Identification,Functional Study,and Promoter Analysis of HbMFT1, a Homolog of MFT from Rubber Tree (Hevea brasiliensis)

A homolog of MOTHER OF FT AND TFL1 (MFT) was isolated from Hevea brasiliensis and its biological function was investigated. Protein multiple sequence alignment and phylogenetic analysis revealed that HbMFT1 conserved critical amino acid residues to distinguish MFT, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1)-like proteins and showed a closer genetic relationship to the MFT-like group. The accumulation of HbMFT1 was generally detected in various tissues except pericarps, with the highest expression in embryos and relatively higher expression in roots and stems of seedlings, flowering inflorescences, and male and female flowers. HbMFT1 putative promoter analysis showed that tissue-specific, environmental change responsive and hormone-signaling responsive elements were generally present. HbMFT1 was strongly induced under a short-day condition at 28 °C, with the highest expression after the onset of a day. Overexpression of HbMFT1 inhibited seed germination, seedling growth, and flowering in transgenic Arabidopsis. The qRT-PCR further confirmed that APETALA1 (AP1) and FRUITFULL (FUL) were drastically down-regulated in 35S::HbMFT1 plants. A histochemical β-glucuronidase (GUS) assay showed that HbMFT1::GUS activity was mainly detected in stamens and mature seeds coinciding with its original expression and notably induced in rosette leaves and seedlings of transgenic Arabidopsis by exogenous abscisic acid (ABA) due to the presence of ABA cis-elements in HbMFT1 promoter. These results suggested that HbMFT1 was mainly involved in maintenance of seed maturation and stamen development, but negatively controlled germination, growth and development of seedlings and flowering. In addition, the HbMFT1 promoter can be utilized in controlling transgene expression in stamens and seeds of rubber tree or other plant species.     

Core RNAi Machinery and Sid1, a Component for Systemic RNAi,in the Hemipteran Insect,Aphis glycines

RNA interference (RNAi) offers a novel tool to manage hemipteran pests. For the success of RNAi based pest control in the field, a robust and systemic RNAi response is a prerequisite. We identified and characterized major genes of the RNAi machinery, Dicer2 (Dcr2), Argonaute2 (Ago2), and R2d2 in Aphis glycines, a serious pest of soybean. The A. glycines genome encodes for at least one copy of Dcr2, R2d2 and Ago2. Comparative and molecular evolution analyses (dN/dS) showed that domain regions of encoded proteins are highly conserved, whereas linker (non-domain) regions are diversified. Sequence homology and phylogenetic analyses suggested that the RNAi machinery of A. glycines is more similar to that of Tribolium casteneum as compared to that of Drosophila melanogaster. We also characterized Sid1, a major gene implicated in the systemic response for RNAi-mediated gene knockdown. Through qPCR, Dcr2, R2d2, Ago2, and Sid1 were found to be expressed at similar levels in various tissues, but higher expression of Dcr2, R2d2, and Ago2 was seen in first and second instars. Characterization of RNAi pathway and Sid1 in A. glycines will provide the foundation of future work for controlling one of the most important insect pests of soybean in North America.     

ZmCIPK21, A Maize CBL-Interacting Kinase,Enhances Salt Stress Tolerance in Arabidopsis thaliana

Salt stress represents an increasing threat to crop growth and yield in saline soil. In this study, we identified a maize calcineurin B-like protein-interacting protein kinase (CIPK), ZmCIPK21, which was primarily localized in the cytoplasm and the nucleus of cells and displayed enhanced expression under salt stress. Over-expression of ZmCIPK21 in wild-type Arabidopsis plants increased their tolerance to salt, as supported by the longer root lengths and improved growth. The downstream stress-response genes, including dehydration-responsive element-binding (DREB) genes were also activated in transgenic plants over-expressing ZmCIPK21. In addition, introduction of the transgenic ZmCIPK21 gene into the Arabidopsis mutant cipk1-2 rescued the salt-sensitive phenotype under high salt stress. Measurement of Na⁺ and K⁺ content in transgenic plants showed that over-expression of ZmCIPK21 decreased accumulation of Na⁺ and allowed retention of relatively high levels of K⁺, thereby enhancing plant tolerance to salt conditions.     

Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana

Flowering is a pivotal event in the life cycle of plants. miR172 has been widely confirmed to play critical roles in flowering time control by regulating its target gene expression in Arabidopsis. However, the role of its counterpart in soybean remains largely unclear. In the present study, we found that the gma-miR172a was regulated by a GIGANTEA ortholog, GmGIa, in soybean through miRNA metabolism. The expression analysis revealed that gma-miR172a has a pattern of diurnal rhythm expression and its abundance increased rapidly as plants grew until the initiation of flowering phase in soybean. One target gene of gma-miR172a, Glyma03g33470, was predicted and verified using a modified RLM 5′-RACE (RNA ligase-mediated rapid amplification of 5′ cDNA ends) assay. Overexpression of gma-miR172a exhibited an early flowering phenotype and the expression of FT, AP1 and LFY were simultaneously increased in gma-miR172a-transgenic Arabidopsis plants, suggesting that the early flowering phenotype was associated with up-regulation of these genes. The overexpression of the gma-miR172a-resistant version of Glyma03g33470 weakened early flowering phenotype in the toe1 mutant of Arabidopsis. Taken together, our results suggested that gma-miR172a played an important role in GmGIa-mediated flowering by repressing Glyma03g33470, which in turn increased the expression of FT, AP1 and LFY to promote flowering in soybean.     

Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective

Maize (Zea mays L.) is a host to numerous pathogenic species that impose serious diseases to its ear and foliage, negatively affecting the yield and the quality of the maize crop. A considerable amount of research has been carried out to elucidate mechanisms of maize-pathogen interactions with a major goal to identify defense-associated proteins. In this review, we summarize interactions of maize with its agriculturally important pathogens that were assessed at the proteome level. Employing differential analyses, such as the comparison of pathogen-resistant and susceptible maize varieties, as well as changes in maize proteomes after pathogen challenge, numerous proteins were identified as possible candidates in maize resistance. We describe findings of various research groups that used mainly mass spectrometry-based, high through-put proteomic tools to investigate maize interactions with fungal pathogens Aspergillus flavus, Fusarium spp., and Curvularia lunata, and viral agents Rice Black-streaked Dwarf Virus and Sugarcane Mosaic Virus.     

ZmGLP1, a Germin-like Protein from Maize,Plays an Important Role in the Regulation of Pathogen Resistance

A gene encoding a protein similar to germin-like proteins (GLPs) was obtained from maize (Zea mays) and designated as ZmGLP1. Based on the ZmGLP1 conserved domain and phylogenetic status, ZmGLP1 was grouped into GLP subfamily b and has high similarity to OsGLP8-14 from Oryza sativa. ZmGLP1 is expressed in different maize tissues during different growth stages and is mainly expressed in the stems and leaves. The induced expression patterns confirmed that ZmGLP1 is differentially expressed under abiotic and hormone stress; it had an early response to jasmonic acid (JA) and ethephon (ET) but a late response to salicylic acid (SA) and was significantly upregulated under Bipolaris maydis infection. The overexpression of ZmGLP1 in Arabidopsis improved the resistance to biotrophic Pseudomonas syringae pv. tomato DC3000 (PstDC3000) and necrotrophic Sclerotinia sclerotiorum by inducing the expression of JA signaling-related genes. Moreover, the hydrogen peroxide (H₂O₂) content increased due to the overexpression of ZmGLP1 in Arabidopsis after pathogen infection. Compared to the wild-type control, the H₂O₂ content of ZmGLP1-overexpressing Arabidopsis infected by PstDC3000 increased significantly but was lower in transgenic plants infected with S. sclerotiorum. Furthermore, high-performance liquid chromatography–tandem mass (HPLC-MS/MS) spectrometry showed that the JA contents of ZmGLP1-overexpressing Arabidopsis markedly increased after pathogen infection. However, the improved resistance of ZmGLP1-overexpressing Arabidopsis pretreated with the JA biosynthetic inhibitor, sodium diethyldithiocarbamate trihydrate (DIECA), was suppressed. Based on these findings, we speculate that ZmGLP1 plays an important role in the regulation of Arabidopsis resistance to biotrophic PstDC3000 and necrotrophic S. sclerotiorum; the regulatory effects are achieved by inducing plant oxidative burst activity and activation of the JA signaling pathway.     

LIM Mineralization Protein-1 Inhibits the Malignant Phenotypes of Human Osteosarcoma Cells

Osteosarcoma (OS), also known as osteogenic sarcoma, is the most common primary malignancy of bone tumor in children and adolescents. However, its underlying molecular pathogenesis is still only vaguely understood. Recently, LIM mineralization protein-1 (LMP-1) was reported to be an essential positive regulator of osteoblast differentiation. In the present study, we found that the expression of LMP-1 is downregulated in OS tissues compared with adjacent normal tissues. Moreover, we restored the expression of LMP-1 through a recombinant adenovirus. Overexpression of LMP-1 inhibited cell proliferation and invasion, arrested cell cycle progression, and induced apoptosis in vitro. Finally, ectopic LMP-1 expression suppressed the expression of Runx2 and BMP-2 in OS cells. These data demonstrate that LMP-1 is an essential tumor suppressor in the OS pathological process, which will provide a new opportunity for discovering and identifying novel effective treatment strategies.     

A Heavy Metal-Associated Protein (AcHMA1) from the Halophyte,Atriplex canescens (Pursh) Nutt., Confers Tolerance to Iron and Other Abiotic Stresses When Expressed in Saccharomyces cerevisiae

Many heavy metals are essential for metabolic processes, but are toxic at elevated levels. Metal tolerance proteins provide resistance to this toxicity. In this study, we identified and characterized a heavy metal-associated protein, AcHMA1, from the halophyte, Atriplex canescens. Sequence analysis has revealed that AcHMA1 contains two heavy metal binding domains. Treatments with metals (Fe, Cu, Ni, Cd or Pb), PEG6000 and NaHCO₃ highly induced AcHMA1 expression in A. canescens, whereas NaCl and low temperature decreased its expression. The role of AcHMA1 in metal stress tolerance was examined using a yeast expression system. Expression of the AcHMA1 gene significantly increased the ability of yeast cells to adapt to and recover from exposure to excess iron. AcHMA1 expression also provided salt, alkaline, osmotic and oxidant stress tolerance in yeast cells. Finally, subcellular localization of an AcHMA1/GFP fusion protein expressed in tobacco cells showed that AcHMA1 was localized in the plasma membrane. Thus, our results suggest that AcHMA1 encodes a membrane-localized metal tolerance protein that mediates the detoxification of iron in eukaryotes. Furthermore, AcHMA1 also participates in the response to abiotic stress.     

TP53inp1 Gene Is Implicated in Early Radiation Response in Human Fibroblast Cells

Tumor protein 53-induced nuclear protein-1 (TP53inp1) is expressed by activation via p53 and p73. The purpose of our study was to investigate the role of TP53inp1 in response of fibroblasts to ionizing radiation. γ-Ray radiation dose-dependently induces the expression of TP53inp1 in human immortalized fibroblast (F11hT) cells. Stable silencing of TP53inp1 was done via lentiviral transfection of shRNA in F11hT cells. After irradiation the clonogenic survival of TP53inp1 knockdown (F11hT-shTP) cells was compared to cells transfected with non-targeting (NT) shRNA. Radiation-induced senescence was measured by SA-β-Gal staining and autophagy was detected by Acridine Orange dye and microtubule-associated protein-1 light chain 3 (LC3B) immunostaining. The expression of TP53inp1, GDF-15, and CDKN1A and alterations in radiation induced mitochondrial DNA deletions were evaluated by qPCR. TP53inp1 was required for radiation (IR) induced maximal elevation of CDKN1A and GDF-15 expressions. Mitochondrial DNA deletions were increased and autophagy was deregulated following irradiation in the absence of TP53inp1. Finally, we showed that silencing of TP53inp1 enhances the radiation sensitivity of fibroblast cells. These data suggest functional roles for TP53inp1 in radiation-induced autophagy and survival. Taken together, we suppose that silencing of TP53inp1 leads radiation induced autophagy impairment and induces accumulation of damaged mitochondria in primary human fibroblasts.     

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.     

AaZFP3, a Novel CCCH-Type Zinc Finger Protein from Adonis amurensis,Promotes Early Flowering in Arabidopsis by Regulating the Expression of Flowering-Related Genes

CCCH-type zinc finger proteins (ZFP) are a large family of proteins that play various important roles in plant growth and development; however, the functions of most proteins in this family are uncharacterized. In this study, a CCCH-type ZFP, AaZFP3, was identified in the floral organ of Adonis amurensis. Quantitative real-time PCR (qPCR) analysis revealed that AaZFP3 was widely expressed in the flowers of A. amurensis. Subcellular localization analysis showed that the AaZFP3 protein was mainly localized to the cytoplasm in tobacco and Arabidopsis. Furthermore, the overexpression of AaZFP3 promoted early flowering in Arabidopsis under both normal and relatively low-temperature conditions. RNA-sequencing and qPCR analyses revealed that the expression of multiple key flowering-time genes was altered in transgenic Arabidopsis overexpressing AaZFP3 compared to wild-type. Of these genes, FLOWERING LOCUS T (AtFT) expression was most significantly up-regulated, whereas FLOWERING LOCUS C (AtFLC) was significantly down-regulated. These results suggest that the overexpression of AaZFP3 promotes early flowering in Arabidopsis by affecting the expression of flowering-time genes. Overall, our study indicates that AaZFP3 may be involved in flowering regulation in A. amurensis and may represent an important genetic resource for improving flowering-time control in other ornamental plants or crops.