A complex role of Arabidopsis CDKD;3 in meiotic progression and cytokinesis.

Meiosis is a specialized cell division that halves the number of chromosomes in two consecutive rounds of chromosome segregation. In angiosperm plants is meiosis followed by mitotic divisions to form rudimentary haploid gametophytes. In Arabidopsis, termination of meiosis and transition to gametophytic development are governed by TDM1 and SMG7 that mediate inhibition of translation. Mutants deficient in this mechanism do not form tetrads but instead undergo multiple cycles of aberrant nuclear divisions that are likely caused by the failure to downregulate cyclin dependent kinases during meiotic exit. A suppressor screen to identify genes that contribute to meiotic exit uncovered a mutation in cyclin-dependent kinase D;3 (CDKD;3) that alleviates meiotic defects in smg7 deficient plants. The CDKD;3 deficiency prevents aberrant meiotic divisions observed in smg7 mutants or delays their onset after initiation of cytokinesis, which permits formation of functional microspores. Although CDKD;3 acts as an activator of cyclin-dependent kinase A;1 (CDKA;1), the main cyclin dependent kinase that regulates meiosis, cdkd;3 mutation appears to promote meiotic exit independently of CDKA;1. Furthermore, analysis of CDKD;3 interactome revealed enrichment for proteins implicated in cytokinesis, suggesting a more complex function of CDKD;3 in cell cycle regulation.

Meiotic exit in Arabidopsis is driven by P-body-mediated inhibition of translation.

Meiosis, at the transition between diploid and haploid life cycle phases, is accompanied by reprograming of cell division machinery and followed by a transition back to mitosis. We show that, in Arabidopsis, this transition is driven by inhibition of translation, achieved by a mechanism that involves processing bodies (P-bodies). During the second meiotic division, the meiosis-specific protein THREE-DIVISION MUTANT 1 (TDM1) is incorporated into P-bodies through interaction with SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA 7 (SMG7). TDM1 attracts eIF4F, the main translation initiation complex, temporarily sequestering it in P-bodies and inhibiting translation. The failure of tdm1 mutants to terminate meiosis can be overcome by chemical inhibition of translation. We propose that TDM1-containing P-bodies down-regulate expression of meiotic transcripts to facilitate transition of cell fates to postmeiotic gametophyte differentiation.

Application of Chemical Inhibitors in Live Cell Imaging of Plant Meiosis Using Light Sheet Fluorescence Microscopy.

Live imaging combined with the application of chemical inhibitors is a powerful research tool that enables researchers to precisely time the inhibition of cellular processes and study the consequences of these perturbations. This approach is usually applied to in vitro cultivated cells that are easily accessible to chemical treatments and microscopic observations. Here we describe a method for live cell imaging of Arabidopsis meiocytes embedded within floral organs combined with the application of a chemical drug at desired timepoints during meiosis. We describe a customized solution for the Zeiss Z.1 light sheet microscope, including sample preparation and data processing, and demonstrate its utility for the analysis of meiotic progression upon spindle inhibition.

Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy.

In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.

Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants.

Telomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. Expression of the reverse-transcriptase telomerase allows for extension of telomeric repeats to counteract natural telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly nondegenerate and that the telomeres form a protective structure, with a subset ending with a 3' overhang and another subset presenting a blunt end. Although telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that telomeres shorten progressively in mutants of this gene. Telomerase mutants eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.

Protection of Arabidopsis Blunt-Ended Telomeres Is Mediated by a Physical Association with the Ku Heterodimer.

Telomeres form specialized chromatin that protects natural chromosome termini from being recognized as DNA double-strand breaks. Plants possess unusual blunt-ended telomeres that are unable to form t-loops or complex with single-strand DNA binding proteins, raising the question of the mechanism behind their protection. We have previously suggested that blunt-ended telomeres in Arabidopsis thaliana are protected by Ku, a DNA repair factor with a high affinity for DNA ends. In nonhomologous end joining, Ku loads onto broken DNA via a channel consisting of positively charged amino acids. Here, we demonstrate that while association of Ku with plant telomeres also depends on this channel, Ku's requirements for DNA binding differ between DNA repair and telomere protection. We show that a Ku complex proficient in DNA loading but impaired in translocation along DNA is able to protect blunt-ended telomeres but is deficient in DNA repair. This suggests that Ku physically sequesters blunt-ended telomeres within its DNA binding channel, shielding them from other DNA repair machineries.

A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement.

Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions.

Germline replications and somatic mutation accumulation are independent of vegetative life span in Arabidopsis.

In plants, gametogenesis occurs late in development, and somatic mutations can therefore be transmitted to the next generation. Longer periods of growth are believed to result in an increase in the number of cell divisions before gametogenesis, with a concomitant increase in mutations arising due to replication errors. However, there is little experimental evidence addressing how many cell divisions occur before gametogenesis. Here, we measured loss of telomeric DNA and accumulation of replication errors in Arabidopsis with short and long life spans to determine the number of replications in lineages leading to gametes. Surprisingly, the number of cell divisions within the gamete lineage is nearly independent of both life span and vegetative growth. One consequence of the relatively stable number of replications per generation is that older plants may not pass along more somatically acquired mutations to their offspring. We confirmed this hypothesis by genomic sequencing of progeny from young and old plants. This independence can be achieved by hierarchical arrangement of cell divisions in plant meristems where vegetative growth is primarily accomplished by expansion of cells in rapidly dividing meristematic zones, which are only rarely refreshed by occasional divisions of more quiescent cells. We support this model by 5-ethynyl-2'-deoxyuridine retention experiments in shoot and root apical meristems. These results suggest that stem-cell organization has independently evolved in plants and animals to minimize mutations by limiting DNA replication.

If the cap fits, wear it: an overview of telomeric structures over evolution.

Genome organization into linear chromosomes likely represents an important evolutionary innovation that has permitted the development of the sexual life cycle; this process has consequently advanced nuclear expansion and increased complexity of eukaryotic genomes. Chromosome linearity, however, poses a major challenge to the internal cellular machinery. The need to efficiently recognize and repair DNA double-strand breaks that occur as a consequence of DNA damage presents a constant threat to native chromosome ends known as telomeres. In this review, we present a comparative survey of various solutions to the end protection problem, maintaining an emphasis on DNA structure. This begins with telomeric structures derived from a subset of prokaryotes, mitochondria, and viruses, and will progress into the typical telomere structure exhibited by higher organisms containing TTAGG-like tandem sequences. We next examine non-canonical telomeres from Drosophila melanogaster, which comprise arrays of retrotransposons. Finally, we discuss telomeric structures in evolution and possible switches between canonical and non-canonical solutions to chromosome end protection.

Studies on recombination processes in two Chlamydomonas reinhardtii endogenous genes, NIT1 and ARG7.

Integration of exogenous DNA in the unicellular green alga Chlamydomonas reinhardtii is principally carried out by mechanisms involving non-homologous recombination (NHR), rather than homologous recombination (HR). Homologous recombination is, however, the mechanism of choice when it comes to gene targeting. Unfortunately, attempts to establish this method in Chlamydomonas have had limited success. In this study we compared two endogenous genes, NIT1 and ARG7, and their HR/NHR ratios when different types of fragments were used as donors of homologous sequences. Transformation of the auxotrophic strain containing the inactivating point mutation arg7-8 with nonfunctional ARG7 gene fragments overlapping this mutation showed increased HR efficiencies when linearized plasmids were used. Efficiency went down rapidly with decreasing length of ARG7 homology. After identification of the inactivating 6726(G→A) point mutation in nit1-305 strains, an analogous set of experiments was performed. In the case of NIT1, overall efficiency of recombination was 10 to 100 fold lower than with ARG7. In order to better demonstrate HR we introduced three silent mutations close to the position of the point mutations in our transforming plasmids. Sequencing of transformants indicated homologous recombination over a short interval.