Regulation of flowering time via miR172-mediated APETALA2-like expression in ornamental gloxinia (Sinningia speciosa).

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.

Progress on the autophagic regulators and receptors in plants.

Autophagy is an evolutionarily highly conserved catabolic pathway among eukaryotic cells that protects the organisms against environmental stress. Normally, autophagy is mainly involved with autophagy-related proteins(ATGs) and autophagic regulators including a series of cytoplasmic proteins and small molecules. Besides, the selective autophagy, which targets damaged organalles or protein aggregates, is mediated by the additional receptors to help the ATGs recognize different substrates. In this review, we summarize recent advances in autophagic regulators like ROS(Reactive oxygen species), TOR(Target of rapamycin) and receptors like NBR1(Neighbor of BRCA1 gene protein), RPN10(Regulatory particle non-ATPase 10) as well as their functional mechanisms mainly in Arabidopsis thaliana.

Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions.

[The regulatory roles of small RNAs in phytohormone signaling pathways].

Phytohormones are signaling molecules that control plant growth and development. Recent studies revealed that non-coding small RNAs play critical roles in plant development and stress responses via phytohormone signaling pathways. In this review, we summarize the present knowledge on the microRNAs (miRNAs) and secondary short interfering RNAs (siRNAs) involved in phytohormone signaling pathways, which include auxin, gibberellic acid, brassinosteroid and abscisic acid pathways. We also discuss their possible implications in phytohormone crosstalk during specific developmental processes.

Purification and characterization of Plantaricin ZJ5, a new bacteriocin produced by Lactobacillus plantarum ZJ5.

The aim of this study is to investigate the antimicrobial potential of Lactobacillus plantarum ZJ5, a strain isolated from fermented mustard with a broad range of inhibitory activity against both Gram-positive and Gram-negative bacteria. Here we present the peptide plantaricin ZJ5 (PZJ5), which is an extreme pH and heat-stable. However, it can be digested by pepsin and proteinase K. This peptide has strong activity against Staphylococcus aureus. PZJ5 has been purified using a multi-step process, including ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic interactions and reverse-phase chromatography. The molecular mass of the peptide was found to be 2572.9 Da using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The primary structure of this peptide was determined using amino acid sequencing and DNA sequencing, and these analyses revealed that the DNA sequence translated as a 44-residue precursor containing a 22-amino-acid N-terminal extension that was of the double-glycine type. The bacteriocin sequence exhibited no homology with known bacteriocins when compared with those available in the database, indicating that it was a new class IId bacteriocin. PZJ5 from a food-borne strain may be useful as a promising probiotic candidate.

Protective effects of ETC complex III and cytochrome c against hydrogen peroxide-induced apoptosis in yeast.

In mammals, the mitochondrial electron transfer components (ETC) complex III and cytochrome c (cyt c) play essential roles in reactive oxygen species (ROS)-induced apoptosis. However, in yeast, the functions of cyt c and other ETC components remain unclear. In this study, three ETC-defective yeast mutants qcr7Δ, cyc1Δcyc7Δ, and cox12Δ, lacking cyt c oxidoreductase (complex III), cyt c, and cyt c oxidase (complex IV), respectively, were used to test the roles of these proteins in the response of cells to hydrogen peroxide (H2O2). Mutants qcr7Δ and cyc1Δcyc7Δ displayed greater H2O2 sensitivity than the wild-type or cox12Δ mutant. Consistent with this, qcr7Δ and cyc1Δcyc7Δ produced higher ROS levels, displayed derepressed expression of the proapoptotic genes AIF1, NUC1, and NMA111, but not YCA1, at the mRNA level, and were more vulnerable to H2O2-induced apoptosis. Interestingly, mutants lacking these proapoptotic genes displayed enhanced H2O2 tolerance, but unaffected ROS accumulation. Furthermore, the overexpression of antiapoptotic genes (Bcl-2, Ced-9, AtBI-1, and PpBI-1) reduced the levels of AIF1, NUC1, and NMA111 mRNAs, and reduced H2O2-induced cell death. Our findings identify two ETC components as early-inhibitory members of the ROS-mediated apoptotic pathway, suggesting their essential roles in metabolizing H2O2, probably by providing reduced cyt c, allowing cyt c peroxidase to remove H2O2 from the cells.

[Recent advances in the techniques of protein-protein interaction study].

Protein-protein interactions play key roles in the development of organisms and the response to biotic and abiotic stresses. Several wet-lab methods have been developed to study this challenging area,including yeast two-hybrid system, tandem affinity purification, Co-immunoprecipitation, GST Pull-down, bimolecular fluorescence complementation, fluorescence resonance energy transfer and surface plasmon resonance analysis. In this review, we discuss theoretical principles and relative advantages and disvantages of these techniques,with an emphasis on recent advances to compensate for limitations.

Association of specific pectin methylesterases with Al-induced root elongation inhibition in rice.

The negative charges of cell wall pectin molecules attributed by pectin methylesterase (PME, EC 3.1.1.11) contribute to Al binding capacity. We examined the expression profiles of 35 members of the PME gene family in the root apex of an Al-sensitive rice 'Zhefu802' under Al stress. While root elongation was inhibited by 40% after 3-h exposure to 25 µM Al, cell wall PME activity and the abundance of eight PME genes transcripts were increased. The same Al treatment which had almost no effect on root elongation of an Al-resistant rice ssp. japonica 'Nipponbare' did not change the expression patterns of these eight PME genes. However, when Al concentration was increased to 50 µM, by which the root elongation of 'Nipponbare' was inhibited by 40% too, the expression of these PME genes were also upregulated except two genes with no signal. These suggest a possible correlation between the upregulated genes and Al-induced inhibition of root elongation in rice. Furthermore, these eight PME genes behaved differently when subjected to CdCl2 and LaCl3 treatments, implying the specificity of different PME genes in response to different metal toxicities. The transgenic rice overexpressing one of these eight PME genes OsPME14 showed higher PME activity and Al content in root tip cell wall, and became more sensitive to Al stress, verifying the involvement of the specific PME gene in Al toxicity. Therefore, our results provided the molecular evidence to connect the expression of specific PME genes with the Al-induced inhibition of root elongation in rice.

De novo characterization of the Chinese fir (Cunninghamia lanceolata) transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.

BACKGROUND: Chinese fir (Cunninghamia lanceolata) is an important timber species that accounts for 20-30% of the total commercial timber production in China. However, the available genomic information of Chinese fir is limited, and this severely encumbers functional genomic analysis and molecular breeding in Chinese fir. Recently, major advances in transcriptome sequencing have provided fast and cost-effective approaches to generate large expression datasets that have proven to be powerful tools to profile the transcriptomes of non-model organisms with undetermined genomes. RESULTS: In this study, the transcriptomes of nine tissues from Chinese fir were analyzed using the Illumina HiSeq™ 2000 sequencing platform. Approximately 40 million paired-end reads were obtained, generating 3.62 gigabase pairs of sequencing data. These reads were assembled into 83,248 unique sequences (i.e. Unigenes) with an average length of 449 bp, amounting to 37.40 Mb. A total of 73,779 Unigenes were supported by more than 5 reads, 42,663 (57.83%) had homologs in the NCBI non-redundant and Swiss-Prot protein databases, corresponding to 27,224 unique protein entries. Of these Unigenes, 16,750 were assigned to Gene Ontology classes, and 14,877 were clustered into orthologous groups. A total of 21,689 (29.40%) were mapped to 119 pathways by BLAST comparison against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The majority of the genes encoding the enzymes in the biosynthetic pathways of cellulose and lignin were identified in the Unigene dataset by targeted searches of their annotations. And a number of candidate Chinese fir genes in the two metabolic pathways were discovered firstly. Eighteen genes related to cellulose and lignin biosynthesis were cloned for experimental validating of transcriptome data. Overall 49 Unigenes, covering different regions of these selected genes, were found by alignment. Their expression patterns in different tissues were analyzed by qRT-PCR to explore their putative functions. CONCLUSIONS: A substantial fraction of transcript sequences was obtained from the deep sequencing of Chinese fir. The assembled Unigene dataset was used to discover candidate genes of cellulose and lignin biosynthesis. This transcriptome dataset will provide a comprehensive sequence resource for molecular genetics research of C. lanceolata.

[Recent research progress of biogenesis and functions of miRNA*].

MicroRNA*s are about 22nt noncoding RNAs, which are processed from precursors with a characteristic hairpin secondary structure in the biogenesis of microRNAs. Recently, miRNA* strands were shown to mediate post-transcriptional regulatory networks, rather than serve merely as non-functional by-product in general view. Unlike miRNAs bound to AGO1, miRNA* strands are bound to AGO2 to form RISC duplex to mediate RNAi, which is similar to siRNA. This paper mainly reviewed the recent research progresses on miRNA*, such as the biosynthesis, biological characteristics, and functions.

Regulation of auxin response by miR393-targeted transport inhibitor response protein 1 is involved in normal development in Arabidopsis.

miR393, which is encoded by MIR393a and MIR393b in Arabidopsis, post-transcriptionally regulates mRNAs for the F-box auxin receptors TIR1 (Transport Inhibitor Response Protein 1), AFB1 (Auxin Signaling F-box Protein 1), AFB2 and AFB3. However, biological functions of the miR393-TIR1/AFBs module in auxin response and plant development is not fully understood. In the study herein, we demonstrate that miR393 accumulated in response to exogenous IAA treatment, and its induction was due to enhanced MIR393b transcription but not MIR393a. Overexpression of a miR393-resistant form of TIR1 (mTIR1) enhanced auxin sensitivity and led to pleiotropic effects on plant development including inhibition of primary root growth, overproduction of lateral roots, altered leave phenotype and delayed flowering. Furthermore, miR393 level was increased in 35S:mTIR1 plant, suggesting that TIR1 promoted the expression of miR393 by a feedback loop. The interaction between miR393 and its target indicates a fine adjustment to the roles of the miR393-TIR1 module, which is required for auxin responses in plant development.

Identification and characterization of two bamboo (Phyllostachys praecox) AP1/SQUA-like MADS-box genes during floral transition.

Bamboo (Bambusoideae) is by far the largest member of the grass family Poaceae, which is vital to the economy of many countries in the tropics and subtropics. However, the mechanism of flowering of bamboo (Phyllostachys praecox) is still unknown. In this study, we isolated two novel genes from P. praecox and evaluated their functional characteristics. The sequence and phylogenetic analysis indicated that these two genes, named PpMADS1 and PpMADS2, belong to FUL3 and FUL1 clade of Poaceae AP1/SQUA-like genes, respectively. The PpMADS2 possesses a truncated C terminus lacking the highly conserved paleoAP1 motif. It was further confirmed that the truncated C-terminal region was produced by natural sequence deletion in exons, but not by alternative splicing. Ectopic expression of PpMADS1 and PpMADS2 significantly promoted early flowering through upregulation of AP1 in Arabidopsis. Yeast two-hybrid experiments demonstrated that AP1 protein can interact with PpMADS1 but not PpMADS2, suggesting that these two genes may act differently in signaling early flowering of bamboo plants. RT-qPCR and in situ hybridization analysis revealed distinct expression patterns of these two genes in vegetative and reproductive tissues of bamboo. Taken together, our results suggest that both PpMADS1 and PpMADS2 are involved in floral transition, and PpMADS2 might play more important roles than PpMADS1 in floral development of Phyllostachys praecox.

Overexpression of the cucumber LEAFY homolog CFL and hormone treatments alter flower development in gloxinia (Sinningia speciosa).

Leafy (LFY) and LFY-like genes control the initiation of floral meristems and regulate MADS-box genes in higher plants. The Cucumber-FLO-LFY (CFL) gene, a LFY homolog in Cucumis sativus L. is expressed in the primordia, floral primordia, and each whirl of floral organs during the early stage of flower development. In this study, functions of CFL in flower development were investigated by overexpressing the CFL gene in gloxinia (Sinningia speciosa). Our results show that constitutive CFL overexpression significantly promote early flowering without gibberellin (GA(3)) supplement, suggesting that CFL can serve functionally as a LFY homolog in gloxinia. Moreover, GA(3) and abscisic acid (ABA) treatments could modulate the expression of MADS-box genes in opposite directions. GA(3) resembles the overexpression of CFL in the expression of MADS-box genes and the regeneration of floral buds, but ABA inhibits the expression of MADS-box genes and flower development. These results suggest that CFL and downstream MADS-box genes involved in flower development are regulated by GA(3) and ABA.

Molecular cloning, expression analyses and primary evolution studies of REV- and TB1-like genes in bamboo.

Most cultured bamboos are perennial woody evergreens that reproduce from rhizomes. It is unclear why some rhizome buds develop into aerial bamboo shoots instead of new rhizomes. REVOLUTA (REV)-like Class III homeodomain leucine-zipper (HD-Zip) proteins and TEOSINTE BRANCHED1 (TB1)-like transcription factors have been shown to play regulatory roles in meristem initiation and outgrowth. We cloned and analyzed the bamboo (Phyllostachys praecox C.D. Chu & C.S. Chao.) REV- (PpHB1) and TB1-like (PpTB1) gene. Gene expression was mainly detected by in situ hybridization. PpHB1 expression was detected in the tips of lateral buds, on the adaxial portion of the leaf and within the developing procambium, indicating its close correlation to rhizome bud formation and procambial development. PpTB1 expression was mainly detected on the top of buds at later developmental stages, suggesting it was more likely involved in bud outgrowth. Meristem genes might therefore serve as specific molecular markers of rhizome bud development and could be useful in studies designed to elucidate the mechanisms underlying bamboo shoot development. In addition, meristem genes such as TB1-like sequences may be useful in phylogenetic analyses of bamboo species.

Programmed cell death-involved aluminum toxicity in yeast alleviated by antiapoptotic members with decreased calcium signals.

The molecular mechanisms of aluminum (Al) toxicity and tolerance in plants have been the focus of ongoing research in the area of stress phytophysiology. Recent studies have described Al-induced apoptosis-like cell death in plant and animal cells. In this study, we show that yeast (Saccharomyces cerevisiae) exposed to low effective concentrations of Al for short times undergoes enhanced cell division in a manner that is dose and cell density dependent. At higher concentrations of Al or longer exposure times, Al induces cell death and growth inhibition. Several apoptotic features appear during Al treatment, including cell shrinkage, vacuolation, chromatin marginalization, nuclear fragmentation, DNA degradation, and DNA strand breaks, as well as concomitant cell aggregation. Yeast strains expressing Ced-9, Bcl-2, and PpBI-1 (a plant Bax inhibitor-1 isolated from Phyllostachys praecox), respectively, display more resistance to Al toxicity compared with control cells. Data from flow cytometric studies show these three antiapoptotic members do not affect reactive oxygen species levels, but decrease calcium ion (Ca(2+)) signals in response to Al stress, although both intracellular reactive oxygen species and Ca(2+) levels were increased. The data presented suggest that manipulation of the negative regulation process of programmed cell death may provide a novel mechanism for conferring Al tolerance.

[Effects of cu2+ on biosynthesis of camptothecin in cell cultures of Camptotheca acuminata].

Camptothecin is a strong anti-tumor compound isolated from Camptotheca acuminata. One of the most important way for the production of Camptothecin is by cell cultures of Camptotheca acuminata. The effect of Cu2+ on camptothecin accumulation in Camptotheca acuminata cell line was described in this paper. The results showed that the optimum CuCl2 concentration in B5 medium was 0.008 mg/mL, which increased camptothecin production for 30 times compare to the control while has no inhibitive effects on cell growth, at the same time, the peroxidase activity was increased and the anthocyanidin accumulation was inhibited. The promotive effects of Cu2+ on camptothecin accumulation in light was higher than that in dark.

Cloning, characterization and tissue specific expression of Amur tiger (Panthera tigris altaica) IGF-I.

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli.

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.

Nitric oxide mediates the fungal elicitor-induced hypericin production of Hypericum perforatum cell suspension cultures through a jasmonic-acid-dependent signal pathway.

Fungal elicitor prepared from the cell walls of Aspergillum niger induces multiple responses of Hypericum perforatum cells, including nitric oxide (NO) generation, jasmonic acid (JA) biosynthesis, and hypericin production. To determine the role of NO and JA in elicitor-induced hypericin production, we study the effects of NO scavenger 2- to 4-carboxyphenyl-4,4, 5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPITO), nitric oxide synthase inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea, and inhibitors of the octadecanoid pathway on elicitor-induced NO generation, JA biosynthesis, and hypericin production. Pretreatment of the cells with cPITO and JA biosynthesis inhibitors suppresses not only the elicitor-induced NO generation and JA accumulation but also the elicitor-induced hypericin production, which suggests that both NO and JA are involved in elicitor-induced hypericin biosynthesis. S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea and cPITO inhibit both elicitor-induced NO generation and JA biosynthesis, while JA biosynthesis inhibitors do not affect the elicitor-induced NO generation, indicating that JA acts downstream of NO generation and that its biosynthesis is regulated by NO. External application of NO via its donor sodium nitroprusside induces hypericin production in the absence of fungal elicitor. Sodium-nitroprusside-induced hypericin production is blocked by JA biosynthesis inhibitors, showing that JA biosynthesis is essential for NO-induced hypericin production. The results demonstrate a causal relationship between elicitor-induced NO generation, JA biosynthesis, and hypericin production in H. perforatum cells and indicate a sequence of signaling events from NO to hypericin production, within which NO mediates the elicitor-induced hypericin biosynthesis at least partially via a JA-dependent signaling pathway.

[Cloning and expression of pituitary prolactin gene in Ailuropoda melanoleuca].

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at molecular level. In this study,the pituitary prolactin (PRL) cDNA of giant panda was amplified by RT-PCR from pituitary total RNA and then cloned, sequenced and submitted to GenBank (GenBank accession No. AY161285). The sequence analysis revealed that the giant panda prolactin cDNA contains a 687-nucleotide open reading frame encoding the prolactin prohormone of 229 amino acid residues. The signal peptide contains 30 amino acid residues and the mature prolactin is composed of 199 amino acid residues. Then the DNA fragment amplified was subcloned into pGEX-4T-1 procaryotic expression plasmid and protein expression was induced by IPTG in Escherichia coil BL21. SDS-PAGE analysis revealed the PRL protein is infusible. The multiple sequence alignments revealed that the homology of giant panda is 95% to cat and pig, 80% - 70% to human, cow and goat, 52% to rat and 45.9% to mouse at the amino acid level. The 64th amino acid of giant panda prolactin is hydrophilic serine instead of hydrophobic proline of cat, goat, and cow or hydrophobic alanine of human.