A versatile CRISPR-based system for lineage tracing in living plants.

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.

Reduced coenzyme Q synthesis confers non-target site resistance to the herbicide thaxtomin A.

Herbicide resistance in weeds is a growing threat to global crop production. Non-target site resistance is problematic because a single resistance allele can confer tolerance to many herbicides (cross resistance), and it is often a polygenic trait so it can be difficult to identify the molecular mechanisms involved. Most characterized molecular mechanisms of non-target site resistance are caused by gain-of-function mutations in genes from a few key gene families-the mechanisms of resistance caused by loss-of-function mutations remain unclear. In this study, we first show that the mechanism of non-target site resistance to the herbicide thaxtomin A conferred by loss-of-function of the gene PAM16 is conserved in Marchantia polymorpha, validating its use as a model species with which to study non-target site resistance. To identify mechanisms of non-target site resistance caused by loss-of-function mutations, we generated 107 UV-B mutagenized M. polymorpha spores and screened for resistance to the herbicide thaxtomin A. We isolated 13 thaxtomin A-resistant mutants and found that 3 mutants carried candidate resistance-conferring SNPs in the MpRTN4IP1L gene. Mprtn4ip1l mutants are defective in coenzyme Q biosynthesis and accumulate higher levels of reactive oxygen species (ROS) than wild-type plants. Mutants are weakly resistant to thaxtomin A and cross resistant to isoxaben, suggesting that loss of MpRTN4IP1L function confers non-target site resistance. Mutants are also defective in thaxtomin A metabolism. We conclude that loss of MpRTN4IP1L function is a novel mechanism of non-target site herbicide resistance and propose that other mutations that increase ROS levels or decrease thaxtomin A metabolism could contribute to thaxtomin A resistance in the field.

Meristem dormancy in Marchantia polymorpha is regulated by a liverwort-specific miRNA and a clade III SPL gene.

The shape of modular organisms depends on the branching architecture, which in plants is determined by the fates of generative centers called meristems. The branches of the liverwort Marchantia polymorpha are derived from two adjacent meristems that develop at thallus apices. These meristems may be active and develop branches or may be dormant and do not form branches. The relative number and position of active and dormant meristems define the overall shape and form of the thallus. We show that the clade III SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor MpSPL1 is required for meristem dormancy. The activity of MpSPL1 is regulated by the liverwort-specific Mpo-MR13 miRNA, which, in turn, is regulated by PIF-mediated signaling. An unrelated PIF-regulated miRNA, MIR156, represses a different SPL gene (belonging to clade IV) that inhibits branching during the shade avoidance response in Arabidopsis thaliana. This suggests that a conserved light signaling mechanism modulates branching architecture in liverworts and angiosperms and therefore is likely operated in the last common ancestor. However, PIF-mediated signaling represses the expression of different miRNA genes with different SPL targets during dichotomous, apical branching in liverworts and during lateral, subapical branching in angiosperms. We speculate that the mechanism that acts downstream of light and regulates meristem dormancy evolved independently in liverworts and angiosperms.

Gene expression variation in Arabidopsis embryos at single-nucleus resolution.

Soon after fertilization of egg and sperm, plant genomes become transcriptionally activated and drive a series of coordinated cell divisions to form the basic body plan during embryogenesis. Early embryonic cells rapidly diversify from each other, and investigation of the corresponding gene expression dynamics can help elucidate underlying cellular differentiation programs. However, current plant embryonic transcriptome datasets either lack cell-specific information or have RNA contamination from surrounding non-embryonic tissues. We have coupled fluorescence-activated nuclei sorting together with single-nucleus mRNA-sequencing to construct a gene expression atlas of Arabidopsis thaliana early embryos at single-cell resolution. In addition to characterizing cell-specific transcriptomes, we found evidence that distinct epigenetic and transcriptional regulatory mechanisms operate across emerging embryonic cell types. These datasets and analyses, as well as the approach we devised, are expected to facilitate the discovery of molecular mechanisms underlying pattern formation in plant embryos. This article has an associated 'The people behind the papers' interview.

Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos.

Small RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.

MicroRNA Dynamics and Functions During Arabidopsis Embryogenesis.

MicroRNAs (miRNAs) are short noncoding RNAs that mediate the repression of target transcripts in plants and animals. Although miRNAs are required throughout plant development, relatively little is known regarding their embryonic functions. To systematically characterize embryonic miRNAs in Arabidopsis (Arabidopsis thaliana), we developed or applied high-throughput sequencing-based methods to profile hundreds of miRNAs and associated targets throughout embryogenesis. We discovered dozens of miRNAs that dynamically cleave and repress target transcripts, including 30 that encode transcription factors. Transcriptome analyses indicated that these miRNA:target interactions have profound effects on embryonic gene expression programs. Moreover, we demonstrated that the miRNA-mediated repression of six transcription factors are individually required for proper division patterns of various embryonic cell lineages. These data indicate that the miRNA-directed repression of multiple transcription factors is critically important for the establishment of the plant body plan, and they provide a foundation to further investigate how miRNAs contribute to these initial cellular differentiation events.

Whole Mount in situ Localization of miRNAs and mRNAs During Somatic Embryogenesis in Arabidopsis.

Somatic embryogenesis (SE) results from the transition of differentiated plant somatic cells into embryogenic cells that requires the extensive reprogramming of the somatic cell transcriptome. Commonly, the SE-involved genes are identified by analyzing the heterogeneous population of explant cells and thus, it is necessary to validate the expression of the candidate genes in the cells that are competent for embryogenic transition. Here, we optimized and implemented the whole mount in situ hybridization (WISH) method (Bleckmann and Dresselhaus, 2016; Dastidar et al., 2016) in order to analyze the spatiotemporal localization of miRNAs (miR156, miR166, miR390, miR167) and mRNAs such as WOX5 and PHABULOSA-target of miR165/166 during the SE that is induced in Arabidopsis explants. This study presents a detailed step-by-step description of the WISH procedure in which DIG-labeled LNA and RNA probes were used to detect miRNAs and mRNAs, respectively. The usefulness of the WISH in the functional analysis of the SE-involved regulatory pathways is demonstrated and the advantages of this method are highlighted: (i) the ability to analyze intact non-sectioned plant tissue; (ii) the specificity of transcript detection; (iii) the detection of miRNA; and (iv) a semi-quantitative assessment of the RNA abundance.

Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation.

Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer, and reduced fertility. So far, 19 genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homolog of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here, we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE81 (AtMUS81), known for its role in the formation of noninterfering COs. AtFANCD2 promotes noninterfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously.

Sensitive whole mount in situ localization of small RNAs in plants.

Small RNAs, such as microRNAs (miRNAs), regulate gene expression and play important roles in many plant processes. Although our knowledge of their biogenesis and mode of action has significantly progressed, we still have comparatively little information about their biological functions. In particular, knowledge about their spatio-temporal expression patterns rely on either indirect detection by use of reporter constructs or labor-intensive direct detection by in situ hybridization on sectioned material. None of the current approaches allows a systematic investigation of small RNA expression patterns. Here, we present a sensitive method for in situ detection of miRNAs and siRNAs in intact plant tissues that utilizes both double-labeled probes and a specific cross-linker. We determined the expression patterns of several small RNAs in diverse plant tissues.

Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS.

One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 106 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.

Contrasting evolution of a satellite DNA and its ancestral IGS rDNA in Phaseolus (Fabaceae).

CC4 is a satellite DNA from common bean (Phaseolus vulgaris L.) that is similar to its intergenic spacer (IGS) rDNA. CC4 was originally hypothesized to be an old, fast evolving satellite family that has invaded common bean rDNA. To test this hypothesis and contribute to the understanding of IGS-like satellite DNA evolution, we have investigated its distribution in the genus Phaseolus and related species. CC4 was cloned and used as probe for Southern blot and FISH experiments. CC4 was observed as an independent satellite in common bean, forming two to three major and a few minor pericentromeric clusters. In Phaseolus coccineus, CC4 was present in four major clusters, also not co-localized with the 45S rDNA sites. Remarkably, in the less related species of the genus, signals were detected co-localized with the 45S rDNA sites, but co-localization was not observed in the species where CC4 is present as an independent satellite. No signal was detected in species from related genera. Altogether, the data suggest that CC4 has originated from the IGS rDNA in the P. vulgaris-P. coccineus lineage and has evolved slower than the IGS rDNA from this lineage.

Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte.

In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. Here we show that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes (MEA and FWA) and a repetitive element (Mu1a) are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. Although the genome of the vegetative cell does not participate in double fertilization, its DME-mediated demethylation is important for male fertility and may contribute to the reconfiguration of the methylation landscape that occurs in the vegetative cell genome.

Cytogenetic map of common bean (Phaseolus vulgaris L.).

A cytogenetic map of common bean was built by in situ hybridization of 35 bacterial artificial chromosomes (BACs) selected with markers mapping to eight linkage groups, plus two plasmids for 5S and 45S ribosomal DNA and one bacteriophage. Together with three previously mapped chromosomes (chromosomes 3, 4, and 7), 43 anchoring points between the genetic map and the cytogenetic map of the species are now available. Furthermore, a subset of four BAC clones was proposed to identify the 11 chromosome pairs of the standard cultivar BAT93. Three of these BACs labelled more than a single chromosome pair, indicating the presence of repetitive DNA in their inserts. A repetitive distribution pattern was observed for most of the BACs; for 38% of them, highly repetitive pericentromeric or subtelomeric signals were observed. These distribution patterns corresponded to pericentromeric and subtelomeric heterochromatin blocks observed with other staining methods. Altogether, the results indicate that around half of the common bean genome is heterochromatic and that genes and repetitive sequences are intermingled in the euchromatin and heterochromatin of the species.

Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin.

Centromeric constitutive heterochromatin is marked by DNA methylation and dimethylated histone H3 Lys 9 (H3K9me2) in Arabidopsis. RNA-directed DNA methylation (RdDM) is a process that uses 24-nucleotide (nt) small interfering RNAs (siRNAs) to induce de novo methylation to its homologous DNA sequences. Despite the presence of centromeric 24-nt siRNAs, mutations in genes required for RdDM do not appreciably influence the methylation of centromeric repeats. The mechanism by which constitutive heterochromatin is protected from RdDM remains puzzling. Here, we report that the vegetative cell nuclei (VN) of the male gametophyte (pollen) invariably undergo extensive decondensation of centromeric heterochromatin and lose centromere identity. VN show greatly reduced H3K9me2, phenocopying nuclei carrying a mutation in the chromatin remodeller DECREASE IN DNA METHYLATION 1 (DDM1). However, unlike the situation in ddm1 nuclei, the decondensed heterochromatin retains dense CG methylation and transcriptional silencing, and, unexpectedly, is subjected to RdDM-dependent hypermethylation in non-CG contexts. These findings reveal two assembly orders of silent heterochromatin and implicate the condensed form in blocking the RdDM machinery.

Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution.

The extent of 5S and 45S ribosomal DNA (rDNA) variation was investigated in wild and domesticated common beans (Phaseolus vulgaris) chosen to represent the known genetic diversity of the species. 5S and 45S rDNA probes were localized on mitotic chromosomes of 37 accessions by fluorescent in situ hybridization (FISH). The two 5S rDNA loci were largely conserved within the species, whereas a high variation in the number of 45S rDNA loci and changes in position of loci and number of repeats per locus were observed. Domesticated accessions from the Mesoamerican gene pool frequently had three 45S rDNA loci per haploid genome, and rarely four. Domesticated accessions from Andean gene pool, particularly from the race Peru, showed six, seven, eight or nine loci, but seven loci were found in all three races of this gene pool. Between three and eight loci were observed in accessions resulting from crosses between Andean and Mesoamerican genotypes. The presence of two to eight 45S rDNA loci in wild common beans from different geographic locations indicates that the 45S rDNA amplification observed in the Andean lineage took place before domestication. Our data suggest that ectopic recombination between terminal chromosomal regions might be the mechanism responsible for this variation.

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.