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In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains. We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains. Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transportrelated genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots. OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.

The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca 2+ -imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism. Products of the mevalonate pathway support plant development. Here the authors show that the extracellular ATP receptor P2K1 phosphorylates mevalonate kinase and this affects the mevalonate pathway.

The plant-specific WUSCHEL-related homeobox (WOX) nuclear proteins have important roles in the transcriptional regulation of many developmental processes. Among the rice (Oryza sativa) WOX proteins, a loss of OsWOX3A function in narrow leaf2 (nal2) nal3 double mutants (termed nal2/3) causes pleiotropic effects, such as narrow and curly leaves, opened spikelets, narrow grains, more tillers, and fewer lateral roots, but almost normal plant height. To examine OsWOX3A function in more detail, transgenic rice overexpressing OsWOX3A (OsWOX3A-OX) were generated; unexpectedly, all of them consistently exhibited severe dwarfism with very short and wide leaves, a phenotype that resembles that of gibberellic acid (GA)-deficient or GA-insensitive mutants. Exogenous GA₃ treatment fully rescued the developmental defects of OsWOX3A-OX plants, suggesting that constitutive overexpression of OsWOX3A downregulates GA biosynthesis. Quantitative analysis of GA intermediates revealed significantly reduced levels of GA20 and bioactive GA₁ in OsWOX3A-OX, possibly due to downregulation of the expression of KAO, which encodes ent-kaurenoic acid oxidase, a GA biosynthetic enzyme. Yeast one-hybrid and electrophoretic mobility shift assays revealed that OsWOX3A directly interacts with the KAO promoter. OsWOX3A expression is drastically and temporarily upregulated by GA₃ and downregulated by paclobutrazol, a blocker of GA biosynthesis. These data indicate that OsWOX3A is a GA-responsive gene and functions in the negative feedback regulation of the GA biosynthetic pathway for GA homeostasis to maintain the threshold levels of endogenous GA intermediates throughout development.

The virescent3 (v3) and stripe1 (st1) mutants in rice (Oryza sativa) produce chlorotic leaves in a growth stage-dependent manner under field conditions. They are temperature-conditional mutants that produce bleached leaves at a constant 20°C or 30°C but almost green leaves under diurnal 30°C/20°C conditions. Here, we show V3 and St1, which encode the large and small subunits of ribonucleotide reductase (RNR), RNRL1, and RNRS1, respectively. RNR regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. RNRL1 and RNRS1 are highly expressed in the shoot base and in young leaves, and the expression of the genes that function in plastid transcription/translation and in photosynthesis is altered in v3 and st1 mutants, indicating that a threshold activity of RNR is required for chloroplast biogenesis in developing leaves. There are additional RNR homologs in rice, RNRL2 and RNRS2, and eukaryotic RNRs comprise α₂β₂ heterodimers. In yeast, RNRL1 interacts with RNRS1 (RNRL1:RNRS1) and RNRL2:RNRS2, but no interaction occurs between other combinations of the large and small subunits. The interacting activities are RNRL1:RNRS1 > RNRL1:rnrs1(st1) > rnrl1(v3):RNRS1 > rnrl1(v3):rnrs1(st1), which correlate with the degree of chlorosis for each genotype. This suggests that missense mutations in rnrl1(v3) and rnrs1(st1) attenuate the first αβ dimerization. Moreover, wild-type plants exposed to a low concentration of an RNR inhibitor, hydroxyurea, produce chlorotic leaves without growth retardation, reminiscent of v3 and st1 mutants. We thus propose that upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves, leading to continuous plant growth.

Senescence-induced NAC (senNAC) TFs play a crucial role in senescence during the final stage of leaf development. In this study, we identified a rice senNAC, ONAC016, which functions as a positive regulator of leaf senescence. The expression of ONAC016 increased rapidly in rice leaves during the progression of dark-induced and natural senescence. The onac016-1 knockout mutant showed a delayed leaf yellowing phenotype, whereas the overexpression of ONAC016 accelerated leaf senescence. Notably, ONAC016 expression was upregulated by abscisic acid (ABA), and thus detached leaves of the onac016-1 mutant remained green much longer under ABA treatment. Quantitative RT-PCR analysis showed that ONAC016 upregulates the genes associated with chlorophyll degradation, senescence, and ABA signaling. Yeast one-hybrid and dual-luciferase assays revealed that ONAC016 binds directly to the promoter regions of OsNAP, a key gene involved in chlorophyll degradation and ABA-induced senescence. Taken together, these results suggest that ONAC016 plays an important role in promoting leaf senescence through the ABA signaling pathway involving OsNAP.

The number of patients with heart failure with reduced ejection fraction (HFrEF) is increasing. These patients have a reduced cardiorespiratory reserve. Therefore, preoperative evaluation is essential to determine the best type of anaesthesia to use in patients with HFrEF. A 70-year-old man with HFrEF was scheduled to undergo debridement of skin necrosis due to thrombotic occlusion of the right common iliac artery. He had undergone wound dressing changes under local anaesthesia every other day for several months, and treatment for heart failure was on-going. A sciatic nerve and fascia iliaca compartment block was performed under ultrasound guidance because of the patient’s cardiopulmonary function. After confirming adequate sensory blockage, surgery was performed without any haemodynamic instability or complications. Thereafter, debridement was performed twice more using the same block technique, and a skin autograft was also successfully performed. We successfully performed an ultrasound-guided sciatic nerve and fascia iliaca compartment block in a patient with HFrEF who was scheduled to undergo lower limb surgery. Peripheral nerve block is an alternative option for patients with HFrEF.

Background/Objectives: Rigid bronchoscopy poses safety challenges due to airway leakage. Although apnoeic oxygenation is a potential strategy, concerns over carbon dioxide (CO2) retention have limited its adoption. The introduction of high-flow nasal cannula (HFNC) has renewed interest by potentially mitigating CO2 accumulation during prolonged apnoea. This study investigated changes in the arterial partial pressure of CO2 (PaCO2) during apnoeic oxygenation using Optiflow™. Methods: We retrospectively analysed patients undergoing rigid bronchoscopy with HFNC (70 L·min−1) from 2020 to 2022. The apnoeic period was defined from the onset of apnoeic oxygenation to ventilation resumption. Arterial blood gas levels and complications, including arrhythmia and desaturation, were evaluated. Regression analysis was used to evaluate changes over time. Results: Apnoeic oxygenation was performed in 10 male patients (mean age 65 ± 14 years; body mass index 24.75 ± 4.18 kg·m−2). The mean duration of apnoea was 33.7 ± 13.7 min, with PaCO2 rising linearly at 1.50 mmHg/min. No interventions were required to maintain SpO2 above 91% for all patients. Except for one case of atrial fibrillation that occurred during emergence rather than the apnoeic period, no significant complications were observed. Conclusions: The observed increase in PaCO2 was lower than in previously reported studies using HFNC via the nares, suggesting that direct delivery of oxygen to the distal airway via bronchoscopy may enhance CO2 clearance through more effective washout. Apnoeic oxygenation with HFNC could potentially overcome airway leakage for selected patients, but vigilant monitoring remains essential throughout the apnoeic period. Further research is warranted to enhance patient safety.

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or “clients”, remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1’s substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

The transition from the vegetative to reproductive stage (flowering) is a critical stage in the life cycle of plants. Transition to flowering is controlled by a complex photoperiod-dependent network. Here, we report the functional analysis of NAC transcription factor ONAC005 as a positive regulator of flowering in rice. An onac005-1 T-DNA insertion mutant showed late flowering only under long-day (LD) conditions, indicating that ONAC005 is an LD-dependent flowering activator. Expression analysis of flowering time genes revealed that ONAC005 negatively regulates expression of OsLFL1, which is a LD-specific repressor of flowering in rice. Consequently, ONAC005 up-regulates expression of downstream genes Ehd1, Hd3a, and RFT1 under LD conditions. ONAC005 physically interacts with previously reported upstream regulators of OsLFL1, OsMADS50 and OsMADS56. Binding assays showed that ONAC005 binds to the promoter regions of OsLFL1. We further found that the ONAC005-OsMADS50-OsMADS56 complex weakly repressed OsLFL1 at the early vegetative stage and then gradually repressed it at the transcriptional level as the plant developed. Taken together, our results suggest that ONAC005 specifically affects flowering under LD-dependent conditions by interacting with an antagonistic protein complex.

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.