Molecular Cloning and Characterization of Two 9-Lipoxygenase Genes from Taxus chinensis

Plant lipoxygenases (LOXs) are functionally diverse class of dioxygenases involved in multiple physiological processes such as plant growth, biotic and abiotic stress responses, and secondary metabolite accumulation. In this paper, two LOX genes, TcLOX1 and TcLOX2, were cloned from Taxus chinensis cells. Multiple alignment of the deduced amino acid sequences with those of other plants demonstrated the putative LH2/PLAT domain, lipoxygenase iron-binding catalytic domain, lipoxygenase_1 and lipoxygenase_2 signature sequences. Phylogenetic analysis suggested that TcLOX1 and TcLOX2 putative proteins are most probably 9-LOXs, and shared the highest identity with the tea plant CsLOX1 and Picea sitchensis LOX genes, respectively. Semiquantitative RT-PCR analysis showed that TcLOX1 was preferentially expressed in stem and root, while TcLOX2 was preferentially expressed in root. Real-time quantitative PCR analysis showed that a strong upregulation of TcLOX1 was observed in response to methyl jasmonate and abscisic acid (ABA), while TcLOX2 was strongly upregulated by ABA. However, TcLOX1 and TcLOX2 were nearly not responding to salicylic acid. These data suggest both TcLOX1 and TcLOX2 play an important role in T. chinensis, and they are required in different physiological processes involved in different plant signals in vivo.     

Characterization and functional annotation of nested transposable elements in eukaryotic genomes

The movement of transposable elements (TE) in eukaryotic genomes can often result in the occurrence of nested TEs (the insertion of TEs into pre-existing TEs). We performed a general TE assessment using available databases to detect nested TEs and analyze their characteristics and putative functions in eukaryote genomes. A total of 802 TEs were found to be inserted into 690 host TEs from a total number of 11,329 TEs. We reveal that repetitive sequences are associated with an increased occurrence of nested TEs and sequence biased of TE insertion. A high proportion of the genes which were associated with nested TEs are predicted to localize to organelles and participate in nucleic acid and protein binding. Many of these function in metabolic processes, and encode important enzymes for transposition and integration. Therefore, nested TEs in eukaryotic genomes may negatively influence genome expansion, and enrich the diversity of gene expression or regulation.     

Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA

microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3'-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT-PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.     

Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g(-1) and 13 μg g(-1) fresh weight (FW), respectively, and for BS 19 μg g(-1) and 0.27 μg g(-1) FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.     

Involvement of the N-terminal B-box Domain of Arabidopsis BBX32 Protein in Interaction with Soybean BBX62 Protein

Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn(2+)-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.     

Topologically conserved residues direct heme transport in HRG-1-related proteins

Caenorhabditis elegans and human HRG-1-related proteins are conserved, membrane-bound permeases that bind and translocate heme in metazoan cells via a currently uncharacterized mechanism. Here, we show that cellular import of heme by HRG-1-related proteins from worms and humans requires strategically located amino acids that are topologically conserved across species. We exploit a heme synthesis-defective Saccharomyces cerevisiae mutant to model the heme auxotrophy of C. elegans and demonstrate that, under heme-deplete conditions, the endosomal CeHRG-1 requires both a specific histidine in the predicted second transmembrane domain (TMD2) and the FARKY motif in the C terminus tail for heme transport. By contrast, the plasma membrane CeHRG-4 transports heme by utilizing a histidine in the exoplasmic (E2) loop and the FARKY motif. Optimal activity under heme-limiting conditions, however, requires histidine in the E2 loop of CeHRG-1 and tyrosine in TMD2 of CeHRG-4. An analogous system exists in humans, because mutation of the synonymous histidine in TMD2 of hHRG-1 eliminates heme transport activity, implying an evolutionary conserved heme transport mechanism that predates vertebrate origins. Our results support a model in which heme is translocated across membranes facilitated by conserved amino acids positioned on the exoplasmic, cytoplasmic, and transmembrane regions of HRG-1-related proteins. These findings may provide a framework for understanding the structural basis of heme transport in eukaryotes and human parasites, which rely on host heme for survival.     

Enhanced sensitivity to higher ozone in a pathogen-resistant tobacco cultivar

Investigations of the effects of elevated ozone (O(3)) on the virus-plant system were conducted to inform virus pathogen management strategies better. One susceptible cultivar of tobacco (Nicotiana tabacum L. cv. Yongding) and a resistant cultivar (Nicotiana tabacum L. cv. Vam) to Potato virus Y petiole necrosis strain (PVY(N)) infection were grown in open-top chambers under ambient and elevated O(3) concentrations. Above-ground biomass, foliage chlorophyll, nitrogen and total non-structural carbohydrate (TNCs), soluble protein, total amino acid (TAA) and nicotine content, and peroxidase (POD) activity were measured to estimate the effects of elevated O(3) on the impact of PVY(N) in the two cultivars. Results showed that under ambient O(3), the resistant cultivar possessed greater biomass and a lower C/N ratio after infection than the susceptible cultivar; however, under elevated O(3), the resistant cultivar lost its biomass advantage but maintained a lower C/N ratio. Variation of foliar POD activity could be explained as a resistance cost which was significantly correlated with biomass and C/N ratio of the tobacco cultivar. Chlorophyll content remained steady in the resistant cultivar but decreased significantly in the susceptible cultivar when stressors were applied. Foliar soluble protein and free amino acid content, which were related to resistance cost changes, are also discussed. This study indicated that a virus-resistant tobacco cultivar showed increased sensitivity to elevated O(3) compared to a virus-sensitive cultivar.     

Transformation with a gene for myo-inositol O-methyltransferase enhances the cold tolerance of Arabidopsis thaliana

In this study, we report a function of myo-inositol-O-methyltransferase (Imt1) in response to low temperature stress using transgenic Arabidopsis thaliana. Imt1 gene was constructed identical to the Imt1 gene from a halophyte Mesembryanthemum crystallinum. After cold stress, the Imt1 transgenic plants exhibited stronger growth than the wild type plants. The elevated cold tolerance of the Imt1 over-expressing plants was confirmed by the lower electrolyte leakage and accumulation of malondialdehyde, but higher proline and soluble sugar contents in transgenic than wild type plants.     

Electrostatic interaction between inactivation ball and T1-S1 linker region of Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a "ball and chain" inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the "ball and chain" inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1-S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1-S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.