Analysis of the local organization and dynamics of cellular actin networks

A ctin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.     

Differences in capacities of in vitro organ regeneration between two Arabidopsis ecotypes Wassilewskija and Columbia

In vitro organogenesis is well-controlled and thus provides an ideal system to study mechanisms of plant organ development. Although it has been well investigated for a long time that exogenous hormones play important roles in determining the types of organs regenerated in vitro, there is currently limited information available for other key factors that mediate de novo organ regeneration. Here, we reported simple and efficient one-step processes for evaluating capacities of inflorescence stem-derived in vitro organogenesis between two different ecotypes in Arabidopsis. Different types of organs, including shoots and roots were initiated from inflorescence stem explants cultured on the media containing 216 combinations of exogenous auxin and cytokinin. Further, we showed that Wassilewskija ecotype had the much higher shoot regeneration capacity than Columbia with different combinations of hormones, indicating that the ecotype is an essential factor determining de novo organogenesis. Our results also suggested that the defined expression patterns of genes involved in auxin and cytokinin biosynthesis were correlated with the variations in organogenesis capacities between the two ecotypes. Thus, in vitro organogenesis is likely regulated by ecotypes through mediating endogenous hormonal biosynthesis.     

Abscisic acid is required for somatic embryo initiation through mediating spatial auxin response in Arabidopsis

Abscisic acid (ABA) regulates many aspects of plant development, including somatic embryo (SE) initiation. However, mechanisms of ABA functions on SE initiation have remained to be investigated. In this study, we examined the endogenous ABA contents of calli in Arabidopsis during the SE inductive process. We further found that the capacity for SE initiation was strongly impaired by treatment of fluridone, a potent inhibitor of ABA biosynthesis, as well as by mutation of ABA biosynthetic gene ABA2, suggesting that ABA is required for SE initiation. Furthermore, treatment of fluridone inhibited local auxin biosynthesis and auxin polar transport in the embryonic calli, resulting in the disturbance of auxin response pattern and the decreased regeneration frequency of SEs. However, application of exogenous ABA in the medium almost recovered patterns of auxin response and SE initiation. Thus, the results suggest that ABA functions on SE initiation through mediating both auxin biosynthesis and polar transport for establishment of auxin response pattern in callus. Our study provides new information for understanding mechanisms of SE initiation.     

Comparative proteomics and physiological characterization of Arabidopsis thaliana seedlings in responses to Ochratoxin A

Ochratoxin A (OTA) is a mycotoxin that is primarily produced by Aspergillus ochraceus and Penicillium verrucosum. This mycotoxin is a contaminant of food and feedstock worldwide and may induce cell death in plants. To investigate the dynamic growth process of Arabidopsis seedlings in response to OTA stress and to obtain a better understanding of the mechanism of OTA toxicity towards Arabidopsis, a comparative proteomics study using 2-DE and MALDI-TOF/TOF MS/MS was performed. Mass spectrometry analysis identified 59 and 51 differentially expressed proteins in seedlings exposed to 25 and 45 μM OTA for 7 days, respectively. OTA treatment decreased root elongation and leaf area, increased anthocyanin accumulation, damaged the photosynthetic apparatus and inhibited photosynthesis. Treatment of the seedlings with 25 μM OTA enhanced energy metabolism, whereas higher concentration of OTA (45 μM) inhibited energy metabolism in the seedlings. OTA treatment caused an increase of ROS, an enhancement of antioxidant enzyme defense responses, disturbance of redox homeostasis and activation of lipid oxidation. Glutamine and S-adenosylmethionine metabolism may also play important roles in the response to OTA. In conclusion, our study provided novel insights regarding the response of Arabidopsis to OTA at the level of the proteome. These results are expected to be highly useful for understanding the physiological responses and dissecting the OTA response pathways in higher plants.     

Bacteria-mediated bisphenol A degradation

Bisphenol A (BPA) is an important monomer in the manufacture of polycarbonate plastics, food cans, and other daily used chemicals. Daily and worldwide usage of BPA and BPA-contained products led to its ubiquitous distribution in water, sediment/soil, and atmosphere. Moreover, BPA has been identified as an environmental endocrine disruptor for its estrogenic and genotoxic activity. Thus, BPA contamination in the environment is an increasingly worldwide concern, and methods to efficiently remove BPA from the environment are urgently recommended. Although many factors affect the fate of BPA in the environment, BPA degradation is mainly depended on the metabolism of bacteria. Many BPA-degrading bacteria have been identified from water, sediment/soil, and wastewater treatment plants. Metabolic pathways of BPA degradation in specific bacterial strains were proposed, based on the metabolic intermediates detected during the degradation process. In this review, the BPA-degrading bacteria were summarized, and the (proposed) BPA degradation pathway mediated by bacteria were referred.     

Defense against Pieris rapae in cabbage plants induced by Bemisia tabaci biotype B

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and Pieris rapae L. (Lepidoptera: Pieridae) are serious pests of vegetables, that can occur simultaneously on a single cabbage plant [Brassica oleracea var. capitata L. (Brassicaceae)]. We determined whether pre‐feeding or infestation by B. tabaci on cabbage could induce physiological and biochemical responses of the plant against P. rapae. Developmental time, length, and weight of immature P. rapae, and defense‐related plant compounds (SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; APX, ascorbate peroxidase) were measured. Development of P. rapae larvae was 21% slower on B. tabaci‐pre‐infested plants than on plants without B. tabaci infestation. When feeding on B. tabaci‐pre‐infested plants, 22% of P. rapae larvae pupated as compared with 83% on B. tabaci‐free plants. Weights of P. rapae from first to fourth instars that fed on B. tabaci‐pre‐infested plants were also reduced, whereas those of fifth instars and pupae were not. Similarly, body length of P. rapae from first to fourth instars was affected by B. tabaci pre‐infestation, whereas that of the fifth instars was not. Peroxidase and APX activities of the B. tabaci‐pre‐infested plants increased more than SOD and CAT. Peroxidase and SOD activities of B. tabaci and P. rapae co‐infested plants increased as compared with those of P. rapae‐infested plants; however, CAT and APX activities were not different between B. tabaci‐ and P. rapae‐infested plants. These results showed that B. tabaci infestation had a negative effect on P. rapae when they occurred simultaneously on the same host plant. The implications of the induced plant changes on the herbivore are discussed.     

嗜碱单胞菌的新型羧基转移酶α亚基基因Aa-accA及其抗盐碱性能

[目的]探讨解淀粉嗜碱单胞菌(Alkalimonas amylolytica)N10来源的羧基转移酶α亚基(Acetyl-coenzyme A carboxylase subunit alpha,AccA)基因Aa-accA对细菌及植物细胞耐盐碱性的作用.[方法]通过PCR方法从嗜碱菌N10基因组中扩增基因Aa-accA,并在大肠杆菌(Escherichia coli)K12中表达,通过测定工程菌及对照菌在不同盐浓度[0%,2%,4%,6%(W/V) NaCl]及不同碱性pH(8.0,8.5,9.0,9.5)的LB中生长12 h后的OD600值,以及二者在分别含6%(W/V) NaCl及pH 9的LB中的生长曲线,评价Aa-accA对大肠杆菌耐盐碱性的影响.同时以pPZP111为载体,构建了植物细胞重组表达载体,通过农杆菌介导方法将该基因转入烟草BY-2悬浮细胞表达,利用FDA染色方法测定经盐碱溶液处理后残存的活细胞数量评价该基因对植物细胞耐盐碱性的影响.[结果]PCR扩增得到基因Aa-accA,其ORF含957 bp,编码318个氨基酸的多肽,BLAST比对显示该基因为羧基转移酶α亚基(AccA)家族中的成员,其氨基酸序列与E.coli的AccA具有76%同源性;含有Aa-accA的E.coli K12相较于对照组在不同NaCl浓度及不同碱性pH的LB中表现出了明显的生长优势,特别是在6%(W/V) NaCl及pH 9的LB中培养12 h后,终OD600分别是对照菌的2.6倍和3.5倍;缺失体实验结果显示基因缺失的突变体E.coli K12△accA在6%(W/V) NaCl及pH 9的LB中不能正常生长,而含有Aa-accA基因的重组质粒使得E.coli K12△accA在同样条件下OD600值达到0.5和0.2;转入此基因的烟草BY-2细胞,经盐碱溶液处理后,其存活细胞比例高于野生型.[结论]本研究首次发现了Aa-accA基因与盐碱性的相关性,可提高大肠杆菌及烟草BY-2细胞的耐盐碱能力.     

高浓度氯苯优势降解菌的筛选及其降解酶的纯化

[目的]分离纯化出一株高浓度氯苯优势降解菌株,对其所产氯苯降解酶进行分离与纯化,为该菌株及其氯苯降解酶的研究提供理论参考.[方法]利用梯度富集培养技术和无菌滤纸片平板法分离菌株,通过形态特征及16S rRNA基因序列分析初步鉴定菌株,用气相色谱法测定培养液中氯苯浓度,以单位细胞氯苯降解率评价菌株对氯苯的降解能力,以氯苯降解率表示氯苯降解酶的活性.取纯化菌株的发酵酶液制备粗酶液,经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后,进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量.[结果]从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13,该菌株在以2000 mg/L氯苯为唯一碳源的无机盐培养基中仍能正常生长,其单位细胞氯苯降解率可达1.37 ×10-10.扫描电镜观察到该菌株细胞大小约为2.3 ×0.8μm,长有数根端生鞭毛.16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5％.所纯化的氯苯降解酶为胞外酶,带正电荷,其分子大小约为57 kDa.整个纯化过程中酶纯化倍数化达8.0倍,酶活回收率达52.51％,酶量回收率达6.57％.纯化后的氯苯降解酶在30℃-55℃和pH在6.0-8.0之间都保持较高的酶活性,其最适反应温度和pH分别在40℃和pH8.0左右.[结论]所分离的氯苯优势降解菌属于Lysinibacillus属菌株,该菌株能有效降解高浓度(500-2000 mg/L)氯苯废水,通过逐级分离纯化,可获得氯苯降解酶纯酶,纯化指标符合分离纯化基本规律,纯化效果较为理想.     

Genomic Resources for Gene Discovery,Functional Genome Annotation,and Evolutionary Studies of Maize and Its Close Relatives

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.     

River islands,refugia and genetic structuring in the endemic brown frog Rana kukunoris (Anura,Ranidae) of the Qinghai‐Tibetan Plateau

Frequently, Pleistocene climatic cycling has been found to be the diver of genetic structuring in populations, even in areas that did not have continental ice sheets, such as on the Qinghai‐Tibetan Plateau (QTP). Typically, species distributed on the plateau have been hypothesized to re‐treat to south‐eastern refugia, especially during the Last Glacial Maximum (LGM). We evaluated sequence variation in the mitochondrial DNA gene Cytb and the nuclear DNA gene RAG‐1 in Rana kukunoris, a species endemic to the QTP. Two major lineages, N and S, were identified, and lineage N was further subdivided into N1 and N2. The geographical distribution and genealogical divergences supported the hypothesis of multiple refugia. However, major lineages and sublineages diverged prior to the LGM. Demographical expansion was detected only in lineage S and sublineage N2. Sublineage N1 might have survived several glacial cycles in situ and did not expand after the LGM because of the absence of suitable habitat; it survived in river islands. Genetic analysis and environment modelling suggested that the north‐eastern edge of QTP contained a major refugium for R. kukunoris. From here, lineage S dispersed southwards after the LGM. Two microrefugia in northern Qilian Mountains greatly contributed to current level of intraspecific genetic diversity. These results were found to have important implications for the habitat conservation in Northwest China.