Non-specific Phospholipase C4 Improves Phosphorus Remobilization From Old to Young Leaves in Camelina.

Camelina sativa is regarded as a low-input oilseed crop for versatile food, biofuels and industrial applications with potential production on marginal lands, whereas phosphate (Pi) deficiency greatly reduces camelina seed production. To improve camelina resilience to low P conditions, here we overexpressed the Pi deficiency-induced non-specific phospholipase C4 (NPC4) to test its effect on camelina seed production under different levels of Pi availability. NPC4-overexpressing (OE) plants displayed increased seed yield and oil production, with a greater magnitude of increases under Pi-deficient than Pi-sufficient conditions. NPC4-OE camelina had a higher level of total P and free Pi in young leaves but a lower level in old leaves than in wild-type plants. More Pi was moved from old leaves to young leaves in NPC4-OE than in wild-type plants. NPC4-OE increased the expression of Pi transporter genes, and the increase was greater in old leaves and under Pi-deficient conditions. These data indicate that NPC4 improves camelina growth by promoting Pi remobilization from old to young tissues, revealing a mechanism by which NPC4 mediates plant response to Pi deficiency.

Astrocytic RARγ mediates hippocampal astrocytosis and neurogenesis deficits in chronic retinoic acid-induced depression.

Accumulating clinical evidence indicates that chronic exposure to retinoic acid (RA) may lead to depressive symptoms and even increase the risk of suicidal behavior, which severely limits the clinical long-term application of RA. The exact mechanisms through which RA contributes to the onset of depression remain largely unclear. Here, we administered intraperitoneal injections of all-trans RA to male C57BL/6 J mice over a period of 21 days. Mice subjected to chronic RA exposure displayed depressive-like behaviors, accompanied by impaired hippocampal neurogenesis and heightened RA receptor gamma (RARγ) levels in the ventral hippocampus (vHip). The administration of an RARγ antagonist effectively mitigated these RA-induced neurogenesis impairments and depressive-like behaviors. Chronic exposure to RA was also observed to promote hippocampal astrocytosis and increase astrocytic Rarγ expression in the ventral dentate gyrus (vDG) of hippocampus. Notably, astrocytic RARγ in the vDG was found to be a key factor in the observed hippocampal astrocytosis and neurogenesis impairments, and depressive-like behaviors. Chronic exposure to RA resulted in increased extracellular glutamate levels in neural stem cells (NSCs), accompanied by a decrease in glutamate transporter 1 (GLT-1) expression. Enhancing astrocytic GLT-1 expression was found to alleviate both hippocampal astrocytosis and depressive-like behaviors caused by RA. These findings underscore the critical role of astrocytic RARγ-GLT-1 axis in the development of hippocampal astrocytosis, neurogenesis impairments, and depressive symptoms, suggesting that targeting RARγ-GLT-1 could potentially offer an effective therapeutic approach for depression.

Photocatalytic Hydrogen Peroxide Production by a Mixed Ligand-Functionalized Uranyl-Organic Framework.

Hydrogen peroxide (H2O2) production driven by solar energy has received enormous attention due to its high efficiency, low cost, and environmental friendliness characteristics. Searching for new photocatalytic materials for H2O2 production is one of the most important targets. In this work, a new three-dimensional (3D) uranyl-organic framework material was constructed with mixed ligands via a solvothermal reaction and used for photocatalytic H2O2 production. The mixed ligand strategy not only benefits the construction of a 3D uranyl-organic framework but also introduces strong photon absorption groups into the framework. The thiophene and pyridine rings in the framework enhance photon absorption and carrier transfer. In addition, with the assistance of the hydrogen abstraction reaction of uranyl centers, the H2O2 production rate reaches 345 µmol h-1 g-1. This study provides a new blueprint for exploring the artificial photosynthesis of H2O2 through uranium-based metal-organic frameworks.

Ultrafine Nanoclusters Unlocked 3d-4f Electronic Ladders for Efficient Electrocatalytic Water Oxidation.

The vast extensional planes of two-dimensional (2D) nanomaterials are recognized as desirable ground for electrocatalytic reactions. However, they tend to exhibit catalytic inertia due to their surface-ordered coordination configurations. Herein, an in situ autoxidation strategy enables high-density grafting of ultrafine CeO2 nanoclusters on 2D Co(OH)2. Affluent active units were activated at the inert interface of Co(OH)2 via the formation of Co-O-Ce units. The optimized catalyst exhibits oxygen evolution reaction activity with an overpotential of 83 mV lower than that of Co(OH)2 at 10 mA cm-2. The cascade orbital coupling between Co (3d) and Ce (4f) in Co-O-Ce units drives electron transfer by unlocking a "d-f electron ladder". Meanwhile, the bond-order theorem analyses and the d-band center show that the occupancy of Co-3d-eg is optimized to balance the adsorption-desorption process of active sites to the key reaction intermediate *OOH, thereby making it easier to release oxygen. This work will drive the development of wider area electron modulation methods and provide guidance for the surface engineering of 2D nanomaterials.

Phospholipase-mediated phosphate recycling during plant leaf senescence.

BACKGROUND: Phosphorus is a macronutrient necessary for plant growth and development and its availability and efficient use affect crop yields. Leaves are the largest tissue that uses phosphorus in plants, and membrane phospholipids are the main source of cellular phosphorus usage. RESULTS: Here we identify a key process for plant cellular phosphorus recycling mediated by membrane phospholipid hydrolysis during leaf senescence. Our results indicate that over 90% of lipid phosphorus, accounting for more than one-third of total cellular phosphorus, is recycled from senescent leaves before falling off the plants. Nonspecific phospholipase C4 (NPC4) and phospholipase Dζ2 (PLDζ2) are highly induced during leaf senescence, and knockouts of PLDζ2 and NPC4 decrease the loss of membrane phospholipids and delay leaf senescence. Conversely, overexpression of PLDζ2 and NPC4 accelerates the loss of phospholipids and leaf senescence, promoting phosphorus remobilization from senescent leaves to young tissues and plant growth. We also show that this phosphorus recycling process in senescent leaves mediated by membrane phospholipid hydrolysis is conserved in plants. CONCLUSIONS: These results indicate that PLDζ2- and NPC4-mediated membrane phospholipid hydrolysis promotes phosphorus remobilization from senescent leaves to growing tissues and that the phospholipid hydrolysis-mediated phosphorus recycling improves phosphorus use efficiency in plants.

Enhanced properties of the mid-infrared superluminescent emitter with a composite waveguide.

This study reports on a composite structure composing tilted taper, and tilted and curved waveguides with the aim of enhancing the spectral width and output power of mid-infrared quantum cascade superluminescent emitters (QC-SLEs). The computational results indicate that a tilt angle of 10° and a curved angle of 20° can avoid the selectivity of a certain wavelength due to interference effects at tilt angles of 6° and 8°, resulting in the minimum reflectivity of 1.3×10-4 and 4.4×10-4 for each wide and narrow cavity surface. Simultaneously, the modes propagating perpendicular to the cavity surface exist the least. The corresponding experimental results show a significant enhancement in the spectral width to 168.5c m -1 and a high power output of 5.1 mW for the device. This study presents what we believe to be a novel concept for the designing of superluminescent emitters with both a broadband and high power output.

Fine-tuning phosphatidic acid production for optimal plant stress responses.

Phosphatidic acid (PA) is involved in biotic and abiotic stress responses in plants. Here, we summarize quantitative lipidomics and real-time imaging used in PA studies and highlight recent studies of diacylglycerol (DAG) kinase (DGK) 5, an enzyme involved in PA biosynthesis, facilitating fine-tuning PA production for optimal stress responses in plants.

Nucleus-targeted carbon dots as peroxidase nanozyme for photoacoustic imaging and phototherapy of tumor.

High-purity carbon dots (CDs) with a highly π-conjugated sp2-hybridized graphite structure were prepared by the pulse electrolysis method using the graphite plate as raw material. Photoacoustic signal together with photothermal effect was found in the CDs-dispersed suspensions under near-infrared (NIR) irradiation. For the suspension with the CDs concentration of 500 µg/mL, the photothermal conversion efficiency is high up 64.3% and the solution's temperature can be increased to 82.2 °C under NIR irradiation. Moreover, CDs can be effectively endocytosed by human hepatoma (HepG2) cells with a few hours, act as peroxidase nanozyme to decompose H2O2 and facilitate the production of reactive oxygen species. Under NIR irradiation, CDs exhibit an outstanding apoptosis-inducing effect on HepG2 cells by the photothermal effect. In addition, in vivo experiments show that CDs can be used in photoacoustic imaging (PAI) and guiding the tumor treatment. As a result, the nucleus-targeted CDs with an unique combination of PAI and photothermal effect have potential in cancer diagnosis and treatment.

Hepatic selective insulin resistance at the intersection of insulin signaling and metabolic dysfunction-associated steatotic liver disease.

Insulin resistance (IR) is a major pathogenic factor in the progression of MASLD. In the liver, insulin suppresses gluconeogenesis and enhances de novo lipogenesis (DNL). During IR, there is a defect in insulin-mediated suppression of gluconeogenesis, but an unrestrained increase in hepatic lipogenesis persists. The mechanism of increased hepatic steatosis in IR is unclear and remains controversial. The key discrepancy is whether insulin retains its ability to directly regulate hepatic lipogenesis. Blocking insulin/IRS/AKT signaling reduces liver lipid deposition in IR, suggesting insulin can still regulate lipid metabolism; hepatic glucose metabolism that bypasses insulin's action may contribute to lipogenesis; and due to peripheral IR, other tissues are likely to impact liver lipid deposition. We here review the current understanding of insulin's action in governing different aspects of hepatic lipid metabolism under normal and IR states, with the purpose of highlighting the essential issues that remain unsettled.

A Four-Week High-Fat Diet Induces Anxiolytic-like Behaviors through Mature BDNF in the mPFC of Mice.

The effect of a high-fat diet (HFD) on mood is a widely debated topic, with the underlying mechanisms being poorly understood. This study explores the anxiolytic effects of a four-week HFD in C57BL/6 mice. Five-week-old mice were exposed to either an HFD (60% calories from fat) or standard chow diet (CD) for four weeks, followed by cannula implantation, virus infusion, behavioral tests, and biochemical assays. Results revealed that four weeks of an HFD induced anxiolytic-like behaviors and increased the protein levels of mature brain-derived neurotrophic factor (mBDNF) and phosphorylated tyrosine kinase receptor B (p-TrkB) in the medial prefrontal cortex (mPFC). Administration of a BDNF-neutralizing antibody to the mPFC reversed HFD-induced anxiolytic-like behaviors. Elevated BDNF levels were observed in both neurons and astrocytes in the mPFC of HFD mice. Additionally, these mice exhibited a higher number of dendritic spines in the mPFC, as well as upregulation of postsynaptic density protein 95 (PSD95). Furthermore, mRNA levels of the N6-methyladenosine (m6A) demethylase, fat mass and obesity-associated protein (FTO), and the hydrolase matrix metalloproteinase-9 (MMP9), also increased in the mPFC. These findings suggest that an HFD may induce FTO and MMP9, which could potentially regulate BDNF processing, contributing to anxiolytic-like behaviors. This study proposes potential molecular mechanisms that may underlie HFD-induced anxiolytic behaviors.

Nonspecific phospholipases C3 and C4 interact with PIN-FORMED2 to regulate growth and tropic responses in Arabidopsis.

The dynamic changes in membrane phospholipids affect membrane biophysical properties and cell signaling, thereby influencing numerous biological processes. Nonspecific phospholipase C (NPC) enzymes hydrolyze common phospholipids to release diacylglycerol (DAG), which is converted to phosphatidic acid (PA) and other lipids. In this study, 2 Arabidopsis (Arabidopsis thaliana) tandemly arrayed genes, NPC3 and NPC4, were identified as critical factors modulating auxin-controlled plant growth and tropic responses. Moreover, NPC3 and NPC4 were shown to interact with the auxin efflux transporter PIN-FORMED2 (PIN2). The loss of NPC3 and NPC4 enhanced the endocytosis and vacuolar degradation of PIN2, which disrupted auxin gradients and slowed gravitropic and halotropic responses. Furthermore, auxin-triggered activation of NPC3 and NPC4 is required for the asymmetric PA distribution that controls PIN2 trafficking dynamics and auxin-dependent tropic responses. Collectively, our study reveals an NPC-derived PA signaling pathway in Arabidopsis auxin fluxes that is essential for fine-tuning the balance between root growth and environmental responses.

Qinzhuliangxue mixture ameliorates psoriasis by restraining apoptosis in psoriasis via downregulating the MDA-5 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoriasis is characterized by hyperkeratosis that produces the classic silvery scales, and the pathogenesis of psoriasis involves abnormal proliferation of keratinocytes. Emerging evidence supports that apoptosis regulates keratinocyte proliferation and formation of stratum corneum, which maintains the homeostasis of the skin. Qinzhuliangxue mixture (QZLX) is a representative formula for the treatment of psoriasis, which was earliest recorded in the classic Chinese medicine book Xia's Surgery. In our previous clinical studies, QZLX demonstrated 83.33% efficacy with few side effects in the treatment of psoriasis. Furthermore, our published basic research has also proved that the QZLX mixture effectively inhibits the hyperproliferation of keratinocytes, thus exerting therapeutic effects on psoriasis. However, whether QZLX mixture can regulate keratinocytes apoptosis requires further clarification. OBJECTIVE OF THE STUDY: To investigate the mechanism of QZLX in the treatment of psoriasis from the perspective of keratinocyte apoptosis. MATERIALS AND METHODS: First, psoriasis-like mice with imiquimod (IMQ)-induced were given QZLX intragastric administration and Psoriasis Area Severity Index (PASI) scores were recored for 11 consecutive days to appraise the efficacy. Then, tissue samples were collected for transcriptome analysis. The DEseq2 method detected significantly differentially expressed genes (DEGs), Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway databases were used to analyze the functions and pathway enrichment of DEGs. After that, the therapeutic mechanisms of QZLX in intervening with psoriasis were explored using TUNEL, immunohistochemical staining, and western blotting. RESULTS: QZLX ameliorated the symptoms and pathological characteristics of IMQ-induced psoriasis in mice. The epidermal cell hyperplasia in the skin was inhibited, in accordance with the suppressed expression of PCNA and Ki67 after treatment. Transcriptome sequencing showed that melanoma differentiation associated gene-5 (MDA-5) was downregulated. GO and KEGG enrichment analysis of the signaling pathways indicated that the differentially expressed genes were significantly enriched in apoptosis pathways. Besides, QZLX treatment decreased the apoptosis of keratinocyte as shown by reduced TUNEL-positive cells. As MDA-5 protein levels decreased, so did the expression of the downstream protein Caspase-8, which indicates that the apoptotic pathway was triggered. Furthermore, QZLX therapy might also help to balance the apoptotic Bcl-2 family expression. CONCLUSION: QZLX restrains the apoptosis of keratinocyte in psoriasis-like mice by downregulating the MDA-5 pathway. The restoration of the balance between cell apoptosis and proliferation in the skin may lead to considerable psoriasis relief. Our study reveals the possible molecular processes behind the effects of QZLX therapy on the skin lesions of psoriasis, and lends support to its clinical efficacy.

Construction of Porphyrin-Based Bimetallic Nanomaterials with Photocatalytic Properties.

The efficient synthesis of nanosheets containing two metal ions is currently a formidable challenge. Here, we attempted to dope lanthanide-based bimetals into porphyrin-based metal-organic skeleton materials (MOFs) by microwave-assisted heating. The results of the EDX, ICP, and XPS tests show that we have successfully synthesized porphyrin-based lanthanide bimetallic nanosheets (Tb-Eu-TCPP) using a household microwave oven. In addition, it is tested and experimentally evident that these nanosheets have a thinner thickness, a larger BET surface area, and higher photogenerated carrier separation efficiency than bulk porphyrin-based bimetallic materials, thus exhibiting enhanced photocatalytic activity and n-type semiconductor properties. Furthermore, the prepared Tb-Eu-TCPP nanomaterials are more efficient in generating single-linear state oxygen under visible light irradiation compared to pristine monometallic nanosheets due to the generation of bimetallic nodes. The significant increase in catalytic activity is attributed to the improved separation and transfer efficiency of photogenerated carriers. This study not only deepens our understanding of lanthanide bimetallic nanosheet materials but also introduces an innovative approach to improve the photocatalytic performance of MOFs.

Protective role of arachidonic acid against diabetic myocardial ischemic injury: a translational study of pigs, rats, and humans.

AIM: Patients with diabetes mellitus have poor prognosis after myocardial ischemic injury. However, the mechanism is unclear and there are no related therapies. We aimed to identify regulators of diabetic myocardial ischemic injury. METHODS AND RESULTS: Mass spectrometry-based, non-targeted metabolomic approach was used to profile coronary sinus blood from diabetic and non-diabetic Bama-mini pigs at 0.5-h post coronary artery ligation. Six metabolites had a |log2 (Fold Change)|> 1.3. Among them, the most changed is arachidonic acid (AA), levels of which were 32 times lower in diabetic pigs than in non-diabetic pigs. The AA-derived products, PGI2 and 6-keto-PGF1α, were also significantly reduced. AA treatment of cultured cardiomyocytes protected against cell death by 30% at 48 h of high glucose and oxygen deprivation, which coincided with increased mitophagic activity (as indicated by increased LC3II/LC3I, decreased p62 and increased parkin & PINK1), improved mitochondrial renewal (upregulation of Drp1 and FIS1), reduced ROS generation and increased ATP production. These cardioprotective effects were abolished by PINK1(a crucial mitophagy protein) knockdown or the autophagy inhibitor 3-Methyladenine. The protective effect of AA was also inhibited by indomethacin and Cay10441, a prostacyclin receptor antagonist. Furthermore, diabetic Sprague Dawley rats were subjected to coronary ligation for 40 min and AA treatment (10 mg/day per animal gavaged) decreased myocardial infarct size, cell apoptosis index, inflammatory cytokines and improved heart function. Scanning electron microscopy showed more intact mitochondria in the border zone of infarcted myocardium in AA treated rats. Lastly, diabetic patients after myocardial infarction had lower plasma levels of AA and 6-keto-PGF1α and reduced cardiac ejection fraction, compared with non-diabetic patients after myocardial infarction. Plasma AA level was inversely correlated with fasting blood glucose. CONCLUSIONS: AA protects against diabetic ischemic myocardial damage by promoting mitochondrial autophagy and renewal, which is related to AA derived PGI2 signaling. AA may represent a new strategy to treat diabetic myocardial ischemic injury.

Ferredoxin-Inspired Design of S-Synergized Fe-Fe Dual-Metal Center Catalysts for Enhanced Electrocatalytic Oxygen Reduction Reaction.

Dual-metal center catalysts (DMCs) have shown the ability to enhance the oxygen reduction reaction (ORR) owing to their distinctive structural configurations. However, the precise modulation of electronic structure and the in-depth understanding of synergistic mechanisms between dual metal sites of DMCs at the atomic level remain challenging. Herein, mimicking the ferredoxin, Fe-based DMCs (Fe2N6-S) are strategically designed and fabricated, in which additional Fe and S sites are synchronously installed near the Fe sites and serve as "dual modulators" for coarse- and fine-tuning of the electronic modulation, respectively. The as-prepared Fe2N6-S catalyst exhibits enhanced ORR activity and outstanding Zinc-air (Zn-air) battery performance compared to the conventional single Fe site catalysts. The theoretical and experimental results reveal that introducing the second metal Fe creates a dual adsorption site that alters the O2 adsorption configuration and effectively activates the O─O bond, while the synergistic effect of dual Fe sites results in the downward shift of the d-band center, facilitating the release of OH*. Additionally, local electronic engineering of heteroatom S for Fe sites further facilitates the formation of the rate-determining step OOH*, thus accelerating the reaction kinetics.

Realization of white-light-emitting diodes from a high-brightness zirconium-based metal-organic gel driven by the AIE effect.

Developing luminescent materials with suitable correlated color temperature (CCT) and sufficient color-rendering index (CRI) is a challenging problem in the field of commercialized warm white LED lighting. Herein, a novel metal-organic gel (MOG) material named YTU-G-1(SE) was synthesized, consisting of zirconium metal coordinated with 1,1,2,2-tetrakis(4-carboxyphenyl) ethylene. YTU-G-1(SE) exhibits strong fluorescent properties with an aggregation-induced emission (AIE) effect, emitting yellow-green fluorescence at 515 nm. The internal and external quantum efficiencies (IQE/EQE) of YTU-G-1(SE) are close to unity, with values of 95.74 ± 0.5% and 88.67 ± 0.5%, respectively. Finally, we combined YTU-G-1(SE) with a commercial blue chip and a commercial red phosphor (Sr,Ca)AlSiN3:Eu2+ to fabricate a warm white light LED with a color temperature of 3736 K, a color-rendering index Ra of 88.2, and a lumen efficiency of 79.42 lm W-1. This work provides a new approach to regulating the emission of AIE and offers a novel idea for developing high-performance warm-white pc-WLEDs.

Immunoregulation of bovine lactoferrin together with osteopontin promotes immune system development and maturation.

The immune system of infants is partly weak and immature, and supplementation of infant formula can be of vital importance to boost the development of the immune system. Lactoferrin (LF) and osteopontin (OPN) are essential proteins in human milk with immunoregulation function. An increasing number of studies indicate that proteins have interactions with each other in milk, and our previous study found that a ratio of LF : OPN at 1 : 5 (w/w, denoted as LOP) had a synergistic effect on intestinal barrier protection. It remains unknown whether LOP can also exert a stronger effect on immunoregulation. Hence, we used an in vitro model of LPS-induced macrophage inflammation and in vivo models of LPS-induced intestinal inflammation and early life development. We showed that LOP increased the secretion of the granulocyte-macrophage colony-stimulating factor (132%), stem cell factor (167%) and interleukin-3 (176%) in bone marrow cells, as well as thymosin (155%) and interleukin-10 (161%) in the thymus, more than LF or OPN alone during development, and inhibited changes in immune cells and cytokines during the LPS challenge. In addition, analysis of the components of digested proteins in vitro revealed that differentially expressed peptides may provide immunoregulation. Lastly, LOP increased the abundance of Rikenellaceae, Muribaculum, Faecalibaculum, and Elisenbergiella in the cecum content. These results imply that LOP is a potential immunomodifier for infants and offers a new theoretical basis for infant formula innovation.

Lipid phosphorylation by a diacylglycerol kinase suppresses ABA biosynthesis to regulate plant stress responses.

Lipid phosphorylation by diacylglycerol kinase (DGK) that produces phosphatidic acid (PA) plays important roles in various biological processes, including stress responses, but the underlying mechanisms remain elusive. Here, we show that DGK5 and its lipid product PA suppress ABA biosynthesis by interacting with ABA-DEFICIENT 2 (ABA2), a key ABA biosynthesis enzyme, to negatively modulate plant response to abiotic stress tested in Arabidopsis thaliana. Loss of DGK5 function rendered plants less damaged, whereas overexpression (OE) of DGK5 enhanced plant damage to water and salt stress. The dgk5 mutant plants exhibited decreased total cellular and nuclear levels of PA with increased levels of diacylglycerol, whereas DGK5-OE plants displayed the opposite effect. Interestingly, we found that both DGK5 and PA bind to the ABA-synthesizing enzyme ABA2 and suppress its enzymatic activity. Consistently, the dgk5 mutant plants exhibited increased levels of ABA, while DGK5-OE plants showed reduced ABA levels. In addition, we showed that both DGK5 and ABA2 are detected in and outside the nuclei, and loss of DGK5 function decreased the nuclear association of ABA2. We found that both DGK5 activity and PA promote nuclear association of ABA2. Taken together, these results indicate that both DGK5 and PA interact with ABA2 to inhibit its enzymatic activity and promote its nuclear sequestration, thereby suppressing ABA production in response to abiotic stress. Our study reveals a sophisticated mechanism by which DGK5 and PA regulate plant stress responses.

Identification of phospholipase Ds and phospholipid species involved in circadian clock alterations using CRISPR/Cas9-based multiplex editing of Arabidopsis.

Reciprocal regulation between the circadian clock and lipid metabolism is emerging, but its mechanisms remain elusive. We reported that a lipid metabolite phosphatidic acid (PA) bound to the core clock transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and chemical suppression of phospholipase D (PLD)-catalyzed PA formation perturbed the clock in Arabidopsis. Here, we identified, among 12 members, specific PLDs critical to regulating clock function. We approached this using a multiplex CRISPR/Cas9 system to generate a library of plants bearing randomly mutated PLDs, then screening the mutants for altered rhythmic expression of CCA1 . All PLD s, except for ß2 , were effectively edited, and the mutations were heritable. Screening of T2 plants identified some with an altered rhythm of CCA1 expression, and this trait was observed in many of their progenies. Genotyping revealed that at least two of six PLD s ( α1, α3 , γ1 , δ , ε and ζ2 ) were mutated in the clock-altered plants. Those plants also had reduced levels of PA molecular species that bound LHY and CCA1. This study identifies combinations of two or more PLDs and changes in particular phospholipid species involved in clock outputs and also suggests a functional redundancy of the six PLDs for regulating the plant circadian clock. One sentence summary: This study identifies combinations of two or more phospholipase Ds involved in altering clock outputs and the specific phosphatidic acid species impacting the clock rhythms.