Zinc deficiency-induced defensin-like proteins are involved in the inhibition of root growth in Arabidopsis.

The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions. DEFLs are small cysteine-rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn-deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202-DEFL208) were categorized into one subgroup. Among the seven DEFLs, the transcripts of five (not DEFL204 and DEFL205) were upregulated by Zn deficiency, consistent with the presence of cis-elements for basic-region leucine-zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP-tagged DEFL203 showed that DEFL203-sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn-deficient conditions through a reduction in root meristem length and cell number.

Single-Cell RNA Sequencing of Arabidopsis Leaf Tissues Identifies Multiple Specialized Cell Types: Idioblast Myrosin Cells and Potential Glucosinolate-Producing Cells.

The glucosinolate-myrosinase defense system (GMDS), characteristic of Brassicales, is involved in plant defense. Previous single-cell transcriptomic analyses have reported the expression profiles of multiple GMDS-related cell types (i.e. myrosinase-rich myrosin idioblasts and multiple types of potential glucosinolate synthetic cells as well as a candidate S-cell for glucosinolate accumulation). However, differences in plant stages and cell-type annotation methods have hindered comparisons among studies. Here, we used the single-cell transcriptome profiles of extended Arabidopsis leaves and verified the distribution of previously used markers to refine the expression profiles of GMDS-associated cell types. Moreover, we performed beta-glucuronidase promoter assays to confirm the histological expression patterns of newly obtained markers for GMDS-associated candidates. As a result, we found a set of new specific reporters for myrosin cells and potential glucosinolate-producing cells.

DNA-functionalized colloidal crystals for macromolecular encapsulation.

Novel DNA-based structures with the ability to encapsulate nanoscale molecules, such as proteins, can be applied to a wide range of areas, including reaction fields and micro/nano drug carriers. DNA-functionalized nanoparticle (DNA-NP) colloidal crystals have emerged as a new class of programmable DNA-based structures harboring metal nanoparticles with improved mechanical properties. The encapsulation of guest molecules into empty spaces in lattice structures is theoretically possible. However, due to the lack of a strategy for versatile encapsulation of guest molecules, the feasibility of nanoscale encapsulation by DNA-NP crystals is unclear. In this study, we developed DNA-functionalized gold nanoparticle (DNA-AuNP) crystals with tunable interparticle spacing for molecular encapsulation. We demonstrated that the modification of DNA-AuNP crystals with functional moieties, that is, biotin molecules, was effective in retaining molecules in the crystals. The crystallinities before and after encapsulation of the molecules were confirmed using small-angle X-ray scattering. We also succeeded in encapsulating CRISPR/Cas9 ribonucleoproteins into DNA-AuNP crystals by harnessing their affinity for target molecules. These findings demonstrated the potential use of metal-DNA hybrid crystals as carriers for direct protein delivery via biolistic bombardment. Thus, this study provides an attractive strategy for creating a new class of DNA-based structures for macromolecular encapsulation, and an alternative research direction toward colloidal crystal engineering using DNA.

Manganese Treatment Alleviates Zinc Deficiency Symptoms in Arabidopsis Seedlings.

Plant phenotypes caused by mineral deficiencies differ depending on growth conditions. We recently reported that the growth of Arabidopsis thaliana was severely inhibited on MGRL-based zinc (Zn)-deficient medium but not on Murashige-Skoog-based Zn-deficient medium. Here, we explored the underlying reason for the phenotypic differences in Arabidopsis grown on the different media. The root growth and chlorophyll contents reduced by Zn deficiency were rescued by the addition of extra manganese (Mn) during short-term growth (10 or 14 d). However, this treatment did not affect the growth recovery after long-term growth (38 d). To investigate the reason for plant recovery from Zn deficiency, we performed the RNA-seq analysis of the roots grown on the Zn-basal medium and the Zn-depleted medium with/without additional Mn. Principal component analysis of the RNA-seq data showed that the gene expression patterns of plants on the Zn-basal medium were similar to those on the Zn-depleted medium with Mn, whereas those on the Zn-depleted medium without Mn were different from the others. The expression of several transcription factors and reactive oxygen species (ROS)-related genes was upregulated in only plants on the Zn-depleted medium without Mn. Consistent with the gene expression data, ROS accumulation in the roots grown on this medium was higher than those grown in other conditions. These results suggest that plants accumulate ROS and reduce their biomass under undesirable growth conditions, such as Zn depletion. Taken together, this study shows that the addition of extra Mn to the Zn-depleted medium induces transcriptional changes in ROS-related genes, thereby alleviating short-term growth inhibition due to Zn deficiency.

The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.

Stem cell polarization is a crucial step in asymmetric cell division, which is a universal system for generating cellular diversity in multicellular organisms. Several conventional genetics studies have attempted to elucidate the mechanisms underlying cell polarization in plants, but it remains largely unknown. In plants, stomata, which are valves for gas exchange, are generated through several rounds of asymmetric divisions. In this study, we identified and characterized a chemical compound that affects stomatal stem cell polarity. High-throughput screening for bioactive molecules identified a pyridine-thiazole derivative, named bubblin, which induced stomatal clustering in Arabidopsis epidermis. Bubblin perturbed stomatal asymmetric division, resulting in the generation of two identical daughter cells. Both cells continued to express the stomatal fate determinant SPEECHLESS, and then differentiated into mispatterned stomata. Bubblin-treated cells had a defect in the polarized localization of BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which is required for asymmetric cell fate determination. Our results suggest that bubblin induces stomatal lineage cells to divide without BASL-dependent pre-mitotic establishment of polarity. Bubblin is a potentially valuable tool for investigating cell polarity establishment in stomatal asymmetric division.

A direction-selective local-thresholding method, DSLT, in combination with a dye-based method for automated three-dimensional segmentation of cells and airspaces in developing leaves.

Quantifying the anatomical data acquired from three-dimensional (3D) images has become increasingly important in recent years. Visualization and image segmentation are essential for acquiring accurate and detailed anatomical data from images; however, plant tissues such as leaves are difficult to image by confocal or multi-photon laser scanning microscopy because their airspaces generate optical aberrations. To overcome this problem, we established a staining method based on Nile Red in silicone-oil solution. Our staining method enables color differentiation between lipid bilayer membranes and airspaces, while minimizing any damage to leaf development. By repeated applications of our staining method we performed time-lapse imaging of a leaf over 5 days. To counteract the drastic decline in signal-to-noise ratio at greater tissue depths, we also developed a local thresholding method (direction-selective local thresholding, DSLT) and an automated iterative segmentation algorithm. The segmentation algorithm uses the DSLT to extract the anatomical structures. Using the proposed methods, we accurately segmented 3D images of intact leaves to single-cell resolution, and measured the airspace volumes in intact leaves.

Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana.

Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms.

Stomagen positively regulates stomatal density in Arabidopsis.

Stomata in the epidermal tissues of leaves are valves through which passes CO(2), and as such they influence the global carbon cycle. The two-dimensional pattern and density of stomata in the leaf epidermis are genetically and environmentally regulated to optimize gas exchange. Two putative intercellular signalling factors, EPF1 and EPF2, function as negative regulators of stomatal development in Arabidopsis, possibly by interacting with the receptor-like protein TMM. One or more positive intercellular signalling factors are assumed to be involved in stomatal development, but their identities are unknown. Here we show that a novel secretory peptide, which we designate as stomagen, is a positive intercellular signalling factor that is conserved among vascular plants. Stomagen is a 45-amino-rich peptide that is generated from a 102-amino-acid precursor protein designated as STOMAGEN. Both an in planta analysis and a semi-in-vitro analysis with recombinant and chemically synthesized stomagen peptides showed that stomagen has stomata-inducing activity in a dose-dependent manner. A genetic analysis showed that TMM is epistatic to STOMAGEN (At4g12970), suggesting that stomatal development is finely regulated by competitive binding of positive and negative regulators to the same receptor. Notably, STOMAGEN is expressed in inner tissues (the mesophyll) of immature leaves but not in the epidermal tissues where stomata develop. This study provides evidence of a mesophyll-derived positive regulator of stomatal density. Our findings provide a conceptual advancement in understanding stomatal development: inner photosynthetic tissues optimize their function by regulating stomatal density in the epidermis for efficient uptake of CO(2).

CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.

Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

Enhancement of leaf photosynthetic capacity through increased stomatal density in Arabidopsis.

Photosynthetic rate is determined by CO2 fixation and CO2 entry into the plant through pores in the leaf epidermis called stomata. However, the effect of increased stomatal density on photosynthetic rate remains unclear. This work investigated the effect of alteration of stomatal density on leaf photosynthetic capacity in Arabidopsis thaliana. Stomatal density was modulated by overexpressing or silencing STOMAGEN, a positive regulator of stomatal development. Leaf photosynthetic capacity and plant growth were examined in transgenic plants. Increased stomatal density in STOMAGEN-overexpressing plants enhanced the photosynthetic rate by 30% compared to wild-type plants. Transgenic plants showed increased stomatal conductance under ambient CO2 conditions and did not show alterations in the maximum rate of carboxylation, indicating that the enhancement of photosynthetic rate was caused by gas diffusion changes. A leaf photosynthesis-intercellular CO2 concentration response curve showed that photosynthetic rate was increased under high CO2 conditions in association with increased stomatal density. STOMAGEN overexpression did not alter whole plant biomass, whereas its silencing caused biomass reduction. Our results indicate that increased stomatal density enhanced leaf photosynthetic capacity by modulating gas diffusion. Stomatal density may be a target trait for plant engineering to improve photosynthetic capacity.

The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice.

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYß genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYß mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

LysM-mediated signaling in Marchantia polymorpha highlights the conservation of pattern-triggered immunity in land plants.

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in angiosperms. The model liverwort Marchantia polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha and the significance of PTI in bryophytes have not yet been described. We here show that M. polymorpha has four lysin motif (LysM)-domain-containing receptor homologs, two of which, LysM-receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, are responsible for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and highlights conserved elements and new aspects of pattern-triggered immunity in land plants.

Positive and negative peptide signals control stomatal density.

The stoma is a micro valve found on aerial plant organs that promotes gas exchange between the atmosphere and the plant body. Each stoma is formed by a strict cell lineage during the early stages of leaf development. Molecular genetics research using the model plant Arabidopsis has revealed the genes involved in stomatal differentiation. Cysteine-rich secretory peptides of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family play crucial roles as extracellular signaling factors. Stomatal development is orchestrated by the positive factor STOMAGEN/EPFL9 and the negative factors EPF1, EPF2, and CHALLAH/EPFL6 in combination with multiple receptors. EPF1 and EPF2 are produced in the stomatal lineage cells of the epidermis, whereas STOMAGEN and CHALLAH are derived from the inner tissues. These findings highlight the complex cell-to-cell and intertissue communications that regulate stomatal development. To optimize gas exchange, particularly the balance between the uptake of carbon dioxide (CO(2)) and loss of water, plants control stomatal activity in response to environmental conditions. The CO(2) level and light intensity influence stomatal density. Plants sense environmental cues in mature leaves and adjust the stomatal density of newly forming leaves, indicating the involvement of long-distance systemic signaling. This review summarizes recent research progress in the peptide signaling of stomatal development and discusses the evolutionary model of the signaling machinery.

Viscosity-aided electromechanical poration of cells for transfecting molecules.

Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. Here, we demonstrate electromechanical poration using a core-shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.

Comprehensive analysis of peptide-coding genes and initial characterization of an LRR-only microprotein in Marchantia polymorpha.

In the past two decades, many plant peptides have been found to play crucial roles in various biological events by mediating cell-to-cell communications. However, a large number of small open reading frames (sORFs) or short genes capable of encoding peptides remain uncharacterized. In this study, we examined several candidate genes for peptides conserved between two model plants: Arabidopsis thaliana and Marchantia polymorpha. We examined their expression pattern in M. polymorpha and subcellular localization using a transient assay with Nicotiana benthamiana. We found that one candidate, MpSGF10B, was expressed in meristems, gemma cups, and male reproductive organs called antheridiophores. MpSGF10B has an N-terminal signal peptide followed by two leucine-rich repeat (LRR) domains and was secreted to the extracellular region in N. benthamiana and M. polymorpha. Compared with the wild type, two independent Mpsgf10b mutants had a slightly increased number of antheridiophores. It was revealed in gene ontology enrichment analysis that MpSGF10B was significantly co-expressed with genes related to cell cycle and development. These results suggest that MpSGF10B may be involved in the reproductive development of M. polymorpha. Our research should shed light on the unknown role of LRR-only proteins in land plants.

Higher Stomatal Density Improves Photosynthetic Induction and Biomass Production in Arabidopsis Under Fluctuating Light.

Stomatal density (SD) is closely associated with photosynthetic and growth characteristics in plants. In the field, light intensity can fluctuate drastically within a day. The objective of the present study is to examine how higher SD affects stomatal conductance (g s ) and CO2 assimilation rate (A) dynamics, biomass production and water use under fluctuating light. Here, we compared the photosynthetic and growth characteristics under constant and fluctuating light among three lines of Arabidopsis thaliana (L.): the wild type (WT), STOMAGEN/EPFL9-overexpressing line (ST-OX), and EPIDERMAL PATTERNING FACTOR 1 knockout line (epf1). ST-OX and epf1 showed 268.1 and 46.5% higher SD than WT (p < 0.05). Guard cell length of ST-OX was 10.0% lower than that of WT (p < 0.01). There were no significant variations in gas exchange parameters at steady state between WT and ST-OX or epf1, although these parameters tended to be higher in ST-OX and epf1 than WT. On the other hand, ST-OX and epf1 showed faster A induction than WT after step increase in light owing to the higher g s under initial dark condition. In addition, ST-OX and epf1 showed initially faster g s induction and, at the later phase, slower g s induction. Cumulative CO2 assimilation in ST-OX and epf1 was 57.6 and 78.8% higher than WT attributable to faster A induction with reduction of water use efficiency (WUE). epf1 yielded 25.6% higher biomass than WT under fluctuating light (p < 0.01). In the present study, higher SD resulted in faster photosynthetic induction owing to the higher initial g s . epf1, with a moderate increase in SD, achieved greater biomass production than WT under fluctuating light. These results suggest that higher SD can be beneficial to improve biomass production in plants under fluctuating light conditions.

Author Correction: Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Two Y-chromosome-encoded genes determine sex in kiwifruit.

Dioecy, the presence of male and female individuals, has evolved independently in multiple flowering plant lineages1-3. Although theoretical models for the evolution of dioecy, such as the 'two-mutations' model, are well established4,5, little is known about the specific genes determining sex and their evolutionary history3. Kiwifruit, a major tree crop consumed worldwide, is a dioecious species. In kiwifruit we previously identified a Y-encoded sex-determinant candidate gene acting as the suppressor of feminization (SuF), named Shy Girl (SyGI)6. Here, we identify a second Y-encoded sex-determinant that we named Friendly Boy (FrBy), which exhibits strong expression in tapetal cells. Gene-editing and complementation analyses in Arabidopsis thaliana and Nicotiana tabacum indicated that FrBy acts for the maintenance of male (M) functions, independently of SyGI, and that these functions are conserved across angiosperm species. We further characterized the genomic architecture of the small (<1 megabase pairs (Mb)) male-specific region of the Y chromosome (MSY), which harbours only two genes expressed extensively in developing gynoecia and androecia, respectively: SyGI and FrBy. Re-sequencing of the genome of a natural hermaphrodite kiwifruit revealed that this individual is genetically male but carries deletion(s) of parts of the Y chromosome, including SyGI. Additionally, expression of FrBy in female kiwifruit resulted in hermaphrodite plants. These results clearly indicate that Y-encoded SyGI and FrBy act independently as the SuF and M factors in kiwifruit, respectively, and provide insight into not only the evolutionary path leading to a two-factor sex-determination system, but also a new breeding approach for dioecious species.

CRISPR/Cas9-Based Genome Editing of Transcription Factor Genes in Marchantia polymorpha.

Plant transcription factors (TFs) belong to a wide variety of gene families. The systematic and rapid establishment of knockout lines of TF genes is critical for functional genetics. Genome engineering techniques for dissecting out the molecular function of TFs have been dramatically improved by CRISPR/Cas9-based genome editing technology. In the CRISPR/Cas9 system, Cas9 functions as a Cas9-gRNA ribonucleoprotein complex and uses its DNA endonuclease activity to induce the cleavage of the genome, which is targeted by gRNA. Double-strand breaks sometimes induce insertions and deletions at the target site, leading to frameshift mutations of TF genes. In this chapter, we describe a detailed protocol for the targeted mutagenesis of TFs using the CRISPR/Cas9 system, specifically in the case of Marchantia polymorpha, an emerging model plant for functional genomics. The CRISPR/Cas9 system is highly versatile for targeting genomic sequences because only an alteration of the gRNA sequence is needed to change target sequences. The labor and cost required to establish genome-edited lines are low enough that multiple mutants of TF genes can be generated in one laboratory. The CRISPR/Cas9-based genome editing technique consists of four steps: (1) gRNA design; (2) vector construction; (3) transformation; and (4) isolation of genome-edited lines. This manuscript focuses mainly on the strategy of gRNA design, the workflow for off-target searches, and the selection and identification of genome-edited lines by genotyping. Although we describe a protocol for M. polymorpha, the basic strategy of generating genome-edited lines of TF genes should be applicable widely to other plants.