Symmetry, asymmetry, and kinetics of silencing establishment in Saccharomyces cerevisiae revealed by single-cell optical assays.

In Saccharomyces cerevisiae, silent chromatin inhibits the expression of genes at the HML, HMR, and telomeric loci. When silent chromatin forms de novo, the rate of its establishment is influenced by different chromatin states. In particular, loss of the enzyme Dot1, an H3 K79 methyltransferase, leads to rapid silencing establishment. We tested whether silencing establishment was antagonized by H3 K79 methylation or by the Dot1 protein itself competing with Sir3 for binding sites on nucleosomes. To do so, we monitored fluorescence activity in cells containing a GFP gene within the HML locus during silencing establishment in a series of dot1 and histone mutant backgrounds. Silencing establishment rate was correlated with Dot1's enzymatic function rather than with the Dot1 protein itself. In addition, histone mutants that mimicked the conformation of unmethylated H3 K79 increased the rate of silencing establishment, indicating that the H3 K79 residue affected silencing independently of Dot1 abundance. Using fluorophore-based reporters, we confirmed that mother and daughter cells often silence in concert, but in instances where asymmetric silencing occurs, daughter cells established silencing earlier than their mothers. This noninvasive technique enabled us to demonstrate an asymmetry in silencing establishment of a key regulatory locus controlling cell fate.

The establishment of gene silencing at single-cell resolution.

The establishment of silencing in Saccharomyces cerevisiae is similar to heterochromatin formation in multicellular eukaryotes. Previous batch culture studies determined that the de novo establishment of silencing initiates during S phase and continues for up to five cell divisions for completion. To track silencing phenotypically, we developed an assay that introduces Sir3 protein into individual sir3Delta mutant cells synchronously and then detects the onset of silencing with single-cell resolution. Silencing was completed within the first one to two cell divisions in most cells queried. Moreover, we uncovered unexpected complexity in the contributions of a histone acetyltransferase (Sas2), two histone methytransferases (Dot1 and Set1) and one histone demethylase (Jhd2) to the dynamics of silencing. Our findings showed that removal of methyl modifications at H3K4 and H3K79 were important steps in silent chromatin formation and that Jhd2 and Set1 had competing roles in the process.

Group II intron splicing factors derived by diversification of an ancient RNA-binding domain.

Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosome. Many group II introns have lost the ability to splice autonomously as the result of an evolutionary process in which the loss of self-splicing activity was compensated by the recruitment of host-encoded protein cofactors. Genetic screens previously identified CRS1 and CRS2 as host-encoded proteins required for the splicing of group II introns in maize chloroplasts. Here, we describe two additional host-encoded group II intron splicing factors, CRS2-associated factors 1 and 2 (CAF1 and CAF2). We show that CRS2 functions in the context of intron ribonucleoprotein particles that include either CAF1 or CAF2, and that CRS2-CAF1 and CRS2-CAF2 complexes have distinct intron specificities. CAF1, CAF2 and the previously described group II intron splicing factor CRS1 are characterized by similar repeated domains, which we name here the CRM (chloroplast RNA splicing and ribosome maturation) domains. We propose that the CRM domain is an ancient RNA-binding module that has diversified to mediate specific interactions with various highly structured RNAs.

WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis.

Epicuticular wax forms a layer of hydrophobic material on plant aerial organs, which constitutes a protective barrier between the plant and its environment. We report here the identification of WIN1, an Arabidopsis thaliana ethylene response factor-type transcription factor, which can activate wax deposition in overexpressing plants. We constitutively expressed WIN1 in transgenic Arabidopsis plants, and found that leaf epidermal wax accumulation was up to 4.5-fold higher in these plants than in control plants. A significant increase was also found in stems. Interestingly, approximately 50% of the additional wax could only be released by complete lipid extractions, suggesting that not all of the wax is superficial. Gene expression analysis indicated that a number of genes, such as CER1, KCS1, and CER2, which are known to be involved in wax biosynthesis, were induced in WIN1 overexpressors. This observation indicates that induction of wax accumulation in transgenic plants is probably mediated through an increase in the expression of genes encoding enzymes of the wax biosynthesis pathway.

Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation.

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.

Maize mutants lacking chloroplast FtsY exhibit pleiotropic defects in the biogenesis of thylakoid membranes.

A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a "leaky" allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.

Toward a systems approach to understanding plant cell walls.

One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses of the polysaccharide components, combined with high-resolution imaging, have provided the basis for much of the current understanding of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into a useful model will require a system-based approach.