Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.

The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis.

The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly.

The RAD23 family provides an essential connection between the 26S proteasome and ubiquitylated proteins in Arabidopsis.

The ubiquitin (Ub)/26S proteasome system (UPS) directs the turnover of numerous regulatory proteins, thereby exerting control over many aspects of plant growth, development, and survival. The UPS is directed in part by a group of Ub-like/Ub-associated (UBL/UBA) proteins that help shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Here, we describe the collection of UBL/UBA proteins in Arabidopsis thaliana, including four isoforms that comprise the RADIATION SENSITIVE23 (RAD23) family. The nuclear-enriched RAD23 proteins bind Ub conjugates, especially those linked internally through Lys-48, via their UBA domains, and associate with the 26S proteasome Ub receptor RPN10 via their N-terminal UBL domains. Whereas homozygous mutants individually affecting the four RAD23 genes are without phenotypic consequences (rad23a, rad23c, and rad23d) or induce mild phyllotaxy and sterility defects (rad23b), higher-order mutant combinations generate severely dwarfed plants, with the quadruple mutant displaying reproductive lethality. Both the synergistic effects of a rad23b-1 rpn10-1 combination and the response of rad23b plants to mitomycin C suggest that RAD23b regulates cell division. Taken together, RAD23 proteins appear to play an essential role in the cell cycle, morphology, and fertility of plants through their delivery of UPS substrates to the 26S proteasome.

Dietary RNAs: New Stories Regarding Oral Delivery.

microRNAs (miRNAs), a class of small RNAs, are important regulators of various developmental processes in both plants and animals. Several years ago, a report showed the detection of diet-derived plant miRNAs in mammalian tissues and their regulation of mammalian genes, challenging the traditional functions of plant miRNAs. Subsequently, multiple efforts have attempted to replicate these findings, with the results arguing against the uptake of plant dietary miRNAs in healthy consumers. Moreover, several reports suggest the potential for "false positive" detection of plant miRNAs in human tissues. Meanwhile, some research continues to suggest both the presence and function of dietary miRNAs in mammalian tissues. Here we review the recent literature and discuss the strengths and weaknesses of emerging work that suggests the feasibility of dietary delivery of miRNAs. We also discuss future experimental approaches to address this controversial topic.

Detection of an Abundant Plant-Based Small RNA in Healthy Consumers.

The mechanisms of delivery of plant small RNAs to consumers must be investigated in order to harness this technology to positively impact biotechnology. Two groups have used honeysuckle (Lonicera japonica) feeding regimes to detect a plant-based small RNA, termed MIR2911, in sera. Meanwhile, numerous groups have failed to detect dietary plant-based small RNAs in consumers. Here we catalog levels of MIR2911 in different herbs, and suggest that in particular herb MIR2911 levels are elevated. Feeding these different herb-based diets to mice, we found MIR2911 levels in the sera and urine were associated with dietary intake levels. Abundance was not the sole determinate of apparent RNA bioavailability, as gavage-feeding large-doses of synthetic MIR2911 permitted only small transient increases in serum levels. Dietary MIR2911 were not modified in circulation by association with the host's RNA-induced silencing complex, as the RNA did not co-immunoprecipitate with AGO2. The stability of dietary MIR2911 in circulation differed from synthesized small RNAs, as tail vein administration of various synthetic plant-based small RNAs resulted in rapid clearance. However, synthetic MIR2911 appeared to be more stable than the other plant miRNAs tested. Notably, this uptake of dietary MIR2911 was not related to perturbations in the host's microbiome or gut permeability. We suggest dietary uptake of MIR2911 is commonplace in healthy consumers, and reproducible detection of plant-based small RNAs in consumers depends on dietary abundance, RNA stability and digestion from within the food-matrix.

Mutation of the Arabidopsis LON2 peroxisomal protease enhances pexophagy.

Peroxisomes are critical organelles housing various, often oxidative, reactions. Pexophagy, the process by which peroxisomes are selectively targeted for destruction via autophagy, is characterized in yeast and mammals but had not been reported in plants. In this article, we describe how the peroxisome-related aberrations of a mutant defective in the LON2 peroxisomal protease are suppressed when autophagy is prevented by mutating any of several key autophagy-related (ATG) genes. Our results reveal that plant peroxisomes can be degraded by selective autophagy and suggest that pexophagy is accelerated when the LON2 protease is disabled.

KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling.

Analysis of the Arabidopsis thaliana RING-ANK (for Really Interesting New Gene-Ankyrin) family, a subgroup of RING-type E3 ligases, identified KEEP ON GOING (KEG) as essential for growth and development. In addition to the RING-HCa and ankyrin repeats, KEG contains a kinase domain and 12 HERC2-like repeats. The RING-HCa and kinase domains were functional in in vitro ubiquitylation and phosphorylation assays, respectively. Seedlings homozygous for T-DNA insertions in KEG undergo growth arrest immediately after germination, suggestive of increased abscisic acid (ABA) signaling, a major phytohormone that plays a key role in plant development and survival under unfavorable conditions. Here, we show that KEG is a negative regulator of ABA signaling. keg roots are extremely sensitive to the inhibitory effects of ABA and exhibit hypersensitivity to exogenous glucose, consistent with the known interaction between glucose and ABA signaling. The observations that KEG accumulates high levels of ABSCISIC ACID-INSENSITIVE5 (ABI5) without exogenous ABA, interacts with ABI5 in vitro, and that loss of ABI5 rescues the growth-arrest phenotype of keg mutant seedlings indicate that KEG is required for ABI5 degradation. In this capacity, KEG is central to ABA signaling by maintaining low levels of ABI5 in the absence of stress.

SIRE1, an endogenous retrovirus family from Glycine max, is highly homogeneous and evolutionarily young.

SIRE1 is unusual among Ty1-copia retrotransposons in that it has an additional open reading frame with structural features similar to retroviral envelope proteins between pol and the 3' LTR. Here we report the characterization and comparison of eight different SIRE1 elements derived from a soybean genomic library, as well as SIRE1 reverse transcriptases from Glycine soja. The DNA sequences of the eight SIRE1 elements are highly homogeneous and share greater than 95% nucleotide identity. Partial sequences obtained from BAC ends are similarly conserved. Phylogenetic analyses resolve two closely related SIRE1 lineages, and nucleotide changes within and between SIRE1 lineages have occurred to preserve function. Both the gag and the env-like genes are evolving under similar levels of functional constraint. Considerable sequence heterogeneity in the form of short duplications was found within the LTRs and in the region between the envelope-like ORF and the 3' LTR. These duplications are suggestive of slippage by reverse transcriptase during replication. Sequence identity between LTRs of individual insertions suggests that they transposed within the past 70,000 years. Two of 10 SIRE1 insertions examined abut Ty3-gypsy retroelements. Since the soybean genome harbors more than 1,000 SIRE1 insertions, the collective data suggest that SIRE1 has undergone a very recent and robust amplification in soybean.