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.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Multi‐color FISH facilitates analysis of cell‐type diversification and developmental gene regulation in the Parasteatoda spider embryo

The simultaneous and quantitative analysis of the expression of multiple genes helps to shed light on gene regulatory networks. We established a method for multi‐color fluorescence in situ hybridization (mFISH) for the analysis of cell‐type diversification and developmental gene regulation in the embryo of the spider Parasteatoda tepidariorum. This mFISH technique allowed quadruple staining using four types of labels for RNA probes, digoxigenin, fluorescein, biotin, and dinitrophenyl, together with different fluorescent tyramides. To validate the usability of mFISH, we conducted four experiments. First, we distinguished similar gene expression patterns with mFISH, which showed overlaps and differences in the expression domains of anterior patterning hedgehog (hh), orthodenticle (otd), and labial genes at a cellular resolution. Second, we used mFISH to identify early cell types that are internalized on the anterior side. We found that fork head‐positive cells were subdivided into two cell types, 012_A08‐positive endoderm cells and twist‐positive mesoderm cells. Third, we quantified the ratio of expression levels of the odd‐paired (opa) gene in the chelicera and pedipalp segments based on the intensity of mFISH signals. Finally, we combined mFISH with embryonic RNA interference. It was possible to identify opa knockdown cell clones and detect the specific reduction of opa and the upregulation of otd and hh expression levels in the same cell clone that formed in the head region. This study proposes that mFISH is a powerful tool for the cell‐level analysis of gene regulation and quantification in the spider model.     

Rolling‐circle amplification of centromeric Helitrons in plant genomes

The unusual eukaryotic Helitron transposons can readily capture host sequences and are, thus, evolutionarily important. They are presumed to amplify by rolling‐circle replication (RCR) because some elements encode predicted proteins homologous to RCR prokaryotic transposases. In support of this replication mechanism, it was recently shown that transposition of a bat Helitron generates covalently closed circular intermediates. Another strong prediction is that RCR should generate tandem Helitron concatemers, yet almost all Helitrons identified to date occur as solo elements in the genome. To investigate alternative modes of Helitron organization in present‐day genomes, we have applied the novel computational tool HelitronScanner to 27 plant genomes and have uncovered numerous tandem arrays of partially decayed, truncated Helitrons in all of them. Strikingly, most of these Helitron tandem arrays are interspersed with other repeats in centromeres. Many of these arrays have multiple Helitron 5′ ends, but a single 3′ end. The number of repeats in any one array can range from a handful to several hundreds. We propose here an RCR model that conforms to the present Helitron landscape of plant genomes. Our study provides strong evidence that plant Helitrons amplify by RCR and that the tandemly arrayed replication products accumulate mostly in centromeres.     

One‐pot sample preparation approach for profiling spatial distribution of gibberellins in a single shoot of germinating cereal seeds

Sample preparation remains a bottleneck in the rapid and reliable quantification of gibberellins (GAs) for obtaining an insight into the physiological processes mediated by GAs. The challenges arise from not only the extremely low content of GAs in complex plant matrices, but the poor detectability of GAs by mass spectrometry (MS) in negative ion mode. In an effort to solve these urgent difficulties, we present a spatial‐resolved analysis method to investigate the distribution of GAs in tiny plant tissues based on a simplified one‐pot sample preparation approach coupled with ultrahigh‐performance liquid chromatography‐tandem MS. By integrating extraction and derivatization into one step, target GAs were effectively extracted from plant materials and simultaneously reacted with N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide, the sample preparation time was largely shortened, the probability of sample loss was minimized and the detection sensitivity of MS was also greatly improved compared with underivatized GAs. Under optimal conditions, the method was validated from the quantification linearity, limits of detection and limits of quantification in the presence of plant matrices, recoveries, and precision. With the proposed method, 15 endogenous GAs were detected and, among these, 11 GAs could be quantified in 0.50 mg fresh weight (FW) wheat shoot samples, and five GAs were quantified in only 0.15 mg FW developing seed samples of Arabidopsis thaliana. The distribution patterns of GAs along both the non‐13‐hydroxylation pathway and the early 13‐hydroxylation pathway in a single shoot of germinating wheat, rice and maize seeds were finally profiled with a spatial resolution down to approximately 1 mm².     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

Bioimaging of multiple elements by high‐resolution LA‐ICP‐MS reveals altered distribution of mineral elements in the nodes of rice mutants

Inter‐vascular transfer in rice (Oryza sativa) nodes is required for delivering mineral elements to developing tissues, which is mediated by various transporters in the nodes. However, the effect of these transporters on distribution of mineral elements in the nodes at a cellular level is still unknown. Here, we established a protocol for bioimaging of multiple elements at a cellular level in rice node by laser ablation‐inductively coupled plasma‐mass spectrometry (LA‐ICP‐MS), and compared the mineral distribution profile between wild‐type (WT) rice and mutants. Both relative comparison of mineral distribution normalized by endogenous ¹³C and quantitative analysis using spiked standards combined with soft ablation gave valid results. Overall, macro‐nutrients such as K and Mg were accumulated more in the phloem region, while micro‐nutrients such as Fe and Zn were highly accumulated at the inter‐vascular tissues of the node. In mutants of nodal Zn transporter OsHMA2, Zn localization pattern in the node tissues did not differ from that of WT; however, Zn accumulation in the inter‐vascular tissues was lower in uppermost node I but higher in the third upper node III compared with the WT. In contrast, Si deposition in the mutants of three nodal Si transporters Lsi2, Lsi3 and Lsi6 showed different patterns, which are consistent with the localization of these transporters. This improved LA‐ICP‐MS analysis combined with functional characterization of transporters will provide further insight into mineral element distribution mechanisms in rice and other plant species.     

In situ marker‐based assessment of leaf trait evolutionary potential in a marginal European beech population

Evolutionary processes are expected to be crucial for the adaptation of natural populations to environmental changes. In particular, the capacity of rear edge populations to evolve in response to the species limiting conditions remains a major issue that requires to address their evolutionary potential. In situ quantitative genetic studies based on molecular markers offer the possibility to estimate evolutionary potentials manipulating neither the environment nor the individuals on which phenotypes are measured. The goal of this study was to estimate heritability and genetic correlations of a suite of leaf functional traits involved in climate adaptation for a natural population of the tree Fagus sylvatica, growing at the rear edge of the species range. Using two marker‐based quantitative genetics approaches, we obtained consistent and significant estimates of heritability for leaf phenological (phenology of leaf flush), morphological (mass, area, ratio mass/area) and physiological (δ¹³C, nitrogen content) traits. Moreover, we found only one significant positive genetic correlation between leaf area and leaf mass, which likely reflected mechanical constraints. We conclude first that the studied population has considerable genetic diversity for important ecophysiological traits regarding drought adaptation and, second, that genetic correlations are not likely to impose strong genetic constraints to future population evolution. Our results bring important insights into the question of the capacity of rear edge populations to evolve.     

Discovery of rice essential genes by characterizing a CRISPR‐edited mutation of closely related rice MAP kinase genes

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co‐expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA‐gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen‐activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR‐edited mutants. The true knock‐out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR‐induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45–86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene‐free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.     

Epitope detection chromatography: a method to dissect the structural heterogeneity and inter‐connections of plant cell‐wall matrix glycans

Plant cell walls are complex, multi‐macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell‐wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell‐wall matrix glycan sub‐populations and inter‐connections. The method combines chromatographic separations with use of glycan‐directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell‐wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub‐populations and their modulation in A. thaliana organs.