Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei.

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

SWEET13 transport of sucrose, but not gibberellin, restores male fertility in Arabidopsis sweet13;14.

SWEET sucrose transporters play important roles in the allocation of sucrose in plants. Some SWEETs were shown to also mediate transport of the plant growth regulator gibberellin (GA). The close physiological relationship between sucrose and GA raised the questions of whether there is a functional connection and whether one or both of the substrates are physiologically relevant. To dissect these two activities, molecular dynamics were used to map the binding sites of sucrose and GA in the pore of SWEET13 and predicted binding interactions that might be selective for sucrose or GA. Transport assays confirmed these predictions. In transport assays, the N76Q mutant had 7x higher relative GA3 activity, and the S142N mutant only transported sucrose. The impaired pollen viability and germination in sweet13;14 double mutants were complemented by the sucrose-selective SWEET13S142N, but not by the SWEET13N76Q mutant, indicating that sucrose is the physiologically relevant substrate and that GA transport capacity is dispensable in the context of male fertility. Therefore, GA supplementation to counter male sterility may act indirectly via stimulating sucrose supply in male sterile mutants. These findings are also relevant in the context of the role of SWEETs in pathogen susceptibility.

OsSWEET11b, a potential sixth leaf blight susceptibility gene involved in sugar transport-dependent male fertility.

SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator-Like effectors (TALe) of Xanthomonas ssp. is key for virulence in rice, cassava and cotton. We identified OsSWEET11b with roles in male fertility and potential bacterial blight (BB) susceptibility in rice. While single ossweet11a or 11b mutants were fertile, double mutants were sterile. As clade III SWEETs can transport gibberellin (GA), a key hormone for spikelet fertility, sterility and BB susceptibility might be explained by GA transport deficiencies. However, in contrast with the Arabidopsis homologues, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility therefore are likely to depend on sucrose transport activity. Ectopic induction of OsSWEET11b by designer TALe enabled TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b is relevant for fertility and for protecting rice against emerging Xoo strains that target OsSWEET11b.

Finding a right place to cut: How katanin is targeted to cellular severing sites.

Microtubule severing by katanin plays key roles in generating various array patterns of dynamic microtubules, while also responding to developmental and environmental stimuli. Quantitative imaging and molecular genetic analyses have uncovered that dysfunction of microtubule severing in plant cells leads to defects in anisotropic growth, division and other cell processes. Katanin is targeted to several subcellular severing sites. Intersections of two crossing cortical microtubules attract katanin, possibly by using local lattice deformation as a landmark. Cortical microtubule nucleation sites on preexisting microtubules are targeted for katanin-mediated severing. An evolutionary conserved microtubule anchoring complex not only stabilises the nucleated site, but also subsequently recruits katanin for timely release of a daughter microtubule. During cytokinesis, phragmoplast microtubules are severed at distal zones by katanin, which is tethered there by plant-specific microtubule-associated proteins. Recruitment and activation of katanin are essential for maintenance and reorganisation of plant microtubule arrays.

Consideration about the society after the COVID-19.

This paper reviews three viewpoints regarding the society after the COVID-19 infection on the concept of safety management. The first is the relationship between With COVID-19 and a zero risk. As a result of coexistence with COVID-19 for more than one year, the Japanese society thought that a zero risk is difficult to accomplish, and some risks will be accepted to maintain social activities. This leads a change in a way of thinking from zero risk to risk-based safety management. The second is the change in the way of working. As a result of having experienced remote work forcibly, it will become the hybrid model that incorporated remote work in a conventional method. Personnel evaluation changes from the seniority system to the job evaluation type, and each person's professional ability will be more focused on. The third is the review of the Japanese society system. In Japan, although the infection level was controlled to some extent by the groupism of the self-restraint of actions by mutual monitoring, there is a limit of managing based on groupism. Moreover, as seen in the delay of vaccine development and the medical care collapse, these problems should be improved by changing Japanese society system.

Advances in Synthetic Fluorescent Probe Labeling for Live-Cell Imaging in Plants.

Fluorescent probes are powerful tools for visualizing cellular and subcellular structures, their dynamics and cellular molecules in living cells and enable us to monitor cellular processes in a spatiotemporal manner within complex and crowded systems. In addition to popular fluorescent proteins, a wide variety of small-molecule dyes have been synthesized through close association with the interdisciplinary field of chemistry and biology, ranging from those suitable for labeling cellular compartments such as organelles to those for labeling intracellular biochemical and biophysical processes and signaling. In recent years, self-labeling technologies including the SNAP-tag system have allowed us to attach these dyes to cellular domains or specific proteins and are beginning to be employed in plant studies. In this mini review, we will discuss the current range of synthetic fluorescent probes that have been exploited for live-cell imaging and the recent advances in the application that enable genetical tagging of synthetic probes in plant research.

An anchoring complex recruits katanin for microtubule severing at the plant cortical nucleation sites.

Microtubules are severed by katanin at distinct cellular locations to facilitate reorientation or amplification of dynamic microtubule arrays, but katanin targeting mechanisms are poorly understood. Here we show that a centrosomal microtubule-anchoring complex is used to recruit katanin in acentrosomal plant cells. The conserved protein complex of Msd1 (also known as SSX2IP) and Wdr8 is localized at microtubule nucleation sites along the microtubule lattice in interphase Arabidopsis cells. Katanin is recruited to these sites for efficient release of newly formed daughter microtubules. Our cell biological and genetic studies demonstrate that Msd1-Wdr8 acts as a specific katanin recruitment factor to cortical nucleation sites (but not to microtubule crossover sites) and stabilizes the association of daughter microtubule minus ends to their nucleation sites until they become severed by katanin. Molecular coupling of sequential anchoring and severing events by the evolutionarily conserved complex renders microtubule release under tight control of katanin activity.

Designs, applications, and limitations of genetically encoded fluorescent sensors to explore plant biology.

The understanding of signaling and metabolic processes in multicellular organisms requires knowledge of the spatial dynamics of small molecules and the activities of enzymes, transporters, and other proteins in vivo, as well as biophysical parameters inside cells and across tissues. The cellular distribution of receptors, ligands, and activation state must be integrated with information about the cellular distribution of metabolites in relation to metabolic fluxes and signaling dynamics in order to achieve the promise of in vivo biochemistry. Genetically encoded sensors are engineered fluorescent proteins that have been developed for a wide range of small molecules, such as ions and metabolites, or to report biophysical processes, such as transmembrane voltage or tension. First steps have been taken to monitor the activity of transporters in vivo. Advancements in imaging technologies and specimen handling and stimulation have enabled researchers in plant sciences to implement sensor technologies in intact plants. Here, we provide a brief history of the development of genetically encoded sensors and an overview of the types of sensors available for quantifying and visualizing ion and metabolite distribution and dynamics. We further discuss the pros and cons of specific sensor designs, imaging systems, and sample manipulations, provide advice on the choice of technology, and give an outlook into future developments.

Sensors for the quantification, localization and analysis of the dynamics of plant hormones.

Plant hormones play important roles in plant growth and development and physiology, and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to monitor their distribution quantitatively, ideally in a non-invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available, including transcriptional reporters, degradation sensors, and luciferase and fluorescent sensors, and compare the tools and their suitability for different purposes.

Using Genetically Encoded Fluorescent Biosensors for Quantitative In Vivo Imaging.

Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)-based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca2+ in Arabidopsis thaliana using a ratiometric genetically encoded Ca2+ indicator, MatryoshCaMP6s.

Covalent Self-Labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings.

Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.

The sucrose transporter MdSUT4.1 participates in the regulation of fruit sugar accumulation in apple.

BACKGROUND: Sugar content is an important determinant of fruit sweetness, but details on the complex molecular mechanism underlying fruit sugar accumulation remain scarce. Here, we report the role of sucrose transporter (SUT) family in regulating fruit sugar accumulation in apple. RESULTS: Gene-tagged markers were developed to conduct candidate gene-based association study, and an SUT4 member MdSUT4.1 was found to be significantly associated with fruit sugar accumulation. MdSUT4.1 encodes a tonoplast localized protein and its expression level had a negative correlation with fruit sugar content. Overexpression of MdSUT4.1 in strawberry and apple callus had an overall negative impact on sugar accumulation, suggesting that it functions to remobilize sugar out of the vacuole. In addition, MdSUT4.1 is located on chromosomal region harboring a previously reported QTL for sugar content, suggesting that it is a candidate gene for fruit sugar accumulation in apple. CONCLUSIONS: MdSUT4.1 is involved in the regulation of fruit sugar accumulation in apple. This study is not only helpful for understanding the complex mechanism of fruit sugar accumulation, but it also provides molecular tools for genetic improvement of fruit quality in breeding programs of apple.

A Novel Katanin-Tethering Machinery Accelerates Cytokinesis.

Cytokinesis is fundamental for cell proliferation [1, 2]. In plants, a bipolar short-microtubule array forms the phragmoplast, which mediates vesicle transport to the midzone and guides the formation of cell walls that separate the mother cell into two daughter cells [2]. The phragmoplast centrifugally expands toward the cell cortex to guide cell-plate formation at the cortical division site [3, 4]. Several proteins in the phragmoplast midzone facilitate the anti-parallel bundling of microtubules and vesicle accumulation [5]. However, the mechanisms by which short microtubules are maintained during phragmoplast development, in particular, the behavior of microtubules at the distal zone of phragmoplasts, are poorly understood. Here, we show that a plant-specific protein, CORTICAL MICROTUBULE DISORDERING 4 (CORD4), tethers the conserved microtubule-severing protein katanin to facilitate formation of the short-microtubule array in phragmoplasts. CORD4 was specifically expressed during mitosis and localized to preprophase bands and phragmoplast microtubules. Custom-made two-photon spinning disk confocal microscopy revealed that CORD4 rapidly localized to microtubules in the distal phragmoplast zone during phragmoplast assembly at late anaphase and persisted throughout phragmoplast expansion. Loss of CORD4 caused abnormally long and oblique phragmoplast microtubules and slow expansion of phragmoplasts. The p60 katanin subunit, KTN1, localized to the distal phragmoplast zone in a CORD4-dependent manner. These results suggest that CORD4 tethers KTN1 at phragmoplasts to modulate microtubule length, thereby accelerating phragmoplast growth. This reveals the presence of a distinct machinery to accelerate cytokinesis by regulating the action of katanin.

CLASP stabilization of plus ends created by severing promotes microtubule creation and reorientation.

Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP's function in promoting microtubule amplification by severing and array reorientation.

SPR2 protects minus ends to promote severing and reorientation of plant cortical microtubule arrays.

The cortical microtubule arrays of higher plants are organized without centrosomes and feature treadmilling polymers that are dynamic at both ends. The control of polymer end stability is fundamental for the assembly and organization of cytoskeletal arrays, yet relatively little is understood about how microtubule minus ends are controlled in acentrosomal microtubule arrays, and no factors have been identified that act at the treadmilling minus ends in higher plants. Here, we identify Arabidopsis thaliana SPIRAL2 (SPR2) as a protein that tracks minus ends and protects them against subunit loss. SPR2 function is required to facilitate the rapid reorientation of plant cortical arrays as stimulated by light perception, a process that is driven by microtubule severing to create a new population of microtubules. Quantitative live-cell imaging and computer simulations reveal that minus protection by SPR2 acts by an unexpected mechanism to promote the lifetime of potential SPR2 severing sites, increasing the likelihood of severing and thus the rapid amplification of the new microtubule array.

Microtubule nucleating and severing enzymes for modifying microtubule array organization and cell morphogenesis in response to environmental cues.

In higher plants, reorientation of cortical microtubule arrays has been postulated to be of importance for modifying cell growth to adapt to environmental conditions. However, the process of microtubule reorientation is largely unknown. Recent genetic and live cell imaging studies of microtubule dynamics shed light on the regulatory mechanisms of microtubule molecular nucleation and severing apparatuses, which are required for array reorientation in response to blue light signaling. Branching nucleation from γ-tubulin complexes creates a small population of discordant microtubules that are acted on by KATANIN-mediated severing in two ways. KATANIN releases microtubules from nucleation sites and rapidly amplifies discordant microtubules by severing at microtubule crossovers. In this review, I focus on the molecular details of these two enzymes, which enable microtubule array transition.

GCP-WD mediates γ-TuRC recruitment and the geometry of microtubule nucleation in interphase arrays of Arabidopsis.

Many differentiated animal cells, and all higher plant cells, build interphase microtubule arrays of specific architectures without benefit of a central organizer, such as a centrosome, to control the location and geometry of microtubule nucleation. These acentrosomal arrays support essential cell functions such as morphogenesis, but the mechanisms by which the new microtubules are positioned and oriented are poorly understood. In higher plants, nucleation of microtubules arises from distributed γ-tubulin ring complexes (γ-TuRCs) at the cell cortex that are associated primarily with existing microtubules and from which new microtubules are nucleated in a geometrically bimodal fashion, either in parallel to the mother microtubule or as a branching event at a mean angle of approximately 40° to the mother microtubule. By imaging the dynamics of individual nucleation events in Arabidopsis, we found that a conserved peripheral protein of the γ-TuRC, GCP-WD/NEDD1, associated with motile γ-TuRCs and localized to nucleation events. Knockdown of this essential protein resulted in reduction of γ-TuRC recruitment to cortical microtubules and total nucleation frequency, showing that GCP-WD controls γ-TuRC positioning and function in these interphase arrays. Further, we discovered an unexpected role for GCP-WD in determining the geometry of microtubule-dependent microtubule nucleation, where it acts to increase the likelihood of branching over parallel nucleation. Cells with normally complex patterns of cortical array organization constructed simpler arrays with cell-wide ordering, suggesting that control of nucleation frequency, positioning, and geometry by GCP-WD allows plant cells to build alternative cortical array architectures.

A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing.

Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes.

RNA processing bodies, peroxisomes, Golgi bodies, mitochondria, and endoplasmic reticulum tubule junctions frequently pause at cortical microtubules.

Organelle motility, essential for cellular function, is driven by the cytoskeleton. In plants, actin filaments sustain the long-distance transport of many types of organelles, and microtubules typically fine-tune the motile behavior. In shoot epidermal cells of Arabidopsis thaliana seedlings, we show here that a type of RNA granule, the RNA processing body (P-body), is transported by actin filaments and pauses at cortical microtubules. Interestingly, removal of microtubules does not change the frequency of P-body pausing. Similarly, we show that Golgi bodies, peroxisomes, and mitochondria all pause at microtubules, and again the frequency of pauses is not appreciably changed after microtubules are depolymerized. To understand the basis for pausing, we examined the endoplasmic reticulum (ER), whose overall architecture depends on actin filaments. By the dual observation of ER and microtubules, we find that stable junctions of tubular ER occur mainly at microtubules. Removal of microtubules reduces the number of stable ER tubule junctions, but those remaining are maintained without microtubules. The results indicate that pausing on microtubules is a common attribute of motile organelles but that microtubules are not required for pausing. We suggest that pausing on microtubules facilitates interactions between the ER and otherwise translocating organelles in the cell cortex.