SANT proteins modulate gene expression by coordinating histone H3KAc and Khib levels and regulate plant heat tolerance.

Histone post-translational modifications (PTMs), such as acetylation and recently identified lysine 2-hydroxyisobutyrylation (Khib), act as active epigenomic marks in plants. SANT domain-containing proteins SANT1, SANT2, SANT3 and SANT4 (SANT1/2/3/4), derived from PIF/Harbinger transposases, form a complex with HISTONE DEACETYLASE 6 (HDA6) to regulate gene expression via histone deacetylation. However, whether SANT1/2/3/4 coordinate different types of PTMs to regulate transcription and mediate responses to specific stresses in plants remains unclear. Here, in addition to modulating histone deacetylation, we found that SANT1/2/3/4 proteins acted like HDA6 or HDA9 in regulating the removal of histone Khib in Arabidopsis (Arabidopsis thaliana). Histone H3 lysine acetylation (H3KAc) and histone Khib were coordinated by SANT1/2/3/4 to regulate gene expression, with H3KAc playing a predominant role and Khib acting complementarily to H3KAc. SANT1/2/3/4 mutation significantly increased the expression of heat-inducible genes with concurrent change of H3KAc levels under normal and heat stress conditions, resulting in enhanced thermotolerance. This study revealed the critical roles of Harbinger transposon-derived SANT domain-containing proteins in transcriptional regulation by coordinating different types of histone PTMs and in the regulation of plant thermotolerance by mediating histone acetylation modification.

Disruption of the Contents of Endogenous Hormones Cause Pollen Development Obstruction and Abortion in Male-Sterile Hybrid Lily Populations.

Lilies are well-known flowers with large anthers and a high quantity of pollen that easily contaminates clothing and tepals. The anthers need to be artificially removed, leading to production problems. Cultivating male-sterile or pollen-free lilies could solve these problems. The key period of male sterility in a specific male-sterile hybrid lily population was determined through cytological observation. The contents of hormones, soluble sugar, soluble protein, and proline were determined by high-performance liquid chromatography, tandem mass spectrometry and colorimetry. Transcriptome sequencing was used to identify the genes with altered expression. The key period of male sterility was determined to be the microspore mother and tetrad stages. The hormone contents were abnormal in the sterile line compared with the fertile line. The indole-3-acetic acid (IAA) content was higher in the sterile line than in the fertile line at all stages, while the gibberellic acid 4 (GA4) content showed the opposite result. Abscisic acid (ABA) accumulated in the sterile line in both the microspore mother and tetrad stages, and the zeatin riboside (ZR) content in the sterile line increased at the microspore mother stage but decreased at the tetrad stage. The contents of soluble sugar, soluble protein and proline were higher in the fertile line than in the sterile line. Genes involved in auxin and ABA synthesis and signalling pathways were highly expressed in the male-sterile line. Our data suggested that abnormal contents of hormones in the microspore mother and tetrad stages resulted in pollen abortion in a male-sterile hybrid lily population, which indicated that the hormone balance in specific stages plays critical functions in pollen development in lilies.

Comparative genomics reveals a unique nitrogen-carbon balance system in Asteraceae.

The Asteraceae (daisy family) is one of the largest families of plants. The genetic basis for its high biodiversity and excellent adaptability has not been elucidated. Here, we compare the genomes of 29 terrestrial plant species, including two de novo chromosome-scale genome assemblies for stem lettuce, a member of Asteraceae, and Scaevola taccada, a member of Goodeniaceae that is one of the closest outgroups of Asteraceae. We show that Asteraceae originated ~80 million years ago and experienced repeated paleopolyploidization. PII, the universal regulator of nitrogen-carbon (N-C) assimilation present in almost all domains of life, has conspicuously lost across Asteraceae. Meanwhile, Asteraceae has stepwise upgraded the N-C balance system via paleopolyploidization and tandem duplications of key metabolic genes, resulting in enhanced nitrogen uptake and fatty acid biosynthesis. In addition to suggesting a molecular basis for their ecological success, the unique N-C balance system reported for Asteraceae offers a potential crop improvement strategy.

Transcriptomic analysis reveals candidate genes associated with anther development in Lilium Oriental Hybrid 'Siberia'.

Lily (Lilium spp. and hybrids) is an important cut flower crop worldwide. Lily flowers have large anthers, which release a large amount of pollen that stains the tepals or clothing and thus can affect the commercial value of cut flowers. In this study, lily Oriental 'Siberia' was used to investigate the regulatory mechanism of lily anther development, which may provide information to prevent pollen pollution in the future. Based on the flower bud length, anther length and color, and anatomical observations, lily anther development was categorized into five stages: green (G), green-to-yellow 1 (GY1), green-to-yellow 2 (GY2), yellow (Y), and purple (P). Total RNA was extracted from the anthers at each stage for transcriptomic analysis. A total of 268.92-Gb clean reads were generated, and 81,287 unigenes were assembled and annotated. The number of differentially expressed genes (DEGs) and unique genes were largest for the pairwise comparison between the G and GY1 stages. The G and P samples were clustered separately, whereas the GY1, GY2, and Y samples were clustered together in scatter plots from a principal component analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs detected in the GY1, GY2, and Y stages revealed that the pectin catabolic process, hormone levels, and phenylpropanoid biosynthesis were enriched. The DEGs associated with jasmonic acid biosynthesis and signaling were highly expressed at the early stages (G and GY1), whereas the DEGs associated with phenylpropanoid biosynthesis were mainly expressed in the intermediate stages (GY1, GY2, and Y). The DEGs involved in the pectin catabolic process were expressed at advanced stages (Y and P). Cucumber mosaic virus-induced gene silencing of LoMYB21 and LoAMS caused a strongly inhibited anther dehiscence phenotype, but without affecting the development of other floral organs. These results provide novel insights for understanding the regulatory mechanism of anther development in lily and other plants.

The unique sweet potato NAC transcription factor IbNAC3 modulates combined salt and drought stresses.

Plants often simultaneously experience combined stresses rather than a single stress, causing more serious damage, but the underlying mechanisms remain unknown. Here, we identified the stress-induced IbNAC3 from sweet potato (Ipomoea batatas) as a nucleus-localized transcription activator. IbNAC3 contains a unique activation domain whose MKD sequence confers transactivation activities to multiple other TFs and is essential for the activated expression of downstream target genes. Ectopic expression of IbNAC3 conferred tolerance to single and combined salt and drought stresses in Arabidopsis (Arabidopsis thaliana), and a group of NAM, ATAF1/2, and CUC2 (NAC) TFs, including ANAC011, ANAC072, ANAC083, ANAC100, and NAP, interacted with IbNAC3, and the specific domains responsible for each interaction varied. Intriguingly, IbNAC3 repressed the interaction among the five NACs, and knockout or mutation of ANAC011 and ANAC072 dramatically impaired combined stress tolerance. IbNAC3-ANAC072 and IbNAC3-NAP modules synergistically activated the MICROTUBULE-RELATED E3 LIGASE57 (MREL57) gene. Consistently, mutation of MREL57 and overexpression of WAVE-DAM-PENED2-LIKE7, encoding a target protein of MREL57, both remarkably impaired combined stress tolerance. Moreover, transgenic plants displayed abscisic acid (ABA) hyposensitivity by directly promoting the transcription of ENHANCED RESPONSE TO ABA 1, a key negative regulator of ABA signaling. The data unravel the unique IbNAC3 TF functions as a pivotal component in combined stress tolerance by integrating multiple regulatory events and ubiquitin pathways, which is essential for developing high-tolerant plants in natural environments.

Regulatory Mechanisms of Heat Stress Response and Thermomorphogenesis in Plants.

As worldwide warming intensifies, the average temperature of the earth continues to increase. Temperature is a key factor for the growth and development of all organisms and governs the distribution and seasonal behavior of plants. High temperatures lead to various biochemical, physiological, and morphological changes in plants and threaten plant productivity. As sessile organisms, plants are subjected to various hostile environmental factors and forced to change their cellular state and morphological architecture to successfully deal with the damage they suffer. Therefore, plants have evolved multiple strategies to cope with an abnormal rise in temperature. There are two main mechanisms by which plants respond to elevated environmental temperatures. One is the heat stress response, which is activated under extremely high temperatures; the other is the thermomorphogenesis response, which is activated under moderately elevated temperatures, below the heat-stress range. In this review, we summarize recent progress in the study of these two important heat-responsive molecular regulatory pathways mediated, respectively, by the Heat Shock Transcription Factor (HSF)-Heat Shock Protein (HSP) pathway and PHYTOCHROME INTER-ACTING FACTOR 4 (PIF4) pathways in plants and elucidate the regulatory mechanisms of the genes involved in these pathways to provide comprehensive data for researchers studying the heat response. We also discuss future perspectives in this field.

Ethylene Response Factor LlERF110 Mediates Heat Stress Response via Regulation of LlHsfA3A Expression and Interaction with LlHsfA2 in Lilies (Lilium longiflorum).

Heat stress seriously affects the quality of cut lily flowers. The ethylene response factors (ERFs) participate in heat stress response in many plants. In this study, heat treatment increased the production of ethylene in lily leaves, and exogenous ethylene treatment enhanced the heat resistance of lilies. LlERF110, an important transcription factor in the ethylene signaling pathway, was found in the high-temperature transcriptome. The coding region of LlERF110 (969 bp) encodes 322 amino acids and LlERF110 contains an AP2/ERF typical domain belonging to the ERF subfamily group X. LlERF110 was induced by ethylene and was expressed constitutively in all tissues. LlERF110 is localized in the nucleus and has transactivation activity. Virus-induced gene silencing of LlERF110 in lilies reduced the basal thermotolerance phenotypes and significantly decreased the expression of genes involved in the HSF-HSP pathway, such as LlHsfA2, LlHsfA3A, and LlHsfA5, which may activate other heat stress response genes; and LlHsp17.6 and LlHsp22, which may protect proteins under heat stress. LlERF110 could directly bind to the promoter of LlHsfA3A and activate its expression according to the yeast one hybrid and dual-luciferase reporter assays. LlERF110 interacts with LlHsfA2 in the nucleus according to BiFC and the yeast two-hybrid assays. In conclusion, these results indicate that LlERF110 plays an important role in the basal thermotolerance of lilies via regulation of the HSF-HSP pathway, which could be the junction of the heat stress response pathway and the ethylene signaling pathway.

Genome-wide identification and expression analysis of MIKCC genes in rose provide insight into their effects on flower development.

The MIKCC-type gene family plays important roles in plant growth, development, and tolerance of biotic and abiotic stress, especially during floral organ differentiation. However, there have been no studies of MIKCC-type genes in rose, and functional differentiation of family members has not been explored. In this study, we identified 42 MIKCC-type genes in rose, classified the genes into 12 subfamilies, and constructed a phylogenetic tree. We performed expression analysis of these genes, and found that expression patterns correlated with the predicted subfamily, indicating that the features of MIKCC-type genes were broadly conserved during evolution. Collinear analysis of MIKCC genes among Rosaceae species confirmed the occurrence of whole genome duplications (WGD) and revealed some species-specific MIKCC genes. Transcriptome analysis showed that the expression of some MIKCC-type genes responded to low temperatures (4°C, 24 h) during flower organ differentiation. These conserved, duplicated, and novel expression patterns of MIKCC-type genes may have facilitated the adaptation of rose to various internal and external environmental changes. The results of this study provide a theoretical basis for future functional analysis of the MIKCC genes in rose and investigation of the evolutionary pattern of the MIKCC gene family in the Rosaceae genome.

The transcriptional co-repressor SEED DORMANCY 4-LIKE (AtSDR4L) promotes the embryonic-to-vegetative transition in Arabidopsis thaliana.

Repression of embryonic traits during the seed-to-seedling phase transition requires the inactivation of master transcription factors associated with embryogenesis. How the timing of such inactivation is controlled is unclear. Here, we report on a novel transcriptional co-repressor, Arabidopsis thaliana SDR4L, that forms a feedback inhibition loop with the master transcription factors LEC1 and ABI3 to repress embryonic traits post-imbibition. LEC1 and ABI3 regulate their own expression by inducing AtSDR4L during mid to late embryogenesis. AtSDR4L binds to sites upstream of LEC1 and ABI4, and these transcripts are upregulated in Atsdr4l seedlings. Atsdr4l seedlings phenocopy a LEC1 overexpressor. The embryonic traits of Atsdr4l can be partially rescued by impairing LEC1 or ABI3. The penetrance and expressivity of the Atsdr4l phenotypes depend on both developmental and external cues, demonstrating the importance of AtSDR4L in seedling establishment under suboptimal conditions.

Disruption of transcription factor RhMYB123 causes the transformation of stamen to malformed petal in rose (Rosa hybrida).

KEY MESSAGE: We find that the R2R3 MYB transcription factor RhMYB123 has a novel function to regulate stamen-petal organ specification in rose. Rose is one of the ornamental plants with economic importance worldwide. Malformed flower seriously affects the ornamental value and fertility of rose. However, the regulatory mechanism is largely unknown. In this work, we identified a R2R3 MYB transcription factor RhMYB123 from rose, the expression of which significantly decreased from flower differentiation stage to floral organ development stage. Phylogenetic analysis indicated that it belongs to the same subgroup as MYB123 of Arabidopsis and located in nucleus. In addition, RhMYB123 was confirmed to have transcriptional activation function by dual luciferase assay. Silencing RhMYB123 using Virus-Induced Gene Silencing (VIGS) in rose could increase the number of malformed petaloid stamen. Furthermore, we identified 549 differential expressed genes (DEGs) in TRV-RhMYB123 lines compared to TRV controls by RNA-seq of floral buds (flower differentiation stage). Among of those genes, expression of 3 MADS box family genes related to floral organ development reduced in TRV-RhMYB123 lines, including AGAMOUS (RhAG), AGAMOUS LIKE 15 (RhAGL15), and SHATTERPROOF 1 (RhSHP1). Given, previous studies have shown that auxin plays a crucial role in floral meristem initiation and stamen-petal organ specification. We also found 6 DEGs were involved in auxin signal transduction, of which five were reduced expression in TRV-RhMYB123. Taken together, our findings suggested that RhMYB123 may govern the development of malformed petaloid stamen by regulating the expressions of some MADS box family members and auxin signaling pathway elements.

Small HSPs play an important role in crosstalk between HSF-HSP and ROS pathways in heat stress response through transcriptomic analysis in lilies (Lilium longiflorum).

BACKGROUND: High temperature seriously limits the annual production of fresh cut lilies, which is one of the four major cut flowers in the global cut flower market. There were few transcriptomes focused on the gene expression of lilies under heat stress. In order to reveal the potential heat response patterns in bulbous plants and provide important genes for further genetic engineering techniques to improve thermotolerance of lily, RNA sequencing of lilies under heat treatments were conducted. RESULTS: In this study, seedlings of Lilium longiflorum 'White Heaven' were heat-treated at 37 °C for different lengths of time (0 h, 0.5 h, 1 h, 3 h, 6 h, and 12 h with a 12 h-light/12 h-dark cycle). The leaves of these lily seedlings were immediately collected after heat treatments and quickly put into liquid nitrogen for RNA sequencing. 109,364,486-171,487,430 clean reads and 55,044 unigenes including 21,608 differentially expressed genes (DEGs) (fold change ≥2) were obtained after heat treatment. The number of DEGs increased sharply during the heat treatments of 0.5 h-1 h and 1 h-3 h compared to that of other periods. Genes of the heat stress transcription factor (HSF) family and the small heat shock proteins (small HSPs, also known as HSP20) family responded to heat stress early and quickly. Compared to that of the calcium signal and hormone pathways, DEGs of the HSF-HSP pathway and reactive oxygen species (ROS) pathway were significantly and highly induced. Moreover, they had the similar expression pattern in response to heat stress. Small HSPs family genes were the major components in the 50 most highly induced genes at each heat stress treatment and involved in ROS pathway in the rapid response to heat stress. Furthermore, the barley stripe mosaic virus induced gene silencing (BSMV-VIGS) of LlHsfA2 caused a significantly reduced thermotolerance phenotype in Lilium longiflorum 'White Heaven', meanwhile decreasing the expression of small HSPs family genes and increasing the ROS scavenging enzyme ascorbate peroxidase (APX) genes, indicating the potential interplay between these two pathways. CONCLUSIONS: Based on our transcriptomic analysis, we provide a new finding that small HSPs play important roles in crosstalk between HSF-HSP and ROS pathways in heat stress response of lily, which also supply the groundwork for understanding the mechanism of heat stress in bulbous plants.

The Heat Stress Transcription Factor LlHsfA4 Enhanced Basic Thermotolerance through Regulating ROS Metabolism in Lilies (Lilium Longiflorum).

Heat stress severely affects the annual agricultural production. Heat stress transcription factors (HSFs) represent a critical regulatory juncture in the heat stress response (HSR) of plants. The HsfA1-dependent pathway has been explored well, but the regulatory mechanism of the HsfA1-independent pathway is still under-investigated. In the present research, HsfA4, an important gene of the HsfA1-independent pathway, was isolated from lilies (Lilium longiflorum) using the RACE method, which encodes 435 amino acids. LlHsfA4 contains a typical domain of HSFs and belongs to the HSF A4 family, according to homology comparisons and phylogenetic analysis. LlHsfA4 was mainly expressed in leaves and was induced by heat stress and H2O2 using qRT-PCR and GUS staining in transgenic Arabidopsis. LlHsfA4 had transactivation activity and was located in the nucleus and cytoplasm through a yeast one hybrid system and through transient expression in lily protoplasts. Over expressing LlHsfA4 in Arabidopsis enhanced its basic thermotolerance, but acquired thermotolerance was not achieved. Further research found that heat stress could increase H2O2 content in lily leaves and reduced H2O2 accumulation in transgenic plants, which was consistent with the up-regulation of HSR downstream genes such as Heat stress proteins (HSPs), Galactinol synthase1 (GolS1), WRKY DNA binding protein 30 (WRKY30), Zinc finger of Arabidopsis thaliana 6 (ZAT6) and the ROS-scavenging enzyme Ascorbate peroxidase 2 (APX2). In conclusion, these results indicate that LlHsfA4 plays important roles in heat stress response through regulating the ROS metabolism in lilies.

A domesticated Harbinger transposase forms a complex with HDA6 and promotes histone H3 deacetylation at genes but not TEs in Arabidopsis.

In eukaryotes, histone acetylation is a major modification on histone N-terminal tails that is tightly connected to transcriptional activation. HDA6 is a histone deacetylase involved in the transcriptional regulation of genes and transposable elements (TEs) in Arabidopsis thaliana. HDA6 has been shown to participate in several complexes in plants, including a conserved SIN3 complex. Here, we uncover a novel protein complex containing HDA6, several Harbinger transposon-derived proteins (HHP1, SANT1, SANT2, SANT3, and SANT4), and MBD domain-containing proteins (MBD1, MBD2, and MBD4). We show that mutations of all four SANT genes in the sant-null mutant cause increased expression of the flowering repressors FLC, MAF4, and MAF5, resulting in a late flowering phenotype. Transcriptome deep sequencing reveals that while the SANT proteins and HDA6 regulate the expression of largely overlapping sets of genes, TE silencing is unaffected in sant-null mutants. Our global histone H3 acetylation profiling shows that SANT proteins and HDA6 modulate gene expression through deacetylation. Collectively, our findings suggest that Harbinger transposon-derived SANT domain-containing proteins are required for histone deacetylation and flowering time control in plants.

Inhibition of Acyl-CoA Synthetase Long-Chain Family Member 4 Facilitates Neurological Recovery After Stroke by Regulation Ferroptosis.

BACKGROUND: Ischemic stroke is the main cause of disability worldwide, leading to a serious socioeconomic burden. Ferroptosis is a non-apoptotic form of programmed cell death and is related to various diseases. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is considered a target of ferroptosis, but its specific role in ischemic stroke remains unclear. In this study, we investigate whether the inhibition of ACSL4 promotes the recovery of neurological function in a way that prevents ferroptosis. METHODS: A transient cerebral ischemia model was established for mice by middle cerebral artery occlusion (MCAO); glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) were detected by Western blot, and changes to mitochondria were observed by a transmission electron microscope. A kit was used to determine iron levels and lipid peroxide indicators, such as glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde. Following MCAO, a ferroptosis inhibitor, liproxstatin-1, was administered intranasally immediately at a concentration of 10 mg/kg. Rosiglitazone was used to inhibit ACSL4 and was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg. Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke. Brain infarct volume was determined by 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining at 72 h after stroke. RESULTS: After MCAO, GPx4 protein expression decreased, ACSL4 and COX2 protein expression increased, GPx activity decreased and iron accumulation. Transmission electron microscopy confirmed that the outer mitochondrial membrane of neurons had ruptured and mitochondrial cristae had decreased or disappeared. Liproxstatin-1 could significantly attenuate the decrease of GPx4 and the increase of COX2 after MCAO, dramatically reducing iron accumulation and decreasing GPx activity, accompanied by a marked reduction in changes in lipid peroxidation indicators. The use of rosiglitazone to inhibit ACSL4 could significantly improve neurological function and reduce the brain infarct volume at 72 h after stroke. Importantly, inhibiting ACSL4 could significantly attenuate the decline of GPx4 after MCAO and markedly attenuate iron accumulation and a decrease in GPx activity. Additionally, changes in lipid peroxidation indicators were also significantly inhibited. CONCLUSION: This study indicates that inhibiting ACSL4 can promote the recovery of neurological function after stroke by suppression of ferroptosis.

Neuroprotective effects of zonisamide on cerebral ischemia injury via inhibition of neuronal apoptosis.

It is known that neuronal apoptosis contributes to pathology of cerebral ischemia injury. Zonisamide (ZNS) has shown anti-apoptosis effects in recent studies. The present study investigated whether the anti-apoptotic effect can account for the neuroprotective action of ZNS on cerebral ischemia. Neuronal cells were maintained under oxygen-glucose deprivation conditions to simulate cerebral ischemia and treated with ZNS simultaneously. The apoptosis of the cells and expression of apoptosis-related proteins were investigated by flow cytometry and western blot analysis, respectively. A cerebral ischemia mouse model was created via middle cerebral artery occlusion, and the mice were treated with ZNS. Neurological deficit scores and infarct volumes of the cerebral ischemia mice were measured. The apoptosis status of the neuronal cells was evaluated by TUNEL staining. In vitro, the ZNS treatment inhibited both the apoptosis of the neuronal cells and apoptosis-related protein expression (caspase-3, caspase-8, and calpain-1) induced by the oxygen-glucose deprivation. The anti-apoptosis effect of ZNS could occur through the blocking of reactive oxygen species. Moreover, ZNS treatment significantly ameliorated neurological deficits and reduced infarct volumes in the cerebral ischemia mice model. In this study, ZNS exerted neuroprotective effects by inhibition of apoptosis in neuronal cells in cerebral ischemia. Therefore, ZNS might be a promising therapy for cerebral ischemia.

Neuroprotective effects of zonisamide on cerebral ischemia injury via inhibition of neuronal apoptosis

It is known that neuronal apoptosis contributes to pathology of cerebral ischemia injury. Zonisamide (ZNS) has shown anti-apoptosis effects in recent studies. The present study investigated whether the anti-apoptotic effect can account for the neuroprotective action of ZNS on cerebral ischemia. Neuronal cells were maintained under oxygen-glucose deprivation conditions to simulate cerebral ischemia and treated with ZNS simultaneously. The apoptosis of the cells and expression of apoptosis-related proteins were investigated by flow cytometry and western blot analysis, respectively. A cerebral ischemia mouse model was created via middle cerebral artery occlusion, and the mice were treated with ZNS. Neurological deficit scores and infarct volumes of the cerebral ischemia mice were measured. The apoptosis status of the neuronal cells was evaluated by TUNEL staining. In vitro, the ZNS treatment inhibited both the apoptosis of the neuronal cells and apoptosis-related protein expression (caspase-3, caspase-8, and calpain-1) induced by the oxygen-glucose deprivation. The anti-apoptosis effect of ZNS could occur through the blocking of reactive oxygen species. Moreover, ZNS treatment significantly ameliorated neurological deficits and reduced infarct volumes in the cerebral ischemia mice model. In this study, ZNS exerted neuroprotective effects by inhibition of apoptosis in neuronal cells in cerebral ischemia. Therefore, ZNS might be a promising therapy for cerebral ischemia.

miR-668 inhibitor attenuates mitochondrial membrane potential and protects against neuronal apoptosis in cerebral ischemic stroke.

Cerebral stroke is a major cause of brain injury due to the production of hypoxic conditions, and new therapeutic interventions are required for its management. Here, we evaluated the protective effect of miR-668 inhibitor against ischemia/reperfusion (I/R)-induced stroke. Cerebral stroke was induced by cerebral artery occlusion in rats, followed by treatment with miR-668 inhibitor for 10 minutes before reperfusion. The neuroprotective effect of miR-668 inhibitor was determined by estimating the neurological deficit score, cerebral infarct area and blood-brain barrier (BBB) permeability. In addition, the levels of inflammatory cytokines, and the expression of NLRP3, zonula occludens-1 (ZO-1), dynamin-related protein 1 (Drp1) and occludin proteins, were estimated by ELISA and Western blotting, respectively. TUNEL assay and immunohistochemical analyses were performed to examine the effects of miR-668 inhibitor against I/R-induced stroke rats. The miR-668 inhibitor treatment group showed reductions in the infarct area, BBB permeability and neurological score compared to the stroke group. The levels of cytokines and reactive oxygen species were reduced in the miR-668 inhibitor treatment group compared to the stroke group. These observations suggested that inhibition of miR-668 reduces neuronal apoptosis by ameliorating the expression of caspase 3, Bax and Bcl-2 protein in I/R stroke rats. The expression of NLRP3, ZO-1 and occludin proteins was attenuated in the brain tissue of the miR-668 inhibitor treatment group compared to the stroke group. Moreover, the phosphorylation of Drp1 protein was reduced in the miR-668 inhibitor group compared to the stroke group. In conclusion, the results of the present study indicated that inhibition of miR-668 prevented neuronal apoptosis in cerebral I/R-induced stroke by modulating mitochondrial function and regulating NLRP3 signalling.

The Arabidopsis Nodulin Homeobox Factor AtNDX Interacts with AtRING1A/B and Negatively Regulates Abscisic Acid Signaling.

The phytohormone abscisic acid (ABA) and the Polycomb group proteins have key roles in regulating plant growth and development; however, their interplay and underlying mechanisms are not fully understood. Here, we identified an Arabidopsis (Arabidopsis thaliana) nodulin homeobox (AtNDX) protein as a negative regulator in the ABA signaling pathway. AtNDX mutants are hypersensitive to ABA, as measured by inhibition of seed germination and root growth, and the expression of AtNDX is downregulated by ABA. AtNDX interacts with the Polycomb Repressive Complex1 (PRC1) core components AtRING1A and AtRING1B in vitro and in vivo, and together, they negatively regulate the expression levels of some ABA-responsive genes. We identified ABA-INSENSITIVE (ABI4) as a direct target of AtNDX. AtNDX directly binds the downstream region of ABI4 and deleting this region increases the ABA sensitivity of primary root growth. Furthermore, ABI4 mutations rescue the ABA-hypersensitive phenotypes of ndx mutants and ABI4-overexpressing plants are hypersensitive to ABA in primary root growth. Thus, our work reveals the critical functions of AtNDX and PRC1 in some ABA-mediated processes and their regulation of ABI4.

Effects of compound organic acid calcium on growth performance, hepatic antioxidation and intestinal barrier of male broilers under heat stress.

OBJECTIVE: The aim of this study was to evaluate the effects of compound organic acid calcium (COAC) on growth performance, hepatic antioxidant status and intestinal barrier of male broilers under high ambient temperature (32.7°C). METHODS: Nine hundred healthy one-d-old Cobb-500 male broiler chicks were randomly assigned into three groups with six replicates of 50 birds each. A basal diet supplemented with 0% (control), 0.4% and 0.8% COAC, respectively were fed to birds for 6 weeks. All treatments were under high ambient indoor temperature of 32.7°C, and had a constant calcium and available phosphorus ratio. RESULTS: The results showed that, compared with control, the average daily gain of broilers in 0.4% and 0.8% was significantly increased and the ratio of feed to gain in in 0.4% and 0.8% was significantly decreased at 1 to 21, 22 to 42 and 1 to 42 days of age (p<0.05). Compared with control, 0.8% COAC slightly decreased (p = 0.093) the content of malondialdehyde in liver at 42 days of age while 0.4% COAC significantly decreased (p<0.05) the activity of alkaline phosphatase. Furthermore, 0.4% COAC significantly enhanced the intestinal barrier function via increasing jejunal and ileal ocln transcription, promoting jejunal mucin 2 transcription at 42 days of age (p<0.05), and decreasing jejunal toll-like receptor 2 (TLR-2) and ileal TLR-15, inducible nitric oxide synthase compared with control group (p<0.05). Whereas, no significant differences on the transcription of interleukin-1ß in jejunum and ileum were observed among three treatments (p>0.05). Overall, heat stress caused by high natural environment temperature may induce the damage to hepatic antioxidation and intestinal barrier. CONCLUSION: Dietary inclusion of COAC can improve the tolerance of broilers to thermal environment through the modification of antioxidative parameters in liver and the mRNA expression of genes in intestinal barrier, resulting in an optimal inclusion level of 0.4%.

Low-dose of organic trace minerals reduced fecal mineral excretion without compromising performance of laying hens.

OBJECTIVE: The objective of this study was to investigate the effects of low doses of organic trace minerals (iron, copper, manganese, and zinc) on productive performance, egg quality, yolk and tissue mineral retention, and fecal mineral excretion of laying hens during the late laying period. METHODS: A total of 405 healthy hens (HY-Line White, 50-week-old) were randomly divided into 3 treatments, with 9 replicates per treatment and 15 birds per replicate. The dietary treatments included feeding a basal diet + inorganic trace minerals at commercial levels (CON), a basal diet + inorganic trace minerals at 1/3 commercial levels (ITM), and a basal diet + proteinated trace minerals at 1/3 commercial levels (TRT). The trial lasted for 56 days. RESULTS: Compared to CON, ITM decreased (p<0.05) egg production, daily egg mass, albumen height, eggshell strength, yolk Fe concentration, serum alkaline phosphatase activity and total protein, and increased (p<0.05) egg loss and feed to egg ratio. Whereas with productive performance, egg quality, yolk mineral retention, and serum indices there were no differences (p>0.05) between CON and TRT. The concentrations of Fe and Mn in the tissue and tibia were changed notably in ITM relative to CON and TRT. Both ITM and TRT reduced (p<0.05) fecal mineral excretion compared to CON. CONCLUSION: These results indicate that dietary supplementation of low-dose organic trace minerals reduced fecal mineral excretion without negatively impacting hen performance and egg quality.