Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases.

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double-strand breaks, making it challenging to generate knock-in events. In this study, we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrated stable and heritable knock-ins in wheat in 1% of the primary transformants. Taken together, our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

Genome-wide analysis of plant miRNA action clarifies levels of regulatory dynamics across developmental contexts.

Development of complex organisms requires the delicate and dynamic spatiotemporal regulation of gene expression. Central to this are microRNAs (miRNAs). These mobile small RNAs offer specificity in conveying positional information and versatility in patterning the outcomes of gene expression. However, the parameters that shape miRNA output during development are still to be clarified. Here, we address this question on a genome-wide scale, using the maize shoot apex as a model. We show that patterns and levels of miRNA accumulation are largely determined at the transcriptional level, but are finessed post-transcriptionally in a tissue-dependent manner. The stem cell environments of the shoot apical meristem and vasculature appear particularly liable to this. Tissue-specific effects are also apparent at the level of target repression, with target cleavage products in the vasculature exceeding those of other tissues. Our results argue against a clearance mode of regulation purely at the level of transcript cleavage, leading us to propose that transcript cleavage provides a baseline level of target repression, onto which miRNA-driven translational repression can act to toggle the mode of target regulation between clearance and rheostat. Our data show how the inherent complexities of miRNA pathways allow the accumulation and activity of these small RNAs to be tailored in space and time to bring about the gene expression versatility needed during development.

Transcriptome profiling of laser-captured crown root primordia reveals new pathways activated during early stages of crown root formation in rice.

Crown roots constitute the main part of the rice root system. Several key genes involved in crown root initiation and development have been identified by functional genomics approaches. Nevertheless, these approaches are impaired by functional redundancy and mutant lethality. To overcome these limitations, organ targeted transcriptome analysis can help to identify genes involved in crown root formation and early development. In this study, we generated an atlas of genes expressed in developing crown root primordia in comparison with adjacent stem cortical tissue at three different developmental stages before emergence, using laser capture microdissection. We identified 3975 genes differentially expressed in crown root primordia. About 30% of them were expressed at the three developmental stages, whereas 10.5%, 19.5% and 12.8% were specifically expressed at the early, intermediate and late stages, respectively. Sorting them by functional ontology highlighted an active transcriptional switch during the process of crown root primordia formation. Cross-analysis with other rice root development-related datasets revealed genes encoding transcription factors, chromatin remodeling factors, peptide growth factors, and cell wall remodeling enzymes that are likely to play a key role during crown root primordia formation. This atlas constitutes an open primary data resource for further studies on the regulation of crown root initiation and development.

A high-resolution gene expression atlas links dedicated meristem genes to key architectural traits.

The shoot apical meristem (SAM) orchestrates the balance between stem cell proliferation and organ initiation essential for postembryonic shoot growth. Meristems show a striking diversity in shape and size. How this morphological diversity relates to variation in plant architecture and the molecular circuitries driving it are unclear. By generating a high-resolution gene expression atlas of the vegetative maize shoot apex, we show here that distinct sets of genes govern the regulation and identity of stem cells in maize versus Arabidopsis. Cell identities in the maize SAM reflect the combinatorial activity of transcription factors (TFs) that drive the preferential, differential expression of individual members within gene families functioning in a plethora of cellular processes. Subfunctionalization thus emerges as a fundamental feature underlying cell identity. Moreover, we show that adult plant characters are, to a significant degree, regulated by gene circuitries acting in the SAM, with natural variation modulating agronomically important architectural traits enriched specifically near dynamically expressed SAM genes and the TFs that regulate them. Besides unique mechanisms of maize stem cell regulation, our atlas thus identifies key new targets for crop improvement.

A protodermal miR394 signal defines a region of stem cell competence in the Arabidopsis shoot meristem.

A long-standing question in plants and animals is how spatial patterns are maintained within stem cell niches despite ongoing cell divisions. Here we address how, during shoot meristem formation in Arabidopsis thaliana, the three apical cell layers acquire stem cell identity. Using a sensitized mutant screen, we identified miR394 as a mobile signal produced by the surface cell layer (the protoderm) that confers stem cell competence to the distal meristem by repressing the F box protein LEAF CURLING RESPONSIVENESS. This repression is required to potentiate signaling from underneath the stem cells by the transcription factor WUSCHEL, maintaining stem cell pluripotency. The interaction of two opposing signaling centers provides a mechanistic framework of how stem cells are localized at the tip of the meristem. Although the constituent cells change, the surface layer provides a stable point of reference in the self-organizing meristem.

In situ localization of small RNAs in plants by using LNA probes.

Small RNAs have crucial roles in numerous aspects of plant biology. Despite our current understanding of their biogenesis and mechanisms of action, the biological function of small RNAs, particularly miRNAs, remains largely unknown. To decipher small RNA function, knowledge about their spatiotemporal patterns of expression is essential. Here we report an in situ hybridization method for the precise localization of small RNAs in plants by using locked nucleic acid (LNA) oligonucleotide probes. This method has been adapted from protocols used to detect messenger RNAs in formaldehyde-fixed and paraffin-embedded tissue sections, but it includes essential optimizations in key prehybridization, hybridization and posthybridization steps. Most importantly, optimization of probe concentration and hybridization temperature is required for each unique LNA probe. We present the detailed protocol starting from sectioned tissues, and we include troubleshooting tips and recommended controls. This method has been used successfully in several plant species and can be completed within 2-6 d.

In situ hybridization for the precise localization of transcripts in plants.

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks(1-3). While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting(4-7). However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs(8-14).

Genome-wide characterization of the HD-ZIP IV transcription factor family in maize: preferential expression in the epidermis.

Transcription factors of the plant-specific homeodomain leucine zipper IV (HD-ZIP IV) family have been found from moss to higher plants, and several family members have been associated with epidermis-related expression and/or function. In maize (Zea mays), four of the five characterized HD-ZIP IV family members are expressed specifically in the epidermis, one contributes to trichome development, and target genes of another one are involved in cuticle biosynthesis. Assessing the phylogeny, synteny, gene structure, expression, and regulation of the entire family in maize, 12 novel ZmHDZIV genes were identified in the recently sequenced maize genome. Among the 17 genes, eight form homeologous pairs duplicated after the split of maize and sorghum (Sorghum bicolor), whereas a fifth duplication is shared with sorghum. All 17 ZmHDZIV genes appear to be derived from a basic module containing seven introns in the coding region. With one possible exception, all 17 ZmHDZIV genes are expressed and show preferential expression in immature reproductive organs. Fourteen of 15 ZmHDZIV genes with detectable expression in laser-dissected tissues exhibit a moderate to very strong expression preference for the epidermis, suggesting that at least in maize, the majority of HD-ZIP IV family members may have epidermis-related functions. Thirteen ZmHDZIV genes carry conserved motifs of 19 and 21 nucleotides in their 3' untranslated region. The strong evolutionary conservation and the size of the conserved motifs in the 3' untranslated region suggest that the expression of HD-ZIP IV genes may be regulated by small RNAs.

Functional characterization of the HD-ZIP IV transcription factor OCL1 from maize.

OCL1 (OUTER CELL LAYER1) encodes a maize HD-ZIP class IV transcription factor (TF) characterized by the presence of a homeo DNA-binding domain (HD), a dimerization leucine zipper domain (ZIP), and a steroidogenic acute regulatory protein (StAR)-related lipid transfer domain (START) involved in lipid transport in animals but the function of which is still unknown in plants. By combining yeast and plant trans-activation assays, the transcriptional activation domain of OCL1 was localized to 85 amino acids in the N-terminal part of the START domain. Full-length OCL1 devoid of this activation domain is unable to trans-activate a reporter gene under the control of a minimal promoter fused to six repeats of the L1 box, a cis-element present in target genes of HD-ZIP IV TFs in Arabidopsis. In addition, ectopic expression of OCL1 leads to pleiotropic phenotypic aberrations in transgenic maize plants, the most conspicuous one being a strong delay in flowering time which is correlated with the misexpression of molecular markers for floral transition such as ZMM4 (Zea Mays MADS-box4) or DLF1 (DELAYED FLOWERING1). As suggested by the interaction in planta between OCL1 and SWI3C1, a bona fide subunit of the SWI/SNF complex, OCL1 may modulate transcriptional activity of its target genes by interaction with a chromatin remodelling complex.

Epidermis: the formation and functions of a fundamental plant tissue.

Epidermis differentiation and maintenance are essential for plant survival. Constant cross-talk between epidermal cells and their immediate environment is at the heart of epidermal cell fate, and regulates epidermis-specific transcription factors. These factors in turn direct epidermal differentiation involving a whole array of epidermis-specific pathways including specialized lipid metabolism necessary to build the protective cuticle layer. An intact epidermis is crucial for certain key processes in plant development, shoot growth and plant defence. Here, we discuss the control of epidermal cell fate and the function of the epidermal cell layer in the light of recent advances in the field.

Overexpression of the epidermis-specific homeodomain-leucine zipper IV transcription factor Outer Cell Layer1 in maize identifies target genes involved in lipid metabolism and cuticle biosynthesis.

Transcription factors of the homeodomain-leucine zipper IV (HD-ZIP IV) family play crucial roles in epidermis-related processes. To gain further insight into the molecular function of OUTER CELL LAYER1 (OCL1), 14 target genes up- or down-regulated in transgenic maize (Zea mays) plants overexpressing OCL1 were identified. The 14 genes all showed partial coexpression with OCL1 in maize organs, and several of them shared preferential expression in the epidermis with OCL1. They encoded proteins involved in lipid metabolism, defense, envelope-related functions, or cuticle biosynthesis and include ZmWBC11a (for white brown complex 11a), an ortholog of AtWBC11 involved in the transport of wax and cutin molecules. In support of the annotations, OCL1-overexpressing plants showed quantitative and qualitative changes of cuticular wax compounds in comparison with wild-type plants. An increase in C24 to C28 alcohols was correlated with the transcriptional up-regulation of ZmFAR1, coding for a fatty acyl-coenzyme A reductase. Transcriptional activation of ZmWBC11a by OCL1 was likely direct, since transactivation in transiently transformed maize kernels was abolished by a deletion of the activation domain in OCL1 or mutations in the L1 box, a cis-element bound by HD-ZIP IV transcription factors. Our data demonstrate that, in addition to AP2/EREBP and MYB-type transcription factors, members of the HD-ZIP IV family contribute to the transcriptional regulation of genes involved in cuticle biosynthesis.