral pH optimum, and the endomembrane organelles are connected by vesicle transport, the site within the endomembrane system at which RNS2 functions is unclear. Here we demonstrate that localization to the vacuole is essential for the physiological function of RNS2. A mutant allele of RNS2, rns2-1, results in production of an active RNS2 RNase but with a mutation that removes a putative C-terminal vacuolar targeting signal. The mutant protein is, therefore, secreted from the cell. This results in a constitutive autophagy phenotype similar to that observed in rns2 null mutants. These findings illustrate that the intracellular retention of RNS2 and localization within the vacuole are critical for its cellular function.

NnSR1, a class III non-S-RNase specifically induced in Nicotiana alata under phosphate deficiency, is localized in endoplasmic reticulum compartments.

A combined strategy of phosphate (Pi) remobilization from internal and external RNA sources seems to be conserved in plants exposed to Pi starvation. Thus far, the only ribonucleases (RNases) reported to be induced in Nicotiana alata undergoing Pi deprivation are extracellular S-like RNase NE and NnSR1. NnSR1 is a class III non S-RNase of unknown subcellular location. Here, we examine the hypothesis that NnSR1 is an intracellular RNase derived from the self-incompatibility system with specific expression in self-incompatible Nicotiana alata. NnSR1 was not induced in self-compatible Nicotiana species exposed to Pi deprivation. NnSR1 conjugated with a fluorescent protein and transiently expressed in Arabidopsis protoplasts and Nicotiana leaves showed that the fusion protein co-localized with an endoplasmic reticulum (ER) marker. Subcellular fractionation by ultracentrifugation of roots exposed to Pi deprivation revealed that the native NnSR1 migrated in parallel with the BiP protein, a typical ER marker. To our knowledge, NnSR1 is the first class III RNase reported to be localized in ER compartments. The induction of NnSR1 was detected earlier than the extracellular RNase NE, suggesting that intracellular RNA may be the first source of Pi used by the cell under Pi stress.

Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis.

Ribosomes account for a majority of the cell's RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2-2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2-2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2-2, atg9-4, atg5-1, rns2-2 atg9-4, and rns2-2 atg5-1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2-2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2-2 atg5-1 double mutant but not by an rns2-2 atg9-4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.

What to eat: evidence for selective autophagy in plants.

Autophagy is a macromolecular degradation pathway by which cells recycle their contents as a developmental process, housekeeping mechanism, and response to environmental stress. In plants, autophagy involves the sequestration of cargo to be degraded, transport to the cell vacuole in a double-membrane bound autophagosome, and subsequent degradation by lytic enzymes. Autophagy has generally been considered to be a non-selective mechanism of degradation. However, studies in yeast and animals have found numerous examples of selective autophagy, with cargo including proteins, protein aggregates, and organelles. Recent work has also provided evidence for several types of selective autophagy in plants. The degradation of protein aggregates was the first selective autophagy described in plants, and, more recently, a hybrid protein of the mammalian selective autophagy adaptors p62 and NBR1, which interacts with the autophagy machinery and may function in autophagy of protein aggregates, was described in plants. Other intracellular components have been suggested to be selectively targeted by autophagy in plants, but the current evidence is limited. Here, we discuss recent findings regarding the selective targeting of cell components by autophagy in plants. [Formula: see text] [ Diane C. Bassham (Corresponding author)].