First Isolation of New Canine Parvovirus 2a from Tibetan Mastiff and Global Analysis of the Full-Length VP2 Gene of Canine Parvoviruses 2 in China

Canine parvovirus 2 (CPV-2) was first identified in 1978, and is responsible for classic parvoviral enteritis. Despite the widespread vaccination of domestic carnivores, CPVs have remained important pathogens of domestic and wild carnivores. In this study, we isolated CPV-2 from Tibetan mastiffs and performed a global analysis of the complete VP2 gene sequences of CPV-2 strains in China. Six isolates were typed as new CPV-2a, according to key amino acid positions. On a phylogenetic tree, these six sequences formed a distinct clade. Five isolates occurred on the same branch as {"type":"entrez-nucleotide","attrs":{"text":"KF785794","term_id":"576295726","term_text":"KF785794"}}KF785794 from China and {"type":"entrez-nucleotide","attrs":{"text":"GQ379049","term_id":"256032181","term_text":"GQ379049"}}GQ379049 from Thailand; CPV-LS-ZA1 formed a separate subgroup with {"type":"entrez-nucleotide","attrs":{"text":"FJ435347","term_id":"215398940","term_text":"FJ435347"}}FJ435347 from China. One hundred ninety-eight sequences from various parts of China and the six sequences isolated here formed seven distinct clusters, indicating the high diversity of CPVs in China. Of 204 VP2 sequences, 183 (91.04%) encoded the mutation Ser297Ala, regardless of the antigenic type, implying that most Chinese CPV-2 strains contain the VP2 mutation Ser297Ala. However, the biological significance of this change from prototype CPV-2a/2b to new CPV-2a/2b types remains unclear. This study is the first to isolate new CPV-2a from the Tibetan mastiff. Our data show that new CPV-2a/2b variants are now circulating in China.     

Molecular Cloning and Characterization of DXS and DXR Genes in the Terpenoid Biosynthetic Pathway of Tripterygium wilfordii

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879187","term_id":"871331037","term_text":"KM879187"}}KM879187), 2148 bp (TwDXS2, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879186","term_id":"871331021","term_text":"KM879186"}}KM879186), 1410 bp (TwDXR, GenBank accession no.{"type":"entrez-nucleotide","attrs":{"text":"KM879185","term_id":"871330999","term_text":"KM879185"}}KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g⁻¹, a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f.     

Rationale for a Combination Therapy with the STAT5 Inhibitor AC-4-130 and the MCL1 Inhibitor S63845 in the Treatment of FLT3-Mutated or TET2-Mutated Acute Myeloid Leukemia

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in one-third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT kinase, MAP kinases, and STAT5. FLT3 inhibitors have been approved for the treatment of FLT3-mutated AML. However, treatment response to FLT3 inhibitors may be short-lived, and resistance may emerge. Compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with FLT3-mutated AML. Here STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin (PKC412), BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845, and BCL-2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Synergistic effects on cell viability were detected in both FLT3-mutated and FLT3-wild-type AML cells treated with AC-4-130 in combination with the MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845. AML patient samples with a strong response to AC-4-130 and {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 combination treatment were characterized by mutated FLT3 or mutated TET2 genes. Susceptibility of AML cells to AC-4-130, PTC596, trametinib, PKC412, and venetoclax was altered in the presence of HS-5 stroma. Only the MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 induced cell death with equal efficacy in the absence or presence of bone marrow stroma. The combination of the STAT5-inhibitor AC-4-130 and the MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 may be an effective treatment targeting FLT3-mutated or TET2-mutated AML.     

Detoxification of Toxic Phorbol Esters from Malaysian Jatropha curcas Linn. Kernel by Trichoderma spp. and Endophytic Fungi

The presence of phorbol esters (PEs) with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum {"type":"entrez-nucleotide","attrs":{"text":"JQ350879.1","term_id":"384096340","term_text":"JQ350879.1"}}JQ350879.1, T. harzianum {"type":"entrez-nucleotide","attrs":{"text":"JQ517493.1","term_id":"380258734","term_text":"JQ517493.1"}}JQ517493.1, Paecilomyces sinensis {"type":"entrez-nucleotide","attrs":{"text":"JQ350881.1","term_id":"384096342","term_text":"JQ350881.1"}}JQ350881.1, Cladosporium cladosporioides {"type":"entrez-nucleotide","attrs":{"text":"JQ517491.1","term_id":"380258732","term_text":"JQ517491.1"}}JQ517491.1, Fusarium chlamydosporum {"type":"entrez-nucleotide","attrs":{"text":"JQ350882.1","term_id":"384096343","term_text":"JQ350882.1"}}JQ350882.1, F. chlamydosporum {"type":"entrez-nucleotide","attrs":{"text":"JQ517492.1","term_id":"380258733","term_text":"JQ517492.1"}}JQ517492.1 and F. chlamydosporum {"type":"entrez-nucleotide","attrs":{"text":"JQ350880.1","term_id":"384096341","term_text":"JQ350880.1"}}JQ350880.1) was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS) and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p < 0.05) removed PEs with percentage losses of 96.9%–99.7%, while F. chlamydosporum strains showed percentage losses of 88.9%–92.2%. All fungal strains could utilize the PEs-rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%–96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs.     

Downregulation of lncRNA PpL-T31511 and Pp-miRn182 Promotes Hydrogen Cyanamide-Induced Endodormancy Release through the PP2C-H2O2 Pathway in Pear (Pyrus pyrifolia)

Bud endodormancy is an important, complex process subject to both genetic and epigenetic control, the mechanism of which is still unclear. The endogenous hormone abscisic acid (ABA) and its signaling pathway play important roles in the endodormancy process, in which the type 2C protein phosphatases (PP2Cs) is key to the ABA signal pathway. Due to its excellent effect on endodormancy release, hydrogen cyanamide (HC) treatment is considered an effective measure to study the mechanism of endodormancy release. In this study, RNA-Seq analysis was conducted on endodormant floral buds of pear (Pyrus pyrifolia) with HC treatment, and the HC-induced PP2C gene PpPP2C1 was identified. Next, software prediction, expression tests and transient assays revealed that lncRNA PpL-{"type":"entrez-nucleotide","attrs":{"text":"T31511","term_id":"613609"}}T31511-derived Pp-miRn182 targets PpPP2C1. The expression analysis showed that HC treatment upregulated the expression of PpPP2C1 and downregulated the expression of PpL-{"type":"entrez-nucleotide","attrs":{"text":"T31511","term_id":"613609"}}T31511 and Pp-miRn182. Moreover, HC treatment inhibited the accumulation of ABA signaling pathway-related genes and hydrogen peroxide (H₂O₂). Furthermore, overexpression of Pp-miRn182 reduced the inhibitory effect of PpPP2C1 on the H₂O₂ content. In summary, our study suggests that downregulation of PpL-{"type":"entrez-nucleotide","attrs":{"text":"T31511","term_id":"613609"}}T31511-derived Pp-miRn182 promotes HC-induced endodormancy release in pear plants through the PP2C-H₂O₂ pathway.     

Group II Metabotropic Glutamate Receptors Reduce Apoptosis and Regulate BDNF and GDNF Levels in Hypoxic-Ischemic Injury in Neonatal Rats

Birth asphyxia causes brain injury in neonates, but a fully successful treatment has yet to be developed. This study aimed to investigate the effect of group II mGlu receptors activation after experimental birth asphyxia (hypoxia-ischemia) on the expression of factors involved in apoptosis and neuroprotective neurotrophins. Hypoxia-ischemia (HI) on 7-day-old rats was used as an experimental model. The effects of intraperitoneal application of mGluR2 agonist {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 (5 mg/kg) and the specific mGluR3 agonist NAAG (5 mg/kg) (1 h or 6 h after HI) on apoptotic processes and initiation of the neuroprotective mechanism were investigated. {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 and NAAG applied shortly after HI prevented brain damage and significantly decreased pro-apoptotic Bax and HtrA2/Omi expression, increasing expression of anti-apoptotic Bcl-2. NAAG or {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 applied at both times also decreased HIF-1α formation. HI caused a significant decrease in BDNF concentration, which was restored after {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 or NAAG administration. HI-induced increase in GDNF concentration was decreased after administration of {"type":"entrez-nucleotide","attrs":{"text":"LY379268","term_id":"1257807854","term_text":"LY379268"}}LY379268 or NAAG. Our results show that activation of mGluR2/3 receptors shortly after HI prevents brain damage by the inhibition of excessive glutamate release and apoptotic damage decrease. mGluR2 and mGluR3 agonists produced comparable results, indicating that both receptors may be a potential target for early treatment in neonatal HI.     

Generation and Analysis of Expressed Sequence Tags (ESTs) from Halophyte Atriplex canescens to Explore Salt-Responsive Related Genes

Little information is available on gene expression profiling of halophyte A. canescens. To elucidate the molecular mechanism for stress tolerance in A. canescens, a full-length complementary DNA library was generated from A. canescens exposed to 400 mM NaCl, and provided 343 high-quality ESTs. In an evaluation of 343 valid EST sequences in the cDNA library, 197 unigenes were assembled, among which 190 unigenes (83.1% ESTs) were identified according to their significant similarities with proteins of known functions. All the 343 EST sequences have been deposited in the dbEST GenBank under accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"JZ535802 to JZ536144","start_term":"JZ535802","end_term":"JZ536144","start_term_id":"584304637","end_term_id":"584305012"}}JZ535802 to JZ536144. According to Arabidopsis MIPS functional category and GO classifications, we identified 193 unigenes of the 311 annotations EST, representing 72 non-redundant unigenes sharing similarities with genes related to the defense response. The sets of ESTs obtained provide a rich genetic resource and 17 up-regulated genes related to salt stress resistance were identified by qRT-PCR. Six of these genes may contribute crucially to earlier and later stage salt stress resistance. Additionally, among the 343 unigenes sequences, 22 simple sequence repeats (SSRs) were also identified contributing to the study of A. canescens resources.     

New Radioligands for Describing the Molecular Pharmacology of MT1 and MT2 Melatonin Receptors

Melatonin receptors have been studied for several decades. The low expression of the receptors in tissues led the scientific community to find a substitute for the natural hormone melatonin, the agonist 2-[¹²⁵I]-iodomelatonin. Using the agonist, several hundreds of studies were conducted, including the discovery of agonists and antagonists for the receptors and minute details about their molecular behavior. Recently, we attempted to expand the panel of radioligands available for studying the melatonin receptors by using the newly discovered compounds SD6, DIV880, and {"type":"entrez-nucleotide","attrs":{"text":"S70254","term_id":"546525","term_text":"S70254"}}S70254. These compounds were characterized for their affinities to the hMT₁ and hMT₂ recombinant receptors and their functionality in the classical GTPγS system. SD6 is a full agonist, equilibrated between the receptor isoforms, whereas {"type":"entrez-nucleotide","attrs":{"text":"S70254","term_id":"546525","term_text":"S70254"}}S70254 and DIV880 are only partial MT₂ agonists, with K_i in the low nanomolar range while they have no affinity to MT₁ receptors. These new tools will hopefully allow for additions to the current body of information on the native localization of the receptor isoforms in tissues.     

Ginsenoside-Rd Promotes Neurite Outgrowth of PC12 Cells through MAPK/ERK- and PI3K/AKT-Dependent Pathways

Panax ginseng is a famous herbal medicine widely used in Asia. Ginsenosides have been identified as the principle active ingredients for Panax ginseng’s biological activity, among which ginsenoside Rd (Rd) attracts extensive attention for its obvious neuroprotective activities. Here we investigated the effect of Rd on neurite outgrowth, a crucial process associated with neuronal repair. PC12 cells, which respond to nerve growth factor (NGF) and serve as a model for neuronal cells, were treated with different concentrations of Rd, and then their neurite outgrowth was evaluated. Our results showed that 10 μM Rd significantly increased the percentages of long neurite- and branching neurite-bearing cells, compared with respective controls. The length of the longest neurites and the total length of neurites in Rd-treated PC12 cells were much longer than that of respective controls. We also showed that Rd activated ERK1/2 and AKT but not PKC signalings, and inhibition of ERK1/2 by PD98059 or/and AKT by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 effectively attenuated Rd-induced neurite outgrowth. Moreover, Rd upregulated the expression of GAP-43, a neuron-specific protein involved in neurite outgrowth, while PD98059 or/and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased Rd-induced increased GAP-43 expression. Taken together, our results provided the first evidence that Rd may promote the neurite outgrowth of PC12 cells by upregulating GAP-43 expression via ERK- and ARK-dependent signaling pathways.     

Inhibition of Autophagy via Activation of PI3K/Akt Pathway Contributes to the Protection of Ginsenoside Rb1 against Neuronal Death Caused by Ischemic Insults

Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1) on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD) in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO) and Monodansylcadaverine (MDC) staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.     

Post Zygotic,Somatic, Deletion in KERATIN 1 V1 Domain Generates Structural Alteration of the K1/K10 Dimer,Producing a Monolateral Palmar Epidermolytic Nevus

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion {"type":"entrez-nucleotide","attrs":{"text":"NM_006121.4","term_id":"1519311739"}}NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation {"type":"entrez-protein","attrs":{"text":"NP_006112.3","term_id":"119395750"}}NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.     

Therapeutic Targeting of Ovarian Cancer Stem Cells Using Estrogen Receptor Beta Agonist

Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest ovarian cancer stem cells (OCSCs) contribute to chemotherapy resistance and tumor relapse. Recent studies demonstrated estrogen receptor beta (ERβ) exerts tumor suppressor functions in OCa. However, the status of ERβ expression in OCSCs and the therapeutic utility of the ERβ agonist {"type":"entrez-nucleotide","attrs":{"text":"LY500307","term_id":"1371032831","term_text":"LY500307"}}LY500307 for targeting OCSCs remain unknown. OCSCs were enriched from ES2, OV90, SKOV3, OVSAHO, and A2780 cells using ALDEFLUOR kit. RT-qPCR results showed ERβ, particularly ERβ isoform 1, is highly expressed in OCSCs and that ERβ agonist {"type":"entrez-nucleotide","attrs":{"text":"LY500307","term_id":"1371032831","term_text":"LY500307"}}LY500307 significantly reduced the viability of OCSCs. Treatment of OCSCs with {"type":"entrez-nucleotide","attrs":{"text":"LY500307","term_id":"1371032831","term_text":"LY500307"}}LY500307 significantly reduced sphere formation, self-renewal, and invasion, while also promoting apoptosis and G2/M cell cycle arrest. Mechanistic studies using RNA-seq analysis demonstrated that {"type":"entrez-nucleotide","attrs":{"text":"LY500307","term_id":"1371032831","term_text":"LY500307"}}LY500307 treatment resulted in modulation of pathways related to cell cycle and apoptosis. Western blot and RT-qPCR assays demonstrated the upregulation of apoptosis and cell cycle arrest genes such as FDXR, p21/CDKN1A, cleaved PARP, and caspase 3, and the downregulation of stemness markers SOX2, Oct4, and Nanog. Importantly, treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY500307","term_id":"1371032831","term_text":"LY500307"}}LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopic OCa murine xenograft models. Our results demonstrate that ERβ agonist {"type":"entrez-nucleotide","attrs":{"text":"LY500307","term_id":"1371032831","term_text":"LY500307"}}LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and shows significant promise as a novel therapeutic agent in treating OCa.     

HBx Protein Promotes Oval Cell Proliferation by Up-Regulation of Cyclin D1 via Activation of the MEK/ERK and PI3K/Akt Pathways

Growing evidence has shown that hepatic oval cells, also named liver progenitor cells, play an important role in the process of liver regeneration in various liver diseases. Oval cell proliferation has been reported in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and chronic liver disease. Studies have found expression of HBV surface and core antigens in oval cells in the livers of patients with HCC, suggesting that HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. In addition, there is evidence of multiplication of HBV in oval cell culture. However, little research has been performed to explore the role of HBV-encoded proteins in the proliferation of hepatic oval cells. Previously, we successfully transfected the HBV x (HBx) gene, one of the four genes in the HBV genome, into a rat LE/6 oval cell line. In this study, we tested whether or not the transfected HBx gene could affect oval cell proliferation in vitro. Our results show that overexpression of HBx promotes the proliferation of oval cells and increases cyclin D1 expression, assessed at both the mRNA and protein levels. We also found that HBx activated the PI-3K/Akt and MEK/ERK1/2 pathways in HBx-transfected oval cells. Furthermore, the HBx-induced increases in cyclin D1 expression and oval cell proliferation were completely abolished by treatment with either MEK inhibitor PD184352 or PI-3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These results demonstrated that HBx has the ability to promote oval cell proliferation in vitro, and its stimulatory effects on cell proliferation and expression of cyclin D1 depend on the activation of the MEK/ERK and PI3K/Akt signaling pathways in cultured oval cells.     

Isolation and Functional Characterization of a LEAFY Gene in Mango (Mangifera indica L.)

LEAFY (LFY) plays an important role in the flowering process of plants, controlling flowering time and mediating floral meristem differentiation. Owing to its considerable importance, the mango LFY gene (MiLFY; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"HQ585988","term_id":"323650468"}}HQ585988) was isolated, and its expression pattern and function were characterized in the present study. The cDNA sequence of MiLFY was 1152 bp, and it encoded a 383 amino acid protein. MiLFY was expressed in all tested tissues and was highly expressed in flowers and buds. Temporal expression analysis showed that MiLFY expression was correlated with floral development stage, and two relative expression peaks were detected in the early stages of floral transition and floral organ differentiation. Moreover, 35S::GFP-MiLFY fusion protein was shown to be localized to the nucleus of cells. Overexpression of MiLFY in Arabidopsis promoted early flowering and the conversion of lateral meristems into terminal flowers. In addition, transgenic plants exhibited obvious morphological changes, such as differences in cauline leaf shape, and the number of lateral branches. When driven by the MiLFY promoter, GFP was highly expressed in leaves, floral organs, stems, and roots, during the flowering period. Exogenous gibberellin (GA₃) treatment downregulated MiLFY promoter expression, but paclobutrazol (PPP₃₃₃) upregulated it. Bimolecular fluorescence complementation (BiFC) assays showed that the MiLFY protein can interact with zinc-finger protein 4 (ZFP4) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (MiSOC1D). Taken together, these results indicate that MiLFY plays a pivotal role in controlling mango flowering, and that it is regulated by gibberellin and paclobutrazol.