45S rDNA Diversity In Natura as One Step towards Ribosomal Heterogeneity in Arabidopsis thaliana.

The keystone of ribosome biogenesis is the transcription of 45S rDNA. The Arabidopsis thaliana genome contains hundreds of 45S rDNA units; however, they are not all transcribed. Notably, 45S rDNA units contain insertions/deletions revealing the existence of heterogeneous rRNA genes and, likely, heterogeneous ribosomes for rRNAs. In order to obtain an overall picture of 45S rDNA diversity sustaining the synthesis of rRNAs and, subsequently, of ribosomes in natura, we took advantage of 320 new occurrences of Arabidopsis thaliana as a metapopulation named At66, sampled from 0 to 1900 m of altitude in the eastern Pyrenees in France. We found that the 45S rDNA copy number is very dynamic in natura and identified new genotypes for both 5' and 3' External Transcribed Spacers (ETS). Interestingly, the highest 5'ETS genotype diversity is found in altitude while the highest 3'ETS genotype diversity is found at sea level. Structural analysis of 45S rDNA also shows conservation in natura of specific 5'ETS and 3'ETS sequences/features required to control rDNA expression and the processing of rRNAs. In conclusion, At66 is a worthwhile natural laboratory, and unraveled 45S rDNA diversity represents an interesting starting material to select subsets for rDNA transcription and alter the rRNA composition of ribosomes both intra- and inter-site.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

Arabidopsis SMO2 modulates ribosome biogenesis by maintaining the RID2 abundance during organ growth.

Ribosome biogenesis is a process of making ribosomes that is tightly linked with plant growth and development. Here, through a suppressor screen for the smo2 mutant, we found that lack of a ribosomal stress response mediator, ANAC082 partially restored growth defects of the smo2 mutant, indicating SMO2 is required for the repression of nucleolar stress. Consistently, the smo2 knock-out mutant exhibited typical phenotypes characteristic of ribosome biogenesis mutants, such as pointed leaves, aberrant leaf venation, disrupted nucleolar structure, abnormal distribution of rRNA precursors, and enhanced tolerance to aminoglycoside antibiotics that target ribosomes. SMO2 interacted with ROOT INITIATION DEFECTIVE 2 (RID2), a methyltransferase-like protein required for pre-rRNA processing. SMO2 enhanced RID2 solubility in Escherichia coli and the loss of function of SMO2 in plant cells reduced RID2 abundance, which may result in abnormal accumulation of FIBRILLARIN 1 (FIB1) and NOP56, two key nucleolar proteins, in high-molecular-weight protein complex. Taken together, our results characterized a novel plant ribosome biogenesis factor, SMO2 that maintains the abundance of RID2, thereby sustaining ribosome biogenesis during plant organ growth.

Light signals counteract alterations caused by simulated microgravity in proliferating plant cells.

PREMISE: Light and gravity are fundamental cues for plant development. Our understanding of the effects of light stimuli on plants in space, without gravity, is key to providing conditions for plants to acclimate to the environment. Here we tested the hypothesis that the alterations caused by the absence of gravity in root meristematic cells can be counteracted by light. METHODS: Seedlings of wild-type Arabidopsis thaliana and two mutants of the essential nucleolar protein nucleolin (nuc1, nuc2) were grown in simulated microgravity, either under a white light photoperiod or under continuous darkness. Key variables of cell proliferation (cell cycle regulation), cell growth (ribosome biogenesis), and auxin transport were measured in the root meristem using in situ cellular markers and transcriptomic methods and compared with those of a 1 g control. RESULTS: The incorporation of a photoperiod regime was sufficient to attenuate or suppress the effects caused by gravitational stress at the cellular level in the root meristem. In all cases, values for variables recorded from samples receiving light stimuli in simulated microgravity were closer to values from the controls than values from samples grown in darkness. Differential sensitivities were obtained for the two nucleolin mutants. CONCLUSIONS: Light signals may totally or partially replace gravity signals, significantly improving plant growth and development in microgravity. Despite that, molecular alterations are still compatible with the expected acclimation mechanisms, which need to be better understood. The differential sensitivity of nuc1 and nuc2 mutants to gravitational stress points to new strategies to produce more resilient plants to travel with humans in new extraterrestrial endeavors.

An In Vitro Approach To Study RNase III Activities of Plant RTL Proteins.

RTL (RNase three-like) proteins belong to a distinct family of endonucleases that cleave double-stranded RNAs in plants. RTL1 to 3 are structurally related to the RNAse III from E. coli and formally belong to the class 1 of RNase III proteins. RTLs have conserved RNase III signature motif(s) and up to two dsRNA binding (DRB) domains. RTLs target and cleave coding and noncoding dsRNAs, including precursors of ribosomal (rRNA), small interference (siRNA), and micro (miRNA) RNAs. Interestingly, RTL proteins have stronger affinity than RNase III-Dicer proteins for dsRNA precursors of siRNAs, but not for miRNAs. However, very little is known of the structural and molecular bases directing and controlling RTL-RNA binding and activity. To address these questions, we have developed in vitro cleavage assays that combine recombinant RTL1 protein and in vitro transcribed or plant-extracted RNAs, RT-PCR, and primer extension experiments or analysis.

SMALL ORGAN4 Is a Ribosome Biogenesis Factor Involved in 5.8S Ribosomal RNA Maturation.

Ribosome biogenesis is crucial for cellular metabolism and has important implications for disease and aging. Human (Homo sapiens) glioma tumor-suppressor candidate region gene2 (GLTSCR2) and yeast (Saccharomyces cerevisiae) Nucleolar protein53 (Nop53) are orthologous proteins with demonstrated roles as ribosome biogenesis factors; knockdown of GLTSCR2 impairs maturation of 18S and 5.8S ribosomal RNAs (rRNAs), and Nop53 is required for maturation of 5.8S and 25S rRNAs. Here, we characterized SMALL ORGAN4 (SMO4), the most likely ortholog of human GLTSCR2 and yeast Nop53 in Arabidopsis (Arabidopsis thaliana). Loss of function of SMO4 results in a mild morphological phenotype; however, we found that smo4 mutants exhibit strong cytological and molecular phenotypes: nucleolar hypertrophy and disorganization, overaccumulation of 5.8S and 18S rRNA precursors, and an imbalanced 40S:60S ribosome subunit ratio. Like yeast Nop53 and human GLTSCR2, Arabidopsis SMO4 participates in 5.8S rRNA maturation. In yeast, Nop53 cooperates with mRNA transport4 (Mtr4) for 5.8S rRNA maturation. In Arabidopsis, we found that SMO4 plays similar roles in the 5.8S rRNA maturation pathway than those described for MTR4. However, SMO4 seems not to participate in the degradation of by-products derived from the 5'-external transcribed spacer (ETS) of 45S pre-rRNA, as MTR4 does.

The siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain.

RNase III enzymes cleave double stranded (ds)RNA. This is an essential step for regulating the processing of mRNA, rRNA, snoRNA and other small RNAs, including siRNA and miRNA. Arabidopsis thaliana encodes nine RNase III: four DICER-LIKE (DCL) and five RNASE THREE LIKE (RTL). To better understand the molecular functions of RNase III in plants we developed a biochemical assay using RTL1 as a model. We show that RTL1 does not degrade dsRNA randomly, but recognizes specific duplex sequences to direct accurate cleavage. Furthermore, we demonstrate that RNase III and dsRNA binding domains (dsRBD) are both required for dsRNA cleavage. Interestingly, the four DCL and the three RTL that carry dsRBD share a conserved cysteine (C230 in Arabidopsis RTL1) in their dsRBD. C230 is essential for RTL1 and DCL1 activities and is subjected to post-transcriptional modification. Indeed, under oxidizing conditions, glutathionylation of C230 inhibits RTL1 cleavage activity in a reversible manner involving glutaredoxins. We conclude that the redox state of the dsRBD ensures a fine-tune regulation of dsRNA processing by plant RNase III.

A duplicated NUCLEOLIN gene with antagonistic activity is required for chromatin organization of silent 45S rDNA in Arabidopsis.

In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.