Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice.

BACKGROUND: High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites. RESULTS: We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired. CONCLUSIONS: This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

Ca2+ deficiency triggers panicle degeneration in rice mediated by Ca2+ /H+ exchanger OsCAX1a.

Increasing rice yield has always been one of the primary objectives of rice breeding. However, panicle degeneration often occurs in rice-growing regions and severely curbs rice yield. In this study, we obtained a new apical panicle degeneration mutant, which induces a marked degeneration rate and diminishes the final grain yield. Cellular and physiological analyses revealed that the apical panicle undergoes programmed cell death, accompanied by excessive accumulations of peroxides. Following, the panicle degeneration gene OsCAX1a was identified in the mutant, which was involved in Ca2+ transport. Hydroponics assays and Ca2+ quantification confirmed that Ca2+ transport and distribution to apical tissues were restricted and over-accumulated in the mutant sheath. Ca2+ transport between cytoplasm and vacuole was affected, and the reduced Ca2+ content in the vacuole and cell wall of the apical panicle and the decreased Ca2+ absorption appeared in the mutant. RNA-Seq data indicated that the abnormal CBL (calcineurin b-like proteins) pathway mediated by deficient Ca2+ might occur in the mutant, resulting in the burst of ROS and programmed cell death in panicles. Our results explained the key role of OsCAX1a in Ca2+ transport and distribution and laid a foundation to further explore the genetic and molecular mechanisms of panicle degeneration in rice.

The tonoplast-localized transporter OsNRAMP2 is involved in iron homeostasis and affects seed germination in rice.

Vacuolar storage of iron (Fe) is important for Fe homeostasis in plants. When sufficient, excess Fe could be stored in vacuoles for remobilization in the case of Fe deficiency. Although the mechanism of Fe remobilization from vacuoles is critical for crop development under low Fe stress, the transporters that mediate vacuolar Fe translocation into the cytosol in rice remains unknown. Here, we showed that under high Fe2+ concentrations, the Δccc1 yeast mutant transformed with the rice natural resistance-associated macrophage protein 2 gene (OsNRAMP2) became more sensitive to Fe toxicity. In rice protoplasts and transgenic plants expressing Pro35S:OsNRAMP2-GFP, OsNRAMP2 was localized to the tonoplast. Vacuolar Fe content in osnramp2 knockdown lines was higher than in the wild type, while the growth of osnramp2 knockdown plants was significantly influenced by Fe deficiency. Furthermore, the germination of osnramp2 knockdown plants was arrested. Conversely, the vacuolar Fe content of Pro35S:OsNRAMP2-GFP lines was significantly lower than in the wild type, and overexpression of OsNRAMP2 increased shoot biomass under Fe deficiency. Taken together, we propose that OsNRAMP2 transports Fe from the vacuole to the cytosol and plays a pivotal role in seed germination.

Expression of a Brassica napus metal transport protein (BnMTP3) in Arabidopsis thaliana confers tolerance to Zn and Mn.

The essential micronutrient elements zinc (Zn) and manganese (Mn) are crucial for plant growth and development. As an important oil crop, the yield and quality of rapeseed are affected by Zn and Mn toxicity. The cation diffusion facilitator (CDF) family of proteins play significant roles in maintaining intracellular ionic homeostasis and tolerance in plants. However, research on CDF proteins in rapeseed is lacking. In this study, the function of a Brassica napus cation diffusion facilitator/ metal tolerance protein (CDF/MTP) was investigated. The protein, abbreviated BnMTP3 is homologous to the Arabidopsis thaliana MTP3 (AtMTP3). Heterologous expression of BnMTP3 in yeast enhanced tolerance and intracellular sequestration of Zn and Mn. Expression of BnMTP3 in A. thaliana increased Zn and Mn tolerance and markedly increased Zn accumulation in roots. Quantitative RT-PCR analysis showed that BnMTP3 is primarily expressed in roots, and subcellular localization suggested that BnMTP3 is localized in the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) in Arabidopsis and rape protoplast. After treatment with Zn and Mn, BnMTP3 was observed on the vacuolar membrane in transgenic Arabidopsis lines. These findings suggest that BnMTP3 confers Zn and Mn tolerance by sequestering Zn and/or Mn into the vacuole.

Pear metal transport protein PbMTP8.1 confers manganese tolerance when expressed in yeast and Arabidopsis thaliana.

Manganese (Mn) is demonstrated to be essential for plants. Ion homeostasis is maintained in plant cells by specialized transporters. PbMTP8.1, which encodes a putative Mn-CDF transporter in Pyrus bretschneideri Rehd, was expressed mainly in leaves and complemented the Mn hypersensitivity of the Mn-sensitive yeast mutant △pmr1 in previous research conducted by our laboratory. In the present study, we report that the expression of PbMTP8.1 can enhance Mn tolerance and accumulation in Saccharomyces cerevisiae. Subcellular localization analysis of the PbMTP8.1-GFP fusion protein indicated that PbMTP8.1 was targeted to the pre-vacuolar compartment (PVC). In addition, the overexpression of PbMTP8.1 in Arabidopsis thaliana conferred increased resistance to plants under toxic Mn levels, as indicated by increased fresh and dry weights of shoots and roots. Mn accumulation in vacuoles of PbMTP8.1-overexpressing plants was significantly increased when compared with that in wild-type plants under Mn stress. This suggests that a considerable proportion of Mn enters into the vacuoles through a PbMTP8.1-dependent mechanism. Taken together, these results indicate PbMTP8.1 is a Mn-specific transporter that is localized to the PVC, and confers Mn tolerance by sequestering Mn into the vacuole.

The Intracellular Transporter AtNRAMP6 Is Involved in Fe Homeostasis in Arabidopsis.

Natural resistance-associated macrophage proteins (NRAMPs) have been shown to transport a wide range of divalent metal ions, such as manganese (Mn), cadmium (Cd), and Iron (Fe). Iron is an essential micronutrient for plants and Fe deficiency can lead to chlorosis or decreased biomass. AtNRAMP6 has demonstrated the capability to transport Cd, but its physiological function is currently unclear. This study demonstrates that AtNRAMP6 is localized to the Golgi/trans-Golgi network and plays an important role in intracellular Fe homeostasis in the flowering plant genus Arabidopsis. GUS tissue-specific expression revealed that AtNRAMP6 is highly expressed in the lateral roots and young leaves (three to four top leaves) of Arabidopsis. Moreover, knocking out AtNRAMP6 was shown to impair lateral root growth without having a differential effect on the main root under Fe-deficient conditions. Lastly, the expression of AtNRAMP6 was found to exacerbate the sensitivity of the yeast mutant Δccc1 to an excessive amount of Fe. These findings indicate that AtNRAMP6 plays an important role in the growth of Arabidopsis in Fe-deficient conditions.

OsMTP11, a trans-Golgi network localized transporter, is involved in manganese tolerance in rice.

Metal tolerance proteins (MTPs) belong to the cation diffusion facilitator family (CDF) and have been implicated in metal transport and homeostasis in different plant species. Here we report on the rice gene OsMTP11 that encodes a putative CDF transporter that is homologous to members of the Mn-CDF cluster. The expression of OsMTP11 was found to enhance Mn tolerance in the Mn-sensitive yeast mutant pmr1. Knockdown of OsMTP11 resulted in growth inhibition in the presence of high concentrations of Mn, and also led to increased accumulation of Mn in the shoots and roots. The overexpression of OsMTP11 was found to enhance Mn tolerance in rice, and under supplementation with a toxic level of Mn, decreased Mn concentration was observed in the shoots and roots. Subcellular localization in rice protoplasts and tobacco epidermal cells revealed that OsMTP11 localizes to the trans-Golgi network (TGN), and a significant relocalization to the plasma membrane can be triggered by high extracellular Mn in tobacco epidermal cells. These findings suggest that OsMTP11 is a TGN-localized Mn transporter that is required for Mn homeostasis and contributes towards Mn tolerance in rice.

Increased 4-hydroxynonenal formation contributes to obesity-related lipolytic activation in adipocytes.

Oxidative stress in adipose tissue plays an etiological role in a variety of obesity-related metabolic disorders. We previously reported that increased adipose tissue 4-hydroxynonenal (4-HNE) contents contributed to obesity-related plasma adiponectin decline in mice. In the present study, we investigated the effects of intracellular 4-HNE accumulation on lipolytic response in adipocytes/adipose tissues and underlying mechanisms. In both fully-differentiated 3T3-L1 and primary adipocytes, a 5-hour 4-HNE exposure elevated lipolytic reaction in a dose-dependent manner at both basal and isoproterenol-stimulated conditions, evidenced by significantly increased glycerol and fatty acids releases. This conclusion was corroborated by the comparable observations when the minced human visceral adipose tissues were used. Mechanistic investigations revealed that 4-HNE-stimulated lipolytic activation is multifactorial. 4-HNE exposure quickly increased intracellular cyclic AMP (cAMP) level, which was concomitant with increased phosphorylations of protein kinase A (PKA) and its direct downstream target, hormone sensitive lipase (HSL). Pre-incubation with H89, a potent PKA inhibitor, prevented 4-HNE stimulated glycerol release, suggesting that enhanced lipolytic action in response to 4-HNE increase is mediated mainly by cAMP/PKA signal pathway in adipocytes. In addition to activating cAMP/PKA/HSL pathway, 4-HNE exposure also suppresses AMP-activated protein kinase (AMPK), a suppressive pathway for lipolysis, measured by both Western blotting for phosphorylated form of AMPK and ELISA for enzyme activity. Furthermore, 5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), a pharmacological AMPK activator, alleviated 4-HNE-induced lipolysis, suggesting that AMPK suppression also contributes to 4-HNE elicited lipolytic response. In conclusion, our findings indicate that increased intracellular 4-HNE accumulation in adipocytes/adipose tissues contributes to obesity-related lipolytic activation.

Inhibition of ERK1/2 pathway suppresses adiponectin secretion via accelerating protein degradation by Ubiquitin-proteasome system: relevance to obesity-related adiponectin decline.

OBJECTIVE: Predominantly secreted by adipose tissue, adiponectin possesses insulin-sensitizing, anti-atherogenic, anti-inflammatory, and anti-angiogenic properties. Paradoxically, obesity is associated with declined plasma adiponectin levels; however, the underlying mechanisms remain elusive. In this study, we investigated the mechanistic involvement of MEK/ERK1/2 pathway in obesity-related adiponectin decrease. MATERIALS/METHODS: C57 BL/6 mice exposed to a high-fat diet (HFD) were employed as animal obesity model. Both fully-differentiated 3T3-L1 and mouse primary adipocytes were used in the in vitro experiments. RESULTS: Obesity and plasma adiponectin decline induced by prolonged HFD exposure were associated with suppressed ERK1/2 activation in adipose tissue. In adipocytes, specific inhibition of MEK/ERK1/2 pathway decreased intracellular and secretory adiponectin levels, whereas adiponectin gene expression was increased, suggesting that MEK/ERK1/2 inhibition may promote adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that MEK/ERK1/2 inhibition accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that intracellular MEK/ERK1/2 activity was negatively associated with ubiquitinated adiponectin protein levels. Consistently, long-term HFD feeing in mice increased ubiquitinated adiponectin levels in the epididymal fat pads. CONCLUSIONS: Adipose tissue MEK/ERK1/2 activity can differentially regulate adiponectin gene expression and protein abundance and its suppression in obesity may play a mechanistic role in obesity-related plasma adiponectin decline.

Inhibition of NF-κB activation by 4-hydroxynonenal contributes to liver injury in a mouse model of alcoholic liver disease.

Long-term alcohol exposure sensitizes hepatocytes to tumor necrosis factor-α (TNF) cytotoxicity. 4-Hydroxynonenal (4-HNE) is one of the most abundant and reactive lipid peroxides. Increased hepatic 4-HNE contents present in both human alcoholics and alcohol-fed animals. In the present study, we investigated the effects of intracellular 4-HNE accumulation on TNF-induced hepatotoxicity and its potential implication in the pathogenesis of alcoholic liver disease. Male C57BL/6 mice were fed an ethanol-containing or a control diet for 5 weeks. Long-term alcohol exposure increased hepatic 4-HNE and TNF levels. Cell culture studies revealed that 4-HNE, at nontoxic concentrations, sensitized hepatocytes to TNF killing, which was associated with suppressed NF-κB transactivity. Further investigation demonstrated that 4-HNE prevented TNF-induced inhibitor of κBα phosphorylation without affecting upstream IκB kinase activity. An immunoprecipitation assay revealed that increased 4-HNE content was associated with increased formation of 4-HNE-inhibitor of κBα adduction in both 4-HNE-treated hepatocytes and in the livers of alcohol-fed mice. Prevention of intracellular 4-HNE accumulation by bezafibrate, a peroxisome proliferator-activated receptor-α agonist, protected hepatocytes from TNF killing via NF-κB activation. Supplementation of N-acetylcysteine, a glutathione precursor, conferred a protective effect on alcohol-induced liver injury in mice, was associated with decreased hepatic 4-HNE formation, and improved hepatic NF-κB activity. In conclusion, increased 4-HNE accumulation represents a potent and clinically relevant sensitizer to TNF-induced hepatotoxicity. These data support the notion that removal of intracellular 4-HNE can serve as a potential therapeutic option for alcoholic liver disease.

Comparative effects of liensinine and neferine on the human ether-a-go-go-related gene potassium channel and pharmacological activity analysis.

Liensinine and neferine, a kind of isoquinoline alkaloid, can antagonize the ventricular arrhythmias. The human ether-a-go-go-related gene (hERG) is involved in repolarization of cardiac action potential. We investigated the effects of liensinine and neferine on the biophysical properties of hERG channel and the underlying structure-activity relationships. The effects of liensinine and neferine were examined on the hERG channels in the stable transfected HEK293 cells using a whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. The pharmacokinetics and tissue distribution determination of liensinine and neferine in rats were determined by a validated RP-HPLC method. Liensinine and neferine induced decrease of current amplitude in dose-dependent. Liensinine reduced hERG tail current from 70.3±6.3 pA/pF in control group to 56.7±2.8 pA/pF in the 1 µM group, 53.0±2.3 pA/pF (3 µM) and 17.8±0.7 pA/pF (30 µM); the corresponding current densities of neferine-treated cells were 41.9±3.1 pA/pF, 32.3±3.1 pA/pF and 16.2±0.6 pA/pF, respectively. Neferine had binding affinity for the open and inactivated state of hERG channel, liensinine only bound to the open state. The inhibitory effects of liensinine and neferine on hERG current were attenuated in the F656V or Y652A mutant channels. Neferine distributed more quickly than liensinine in rats, which was found to be in higher concentration than liensinine. Both liensinine and neferine had no effect on the generation and expression of hERG channels. In conclusion, neferine is a more potent blocker of hERG channels than liensinine at low concentration (<10 µM), which may be due to higher hydrophobic nature of neferine compared with liensinine. Neferine may be safety even for long-term treatment as an antiarrhythmic drug.

4-Hydroxynonenal differentially regulates adiponectin gene expression and secretion via activating PPARγ and accelerating ubiquitin-proteasome degradation.

Although well-established, the underlying mechanisms involved in obesity-related plasma adiponectin decline remain elusive. Oxidative stress is associated with obesity and insulin resistance and considered to contribute to the progression toward obesity-related metabolic disorders. In this study, we investigated the effects of 4-hydroxynonenal (4-HNE), the most abundant lipid peroxidation end product, on adiponectin production and its potential implication in obesity-related adiponectin decrease. Long-term high-fat diet feeding led to obesity in mouse, accompanied by decreased plasma adiponectin and increased adipose tissue 4-HNE content. Exposure of adipocytes to exogenous 4-HNE resulted in decreased adiponectin secretion in a dose-dependent manner, which was consistent with significantly decreased intracellular adiponectin protein abundance. In contrast, adiponectin gene expression was significantly elevated by 4-HNE treatment, which was concomitant with increased peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression and transactivity. The effect was abolished by T0070907, a PPAR-γ antagonist, suggesting that PPAR-γ activation plays a critical role in this process. To gain insight into mechanisms involved in adiponectin protein decrease, we examined the effects of 4-HNE on adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that 4-HNE exposure accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that 4-HNE exposure increased ubiquitinated adiponectin protein levels. These data altogether indicated that 4-HNE enhanced adiponectin protein degradation via ubiquitin-proteasome system. Finally, we demonstrated that supplementation of HF diet with betaine, an antioxidant and methyl donor, alleviated high-fat-induced adipose tissue 4-HNE increase and attenuated plasma adiponectin decline. Taken together, our findings suggest that the lipid peroxidation product 4-HNE can differentially regulates adiponectin gene expression and protein abundance and may play a mechanistic role in obesity-related plasma adiponectin decline.

The novel mechanism of oxymatrine affecting HERG currents at different temperatures.

BACKGROUND/AIMS: Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Our studies were to investigate the effects of oxymatrine (one of the natural constituents extracted from Chinese herb Sophora flavescens Ait) on hERG-encoded K(+) channels at different temperatures and its underlying mechanism. METHODS: The effects of oxymatrine were examined on hERG channels stably expressed in HEK293 cells using a whole-cell patch clamp technique. RESULTS: At the temperature 30°C, oxymatrine inhibited hERG current in a concentration-dependent manner and the IC(50) was ∼665 µM. However at the temperature of 20°C, low concentration oxymatrine C≤100 µM increased hERG current density. However, high concentration oxymatrine C>100 µM inhibited the hERG current density significantly. Oxymatrine only affected the activation kinetic of hERG channels at all temperatures and had a high binding affinity for open state of hERG channels except the 300 µM-20°C group which had a high binding affinity for inactive state of hERG channels. CONCLUSION: Oxymatrine is a low potency blocker of hERG K+ channels at 30°C, low concentration oxymatrine affect the hERG activation gating with accelerating hERG tail current at 20°C, oxymatrine is a potential hERG activator at low temperatures.

Blockade of HERG K+ channel by isoquinoline alkaloid neferine in the stable transfected HEK293 cells.

We studied the effects of isoquinoline alkaloid neferine (Nef) extracted from the seed embryo of Nelumbo nucifera Gaertn on Human ether-à-go-go-related gene (HERG) channels stably expressed in human embryonic kidney (HEK293) cells using whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. Nef induced a concentration-dependent decrease in current amplitude according to the voltage steps and tail currents of HERG with an IC(50) of 7.419 microM (n(H) -0.5563). Nef shifted the activation curve in a significantly negative direction and accelerated recovery from inactivation and onset of inactivation, however, slowed deactivation. In addition, it had no significant influence on steady-state inactivation curve. Western blot and immunofluorescence results suggested Nef had no significant effect on the expression of HERG protein. In summary, Nef can block HERG K(+) channels that functions by changing the channel activation and inactivation kinetics. Nef has no effect on the generation and trafficking of HERG protein. A blocked-off HERG channel was one mechanism of the anti-arrhythmic effects by Nef.