MtPAP1表达特性及异源表达对拟南芥有机态磷吸收的影响

高磷条件下,MtPAP1主要在叶片中表达;在低磷条件下,MtPAP1在叶片中表达水平较低,在根系中的表达量则较高,超过了其它器官中的表达量。在以植酸盐为唯一磷供源的条件下,与对照相比,超量表达MtPAP1的拟南芥转基因植株中,根系细胞间隙中的酸性磷酸化酶(APase)活性明显提高。HPLC分析表明,液体培养基中的有机态磷可被转基因拟南芥分泌的APase快速降解。在以植酸盐为唯一磷供源条件下,超量表达MtPAP1的拟南芥转基因植株的生物学产量、植株无机磷含量和全磷含量明显高于野生种。

豆奶甲醇提取物中抗氧化活性物质的鉴定

本文主要在研究了豆奶甲醇提取物有无抗氧化活性的基础上,分析鉴定了其可能存在的抗氧化物质,为相关研究提供理论依据。测定抗氧化能力的方法包括1,1-二苯基-2-三硝基苯肼(1,1-Diphenyl-2-picrylhydrazyl radical,DPPH)法和铁离子还原能力(Ferricreducing antioxidant power,FRAP)法。比色法测定总酚含量和总黄酮含量。通过超高效液相-四极杆飞行时间串联质谱(Ultra-highperformance liquid chromatography-quadruple time-of-flight-mass spectrometry,UHPLC-QTOF-MS)测定豆奶甲醇提取液中活性成分的种类和含量。实验表明,豆奶甲醇提取液具有一定的抗氧化性,且DPPH法和FRAP法测定豆奶抗氧化值不存在显著性差异。豆奶甲醇提取物的总酚含量(285.77±2.93μg/mL)远高于总黄酮含量(11.27±0.51μg/mL)。鉴定出5种化合物,其中只有6,7,4'-三羟基黄酮和Rha-Ara-GlcA-SoyB与抗氧化值存在正相关性。异黄素和大豆甙元两种异黄酮与抗氧化值存在着负相关,6,7,4'-三羟基黄酮可能是主要的抗氧化物质。

市售橙汁抗氧化活性物质的研究

利用1,1-二苯基-2-三硝基苯肼(1,1-Diphenyl-2-picrylhydrazyl radical,DPPH)法和铁离子还原能力(Ferric reducing antioxidant power,FRAP)法、福林酚法以及超高效液相-四极杆飞行时间串联质谱(Ultra-high performance liquid chromatography-quadruple time-of-flight-mass spectrometry,UPLC-QTOF-MS)法,分别对市售橙汁的抗氧化活性、总酚含量以及抗氧化活性物质种类进行了研究,以达到明确橙汁的抗氧化活性有无,以及研究其主要抗氧化活性物质种类的目的。实验结果表明橙汁甲醇提取液具有清除DPPH自由基的能力[(1023±9.56)μg/m L,VCEAC(Vitamin cequivalent antioxidant capaciy)]和还原铁离子能力[(804.49±7.57)μg/m L,VCEAC],并且总酚含量丰富[(601.57±9.92)μg/m L,阿魏酸当量]。橙汁中鉴定出的主要的酚类化合物为柚皮素和橙皮苷,其中柚皮素是主要的抗氧化活性物质之一。

Characterization of the Formation of Branched Short-Chain Fatty Acid：CoAs for Bitter Acid Biosynthesis in Hop Glandular Trichomes

Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop （Humulus lupulus） glandular trichomes. Branched short-chain acyI-CoAs （e.g. isobutyryI-CoA, isovaleryl- CoA and 2-methylbutyryI-CoA）, derived from the degradation of branched-chain amino acids （BCAAs）, are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyI-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HICCL2 （carboxyl CoA ligase） protein had high specific activity for isovaleric acid as a substrate （Kcat/Km = 4100 s-~ M-l）, whereas recombinant HICCL4 specifically utilized isobutyric acid （Kcat/Km = 1800 s-1 M-1） and 2-methylbutyric acid （Kcat/ Km = 6900 s-1 M-~） as substrates. Both HICCLs, like hop valerophenone synthase （HIVPS）, were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HICCL2 and HICCL4 with HIVPS in yeast led to significant production of acylphloroglucinols （the direct precursors for bitter acid biosynthesis）, which further confirmed the biochemical function of these two HICCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid：CoA ligases in planta are also discussed.

Intracellular Transport of Plant Viruses： Finding the Door out of the Cell

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein （MP）, a protein dedicated to enlarging the pore size of plasmodesmata （PD） and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread.

A Transcriptome Atlas of Physcomitrella patens Provides Insights into the Evolution and Development of Land Plants

Identifying the genetic mechanisms that underpin the evolution of new organ and tissue systems is an aim of evolutionary developmental biology. Comparative functional genetic studies between angiosperms and bryophytes can define those genetic changes that were responsible for developmental innovations. Here, we report the generation of a transcriptome atlas covering most phases in the life cycle of the model bryo- phyte Physcomitrella patens, including detailed sporophyte developmental progression. We identified a comprehensive set of sporophyte-specific transcription factors, and found that many of these genes have homologs in angiosperms that function in developmental processes such as flowering and shoot branching. Deletion of the PpTCP5 transcription factor results in development of supernumerary sporangia attached to a single seta, suggesting that it negatively regulates branching in the moss sporophyte. Given that TCP genes repress branching in angiosperms, we suggest that this activity is ancient. Finally, compar- ison of P. patens and Arabidopsis thaliana transcriptomes led us to the identification of a conserved core of transcription factors expressed in tip-growing cells. We identified modifications in the expression patterns of these genes that could account for developmental differences between P. patens tip-growing cells and A. thaliana pollen tubes and root hairs.

Roles of Ubiquitination in the Control of Phosphate Starvation Responses in Plants

Throughout evolution, plants have evolved sophisticated adaptive responses that allow them to grow with a limited supply of phos-phate, the preferential form in which the essential macronutrient phosphorus is absorbed by plants. Most of these responses are aimed to increase phosphate availability and acquisition through the roots, to optimize its usage in metabolic processes, and to protect plants from the deleterious effects of phosphate deficiency stress. Regulation of these adaptive responses requires fine percep- tion of the external and internal phosphate levels, and a complex signal transduction pathway that integrates information on the phosphate status at the whole-plant scale. The molecular mecha-nisms that participate in phosphate homeostasis include transcriptional control of gene expression, RNA silencing mediated by microRNAs, regulatory non-coding RNAs of miRNA activity, phosphate transporter trafficking, and post-translational modification of proteins, such as phosphorylation, sumoylation and ubiquitination. Such a varied regulatory repertoire reflects the complexity intrinsic to phosphate surveying and signaling pathways. Here, we describe these regulatory mechanisms, emphasizing the increasing importance of ubiquitination in the control of phosphate starvation responses.

Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts

Blue native-PAGE （BN-PAGE） resolves protein complexes in their native state. When combined with immu- noblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry （MS/MS） approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed iden- tification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteo- mic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facil- itate scaled-up comparative analyses, as we demonstrate with the reticulata chloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illus- trate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments.

Ectopic Expression of a Phytase Gene from Medicago truncatula Barrel Medic Enhances Phosphorus Absorption in Plants

In the present study, the phosphorus-absorption capacity of transgenic Arabidopsis plants ectopically ex- pressing a novel phytase gene from Medicago truncatula Barrel Medic was evaluated. A full-length cDNA encoding an extracellular form of phytase was isolated from the model legume M. truncatula. The phytase gene （MtPHY1） has an open reading frame of I 632 bp predicted to encode 543 amino acids, including an N- terminal signal peptide of 27 amino acids. The genomic sequence of the MtPHY1 gene is 5 151 bp, containing seven exons interrupted by six introns. Under high-Pi （2 mmol/L） growth conditions, higher levels of MtPHY1 transcripts accumulated in the leaf and stem than in the root. The transcript level was reduced in the stem and increased in the root, with no obvious changes in the hybridization signal detected in the leaf under IowPi （10 pmol/L） conditions. Chimeric transgenes were constructed by placing MtPHY1 under the control of the constitutive CaMV35S promoter and the root-specific MtPT1 promoter. Phytase activities in root apoplast of transgenic Arabidopsis were 12.3- to 16.2-fold of that in control plants. The phytase expressed was secreted into the rhizosphere, as demonstrated by HPLC analysis of phytate degradation by root exudates. Ectopic expression of MtPHY1 in Arabidopsis, leading to significant improvement in organic phosphorus absorption and plant growth, indicated that MtPHY1 has great potential for improving plant phosphorus absorption and phytoremediation.

Plasma Membrane-Associated SCAR Complex Subunits Promote Cortical F-Actin Accumulation and Normal Growth Characteristics in Arabidopsis Roots

The ARP2/3 complex, a highly conserved nucleator of F-actin polymerization, and its activator, the SCAR complex, have been shown to play important roles in leaf epidermal cell morphogenesis in Arabidopsis. However, the intracellular site（s） and function（s） of SCAR and ARP2/3 complex-dependent actin polymerization in plant cells remain unclear. We demonstrate that putative SCAR complex subunits BRK1 and SCAR1 are localized to the plasma membrane at sites of cell growth and wall deposition in expanding cells of leaves and roots. BRK1 localization is SCAR-dependent, providing further evidence of an association between these proteins in vivo. Consistent with plasma membrane localization of SCAR complex subunits, cortical F-actin accumulation in root tip cells is reduced in brkl mutants. Moreover, mutations disrupting the SCAR or ARP2/3 complex reduce the growth rate of roots and their ability to penetrate semi-solid medium, suggesting reduced rigidity. Cell walls of mutant roots exhibit abnormal structure and composition at intercellular junctions where BRK1 and SCAR1 are enriched in the adjacent plasma membrane. Taken together, our results suggest that SCAR and ARP2/3 complex-dependent actin polymerization promotes processes at the plasma membrane that are important for normal growth and wall assembly.

Cloning and Characterization of a Novel Purple Acid Phosphatase Gene （MtPAP1） from Medicago truncatula Barrel Medic

A novel purple acid phosphatase gene （MtPAP1） was Isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp In length with an open reading frame （ORF） of 1 398 bp capable of encoding an N-terminal signal peptlde of 23 amino acids. The transcripts of MtPAP1 were mainly detected In leaves under high-phosphate conditions, whereas under low-phosphate conditions the transcript level was reduced In leaves and Increased In roots, with the strongest hybridization signal detected In roots. A chimeric gene construct fusing MtPAP1 and GFPwas made In which the fusion was driven by the CaMV35S promoter. Transgenlc Arabidopsis plants carrying the chimeric gene constructs showed that the fusion protein was mainly located at the apoplast based on confocal mlcroecoplc analysis, showing that MtPAP1 could be secreted to the outside of the cell directed by the signal peptlde at the N-terminal. The coding region of MtPAP1 without signal peptlde was Inserted Into the prokaryotlc expression vector pET-30a （＋） and overexpressed In Escherichla coil BL21 （DE3）. The acid phosphatase （APase） proteins extracted from bacterial culture were found largely based on sodium dodecyl sulfate-polyecrylamlde gel electrophoresls. An enzyme activity assay demonstrated that the APase activity In the transformed bacteria was 3.16-fold higher than that of control. The results Imply that MtPAP1 functions to Improve phosphorus acquisition In plants under conditions of phosphorus （P） stress.

Annual survival and site fidelity of free-ranging white-tailed deer (Odocoileus virginianus): comparative demography before (1983- 1992) and after (1993-2005) spatial confinement

Survival and movement are important demographic variables influencing the dynamics of large herbivores with implications for management and evolution of life-history strategies.Management practices such as spatial con-finement and harvest regulation attempt to control survival and movement for the sustainability of harvested deer populations,but a paucity of long-term data exists on these management practices.We examined annual survival and site fidelity of free-ranging white-tailed deer(Odocoileus virginianus)over 10 years(1983-1992)to compare demographic parameters after spatial confinement(1993-2005).We used capture records(n=174;104 females,70 males),marked deer recaptures(n=42),and dead recoveries(n=68)to estimate sex-specif-ic,age-specific and time-specific parameters.We found that annual female survival was 50%from 1983-1987 during a period of intense harvest,but increased to 93.7%after intense harvesting was eliminated.Prior to spa-tial confinement,annual survival of marked male deer averaged 36.7%-42.5%.After spatial confinement,annu-al survival increased on average for males(58%-99%)and females(77%-98%).Females showed high levels of site fidelity(>99%)prior to spatial confinement,whereas males showed much less site fidelity(≤4.5%for the 2 top-ranking models).During spatial confinement,the semi-impermeable fence effectively increased site fidelity of males(≥56%).These results stem from long-term study(23 years)of a large herbivore experiencing changes to life-history,resulting from changes in management that were applied to the population and aimed at altering population demographics,for sustainability of a harvestable population of deer.

Incorporating within- and between-patch resource selection in identification of critical habitat for brood-rearing greater sage-grouse

Introduction:Incorporating information on animal behavior in resource-based predictive modeling(e.g.,occurrence mapping)can elucidate the relationship between process and spatial pattern and depict habitat in terms of its structure as well as its function.In this paper,we assigned location data on brood-rearing greater sage-grouse(Centrocercus urophasianus)to either within-patch(encamped)or between-patch(traveling)behavioral modes by estimating a movement-based relative displacement index.Objectives were to estimate and validate spatially explicit models of within-versus between-patch resource selection for application in habitat management and compare these models to a non-behaviorally adjusted model.Results:A single model,the vegetation and water resources model,was most plausible for both the encamped and traveling modes,including the non-behaviorally adjusted model.When encamped,sage-grouse selected for taller shrubs,avoided bare ground,and were closer to mesic areas.Traveling sage-grouse selected for greater litter cover and herbaceous vegetation.Preference for proximity to mesic areas was common to both encamped and traveling modes and to the non-behaviorally adjusted model.The non-behaviorally adjusted map was similar to the encamped model and validated well.However,we observed different selection patterns during traveling that could have been masked had behavioral state not been accounted for.Conclusions:Characterizing habitat that structured between-patch movement broadens our understanding of the habitat needs of brood-rearing sage-grouse,and the combined raster surface offers a reliable habitat management tool that is readily amenable to application by GIS users in efforts to focus sustainable landscape management.

Functional characterization of caffeic acid O-methyltransferase in internode lignification of switchgrass(Panicum virgatum)

Caffeic acid O-methyltransferase(COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1,was isolated from switchgrass(Panicum virgatum), a C4 warm-season dual-purpose forage and bioenergy crop. Our results showed that recombinant PvCOMT1 enzyme protein catalyzed the methylation of 5-OH coniferyl alcohol, 5-OH coniferaldehyde(CAld5H) and 5-OH ferulic acid. Further in vitro studies indicate that CAld5H can dominate COMT-mediated reactions by inhibiting the methylation of the other substrates. Transgenic switchgrass plants generated by an RNAi approach were further employed to study the function of COMT in internode lignification. A dramatic decrease in syringyl lignin units coupled with an obvious incorporation in 5-OH guaiacyl lignin units were observed in the COMT-RNAi transgenic plants. However, the constitutive suppression of COMT in switchgrass plants altered neither the pattern of lignin deposition along the stem nor the anatomical structure of internodes. Consistent with the biochemical characterization of PvCOMT1, a significant decrease in sinapaldehyde was found in the COMT-RNAi transgenic switchgrass plants, suggesting that CAld5H could be the optimal intermediate in the biosynthesis syringyl lignin.