Search

The gaseous hormone ethylene is one of the master regulators of development and physiology throughout the plant life cycle. Ethylene biosynthesis is stringently regulated to permit maintenance of low levels during most phases of vegetative growth but to allow for rapid peaks of high production at developmental transitions and under stress conditions. In most tissues ethylene is a negative regulator of cell expansion, thus low basal levels of ethylene biosynthesis in dark-grown seedlings are critical for optimal cell expansion during early seedling development. The committed steps in ethylene biosynthesis are performed by the enzymes 1-aminocyclopropane 1-carboxylate synthase (ACS) and 1-aminocyclopropane 1-carboxylate oxidase (ACO). The abundance of different ACS enzymes is tightly regulated both by transcriptional control and by post-translational modifications and proteasome-mediated degradation. Here we show that specific ACS isozymes are targets for regulation by protein phosphatase 2A (PP2A) during Arabidopsis thaliana seedling growth and that reduced PP2A function causes increased ACS activity in the roots curl in 1-N-naphthylphthalamic acid 1 (rcn1) mutant. Genetic analysis reveals that ethylene overproduction in PP2A-deficient plants requires ACS2 and ACS6, genes that encode ACS proteins known to be stabilized by phosphorylation, and proteolytic turnover of the ACS6 protein is retarded when PP2A activity is reduced. We find that PP2A and ACS6 proteins associate in seedlings and that RCN1-containing PP2A complexes specifically dephosphorylate a C-terminal ACS6 phosphopeptide. These results suggest that PP2A-dependent destabilization requires RCN1-dependent dephosphorylation of the ACS6 C-terminus. Surprisingly, rcn1 plants exhibit decreased accumulation of the ACS5 protein, suggesting that a regulatory phosphorylation event leads to ACS5 destabilization. Our data provide new insight into the circuitry that ensures dynamic control of ethylene synthesis during plant development, showing that PP2A mediates a finely tuned regulation of overall ethylene production by differentially affecting the stability of specific classes of ACS enzymes., Author Summary Like animals, plants produce a number of substances that regulate growth and coordinate developmental transitions and responses to environmental signals. Ethylene gas is one such regulator of the plant life cycle, playing important roles in fruit ripening, pathogen defenses, and the regulation of cell expansion. Because overall plant form is determined largely by the degree and directionality of cell expansion, ethylene is a crucial regulator of morphology, and ethylene production must be maintained at low levels during phases of rapid cell expansion, such as early seedling growth. Recent work has identified molecular mechanisms that target ethylene biosynthetic enzymes for proteolytic degradation; this degradation plays a key role in controlling ethylene production. Here we exploit the molecular genetic resources available in the Arabidopsis thaliana system to identify a highly conserved protein complex that dephosphorylates target proteins as a new component of the mechanism that regulates degradation of ethylene-producing enzymes. Our findings show that protein phosphatase 2A plays a nuanced role in this regulatory circuit, with both positive and negative inputs into the stability of specific proteins that drive ethylene biosynthesis. This work enhances our understanding of the mechanisms that enforce adaptive levels of hormone production in plants.

Histone H3 lysine-4 (H3K4) methylation is associated with transcribed genes in eukaryotes. In Drosophila and mammals, both di- and tri-methylation of H3K4 are associated with gene activation. In contrast to animals, in Arabidopsis H3K4 trimethylation, but not mono- or di-methylation of H3K4, has been implicated in transcriptional activation. H3K4 methylation is catalyzed by the H3K4 methyltransferase complexes known as COMPASS or COMPASS-like in yeast and mammals. Here, we report that Arabidopsis homologs of the COMPASS and COMPASS-like complex core components known as Ash2, RbBP5, and WDR5 in humans form a nuclear subcomplex during vegetative and reproductive development, which can associate with multiple putative H3K4 methyltransferases. Loss of function of ARABIDOPSIS Ash2 RELATIVE (ASH2R) causes a great decrease in genome-wide H3K4 trimethylation, but not in di- or mono-methylation. Knockdown of ASH2R or the RbBP5 homolog suppresses the expression of a crucial Arabidopsis floral repressor, FLOWERING LOCUS C (FLC), and FLC homologs resulting in accelerated floral transition. ASH2R binds to the chromatin of FLC and FLC homologs in vivo and is required for H3K4 trimethylation, but not for H3K4 dimethylation in these loci; overexpression of ASH2R causes elevated H3K4 trimethylation, but not H3K4 dimethylation, in its target genes FLC and FLC homologs, resulting in activation of these gene expression and consequent late flowering. These results strongly suggest that H3K4 trimethylation in FLC and its homologs can activate their expression, providing concrete evidence that H3K4 trimethylation accumulation can activate eukaryotic gene expression. Furthermore, our findings suggest that there are multiple COMPASS-like complexes in Arabidopsis and that these complexes deposit trimethyl but not di- or mono-methyl H3K4 in target genes to promote their expression, providing a molecular explanation for the observed coupling of H3K4 trimethylation (but not H3K4 dimethylation) with active gene expression in Arabidopsis., Author Summary Histones can be covalently modified and histone modifications regulate chromatin structure and gene transcription. One such modification is histone H3 lysine-4 (H3K4) methylation, which can be mono-, di-, or tri-methylated. In animals such as fruitfly and mammals, both di- and tri-methylation of H3K4 are associated with active gene expression. In contrast to animals, in the flowering plant Arabidopsis only H3K4 trimethylation has been implicated in gene transcriptional activation. H3K4 methylation is catalyzed by the H3K4 methyltransferase complexes known as COMPASS-like in mammals. Here, we report that COMPASS-like H3K4 methyltransferase complexes exist in Arabidopsis. Loss of function of a core complex protein causes a great decrease in Arabidopsis genome-wide H3K4 trimethylation, but not in di- or mono-methylation. Our analyses of several direct target genes of these COMPASS-like complexes show that they mediate deposition of trimethyl but not dimethyl H3K4 in these loci to activate their expression, providing concrete evidence for the notion that H3K4 trimethylation accumulation can activate eukaryotic gene expression. Furthermore, our findings provide a molecular explanation for the observed coupling of trimethylation but not dimethylation of H3K4 with active gene expression in Arabidopsis. In addition, we found that H3K4 trimethylation regulates leaf growth and development, flowering, and embryo development.

Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, we reveal a role of the type III HD-Zip genes, previously known to specify lateral organ polarity, in stem cell termination. Both reduction in HD-Zip expression by over-expression of miR165/166 and mis-expression of HD-Zip genes by rendering them resistant to miR165/166 lead to prolonged floral stem cell activity, indicating that the expression of HD-Zip genes needs to be precisely controlled to achieve floral stem cell termination. We also show that both the ubiquitously expressed ARGONAUTE1 (AGO1) gene and its homolog AGO10, which exhibits highly restricted spatial expression patterns, are required to maintain the correct temporal program of floral stem cells. We provide evidence that AGO10, like AGO1, associates with miR172 and miR165/166 in vivo and exhibits “slicer” activity in vitro. Despite the common biological functions and similar biochemical activities, AGO1 and AGO10 exert different effects on miR165/166 in vivo. This work establishes a network of microRNAs and transcription factors governing the temporal program of floral stem cells and sheds light on the relationships among different AGO genes, which tend to exist in gene families in multicellular organisms., Author Summary Stem cells have the capacity to self renew while producing daughter cells that undergo differentiation. While some stem cells remain as stem cells throughout the life of an organism, others are programmed to terminate within developmental contexts. It is presumed that stem cell termination is simply the differentiation of stem cells into a specific cell type(s). Using floral stem cells as a model, we show that the temporally regulated termination of floral stem cells is genetically separable from stem cell differentiation, and thus we reveal the presence of a temporal program of stem cell regulation. We show that two microRNAs, miR172 and miR165/166, and two argonaute family proteins, ARGONAUTE1 (AGO1) and AGO10, regulate the termination of floral stem cells. We establish the homeodomain-leucine zipper (DH-Zip) genes, targets of miR165/166, as crucial factors in floral stem cell termination. While AGO1 is the major miRNA effector, the molecular function of AGO10 has been elusive. Here we demonstrate that AGO10 is also a miRNA effector in that AGO10 is associated with miRNAs in vivo and exhibits “slicer” activity in vitro. Despite the similar biochemical activities, AGO1 and AGO10 promote floral stem cell termination by exerting opposite effects on miR165/166.

Background Alternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development. Methodology/Principal Findings To identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data—variations caused by differential probe affinity—was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation. Conclusion/Significance We identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term “regulation of flower development” among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request.

[Abstract] Cell-to-cell communication is crucial for the development of multicellular organisms, especially during the generation of new tissues and organs. Secondary growth—the lateral expansion of plant growth axes—is a highly dynamic process that depends on the activity of the cambium. The cambium is a stem cell–like tissue whose activity is responsible for wood production and, thus, for the establishment of extended shoot and root systems. Attempts to study cambium regulation at the molecular level have been hampered by the limitations of performing genetic analyses in trees and by the difficulty of accessing this tissue in model systems such as Arabidopsis thaliana. Here, we describe the roles of two receptor-like kinases, REDUCED IN LATERAL GROWTH1 (RUL1) and MORE LATERAL GROWTH1 (MOL1), as opposing regulators of cambium activity. Their identification was facilitated by a novel in vitro system in which cambium formation is induced in isolated Arabidopsis stem fragments. By combining this system with laser capture microdissection, we characterized transcriptome remodeling in a tissue- and stage-specific manner and identified series of genes induced during different phases of cambium formation. In summary, we provide a means for investigating cambium regulation in unprecedented depth and present two signaling components that control a process responsible for the accumulation of a large proportion of terrestrial biomass., [Author Summary] In contrast to animals, plants have the capacity to grow and form new organs throughout their entire life cycle, thereby building up some of the largest organisms on earth. This remarkable capacity is based on the activity of stem cell–like tissues—the meristems—located at shoot and root apices and, in a large repertoire of species, in lateral positions at the flanks of growth axes. In comparison to apical meristems, our knowledge of the molecular mechanisms controlling the activity of lateral meristems like the cambium is very limited. This is despite the fact that lateral growth is responsible for wood formation, and thus for the accumulation of large amounts of terrestrial biomass, and for fixation of atmospheric CO2. Here, we introduce an in vitro system by which cambium initiation can be stimulated under controlled conditions in stems of the reference plant Arabidopsis thaliana. By revealing genome-wide and tissue-specific alterations in transcript accumulation during cambium initiation, we identify two novel receptor-like kinases, namely MOL1 and RUL1, as opposing cambium regulators. These findings demonstrate that our in vitro system represents a valuable tool for studying cambium regulation and open up possibilities to dissect lateral growth in plants from novel perspectives., This work was supported by grant P21258-B03 of the Austrian Science Fund (FWF, http://www.fwf.ac.at/en/index.asp).

Seed development in angiosperms is dependent on the interplay among different transcriptional programs operating in the embryo, the endosperm, and the maternally-derived seed coat. In angiosperms, the embryo and the endosperm are products of double fertilization during which the two pollen sperm cells fuse with the egg cell and the central cell of the female gametophyte. In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed. Here we have exploited cdka;1 fertilization as a novel tool for the identification of seed regulators and factors involved in parent-of-origin–specific regulation during seed development. We have generated genome-wide transcription profiles of cdka;1 fertilized seeds and identified approximately 600 genes that are downregulated in the absence of a paternal genome. Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented. Here, AGL36 was chosen for an in-depth study and shown to be imprinted. We demonstrate that AGL36 parent-of-origin–dependent expression is controlled by the activity of METHYLTRANSFERASE1 (MET1) maintenance DNA methyltransferase and DEMETER (DME) DNA glycosylase. Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds., Author Summary Seeds of flowering plants consist of three different organisms that develop in parallel. In contrast to animals, a double fertilization event takes place in plants, producing two fertilization products, the embryo and the endosperm. Imprinting, the parent-of-origin–specific expression of genes, typically takes place in the mammalian placenta and in the plant endosperm. A prevailing hypothesis predicts that a parental tug-of-war on the allocation of available recourses to the developing progeny has led to the evolution of imprinting systems where genes expressed from the mother dampen growth whereas genes expressed from the father are growth enhancers. The number of imprinted genes identified in plants is low compared to mammals, and this precludes the elucidation of the epigenetic mechanisms responsible for this specialized expression system. Here, we have used genome-wide transcript profiling of endosperm without paternal contribution to identify seed regulators and, among these, imprinted genes. We identified a cluster of downregulated MADS-box transcription factors, including AGL36, that was subsequently shown to be imprinted by an epigenetic mechanism involving the DNA methylase MET1 and the glycosylase DME. In addition, the expression of the active AGL36 allele was dampened by the FIS Polycomb Repressive Complex, identifying a novel mode of regulation of imprinted genes.

Discovering links between the genotype of an organism and its metabolite levels can increase our understanding of metabolism, its controls, and the indirect effects of metabolism on other quantitative traits. Recent technological advances in both DNA sequencing and metabolite profiling allow the use of broad-spectrum, untargeted metabolite profiling to generate phenotypic data for genome-wide association studies that investigate quantitative genetic control of metabolism within species. We conducted a genome-wide association study of natural variation in plant metabolism using the results of untargeted metabolite analyses performed on a collection of wild Arabidopsis thaliana accessions. Testing 327 metabolites against >200,000 single nucleotide polymorphisms identified numerous genotype–metabolite associations distributed non-randomly within the genome. These clusters of genotype–metabolite associations (hotspots) included regions of the A. thaliana genome previously identified as subject to recent strong positive selection (selective sweeps) and regions showing trans-linkage to these putative sweeps, suggesting that these selective forces have impacted genome-wide control of A. thaliana metabolism. Comparing the metabolic variation detected within this collection of wild accessions to a laboratory-derived population of recombinant inbred lines (derived from two of the accessions used in this study) showed that the higher level of genetic variation present within the wild accessions did not correspond to higher variance in metabolic phenotypes, suggesting that evolutionary constraints limit metabolic variation. While a major goal of genome-wide association studies is to develop catalogues of intraspecific variation, the results of multiple independent experiments performed for this study showed that the genotype–metabolite associations identified are sensitive to environmental fluctuations. Thus, studies of intraspecific variation conducted via genome-wide association will require analyses of genotype by environment interaction. Interestingly, the network structure of metabolite linkages was also sensitive to environmental differences, suggesting that key aspects of network architecture are malleable., Author Summary Understanding how genetic variation can control phenotypic variation is a fundamental goal of modern biology. We combined genome-wide association mapping with metabolomics in the plant Arabidopsis thaliana to explore how species-wide genetic variation controls metabolism. We identified numerous naturally-variable genes that may influence plant metabolism, often clustering in “hotspots.” These hotspots were proximal to selective sweeps, regions of the genome showing decreased diversity possibly from a strong selective advantage of specific variants within the region. This suggests that metabolism may be connected to the selective advantage. Interestingly, metabolite variation in wild Arabidopsis is highly constrained despite the significant genetic variation, thus providing the plant un-sampled metabolic space if the environment shifts. The observed structuring of genetic and metabolic variation suggests individual convergence upon similar phenotypes via different genotypes, possibly intra-specific parallel evolution. This phenotypic convergence couples with a pattern of genotype—phenotype association consistent with metabolite variation largely controlled by numerous small effect genetic variants. This supports the supposition that large magnitude variation is likely unstable in a complex and interconnected metabolism. If this pattern proves generally applicable to other species, it could present a significant hurdle to identifying genes controlling metabolic trait variation via genome-wide association studies.

Pontvianne, Frédéric et al. 13 p.-8 fig., In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes.Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis., This work was supported by the Centre National de la Recherche Scientifique(CNRS). Work performed in FJM laboratory was financed by a grant of the Spanish National Plan for R&D, N u ESP2006-13600-C02-02.

Background: The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. Methodology/Principal Findings: We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. Conclusions/Significance: We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.

Polycomb group (PcG) proteins act as evolutionary conserved epigenetic mediators of cell identity because they repress transcriptional programs that are not required at particular developmental stages. Each tissue is likely to have a specific epigenetic profile, which acts as a blueprint for its developmental fate. A hallmark for Polycomb Repressive Complex 2 (PRC2) activity is trimethylated lysine 27 on histone H3 (H3K27me3). In plants, there are distinct PRC2 complexes for vegetative and reproductive development, and it was unknown so far whether these complexes have target gene specificity. The FERTILIZATION INDEPENDENT SEED (FIS) PRC2 complex is specifically expressed in the endosperm and is required for its development; loss of FIS function causes endosperm hyperproliferation and seed abortion. The endosperm nourishes the embryo, similar to the physiological function of the placenta in mammals. We established the endosperm H3K27me3 profile and identified specific target genes of the FIS complex with functional roles in endosperm cellularization and chromatin architecture, implicating that distinct PRC2 complexes have a subset of specific target genes. Importantly, our study revealed that selected transposable elements and protein coding genes are specifically targeted by the FIS PcG complex in the endosperm, whereas these elements and genes are densely marked by DNA methylation in vegetative tissues, suggesting that DNA methylation prevents targeting by PcG proteins in vegetative tissues., PLoS Genetics, 6 (10), ISSN:1553-7390, ISSN:1553-7404

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding.

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA–directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term DOMAINS REARRANGED METHYLTRANSFERASE3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA–directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA–directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA–directed DNA methylation systems consist of a combination of ancestral and convergent features., Author Summary Nuclear DNA quantity varies widely between species and is poorly correlated with gene number. Variation in genome size can be explained by differing amounts of repetitive DNA. Repetitive DNA may be mobile, meaning it can increase its copy number within genomes. To prevent this, plants and animals suppress expression of repeats, often by marking the repeated sequence with a methyl group on cytosine bases. DRM2 is an enzyme capable of establishing this methylation, which can be guided to target sequences by short complementary RNA guides. As repeated sequences are prone to generate short RNAs they are efficiently recognized and silenced. We show that DRM2 requires a related inactive DRM3 protein to normally silence repeated sequences. A similar situation exists in mammals, where active and inactive DNA methyltransferases act together to silence repeats. We also demonstrate that non-catalytic regions of the DRM2 enzyme are functionally important, which we speculate to be involved in targeting the enzyme to the genome. Although plant and mammal RNA–directed DNA methylation systems share key similarities, there are important mechanistic differences, meaning that they are likely to have arisen convergently.

Rice NH1 (NPR1 homolog 1) is a key mediator of innate immunity. In both plants and animals, the innate immune response is often accompanied by rapid cell death at the site of pathogen infection. Over-expression of NH1 in rice results in resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo), constitutive expression of defense related genes and enhanced benzothiadiazole (BTH)- mediated cell death. Here we describe a forward genetic screen that identified a suppressor of NH1-mediated lesion formation and resistance, snl6. Comparative genome hybridization and fine mapping rapidly identified the genomic location of the Snl6 gene. Snl6 is a member of the cinnamoyl-CoA reductase (CCR)-like gene family. We show that Snl6 is required for NH1-mediated resistance to Xoo. Further, we show that Snl6 is required for pathogenesis-related gene expression. In contrast to previously described CCR family members, disruption of Snl6 does not result in an obvious morphologic phenotype. Snl6 mutants have reduced lignin content and increased sugar extractability, an important trait for the production of cellulosic biofuels. These results suggest the existence of a conserved group of CCR-like genes involved in the defense response, and with the potential to alter lignin content without affecting development., Author Summary Plants possess potent and effective endogenous methods for responding to pathogen attacks, referred to as plant innate immunity. In this report we further our understanding of rice innate immunity through characterization of the Snl6 gene. The snl6 mutant was identified from a mutant screen for positive regulators of immunity. While innate immunity represents a powerful agronomic tool, identification of desirable genes from crop species is limited by the slow and laborious nature of map-based cloning. Here we describe our methodology of combining comparative genome hybridization and fine mapping to rapidly identify the Snl6 gene. Snl6 is distantly related to members of the cinnamoyl-CoA reductase gene family, is required for pathogenesis gene expression and resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. Snl6 mutants have reduced lignin content and increased sugar extractability, an important trait for the production of cellulosic biofuels.

Quantitative Trait Loci (QTL) analyses in immortal populations are a powerful method for exploring the genetic mechanisms that control interactions of organisms with their environment. However, QTL analyses frequently do not culminate in the identification of a causal gene due to the large chromosomal regions often underlying QTLs. A reasonable approach to inform the process of causal gene identification is to incorporate additional genome-wide information, which is becoming increasingly accessible. In this work, we perform QTL analysis of the shade avoidance response in the Bayreuth-0 (Bay-0, CS954) x Shahdara (Sha, CS929) recombinant inbred line population of Arabidopsis. We take advantage of the complex pleiotropic nature of this trait to perform network analysis using co-expression, eQTL and functional classification from publicly available datasets to help us find good candidate genes for our strongest QTL, SAR2. This novel network analysis detected EARLY FLOWERING 3 (ELF3; AT2G25930) as the most likely candidate gene affecting the shade avoidance response in our population. Further genetic and transgenic experiments confirmed ELF3 as the causative gene for SAR2. The Bay-0 and Sha alleles of ELF3 differentially regulate developmental time and circadian clock period length in Arabidopsis, and the extent of this regulation is dependent on the light environment. This is the first time that ELF3 has been implicated in the shade avoidance response and that different natural alleles of this gene are shown to have phenotypic effects. In summary, we show that development of networks to inform candidate gene identification for QTLs is a promising technique that can significantly accelerate the process of QTL cloning., Author Summary A major interest in evolutionary biology is to understand the genetic mechanisms that underlie phenotypic variation in nature and how they interact with the environment. A good example of adaptive genetic variation in response to the environment is the shade avoidance response. Although some plant groups try to outgrow their competitors when shade cues are detected others do not, as they are adapted to live in constitutive shade, such as a forest canopy. We used a segregating population derived from two Arabidopsis ecotypes to investigate this variation and found a chromosomal region affecting the shade avoidance response. We developed a network analysis method that combines genomic information from publicly available databases to identify the causative gene in that interval as ELF3. Using genetic and transgenic methods we confirmed the effect of ELF3 in the shade avoidance response, and showed that different alleles of this gene in natural populations of Arabidopsis result in different developmental times and circadian periodicity depending on the environmental conditions.

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation–dependent and –independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity., Author Summary The characteristics of the diverse cell types in multicellular organisms result from differential gene expression that is dependent on the level of DNA packaging. Genes that are essential for the function of the cell are expressed; while unessential genes, and DNA elements (transposons or “jumping genes”) that can move from one position to another within a genome and potentially cause deleterious mutations, are repressed. The mechanisms evolved in eukaryotes to avoid unwanted gene expression and transposon movement include DNA methylation and specific combinations of post translational modifications (PTMs) of the histones that package DNA. Here we show that the SUVR4 enzyme binds the signaling protein ubiquitin and that ubiquitin enables the enzyme to trimethylate lysine 9 (H3K9me3) of histone H3. In contrast to other reports demonstrating an activating role on expressed genes, we show that H3K9me3 has a locus-specific repressive effect on the expression of transposons. The specificity is maintained by the communication with other PTMs on transposons and euchromatic genes, which has a stimulating or repressing effect on enzyme activity, respectively. Our results demonstrate how repression of transcription can be restricted to specific targets and demonstrate that this repression involves a context-dependent read-out of different PTMs.

Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for ∼20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA–resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants., Author Summary MiRNAs are small RNA molecules that play an important role in regulating gene function, both in animals and in plants. In plants, miRNA target mimicry is an endogenous mechanism used to negatively regulate the activity of a specific miRNA family, through the production of a false target transcript that cannot be cleaved. This mechanism can be engineered to target different miRNA families. Using this technique, we have generated artificial target mimics predicted to reduce the activity of most of the miRNA families in Arabidopsis thaliana and have observed their effects on plant development. We found that deeply conserved miRNAs tend to have a strong impact on plant growth, while more recently evolved ones had generally less obvious effects, suggesting either that they primarily affect processes other than development, or else that they have more subtle or conditional functions or are even dispensable. In several cases, the effects on plant development that we observed closely resembled those seen in plants expressing miRNA–resistant versions of the major predicted targets, indicating that a limited number of targets mediates most effects of these miRNAs. Analyses of mimic expressing plants also support that plant miRNAs affect both transcript stability and protein accumulation. The artificial target mimic collection will be a useful resource to further investigate the function of individual miRNA families.

A significant fraction of a plant's nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development., Author Summary Understanding the convergent developmental mechanisms of core cell cycle genes is highly instructive in biology. When these genes are essential in development, lethality precludes mutation analysis throughout the life cycle of an organism. We subjected a homozygous lethal mutation in RETINOBLASTOMA RELATED (RBR) of Arabidopsis for tetraploid genetic analysis to study the function of RBR during the plant life cycle. In diploids, while RBR–deficient female gametophytes with features of aberrant cell fate and differentiation were analogous to what was previously reported for male gametophytes, we provide evidence that RBR controls gametic genome duplication, thus genome integrity in the gametophyte-derived progeny. Quantitative reduction of RBR in tetraploids led to identification of rbr heterozygous plants that displayed novel RBR dosage-dependent phenotypes in differentiation and development of the sporophyte albeit the absence of cell cycle defects. These phenotypes coincided with deregulation of conserved epigenetic factors such as Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the sporophyte, as shown for the gametophytes as well. However, unlike the repression by the PRC2 in gametophytes, RBR is activated by the sporophytic PRC2 subunits, suggesting that distinct modules of the conserved RBR-PRC2-MET1 loop control gametophyte and sporophyte generations in plants.

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.

Gene duplication is a major mechanism facilitating adaptation to changing environments. From recent genomic analyses, the acquisition of zinc hypertolerance and hyperaccumulation characters discriminating Arabidopsis halleri from its zinc sensitive/non-accumulator closest relatives Arabidopsis lyrata and Arabidopsis thaliana was proposed to rely on duplication of genes controlling zinc transport or zinc tolerance. Metal Tolerance Protein 1 (MTP1) is one of these genes. It encodes a Zn2+/H+ antiporter involved in cytoplasmic zinc detoxification and thus in zinc tolerance. MTP1 was proposed to be triplicated in A. halleri, while it is present in single copy in A. thaliana and A. lyrata. Two of the three AhMTP1 paralogues were shown to co-segregate with zinc tolerance in a BC1 progeny from a cross between A. halleri and A. lyrata. In this work, the MTP1 family was characterized at both the genomic and functional levels in A. halleri. Five MTP1 paralogues were found to be present in A. halleri, AhMTP1-A1, -A2, -B, -C, and -D. Interestingly, one of the two newly identified AhMTP1 paralogues was not fixed at least in one A. halleri population. All MTP1s were expressed, but transcript accumulation of the paralogues co-segregating with zinc tolerance in the A. halleri X A. lyrata BC1 progeny was markedly higher than that of the other paralogues. All MTP1s displayed the ability to functionally complement a Saccharomyces cerevisiæ zinc hypersensitive mutant. However, the paralogue showing the least complementation of the yeast mutant phenotype was one of the paralogues co-segregating with zinc tolerance. From our results, the hypothesis that pentaplication of MTP1 could be a major basis of the zinc tolerance character in A. halleri is strongly counter-balanced by the fact that members of the MTP1 family are likely to experience different evolutionary fates, some of which not concurring to increase zinc tolerance., Author Summary Arabidopsis halleri has developed the characters of zinc hypertolerance and hyperaccumulation as compared to its close relatives Arabidopsis thaliana and Arabidopsis lyrata. Different candidate genes were proposed to account for the appearance of these characters in A. halleri. One of them is MTP1 (Metal Tolerance Protein 1), which is involved in cytoplasmic zinc detoxification. We found that A. halleri harbored five gene copies of MTP1, whereas A. thaliana and A. lyrata possess a single copy. It is thus tempting to associate the zinc hypertolerance character of A. halleri to the pentaplication of MTP1. However, we observed that one of the five MTP1 copies is not fixed in a population growing on metal contaminated soil. Also, the different MTP1 genes are markedly differentially expressed in A. halleri, two of them being poorly expressed and repressed in response to zinc constraint. AhMTP1 copies were also demonstrated to display a differential ability to complement the zinc hypersensitivity of a yeast mutant that is dysfunctional in vacuolar zinc transporters. Our findings suggest that different evolutionary fates, some of them not concurring to increase zinc tolerance, are likely to take place for the members of the MTP1 family in A. halleri.

Despite the described central role of jasmonate signaling in plant defense against necrotrophic pathogens, the existence of intraspecific variation in pathogen capacity to activate or evade plant jasmonate-mediated defenses is rarely considered. Experimental infection of jasmonate-deficient and jasmonate-insensitive Arabidopsis thaliana with diverse isolates of the necrotrophic fungal pathogen Botrytis cinerea revealed pathogen variation for virulence inhibition by jasmonate-mediated plant defenses and induction of plant defense metabolites. Comparison of the transcriptional effects of infection by two distinct B. cinerea isolates showed only minor differences in transcriptional responses of wild-type plants, but notable isolate-specific transcript differences in jasmonate-insensitive plants. These transcriptional differences suggest B. cinerea activation of plant defenses that require plant jasmonate signaling for activity in response to only one of the two B. cinerea isolates tested. Thus, similar infection phenotypes observed in wild-type plants result from different signaling interactions with the plant that are likely integrated by jasmonate signaling., Author Summary While many important elements of plant defense signaling have been identified, the function of these defense signaling pathways may mask additional variation in the plant–pathogen interaction, including both pathogen variation and variation in downstream plant defense responses. Jasmonate plant hormones contribute to both plant development and defense, including plant defense against necrotrophic fungal pathogens such as the grey mold Botrytis cinerea. Ten diverse B. cinerea isolates all showed increased virulence and decreased induction of a plant antimicrobial metabolite in experimental infections of Arabidopsis thaliana lacking functional jasmonate signaling. Yet within this consistent result, B. cinerea isolates varied considerably. Through comparing the transcript profiles of A. thaliana infected with the two most disparate B. cinerea isolates, we found that wild-type plants showed similar transcriptional responses to infection with these two isolates, but the absence of functional jasmonate signaling revealed dramatic differences in plant response, including groups of co-regulated genes that may participate in undescribed plant response networks. Jasmonate signaling appears to integrate plant responses to diverse pathogen inputs, and its absence may reveal novel aspects of plant–pathogen interaction.

BackgroundIn plants and animals there are many classes of short RNAs that carry out a wide range of functions within the cell; short silencing RNAs (ssRNAs) of 21-25 nucleotides in length are produced from double-stranded RNA precursors by the protein Dicer and guide nucleases and other proteins to their RNA targets through base pairing interactions. The consequence of this process is degradation of the targeted RNA, suppression of its translation or initiation of secondary ssRNA production. The secondary ssRNAs in turn could then initiate further layers of ssRNA production to form extensive cascades and networks of interacting RNA [1]. Previous empirical analysis in plants established the existence of small secondary ssRNA cascade [2], in which a single instance of this event occurred but it was not known whether there are other more extensive networks of secondary sRNA production.Methodology/principal findingsWe generated a network by predicting targets of ssRNA populations obtained from high-throughput sequencing experiments. The topology of the network shows it to have power law connectivity distribution, to be dissortative, highly clustered and composed of multiple components. We also identify protein families, PPR and ULP1, that act as hubs within the network. Comparison of the repetition of genomic sub-sequences of ssRNA length between Arabidopsis and E.coli suggest that the network structure is made possible by the underlying repetitiveness in the genome sequence.Conclusions/significanceTogether our results provide good evidence for the existence of a large, robust ssRNA interaction network with distinct regulatory function. Such a network could have a massive effect on the regulation of gene expression via mediation of transcript levels.

BackgroundThe genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels.Methodology/principal findingsWe designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource).Conclusions/significanceOur comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.

BACKGROUND: The SWR1 complex is important for the deposition of histone variant H2A.Z into chromatin necessary to robustly regulate gene expression during growth and development. In Arabidopsis thaliana, the catalytic subunit of the SWR1-like complex, encoded by PIE1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1), has been shown to function in multiple developmental processes including flowering time pathways and petal number regulation. However, the function of the PIE1 orthologs in monocots remains unknown. METHODOLOGY/FINDINGS: We report the identification of the rice (Oryza sativa) ortholog, OsPIE1. Although OsPIE1 does not exhibit a conserved exon/intron structure as Arabidopsis PIE1, its encoded protein is highly similar to PIE1, sharing 53.9% amino acid sequence identity. OsPIE1 also has a very similar expression pattern as PIE1. Furthermore, transgenic expression of OsPIE1 completely rescued both early flowering and extra petal number phenotypes of the Arabidopsis pie1-2 mutant. However, homozygous T-DNA insertional mutants of OsPIE1 in rice were embryonically lethal, in contrast to the viable mutants in the orthologous genes for yeast, Drosophila and Arabidopsis (Swr1, DOMINO and PIE1, respectively). CONCLUSIONS/SIGNIFICANCE: Taken together, our results suggest that OsPIE1 is the rice ortholog of Arabidopsis PIE1 and plays an essential role in rice embryo development.

The mutation of as few as three genes in a sexual plant transforms meiosis into mitosis and results in diploid gametes that are genetically identical to the mother plant. This phenotype resembles apomeiosis, which is a major step in apomixis., Apomixis, or asexual clonal reproduction through seeds, is of immense interest due to its potential application in agriculture. One key element of apomixis is apomeiosis, a deregulation of meiosis that results in a mitotic-like division. We isolated and characterised a novel gene that is directly involved in controlling entry into the second meiotic division. By combining a mutation in this gene with two others that affect key meiotic processes, we created a genotype called MiMe in which meiosis is totally replaced by mitosis. The obtained plants produce functional diploid gametes that are genetically identical to their mother. The creation of the MiMe genotype and apomeiosis phenotype is an important step towards understanding and engineering apomixis., Author Summary In the life cycle of sexual organisms, a specialized cell division—meiosis—reduces the number of chromosomes from two sets (2n, diploid) to one set (n, haploid), while fertilization restores the original chromosome number. In contrast, mitosis produces two identical daughter cells. Basically, three features distinguish meiosis from mitosis. Meiosis has: (i) a succession of two rounds of division following a single replication, (ii) recombination, and (iii) co-segregation of sister chromatids at the first division. In this study, we identified a gene that controls one of these three features— entry into the second meiotic division—in the sexual plant Arabidopsis thaliana. By combining a mutation in this gene with two other mutations—one that eliminates recombination and another that modifies chromatid segregation—we created a genotype (called MiMe for mitosis instead of meiosis) in which meiosis is totally replaced by mitosis. As a consequence, MiMe plants produced diploid male and female gametes that are genetically identical to their parent, and ploidy doubles at each generation. The replacement of meiosis by mitosis is a key component of apomixis, or clonal reproduction through seeds, which has potential revolutionary application in crop improvement.

Reproductive development of higher plants comprises successive events of organ differentiation and growth which finally lead to the formation of a mature fruit. However, most of the genetic and molecular mechanisms which coordinate such developmental events are yet to be identified and characterized. Arlequin (Alq), a semi-dominant T-DNA tomato mutant showed developmental changes affecting flower and fruit ripening. Sepals were converted into fleshy organs which ripened as normal fruit organs and fruits displayed altered ripening features. Molecular characterization of the tagged gene demonstrated that it corresponded to the previously reported TOMATO AGAMOUS-LIKE 1 (TAGL1) gene, the tomato ortholog of SHATTERPROOF MADS-box genes of Arabidopsis thaliana, and that the Alq mutation promoted a gain-of-function phenotype caused by the ectopic expression of TAGL1. Ectopic overexpression of TAGL1 resulted in homeotic alterations affecting floral organ identity that were similar to but stronger than those observed in Alq mutant plants. Interestingly, TAGL1 RNAi plants yielded tomato fruits which were unable to ripen. They displayed a yellow-orange color and stiffness appearance which are in accordance with reduced lycopene and ethylene levels, respectively. Moreover, pericarp cells of TAGL1 RNAi fruits showed altered cellular and structural properties which correlated to both decreased expression of genes regulating cell division and lignin biosynthesis. Over-expression of TAGL1 is able to rescue the non-ripening phenotype of rin and nor mutants, which is mediated by the transcriptional activation of several ripening genes. Our results demonstrated that TAGL1 participates in the genetic control of flower and fruit development of tomato plants. Furthermore, gene silencing and over-expression experiments demonstrated that the fruit ripening process requires the regulatory activity of TAGL1. Therefore, TAGL1 could act as a linking factor connecting successive stages of reproductive development, from flower development to fruit maturation, allowing this complex process to be carried out successfully., This work was funded by the Spanish Ministry of Science and Innovation (grants AGL2009-13388 and BIO2009-11484). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

International audience; Background: Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes. Methodology/Principal Findings: We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordeiinfected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis. Conclusions/Significance: The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this population.

BackgroundThere is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size ( approximately 23.8 Gb/C).Methodology/principal findingsA 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates).Conclusions/significanceThis study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.

Plants respond to different stresses by inducing or repressing transcription of partially overlapping sets of genes. In Arabidopsis, the PHR1 transcription factor (TF) has an important role in the control of phosphate (Pi) starvation stress responses. Using transcriptomic analysis of Pi starvation in phr1, and phr1 phr1-like (phl1) mutants and in wild type plants, we show that PHR1 in conjunction with PHL1 controls most transcriptional activation and repression responses to phosphate starvation, regardless of the Pi starvation specificity of these responses. Induced genes are enriched in PHR1 binding sequences (P1BS) in their promoters, whereas repressed genes do not show such enrichment, suggesting that PHR1(-like) control of transcriptional repression responses is indirect. In agreement with this, transcriptomic analysis of a transgenic plant expressing PHR1 fused to the hormone ligand domain of the glucocorticoid receptor showed that PHR1 direct targets (i.e., displaying altered expression after GR:PHR1 activation by dexamethasone in the presence of cycloheximide) corresponded largely to Pi starvation-induced genes that are highly enriched in P1BS. A minimal promoter containing a multimerised P1BS recapitulates Pi starvation-specific responsiveness. Likewise, mutation of P1BS in the promoter of two Pi starvation-responsive genes impaired their responsiveness to Pi starvation, but not to other stress types. Phylogenetic footprinting confirmed the importance of P1BS and PHR1 in Pi starvation responsiveness and indicated that P1BS acts in concert with other cis motifs. All together, our data show that PHR1 and PHL1 are partially redundant TF acting as central integrators of Pi starvation responses, both specific and generic. In addition, they indicate that transcriptional repression responses are an integral part of adaptive responses to stress., Author Summary As sessile organisms, plants are often exposed to stress conditions, and have evolved adaptive responses to protect themselves from different types of stress. Some responses are stress type-specific whereas others are common to different stress types. Understanding how these responses are controlled is crucial for rational improvement of stress tolerance, a limiting factor in crop productivity. Here we examined the physiological and molecular responses to phosphate starvation and found that a single transcription factor family, represented by PHOSPHATE STARVATION RESPONSE REGULATOR 1 (PHR1), has a central role in the control of specific and shared phosphate starvation stress responses. In consonance with the importance of PHR1, we found that the PHR1-binding sequence, present in most PHR1 direct targets, is a crucial cis motif for Pi starvation responsiveness. An artificial promoter controlled by PHR1 recapitulates responsiveness to Pi starvation and to modulators of this response, qualifying PHR1 family members as central integrators in Pi starvation signalling. This central integrator system also controls most transcriptional repression responses to Pi starvation, indicating that they are an integral part of the adaptive response, and not a consequence of plant malfunction due to stress.

Flowering time is a key life-history trait in the plant life cycle. Most studies to unravel the genetics of flowering time in Arabidopsis thaliana have been performed under greenhouse conditions. Here, we describe a study about the genetics of flowering time that differs from previous studies in two important ways: first, we measure flowering time in a more complex and ecologically realistic environment; and, second, we combine the advantages of genome-wide association (GWA) and traditional linkage (QTL) mapping. Our experiments involved phenotyping nearly 20,000 plants over 2 winters under field conditions, including 184 worldwide natural accessions genotyped for 216,509 SNPs and 4,366 RILs derived from 13 independent crosses chosen to maximize genetic and phenotypic diversity. Based on a photothermal time model, the flowering time variation scored in our field experiment was poorly correlated with the flowering time variation previously obtained under greenhouse conditions, reinforcing previous demonstrations of the importance of genotype by environment interactions in A. thaliana and the need to study adaptive variation under natural conditions. The use of 4,366 RILs provides great power for dissecting the genetic architecture of flowering time in A. thaliana under our specific field conditions. We describe more than 60 additive QTLs, all with relatively small to medium effects and organized in 5 major clusters. We show that QTL mapping increases our power to distinguish true from false associations in GWA mapping. QTL mapping also permits the identification of false negatives, that is, causative SNPs that are lost when applying GWA methods that control for population structure. Major genes underpinning flowering time in the greenhouse were not associated with flowering time in this study. Instead, we found a prevalence of genes involved in the regulation of the plant circadian clock. Furthermore, we identified new genomic regions lacking obvious candidate genes., Author Summary Dissecting the genetic bases of adaptive traits is of primary importance in evolutionary biology. In this study, we combined a genome-wide association (GWA) study with traditional linkage mapping in order to detect the genetic bases underlying natural variation in flowering time in ecologically realistic conditions in the plant Arabidopsis thaliana. Our study involved phenotyping nearly 20,000 plants over 2 winters under field conditions in a temperate climate. We show that combined linkage and association mapping clearly outperforms each method alone when it comes to identifying true associations. This highlights the utility of combining different methods to localize genes involved in complex trait natural variation. Most candidate genes found in this study are involved in the regulation of the plant circadian clock and, surprisingly, were not associated with flowering time scored under greenhouse conditions. While rapid advances have been made in high-throughput genotyping and sequencing, high-throughput phenotyping of complex traits under natural conditions will be the next challenge for dissecting the genetic bases of adaptive variation in “laboratory” model organisms.

International audience; The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix− nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this “nodule-specific transcriptome” were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.

Background: Higher plants possess a large multigene family encoding secreted class III peroxidase (Prx) proteins. Peroxidases appear to be associated with plant disease resistance based on observations of induction during disease challenge and the presence or absence of isozymes in resistant vs susceptible varieties. Despite these associations, there is no evidence that allelic variation of peroxidases directly determines levels of disease resistance. Methodology/Principal Findings: The current study introduces a new strategy called Prx-Profiling. We showed that with this strategy a large number of peroxidase genes can be mapped on the barley genome. In order to obtain an estimate of the total number of Prx clusters we followed a re-sampling procedure, which indicated that the barley genome contains about 40 peroxidase gene clusters. We examined the association between the Prxs mapped and the QTLs for resistance of barley to homologous and heterologous rusts, and to the barley powdery mildew fungus. We report that 61% of the QTLs for partial resistance to P. hordei, 61% of the QTLs for resistance to B. graminis and 47% of the QTLs for non-host resistance to other Puccinia species co-localize with Prx based markers. Conclusions/Significance: We conclude that Prx-Profiling was effective in finding the genetic location of Prx genes on the barley genome. The finding that QTLs for basal resistance to rusts and powdery mildew fungi tend to co-locate with Prx clusters provides a base for exploring the functional role of Prx-related genes in determining natural differences in levels of basal resistance. © 2010 González et al.

A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora × G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (−90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (−148 to −85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the −149 to −124 and −107 to −83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants.

The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB–type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle., Author Summary A major step in the evolution of land plants was the formation of a cuticular layer on their outer surfaces. Despite the cuticle's key role in organ development and in protecting against a variety of stresses, very little is known about the regulation of the metabolic pathways that generate its building blocks. Flavonoids, often embedded in the cuticle, have been suggested to impact the characteristics of this structure and to provide protection against radiation and pathogens. Flavonoids are an integral part of the human diet and are likely responsible for the observed beneficial effects of a fruit-rich diet. Here, we examine in detail the tomato colorless peel y mutant, which lacks the yellow flavonoid pigment naringenin chalcone, a major constituent of the fruit cuticle. Extensive transcript and metabolite profiling of this mutant revealed SlMYB12 as a key regulator in a transcription network that controls flavonoid accumulation in tomato peel. Moreover, the change in cuticle flavonoid composition enabled us to evaluate the importance of these constituents as barriers in the cuticle layer. Finally, because most commercial cultivars in the Far East are based on the y genetic background, discovery of the y gene will contribute also to tomato breeding programs.

A newly developed maize Illumina GoldenGate Assay with 1536 SNPs from 582 loci was used to genotype a highly diverse global maize collection of 632 inbred lines from temperate, tropical, and subtropical public breeding programs. A total of 1229 informative SNPs and 1749 haplotypes within 327 loci was used to estimate the genetic diversity, population structure, and familial relatedness. Population structure identified tropical and temperate subgroups, and complex familial relationships were identified within the global collection. Linkage disequilibrium (LD) was measured overall and within chromosomes, allelic frequency groups, subgroups related by geographic origin, and subgroups of different sample sizes. The LD decay distance differed among chromosomes and ranged between 1 to 10 kb. The LD distance increased with the increase of minor allelic frequency (MAF), and with smaller sample sizes, encouraging caution when using too few lines in a study. The LD decay distance was much higher in temperate than in tropical and subtropical lines, because tropical and subtropical lines are more diverse and contain more rare alleles than temperate lines. A core set of inbreds was defined based on haplotypes, and 60 lines capture 90% of the haplotype diversity of the entire panel. The defined core sets and the entire collection can be used widely for different research targets.

MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR–RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with ∼35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes., Author Summary MicroRNAs are non-coding RNAs that regulate gene expression post-transcriptionally and play roles in diverse pathways including those acting on development and responses to stress. Here, we describe a genome-wide computational prediction of maize miRNA genes and their characterization with respect to expression, putative targets, evolution following whole genome duplication, and allelic diversity. The structures of unprocessed primary miRNA transcripts were determined by 5′ RACE and 3′ RACE. Expression profiles were surveyed in five tissue types by deep-sequencing of small RNA libraries. We predicted miRNA targets computationally based on the most recent maize protein annotations. Analysis of the predicted functions of target genes, on the basis of gene ontology, supported their roles in regulatory processes. We identified putative orthologs in Sorghum based on an analysis of synteny and found that maize-homoeologous miRNA genes were retained more frequently than expected. We also explored miRNA nucleotide diversity among many maize inbred lines and partially inbred teosinte lines. The results indicated that mature miRNA genes were highly conserved during their evolution. This preliminary characterization based on our findings provides a framework for future analysis of miRNA genes and their roles in key traits of maize as feed, fodder, and biofuel.

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination., Author Summary Genomic insertion sites of Mu transposons were amplified and sequenced via next generation technology, revealing more than 40,000 non-redundant Mu insertion sites that are non-uniformly distributed across the maize genome and within genes. Along chromosomes, frequencies of Mu transposon insertions are strongly correlated with recombination rates. Although both Mu and recombination occur preferentially in genes, gene density does not fully explain these patterns. Instead, the finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination.

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop., Author Summary There is a growing appreciation for the role of genome structural variation in creating phenotypic variation within a species. Comparative genomic hybridization was used to compare the genome structures of two maize inbred lines, B73 and Mo17. The data reinforce the view that maize is a highly polymorphic species, but also show that there are often large genomic regions that have little or no variation. We identify several hundred sequences that, while present in both B73 and Mo17, have copy number differences in the two genomes. In addition, there are several thousand sequences, including at least 180 sequences annotated as single-copy genes, that are present in one genome but entirely missing in the other genome. This genome content variation leads to differences in transcript content between inbred lines and likely contributes to phenotypic diversity and heterosis in maize.

Photosynthetic diatoms are exposed to rapid and unpredictable changes in irradiance and spectral quality, and must be able to acclimate their light harvesting systems to varying light conditions. Molecular mechanisms behind light acclimation in diatoms are largely unknown. We set out to investigate the mechanisms of high light acclimation in Phaeodactylum tricornutum using an integrated approach involving global transcriptional profiling, metabolite profiling and variable fluorescence technique. Algae cultures were acclimated to low light (LL), after which the cultures were transferred to high light (HL). Molecular, metabolic and physiological responses were studied at time points 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h after transfer to HL conditions. The integrated results indicate that the acclimation mechanisms in diatoms can be divided into an initial response phase (0–0.5 h), an intermediate acclimation phase (3–12 h) and a late acclimation phase (12–48 h). The initial phase is recognized by strong and rapid regulation of genes encoding proteins involved in photosynthesis, pigment metabolism and reactive oxygen species (ROS) scavenging systems. A significant increase in light protecting metabolites occur together with the induction of transcriptional processes involved in protection of cellular structures at this early phase. During the following phases, the metabolite profiling display a pronounced decrease in light harvesting pigments, whereas the variable fluorescence measurements show that the photosynthetic capacity increases strongly during the late acclimation phase. We show that P. tricornutum is capable of swift and efficient execution of photoprotective mechanisms, followed by changes in the composition of the photosynthetic machinery that enable the diatoms to utilize the excess energy available in HL. Central molecular players in light protection and acclimation to high irradiance have been identified. © 2009 Nymark et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In West Africa two rice species (Oryza glaberrima Steud. and Oryza sativa L.) co-exist. Although originally it was thought that interspecific hybridization is impossible without biotechnological methods, progenies of hybridization appear to occur in farmer fields. AFLP analysis was used to assess genetic diversity in West Africa (including the countries The Gambia, Senegal, Guinea Bissau, Guinea Conakry, Sierra Leone, Ghana and Togo) using 315 rice samples morphologically classified prior to analysis. We show evidence for farmer interspecific hybrids of African and Asian rice, resulting in a group of novel genotypes, and identify possible mechanisms for in-field hybridization. Spontaneous back-crossing events play a crucial role, resulting in different groups of genetic diversity in different regions developed by natural and cultural selection, often under adverse conditions. These new groups of genotypes may have potential relevance for exploitation by plant breeders. Future advances in crop development could be achieved through co-operation between scientists and marginalized farmer groups in order to address challenges of rapid adaptation in a world of increasing socio-political and climatic uncertainty.

12 páginas, 7 figuras., Dynamic regulation of chromatin structure is of fundamental importance for modulating genomic activities in higher eukaryotes. The opposing activities of Polycomb group (PcG) and trithorax group (trxG) proteins are part of a chromatin-based cellular memory system ensuring the correct expression of specific transcriptional programs at defined developmental stages. The default silencing activity of PcG proteins is counteracted by trxG proteins that activate PcG target genes and prevent PcG mediated silencing activities. Therefore, the timely expression and regulation of PcG proteins and counteracting trxG proteins is likely to be of fundamental importance for establishing cell identity. Here, we report that the chromodomain/helicase/DNA–binding domain CHD3 proteins PICKLE (PKL) and PICKLE RELATED2 (PKR2) have trxG-like functions in plants and are required for the expression of many genes that are repressed by PcG proteins. The pkl mutant could partly suppress the leaf and flower phenotype of the PcG mutant curly leaf, supporting the idea that CHD3 proteins and PcG proteins antagonistically determine cell identity in plants. The direct targets of PKL in roots include the PcG genes SWINGER and EMBRYONIC FLOWER2 that encode subunits of Polycomb repressive complexes responsible for trimethylating histone H3 at lysine 27 (H3K27me3). Similar to mutants lacking PcG proteins, lack of PKL and PKR2 caused reduced H3K27me3 levels and, therefore, increased expression of a set of PcG protein target genes in roots. Thus, PKL and PKR2 are directly required for activation of PcG protein target genes and in roots are also indirectly required for repression of PcG protein target genes. Reduced PcG protein activity can lead to cell de-differentiation and callus-like tissue formation in pkl pkr2 mutants. Thus, in contrast to mammals, where PcG proteins are required to maintain pluripotency and to prevent cell differentiation, in plants PcG proteins are required to promote cell differentiation by suppressing embryonic development., This research was supported by Swiss National Science Foundation (http://www.snf.ch/D/Seiten/default.aspx) grant PP00A-106684/1 to CK, grant 3100AO-116060 to LH, and by the ETH Research Grant (http://www.vpf.ethz.ch/services/TH/index​_EN) TH-12 06-1 to CK.

Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses., Author Summary Multicellular plants possess a unique set of DNA–dependent RNA polymerase complexes (RNAPs) that prevent certain repetitious regions of the genome from being copied into stable RNAs. Two distinct RNAPs, termed Pol IV and Pol V, are required for this type of genome-silencing behavior in the eudicot Arabidopsis thaliana, but the mechanism by which these RNAPs accomplish this function is still relatively unknown. Using genetic and molecular methodologies, we identified a Pol IV–type subunit protein as being involved in a process of meiotically-heritable gene silencing in the maize plant known as paramutation. Our analyses of the available plant genome sequences indicate that monocots have a greater potential for RNAP diversity due to having duplicate variants of this particular subunit. Consistent with this inferred diversity, comparative analyses with plants defective in a different core Pol IV subunit indicate that the Pol IV–type RNAP in maize has distinct functional isoforms. The mechanistic and biological role(s) of these specific RNAPs in mediating genome regulation and heritable gene silencing in large genome cereals should now be tractable by biochemical approaches.

Mutations affecting the maintenance of heritable epigenetic states in maize identify multiple RNA–directed DNA methylation (RdDM) factors including RMR1, a novel member of a plant-specific clade of Snf2-related proteins. Here we show that RMR1 is necessary for the accumulation of a majority of 24 nt small RNAs, including those derived from Long-Terminal Repeat (LTR) retrotransposons, the most common repetitive feature in the maize genome. A genetic analysis of DNA transposon repression indicates that RMR1 acts upstream of the RNA–dependent RNA polymerase, RDR2 (MOP1). Surprisingly, we show that non-polyadenylated transcripts from a sampling of LTR retrotransposons are lost in both rmr1 and rdr2 mutants. In contrast, plants deficient for RNA Polymerase IV (Pol IV) function show an increase in polyadenylated LTR RNA transcripts. These findings support a model in which Pol IV functions independently of the small RNA accumulation facilitated by RMR1 and RDR2 and support that a loss of Pol IV leads to RNA Polymerase II–based transcription. Additionally, the lack of changes in general genome homeostasis in rmr1 mutants, despite the global loss of 24 nt small RNAs, challenges the perceived roles of siRNAs in maintaining functional heterochromatin in the genomes of outcrossing grass species., Author Summary Most eukaryotic genomes are divided into two functional classes of regulation: the euchromatic and the heterochromatic. Heterochromatic regions, often composed of potentially deleterious transposons and retrotransposons, are typically viewed as “silent” or not transcribed. Paradoxically, evidence from multiple organisms indicates that heterochromatic regions must be transcribed to maintain a heterochromatic character. In plants, specialized RNA polymerase complexes are thought to specifically process repetitive regions of the genome into small RNA molecules that facilitate maintenance of a heterochromatic environment. We investigated the role of this specialized polymerase pathway in maintaining maize genome homeostasis with particular focus on RMR1, a novel protein related to a family of DNA repair proteins, whose function in modifying repetitive regions of the genome is unknown. We find most small RNA generation is dependent on RMR1, which appears to function downstream of the specialized polymerase, RNA polymerase IV. However, we provide evidence that the function of RNA polymerase IV is not disrupted by the absence of small RNA generation. Our results suggest the division of the plant genome into euchromatin and heterochromatin is maintained by template competition between the specialized plant polymerases and canonical RNA polymerase II, and not by the subsequent generation of small RNA molecules.

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed., Author Summary DNA replication, repair, and recombination are interrelated processes. Chromatin structure, into which DNA is packaged, is important for regulation of DNA replication, repair, and recombination, as well as gene transcription. After DNA replication and repair, chromatin status including its structure and modification has to be reproduced, and defects in these processes can alter gene expression program because of change in chromatin regulation. Our series of genetic analysis of tebichi (teb) mutant of model plant Arabidopsis thaliana suggest that TEB gene is involved in DNA replication and recombination. We also show here that TEB gene is required for correct expression of many genes including genes regulating development. From these results we propose that TEB gene function is important for maintenance of gene expression pattern after DNA replication and recombination. Furthermore, preferential upregulation of genes near highly duplicated transposons and tandemly arrayed homologous genes are observed in teb mutants, suggesting the interrelationship between homologous recombination and gene transcription around the repetitive sequences.

The flowering repressors SMZ and FLM, members of the AP-2 and MADS domain transcription factor families, unexpectedly work together to regulate flowering time via their effects on expression of the FT gene., A small mobile protein, encoded by the FLOWERING LOCUS T (FT) locus, plays a central role in the control of flowering. FT is regulated positively by CONSTANS (CO), the output of the photoperiod pathway, and negatively by FLC, which integrates the effects of prolonged cold exposure. Here, we reveal the mechanisms of regulation by the microRNA miR172 target SCHLAFMÜTZE (SMZ), a potent repressor of flowering. Whole-genome mapping of SMZ binding sites demonstrates not only direct regulation of FT, but also of many other flowering time regulators acting both upstream and downstream of FT, indicating an important role of miR172 and its targets in fine tuning the flowering response. A role for the miR172/SMZ module as a rheostat in flowering time is further supported by SMZ binding to several other genes encoding miR172 targets. Finally, we show that the action of SMZ is completely dependent on another floral repressor, FLM, providing the first direct connection between two important classes of flowering time regulators, AP2- and MADS-domain proteins., Author Summary Flowering is a pivotal event in the life cycle of many plants and is therefore under tight control. The ability to detect the daily photoperiod is of particular importance in many plant species, as it enables them to enter the reproductive phase in response to seasonal changes in day length. When the photoperiod is permissive to flowering, a signal is produced in leaves that is transported to the shoot meristem, where it initiates the formation of flowers. It is now widely accepted that an important component of this long-distance signal is the flowering protein FT. Here, we show that the AP2-like transcription factor SMZ, which represses flowering and is a target of the regulatory miRNA172 microRNA, functions together with related proteins to directly regulate FT expression. Using chromatin immunoprecipitation coupled to genome tiling arrays, we find that SMZ binds directly to the FT genomic locus and to several other key flowering-related loci. Unexpectedly, the ability of SMZ to repress flowering strictly depends on the presence of the MADS-domain transcription factor FLM. In addition, SMZ binds to its own regulatory sequences and those of three closely related genes, providing evidence of strong negative feedback between SMZ and the other AP2-like miRNA172 targets.

International audience; Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.

Though central to our understanding of how roots perform their vital function of scavenging water and solutes from the soil, no direct genetic evidence currently exists to support the foundational model that suberin acts to form a chemical barrier limiting the extracellular, or apoplastic, transport of water and solutes in plant roots. Using the newly characterized enhanced suberin1 (esb1) mutant, we established a connection in Arabidopsis thaliana between suberin in the root and both water movement through the plant and solute accumulation in the shoot. Esb1 mutants, characterized by increased root suberin, were found to have reduced day time transpiration rates and increased water-use efficiency during their vegetative growth period. Furthermore, these changes in suberin and water transport were associated with decreases in the accumulation of Ca, Mn, and Zn and increases in the accumulation of Na, S, K, As, Se, and Mo in the shoot. Here, we present direct genetic evidence establishing that suberin in the roots plays a critical role in controlling both water and mineral ion uptake and transport to the leaves. The changes observed in the elemental accumulation in leaves are also interpreted as evidence that a significant component of the radial root transport of Ca, Mn, and Zn occurs in the apoplast., Author Summary The root system is a highly specialized plant organ that works to get both water and essential mineral nutrients from the changing chemically and physically complex environment of the soil. Roots do this by both controlling the uptake of water and essential mineral ions, as well as regulating their movement to the central vascular system of the plant for long distance transport to the shoot. To allow the cellular control of water and mineral ion uptake and transport via specialized transport proteins, plant roots contain a waxy layer of suberin that acts to seal connections between cells, preventing uncontrolled leakage of water and mineral ions between cells. By screening thousands of mutant A. thaliana plants, we were able to identify a plant with elevated levels of suberin in the root. Using this mutant, we were able to uncover the importance of suberin in sealing connections between root cells to regulate water movement through the plant and accumulation of various essential and nonessential minerals in leaves, including sodium, sulfur, potassium, calcium, manganese, zinc, arsenic, selenium, and molybdenum.

BACKGROUND: Nitrogen (N(2)) fixation also yields hydrogen (H(2)) at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2) as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. METHODS AND FINDINGS: As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2)-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. CONCLUSIONS: Representative of aerobic N(2)-fixing and H(2)-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2) respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2), specifically that produced by N(2) fixation. To benefit human civilization, H(2) has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such as the A. caulinodans Hyq endo-hydrogenase, offer promise as biocatalytic agents for H(2) production and/or consumption.