Capsicum annuum tobacco mosaic virus-induced clone 1 expression perturbation alters the plant's response to ethylene and interferes with the redox homeostasis

Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.     

Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis

Incompatible plant-pathogen interactions result in the rapid cell death response known as hypersensitive response (HR) and activation of host defense related genes. To understand the cellular mechanism controlling defense response better, a novel pathogenesis-related (PR) gene and putative cell wall protein gene, CaTin2, was isolated through differential screening of a hot pepper cDNA library and characterized. CaTin2 gene was locally and systemically induced in hot pepper plants upon TMV-P₀ inoculation which induces HR. However, CaTin2 gene wasn't regulated by bacterial HR-specific signal pathway. The full-length cDNA for CaTin2, which is 864 nucleotides long, contained the open reading frame of 200 amino acids including cell wall targeting sequences of 26 amino acids. CaTin2 gene has no sequence similarity with other cell wall protein genes except the signal sequence and exists as only one copy in hot pepper genome. CaTin2 gene contains repeated helix-turn-helix motif consisting of 39 amino acids. CaTin2 mRNA accumulation was induced in response to various treatments such as ethylene, SA, MeJA, ABA, methyl viologen, NaCl and wounding at early time points. Subcelluar localization of CaTin2 was confirmed in the cell wall in hot pepper leaves by making CaTin2::smGFP fusion protein. The transgenic plants overexpressing CaTin2 cDNA were resistant to TMV and CMV inoculation. From these results, CaTin2 gene may encode a virus-related new cell wall protein member.     

桃PpMADS1基因启动子的克隆及功能分析

PpMADS1基因属于一类MADS box 基因,在植物的花发育调控中起着重要的作用。通过Genome Walking的方法从桃基因组中分离了长度为1 814bp的PpMADS1基因启动子片段,序列分析表明,在此启动子上不仅含有TATA box 和CAAT box基本元件,而且含有大量的与光调节有关的调控元件,如GT-1,Sp1和as-2-box,另外存在两个CArG-box元件、一个G-box元件和一个TGA-element,说明该启动子可能受光周期和激素的调控。将该启动子通过5′端缺失,分区段与GUS报告基因连接构建表达载体,并转化拟南芥。GUS组织化学染色分析结果表明,在-197到-454bp有促使GUS在花原基中表达的花原基特异性元件,在-454到-678bp之间存在促使GUS在萼片和花瓣表达的特异性元件,在-678到-978bp存在负调控作用元件,阻遏了GUS基因在花药中的表达。     

Production of a heterologous recombinant protein using fragments of the glyceraldehyde-3-phosphate dehydrogenase promoter from Penicillium camemberti

The biotechnological applications of cheese-ripening fungi have been limited by a lack of genetics tools, in particular the identification and characterization of suitable promoters for protein expression. In this study, the suitability of the glyceraldehyde-3-phosphate dehydrogenase (gpdP) promoter from Penicillium camemberti to drive the production of a recombinant protein was evaluated. The gpdP gene and its promoter were isolated using PCR and Genome Walker. The promoter of gpdP has two regions with high identity to the regulatory elements gpd-box and ct-box previously described in Aspergillus nidulans. Two fragments of the promoter containing the gpd- and ct-box or the ct-box alone were used to drive the in vivo production of recombinant β-galactosidase using A. nidulans as host. Our results indicate that larger fragment containing gpd-box enhances the production of β-galactosidase activity levels respect to ct-box alone, and that both boxes are necessary to obtain maximal enzymatic activity production. The smaller fragment (187 nt) containing the ct-box alone was able to trigger up to 27% of β-galactosidase activity, and to our knowledge this is the smallest fragment from a gpd gene used to produce a recombinant protein. Differences were not observed when glycerol, galactose or glucose were used as carbon sources, suggesting that the promoter activity is carbohydrate-independent. This is the first report in which a Penicillium gpd promoter is used for recombinant protein production. Our results open the way for the future development of a system for recombinant proteins expression in the biotechnologically important cheese-ripening fungus P. camemberti.     

Modification of the standing long jump test enhances ability to predict anaerobic performance

The purpose of this study was to investigate whether modifying the standing long jump test would enhance its ability to be a better predictor of anaerobic performance compared to other common anaerobic power tests. Three modified box long jump (MBLJ) tests were performed using 1, 2, or 3 boxes. Subjects consisted of 38 healthy males (age, 21.7 +/- 1.7 years) who performed all the testing procedures. All 3 variations of the MBLJ test showed significant correlations (p < 0.05) with the vertical jump (VJ); standing long jump (SLJ); 50-, 100-, 200-, 400-m runs; long jump; triple jump; and shot put ability (r = 0.362-0.891). All 3 variations of the MBLJ test also showed significant correlations with isokinetic peak torque knee extension and flexion, Wingate mean power (W), and Wingate mean power per kilogram (W/kg) (r = 0.357-0.504). Generally, correlations of the 3 MBLJ tests were stronger than correlations between VJ and SLJ ability to the same measure of power. Generally, the 3-box MBLJ tests showed stronger correlations with measures of power than the 1- and 2-box MBLJ tests. Multiple linear regression models indicated that the 3-box MBLJ test is a major predictor of the track and field performances compared to the other tests of anaerobic power. Along with other independent variables, the 3-box MBLJ test explained 55%, 44%, 51%, 61%, 52%, and 72% of the variance of 50-, 100-, 200-, and 400-m runs; long jump; and triple jump performance, respectively. In conclusion, due to the significant correlations between the MBLJ tests, especially the 3-box version, and other measures of power, these tests are appropriate for testing lower body power.     

TMV-induced expression of tobacco β-glucanase promoter activity is mediated by a single, inverted, GCC motif

Tobacco basic β-1,3-glucanase has been implicated in plant development and defense responses against pathogens. We examined the functional cis-elements involved in the response of basic β-1,3-glucanase promoter (gglb50) to infection with tobacco mosaic virus (TMV). In plants transformed with chimeric gglb50-β-glucuronidase (GUS) reporter gene, significant GUS-activity levels were measured in the leaves and in roots. Activity in the leaves was further induced (10-fold) by TMV infection. Maximal virus-induced activity was directed by a promoter region between −1233 to +19 (gglb-1233). A 5′ deletion to −1038, which removed two TAAGAGCCGCC motifs (GCC-boxes), reduced virus-induced activity. However, when the gglb-1233 promoter was mutated by base substitutions within a third, inverted, GCC-box located between positions −106 and −95, virus-induced promoter activity was abolished. Duplication of a small region containing this inverted GCC-box in the context of the gglb-1233 promoter resulted in increased virus-induced activity. Gel retardation assay demonstrated nuclear-factor binding to the inverted GCC-box. These studies strongly suggest the inverted GCC-box as a regulatory element essential for TMV-induced activity directed by the gglb50 promoter.     

Characterization of Cullin-box sequences that direct recruitment of Cul2-Rbx1 and Cul5-Rbx2 modules to Elongin BC-based ubiquitin ligases

The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown ( Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065 ) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPPhiP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as approximately 80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases.