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

Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles

The northwestern assay is employed to study the interaction between protein and RNA. The RNA binding proteins tend to bind to different kinds of RNA through either known domains or unknown sequences of proteins. Rice LGD1 recombinant protein, a grass-specific novel protein with RNA binding sequences in its C-terminal, was used to probe its function as an RNA binding protein. The LGD1 comprising von Willebrand factor type-A domain (vWA), coiled-coil and nuclear localization signal (NLS) is a class of protein that localizes both in the nucleus and cytoplasm. Although LGD1 does not contain any putative RNA binding domains, we could find high-affinity RNA binding residues at the C-terminus using ‘RNABindR’ prediction software (Terribilini et al., 2007). The LGD1 recombinant protein, purified from bacteria, somehow forms both dimer and monomer even under denaturing conditions. However, only the dimeric form is able to bind to total and mRNAs. Due to its reproducibility and reliability, we believe that this protocol can be used across different organisms

Reduced activity of Arabidopsis chromosome-cohesion regulator gene CTF7/ECO1 alters cytosine methylation status and retrotransposon expression

ORCID IDs: 0000-0003-1729-0561 (P.B.-V.); 0000-0003-3459-1331 (G.-Y.J.)Multicellular organisms such as higher plants require timely regulation of DNA replication and cell division to grow and develop. Recent work in Arabidopsis has shown that chromosome segregation during meiosis and mitosis depends on the activity of several genes that in yeast are involved in the establishment of chromosomal cohesion. In this process, proteins of the STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC) family tether chromosomes and establish inter- and intrachromosomal connections. In Arabidopsis, recruitment of SMC proteins and establishment of cohesion during key stages of the cell cycle depend on the activity of CHROMOSOME TRANSMISSION FIDELITY 7/ ESTABLISHMENT OF COHESION 1 (CTF7/ECO1). Here we show that loss of CTF7/ECO1 activity alters the status of cytosine methylation in both intergenic regions and transposon loci. An increase in expression was also observed for transposon copia28, which suggests a link between CTF7/ECO1 activity, DNA methylation and gene silencing. More work is needed to determine the mechanistic relationships that intervene in this process.Academia Sinica (Taiwan)National Science and Technology Program for Agricultural Biotechnology (NSTP/AB, 098S0030055-AA, Taiwan)Ministry of Science and Technology (99-2321- B-001-036-MY3 and 102-2321-B-001-040-MY3)Universidad de Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM

Rise of the clones: apomixis in plant breeding

artículo -- Universidad de Costa Rica. escuela de Agronomía, 2011. Ese documento es privado debido a limitaciones de derechos de autor.In flowering plants the transfer of traits from one generation to the other involves fertilization of female gametophyte with sperm cells delivered by pollen tube, and the subsequent reassortment of traits (alleles) in the developing progeny. In nature DNA recombination and segregation of traits to the progeny prevents the accumulation of deleterious genes and loss of fitness; however for breeding purposes it is advantageous to fix superior trait combinations (genotypes) and by-pass sexual reproduction. The formation of sexual seed without fertilization of the egg is called apomixis and is considered the holy grail of plant breeding because top-performing varieties can be reproduced indefinitely without changes in the genotypes themselves or in their expression patterns. Apomixis is a dominant trait and consists on several processes working in tandem, separately each process is detrimental for plants, however, as a single unit they allow development of embryo and endosperm from unfertilized eggs. The three processes that constitute apomixis are: 1) apomeiosis, or cell division without DNA recombination in pollen and eggs, 2) parthenogenesis, autonomous development of eggs into fully formed embryos, and 3) stable development of the endosperm, the part of the seed, needed for the embryo to grow. Unfortunately full expression and transmission of apomixis is affected by DNA recombination, therefore introgression of the trait from wild relatives into commercial varieties is extremely difficult. In this review we present genes that have been identified to regulate each step of apomixis and discuss strategies to allow transmission of the trait in full using tools from molecular biology.Academia Sinica (Taiwan). National Science Council (Taiwan). Universidad de Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomí

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

Pollen development: a play with many actors

artículo -- Universidad de Costa Rica. Escuela de Agronomía, 2010. Trabajo de estudiante de posgrado en China. Este documento es privado debido a limitaciones de derechos de autor de la revista.Seeds are a major resource of food supply around the world and their production depends on successful double fertilization during plant sexual reproduction. In higher plants, fertilization of female gametophytes involves fusion of two pollen tube-delivered sperm cells with the egg proper and the central nuclei, giving rise to the embryo and the endosperm, respectively. Double fertilization and functional specialization of the male gametophyte are two essential factors driving the evolutionary success of angiosperms. Understanding the functions of genes involved in pollen development (microsporogenesis and microgametogenesis) and (pollination) can provide new insights into the regulatory elements that control male germline identity and male molecules involved in the interaction with the female partner during fertilization, aspects that are crucial for plant breeding. Additionally, a better understanding of the cellular and molecular factors governing the development and release of the sperm cells from pollen tubes can have a great impact on strategies for crop improvement. In this review, we discuss recent progresses and advances in aspects of microsporogenesis and microgametogenesis, such as microspore meiosis, mitosis, cell cycle regulation, and pollen tube guidance that have implications for crop improvementThis work was supported by research grants from the Academia Sinica (Taiwan), the National Science Council of Taiwan, and the Li Foundation (USA).UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomí

An Actin-Binding Protein, LlLIM1, Mediates Calcium and Hydrogen Regulation of Actin Dynamics in Pollen Tubes1[C][W][OA]

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 Ca2+: LlLIM1 showed a preference for F-actin binding under low pH and low Ca2+ 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