Nicotiana benthamiana XYLEM CYSTEINE PROTEASE genes facilitate tracheary element formation in interfamily grafting.

Grafting is a plant propagation technique widely used in agriculture. A recent discovery of the capability of interfamily grafting in Nicotiana has expanded the potential combinations of grafting. In this study, we showed that xylem connection is essential for the achievement of interfamily grafting and investigated the molecular basis of xylem formation at the graft junction. Transcriptome and gene network analyses revealed gene modules for tracheary element (TE) formation during grafting that include genes associated with xylem cell differentiation and immune response. The reliability of the drawn network was validated by examining the role of the Nicotiana benthamiana XYLEM CYSTEINE PROTEASE (NbXCP) genes in TE formation during interfamily grafting. Promoter activities of NbXCP1 and NbXCP2 genes were found in differentiating TE cells in the stem and callus tissues at the graft junction. Analysis of a Nbxcp1;Nbxcp2 loss-of-function mutant indicated that NbXCPs control the timing of de novo TE formation at the graft junction. Moreover, grafts of the NbXCP1 overexpressor increased the scion growth rate as well as the fruit size. Thus, we identified gene modules for TE formation at the graft boundary and demonstrated potential ways to enhance Nicotiana interfamily grafting.

Regulation of the iron-deficiency response by IMA/FEP peptide.

Iron (Fe) is an essential micronutrient for plant growth and development, participating in many significant biological processes including photosynthesis, respiration, and nitrogen fixation. Although abundant in the earth's crust, most Fe is oxidized and difficult for plants to absorb under aerobic and alkaline pH conditions. Plants, therefore, have evolved complex means to optimize their Fe-acquisition efficiency. In the past two decades, regulatory networks of transcription factors and ubiquitin ligases have proven to be essential for plant Fe uptake and translocation. Recent studies in Arabidopsis thaliana (Arabidopsis) suggest that in addition to the transcriptional network, IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) peptide interacts with a ubiquitin ligase, BRUTUS (BTS)/BTS-LIKE (BTSL). Under Fe-deficient conditions, IMA/FEP peptides compete with IVc subgroup bHLH transcription factors (TFs) to interact with BTS/BTSL. The resulting complex inhibits the degradation of these TFs by BTS/BTSL, which is important for maintaining the Fe-deficiency response in roots. Furthermore, IMA/FEP peptides control systemic Fe signaling. By organ-to-organ communication in Arabidopsis, Fe deficiency in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. IMA/FEP peptides regulate this compensatory response through Fe-deficiency-triggered organ-to-organ communication. This mini-review summarizes recent advances in understanding how IMA/FEP peptides function in the intracellular signaling of the Fe-deficiency response and systemic Fe signaling to regulate Fe acquisition.

Feelings toward medicine in remote areas among medical students aiming to become generalists : A comparison with other specialists.

Geographic maldistribution of physicians is a serious problem in Japan, resulting in a few physicians in remote areas. We distributed questionnaires to 396 medical students at Tokushima University from 2013 to 2016. We examined the intensity of medical students toward medicine in remote areas by using a visual analog scale and investigated what medical specialty they chose after two years of clinical training. The intensity of interest in medicine and the intensity of willingness to contribute to it in remote areas were statistically higher among medical students who intended to choose general medicine. After graduation, only five participants chose to specialize in general medicine. Of the 14 medical students who originally chose general medicine, six chose internal medicine, three chose general medicine, and the others chose surgery, neurosurgery, anesthesiology, orthopedics, or emergency medicine. This study demonstrated that medical students who intend to choose general medicine have a higher intensity of willingness to contribute to medicine in remote areas, indicating that the support program for these students to become general practitioners after graduation increases the number of physicians in remote areas. J. Med. Invest. 70 : 129-134, February, 2023.

PECT1, a rate-limiting enzyme in phosphatidylethanolamine biosynthesis, is involved in the regulation of stomatal movement in Arabidopsis.

An Arabidopsis mutant displaying impaired stomatal responses to CO2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H+ -ATPases in guard cells via phosphorylation. Arabidopsis H+ -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H+ -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H+ -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.].

Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips.

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Development of microfluidic chip for entrapping tobacco BY-2 cells.

The tobacco BY-2 cell line has been used widely as a model system in plant cell biology. BY-2 cells are nearly transparent, which facilitates cell imaging using fluorescent markers. As cultured cells are drifted in the medium, therefore, it was difficult to observe them for a long period. Hence, we developed a microfluidic device that traps BY-2 cells and fixes their positions to allow monitoring the physiological activity of cells. The device contains 112 trap zones, with parallel slots connected in series at three levels in the flow channel. BY-2 cells were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell filaments consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell filaments at 25 out of 112 trap zones (22.3%). The cell numbers increased through cell division from 1 to 4 days after trapping with a peak of mitotic index on day 2. Recovery experiments of fluorescent proteins after photobleaching confirmed cell survival and permeability of plasmodesmata. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe cell activity in real time under controllable conditions.

Cell-cell adhesion in plant grafting is facilitated by ß-1,4-glucanases.

Plant grafting is conducted for fruit and vegetable propagation, whereby a piece of living tissue is attached to another through cell-cell adhesion. However, graft compatibility limits combinations to closely related species, and the mechanism is poorly understood. We found that Nicotiana is capable of graft adhesion with a diverse range of angiosperms. Comparative transcriptomic analyses on graft combinations indicated that a subclade of ß-1,4-glucanases secreted into the extracellular region facilitates cell wall reconstruction near the graft interface. Grafting was promoted by overexpression of the ß-1,4-glucanase. Using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. These findings demonstrate that the process of cell-cell adhesion is a potential target to enhance plant grafting techniques.

Past infections are associated with low levels of anti-citrullinated protein autoantibodies in rheumatoid arthritis.

Background : Rheumatoid arthritis (RA), an autoimmune disease of unknown etiology, is believed to occur as the result of actions of genetic and environmental factors. In this study, we examined the relation of past histories about infectious diseases with the levels anti-citrullinated protein autoantibodies (ACPA) in RA. Methods : Results of a questionnaire about histories of infectious diseases were obtained from 85 patients with RA, and were analyzed. Results : Significantly lower level of ACPA was detected in patients with the history of tonsillitis, otitis media or urinary cystitis than in those without it. There was no difference in the level of ACPA in RA patients between with and without cold / influenza, rubella, chickenpox, herpes labialis or herpes zoster. When RA patients were divided into two groups, high-level and low-level ACPA, multiple logistic regression analysis revealed that the history of otitis media was a significantly independent factor for the low level of ACPA. There was no significant relation between the level of rheumatoid factor and histories of infectious diseases. Conclusion : This study clarified that the past history of otitis media is associated with the low level of ACPA in RA. J. Med. Invest. 67 : 182-188, February, 2020.

Shoot-to-root mobile polypeptides involved in systemic regulation of nitrogen acquisition.

Plants uptake nitrogen (N) from the soil mainly in the form of nitrate. However, nitrate is often distributed heterogeneously in natural soil. Plants, therefore, have a systemic long-distance signalling mechanism by which N starvation on one side of the root leads to a compensatory N uptake on the other N-rich side1,2. This systemic N acquisition response is triggered by a root-to-shoot mobile peptide hormone, C-TERMINALLY ENCODED PEPTIDE (CEP), originating from the N-starved roots3,4, but the molecular nature of the descending shoot-to-root signal remains elusive. Here, we show that phloem-specific polypeptides that are induced in leaves upon perception of root-derived CEP act as descending long-distance mobile signals translocated to each root. These shoot-derived polypeptides, which we named CEP DOWNSTREAM 1 (CEPD1) and CEPD2, upregulate the expression of the nitrate transporter gene NRT2.1 in roots specifically when nitrate is present in the rhizosphere. Arabidopsis plants deficient in this pathway show impaired systemic N acquisition response accompanied with N-deficiency symptoms. These fundamental mechanistic insights should provide a conceptual framework for understanding systemic nutrient acquisition responses in plants.

Long-distance peptide signaling essential for nutrient homeostasis in plants.

Organ-to-organ communication is indispensable for higher organisms to maintain homeostasis over their entire life. Recent findings have uncovered that plants, like animals, mediate organ-to-organ communication by long-distance signaling through the vascular system. In particular, xylem-mobile secreted peptides have attracted much attention as root-to-shoot long-distance signaling molecules in response to fluctuating environmental nutrient status. Several leguminous CLE peptides induced by rhizobial inoculation act as 'satiety' signals in long-distance negative feedback of nodule formation. By contrast, Arabidopsis CEP family peptides induced by local nitrogen (N)-starvation behave as systemic 'hunger' signals to promote compensatory N acquisition in other parts of the roots. Xylem sap peptidomics also implies the presence of still uncharacterized long-distance signaling peptides. This review highlights the current understanding of and new insights into the mechanisms and functions of root-to-shoot long-distance peptide signaling during environmental responses.

BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2 constitute a signaling pathway and modulate CLE peptide-triggered growth inhibition in Arabidopsis root.

Ligand receptor-based signaling is a means of cell-to-cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell-producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV3/EMBRYO SURROUNDING REGION (CLE) peptide shrinks the root meristem through the actions of CLAVATA2 (CLV2) and the RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) pathway in Arabidopsis thaliana. Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM1, which is a member of the leucine-rich repeat receptor-like kinase (LRR-RLK) family, is also involved in this pathway. BAM1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM1 functions together with RPK2. Using coimmunoprecipitation assay, we showed that BAM1 is capable of forming heteromeric complexes with RPK2. These findings suggest that the BAM1 and RPK2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems.

Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling.

Nitrogen (N) is a critical nutrient for plants but is often distributed unevenly in the soil. Plants therefore have evolved a systemic mechanism by which N starvation on one side of the root system leads to a compensatory and increased nitrate uptake on the other side. Here, we study the molecular systems that support perception of N and the long-distance signaling needed to alter root development. Rootlets starved of N secrete small peptides that are translocated to the shoot and received by two leucine-rich repeat receptor kinases (LRR-RKs). Arabidopsis plants deficient in this pathway show growth retardation accompanied with N-deficiency symptoms. Thus, signaling from the root to the shoot helps the plant adapt to fluctuations in local N availability.

Evolutionarily conserved CLE peptide signaling in plant development, symbiosis, and parasitism.

Small polypeptides are widely used as signaling molecules in cell-to-cell communication in animals and plants. The CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family is composed of numerous genes that contain conserved CLE domains in various plant species and plant-parasitic nematodes. Here, we review recent progress in our understanding of CLE signaling during stem cell maintenance in Arabidopsis and grasses. We also summarize the roles of CLE signaling in the legume-Rhizobium symbiosis and infection by plant-parasitic nematodes. CLE signaling is important for diverse aspects of cell-to-cell signaling and long-distance communication, which are critical for survival, and the basic components of the CLE signaling pathway are evolutionarily conserved in both plants and animals.

Treatment responses and their predictors in patients with rheumatoid arthritis treated with biological agents.

Biological agents represent an important advancement in for the treatment of rheumatoid arthritis (RA), but there is a subset of patients who do not improve despite therapy. This study aimed to determine the efficacy of biological agents for RA and to identify clinical factors that are associated with their response. We studied 98 patients with RA who started an initiating biological agent which was selected from infliximab, etanercept, adalimumab and tociliximab at 4 medical institutions. Etanercept was the most frequently used biological agent followed by infliximab although there was a difference in the selection of the biological agents among medical institutions. We found that etanercept achieved the highest treatment response, remission rate and drug survival rate. A high disease activity in the baseline disease activity score-c-reactive protein (CRP) was shown to be a negative predictor of the treatment response, and high patient global assessment was significantly less likely to achieve a good response. At week 4, decreases in 28 swollen joint counts and CRP were useful as predictors for sustaining the efficacy up to week 48. These data demonstrate that assessments of the disease activity at baseline and the early treatment response may be useful in predicting the efficacy and drug survival rate of biological agents.

Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension.

Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.