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Abscisic acid (ABA) regulates plant development mainly through its signaling, in which ABA binds to receptors to inhibit type 2C protein phosphatases (PP2Cs). The exact roles of PP2Cs in fruit development are still unclear. In this work, we verify that tomato SlPP2C5 works as a negative regulator in ABA signaling during fruit development. SlPP2C5 was inhibited by both monomeric and dimeric ABA receptors SlPYLs through ABA dose–dependent way, and it interacted physically with SlPYLs and SlSnRK2s. SlPP2C5 was highly expressed in fruits induced by exogenous ABA. Plants with overexpressed SlPP2C5 had lower sensitivity to ABA, which showed faster seed germination and primary root growth compared to Wild type (WT), while SlPP2C5-suppressed plants were more sensitive to ABA. SlPP2C5-over-expression (OE) delayed fruit ripening onset, while SlPP2C5-RNAi advanced fruit ripening. Alteration of SlPP2C5 expression impacts fruit quality parameters as well, including pericarp thickness, fruit shape index, seed number and weight and the soluble solid content. RNA-seq analysis revealed that there were significant expression differences of genes related to ethylene release and lycopene synthesis between WT and both SlPP2C5-OE and SlPP2C5-RNAi lines with an inversed variation. Taken together, our findings demonstrate that SlPP2C5 plays an important role in the regulation of fruit development, ripening and quality.

Abscisic acid (ABA) plays a vital role in coordinating physiological processes during fresh fruit ripening. Binding of ABA to receptors facilitates the interaction and inhibition of type 2C phosphatase (PP2C) co-receptors. However, the exact mechanism of PP2C during fruit ripening is unclear. In this study, we determined the role of the tomato ABA co-receptor type 2C phosphatase SlPP2C3, a negative regulator of ABA signaling and fruit ripening. SlPP2C3 selectively interacted with monomeric ABA receptors and SlSnRK2.8 kinase in both yeast and tobacco epidermal cells. Expression of SlPP2C3 was ABA-inducible, which was negatively correlated with fruit ripening. Tomato plants with suppressed SlPP2C3 expression exhibited enhanced sensitivity to ABA, while plants overexpressing SlPP2C3 were less sensitive to ABA. Importantly, lack of SlPP2C3 expression accelerated the onset of fruit ripening and affected fruit glossiness by altering the outer epidermis structure. There was a significant difference in the expression of cuticle-related genes in the pericarp between wild-type and SlPP2C3-suppressed lines based on RNA sequencing (RNA-seq) analysis. Taken together, our findings demonstrate that SlPP2C3 plays an important role in the regulation of fruit ripening and fruit glossiness in tomato.

Abscisic acid (ABA) regulates fruit ripening, yet little is known about the exact roles of ABA receptors in fruit. In this study, we reveal the role of SlPYL9, a tomato pyrabactin resistance (PYR)/pyrobactin resistance-like (PYL)/regulatory component of ABA receptors (RCAR) protein, as a positive regulator of ABA signaling and fruit ripening. SlPYL9 inhibits protein phosphatase-type 2C (PP2C2/6) in an ABA dose-dependent way, and it interacts physically with SlPP2C2/3/4/5 in an ABA-dependent manner. Expression of SlPYL9 was observed in the seeds, flowers, and fruits. Overexpression and suppression of SlPYL9 induced a variety of phenotypes via altered expression of ABA signaling genes (SlPP2C1/2/9, SlSnRK2.8, SlABF2), thereby affecting expression of ripening-related genes involved in ethylene release and cell wall modification. SlPYL9-OE/RNAi plants showed a typical ABA hyper-/hypo-sensitive phenotype in terms of seed germination, primary root growth, and response to drought. Fruit ripening was significantly accelerated in SlPYL9-OE by 5–7 d as a result of increased endogenous ABA accumulation and advanced release of ethylene compared with the wild-type. In the SlPYL9-RNAi lines, fruit ripening was delayed, mesocarp thickness was enhanced, and petal abscission was delayed compared with the wild-type, resulting in conical/oblong and gourd-shaped fruits. These results suggest that SlPYL9 is involved in ABA signaling, thereby playing a role in the regulation of flower abscission and fruit ripening in tomato., ABA receptor PYL9 plays a role in the regulation of flower abscission and fruit ripening in tomato by modulating ABA signaling components, which regulates expression of related genes.

Objective. To explore the mechanisms of ursolic acid for treating colon cancer based on network pharmacology. Method. In this study, the potential targets of ursolic acid against colon cancer were predicted and screened through the TCMSP, SYMMAP, Drug Bank, UNI-PROT, and DISGENET databases. The protein interaction (PPI) network was constructed based on the STRING database, and graphs were drawn with the help of Cytoscape software. GO and KEGG enrichment analyses were performed on the targets by using the DAVID database for biological information annotation. Results. Ursolic acid has 113 targets in the treatment of colon cancer. The core targets included interleukin-6 (IL-6), mitogen-activated protein kinase 3 (MAPK3), vascular endothelial growth factor receptor (VEGFA), prostaglandin endoperoxide synthase 2 (PTGS2), caspase-3 (CASP3), mitogen-activated protein kinase 8 (MAPK8), tumor necrosis factor (TNF), cyclin D1 (CCND1), JUN, signal transducer and transcriptional activator 3 (STAT3), and other targets. The first 10 pathways related to colon cancer were screened out. The main signaling pathways included the TNF signaling pathway and the AGE-RAGE signaling pathway in diabetic complications and human colon cancer infections. Conclusion. This study revealed that ursolic acid played a multitarget and multichannel antitumor role by inhibiting the proliferation of tumor cells, inducing apoptosis, and enhancing antiangiogenesis.

Abscisic acid (ABA) plays a vital role in coordinating physiological processes during fresh fruit ripening. ABA can bind to ABA receptors which interacts and inhibits their co-receptors type 2C phosphatases (PP2Cs). However, the dissected mechanism of PP2C during fruit ripening is unclear. In this study, we identify the role of SlPP2C3, a tomato type 2C phosphatase, as a negative regulator of ABA signaling and fruit ripening. SlPP2C3 selectively interacted with monomeric ABA receptors and SlSnRK2.8 kinase in both yeast and tobacco epidermal cells. Expressions of SlPP2C3 were observed in all tissues, and it negatively correlated with the fruit ripening which was induced by exogenous ABA. Tomato plants with suppressed SlPP2C3 expression exhibited enhanced sensitivity to ABA, while SlPP2C3 over-expressed plants were less sensitive to ABA. Meaningfully, lack of SlPP2C3 expression causes the acceleration of fruit ripening onset via the alternation of ABA signaling activity, and the fruit gloss is affected by the changes of outer epidermis structure. RNA-seq analysis found significant different expression of cuticle-related genes in pericarp between wild-type and SlPP2C3 suppressed lines. Taken together, our finding demonstrate that SlPP2C3 plays an important role in the regulation of fruit ripening and fruit appearance quality in tomato.

Context: 1 D is a novel derivative of curcumin and shows very promising antitumor activities in various cancer cell lines. Objective: To characterize its preclinical pharmacokinetic profiles, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of 1 D in rat plasma. Materials and methods: An aliquot of 50 μL plasma sample was processed by protein precipitation with methanol. Chromatographic separation was accomplished on a Zorbax Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 μm) with a gradient elution system (water/0.1% formic acid and methanol). Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. The optimized fragmentation transition for 1 D was m/z 491.2 → 361.2. Results: The method was linear over the concentration range of 5–1000 ng/mL. The intra- and inter-day precisions were less than 9.8% and the accuracy was within ± 14.5%. The mean recovery of 1 D ranged from 102.5 to 105.9%. No matrix effects and significant sample loss during sample processing were observed. The validated method has been successfully applied to a pharmacokinetic study in rats after intravenous administration of 1 D. Non-compartmental pharmacokinetic parameters, including half-life (t1/2), apparent volume of distribution (Vz), clearance (CLz), and area under the concentration-time curve (AUC(0–t)) were 4.92 h, 46.56 L/kg, 6.33 L/h/kg, and 806.70 μg/L/h, respectively. Discussion and conclusions: Results demonstrated that 1 D displayed favourable pharmacokinetic properties for further in vivo pharmacologic evaluation, which could be facilitated by the validated LC-MS/MS method.

Abscisic acid (ABA) regulates plant growth and development, but the role of ABA in the development of reproductive organs in tomato has rarely been addressed. In the present study, the role of ABA in the regulation of male and female gametogenesis as well as pollen development and germination is tested in tomato. qRT-PCR and in situ hybridization analysis of 9-cis-epoxycarotenoid dioxygenase (SlNCED1), a key enzyme in the ABA biosynthetic pathway, showed high expression of SlNCED1 primarily in the meristem during gametogenesis and mainly in ovule, stigma, anther/pollen and vascular tissues during floral organ development. SlNCED1 expression and ABA accumulation in anther peak at stages 13-14, suggesting that ABA plays a role in the primary formation of pollen grains. Over expression and suppression of SlNCED1 led to the abnormal development of anther/pollen, especially in SlNCED1-OE lines, which have serious pollen deterioration. The percentage of pollen germination in wild type is 91.47%, whereas it is 6.85% in OE transgenic lines and 38.4% at anthesis in RNAi lines. RNA-Seq of anthers shows that SlNCED1-OE can significantly enhance the expression of SlPP2Cs and down-regulate the expression of SlMYB108 and SlMYB21, which are anther/flower-specific transcriptional factors in tomato. Finally, anther transcriptome data indicate that SlNCED1 is involved in ABA-mediated regulation in pollen/anther metabolism, cell wall modification, and transcription levels. These results support an important role for ABA in the development of reproductive organs in tomato and contribute to the elucidation of the underlying regulatory mechanisms.

Abscisic acid plays a crucial role in the regulation of fruit development and ripening, however, its role in the floral development and the fruit set is still unclear. In the present study, the ABA accumulation and the expression patterns of genes related to ABA metabolism and signalling in sweet cherry were investigated. The results showed that ABA accumulation increased and peaked at stage V in ovary, at stage VI in stamen, and in young fruit it peaked at 7 days after full bloom. The expression pattern of ABA synthetase PaNCED1 was consistent with the changes of ABA accumulation. Among four ABA degradation enzymes PaCYP707As, PaCYP707A4 was highly expressed in ovary, PaCYP707A1 was mainly in stamen, and PaCYP707A2 was in young fruit, and their expressions were reversed to the trend of PaNCED1. With regard to ABA signalling genes, among three ABA receptors PaPYLs, PaPYL2 and PaPYL3 were high expression genes in ovary and in young fruit with similar expression patterns, while PaPYL3 was the high expression gene in stamen. Within six PaPP2Cs, PaPP2C1/2/3 were highly expressed in ovary and young fruit, while PaPP2C3/4 were mainly in stamen. The six PaSnRK2s showed different expression patterns: PaSnRK2.1/2.2/2.4 were highly expressed in ovary and young fruit, while PaSnRK2.1/2.3 were highly expressed in stamen. In situ hybridization results showed that PaPYL3, PaPP2C3 and PaSnRK2.4 were expressed in seed, pulp and fruit peel during fruit set. In conclusion, ABA and its signaling may play an important role in the regulation of floral development and fruit set.

The VlMYBA subfamily of transcription factors has been known to be the functional regulators in anthocyanin biosynthesis in red grapes. In this study, the expressions of the VlMYBA1-2 and VlMYBA 2 genes, and the responses of the VlMYBA1-2/2 promoters to ABA and ACC treatments in Kyoho grape berries are examined through quantitative real-time PCR analysis and the transient expression assay. The results show that the expressions of VlMYBA1-2/2 increase dramatically after véraison and reach their highest levels when the berries are nearly fully ripe. Exogenous ABA promotes the expressions of VlMYBA1-2/2, whereas the ACC treatment increases the expression of VlMYBA2, however, it has no effect on VlMYBA1-2. The ABA treatment has a faster and stronger effect on berry pigmentation than ACC does. The VlMYBA1-2 promoter sequence contains two ABA response elements (ABRE) but no ethylene response element (ERE), whereas the VlMYBA2 promoter sequence contains two ABRE and one ERE in the upstream region of the start codon. The VlMYBA2 promoter can be activated by both ABA (more effective) and ACC, whereas the VlMYBA1-2 promoter can be activated by ABA only. In sum, ABA can promote the coloring of Kyoho grape by the promotion of VlMYBA1-2/2 transcriptions via activating the response of their promoters to ABA, whereas ethylene only regulates VlMYBA2 through the response activation of its promoter to ACC which partially enhances the coloring.

Abscisic acid (ABA) is an important regulator of many plant developmental processes, although its regulation in the pistil during anthesis is unclear. We investigated the role of 9-cis-epoxycarotenoid dioxygenase (SlNCED1), a key ABA biosynthesis enzyme, through overexpression and transcriptome analysis in the tomato pistil. During pistil development, ABA accumulates and SlNCED1 expression increases continually, peaking one day before full bloom, when the maximum amount of ethylene is released in the pistil. ABA accumulation and SlNCED1 expression in the ovary remained high for three days before and after full bloom, but then both declined rapidly four days after full bloom following senescence and petal abscission and expansion of the young fruits. Overexpression of SlNCED1 significantly increased ABA levels and also up-regulated SlPP2C5 expression, which reduced ABA signaling activity. Overexpression of SlNCED1 caused up-regulation of pistil-specific Zinc finger transcription factor genes SlC3H29, SlC3H66, and SlC3HC4, which may have affected the expression of SlNCED1-mediated pistil development-related genes, causing major changes in ovary development. Increased ABA levels are due to SlNCED1 overexpresson which caused a hormonal imbalance resulting in the growth of parthenocarpic fruit. Our results indicate that SlNCED1 plays a crucial role in the regulation of ovary/pistil development and fruit set.

β-Glucosidases (BG) are present in many plant tissues. Among these, abscisic acid (ABA) β-glucosidases are thought to take part in the adjustment of cellular ABA levels, however the role of ABA-BG in fruits is still unclear. In this study, through RNA-seq analysis of persimmon fruit, 10 full-length DkBG genes were isolated and were all found to be expressed. In particular, DkBG1 was highly expressed in persimmon fruits with a maximum expression 95 days after full bloom (DAFD). We verified that, in vitro, DkBG1 protein can hydrolyze ABA-glucose ester (ABA-GE) to release free ABA. Compared with wild-type, tomato plants that overexpressed DkBG1 significantly upregulated the expression of ABA receptor PYL3/7 genes and showed typical symptoms of ABA hypersensitivity in fruits. DkBG1 overexpression (DkBG1-OE) accelerated fruit ripening onset by 3-4 days by increasing ABA levels at the pre-breaker stage and induced early ethylene release compared with wild-type fruits. DkBG1-OE altered the expression of ripening regulator NON-RIPENING (NOR) and its target genes; this in turn altered fruit quality traits such as coloration. Our results demonstrated that DkBG1 plays an important role in fruit ripening and quality by adjusting ABA levels via hydrolysis of ABA-GE.

Squash is a non-climacteric fruit, so the release of ethylene plays a limited role in the ripening process; however, there is a large accumulation of abscisic acid (ABA) during fruit maturation and ripening. To investigate the contribution of ABA in squash fruit development, the CpNCED1 gene, which encodes the key enzyme in ABA biosynthesis (9-cis-epoxycarotenoid dioxygenase [NCED]), was cloned from squash fruit, and its transcriptional regulation during fruit development, dehydration and pollination was analysed using a quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of CpNCED1 peaked 20 days after full bloom (DAFB) in the pulp and seed, and 25 DAFB in the peel, all of which corresponded with the accumulation of ABA. The application of exogenous ABA increased the expression of CpNCED1 and the release of ethylene, thereby promoting fruit ripening. The expression of CpNCED1 was also induced by dehydration stress and pollination. These findings show that ABA pl...

Studying and understanding the mechanism of inflammation in nucleus pulposus is the key to understand and prevent intervertebral disc degeneration. We propose a model of mechanical sensitive ion channel Piezo1 mediated inflammation of nucleus pulposus cells. Piezo1 can up-regulate the level of interleukin-1β (IL-1β) in nucleus pulposus cells once it is activated. It is suggested that Piezo1 may mediate inflammation by activating Nod-like receptor protein 3 (NLRP3) inflammasome to accelerate the production and maturation of IL-1β. The primary objective of this study was to explore whether Piezo1 activates NLRP3 inflammasome in nucleus pulposus cells. Piezo1 sensitization was induced by mechanical stretch following which we analyzed the priming and assembly of NLRP3 inflammasome and also studied if the downstream Ca2+/NF-κB pathway mediated this activation in nucleus pulposus cells. The expression of Piezo1 and NLRP3 inflammasome increased in a time-dependent manner upon mechanical stretch. Piezo1 activation promoted NLRP3 inflammasome assembly, which was demonstrated by the upregulation of caspase-1 activation and IL-1β production. Transfection of Piezo1-siRNA reversed this process. The inhibition of Ca2+/NF-κB pathway reduced Piezo1-dependent activation of NLRP3 inflammasome. Thus, we propose that activation of NLRP3 inflammasome in nucleus pulposus cells mediated by Piezo1 through the Ca2+/NF-κB pathway is a novel pathogenesis for the progress of intervertebral disc degeneration. As per our knowledge this is the first study which has provided evidence linking Piezo1-mediated inflammation in nucleus pulposus cells with the production of NLRP3 inflammasome.

Highlight PacCYP707A2 plays a primary role in regulating ABA levels during the onset of cherry fruit ripening, while PacCYP707A1 regulates the ABA content in response to dehydration., Sweet cherry is a non-climacteric fruit and its ripening is regulated by abscisic acid (ABA) during fruit development. In this study, four cDNAs (PacCYP707A1–4) encoding 8′-hydroxylase, a key enzyme in the oxidative catabolism of ABA, were identified in sweet cherry fruits using tobacco rattle virus-induced gene silencing (VIGS) and particle bombardment approaches. Quantitative real-time PCR confirmed significant down-regulation of target gene transcripts in VIGS-treated cherry fruits. In PacCYP707A2-RNAi-treated fruits, ripening and fruit colouring were promoted relative to control fruits, and both ABA accumulation and PacNCED1 transcript levels were up-regulated by 140%. Silencing of PacCYP707A2 by VIGS significantly altered the transcripts of both ABA-responsive and ripening-related genes, including the ABA metabolism-associated genes NCED and CYP707A, the anthocyanin synthesis genes PacCHS, PacCHI, PacF3H, PacDFR, PacANS, and PacUFGT, the ethylene biosynthesis gene PacACO1, and the transcription factor PacMYBA. The promoter of PacMYBA responded more strongly to PacCYP707A2-RNAi-treated fruits than to PacCYP707A1-RNAi-treated fruits. By contrast, silencing of PacCYP707A1 stimulated a slight increase in fruit colouring and enhanced resistance to dehydration stress compared with control fruits. These results suggest that PacCYP707A2 is a key regulator of ABA catabolism that functions as a negative regulator of fruit ripening, while PacCYP707A1 regulates ABA content in response to dehydration during fruit development.

The changes in endogenous abscisic acid (ABA) content during ‘Hanagosho’ persimmon (Diospyros kaki Thunb.) development and maturation, as well as the effects of ABA, nordihydroguaiaretic acid (NDGA), 1-methylcyclopropene (1-MCP) application and polyethylene film package treatment on detached young fruit during fruit softening and senescence were investigated. The results showed that the ABA content in both peel and pulp peaked at 30 and 140 days after full bloom (DAFB). Moreover, the ABA content in peel at 30 and 140 DAFB was 17 and 29 times higher than in pulp, respectively. Dehydration in detached fruits induced the production of ABA and ethylene, which further led to fruit softening, and during this process ABA peaked earlier than ethylene. Expressions of the ethylene biosynthesis gene DkACS2 and ethylene oxidase gene DkACO1 were increased by the ABA treatment and their expression peaks also appeared earlier. However, expressions of DkACS2 and DkACO1 were down-regulated by the application of NDGA and 1...

The Piezo1 protein ion channel is a novel mechanical stretch-activated ion channel (SAC) closely related to mechanical signals. Mechanotransduction plays a crucial role in organ development and homeostasis. Previous studies identified Piezo1 and demonstrated that it is distinct from other ion channels with well-established roles in lower organisms. Mechanical stretch-activated ion channels from other organisms are not conserved in mammals or do not act as mechanically activated channels in mammals. In the current study, we explored the role of the Piezo1 ion channel in human nucleus pulposus cell (NP cell) apoptosis through mechanical force-induced mitochondrial dysfunction and endoplasmic reticulum stress. Reverse Transcription Polymerase chain reaction (RT-PCR), immunofluorescence, immunohistochemistry and Annexin V binding and propidium iodide analyses revealed that the Piezo1 protein ion channel was highly expressed in human NP cells, which are the primary cells that comprise the intervertebral disc. In patients with intervertebral disc degeneration (IVDD), the Piezo1 protein may play a crucial role in human NP cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress under abnormal loading conditions. This study also verified that human NP cells have an intimate connection with the cytoskeleton upon treatment of the cells with the Piezo1 blocking peptide GsMTx4 from tarantula venom. In summary, Piezo1 functions in human NP cell apoptosis, which may be one underlying mechanism of apoptosis induced by abnormal loading in IVDD patients.

Although ABA signaling has been widely studied in Arabidopsis, the roles of core ABA signaling components in fruit remain poorly understood. Herein, we characterize SlPP2C1, a group A type 2C protein phosphatase that negatively regulates ABA signaling and fruit ripening in tomato. The SlPP2C1 protein was localized in the cytoplasm close to AtAHG3/AtPP2CA. The SlPP2C1 gene was expressed in all tomato tissues throughout development, particularly in flowers and fruits, and it was up-regulated by dehydration and ABA treatment. SlPP2C1 expression in fruits was increased at 30 d after full bloom and peaked at the B + 1 stage. Suppression of SlPP2C1 expression significantly accelerated fruit ripening which was associated with higher levels of ABA signaling genes that are reported to alter the expression of fruit ripening genes involved in ethylene release and cell wall catabolism. SlPP2C1-RNAi (RNA interference) led to increased endogenous ABA accumulation and advanced release of ethylene in transgenic fruits compared with wild-type (WT) fruits. SlPP2C1-RNAi also resulted in abnormal flowers and obstructed the normal abscission of pedicels. SlPP2C1-RNAi plants were hypersensitized to ABA, and displayed delayed seed germination and primary root growth, and increased resistance to drought stress compared with WT plants. These results demonstrated that SlPP2C1 is a functional component in the ABA signaling pathway which participates in fruit ripening, ABA responses and drought tolerance.

To further understand the basis of abscisic acid (ABA) signal transduction in regulating sweet cherry fruit development and ripening, and also the interaction between ABA and indole acetic acid (IAA) at the onset of fruit ripening, full-length or partial cDNA of three PaPYLs (ABA receptor), six PaPP2Cs (type 2C protein phosphatase) and six PaSnRK2s (subfamily 2 of SNF1-related kinases) were identified from sweet cherry. Multiple alignments and phylogeny analyses showed that most of the functional residues or domains were well conserved within each gene family; all PaPP2Cs belonged to the group A PP2C; PaSnRK2.4 and PaSnRK2.5 belonged to subclass III of SnRK2. PaPYL2/3, PaPP2C3/4/6 and PaSnRK2.4 were highly expressed at the early stages of fruit development. Moreover, PaPYL2, PaPP2C3/4, PaSnRK2.4 also had a high expression level at the onset of fruit ripening. Exogenous ABA treatment at 28 days after full bloom decreased the IAA level and promoted fruit ripening by increasing anthocyanin and soluble solids content. However, although IAA treatment induced the ABA accumulation, it delayed the onset of fruit ripening. Most of the PaPYLs and PaSnRK2s didn’t strongly respond to ABA and IAA treatments at 28 days after full bloom, while expressions of PaPP2C3, PaPP2C5 and PaPP2C6 were significantly induced by exogenous ABA and PaPYL1 was significantly induced by exogenous IAA.

Abscisic acid (ABA) is an important phytohormone that regulates lots of physiological and biochemical processes in plant life cycle, especially in seed germination and stress responses. For exploring the transcriptional regulation of ABA signal transduction during cucumber (Cucumis sativus L.) seed germination and under Cu2+, Zn2+, NaCl and simulated acid rain stresses, nine CsPYLs, three group A CsPP2Cs and two subclass III CsSnRK2s were identified from cucumber genome, which respectively showed high sequence similarities and highly conserved domains with homologous genes in Arabidopsis. Based on Real-time PCR analysis, most of the tested genes' expression decreased during cucumber seed germination, which was in accordance with the ABA level variation. In addition, according to the absolute expression, CsPYL1, CsPYL3, CsPP2C5, CsABI1, CsSnRK2.3 and CsSnRK2.4 were highly expressed, indicating that they may play more important roles in ABA signaling during cucumber seed germination. Moreover, most of these highly expressed genes, except CsPYL3, were up-regulated by ABA treatment. Meanwhile, most of the tested genes' expression dramatically changed at the initial water uptake phase, indicating that this period may be critical in the regulation of ABA on seed germination. Under Cu2+, Zn2+, NaCl and simulated acid rain stresses, cucumber seed germination percentage decreased and ABA content increased. Meanwhile, the expression of ABA signal transduction core components genes showed specific response to a particular stress and was not always consist with ABA variation. Generally, the expression of CsPYL1, CsPYL3, CsABI1, CsSnRK2.3 and CsSnRK2.4 was sensitive to 120 mM NaCl and 0.5 mM Cu2+ treatments.

SummaryChanges in the severity of pulp browning, endogenous abscisic acid (ABA) and ethylene concentrations, and related gene expression in peach (Prunus persica L. ‘Bayuecui’) fruit stored at the biological freezing-point temperature (BFT; 1oC), at 4oC, or at room temperature (20oC; as a control) were investigated.The results showed that fruit stored at room temperature showed climacteric changes in ethylene and ABA concentrations and in levels of expression of the corresponding ethylene synthetase genes (PpACS1, PpACO1) and the gene for a key enzyme of ABA synthesis (PpNCED), concomitant with a significant decrease in fruit firmness and an increase in juice yield. Meanwhile, two sucrose synthase genes (PpSUS1 and PpSUS2) were expressed at relatively low levels. However, fruit firmness decreased slightly and chilling injury (CI) increased significantly 40 d after storage (DAS) at 4oC, with a significant accumulation of proline [from 8.33 µg g–1 (at t = 0 d) to 9.125 µg g–1 (at t = 40 d)]. In contrast, fr...

The purpose of this study was to investigate the role of abscisic acid (ABA) in the pre- and postharvest maturation of the nonclimacteric sweet cherry. It was found that ABA content increased rapidly at the straw-colored stage and reached its highest level 4 days before commercial harvest time. The increase in ABA level was paralleled by an increase in the expression of a gene involved in ABA synthesis [9-cis-epoxycarotenoid dioxygenase (PacNCED1)]. Well before the ABA increase, the expression of a gene involved in ABA catabolism (PacCYP707A2) was downregulated. During the straw-colored stage, the application of exogenous ABA induced ABA accumulation, anthocyanin biosynthesis, and an increase in the maturity index (SSC/TA), thereby promoting fruit ripening. ABA treatment stimulated the expression of PacNCED1 and of genes involved in anthocyanin biosynthesis (PacCHS, PacF3H, PacCHI, PacDFR, and PacUFGT). The application of NiCl2 inhibited the release of ethylene and the expression of PacACO1 encoded 1-aminocyclopropane-1-carboxylic acid oxidase, of which the effect on fruit ripening was opposite that of ABA. Ethephon and nordihydroguaiaretic acid treatments, however, had no effect on fruit ripening. For postharvest fruit, the pedicel was a major route for water loss, and dehydration induced the transcription of PacNCED1 and the accumulation of ABA. Followed by ethylene release, the start of postharvest fruit senescence is triggered. In conclusion, endogenous ABA triggered the ripening of fruit via the modulation of ripening-related metabolism pathways such as anthocyanin accumulation. ABA also induced postharvest fruit senescence through stimulation of ethylene release in cherry fruit.

The phytohormone abscisic acid (ABA) plays a crucial role not only in fruit development and ripening, but also in adaptive responses to biotic and abiotic stresses. In these processes, the actions of ABA are under the control of complex regulatory mechanisms involving ABA metabolism, signal transduction, and transport. The endogenous ABA content is determined by the dynamic balance between biosynthesis and catabolism, processes which are regulated by 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA 8'-hydroxylase (CYP707A), respectively. ABA conjugation by cytosolic UDP-glucosyltransferases, or release by β-glucosidases, is also important for maintaining ABA homeostasis. Recently, multiple putative ABA receptors localized at different subcellular sites have been reported. Among these is a major breakthrough in the field of ABA signalling-the identification of a signalling cascade involving the PYR/PYL/RCAR protein family, the type 2C protein phosphatases (PP2Cs), and subfamily 2 of the SNF1-related kinases (SnRK2s). With regard to transport, two ATP-binding cassette (ABC) proteins and two ABA transporters in the nitrate transporter 1/peptide transporter (NRT1/PTR) family have been identified. In this review, we summarize recent research progress on the role of ABA in fruit ripening, stress response, and transcriptional regulation, and also the functional verification of both ABA-responsive and ripening-related genes. In addition, we suggest possible commercial applications of genetic manipulation of ABA signalling to improve fruit quality and yields.

Summary Abscisic acid (ABA) glucose conjugation mediated by uridine diphosphate glucosyltransferases (UGTs) is an important pathway in regulating ABA homeostasis. In the present study, we investigated three tomato SlUGTs that are highly expressed in fruit during ripening, and these SlUGTs were localized to the cytoplasm and cell nucleus. Among these three UGTs, SlUGT75C1 catalyzes the glucosylation of both ABA and IAA in vitro; SlUGT76E1 can only catalyze the conjugation of ABA; and SlUGT73C4 cannot glycosylate either ABA or IAA. Therefore, SlUGT75C1 was selected for further investigation. SlUGT75C1 RNA interference significantly up-regulated the expression level of SlCYP707A2, which encodes an ABA 8’-hydroxylase but did not affect the expression of SlNCED1, which encodes a key enzyme in ABA biosynthesis. Suppression of SlUGT75C1 significantly accelerated fruit ripening by enhancing ABA levels and promoting the early release of ethylene. SlUGT75C1-RNAi altered the expression of fruit ripening genes (genes involved in ethylene release and cell wall catabolism). SlUGT75C1-RNAi seeds showed delayed germination and root growth compared with wild type as well as increased sensitivity to exogenous ABA. SlUGT75C1-RNAi plants were also more resistant to drought stress. These results demonstrated that SlUGT75C1 plays a crucial role in ABA-mediated fruit ripening, seed germination, and drought responses in tomato. This article is protected by copyright. All rights reserved.

Abscisic acid (ABA) regulates fruit development and ripening via its signaling. However, the exact role of ABA signaling core components in fruit have not yet been clarified. In this study, we investigated the potential interactions of tomato (Solanum lycopersicon) ABA signaling core components using yeast two-hybrid analysis, with or without ABA at different concentrations. The results showed that among 12 PYR/PYL/RCAR ABA receptors (SlPYLs), SlPYL1, SlPYL2, SlPYL4, SlPYL5, SlPYL 7, SlPYL8, SlPYL9, SlPYL10, SlPYL11, and SlPYL13 were ABA-dependent receptors, while SlPYL3 and SlPYL12 were ABA-independent receptors. Among five SlPP2Cs (type 2C protein phosphatases) and seven SlSnRK2s (subfamily 2 of SNF1-related kinases), all SlSnRK2s could interact with SlPP2C2, while SlSnRK2.8 also interacted with SlPP2C3. SlSnRK2.5 could interact with SlABF2/4 (ABA-responsive element binding factors). Expressions of SlPYL1, SlPYL2, SlPYL8, and SlPYL10 were upregulated under exogenous ABA but downregulated under nordihydroguaiaretic acid (NDGA) at the mature green stage of fruit ripening. The expressions of SlPP2C1, SlPP2C2, SlPP2C3, and SlPP2C5 were upregulated in ABA-treated fruit, but downregulated in NDGA-treated fruit at the mature green stage. The expressions of SlSnRK2.4, SlSnRK2.5, SlSnRK2.6, and SlSnRK2.7 were upregulated by ABA, but downregulated by NDGA. However, SlSnRK2.2 was down regulated by ABA. Expression of SlABF2/3/4 was enhanced by ABA but decreased by NDGA. Based on these results, we concluded that the majority of ABA receptor PYLs interact with SlPP2Cs in an ABA-dependent manner. SlPP2C2 and SlPP2C3 can interact with SlSnRK2s. SlSnRK2.5 could interact with SlABF2/4. Most ABA signaling core components respond to exogenous ABA.

The ‘Elizabeth’ melon (Cucumis melo L. cv. Elizabeth) is regarded as a climacteric fruit, but it has characteristics of both climacteric and nonclimacteric fruits during the ripening stage. To clarify whether the ethylene-independent pathways during ripening were related to abscisic acid (ABA), four cDNAs that encode for key enzymes of ABA metabolism (CMNCEDs, CMCYP707A1, and CMBG1) were cloned and their transcription expression levels were analyzed. The levels of endogenous ABA and ethylene production in fruit ripening and the effect of exogenous ABA or nordihydroguaiaretic acid (NDGA) treatment on fruit senescence post-harvest were also investigated. The results showed that the ABA levels in young fruit were initially high then gradually decreased. At 20 days after fruit setting (DAFS) there was a rapid increase that peaked at 30 DAFS. Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) content, and ACC oxidase (ACO) activity were all at low levels at the early stages of fruit setting, and their peaks were observed 5 days after the ABA peak. The CMNCED3 gene and the β-glucosidase gene CMBG1 had similar expression patterns, with expression peaks that occurred at 30 DAFS, which was consistent with the accumulation of ABA. The expression of CMNCED2 was relatively high during the early stages and no peak value occurred in the ripening stage. The CMCYP707A1 gene was expressed mainly at the late ripening stage. The exogenous ABA treatment promoted fruit ripening and softening by the upregulation and expression of the ethylene synthesis genes CmACS1 and CmACO1 and the cell wall catabolic enzyme gene CmPG1. The effect was proportional to the concentration of ABA, but too much ABA (>500 μmol/L) had a negative effect on fruit development. The NDGA treatment also had a negative effect on fruit ripening and development. ABA and ethylene have a synergistic effect on the regulation of melon fruit ripening. ABA mainly acted in the early stages of development, whereas ethylene played a major role in later stages of ripening. The endogenous ABA levels were maintained in a dynamic balance of biosynthesis and catabolism, which were regulated by CMNCEDs, CMBG1, and CMCYP707A1.

Abscisic acid (ABA) plays important roles during tomato fruit ripening. To study the regulation of carotenoid biosynthesis by ABA, the SlNCED1 gene encoding 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the ABA biosynthesis, was suppressed in tomato plants by transformation with an RNA interference (RNAi) construct driven by a fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20-50% of that in control fruit. This significant reduction in NCED activity led to the carbon that normally channels to free ABA as well as the ABA metabolite accumulation during ripening to be partially blocked. Therefore, this 'backlogged' carbon transformed into the carotenoid pathway in the RNAi lines resulted in increased assimilation and accumulation of upstream compounds in the pathway, chiefly lycopene and β-carotene. Fruit of all RNAi lines displayed deep red coloration compared with the pink colour of control fruit. The decrease in endogenous ABA in these transgenics resulted in an increase in ethylene, by increasing the transcription of genes related to the synthesis of ethylene during ripening. In conclusion, ABA potentially regulated the degree of pigmentation and carotenoid composition during ripening and could control, at least in part, ethylene production and action in climacteric tomato fruit.

Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), β-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-β-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp).

SummaryTo reveal details of the role(s) of abscisic acid (ABA) during fruit ripening and the regulation of ABA levels, two 9-cis-epoxycarotenoid dioxygenase genes (DkNCED1 and DkNCED2) and the DkCYP707A1 gene for ABA 8'-hydroxylase were isolated from unripened persimmon fruit. Expression of DkNCED1 increased at the start of fruit maturation and peaked 20 d before harvest, which was consistent with the accumulation of ABA during ripening in persimmon. Expression of the DkNCED2 gene was high in fruit at the immature stage, then declined continuously throughout ripening. Compared to the DkNCED genes, expression of DkCYP707A1 peaked 145 d after full-bloom (DAFB), then declined. Ethylene release decreased gradually from 65 DAFB to 115 DAFB, then peaked at 10 d after harvest. Water-deficit stress enhanced the expression of DkNCED1, but down-regulated transcript levels of DkCYP707A1 in the calyx. These results suggest that endogenous ABA content is modulated by a dynamic balance between ABA biosynthesis and cata...

To investigate the role of abscisic acid (ABA) and ethylene in the maturation of sweet cherry fruit, cloning was carried out on one cDNA (PacNCED1) encoding 9-cis-epoxycarotenoid dioxygenase (NCED)—a key enzyme in ABA biosynthesis—and on one cDNA (PacACO1) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase—an enzyme involved in ethylene biosynthesis. A BLAST homology search revealed that the sequences of DNA and amino acids in both PacNCED1 and PacACO1 showed a high degree of homology to comparable molecules of other plant species. The expression of PacNCED1 increased at the beginning of maturation and peaked 4 days before harvest, which was consistent with ABA accumulation during cherry ripening. Application of exogenous ABA increased the ABA content, inducing the expression of PacNCED1, and promoted ripening via enhancing colour formation and sugar accumulation. The expression of PacACO1 was not observed, and ethylene production was below detection levels, during the entire process of fruit de...

In order to characterize the potential transcriptional regulation of core components of abscisic acid (ABA) signal transduction in tomato fruit development and drought stress, eight SlPYL (ABA receptor), seven SlPP2C (type 2C protein phosphatase), and eight SlSnRK2 (subfamily 2 of SNF1-related kinases) full-length cDNA sequences were isolated from the tomato nucleotide database of NCBI GenBank. All SlPYL, SlPP2C, and SlSnRK2 genes obtained are homologous to Arabidopsis AtPYL, AtPP2C, and AtSnRK2 genes, respectively. Based on phylogenetic analysis, SlPYLs and SlSnRK2s were clustered into three subfamilies/subclasses, and all SlPP2Cs belonged to PP2C group A. Within the SlPYL gene family, SlPYL1, SlPYL2, SlPYL3, and SlPYL6 were the major genes involved in the regulation of fruit development. Among them, SlPYL1 and SlPYL2 were expressed at high levels throughout the process of fruit development and ripening; SlPYL3 was strongly expressed at the immature green (IM) and mature green (MG) stages, while SlPYL6 was expressed strongly at the IM and red ripe (RR) stages. Within the SlPP2C gene family, the expression of SlPP2C, SlPP2C3, and SlPP2C4 increased after the MG stage; SlPP2C1 and SlPP2C5 peaked at the B3 stage, while SlPP2C2 and SlPP2C6 changed little during fruit development. Within the SlSnRK2 gene family, the expression of SlSnRK2.2, SlSnRK2.3, SlSnRK2.4, and SlSnRK2C was higher than that of other members during fruit development. Additionally, most SlPYL genes were down-regulated, while most SlPP2C and SlSnRK2 genes were up-regulated by dehydration in tomato leaf.

Mitochondrial dysfunction plays important roles in the development of diabetes. Elevated nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) has been shown to be closely related to diabetes. But the relationship between them in diabetes has not been determined. This study was to explore the role of ADMA in hepatic mitochondrial dysfunction and its potential mechanisms in diabetic rats and hepatocytes.Respiratory enzymes activities, mitochondrial transmembrane potential and ATP content were measured to evaluate mitochondrial function. The copy number ratio of mitochondrial gene to nuclear gene was used to represent mitochondrial biogenesis. The activity of superoxide dismutase and malondialdehyde content were detected to reflect oxidative stress. Furthermore, changes in ADMA and NO contents, uncoupling protein 2 (UCP2) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) transcriptions were determined.Elevated ADMA levels in serum of diabetic rats were found to be associated with hepatic mitochondrial dysfunction reflected by reductions of respiratory enzyme activities, mitochondrial membrane potential and ATP contents. Similar mitochondrial dysfunction also occurred in ADMA-treated hepatocytes. The mitochondrial dysfunction observed in diabetic rats or hepatocytes was accompanied with suppressions of mitochondrial biogenesis, PGC-1α transcription and NO synthesis as well as enhances of UCP 2 transcription and oxidative stress. These effects of ADMA could be attenuated by treatments with antioxidant or NO donor.These results indicate that elevated endogenous ADMA contributes to hepatic mitochondrial dysfunction in diabetic rats, and underlying mechanisms may be related to the suppression of mitochondrial biogenesis and mitochondrial uncoupling via inhibiting NO synthesis and enhancing oxidative stress.

Abscisic acid (ABA) plays a key role in various aspects of plant growth and development, including adaptation to environmental stress and fruit maturation in sweet cherry fruit. In higher plants, the level of ABA is determined by synthesis and catabolism. In order to gain insight into ABA synthesis and catabolism in sweet cherry fruit during maturation and under stress conditions, four cDNAs of PacCYP707A1 -PacCYP707A4 for 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA, and one cDNA of PacNCED1 for 9-cis-epoxycarotenoid dioxygenase, a key enzyme in the ABA biosynthetic pathway, were isolated from sweet cherry fruit (Prunus avium L.). The timing and pattern of PacNCED1 expression was coincident with that of ABA accumulation, which was correlated to maturation of sweet cherry fruit. All four PacCYP707As were expressed at varying intensities throughout fruit development and appeared to play overlapping roles in ABA catabolism throughout sweet cherry fruit development. The application of ABA enhanced the expression of PacCYP707A1 -PacCYP707A3 as well as PacNCED1, but downregulated the PacCYP707A4 transcript level. Expressions of PacCYP707A1, PacCYP707A3 and PacNCED1 were strongly increased by water stress. No significant differences in PacCYP707A2 and PacCYP707A4 expression were observed between dehydrated and control fruits. The results suggest that endogenous ABA content is modulated by a dynamic balance between biosynthesis and catabolism, which are regulated by PacNCED1 and PacCYP707As transcripts, respectively, during fruit maturation and under stress conditions.

To understand details about the molecular mechanisms of abscisic acid (ABA) action in fruit maturation, one partial cDNA (PaCHLH1) encoding magnesium chelatase H subunit (CHLH) which was proven to be an ABA receptor in Arabidopsis was cloned from sweet cherry fruit. The sequence of PacCHLH1 was 442 bp. The amino acid sequence of PacCHLH1 shows high homology to other CHLHs. PacCHLH1 expression dramatically declined after 25 days after full bloom (DAFB) which is the beginning of the ripening process, in contrast to the changes in ABA levels during this period. The application of ABA promoted endogenous ABA biosynthesis and fruit ripening but significantly decreased the expression of PacCHLH1 in fruits. No significant correlation was observed between the PacCHLH1 transcript and ABA accumulation in dehydrated fruits. In dehydrated leaves, the ABA and PacCHLH1 transcript levels were both up-regulated.

Unlike the typical climacteric fruits, persimmons (Diospyros kaki Thunb.) produce higher levels of ethylene when they are detached from trees at a younger stage. In order to obtain detailed information on the role of abscisic acid (ABA) in ripening, we cloned the DKNCED1, DKACS2, and DKACO1 genes from the calyx. Water loss was first noted in the calyx lobe, and DKNCED1 was highly expressed 1 d after the fruits were detached, coinciding with an increase in the ABA content. Then, the DKACS2 and DKACO1 genes were expressed after some delay. In the calyx, the ABA peak was observed 2 d after the fruits were harvested, and this peak preceded the ethylene peak observed on day 3. The fruit firmness rapidly decreased on day 4, and the fruits softened completely 6 d after they were harvested. The increases in the expressions of ABA, ethylene, and the genes in the calyxes occurred earlier than the corresponding increases in the pulp, although the 3 increases occurred on different days. Exogenous ABA treatment increased ABA concentration, induced expression of both ACS and ACO, and promoted ethylene synthesis and young-fruit softening; by contrast, treatment with NDGA inhibited the gene expressions and ethylene synthesis and delayed young-fruit softening. These results indicate that ethylene biosynthesis in the detached young persimmon fruits is initially triggered by ABA, which is induced by water loss in the calyx, through the induction of DKACS2 and DKACO1 expressions. The ethylene produced in the calyx subsequently diffuses into the pulp tissue, where it induces autocatalytic ethylene biosynthesis, resulting in an abrupt increase in ethylene production.

Ripening and senescence are generally controlled by ethylene in climacteric fruits like peaches, and the ripening process of grape, a non-climacteric fruit, may have some relationship to abscisic acid (ABA) function. In order to better understand the role of ABA in ripening and senescence of these two types of fruits, we cloned the 9-cis-epoxycarotenoid dioxygenase (NCED) gene that encodes a key enzyme in ABA biosynthesis from peaches and grapes using an RT-PCR approach. The NCED gene fragments were cloned from peaches (PpNCED1and PpNCED2, each 740bp) and grapes (VVNCED1, 741bp) using degenerate primers designed based on the conserved amino acids sequence of NCEDs in other plants. PpNCED1 showed 78.54% homology with PpNCED2, 74.90% homology with VVNCED1, and both showed high homology to NCEDs from other plants. The expression patterns of PpNCED1 and VVNCED1 were very similar. Both were highly expressed at the beginning of ripening when ABA content becomes high. The maximum ABA preceded ethylene production in peach fruit. ABA in the grape gradually increased from the beginning of ripening and reached the highest level at 20d before the harvest stage. However, ethylene remained at low levels during the entire process of fruit development, including ripening and senescence. ABA content, and ripening and softening of both types of fruits, were promoted or delayed by exogenous ABA or Fluridone (or NDGA) treatment. The roles of ABA and ethylene in the later ripening of fruit are complex. Based on results obtained in this study, we concluded that PpNCED1 and VVNCED1 initiate ABA biosynthesis at the beginning of fruit ripening, and that ABA accumulation might play a key role in the regulation of ripeness and senescence of both peach and grape fruits.

In order to understand more details about the role of abscisic acid (ABA) in fruit ripening and senescence of tomato, two cDNAs (LeNCED1 and LeNCED2) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) as a key enzyme in ABA biosynthesis, two cDNAs (LeACS2 and LeACS4) which encode 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, and one cDNA (LeACO1) which encodes ACC oxidase involved in ethylene biosynthesis were cloned from tomato fruit using a reverse transcription-PCR (RT-PCR) approach. The relationship between ABA and ethylene during ripening was also investigated. Among six sampling times in tomato fruits, the LeNCED1 gene was highly expressed only at the breaker stage when the ABA content becomes high. After this, the LeACS2, LeACS4, and LeACO1 genes were expressed with some delay. The change in pattern of ACO activity was in accordance with ethylene production reaching its peak at the pink stage. The maximum ABA content preceded ethylene production in both the seeds and the flesh. The peak value of ABA, ACC, and ACC oxidase activity, and ethylene production all started to increase earlier in seeds than in flesh tissues, although they occurred at different ripening stages. Exogenous ABA treatment increased the ABA content in both flesh and seed, inducing the expression of both ACS and ACO genes, and promoting ethylene synthesis and fruit ripening, while treatment with fluridone or nordihydroguaiaretic acid (NDGA) inhibited them, delaying fruit ripening and softening. Based on the results obtained in this study, it was concluded that LeNCED1 initiates ABA biosynthesis at the onset of fruit ripening, and might act as an original inducer, and ABA accumulation might play a key role in the regulation of ripeness and senescence of tomato fruit.

Highlight text SlNCED1 and SlCYP707A2 are key genes in the regulation of ABA synthesis and catabolism, and are involved in fruit ripening as positive and negative regulators, respectively., Abscisic acid (ABA) plays an important role in fruit development and ripening. Here, three NCED genes encoding 9-cis-epoxycarotenoid dioxygenase (NCED, a key enzyme in the ABA biosynthetic pathway) and three CYP707A genes encoding ABA 8′-hydroxylase (a key enzyme in the oxidative catabolism of ABA) were identified in tomato fruit by tobacco rattle virus-induced gene silencing (VIGS). Quantitative real-time PCR showed that VIGS-treated tomato fruits had significant reductions in target gene transcripts. In SlNCED1-RNAi-treated fruits, ripening slowed down, and the entire fruit turned to orange instead of red as in the control. In comparison, the downregulation of SlCYP707A2 expression in SlCYP707A2-silenced fruit could promote ripening; for example, colouring was quicker than in the control. Silencing SlNCED2/3 or SlCYP707A1/3 made no significant difference to fruit ripening comparing RNAi-treated fruits with control fruits. ABA accumulation and SlNCED1transcript levels in the SlNCED1-RNAi-treated fruit were downregulated to 21% and 19% of those in control fruit, respectively, but upregulated in SlCYP707A2-RNAi-treated fruit. Silencing SlNCED1 or SlCYP707A2 by VIGS significantly altered the transcripts of a set of both ABA-responsive and ripening-related genes, including ABA-signalling genes (PYL1, PP2C1, and SnRK2.2), lycopene-synthesis genes (SlBcyc, SlPSY1 and SlPDS), and cell wall-degrading genes (SlPG1, SlEXP, and SlXET) during ripening. These data indicate that SlNCED1 and SlCYP707A2 are key genes in the regulation of ABA synthesis and catabolism, and are involved in fruit ripening as positive and negative regulators, respectively.

Objective Increasing evidence suggested that endoplasmic reticulum (ER) stress contributes to insulin resistance, which plays an important role in the development of type 2 diabetes mellitus (T2DM). Accumulation of endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), is associated with insulin resistance, T2DM, and diabetic cardiovascular complications, although the mechanisms have not been elucidated. This study was to determine whether elevated endogenous ADMA is involved in hepatic ER stress of type 2 diabetic rats, verify their causal relationship, and elucidate the potential mechanism underlying ADMA induced ER stress in rat hepatocytes. Methods Immunoglobulin binding protein (Bip) transcription, eukaryotic initiation factor 2α kinase (eIF2α) phosphorylation, X box-binding protein-1 (XBP-1) mRNA splicing and C/EBP homologues protein (CHOP) expression were measured to reflect ER stress. Contents of ADMA and nitrite/nitrate as well as activities or expression of NOS and dimethylarginine dimethylaminohydrolase (DDAH) were detected to show the changes in DDAH/ADMA/NOS/NO pathway. The lipid peroxidation product malondialdehyde content and antioxidant enzyme superoxide dismutase activity were analyzed to evaluate oxidative stress. Results ER stress was provoked in the liver of type 2 diabetic rats, as expressed by increases of Bip transcription, eIF2α phosphorylation, XBP-1 splicing and CHOP expression, all of which were in parallel with the elevation of serum ADMA, suppression of NO generation, NOS and DDAH activities in the liver. Exposure of hepatocytes to ADMA or hydrogen peroxide also induced ER stress, which was associated with the inhibition of NO production and increase of oxidative stress. Treatment of hepatocytes with antioxidant pyrrolidine dithiocarbamate not only decreased ADMA-induced oxidative stress and inhibition of NO production but also reduced ADMA-triggered ER stress. Conclusions These results indicate that increased endogenous ADMA contributes to hepatic ER stress in type 2 diabetic rats, and the mechanism underlying ADMA-induced ER stress may relate to oxidative stress via NOS uncoupling.

To investigate the contribution of abscisic acid (ABA) in pear 'Gold Nijisseiki' during fruit ripening and under dehydration stress, two cDNAs (PpNCED1 and PpNCED2) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) (a key enzyme in ABA biosynthesis), two cDNAs (PpCYP707A1 and PpCYP707A2) which encode 8'-hydroxylase (a key enzyme in the oxidative catabolism of ABA), one cDNA (PpACS3) which encodes 1-aminocyclopropane-1-carboxylic acid (ACC), and one cDNA (PpACO1) which encodes ACC oxidase involved in ethylene biosynthesis were cloned from 'Gold Nijisseiki' fruit. In the pulp, peel and seed, expressions of PpNCED1 and PpNCED2 rose in two stages which corresponded with the increase of ABA levels. The expression of PpCYP707A1 dramatically declined after 60-90 days after full bloom (DAFB) in contrast to the changes of ABA levels during this period, while PpCYP707A2 stayed low during the whole development of fruit. Application of exogenous ABA at 100 DAFB increased the soluble sugar content and the ethylene release but significantly decreased the titratable acid and chlorophyll contents in fruits. When fruits harvested at 100 DAFB were stored in the laboratory (25 °C, 50% relative humidity), the ABA content and the expressions of PpNCED1/2 and PpCYP707A1 in the pulp, peel and seed increased significantly, while ethylene reached its highest value after the maximum peak of ABA accompanied with the expressions of PpACS3 and PpACO1. In sum the endogenous ABA may play an important role in the fruit ripening and dehydration of pear 'Gold Nijisseiki' and the ABA level was regulated mainly by the dynamics of PpNCED1, PpNCED2 and PpCYP707A1 at the transcriptional level.

Summary In plants, β–glucosidases (BG) have been implicated in developmental and pathogen defense, and are thought to take part in abscisic acid (ABA) synthesis via hydrolysis of ABA glucose ester to release active ABA; however, there is no genetic evidence for the role of BG genes in ripening and biotic/abiotic stress in fruits. To clarify the role of BG genes in fruit, eight Fa/FvBG genes encoding β–glucosidase were isolated using information from the GenBank strawberry nucleotide database. Of the Fa/FvBG genes examined, expression of FaBG3 was the highest, showing peaks at the mature stage, coincident with the changes observed in ABA content. To verify the role of this gene, we suppressed the expression of FaBG3 via inoculation with Agrobacterium tumefaciens containing tobacco rattle virus carrying a FaBG3 fragment (RNAi). The expression of FaBG3 in FaBG3-RNAi-treated fruit was markedly reduced, and the ABA content was lower than that of the control. FaBG3-RNAi-treated fruit did not exhibit full ripening, and were firmer, had lower sugar content, and were pale compared with the control due to down-regulation of ripening-related genes. FaBG3-RNAi-treated fruit with reduced ABA levels were much more resistant to Botrytis cinerea fungus but were more sensitive to dehydration stress than control fruit. These results indicate that FaBG3 may play key roles in fruit ripening, dehydration stress and B. cinerea fungal infection in strawberries via modulation of ABA homeostasis and transcriptional regulation of ripening-related genes.

Cucumber (Cucumis sativus L.), a kind of fruit usually harvested at the immature green stage, belongs to non-climacteric fruit. To investigate the contribution of abscisic acid (ABA) to cucumber fruit development and ripening, variation in ABA level was investigated and a peak in ABA level was found in pulp before fruit get fully ripe. To clarify this point further, exogenous ABA was applied to cucumber fruits at two different development stages. Results showed that ABA application at the turning stage promotes cucumber fruit ripening, while application at the immature green stage had inconspicuous effects. In addition, with the purpose of understanding the transcriptional regulation of ABA, two partial cDNAs of CsNCED1 and CsNCED2 encoding 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthetic pathway; one partial cDNA of CsCYP707A1 for 8′-hydroxylase, a key enzyme in the oxidative catabolism of ABA and two partial cDNAs of CsBG1 and CsBG2 for β-glucosidase (BG) that hydrolyzes ABA glucose ester (ABA-GE) to release active ABA were cloned from cucumber. The DNA and deduced amino acid sequences of these obtained genes respectively showed high similarities to their homologous genes in other plants. Real-time PCR analysis revealed that ABA content may be regulated by its biosynthesis (CsNCEDs), catabolism (CsCYP707A1) and reactivation genes (CsBGs) at the transcriptional level during cucumber fruit development and ripening, in response to ABA application, dehydration and pollination, among which CsNCED1, CsCYP707A1 and CsBG1 were highly expressed in pulp and may play more important roles in regulating ABA metabolism.

The abscisic acid (ABA) signal transduction core components in plants include the ABA receptors (PYR/PYL/RCARs), the group A type 2C protein phosphatases (PP2Cs) and the subclass III SNF1-related protein kinases 2 (SnRK2s). In this study, via homology cloning, three CsPYLs, four CsPP2Cs and two CsSnRK2s partial cDNAs were obtained in cucumber (Cucumis sativus). In silico analysis results indicated that all CsPYL, CsPP2C, and CsSnRK2 genes obtained are homologous to Arabidopsis AtPYL, AtPP2C, and AtSnRK2 genes, respectively. Based on phylogenetic analysis, only CsPP2C2 and CsSnRK2.2 belonged to group A PP2C and subclass III SnRK2, respectively, and may involve in ABA signal transduction. During cucumber fruit development and ripening, CsPYL2 and CsPP2C2 were highly expressed and both reached their peak value at 27 DAF, at which stage the ABA content was also at its highest level, which indicated that the CsPYL2 and CsPP2C2 may involve in transducing ABA signal in fruit and regulating fruit development and ripening. Under drought stress condition in cucumber seedlings, CsPYL1, CsPYL2, CsPP2C2 and CsSnKR2.2 were sensitive and up-regulated in root, stem and leaf; meanwhile, CsPYL3 showed a low sensitivity and were down-regulated in root and stem.

The aim of this study was to obtain new insights into the mechanisms that regulate endogenous abscisic acid (ABA) levels by β-glucosidase genes during the development of watermelons (Citrullus lanatus) and under drought stress conditions. In total, five cDNAs from watermelons were cloned by using reverse transcription-PCR (RT-PCR). They included three cDNAs (ClBG1, ClBG2 and ClBG3) homologous to those that encode β-glucosidase l that hydrolyzes the ABA glucose ester (ABA-GE) to release active ABA, ClNCED4, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis, and ClCYP707A1, encoding ABA 8'-hydroxylase. A BLAST homology search revealed that the sequences of cDNAs and the deduced amino acids of these genes showed a high degree of homology to comparable molecules of other plant species. During fruit development and ripening, the expressions of ClBG1, ClNCED4 and ClCYP707A1 were relatively low at an early stage, increased rapidly along with fruit ripening, and reached the highest levels at 27 days after full bloom (DAFB) at the harvest stage. This trend was consistent with the accumulation of ABA. The ClBG2 gene on the other hand was highly expressed at 5 DAFB, and then decreased gradually with fruit development. Unlike ClBG1 and ClBG2, the expression of ClBG3 was low at an early stage; its expression peak occurred at 15 DAFB and then declined to the lowest point. When watermelon seedlings were subjected to drought stress, expressions of ClBG1 and ClCYP707A1 were significantly down-regulated, while expressions of ClBG2 and ClNCED4 were up-regulated in the roots, stems and leaves. The expression of ClBG3 was down-regulated in root tissue, but was up-regulated in stems and leaves. In conclusion, endogenous ABA content was modulated by a dynamic balance between biosynthesis and catabolism regulated by ClNCED4, ClCYP707A1 and ClBGs during development and under drought stress condition. It seems likely that β-glucosidase genes are important for this regulation process.

The freezing resistances of three Diospyros species and three cultivars of D. kaki were evalu- ated by survival of twigs and electrolyte leakage from the tissues after freezing treatments. Dormant 1-year-old twigs were cooled from -5° to -5°- 30°C within 5 hr, maintained at each temperature for 5 hr and thawed gradually to 5°C for 5 hr. The twigs soaked in water were held for 20 days under 20°C to determine recovery.The hardiest species during the coldest period of the season was D. lotus followed by D. kaki, and the least hardy was D. taitoensis which is distributed in subtropical Taiwan. The critical low temperatures that did not injure the plants were -10°C for D. taitoensis, -15°C for D. kaki and -20°C for D. lotus; the temperatures at which half of the test twigs was killed (LT50) were - 12°, -20° and -25°C, respectively. Their electrolyte leakage at LT50 ranged from 25 to 28%.Among three cultivars of D. kaki, Eiratanenashi' which is native to the northern region of Japan, was most tolerant to freezing during mid-winter ("true freezing resistance"), however, it was the most sensitive to freezing before cold acclimation in early autumn and after cold acclimation in spring.Starch hydrolyzed to sugars in twigs of three cultivars of D. kaki under natural conditions during mid-winter. A close relation was found between the freezing resistance, evaluated by electrolyte leakage from twig cells, and anthocyanin in the cortical tissues. Anthocyanin con- tents in cortical tissues reached a maximum in mid-February; the pigment levels tended to increase when plants were exposed to -10°- 30°C which were below the ambient tem- perature. Moreover, anthocyanin contents in the cortex decreased, and the twigs became less hardy by the deacclimation treatment during the coldest part of the season. Conversely, twigs under the chilling treatment before cold acclimation in early autumn became hardy; the change was accompanied by an elevation of anthocyanin content.

Background The ripening of grape berry is generally regulated by abscisic acid (ABA), and has no relationship with ethylene function. However, functional interaction and synergism between ABA and ethylene during the beginning of grape berry ripening (véraison) has been found recently. Results The expressions of VvNCED1 encoding 9-cis-epoxycarotenoid dioxygenase (NCED) and VvGT encoding ABA glucosyltransferase were all increased rapidly at the stage of véraison and reached the highest level at 9th week after full bloom. However, VvCYP1 encoding ABA 8'-hydroxylase and VvβG1 encoding berry β-glucosidase are different, whose expression peak appeared at the 10th week after full bloom and in especial VvβG1 remained at a high level till harvest. The VvACO1 encoding 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, the VvETR2 (ethylene response 2) and VvCTR1 (constitutive triple response 1) had a transient expression peak at pre-véraison, while the VvEIN4 (ethylene insensitive 4) expression gradually increased from the véraison to one week before harvest stage. The above mentioned changes happened again in the berry after harvest. At one week before véraison, double block treatment with NiCl2 plus 1-methylcyclopropene (1-MCP) not only inhibited the release of ethylene and the expression of related genes but also suppressed the transcription of VvNCED1 and the synthesis of ABA which all might result in inhibiting the fruit ripening onset. Treatment with ABA could relieve the double block and restore fruit ripening course. However, after harvest, double block treatment with NiCl2 plus 1-MCP could not suppress the transcription of VvNCED1 and the accumulation of ABA, and also could not inhibit the start of fruit senescence. Conclusion The trace endogenous ethylene induces the transcription of VvNCED1 and then the generation of ABA followed. Both ethylene and ABA are likely to be important and their interplaying may be required to start the process of berry ripening. When the level of ABA reached the peak value, part of it will be stored in the form of ABA-GE. While after harvest, abiotic stresses principally (such as dehydration, harvest shock) could induce the transcription of VvNCED1 and the accumulation of ABA, thus starting the process of fruit senescence.

Ripening and senescence are generally controlled by ethylene in climacteric fruits like peaches, and the ripening process of grape, a non-climacteric fruit, may have some relationship to abscisic acid (ABA) function. In order to better understand the role of ABA in ripening and senescence of these two types of fruits, we cloned the 9-cis-epoxycarotenoid dioxygenase (NCED) gene that encodes a key enzyme in ABA biosynthesis from peaches and grapes using an RT-PCR approach. The NCED gene fragments were cloned from peaches (PpNCED1and PpNCED2, each 740bp) and grapes (VVNCED1, 741bp) using degenerate primers designed based on the conserved amino acids sequence of NCEDs in other plants. PpNCED1 showed 78.54% homology with PpNCED2, 74.90% homology with VVNCED1, and both showed high homology to NCEDs from other plants. The expression patterns of PpNCED1 and VVNCED1 were very similar. Both were highly expressed at the beginning of ripening when ABA content becomes high. The maximum ABA preceded ethylene production in peach fruit. ABA in the grape gradually increased from the beginning of ripening and reached the highest level at 20d before the harvest stage. However, ethylene remained at low levels during the entire process of fruit development, including ripening and senescence. ABA content, and ripening and softening of both types of fruits, were promoted or delayed by exogenous ABA or Fluridone (or NDGA) treatment. The roles of ABA and ethylene in the later ripening of fruit are complex. Based on results obtained in this study, we concluded that PpNCED1 and VVNCED1 initiate ABA biosynthesis at the beginning of fruit ripening, and that ABA accumulation might play a key role in the regulation of ripeness and senescence of both peach and grape fruits.