Role of Abscisic Acid, Reactive Oxygen Species, and Ca2+ Signaling in Hydrotropism-Drought Avoidance-Associated Response of Roots.

Plant roots exert hydrotropism in response to moisture gradients to avoid drought stress. The regulatory mechanism underlying hydrotropism involves novel regulators such as MIZ1 and GNOM/MIZ2 as well as abscisic acid (ABA), reactive oxygen species (ROS), and Ca2+ signaling. ABA, ROS, and Ca2+ signaling are also involved in plant responses to drought stress. Although the mechanism of moisture gradient perception remains largely unknown, the sensory apparatus has been reported to reside in the root elongation zone rather than in the root cap. In Arabidopsis roots, hydrotropism is mediated by the action of MIZ1 and ABA in the cortex of the elongation zone, the accumulation of ROS at the root curvature, and the variation in the cytosolic Ca2+ concentration in the entire root tip including the root cap and stele of the elongation zone. Moreover, root exposure to moisture gradients has been proposed to cause asymmetric ABA distribution or Ca2+ signaling, leading to the induction of the hydrotropic response. A comprehensive and detailed analysis of hydrotropism regulators and their signaling network in relation to the tissues required for their function is apparently crucial for understanding the mechanisms unique to root hydrotropism. Here, referring to studies on plant responses to drought stress, we summarize the recent findings relating to the role of ABA, ROS, and Ca2+ signaling in hydrotropism, discuss their functional sites and plausible networks, and raise some questions that need to be answered in future studies.

Proton Pump Inhibitors and Cyclin-Dependent Kinase 4/6 Inhibitors in Patients With Breast Cancer.

BACKGROUND: Proton pump inhibitors (PPIs) reduce the bioavailability of several anticancer drugs. The impact of PPIs co-administered with cyclin-dependent kinase 4 and 6 inhibitors is controversial. We aimed to clarify whether the concomitant use of PPIs impacts palbociclib and abemaciclib effectiveness in breast cancer treatment. PATIENTS AND METHODS: This multicenter, retrospective, observational study, conducted across 4 medical institutions in Japan, consecutively included patients with endocrine-resistant metastatic breast cancer, receiving palbociclib or abemaciclib between December 2017 and August 2022. Propensity score-matched analyses were performed. Treatment efficacy and safety with and without PPIs were compared. Progression-free survival and overall survival were estimated using the Kaplan-Meier method and compared using a log-rank test. A Cox proportional hazards model was used to estimate the hazard ratio. RESULTS: The study included 240 patients. After 1:1 matching, 112 patients were treated with and without PPIs. The median progression-free survival period was 1.2 years in the PPI group and 1.3 years in the non-PPI group (hazard ratio, 1.19; 95% CI, 0.70-2.02). The median overall survival period was 3.6 years in the PPI group, whereas it was not reached in the non-PPI group (hazard ratio, 1.23; 95% CI, 0.61-2.47). Consistent results were obtained for subgroups receiving palbociclib (n = 177) and abemaciclib (n = 63) without propensity score matching. Adverse event incidence and severity were similar in both groups. CONCLUSION: The effectiveness of cyclin-dependent kinase 4/6 inhibitors is unlikely to be affected by concomitant PPI use. Future prospective pharmacokinetic studies are warranted.

Therapeutic effect of aged garlic extract on gingivitis in dogs.

Periodontal disease is one of the most common dental health problems in dogs. Clinical studies in humans have shown that aged garlic extract (AGE), which contains stable and water-soluble sulfur-containing bioactive compounds, improves the symptoms of periodontal diseases. Our previous study demonstrated that oral administration of AGE in healthy Beagle dogs at 90 mg/kg/day for 12 weeks had no adverse effects such as hemolytic anemia, which is well known to occur as a result of ingestion of Allium species, including onions and garlic, in dogs. However, the therapeutic potential of AGE in canine periodontal disease remains unclear. Accordingly, we investigated the therapeutic effects of AGE in Beagle dogs with mild gingivitis. Feeding 18 mg/kg/day of AGE for 8 weeks resulted in the improvement of gingival index score, level of volatile sulfur compounds in exhaled air, and enzyme activity of periodontal pathogens without any adverse effects on clinical signs and hematological and serum biochemical parameters. Moreover, AGE increased the concentration of salivary cathelicidin, an antimicrobial peptide that contributes to the oral innate immune response. These results suggest that AGE could be a potential therapeutic agent for canine gingivitis.

Improvement in persistent idiopathic facial pain with comorbid ADHD using the combination of a dopamine system stabilizer and psychostimulant: A case report.

Key Clinical Message: Persistent idiopathic facial pain (PIFP) and attention-deficit/hyperactivity disorder (ADHD) may coexist and can be improved with ADHD medications. Thus, clinicians should screen for ADHD by a multidisciplinary approach when treating PIFP and differentiate between other odontogenic disorders. Abstract: We report a case of a woman with persistent idiopathic facial pain (PIFP) and attention-deficit/hyperactivity disorder (ADHD) that markedly improved with the administration of a combination of aripiprazole (APZ) and methylphenidate (MP) treatment. Screening for ADHD and administration of APZ and/or MP may be considered in treating PIFP.

Diagnosis and treatment of intractable idiopathic orofacial pain with attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) has been reported to be associated with primary chronic pain syndromes, such as fibromyalgia, migraine, and chronic low back pain. Although idiopathic orofacial pain (IOP) is classified as burning mouth syndrome or persistent idiopathic facial or dentoalveolar pain and as a primary chronic pain, the association between IOP and ADHD has not been investigated. This retrospective cohort study investigated the severity of ADHD symptoms measured using the ADHD scale and the effects of treatment using ADHD drugs and the dopamine system stabilizer aripiprazole. The participants were 25 consecutive patients with refractory IOP referred to a psychiatrist and diagnosed with coexisting ADHD according to the Diagnostic and Statistical Manual of Mental Disorders-5. The ADHD scale scores were higher in patients with intractable IOP than those in the general population. Pharmacotherapy used in this study led to clinically significant improvements in pain, anxiety/depression, and pain catastrophizing. Intractable IOP and ADHD were shown to be associated. In the future, screening and pharmacotherapy for ADHD should be considered in the treatment of intractable IOP.

Periodontal ligaments enhance neurite outgrowth in trigeminal ganglion neurons through Wnt5a production induced by mechanical stimulation.

The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

Novel chemical stimulation for geothermal reservoirs by chelating agent driven selective mineral dissolution in fractured rocks.

Improving geothermal systems through hydraulic stimulation to create highly permeable fractured rocks can induce seismicity. Therefore, the technique must be applied at a moderate intensity; this has led to concerns of insufficient permeability enhancement. Adding chemical stimulation can mitigate these issues, but traditional methods using strong mineral acids have challenges in terms of achieving mineral dissolution over long distances and highly variable fluid chemistry. Here, we demonstrate a novel chemical stimulation method for improving the permeability of rock fractures using a chelating agent that substantially enhances the dissolution rate of specific minerals to create voids that are sustained under crustal stress without the challenges associated with the traditional methods. Applying this agent to fractured granite samples under confining stress at 200 °C in conjunction with 20 wt% aqueous solutions of sodium salts of environmentally friendly chelating agents (N-(2-hydroxyethyl)ethylenediamine-N, N', N'-triacetic acid and N, N-bis(carboxymethyl)-L-glutamic acid) at pH 4 was assessed. A significant permeability enhancement of up to approximately sixfold was observed within 2 h, primarily due to the formation of voids based on the selective dissolution of biotite. These results demonstrate a new approach for chemical stimulation.

Capsaicin and Proton Differently Modulate Activation Kinetics of Mouse Transient Receptor Potential Vanilloid-1 Channel Induced by Depolarization.

The transient receptor potential vanilloid type 1 (TRPV1) channel is a non-selective cation channel expressed with transient receptor potential ankyrin type 1 (TRPA1) in small and medial size neurons of the dorsal root ganglions and trigeminal ganglions. TRPV1 is activated by capsaicin, thermal stimuli higher than 43°C, mechanical stress, and protons (H+). Although the TRPV1 channel does not have positively charged residues at regular intervals on its transmembrane segments, alterations in membrane potential also affect the state of TRPV1 channel. In the presence of capsaicin, voltage-dependent probability of opening of the TRPV1 channel and its kinetics have been examined, but the characteristics in the low pH remain unclear. To understand the voltage-dependency of the TRPV1 channel activation, we recorded capsaicin- and proton-induced mouse TRPV1 channel currents in a heterologous expression system. Outward current evoked by depolarizing square pulses in the presence of capsaicin or protons was fitted to a two-exponential function with a time-independent component. The voltage-dependent changes in amplitude of the three components displayed shallow curves and the changes in their ratio to the total current display similar tendencies in the presence of capsaicin and under the low pH. However, the fast and slow time constants in the presence of capsaicin were respectively 5- and 8-fold lower than those obtained under low pH conditions. These results suggest that the TRPV1 channel slowly drives the feed-forward cycle of pain sensation, and capsaicin and protons differently modulate the voltage-dependent TRPV1 channel gating.

Mode-selective inhibitory effects of eugenol on the mouse TRPV1 channel.

The transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal receptor in sensory nerves and involved in pain sensation. TRPV1 has at least three distinct activation modes that are selectively induced by different stimuli capsaicin, noxious heat, and protons. Although many mode-selective TRPV1 antagonists have been developed for their anticipated analgesic effects, there have been few successful reports because of adverse effects due to burn injuries and hyperthermia. Eugenol is a vanilloid that has been used as an analgesic in the dental treatment, and its TRPV1 activation ability has been reported. However, our knowledge about the underlying mechanisms of the antagonistic effects of eugenol on TRPV1 activation induced by three different modes is limited. Here, we show that eugenol dose-dependently inhibited the capsaicin-activated inward currents of mouse TRPV1 expressed in human embryonic kidney 293 (HEK293) cells. Under low pH conditions, low concentrations of eugenol only enhanced the proton-induced TRPV1 currents, whereas high eugenol concentrations initially potentiated but then immediately abrogated TRPV1 currents. Finally, eugenol had no modulatory effects on heat-activated TRPV1 in electrophysiological and Fura-2-based Ca2+ imaging experiments. Our results demonstrate that eugenol is a mode-selective antagonist of TRPV1 and can be evaluated as a lead compound of analgesics targeting TRPV1 without serious side effects.

Blood-brain barrier permeability analysis of plant ceramides.

Ceramides, a type of sphingolipid, are cell membrane components and lipid mediators that modulate a variety of cell functions. In plants, ceramides are mostly present in a glucosylated glucosylceramide (GlcCer) form. We previously showed that oral administration of konjac-derived GlcCer to a mouse model of Alzheimer's disease reduced brain amyloid-ß and amyloid plaques. Dietary plant GlcCer compounds are absorbed as ceramides, but it is unclear whether they can cross the blood-brain barrier (BBB). Herein, we evaluated the BBB permeability of synthetic plant-type ceramides (4, 8-sphingadienine, d18:2) using mouse and BBB cell culture models, and found that they could permeate the BBB both in vivo and in vitro. In addition, administrated ceramides were partially metabolized to other sphingolipid species, namely sphingomyelin (SM) and GlcCer, while crossing the BBB. Thus, plant ceramides can cross the BBB, suggesting that ceramides and their metabolites might affect brain functions.

Phylogenetic diversity in fim and mfa gene clusters between Porphyromonas gingivalis and Porphyromonas gulae, as a potential cause of host specificity.

BACKGROUND: Periodontopathic bacteria Porphyromonas gingivalis in humans and Porphyromonas gulae in animals are phylogenetically close and commonly have FimA and Mfa1 fimbriae. However, little is known about how fimA and mfa1 are phylogenetically different between P. gingivalis and P. gulae. Here, we examined phylogenetic diversity in their fim and mfa gene clusters. METHODS: Twenty P. gulae strains were isolated from the periodontal pocket of 20 dogs. For their genomic information, along with 64 P. gingivalis and 11 P. gulae genomes, phylogenetic relationship between the genotypes of fimA and mfa1 was examined. Variability of amino acid sequences was examined in the three-dimensional structure of FimA. The distance between strains was calculated for fim and mfa genes. RESULTS: Some fimA genotypes in P. gulae were close to particular types in P. gingivalis. Two types of mfa1 were classified as 70-kDa and 53-kDa protein-coding mfa1. The variable amino acid positions were primarily at the outer part of FimA. The genes encoding the structural proteins and the main component were similarly distant from the reference strain in P. gingivalis, but not in P. gulae. CONCLUSIONS: The differences in the gene clusters between P. gingivalis and P. gulae may result in their host specificity.

Two cases of bacterial meningitis due to meropenem-resistant Streptococcus pneumoniae: A threat of serotype 35B, ST 558 lineage.

Although the pneumococcal conjugate vaccine (PCV) has decreased the incidence of invasive pneumococcal disease (IPD) in children, cases of IPD caused by non-PCV serotypes have been increasing. Here, we report two cases of bacterial meningitis caused by meropenem-resistant Streptococcus pneumoniae; in both the cases, 13-valent PCV (PCV13) had been administered. The isolated S. pneumoniae strains were non-PCV13 serotype 35B and resistant to penicillin G, cefotaxime, and meropenem. In addition, multilocus sequence typing (MLST) revealed the sequence type (ST) to be 558. In case 1, a 6-month-old girl recovered without sequelae after antibiotic therapy comprising cefotaxime and vancomycin, whereas in case 2, a 9-month-old boy was treated with an empirical treatment comprising ceftriaxone and vancomycin administration. However, maintaining the blood concentration of vancomycin within the effective range was difficult, due to which the antibiotics were changed to panipenem/betamipron. During the treatment, he presented with seizures, which were effectively controlled with antiepileptic drugs. The rate of incidence of penicillin-susceptible IPD has been substantially increasing after the introduction of PCV. However, an upsurge in IPD cases due to multidrug-resistant (MDR) serotype 35B has been reported in countries where PCV13 was introduced before introducing in Japan. Moreover, an increase in the proportion of MDR serotype 35B and decrease in the susceptibility to broad-spectrum antimicrobials, including meropenem, have been reported. Hence, the number of meningitis cases caused by MDR serotype 35B/ST558 may increase in the future.

Reimplantation of a Pacemaker into a Subpectoral Pocket Via the Lateral Approach in Collaboration with Plastic Surgeons.

An 86-year-old woman had a pacemaker implanted into a subfascial pocket. After four months, the generator became exposed, and the pacemaker was removed. She exhibited a lack of prepectoral tissue. We therefore performed reimplantation in collaboration with plastic surgeons. We placed the leads via the extrathoracic subclavian venous approach, and plastic surgeons created a subpectoral pocket from the low lateral side of the pectoralis major muscle. General cardiologists rarely create subpectoral pockets and they are unable to implant leadless pacemakers at their hospital due to lack of sufficient skill. Our case showed that creating a subpectoral pocket in collaboration with plastic surgeons is quick and safe.

Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-ß pathologies in a mouse model of Alzheimer's disease.

The accumulation of amyloid-ß protein (Aß) in brain is linked to the early pathogenesis of Alzheimer's disease (AD). We previously reported that neuron-derived exosomes promote Aß clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aß levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aß in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aß accumulation by promoting EVs-dependent Aß clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology.

Association between the rs7583431 single nucleotide polymorphism close to the activating transcription factor 2 gene and the analgesic effect of fentanyl in the cold pain test.

BACKGROUND: Activating transcription factor 2 (ATF2) is a member of the leucine zipper family of DNA binding proteins and is widely distributed in tissues. Several recent studies have demonstrated that this protein is involved in mechanisms that are related to pain and inflammation. However, unclear is whether polymorphisms of the ATF2 gene, which encodes the human ATF2 protein, influence pain or analgesic sensitivity. This study examined associations between the analgesic effect of fentanyl in the cold pressor-induced pain test and polymorphisms in the ATF2 gene in 355 Japanese subjects. RESULTS: In this study, 33 single nucleotide polymorphisms (SNPs) were selected, and a total of 2 linkage disequilibrium blocks with 6 Tag SNPs (rs1153702, rs7583431, rs2302663, rs3845744, rs268214, and rs1982235) were observed in the region within and around the ATF2 gene. We further analyzed associations between these Tag SNPs and clinical data. Even after multiple testing with Bonferroni adjustments, an increase in the analgesic effect of fentanyl in the cold pressor-induced pain test was significantly associated with a greater number of the A allele of the rs7583431 SNP (linear regression, P = .001). CONCLUSIONS: The present findings may contribute to adequate pain relief in individual patients. Although more research on the genetic factors that influence opioid sensitivity is needed, analgesic requirements may be predicted by analyzing ATF2SNPs, together with other polymorphisms of genes that are reportedly associated with opioid sensitivity, such as CREB1, OPRM1, and GIRK2.

Association between rs2275913 single-nucleotide polymorphism of the interleukin-17A gene and perioperative analgesic use in cosmetic orthognathic surgery.

AIM: Interleukin-17A (IL-17A) plays an essential role in tissue inflammation by inducing proinflammatory cytokine and chemokine production and is related to innate immune reactions. IL-17A also contributes to neuroinflammation, neuropathic pain, and mechanical hypersensitivity after peripheral nerve injury in rodents. To clarify the contribution of IL-17A to pain-related phenotypes in humans, we investigated the association between pain-related phenotypes and the rs2275913 single-nucleotide polymorphism (SNP) of the IL-17A gene, which has been reported to be associated with rheumatoid arthritis, ulcerative colitis, and some cancers. METHODS: The present study used a correlational design to examine the impact of the rs2275913 SNP on postoperative pain-related phenotypes in a group of patients who underwent cosmetic orthognathic surgery. RESULTS: Carriers of the AA genotype had higher opioid requirements during and after surgery than carriers of the AG and GG genotypes (P = .009). Linear regression analysis indicated that opioid requirements linearly increased as the copy number of the A allele of the SNP increased (P = .008). CONCLUSIONS: Opioid requirements during and after surgery are enhanced in carriers of the AA genotype of the rs2275913 SNP of the IL-17A gene, possibly through an enhancement of IL-17A function that induces inflammation that is related to the inflammatory pain stimulus.

Genome-wide association study identifies polymorphisms associated with the analgesic effect of fentanyl in the preoperative cold pressor-induced pain test.

Opioid analgesics are widely used for the treatment of moderate to severe pain. The analgesic effects of opioids are well known to vary among individuals. The present study focused on the genetic factors that are associated with interindividual differences in pain and opioid sensitivity. We conducted a multistage genome-wide association study in subjects who were scheduled to undergo mandibular sagittal split ramus osteotomy and were not medicated until they received fentanyl for the induction of anesthesia. We preoperatively conducted the cold pressor-induced pain test before and after fentanyl administration. The rs13093031 and rs12633508 single-nucleotide polymorphisms (SNPs) near the LOC728432 gene region and rs6961071 SNP in the tcag7.1213 gene region were significantly associated with the analgesic effect of fentanyl, based on differences in pain perception latency before and after fentanyl administration. The associations of these three SNPs that were identified in our exploratory study have not been previously reported. The two polymorphic loci (rs13093031 and rs12633508) were shown to be in strong linkage disequilibrium. Subjects with the G/G genotype of the rs13093031 and rs6961071 SNPs presented lower fentanyl-induced analgesia. Our findings provide a basis for investigating genetics-based analgesic sensitivity and personalized pain control.

Stable Accumulation of Photosystem II Requires ONE-HELIX PROTEIN1 (OHP1) of the Light Harvesting-Like Family.

The cellular functions of two Arabidopsis (Arabidopsis thaliana) one-helix proteins, OHP1 and OHP2 (also named LIGHT-HARVESTING-LIKE2 [LIL2] and LIL6, respectively, because they have sequence similarity to light-harvesting chlorophyll a/b-binding proteins), remain unclear. Tagged null mutants of OHP1 and OHP2 (ohp1 and ohp2) showed stunted growth with pale-green leaves on agar plates, and these mutants were unable to grow on soil. Leaf chlorophyll fluorescence and the composition of thylakoid membrane proteins revealed that ohp1 deletion substantially affected photosystem II (PSII) core protein function and led to reduced levels of photosystem I core proteins; however, it did not affect LHC accumulation. Transgenic ohp1 plants rescued with OHP1-HA or OHP1-Myc proteins developed a normal phenotype. Using these tagged OHP1 proteins in transgenic plants, we localized OHP1 to thylakoid membranes, where it formed protein complexes with both OHP2 and High Chlorophyll Fluorescence244 (HCF244). We also found PSII core proteins D1/D2, HCF136, and HCF173 and a few other plant-specific proteins associated with the OHP1/OHP2-HCF244 complex, suggesting that these complexes are early intermediates in PSII assembly. OHP1 interacted directly with HCF244 in the complex. Therefore, OHP1 and HCF244 play important roles in the stable accumulation of PSII.

PCoM-DB Update: A Protein Co-Migration Database for Photosynthetic Organisms.

The identification of protein complexes is important for the understanding of protein structure and function and the regulation of cellular processes. We used blue-native PAGE and tandem mass spectrometry to identify protein complexes systematically, and built a web database, the protein co-migration database (PCoM-DB, http://pcomdb.lowtem.hokudai.ac.jp/proteins/top), to provide prediction tools for protein complexes. PCoM-DB provides migration profiles for any given protein of interest, and allows users to compare them with migration profiles of other proteins, showing the oligomeric states of proteins and thus identifying potential interaction partners. The initial version of PCoM-DB (launched in January 2013) included protein complex data for Synechocystis whole cells and Arabidopsis thaliana thylakoid membranes. Here we report PCoM-DB version 2.0, which includes new data sets and analytical tools. Additional data are included from whole cells of the pelagic marine picocyanobacterium Prochlorococcus marinus, the thermophilic cyanobacterium Thermosynechococcus elongatus, the unicellular green alga Chlamydomonas reinhardtii and the bryophyte Physcomitrella patens. The Arabidopsis protein data now include data for intact mitochondria, intact chloroplasts, chloroplast stroma and chloroplast envelopes. The new tools comprise a multiple-protein search form and a heat map viewer for protein migration profiles. Users can compare migration profiles of a protein of interest among different organelles or compare migration profiles among different proteins within the same sample. For Arabidopsis proteins, users can compare migration profiles of a protein of interest with putative homologous proteins from non-Arabidopsis organisms. The updated PCoM-DB will help researchers find novel protein complexes and estimate their evolutionary changes in the green lineage.

Allocation of Heme Is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells.

Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.