Tim Xing

Ectopic Expression of an Arabidopsis Calmodulin-Like Domain Protein Kinase-Enhanced NADPH Oxidase Activity and Oxidative Burst in Tomato Protoplasts

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.

The role of light in the regulation of anthocyanin accumulation in Gerbera hybrida

In the present work, the pigmentation regulated by light was investigated in ray floret (rf) of Gerbera hybrida. When inflorescences from stage 1 were covered with aluminium foil in vivo the pigmentation of the rf petals was strongly blocked and the gene expression of CHS (Chalcone synthase) and DFR (Dihydroflavonol-4-reductase) was inhibited. Similar results were obtained when the detached rfs were cultured in vitro. Covering of the leaves on the plants resulted in reduced pigmentation compared with the covering of inflorescences in vivo. Removal of the green bracts did not affect the pigmentation significantly and the anthocyanin concentration was maintained at a level similar to that of the control. The ultrastructure of the plastids in rf petals was examined to investigate the possible role of photosynthesis in light regulation of flower pigmentation. Plastids within rf epidermal cells showed a characteristic chloroplast morphology in flowers at stage 2, which deteriorated by stage 3. They then changed to a chromoplast-like structure in fully opened rf petals (stage 6). Similar chromoplast-like structures were observed in the plastids of the rf petals from inflorescences both shaded in vivo and in vitro. Additionally, DCMU, a photosynthetic inhibitor, did not show a significant effect on light-induced anthocyanin accumulation. Our data suggest that light is an important factor for pigmentation of rf petal in Gerbera and the petal itself acts as a light sensor site to perceive the light signal. From the different light qualities evaluated, blue light promoted gene expression of CHS and DFR, and red light enhanced the gene expression of CHS, indicating the photoreceptors responding to blue and red light involved in the photoregulation of flower pigmentation in Gerbera.

Genomic analysis of MAP kinase cascades inArabidopsis defense responses

The process of phosphorylation and dephosphorylation is a common mechanism of signal transduction in plants, connecting the perception of extracellular signals with the final responses to those signals. This paper will concentrate on the mitogen-activated protein (MAP) kinase pathway, one of the main phosphorylation pathways that plants use in biotic and abiotic stress resistance. It is a cascade consisting of several classes of kinases, each having a different role in signal integration and divergence. The cascade is regulated by various mechanisms, including not only transcriptional and translational regulations but also post-transcriptional regulations and protein-protein interactions. Recent detailed analysis of certain specific MAP kinase pathways has revealed the specificity of the kinases in the cascade, signal transduction patterns, identity of pathway targets, and the complexity of the cascade. Strategies in the study of phosphorylation pathways are discussed, and approaches integrating various genomics and proteomics technologies are suggested.

Role of HD2 genes in seed germination and early seedling growth in Arabidopsis

The Arabidopsis HD2 family of histone deacetylases consist of 4 members (HD2A, HD2B, HD2C, HD2D) that play diverse roles in plant development and physiology through chromatin remodelling. Here, we show that the transcripts of HD2 family members selectively accumulate in response to glucose through a HXK1-independent signal transduction pathway during the early stages of seedling growth. Germination was enhanced in hd2a null mutants relative to wild-type seeds. In contrast, hd2c mutants were restrained in germination relative to wild-type seeds. In hd2a/hd2c double mutants, germination was restored to wild-type levels. The data suggests that HD2A and HD2C may have different and opposing functions in germination with the glucose/HD2A pathway acting to restrain germination and the HD2C pathway acting to enhance germination. These pathways may function early in the regulation of seedling germination, independently of the glucose/HXK1/ABA signal transduction pathway, to fine tune the onset of germination.

Heat Shock Induces Programmed Cell Death in Wheat Leaves

Programmed cell death (PCD) was triggered in wheat leaves by a heat treatment (42 °C). This treatment caused DNA fragmentation as shown in DNA laddering analysis and in terminal deoxynucleotidyl transferase-mediated deoxyuridine-5′-triphosphate (dUTP) nick end labeling (TUNEL) analysis. Methanol and acetone treatment of leaves significantly blocked PCD. Western analysis indicated that a 65 kDa poly(ADP-ribose) polymerase-like protein was degraded during the treatment. However, high temperature (80 °C) treatment caused necrosis but not PCD.

Revealing plant defense signaling Getting more sophisticated with phosphoproteomics

The regulation mechanisms of any plant-pathogen interaction are complex and dynamic. A proteomic approach is necessary in understanding regulatory networks because it identifies new proteins in relation to their function and ultimately aims to clarify how their expression, accumulation and modification is controlled. One of the major control mechanisms for protein activity in plant-pathogen interactions is protein phosphorylation, and an understanding of the significance of protein phosphorylation in plant-pathogen interaction can be overwhelming. Due to the high number of protein kinases and phosphatases in any single plant genome and specific limitations of any technologies, it is extremely challenging for us to fully delineate the phosphorylation machinery. Current proteomic approaches and technology advances have demonstrated their great potential in identifying new components. Recent studies in well-developed plant-pathogen systems have revealed novel phosphorylation pathways, and some of them are off the core phosphorylation cascades. Additional phosphoproteomic studies are needed to increase our comprehension of the different mechanisms and their fine tuning involved in the host resistance response to pathogen attacks.

Antimicrobial and P450 inhibitory properties of common functional foods.

PURPOSE To study the effect of functional foods on human cytochrome P450 (CYP) and the gut bacterial microflora that may potentially affect drug metabolism and ultimately affect human health and wellness. METHODS This study examined a variety of food plants from the Apiaceae, Fabaceae, and Lamiaceae families for their inhibitory potential on cytochrome 2D6-, 3A4-, 3A5-, and 3A7-mediated metabolism. The antimicrobial effects of these samples were also investigated with 7 selected bacterial surrogate species to determine potential effects on the gut microflora. RESULTS The highest CYP inhibitory activities, based upon visual examination, were observed from extracts of celery seed, cumin, fennel seed, basil, oregano, and rosemary belonging to the Apiaceae and Lamiaceae families, respectively. Likewise, the strongest antimicrobial activities were also observed in the Apiaceae and Lamiaceae. No significant antimicrobial and CYP inhibition was observed in the Fabaceae extracts. CONCLUSION Results demonstrated the possible risk of food-drug interactions from spice and herb plants may affect drug disposition and safety.

A novel stress-associated protein SbSAP14 from Sorghum bicolor confers tolerance to salt stress in transgenic rice

We have characterized a member of the stress-associated protein (SAP) gene family from Sorghum bicolor (SbSAP14) with A20 and AN1 zinc-finger domains. Expression profiling revealed that SbSAP14 is specifically induced in response to dehydration, salt, and oxidative stress as well as abscisic acid treatment. During the early stage of salt stress, overexpression of SbSAP14 was able to prevent yellowing and withering of the leaf tip of rice plants. Measurements of malondialdehyde, ion leakage, and chlorophyll content demonstrated that transgenic rice had an enhanced tolerance to oxidative damage caused by salt stress. Under prolonged salt stress, transgenic rice plants had a higher seed germination rate and higher percentage seedling survival than wild-type (WT) plants. Importantly, in vivo and in situ assays revealed that the accumulation of reactive oxygen species in transgenic rice plants was significantly lower than that in WT plants. Among the six antioxidant genes tested, APX2, CatB, CatC, and SodA1 showed a higher expression level in transgenic rice than in WT rice. Based on these results, we propose that SbSAP14 may play a key role in antioxidant defense systems and possibly be involved in the induction of antioxidant genes in plants, suggesting a possible mechanism of the SAP gene family in stress defense response.

Yeast two-hybrid screening of MAP kinase cascade identifies cytosolic glutamine synthetase 1b as a tMEK2 interactive protein in wheat

The mitogen-activated protein kinase kinase (MAPKK) tMEK2 from tomato (Solanum lycopersicum) is a key component in the defense pathways against pathogen attacks. Because tMEK2-transgenic wheat (Triticum aestivum) has partial resistance to wheat leaf rust (caused by Puccinia triticina), we studied tMEK2 functions in wheat by heterologous screening for tMEK2 interactive proteins in a wheat yeast two-hybrid library. Within the 46 positive colonies examined, four genes encoding tMEK2-interactive proteins were sequenced. One of them was wheat GS1b, a cytosolic glutamine synthetase (GS). Its interaction with tMEK2 was confirmed by cotransformation in yeast (Saccharomyces cerevisiae) and by coimmunoprecipitation. The GS activities remained higher in tMEK2 transgenic wheat than in wild-type control during fungal toxin treatment. Our results provide evidence that tMEK2 plays multiple roles in plant defense against pathogen attacks, not only by activating downstream MAPKs and pathogenesis-related proteins but also possibly by recruiting metabolic pathways. Key words: wheat, MAPKK, yeast two-hybrid, glutamine synthetase, Triticum aestivum. La protéine tMEK2 de la tomate, protéine kinase kinase activée par le mitogène (MAPKK), est un composant essentiel des réactions de défense contre les attaques d’agents pathogènes. Étant donné que la protéine tMEK2 du blé transgénique n’offre qu’une résistance partielle à la rouille brune, nous avons étudié les rôles de tMEK2 chez le blé par échantillonnage hétérologue des protéines interactives tMEK2 dans une bibliothèque de blé en utilisant le système de double hybride chez la levure. Des 46 colonies positives, 4 gènes encodant les protéines interactives tMEK2 ont été séquencés. L’un d’eux était le gène du blé GS1b, une glutamine synthétase (GS) cytosolique. Son interaction avec la protéine tMEK2 a été confirmée par cotransformation en levure et co-immunoprécipitation. Durant le traitement faisant appel aux toxines fongiques, la GS a affiché des taux plus élevés d’activité dans les protéines tMEK2 du blé transgénique que dans celles des variétés sauvages témoins. Nos résultats ont montré que les protéines tMEK2 jouent plusieurs rôles dans la défense des plantes contre les attaques d’agents pathogènes, et ce, non seulement en activant les protéines kinases activées par le mitogène (MAPK) situées en aval et les protéines associées à la pathogenèse, mais aussi, probablement, en sollicitant les voies métaboliques. Mots-clés : blé, MAPKK, double-hybride chez la levure, glutamine synthétase, T. aestivum.

Exogenous ammonium inhibits petal pigmentation and expansion in Gerbera hybrida

Petal pigmentation is the most important aspect in natural flower coloration. In the present study, the inhibition of petal pigmentation by exogenous ammonium was investigated. Ray floret petals detached from inflorescences of Gerbera hybrida (Shenzhen No. 5) were cultured in vitro on media supplied with different forms of nitrogen and its assimilated compounds. The expression of a set of genes involved in anthocyanin biosynthesis and regulation was determined by Northern blotting assay. It was found that ammonium (NH4+), not nitrate (NO3-), in millimolar concentrations inhibited anthocyanin accumulation. The expressions of Gerbera chalcone synthase 1 (GCHS1), Gerbera chalcone synthase 2 (GCHS2) and Gerbera dihydroflavonol-4-reductase (GDFR) decreased, while six other related genes showed no significant changes after NH4+ treatment. Further studies on NH4+ function indicated that glutamine (Gln) acted as a downstream factor of NH4+ to suppress petal pigmentation. Both exogenous Gln and NH4+ were found to inhibit anthocyanin accumulation in the petals, and the application of Gln was also found to inhibit the expressions of GCHS1, GCHS2 and GDFR. The application of NH4+ also resulted in an increase in the activity of Gerbera glutamine synthetase (EC 6.3.1.2) along with a rapid increase of Gln content. When methionine sulfoximine, an inhibitor of glutamine synthetase (GS), was added, it was found to block the NH4+-induced inhibition of pigmentation. From these experiments, we conclude that the NH4+-induced suppression of petal pigmentation is not because of NH4+ toxicity, and the inhibition of pigmentation caused by the addition of exogenous NH4+ is the result of its assimilation into Gln.