Plant selectable markers and reporter genes

In recent years, considerable progress has been made in genetic engineering of various plant species, both agronomically important crops as well as model plants. The bases of this progress were, in addition to efficient transformation methods, the design of appropriate signals regulating transgene expression and the use of selection marker or reporter genes. In most cases, a gene of interest is introduced into plants in association with a selectable marker gene (nptII, hpt, acc3, aadA, bar, pat). Recovery of a transgenic plant is, therefore, facilitated by selection of putative transformants on a medium containing a selection agent, such as antibiotic (nptII, hpt, acc3, aadA), antimetabolite (dhfr), herbicide (bar, pat), etc. On the other hand, use of reporter genes (cat, lacZ, uidA, luc, gfp) allows not only to distinguish transformed and non-transformed plants, but first of all to study regulation of different cellular processes. In particular, by employing vital markers (Luc, GFP) gene expression, protein localization and intracellular protein traffic can be now observed in situ, without the need of destroying plant.     

A critical review of labelling techniques used to quantify rhizosphere carbon-flow

The rhizosphere is a major sink for photo-assimilated carbon and quantifying inputs into this sink is one of the main goals of rhizosphere biology as organic carbon lost from plant roots supports a higher microbial population in the rhizosphere compared to bulk soil. Two fundamentally different¹⁴CO₂ labelling strategies have been developed to estimate carbon fluxes through the rhizosphere — continuous feeding of shoots with labelled carbon dioxide and pulse-chase experiments. The biological interpretation that can be placed on the results of labelling experiments is greatly biased by the technique used. It is the purpose of this paper to assess the advantages, disadvantages and the biological interpretation of both continuous and pulse labelling and to consider how to partition carbon fluxes within the rhizosphere.     

The effect of soil pH on rhizosphere carbon flow of Lolium perenne

Perennial rye-grass plants were grown at 15°C in microcosms containing soil sampled from field plots that had been maintained at constant pH for the last 30 years. Six soil pH values were tested in the experiment, with pH ranging from 4.3–6.5. After 3 weeks growth in the microcosms, plant shoots were exposed to a pulse of ¹⁴C-CO₂. The fate of this label was determined by monitoring ¹⁴C-CO₂ respired by the plant roots/soil and by the shoots. The ¹⁴C remaining in plant roots and shoots was determined when the plants were harvested 7 days after receiving the pulse label. The amount of ¹⁴C (expressed as a percentage of the total ¹⁴C fixed by the plant) lost from the plant roots increased from 12.3 to 30.6% with increasing soil pH from 4.3 to 6. Although a greater percentage of the fixed ¹⁴C was respired by the root/soil as soil pH increased, plant biomass was greater with increasing soil pH. Possible reasons for observed changes in the pattern of ¹⁴C distribution are discussed and, it is suggested that changes in the soil microbial biomass and in plant nitrogen nutrition may, in particular be key factors which led to increased loss of carbon from plant roots with increasing soil pH.     

Bacterial and firefly luciferase genes in transgenic plants: Advantages and disadvantages of a reporter gene

Genes encoding light-emitting luciferase were recently isolated from luminous marine bacteria and fireflies. Expression of luciferase genes in diverse organisms is a unique way for studying gene expression by simple and sensitive measurement of light. Recent advances in application of luciferase reporter genes are reviewed and documented by examples of in vivo visualization of their expression in transgenic plants.     

Quantifying the Effect of Rhizosphere Processes on the Availability of Soil Cadmium and Zinc

Our purpose was to quantify the effect of rhizosphere processes on the availability of soil cadmium and zinc to various plant species. E values for Cd and Zn were measured on two contaminated soils differing mainly in their pH (6.2 and 8.1). L values were measured for six plant species with contrasting metal uptakes. The difference between E and L values quantifies the mobilization of the element which was non-phyto-available prior to cultivation. For Zn, L values were 1.2 to 5.6 times greater than the E values, depending on the plant species. The increase in Zn availability was greater in the basic soil. For Cd, L values in the basic soil were 1.1 to 2 times greater than the E value. In the slightly acid soil, plants did not enhance the cadmium availability. The mobilization of non-labile metal could be due to exudates from roots or microflora, phytosiderophores being responsible for the highest mobilization. The lower availability of Fe or Zn could explain the greater mobilization in the basic soil.     

Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression

The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.     

Luc genes: Introduction of colour into bioluminescence assays

Luminescence assays are generally based on measurements of light intensity alone. Inclusion of colour as an additional parameter of the assay could increase the information content. Colour variation in luminescence is particularly prevalent among beetle luciferases. To study the relationship between enzyme structure and colour, luciferases from a Jamalcan click beetle were examined as a model system. These luciferases emit light ranging from green to orange, though their amino acid sequences differ by less than 5%. Through mutation of their respective cDNA clones, the amino acids responsible for the colour variation were identified. These specific amino acids are few, and they act upon colour independently with respect to the enzyme structure. Analysis of their effects indicates that the potential for colour variation among beetle luciferases is greater than is evident among the click beetle luciferase. Because of the subtle changes of enzyme structure that effect colour, luciferases that emit different colours may be useful as paired genetic reporters. They should interact equivalently with the intracellular environment of a host, but could be distinguished by colour in their assay. Such paired reporters could be used to observed simultaneous events, or to provide internal control for luminescence measurements.     

A simple method to overcome the interference of hemoglobin in the detection of reporter genes in vivo

The determination of chemiluminescent intensity of reporter gene expression in vivo is generally disturbed by the presence of hemoglobin. Current methods consist in using perfusion to eliminate blood from investigated tumors or organs. In this work we propose a simple method to overcome this difficulty. The method consists in establishing an absorbance-dependence plot of the ratio R% = phi/phi(0) between the chemiluminescent intensities measured when hemoglobin is present or absent. For every measurement of the luminescent intensity phi on sample containing blood, if the absorbance A of the hemoglobin is determined, it allows one to have the intensity ratio R% which in turn gives the corrected intensity phi(0) when the absorption by hemoglobin is eliminated. The method is particularly adapted for comparative measurements of transfection levels in tumors where perfusion cannot be easily performed.     

Differential expression of genes of Pseudomonas syringae on leaves and in culture evaluated with random genomic lux fusions

Differential expression of genes of Pseudomonas syringae strain B728a on plants and in culture was assessed by measuring light production by a large collection of mutant strains containing random genomic insertions of a promoterless lux operon. Reporter gene fusions were made using Tn4431 containing lux CDABE from Vibrio fisheri. Light production reproducibly increased seven-fold when n-decanal was added to cells harvested from plant surfaces, to over 800-fold when added to cells cultured on a solidified culture medium, thus increasing the sensitivity of this reporter gene system. One of the 173 mutants tested exhibited significantly higher light production on plants than on solidified culture media compared to other mutants, while one lux fusion-containing strain produced significantly more light on culture media than on plants relative to the other mutants. The plant-inducible genes identified were not required for pathogenicity of this strain. Approximately 2% of the genes of P. syringae are apparently transcribed more actively in cells growing epiphytically on plants than in common culture media indicating that bacterial cells on plants may have substantially different behaviours than that of cultured cells.     

Root Development and Absorption of Ammonium and Nitrate from the Rhizosphere

Plant roots operate in an environment that is extremely heterogeneous, both spatially and temporally. Nonetheless, under conditions of limited diffusion and against intense competition from soil microorganisms, plant roots locate and acquire vital nitrogen resources. Several factors influence the mechanisms by which roots respond to ammonium and nitrate. Nitrogen that is required for cell division and expansion derives primarily from the apex itself absorbing rhizosphere ammonium and nitrate. Root density and extension are greater in nutrient solutions containing ammonium than in those containing nitrate as the sole nitrogen source. Root nitrogen acquisition alters rhizosphere pH and redox potential, which in turn regulate root cell proliferation and mechanical properties. The net result is that roots proliferate in soil zones rich in nitrogen. Moreover, plants develop thinner and longer roots when ammonium is the primary nitrogen source, an appropriate strategy for a relatively immobile nitrogen form. Present address of Alison R. Taylor: The Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK.     

Introduction to beetle luciferases and their applications

All beetle luciferases have evolved from a common ancestor: they all use ATP, O₂, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.     

Fundamentals and Application Potential of Arsenic-Resistant Bacteria for Bioremediation in Rhizosphere: A Review

As a hazardous environmental metalloid toxicant, arsenic (As)—at elevated levels in water and soil—has created a major public health concern through its entry into the food chain by accumulation in crops. Among the various methods reported thus far for reclamation of As-contaminated crop fields, bioremediation using bacteria with plant-growth-promoting traits has been found to be a most promising solution. There is every possibility that bacterial isolates with the ability to remove or immobilize As could be used for successful bioremediation. However, bioremediation needs to define its boundaries between promise and field application, as most studies have been restricted to laboratory results only. Rhizosphere interactions play a critical role in monitoring As bioavailability to crop plants, thus a better understanding of it might improve rhizoremediation technologies. The challenges rely on the application of these novel approaches under field conditions. Despite some limitations, the prospect for successful stimulation and exploitation of microbial metabolism for As rhizoremediation appears to be very promising.     

Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue

We report the investigation of an endosperm-specific promoter from the rsus3 gene from rice (Oryza sativa). The promoter was characterized by deletion analysis and transient expression in guar (Cyamopsis tetragonoloba) seed-tissue. Transient expression was monitored by histochemical GUS assay, and quantitative dual reporter assays comprising firefly luciferase as a test reporter, and Renilla luciferase and GUS as reference reporters. These revealed high expression levels of the reporter genes directed by the rsus3 promoter in guar endosperm. Specificity for this tissue in seeds was apparent from a virtual absence of reporter activity in guar cotyledons. Removal of a putative intron region only slightly raised the expression level, whereas duplication of the minimal promoter region, in a tandem-repeat rsus3 promoter construct, retained endosperm specificity in guar, and displayed three times the reporter activity observed with the single copy construct.     

Rhizosphere geometry and heterogeneity arising from root-mediated physical and chemical processes

The rhizosphere differs from the bulk soil in a range of biochemical, chemical and physical processes that occur as a consequence of root growth, water and nutrient uptake, respiration and rhizodeposition. These processes also affect microbial ecology and plant physiology to a considerable extent. This review concentrates on two features of this unique environment: rhizosphere geometry and heterogeneity in both space and time. Although it is often depicted as a soil cylinder of a given radius around the root, drawing a boundary between the rhizosphere and bulk soil is an impossible task because rhizosphere processes result in gradients of different sizes. For instance, because of diffusional constraints, root uptake can result in a depletion zone extending <1 mm for phosphate to several centimetres for nitrate, while respiration may affect the bulk of the soil. Rhizosphere processes are responsible for spatial and temporal heterogeneities in the soil, although these are sometimes difficult to distinguish from intrinsic soil heterogeneity. A further complexity is that these processes are regulated by plants, microbial communities and soil constituents, and their many interactions. Novel in situ techniques and modelling will help in providing a holistic view of rhizosphere functioning, which is a prerequisite for its management and manipulation.