Dual Role of a SAS10/C1D Family Protein in Ribosomal RNA Gene Expression and Processing Is Essential for Reproduction in Arabidopsis thaliana.

In eukaryotic cells, ribosomal RNAs (rRNAs) are transcribed, processed, and assembled with ribosomal proteins in the nucleolus. Regulatory mechanisms of rRNA gene (rDNA) transcription and processing remain elusive in plants, especially their connection to nucleolar organization. We performed an in silico screen for essential genes of unknown function in Arabidopsis thaliana and identified Thallo (THAL) encoding a SAS10/C1D family protein. THAL disruption caused enlarged nucleoli in arrested embryos, aberrant processing of precursor rRNAs at the 5' External Transcribed Spacer, and repression of the major rDNA variant (VAR1). THAL overexpression lines showed de-repression of VAR1 and overall reversed effects on rRNA processing sites. Strikingly, THAL overexpression also induced formation of multiple nucleoli per nucleus phenotypic of mutants of heterochromatin factors. THAL physically associated with histone chaperone Nucleolin 1 (NUC1), histone-binding NUC2, and histone demethylase Jumonji 14 (JMJ14) in bimolecular fluorescence complementation assay, suggesting that it participates in chromatin regulation. Furthermore, investigation of truncated THAL proteins revealed that the SAS10 C-terminal domain is likely important for its function in chromatin configuration. THAL also interacted with putative Small Subunit processome components, including previously unreported Arabidopsis homologue of yeast M Phase Phosphoprotein 10 (MPP10). Our results uncovering the dual role of THAL in transcription and processing events critical for proper rRNA biogenesis and nucleolar organization during reproduction are the first to define the function of SAS10/C1D family members in plants.

VPS36-Dependent Multivesicular Bodies Are Critical for Plasmamembrane Protein Turnover and Vacuolar Biogenesis.

Most eukaryotic cells target ubiquitinated plasma membrane (PM) proteins for vacuolar degradation in response to environmental and developmental cues. This process involves endosomal sorting complexes required for transport (ESCRT). However, little is known about the cellular mechanisms of ESCRTs in plants. Here, we studied the function of one ESCRT-II component, VPS36, which shows ubiquitin-binding activity and may form a putative ESCRT-II with VPS22 and VPS25 in Arabidopsis (Arabidopsis thaliana). Recessive mutation of the ubiquitously expressed VPS36 causes multiple defects, including delayed embryogenesis, defective root elongation, and limited expansion of cotyledons, and these effects can be complemented by its genomic DNA. Abnormal intracellular compartments containing several membrane transporters, including members of the PIN-FORMEDs, AUXIN RESISTANT 1, and PIP1 families, were found in vps36-1 plants. Employing a genetic approach to cross vps36-1/+ with transgenic plants harboring various fluorescent protein-tagged organelle markers, as well as fluorescent probe and ultrastructural approaches, revealed PM proteins in microsomal fractions from vps36-1 seedlings and demonstrated that VPS36 is critical for forming multivesicular bodies and vacuolar biogenesis for protein degradation. Our study shows that functional VPS36 is essential for a proper endosomal sorting pathway and for vacuolar biogenesis in Arabidopsis.

Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing.

The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg(300)-to-Trp(300) and Pro(313)-to-Ser(313) amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles.

Arabidopsis CHROMOSOME TRANSMISSION FIDELITY 7 (AtCTF7/ECO1) is required for DNA repair, mitosis and meiosis.

The proper transmission of DNA in dividing cells is crucial for the survival of eukaryotic organisms. During cell division, faithful segregation of replicated chromosomes requires their tight attachment, known as sister chromatid cohesion, until anaphase. Sister chromatid cohesion is established during S-phase in a process requiring an acetyltransferase that in yeast is known as Establishment of cohesion 1 (Eco1). Inactivation of Eco1 typically disrupts chromosome segregation and homologous recombination-dependent DNA repair in dividing cells, ultimately resulting in lethality. We report here the isolation and detailed characterization of two homozygous T-DNA insertion mutants for the Arabidopsis thaliana Eco1 homolog, CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION 1 (CTF7/ECO1), called ctf7-1 and ctf7-2. Mutants exhibited dwarfism, poor anther development and sterility. Analysis of somatic tissues by flow cytometry, scanning electron microscopy and quantitative real-time PCR identified defects in DNA repair and cell division, including an increase in the area of leaf epidermal cells, an increase in DNA content and the upregulation of genes involved in DNA repair including BRCA1 and PARP2. No significant change was observed in the expression of genes that influence entry into the endocycle. Analysis of meiocytes identified changes in chromosome morphology and defective segregation; the abundance of chromosomal-bound cohesion subunits was also reduced. Transcript levels for several meiotic genes, including the recombinase genes DMC1 and RAD51C and the S-phase licensing factor CDC45 were elevated in mutant anthers. Taken together our results demonstrate that Arabidopsis CTF7/ECO1 plays important roles in the preservation of genome integrity and meiosis.

Arabidopsis mTERF15 is required for mitochondrial nad2 intron 3 splicing and functional complex I activity.

Mitochondria play a pivotal role in most eukaryotic cells, as they are responsible for the generation of energy and diverse metabolic intermediates for many cellular events. During endosymbiosis, approximately 99% of the genes encoded by the mitochondrial genome were transferred into the host nucleus, and mitochondria import more than 1000 nuclear-encoded proteins from the cytosol to maintain structural integrity and fundamental functions, including DNA replication, mRNA transcription and RNA metabolism of dozens of mitochondrial genes. In metazoans, a family of nuclear-encoded proteins called the mitochondrial transcription termination factors (mTERFs) regulates mitochondrial transcription, including transcriptional termination and initiation, via their DNA-binding activities, and the dysfunction of individual mTERF members causes severe developmental defects. Arabidopsis thaliana and Oryza sativa contain 35 and 48 mTERFs, respectively, but the biological functions of only a few of these proteins have been explored. Here, we investigated the biological role and molecular mechanism of Arabidopsis mTERF15 in plant organelle metabolism using molecular genetics, cytological and biochemical approaches. The null homozygous T-DNA mutant of mTERF15, mterf15, was found to result in substantial retardation of both vegetative and reproductive development, which was fully complemented by the wild-type genomic sequence. Surprisingly, mitochondria-localized mTERF15 lacks obvious DNA-binding activity but processes mitochondrial nad2 intron 3 splicing through its RNA-binding ability. Impairment of this splicing event not only disrupted mitochondrial structure but also abolished the activity of mitochondrial respiratory chain complex I. These effects are in agreement with the severe phenotype of the mterf15 homozygous mutant. Our study suggests that Arabidopsis mTERF15 functions as a splicing factor for nad2 intron 3 splicing in mitochondria, which is essential for normal plant growth and development.

An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes.

Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

Transcriptomic adaptations in rice suspension cells under sucrose starvation.

Sugar is an important resource for energy generation and developmental regulation in plants, and sucrose starvation causes enormous changes in cellular morphology, enzyme activities and gene expression. Genome-wide gene expression profiling provides a comprehensive knowledge of gene expression under nutrient depletion and senescence; however, that of a monocot model plant, rice, under sucrose depletion is still under investigation. Here, the time-course monitoring of gene expression profiles in sucrose-starved rice (Oryza sativa cv Tainung67) suspension cells was investigated by 21495 probes contained in Agilent rice chip. In sucrose-starved cells, the induced vacuolar biogenesis coincided with significantly upregulated transcripts of H+-pyrophosphatase, delta-TIP, one putative alpha-TIP, several vacuolar proteases and proteinase inhibitors, and one OsATG3. To survey the overall metabolic adaptations under sucrose depletion, the genes with significantly altered expression level were incorporated into multiple metabolic pathways. Most genes encoding enzymes involved in biosynthesis and degradation pathways of various macromolecules were comprehensively down-and upregulated, respectively, with sucrose starvation. Transcriptional regulation of gene expression is important for physiological adaptations to environmental stress, and many transcription factors, including bZIPs, NACs, and WRKY, showed significant increase in transcriptional level under sucrose starvation. Concurrently, statistical analysis revealed that their corresponding consensus cis-elements, such as ABA-responsive element, CACG, ACI, ACII and CTTATCC, were frequently found in the promoter regions of many sucrose starvation-upregulated genes. Particle bombardment-mediated and luciferase activity-based transient promoter assays revealed the CTTATCC, derived form TATCCA, and the AC motifs to be promising sucrose-starvation responsive activators in rice suspension cells.