ARGONAUTE1-binding Tudor domain proteins function in small interfering RNA production for RNA-directed DNA methylation.

Transposable elements (TEs) contribute to plant evolution, development, and adaptation to environmental changes, but the regulatory mechanisms are largely unknown. RNA-directed DNA methylation (RdDM) is 1 TE regulatory mechanism in plants. Here, we identified that novel ARGONAUTE 1 (AGO1)-binding Tudor domain proteins Precocious dissociation of sisters C/E (PDS5C/E) are involved in 24-nt siRNA production to establish RdDM on TEs in Arabidopsis thaliana. PDS5 family proteins are subunits of the eukaryote-conserved cohesin complex. However, the double mutant lacking angiosperm-specific subfamily PDS5C and PDS5E (pds5c/e) exhibited different developmental phenotypes and transcriptome compared with those of the double mutant lacking eukaryote-conserved subfamily PDS5A and PDS5B (pds5a/b), suggesting that the angiosperm-specific PDS5C/E subfamily has a unique function in angiosperm plants. Proteome and imaging analyses revealed that PDS5C/E interact with AGO1. The pds5c/e double mutant had defects in 24-nt siRNA accumulation and CHH DNA methylation on TEs. In addition, some lncRNAs that accumulated in the pds5c/e mutant were targeted by AGO1-loading 21-nt miRNAs and 21-nt siRNAs. These results indicate that PDS5C/E and AGO1 participate in 24-nt siRNA production for RdDM in the cytoplasm. These findings indicate that angiosperm plants evolved a new regulator, the PDS5C/E subfamily, to control the increase in TEs during angiosperm evolution.

Lipid droplets in Arabidopsis thaliana leaves contain myosin-binding proteins and enzymes associated with furan-containing fatty acid biosynthesis.

Lipid droplets (LDs) are lipid storage organelles in plant leaves and seeds. Seed LD proteins are well known, and their functions in lipid metabolism have been characterized; however, many leaf LD proteins remain to be identified. We therefore isolated LDs from leaves of the leaf LD-overaccumulating mutant high sterol ester 1 (hise1) of Arabidopsis thaliana by centrifugation or co-immunoprecipitation. We then performed LD proteomics by mass spectrometry and identified 3,206 candidate leaf LD proteins. In this study, we selected 31 candidate proteins for transient expression assays using a construct encoding the candidate protein fused with green fluorescent protein (GFP). Fluorescence microscopy showed that MYOSIN BINDING PROTEIN14 (MYOB14) and two uncharacterized proteins localized to LDs labeled with the LD marker. Subcellular localization analysis of MYOB family members revealed that MYOB1, MYOB2, MYOB3, and MYOB5 localized to LDs. LDs moved along actin filaments together with the endoplasmic reticulum. Co-immunoprecipitation of myosin XIK with MYOB2-GFP or MYOB14-GFP suggested that LD-localized MYOBs are involved in association with the myosin XIK-LDs. The two uncharacterized proteins were highly similar to enzymes for furan fatty acid biosynthesis in the photosynthetic bacterium Cereibacter sphaeroides, suggesting a relationship between LDs and furan fatty acid biosynthesis. Our findings thus reveal potential molecular functions of LDs and provide a valuable resource for further studies of the leaf LD proteome.

Shoot gravitropism and organ straightening cooperate to arrive at a mechanically favorable shape in Arabidopsis.

Gravitropism is the plant organ bending in response to gravity, while a straightening mechanism prevents bending beyond the gravitropic set-point angle. The promotion and prevention of bending occur simultaneously around the inflorescence stem tip. How these two opposing forces work together and what part of the stem they affect are unknown. To understand the mechanical forces involved, we rotated wild type and organ-straightening-deficient mutant (myosin xif xik) Arabidopsis plants to a horizontal position to initiate bending. The mutant stems started to bend before the wild-type stems, which led us to hypothesize that the force preventing bending was weaker in mutant. We modeled the wild-type and mutant stems as elastic rods, and evaluated two parameters: an organ-angle-dependent gravitropic-responsive parameter (ß) and an organ-curvature-dependent proprioceptive-responsive parameter (γ). Our model showed that these two parameters were lower in mutant than in wild type, implying that, unexpectedly, both promotion and prevention of bending are weak in mutant. Subsequently, finite element method simulations revealed that the compressive stress in the middle of the stem was significantly lower in wild type than in mutant. The results of this study show that myosin-XIk-and-XIf-dependent organ straightening adjusts the stress distribution to achieve a mechanically favorable shape.

Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha.

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light.

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.

Plant ESCRT protein ALIX coordinates with retromer complex in regulating receptor-mediated sorting of soluble vacuolar proteins.

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

Aerial (+)-borneol modulates root morphology, auxin signalling and meristematic activity in Arabidopsis roots.

One of the characteristic aspects of odour sensing in humans is the activation of olfactory receptors in a slightly different manner in response to different enantiomers. Here, we focused on whether plants showed enantiomer-specific response similar to that in humans. We exposed Arabidopsis seedlings to methanol (control) and (+)- or (-)-borneol, and found that only (+)-borneol reduced the root length. Furthermore, the root-tip width was more increased upon (+)-borneol exposure than upon (-)-borneol exposure. In addition, root-hair formation was observed near the root tip in response to (+)-borneol. Auxin signalling was strongly reduced in the root tip following exposure to (+)-borneol, but was detected following exposure to (-)-borneol and methanol. Similarly, in the root tip, the activity of cyclin B1:1 was detected on exposure to (-)-borneol and methanol, but not on exposure to (+)-borneol, indicating that (+)-borneol inhibits the meristematic activity in the root. These results partially explain the (+)-borneol-specific reduction in the root length of Arabidopsis. Our results indicate the presence of a sensing system specific for (+)-borneol in Arabidopsis.

A rich and bountiful harvest: Key discoveries in plant cell biology.

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke's discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

Galactoglucomannan structure of Arabidopsis seed-coat mucilage in GDP-mannose synthesis impaired mutants.

Cell-wall polysaccharides are synthesized from nucleotide sugars by glycosyltransferases. However, in what way the level of nucleotide sugars affects the structure of the polysaccharides is not entirely clear. guanosine diphosphate (GDP)-mannose (GDP-Man) is one of the major nucleotide sugars in plants and serves as a substrate in the synthesis of mannan polysaccharides. GDP-Man is synthesized from mannose 1-phosphate and GTP by a GDP-Man pyrophosphorylase, VITAMIN C DEFECTIVE1 (VTC1), which is positively regulated by the interacting protein KONJAC1 (KJC1) in Arabidopsis. Since seed-coat mucilage can serve as a model of the plant cell wall, we examined the influence of vtc1 and kjc1 mutations on the synthesis of mucilage galactoglucomannan. Sugar composition analysis showed that mannose content in adherent mucilage of kjc1 and vtc1 mutants was only 42% and 11% of the wild-type, respectively, indicating a drastic decrease of galactoglucomannan. On the other hand, structural analysis based on specific oligosaccharides released by endo-ß-1,4-mannanase indicated that galactoglucomannan had a patterned glucomannan backbone consisting of alternating residues of glucose and mannose and the frequency of α-galactosyl branches was also similar to the wild type structure. These results suggest that the structure of mucilage galactoglucomannan is mainly determined by properties of glycosyltransferases rather than the availability of nucleotide sugars.

Regulation and Physiological Significance of the Nuclear Shape in Plants.

The shape of plant nuclei varies among different species, tissues, and cell types. In Arabidopsis thaliana seedlings, nuclei in meristems and guard cells are nearly spherical, whereas those of epidermal cells in differentiated tissues are elongated spindle-shaped. The vegetative nuclei in pollen grains are irregularly shaped in angiosperms. In the past few decades, it has been revealed that several nuclear envelope (NE) proteins play the main role in the regulation of the nuclear shape in plants. Some plant NE proteins that regulate nuclear shape are also involved in nuclear or cellular functions, such as nuclear migration, maintenance of chromatin structure, gene expression, calcium and reactive oxygen species signaling, plant growth, reproduction, and plant immunity. The shape of the nucleus has been assessed both by labeling internal components (for instance chromatin) and by labeling membranes, including the NE or endoplasmic reticulum in interphase cells and viral-infected cells of plants. Changes in NE are correlated with the formation of invaginations of the NE, collectively called the nucleoplasmic reticulum. In this review, what is known and what is unknown about nuclear shape determination are presented, and the physiological significance of the control of the nuclear shape in plants is discussed.

In vitro assembly of nuclear envelope in tobacco cultured cells.

The coordinated regulation of the nucelar envelope (NE) reassembly during cell division is an essential event. However, there is little information on the molecular components involved in NE assembly in plant cells. Here we developed an in vitro assay of NE assembly using tobacco BY-2 cultured cells. To start the NE assembly reaction, the demembranated nuclei and the S12 fraction (cytosol and microsomes) were mixed in the presence of GTP and ATP nucleotides. Time-course analysis indicated that tubule structures were extended from the microsomal vesicles that accumulated on the demembranated nuclei, and finally sealed the NE. Immunofluorescence confirmed that the assembled membrane contains a component of nuclear pore complex. The efficiency of the NE assembly is significantly inhibited by GTPγS that suppresses membrane fusion. This in-vitro assay system may elucidate the role of specific proteins and provide important insights into the molecular machinery of NE assembly in plant cells.

Excess sterol accumulation affects seed morphology and physiology in Arabidopsis thaliana.

Sterols are essential lipids for plant growth, and the sterol content is tightly regulated by a fail-safe system consisting of two processes: 1) suppression of excess sterol production by a negative regulator of sterol biosynthesis (HIGR STEROL ESTER 1, HISE1), and 2) conversion of excess sterols to sterol esters by PHOSPHOLIPID STEROL ACYLTRANSFERASE 1 (PSAT1) in Arabidopsis thaliana. The hise1-3 psat1-2 double mutant has a 1.5-fold higher sterol content in leaves than the wild type; this upregulates the expression of stress-responsive genes, leading to disruption of cellular activities in leaves. However, the effects of excess sterols on seeds are largely unknown. Here, we show that excess sterols cause multiple defects in seeds. The seeds of hise1-3 psat1-2 plants had a higher sterol content than wild-type seeds and showed a deeper color than wild-type seeds because of the accumulation of proanthocyanidin. The seed coat in the hise1-3 psat1-2 mutant was abnormally wrinkled. Seed coat formation is accompanied by cell death-mediated shrinkage of the inner integument. In the hise1-3 psat1-2 mutant, transmission electron microscopy showed that shrinkage of the integument was impaired, resulting in a thick seed coat and delayed seed germination. Moreover, psat1-2 and hise1-3 psat1-2 seeds displayed defective imbibition. Taken together, the results suggest that excess sterols impair proper seed coat formation, thereby inhibiting seed germination.

The Arabidopsis transcription factor NAI1 activates the NAI2 promoter by binding to the G-box motifs.

Brassicaceae plants, including Arabidopsis thaliana, develop endoplasmic reticulum (ER)-derived structures called ER bodies, which are involved in chemical defense against herbivores. NAI1 is a basic helix-loop-helix (bHLH) type transcription factor that regulates two downstream genes, NAI2 and BGLU23, that are responsible for the ER body formation and function. Here, we examined the transcription factor function of NAI1, and found that NAI1 binds to the promoter region of NAI2 and activates the NAI2 promoter. The recombinant NAI1 protein recognizes the canonical and non-canonical G-box motifs in the NAI2 promoter. Furthermore, we examined the DNA binding activity of NAI1 toward several E-box motifs in the NAI2 and BGLU23 promoters and found that NAI1 binds to a DNA fragment that includes an E-box motif from the BGLU23 promoter. Subcellular localization of NAI1 was evident in the nucleus, which is consistent with its transcription factor function. Transient expression experiments in Nicotiana benthamiana leaves showed that GFP-NAI1 protein activated the NAI2 promoter by binding to the two G-boxes of the promoter. Disruption of the G-boxes abolished the NAI1-dependent activation of the NAI2 promoter. These results indicate that NAI1 has a DNA binding activity in a motif-dependent manner and suggest that NAI1 regulates NAI2 and BGLU23 gene expressions through binding to these DNA motifs in their promoters.

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.

Subnuclear gene positioning through lamina association affects copper tolerance.

The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated (CA) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions.

Characterization of rhizome transcriptome and identification of a rhizomatous ER body in the clonal plant Cardamine leucantha.

The rhizome is a plant organ that develops from a shoot apical meristem but penetrates into belowground environments. To characterize the gene expression profile of rhizomes, we compared the rhizome transcriptome with those of the leaves, shoots and roots of a rhizomatous Brassicaceae plant, Cardamine leucantha. Overall, rhizome transcriptomes were characterized by the absence of genes that show rhizome-specific expression and expression profiles intermediate between those of shoots and roots. Our results suggest that both endogenous developmental factors and external environmental factors are important for controlling the rhizome transcriptome. Genes that showed relatively high expression in the rhizome compared to shoots and roots included those related to belowground defense, control of reactive oxygen species and cell elongation under dark conditions. A comparison of transcriptomes further allowed us to identify the presence of an ER body, a defense-related belowground organelle, in epidermal cells of the C. leucantha rhizome, which is the first report of ER bodies in rhizome tissue.

The nuclear envelope protein KAKU4 determines the migration order of the vegetative nucleus and sperm cells in pollen tubes.

A putative component protein of the nuclear lamina, KAKU4, modulates nuclear morphology in Arabidopsis thaliana seedlings, but its physiological significance is unknown. KAKU4 was highly expressed in mature pollen grains, each of which has a vegetative cell and two sperm cells. KAKU4 protein was highly abundant on the envelopes of vegetative nuclei and less abundant on the envelopes of sperm cell nuclei in pollen grains and elongating pollen tubes. Vegetative nuclei are irregularly shaped in wild-type pollen. However, KAKU4 deficiency caused them to become more spherical. After a pollen grain germinates, the vegetative nuclei and sperm cells enter and move along the pollen tube. In the wild type, the vegetative nucleus preceded the sperm cell nuclei in >90% of the pollen tubes, whereas, in kaku4 mutants, the vegetative nucleus preceded the sperm cell nuclei in only about half of the pollen tubes. kaku4 pollen was less competitive for fertilization than wild-type pollen after pollination. These results led us to hypothesize that the nuclear shape in vegetative cells of pollen grains affects the orderly migration of the vegetative nucleus and sperm cells in pollen tubes.

Generation of Arabidopsis lines with a red fluorescent marker for endoplasmic reticulum using a tail-anchored protein cytochrome b5 -B.

The endoplasmic reticulum (ER) is a multifunctional organelle that performs multiple cellular activities in eukaryotes. Visualizing ER using fluorescent proteins is a powerful method of analyzing its dynamics and to understand its functions. However, red fluorescent proteins with both an N-terminal signal peptide (SP) and a C-terminal ER retention tetrapeptide (HDEL) often cause mislocalization to vacuoles or extracellular spaces when they are constitutively expressed in Arabidopsis. To obtain a red fluorescent ER marker, we selected Arabidopsis cytochrome b5 -B (Cb5-B), a tail-anchored (TA) protein on the ER membrane. Its localization is determined by the transmembrane domain (TMD) and tail domain at the C-terminus. We fused the TMD and the tail domain of Cb5-B to the C-terminus of a red fluorescent protein, tdTomato (tdTomato-CTT). When tdTomato-CTT was constitutively expressed under the ubiquitin10 promoter in Arabidopsis, the fluorescent signal was exclusively detected at the ER by means of the reliable ER marker SP-GFP-HDEL. Therefore, tdTomato-CTT can accurately visualize the ER in stable Arabidopsis lines. Additionally, transient assays showed that tdTomato-CTT can also be used as an ER marker in onion, rice, and Nicotiana benthamiana. We believe that TA proteins could be used to generate various organellar membrane markers in plants.

Dynamic Capture and Release of Endoplasmic Reticulum Exit Sites by Golgi Stacks in Arabidopsis.

Protein transport from the endoplasmic reticulum (ER) to Golgi stacks is mediated by the coat protein complex COPII, which is assembled at an ER subdomain called ER exit site (ERES). However, the dynamic relationship between ERESs and Golgi stacks is unknown. Here, we propose a dynamic capture-and-release model of ERESs by Golgi stacks in Arabidopsis thaliana. Using variable-angle epifluorescence microscopy with high-temporal-resolution imaging, COPII-component-bound ERESs were detected as punctate structures with sizes of 300-500 nm. Some punctate ERESs are distributed on ER tubules and sheet rims, whereas others gather around a Golgi stack in an ER-network cavity to form a beaded-ring structure. Free ERESs that wander into an ER cavity are captured by a Golgi stack in a cytoskeleton-independent manner. Then, they are released by the Golgi stack for recycling. The dynamic ERES cycling might contribute to efficient transfer of de novo synthesized cargo proteins from the ER to Golgi stacks.