The performance of pathogenic bacterial phytosensing transgenic tobacco in the field

Phytosensors are useful for rapid‐on‐the‐plant detection of contaminants and agents that cause plant stress. Previously, we produced a series of plant pathogen‐inducible synthetic promoters fused to an orange fluorescent protein (OFP) reporter gene and transformed them into tobacco and Arabidopsis thaliana plants; in these transgenic lines, an OFP signal is expressed commensurate with the presence of plant pathogens. We report here the results of 2 years of field experiments using a subset of these bacterial phytosensing tobacco plants. Time‐course analysis of field‐grown phytosensors showed that a subset of plants responded predictably to treatments with Pseudomonas phytopathogens. There was a twofold induction in the OFP fluorescence driven by two distinct salicylic acid‐responsive synthetic promoters, 4 × PR1 and 4 × SARE. Most notably, transgenic plants containing 4 × PR1 displayed the earliest and highest OFP induction at 48 and 72 h postinoculation (h p.i.) upon inoculation with two phytopathogens Pseudomonas syringae pv. tomato and P. syringae pv. tabaci, respectively. These results demonstrate transgenic tobacco harbouring a synthetic inducible promoter‐driven OFP could be used to facilitate monitoring and early‐warning reporting of phytopathogen infections in agricultural fields.     

In Planta visual monitoring of green fluorescent protein in transgenic rice plants

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a widely used reporter that can be directly visualized in the living cells in both animals and plants. We inserted a synthetic gene (sgfp) encoding a modified form of the GFP into expression vector, Act1-sgfp for the direct expression of GFP which is easily detectable in rice plants. Green fluorescence emitted from GFP could be visualized in calli, dry seeds, roots and seedlings with green shoots of transgenic rice plants. In our visualization system with a charge-coupled device camera, band-pass filters and a light source, the presence of red chlorophyll autofluorescence from chloroplasts did not alter the green fluorescence of GFP. These results demonstrate that GFP could be used as a non-destructive visual selection marker for examining gene expression in transformed calli, dry seeds and young plants.     

Evaluation of the use of the tobacco PR-1a promoter to monitor defense gene expression by the luciferase bioluminescence reporter system

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.     

花青素调节基因VlmybA2对番茄遗传转化

以小型番茄 Micro-Tom 为材料,利用农杆菌介导法导入花青素调节基因VlmybA2。对抗性筛选出的再生植株进行 GUS 组织染色和 PCR 检测,证明外源基因已经整合到 Micro-Tom 中,转基因番茄根、茎、叶脉、果皮均呈紫色,花色为黄紫嵌合。而野生型的根为白色,茎、叶脉呈绿色,果皮为红色,花为黄色。对转基因番茄的花青素含量、叶片叶绿素含量和光合速率等生理指标进行测定,花青素含量有显著增加,叶绿素含量降低,VlmybA2基因过量表达会降低植株的光合效率,但对植株正常生长影响并不显著。VlmybA2 基因既可增加抗衰老物质花青素含量,又可作为转基因植株的报告基因。     

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post‐symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early‐warning sentinels potentially have tremendous utility as wide‐area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis‐acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time‐course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.     

Chlorophyll b to chlorophyll a conversion precedes chlorophyll degradation in Hordeum vulgare L.

This study reveals by in vivo deuterium labeling that in higher plants chlorophyll (Chl) b is converted to Chl a before degradation. For this purpose, de-greening of excised green primary leaves of barley (Hordeum vulgare) was induced by permanent darkness in the presence of heavy water (80 atom % (2)H). The resulting Chl a catabolite in the plant extract was subjected to chemical degradation by chromic acid. 3-(2-Hydroxyethyl)-4-methyl-maleimide, the key fragment that originates from the Chl catabolite, was isolated. High resolution (1)H-, (2)H-NMR and mass spectroscopy unequivocally demonstrates that a fraction of this maleimide fragment consists of a mono-deuterated methyl group. These results suggest that Chl b is converted into Chl a before degradation. Quantification proves that the initial ratio of Chl a:Chl b in the green plant is preserved to about 60-70% in the catabolite composition isolated from yellowing leaves. The incorporation of only one deuterium atom indicates the involvement of two distinguishable redox enzymes during the conversion.     

Evaluation of the Use of the Tobacco PR-1a Promoter to Monitor Defense Gene Expression by the Luciferase Bioluminescence Reporter System

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.     

DR5 as a reporter system to study auxin response in Populus

Key message

Auxin responsive promoter DR5 reporter system is functional in Populus to monitor auxin response in tissues including leaves, roots, and stems.

Abstract

We described the behavior of the DR5::GUS reporter system in stably transformed Populus plants. We found several similarities with Arabidopsis, including sensitivity to native and synthetic auxins, rapid induction after treatment in a variety of tissues, and maximal responses in root tissues. There were also several important differences from Arabidopsis, including slower time to maximum response and lower induction amplitude. Young leaves and stem sections below the apex showed much higher DR5 activity than did older leaves and stems undergoing secondary growth. DR5 activity was highest in cortex, suggesting high levels of auxin concentration and/or sensitivity in this tissue. Our study shows that the DR5 reporter system is a sensitive and facile system for monitoring auxin responses and distribution at cellular resolution in poplar.     

The ontogenetic approach to chlorophyll fluorescence studies of plant photosynthetic apparatus under stressful conditions

Based on the analysis of reasons limiting the application of the method of chlorophyll fluorescence induction for estimating the state of the leaf photosynthetic apparatus under prolonged stress, the necessity of the ontogenetic approach consisting in a more exact determination of leaf age was substantiated. A comparison of the calendar and ontogenetic ways of determination of age of cucumber leaves under controlled conditions revealed essential distinctions in the estimation of plant leaf photosynthetic apparatus by the method of chlorophyll fluorescence induction for two variants distinguishing by the cultivation light regime ("white", 400-700 nm, and "red", 600-700 nm). It was shown that, in the case of prolonged effect of the stress factor on the plant, the unambiguity of the interpretation of chlorophyll fluorescence induction parameters in the estimation of the state of their photosynthetic apparatus depends essentially on the choice of the ontogenetic period of leaves of plants being compared and the accuracy of determination of leaf age.     

A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli

To develop a rapid reporter system for the screening of stationary-phase promoters in Escherichia coli, the expression pattern of the green fluorescent protein (GFP) during bacterial cultivation was compared with that of the commonly used β-galactosidase. Using GFP with enhanced fluorescence, the expression pattern of both reporter systems GFP and β-galactosidase were similar and showed a typical induction of gene activity of the reporter genes, i.e. increase of expression at the transition from exponential to stationary phase. The expression was affected by the culture medium, i.e. in contrast to the complex medium (LB medium), the stationary-phase specific induction was only observed in synthetic medium (M9) when amino acids were added, whereas there was generally no induction in MOPS medium. To develop a rapid screening method on agar plates for stationary-phase promoters, a photographic approach was used, continued with computational image treatment. A screening method is presented which enables an on-line monitoring of gene activity.     

Experimental models for creation of transgenic plants resistant to stressors

Experimental models of primary potato transgenic plants that express the cry3aM-licBM2 hybrid gene were created. The molecular analysis and biotests of the experimental models allow a new system of cry genes expression in plants to be proposed. This system is based on the expression of hybrid genes containing the reporter lichenase gene sequence and the use of a light-induced promoter ensuring preferential expression of the regulated genes only in green plant tissues (leaves), the target tissues for pests, as a regulatory element. In is shown that the presence of lichenase in hybrid proteins facilitates selection and analysis of the level of expression of hybrid proteins in transgenic plants. Judging by the properties of the reporter protein lichenase in hybrid proteins, it seems possible to use this reporter system for transgene monitoring in agrocenosis, because this system is fairly simple and precise and does not need considerable material and time expenses.     

Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate

Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control. We have developed a novel reporter assay system in which three luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.     

Virus infection decreases the attractiveness of white clover plants for a non-vectoring herbivore

Plant pathogens and insect herbivores are prone to share hosts under natural conditions. Consequently, pathogen-induced changes in the host plant can affect herbivory, and vice versa. Even though plant viruses are ubiquitous in the field, little is known about plant-mediated interactions between viruses and non-vectoring herbivores. We investigated the effects of virus infection on subsequent infestation by a non-vectoring herbivore in a natural genotype of Trifolium repens (white clover). We tested whether infection with White clover mosaic virus (WClMV) alters (1) the effects of fungus gnat feeding on plant growth, (2) the attractiveness of white clover for adult fungus gnat females, and (3) the volatile emission of white clover plants. We observed only marginal effects of WClMV infection on the interaction between fungus gnat larvae and white clover. However, adult fungus gnat females clearly preferred non-infected over WClMV-infected plants. Non-infected and virus-infected plants could easily be discriminated based on their volatile blends, suggesting that the preference of fungus gnats for non-infected plants may be mediated by virus-induced changes in volatile emissions. The compound β-caryophyllene was exclusively detected in the headspace of virus-infected plants and may hence be particularly important for the preference of fungus gnat females. Our results demonstrate that WClMV infection can decrease the attractiveness of white clover plants for fungus gnat females. This suggests that virus infections may contribute to protecting their hosts by decreasing herbivore infestation rates. Consequently, it is conceivable that viruses play a more beneficial role in plant-herbivore interactions than generally thought.     

Functional analysis of a Golgi-localized Kex2p-like protease in tobacco suspension culture cells

Kex2p is the prototype of a Golgi-resident protease responsible for the processing of prohormones in yeast and mammalian cells. A Kex2p-like pathway was shown to be responsible for processing the fungal KP6 protoxin in transgenic tobacco plants. We previously described a chimeric integral membrane reporter protein that traffics through Golgi to the lytic prevacuole where it was proteolytically processed. As a first step to isolate and clone the Kex2p-like protease in plant cells, we designed and used a similar chimeric reporter protein containing Kex2 cleavage sites to assay the Kex2p-like activity and to determine its substrate specificity in tobacco cells. Here we demonstrate that the Kex2 cleavage sites of the reporter were specifically processed by a protease activity with a substrate specificity characteristic of yeast Kex2p. This Kex2p-like protease in tobacco cells is also a Golgi-resident enzyme. Thus, the reporter protein provides a biochemical marker for studying protein traffic through the Golgi in plant cells. These results additionally should allow the design of synthetic substrates for use in biochemical purification of the plant enzyme.     

大亚湾水质遥感监测指标分析

我国沿海经济高速发展,对近海岸水质环境影响极大。传统的近海岸水质监测手段不仅成本大,而且空间信息有限,难以对相关海域进行全面、准确的监测与评价;利用遥感的手段进行近海岸水质监测可克服这些局限。利用遥感进行水质评价,需要建立相应的指标体系。论文以大亚湾为例,分析了大亚湾的水质环境现状,讨论了具有光学敏感性或热敏感性的水质因子特性,指出水温、悬浮物及叶绿素是大亚湾水质遥感监测与评价的首选指标。讨论了悬浮物、叶绿素的污染表征物特征,以及在近海岸、湖泊及河流水质研究中的重要指示意义,同时建议将叶绿素作为常规监测指标纳入我国海水水质评价体系。