Methylation of a MITE insertion in the MdRFNR1-1 promoter is positively associated with its allelic expression in apple in response to drought stress.

Miniature inverted-repeat transposable elements (MITEs) are widely distributed in the plant genome and can be methylated. However, whether DNA methylation of MITEs is associated with induced allelic expression and drought tolerance is unclear. Here, we identified the drought-inducible MdRFNR1 (root-type ferredoxin-NADP+ oxidoreductase) gene in apple (Malus domestica). MdRFNR1 plays a positive role in drought tolerance by regulating the redox system, including increasing NADP+ accumulation and catalase and peroxidase activities and decreasing NADPH levels. Sequence analysis identified a MITE insertion (MITE-MdRF1) in the promoter of MdRFNR1-1 but not the MdRFNR1-2 allele. MdRFNR1-1 but not MdRFNR1-2 expression was significantly induced by drought stress, which was positively associated with the MITE-MdRF1 insertion and its DNA methylation. The methylated MITE-MdRF1 is recognized by the transcriptional anti-silencing factors MdSUVH1 and MdSUVH3, which recruit the DNAJ domain-containing proteins MdDNAJ1, MdDNAJ2, and MdDNAJ5, thereby activating MdRFNR1-1 expression under drought stress. Finally, we showed that MdSUVH1 and MdDNAJ1 are positive regulators of drought tolerance. These findings illustrate the molecular roles of methylated MITE-MdRF1 (which is recognized by the MdSUVH-MdDNAJ complex) in induced MdRFNR1-1 expression as well as the drought response of apple and shed light on the molecular mechanisms of natural variation in perennial trees.

MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance.

Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole-3-acetic acid (IAA) conjugating enzyme, to be a negative regulator of water-deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water-deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water-deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water-deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water-deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water-deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.

The Apple Receptor-Like Kinase MdSRLK3 Positively Regulates Resistance Against Pathogenic Fungus Valsa mali by Affecting the Ca2+ Signaling Pathway.

Valsa mali is the main pathogenic fungus that causes the apple Valsa canker, a destructive disease severely threatening apple production in the world. However, the underlying key components involved in resistance against V. mali in apple trees remain largely unexplored. Here, we isolated and functionally characterized a G-type lectin S-receptor-like protein kinase MdSRLK3 from the cultivar Royal Gala derivative line GL-3. qRT-PCR showed that the relative expression of MdSRLK3 in apple branches reached its highest level at 24 h post V. mali inoculation, which was 13.42 times higher than without inoculation. Transient overexpression of MdSRLK3 enhanced apple resistance against V. mali, while transient silencing of MdSRLK3 reduced its resistance against the pathogen. More importantly, stable silencing of MdSRLK3 resulted in reduced resistance against this fungus. Furthermore, we demonstrated that MdSRLK3 positively regulated apple resistance by affecting the Ca2+ signaling pathway, and the regulation was also related to the H2O2 and callose signaling pathways. Overall, our data reveal that MdSRLK3 is a positive regulator of apple immunity.

Effects of Betulinic Acid Derivative on Lung Inflammation in a Mouse Model of Chronic Obstructive Pulmonary Disease Induced by Particulate Matter 2.5.

BACKGROUND Chronic obstructive pulmonary disease (COPD) is mainly induced by the increased content of particulate matter 2.5 (PM2.5) in the atmosphere. This study aimed to evaluate the effects of betulinic acid derivative on lung inflammation in a mouse model of chronic obstructive pulmonary disease induced by particulate matter 2.5. MATERIAL AND METHODS The mice were given a PM2.5 (25 µl) suspension for 7 days by the intranasal route to establish a COPD model. The content of TNF-alpha and IL-6 in the BALF samples was measured by commercially available ELISA kits. RESULTS The PM2.5-induced higher LDH and ACP levels were significantly alleviated in mouse lung tissues by treatment with betulinic acid derivative. Treatment with betulinic acid derivative also suppressed PM2.5-induced increase in AKP and ALB levels in mouse lung tissues. Betulinic acid derivative reversed PM2.5-mediated suppression of SOD activity and elevation of NOS level in mouse BALF. Moreover, the PM2.5-induced excessive NO and MDA levels in mouse BALF were significantly reduced (P.

Protective Effect of Colchicine on LPS-Induced Lung Injury in Rats via Inhibition of P-38, ERK1/2, and JNK Activation.

INTRODUCTION: Acute lung injury (ALI), a commonly detected syndrome, is characterized by the accumulation of neutrophils and leucocytes, and inflammation of pulmonary tissues. OBJECTIVE: The present study was designed to investigate the effect and underlying mechanism of colchicine on LPS-induced lung injury. METHODS: The rats were divided randomly into 6 groups of 10 each: normal control, untreated, and 4 colchicine (5, 10, 15, and 20 mg/kg) treatment groups. ALI was induced in rats by the administration of 20 µg LPS intratracheally. Rats in the normal control and untreated groups were injected normal saline, while those in the treatment groups received 5, 10, 15, and 20 mg/kg doses of colchicine daily for 1 month. ELISA was used for determination of interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)-α, superoxide dismutase (SOD), and leucocytes in the rat bronchoalveolar lavage fluid (BALF). The expression of P-38, JNK, and Erk-1/2 was analysed by Western blotting. RESULTS: In LPS-administered TC-1 cells, the levels of IL-1ß, IL-6, and TNF-α were markedly higher. Treatment with colchicine reduced the levels of IL-1ß, IL-6, and TNF-α in LPS-administered TC-1 cells. Colchicine treatment caused a marked reduction in LPS-induced accumulation of inflammatory cells in the rat lungs. The LPS-induced aggregation of leucocytes and neutrophils in the rat BALF was also suppressed markedly on treatment with colchicine. Treatment of the lung injury in rats with colchicine caused a marked decrease in the level of IL-1ß, IL-6, and TNF-α in BALF. The LPS-mediated suppression of SOD in the rat BALF was prevented by treatment with colchicine. Treatment of the rats with colchicine attenuated the LPS-induced activation of P-38, Erk1/2, and JNK in pulmonary tissues. CONCLUSION: In summary, colchicine treatment prevents LPS-induced lung damage in rats through targeting activation of P-38, ERK1/2, and JNK. Therefore, colchicine may be used for the development of treatment for ALI.

Transcriptomic and phosphoproteomic profiling and metabolite analyses reveal the mechanism of NaHCO3-induced organic acid secretion in grapevine roots.

BACKGROUND: Organic acid secretion is a widespread physiological response of plants to alkalinity. However, the characteristics and underlying mechanism of the alkali-induced secretion of organic acids are poorly understood. RESULTS: Oxalate was the main organic acid synthesized and secreted in grapevine (a hybrid of Vitis amurensis, V. berlandieri and V. riparia) roots, while acetate synthesis and malate secretion were also promoted under NaHCO3 stress. NaHCO3 stress enhanced the H+ efflux rate of grapevine roots, which is related to the plasma membrane H+-ATPase activity. Transcriptomic profiling revealed that carbohydrate metabolism was the most significantly altered biological process under NaHCO3 stress; a total of seven genes related to organic acid metabolism were significantly altered, including two phosphoenolpyruvate carboxylases and phosphoenolpyruvate carboxylase kinases. Additionally, the expression levels of five ATP-binding cassette transporters, particularly ATP-binding cassette B19, and two Al-activated malate transporter 2 s were substantially upregulated by NaHCO3 stress. Phosphoproteomic profiling demonstrated that the altered phosphoproteins were primarily related to binding, catalytic activity and transporter activity in the context of their molecular functions. The phosphorylation levels of phosphoenolpyruvate carboxylase 3, two plasma membrane H+-ATPases 4 and ATP-binding cassette B19 and pleiotropic drug resistance 12 were significantly increased. Additionally, the inhibition of ethylene synthesis and perception completely blocked NaHCO3-induced organic acid secretion, while the inhibition of indoleacetic acid synthesis reduced NaHCO3-induced organic acid secretion. CONCLUSIONS: Our results demonstrated that oxalate was the main organic acid produced under alkali stress and revealed the necessity of ethylene in mediating organic acid secretion. Additionally, we further identified several candidate genes and phosphoproteins responsible for organic acid metabolism and secretion.

Heterologous Overexpression of Apple MdKING1 Promotes Fruit Ripening in Tomato.

Fruit ripening is governed by a complex regulatory network, and ethylene plays an important role in this process. MdKING1 is a γ subunit of SNF1-related protein kinases (SnRKs), but the function was unclear. Here, we characterized the role of MdKING1 during fruit ripening, which can promote fruit ripening through the ethylene pathway. Our findings reveal that MdKING1 has higher expression in early-ripening cultivars than late-ripening during the early stage of apple fruit development, and its transcription level significantly increased during apple fruit ripening. Overexpression of MdKING1 (MdKING1 OE) in tomatoes could promote early ripening of fruits, with the increase in ethylene content and the loss of fruit firmness. Ethylene inhibitor treatment could delay the fruit ripening of both MdKING1 OE and WT fruits. However, MdKING1 OE fruits turned fruit ripe faster, with an increase in carotenoid content compared with WT. In addition, the expression of genes involved in ethylene biosynthesis (SlACO1, SlACS2, and SlACS4), carotenoid biosynthesis (SlPSY1 and SlGgpps2a), and fruit firmness regulation (SlPG2a, SlPL, and SlCEL2) was also increased in the fruits of MdKING1 OE plants. In conclusion, our results suggest that MdKING1 plays a key role in promoting tomato fruit ripening, thus providing a theoretical basis for apple fruit quality improvement by genetic engineering in the future.

Application of γ-aminobutyric acid (GABA) improves fruit quality and rootstock drought tolerance in apple.

GABA (γ-aminobutyric acid) plays a multifaceted role in plant growth, fruit quality, and tolerance to abiotic stresses. However, its physiological roles and mechanisms in the fruit quality and response to long-term drought stress in apple remain unelucidated. To investigate the effect of GABA on apple fruit quality and drought tolerance, we sprayed exogenous GABA on apple cultivar "Cripps Pink" and irrigated rootstock M.9-T337 with GABA, respectively. Results showed that exogenous GABA could effectively improve the fruit quality of "Cripps Pink", including increased sugar-to-acid ratio, flesh firmness, pericarp malleability, and GABA content, as well as reduced fruit acidity. In addition, pretreatment of M.9-T337 plants with GABA improved their tolerance to both long- and short-term drought stress. Specifically, 1 mM exogenous GABA increased the net photosynthetic rate, relative leaf water content, root-to-shoot ratio, and water use efficiency under long-term drought stress, and delayed the increased of the relative electrolyte leakage under short-term drought stress. RNA-seq analysis identified 1271 differentially expressed genes (DEGs) between nontreated and GABA-pretreated plants under short-term drought stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these DEGs revealed that GABA may enhance plant drought resistance by upregulating the expression of genes related to "Biosynthesis of secondary metabolites", "MAPK signaling pathway", "Glutathione metabolism", and "Carbon fixation in photosynthetic organisms". In conclusion, these results revealed that exogenous GABA can improve fruit quality and enhance drought tolerance in apple.

Insights into the molecular mechanisms underlying responses of apple trees to abiotic stresses.

Apple (Malus[Formula: see text]domestica) is a popular temperate fruit crop worldwide. However, its growth, productivity, and quality are often adversely affected by abiotic stresses such as drought, extreme temperature, and high salinity. Due to the long juvenile phase and highly heterozygous genome, the conventional breeding approaches for stress-tolerant cultivars are time-consuming and resource-intensive. These issues may be resolved by feasible molecular breeding techniques for apples, such as gene editing and marker-assisted selection. Therefore, it is necessary to acquire a more comprehensive comprehension of the molecular mechanisms underpinning apples' response to abiotic stress. In this review, we summarize the latest research progress in the molecular response of apples to abiotic stressors, including the gene expression regulation, protein modifications, and epigenetic modifications. We also provide updates on new approaches for improving apple abiotic stress tolerance, while discussing current challenges and future perspectives for apple molecular breeding.

[Expression changes of IL-1beta in lung of rats subjected to brain ischemia].

OBJECTIVE: To study the expression changes of interleukin-1beta (IL-1beta) in the lung of rats with brain ischemia. METHODS: Adult SD rats were divided into sham operation group and brain ischemia lung injury (BILI) group randomly. Focal cerebral ischemia inflammatory lung injury model was developed with intraluminal thread technique. Lungs were harvested from rats at different time point respectively. RT-PCR (24 h), Western blot (48 h) and immunohistochemistry (72 h) were employed to detect the expressional changes and the distributions of IL-1beta in the lung tissues. RESULTS: IL-1beta immunohistochemical positive reaction was observed in epithelia cell and neutrophil as well as macrophage. Increased protein level and mRNA expression for IL-1beta were found in lung after brain ischemia compared with those of sham group. CONCLUSION: IL-1beta, as a crucial inflammatory factor, could be associated with airway inflammation in lung following brain ischemia in rats.

Circ-PITX1 Promotes the Progression of Non-Small Cell Lung Cancer Through Regulating the miR-1248/CCND2 Axis.

BACKGROUND: Circular RNA (circRNA) is a key regulator of cancer, and it has been proved to be involved in the regulation of cancer progression including non-small cell lung cancer (NSCLC). Circ-PITX1 was found to be a significantly upregulated circRNA in NSCLC, and its role and potential mechanism in NSCLC progression deserve further investigation. METHODS: The expression levels of circ-PITX1, microRNA (miR)-1248 and cyclin D2 (CCND2) were examined by quantitative real-time PCR (qRT-PCR). Cell proliferation, apoptosis, cell cycle process, migration and invasion were determined using cell counting kit 8 (CCK8) assay, colony formation assay, flow cytometry, wound healing assay and transwell assay. Xenograft models were built to explore the role of circ-PITX1 in NSCLC tumor growth in vivo. The glycolysis and glutamine metabolism of cells were assessed by detecting the consumptions of glucose and glutamine, cell extracellular acidification rate (ECAR), and the productions of lactate, α-ketoglutaric acid (α-KG) and ATP. The protein levels of hexokinase 2 (HK-2), glutaminase 1 (GLS1) and CCND2 were tested by Western blot (WB) analysis. Dual-luciferase reporter assay and RIP assay were employed to verify the interaction between miR-1248 and circ-PITX1 or CCND2. RESULTS: Circ-PITX1 was upregulated in NSCLC and its silencing could inhibit the proliferation, migration, invasion, cell cycle process, glycolysis, glutamine metabolism, and promote the apoptosis of NSCLC cells in vitro, as well as reduced tumor growth in vivo. In the terms of mechanism, we found that circ-PITX1 could act as a sponge of miR-1248, and miR-1248 could target CCND2. In addition, miR-1248 inhibitor reversed the inhibitory effect of circ-PITX1 knockdown on NSCLC progression. Similarly, CCND2 overexpression also reversed the suppressive effect of miR-1248 on NSCLC progression. Moreover, circ-PITX1 positively regulated CCND2 expression by sponging miR-1248. CONCLUSION: Circ-PITX1 served as a sponge of miR-1248 to promote NSCLC progression by upregulating CCND2.

Melatonin promotes ripening of grape berry via increasing the levels of ABA, H2O2, and particularly ethylene.

The role of melatonin in the regulation of fruit ripening and the mechanism involved remain largely unknown. In "Moldova" grape berries, melatonin accumulated rapidly from onset of veraison, reached the maximum at 94 days after bloom (DAB) and then exhibited low levels at late stages of berry ripening. By contrast, abscisic acid (ABA) and hydrogen peroxide (H2O2) exhibited different accumulation patterns, and ethylene was primarily produced immediately before veraison. Further experiments demonstrated that 10 and particularly 100 µM melatonin treatments increased the levels of ABA, H2O2, and ethylene production and promoted berry ripening compared with the control treatment, whereas 0.1 and 1.0 µM melatonin did not lead to clear effects. Additionally, the application of inhibitors indicated that ABA, H2O2, and ethylene participated in the regulation of berry ripening induced by melatonin, and the suppression of ethylene biosynthesis produced the greatest inhibitory effects on melatonin-induced berry ripening compared with those of ABA and H2O2. Melatonin also promoted ethylene production via ABA. In summary, 10 and particularly 100 µM melatonin treatments promoted berry ripening, which was accomplished, at least partially, via the other signaling molecules of ABA, H2O2, and particularly ethylene. This research provides insight into melatonin signaling during berry ripening and may advance the application of melatonin to accelerate berry ripening.

Characterization of Gene Expression Profile, Phenolic Composition, and Antioxidant Capacity in Red-Fleshed Grape Berries and Their Wines.

Gene expression profile, phenolic composition, and antioxidant capacity were evaluated in red-fleshed berries and their wines (RF berries and wines) from new grape genotypes. Transcriptomic analysis revealed that ten metabolic pathways involved in polyphenol synthesis and catabolism were significantly altered, and 13 genes related to the biosynthesis and transport of phenolics were largely upregulated in RF berries compared to that of Cabernet Sauvignon (CS). Expression of MybA1 was associated with anthocyanin accumulation in red flesh. Additionally, RF berries and wines contained higher concentrations of total anthocyanins, phenols, flavonoids, and proanthocyanidins than those in CS berries and wine. Particularly, diglucosides of malvidin, peonidin, delphinidin, and cyanidin were present in red flesh and RF wines, but they were undetectable or present at very low concentrations in CS flesh and wine. Cinnamic acid and ferulic acid were clearly increased in the RF wines compared to those in the CS wine. Additionally, the RF wines had higher antioxidant capacity than that in the CS wine, and total anthocyanin content was significantly correlated to antioxidant capacity. This research provides insight into the mechanisms underlying grape flesh coloration and the composition of phenolic compounds in RF berries and wines.

VvVHP1; 2 Is Transcriptionally Activated by VvMYBA1 and Promotes Anthocyanin Accumulation of Grape Berry Skins via Glucose Signal.

In this work, four vacuolar H+-PPase (VHP) genes were identified in the grape genome. Among them, VvVHP1; 2 was strongly expressed in berry skin and its expression exhibited high correlations to anthocyanin content of berry skin during berry ripening and under ABA and UVB treatments. VvVHP1; 2 was transcriptionally activated directly by VvMYBA1, and VvVHP1; 2 overexpression promoted anthocyanin accumulation in berry skins and Arabidopsis leaves; therefore, VvVHP1; 2 mediated VvMYBA1-regulated berry pigmentation. On the other hand, RNA-Seq analysis of WT and transgenic berry skins revealed that carbohydrate metabolism, flavonoid metabolism and regulation and solute carrier family expression were the most clearly altered biological processes. Further experiments elucidated that VvVHP1; 2 overexpression up-regulated the expression of the genes related to anthocyanin biosynthesis and transport via hexokinase-mediated glucose signal and thereby promoted anthocyanin accumulation in berry skins and Arabidopsis leaves. Additionally, modifications of sugar status caused by enhanced hexokinase activities likely play a key role in VvVHP1; 2-induced sugar signaling.

Melatonin Enhances Phenolics Accumulation Partially via Ethylene Signaling and Resulted in High Antioxidant Capacity in Grape Berries.

This study assessed the primary impacts of exogenous melatonin (MT) treatment on grape berry metabolism. Exogenous MT treatment increased the endogenous MT content and modified berry ripening. Transcriptomic analysis revealed that the processes of polyphenol metabolism, carbohydrate metabolism and ethylene biosynthesis and signaling were the three most significantly altered biological processes upon MT treatment. Further experiments verified that MT treatment increased the contents of total anthocyanins, phenols, flavonoids and proanthocyanidins in berries. Additionally, the contents of 18 of the 22 detected individual phenolic compounds were enhanced by MT treatment; particularly, the resveratrol content was largely increased concomitantly with the up-regulation of STS gene expression. Meanwhile, MT treatment enhanced the antioxidant capacity of berries. On the other hand, it was indicated that ethylene participated in the regulation of polyphenol metabolism and antioxidant capacity under MT treatment in grape berries. In summary, MT enhances the polyphenol content and antioxidant capacity of grape berries partially via ethylene signaling.