Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.     

Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants

The expression of foreign genes in pollen may pose potentialproblems in the field release of transgenic plants, since pollenrepresents a route whereby foreign genes and their productsmay escape into the wider environment. The possible risks posedby cross-hybridization with wild relatives have been extensivelyexplored, but problems that may arise due to the expressionof foreign gene products in pollen have not been so widely studied.The activities of the CaMV 35S and nos promoters in pollen inpopulations of stably transformed plants and in transient expressionanalysis are described. These promoters are commonly used inall areas of plant molecular biology research and their expressionpatterns will be of interest to those involved in field releasestudies. The results show that both promoters had no detectablepollen activity in Arabidopsis, but both showed activity intobacco pollen. The CaMV 35S-gus gene fusion showed heritableexpression levels in tobacco pollen of up to a maximum of 64.6pmol 4-MU min^–1 mg ^–1 total protein. nos promoteractivity in transgenic tobacco pollen was highly variable, withGUS activities ranging from undetectable levels up to 2561 pmol4-MU min^–1 mg^–1 total protein within the transgenicpopulation. Histochemical staining of anther sections from 10–12mm buds revealed that the CaMV 35S promoter had some activityin the vascular bundle, stomium and tapetum, while GUS expressionfrom the nos promoter in sporophytic tissues was confined entirelyto the stomium. Key words: CaMV 35S promoter, nos promoter, pollen, transgenic plant release     

Constitutive expression of the <Emphasis Type="Italic">ARGOS</Emphasis> gene driven by dahlia mosaic virus promoter in tobacco plants

The auxin-inducible gene ARGOS from Arabidopsis thaliana is expressed in growing tissues and controls the plant organ size by regulating cell proliferation and meristematic competence. The promoter of the dahlia (Dahlia pinnata Cav.) mosaic virus (DMV) resembles the well-known cauliflower mosaic virus 35S promoter but shows a higher activity in transgenic tobacco plants (Nicotiana tabacum L.). We obtained transgenic tobacco plants expressing the Arabidopsis ARGOS gene under the control of the DMV promoter. Several of the T0 generation plants exhibited an accelerated transition to flowering, a slight increase in flower size, and a significant increase in the leaf size. The T1 transgenic plants were characterized by faster growth, the increased leaf size, and somewhat enlarged flowers as compared with control plants. These phenotypic traits, as well as stability and inheritance of the transgene were demonstrated also in T2 transgenic plants.     

Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny

Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.     

Functional role of CAAT box element of the nopaline synthase (nos) promoter

The nopaline synthase (nos) promoter is active in a wide range of plant tissues and regulated by various environmental stimuli. It was previously found that the CAAT box region is important for nos promoter activity. In the present study, the location of the CAAT box element was determined by site specific mutation analysis. Point mutations within the conserved CAAT box element significantly reduced the promoter response in transgenic tobacco plants and calli to wounding, H₂O₂, methyl jasmonate, and 2,4-D, but not to salicylic acid. However, mutations immediately upstream from the CAAT box did not affect these responses. These results suggest that the CAAT box element is important in responding to certain stimuli.     

Expression of ipt gene controlled by an ethylene and auxin responsive fragment of the LEACO1 promoter increases flower number in transgenic Nicotiana tabacum

Cytokinins play important roles in regulating plant growth and development. A new genetic construct for regulating cytokinin content in plant cells was cloned and tested. The gene coding for isopentenyl transferase (ipt) was placed under the control of a 0.821 kb fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene promoter from Lycopersicon esculentum (LEACO1) and introduced into Nicotiana tabacum (cv. Havana). Some LEACO1_{0.821 kb}-ipt transgenic plant lines displayed normal shoot morphology but with a dramatic increase in the number of flower buds compared to nontransgenic plants. Other transgenic lines produced excessive lateral branch development but no change in flower bud number. Isolated leaves of transgenic tobacco plants showed a significantly prolonged retention of chlorophyll under dark incubation (25°C for 20 days). Leaves of nontransformed plants senesced gradually under the same conditions. Experiments with LEACO1_{0.821 kb}-gus transgenic tobacco plants suggested auxin and ethylene involvement in induction of LEACO1_{0.821 kb} promoter activity. Multiple copies of nucleotide base sequences associated with either ethylene or auxin response elements were identified in the LEACO1_{0.821 kb} promoter fragment. The LEACO1_{0.821 kb}-ipt fusion gene appears to have potential utility for improving certain ornamental and agricultural crop species by increasing flower bud initiation and altering branching habit.     

A pFBP6::Barnase Construct Resulted in Stigma and Style Ablation and Floral Abscission in Transgenic Tobacco

The potential for transgene dispersal through pollen, fruit, and seed is an important argument against the release of genetically modified plants. One approach toward addressing the concerns of gene flow from transgenic crops into closely related wild species involves in the use of tissue-specific promoters to engineer male and/or female sterility. In this study, we investigated the potential of Barnase ectopic expression for engineering floral sterility. A 2.6?kb promoter region of floral binding protein 6 (FBP6) from Petunia hybrida was isolated and fused to a reporter gene encoding ??-glucuronidase (GUS). The construct was introduced into tobacco plants where GUS staining was detected ubiquitously throughout the various tissues. The expression pattern of FBP6 resembled AG promoters, i.e., weak promoter activity was found in vegetative tissues, and strong activity was found in the various floral organs including the carpels and stigma. Meanwhile,The pFBP6::Barnase construct was then cotransformed into tobacco along with the Barstar gene, encoding an enzymatic inhibitor of Barnase, which was expressed at low but ubiquitous levels. Although cotransformed tobacco plants showed near normal vegetative growth, 74% of transgenic plants exhibited stigma and style ablation, and 98% of flower buds abscised before opening. Further analyses confirmed that stigma and style ablation prevented fertilization of the flower, and abscission of the bud followed rapidly. Thus, this approach has advantages for those ornamental/landscaping species where the pollen and fruit represent pollutants of the urban environment (e.g., platanus and poplar).     

Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Constitutive promoters are the most common promoters used to drive the expression of various genes in monocots and dicots. Therefore, it is of intense interest to ascertain their expression patterns in various plant species, organs and during their ontogenic development. In this study, the activity of the CaMV 35S promoter in transgenic tobacco plants was assessed. In contrast to other studies, performed rather on the primary transformants (T₀ generation), here, individuals of T₁ and T₂ generations were used. The expression profiles of the CaMV 35S promoter were tracked within various plant organs and tissues using the GFP marker. Special attention was given to floral tissues for which the original data regarding the CaMV 35S expression were obtained. As expected, distinct developmental and organ/tissue specific expression patterns in a plant body were observed. CaMV 35S activity was detected in most of the plant tissues and during different developmental stages. The GFP signal was not visible in dry seeds only, but it became clearly apparent within 24–48 h after sowing onto the medium, what, among other things, enables the discrimination of transgenic and non-transgenic seeds/seedlings. Afterwards, the most pronounced GFP fluorescence intensity was usually visible in various vascular tissues of both, T₁ and T₂ plants, indicating the high promoter activity. A stable manifestation of the promoter was retained in the next T₂ generation without any evident changes or losses of activity, showing the expression stability of the CaMV 35S.     

Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber (Cucumis sativus L.) flower buds and fruits

Thaumatin II is an extremely sweet-tasting protein produced by fruits of the West African shrubThaumatococcus daniellii Benth, so it can be used in biotechnology to improve the tastes of various plant products. This study is concerned with the spatial and temporal aspects of expression of the 35S-pre-prothaumatin II chimeric gene in flower buds and fruits of transgenic cucumber (Cucumis sativus L.) line 225. The activity of the 35S promoter in organs of line 225 was compared with its activity in 2 other transgenic lines. The accumulation of recombinant thaumatin varied spatially in flower bud tissues of transgenic lines. We found that these differences in the spatial accumulation of transgenic protein concerned the ovary of female buds and the perianth of male buds. In contrast to flower parts, recombinant thaumatin was found in nearly all parts of the young fruit from the transgenic plants. The pre-prothaumatin II gene expression was detected at a very early developmental stage in male buds, and its pattern was rather conserved as the buds aged. The expression of the transgene was also detected in vascular tissues of examined organs but was undetectable in pollen grains, in agreement with the generally held view that the CaMV 35S promoter is virtually silent in pollen. Immunocytochemical analyses of sections of control organs revealed endogenous homolog(s) of thaumatin when using polyclonal antisera, but not when using monoclonal antibodies for recombinant thaumatin detection in transgenic cucumber.     

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene

Key message

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin.

Abstract

The ubiquitin–26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA₃ conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.     

Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells

Summary An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.     

Two tobacco AP1-like gene promoters drive highly specific,tightly regulated and unique expression patterns during floral transition,initiation and development

The genetic engineering of agronomic traits requires an array of highly specific and tightly regulated promoters that drive expression in floral tissues. In this study, we isolated and characterized two tobacco APETALA1-like (AP1-like) promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using the GUS reporter system, along with tissue-specific ablation analyses. Our results demonstrated that the two promoters are active in floral inflorescences but not in vegetative apical meristems or other vegetative tissues, as reflected by strong GUS staining and DT-A-mediated ablation of apical shoot tips during reproductive but not vegetative growth. We also showed that the NtAP1Lb1 promoter was more active than NtAP1La in inflorescences, as the former yielded higher frequencies and greater phenotypic evidence of tissue ablation compared to the latter. We further revealed that both promoters were uniformly expressed in the meristems of stage 1 and 2 floral buds, but were differentially expressed in floral organs later during development. While NtAP1La was found to be active in stage 4–5 carpels, later becoming confined to ovary tissue from stage 9 onwards, NtAP1Lb1 activity was apparent in all floral organs from stages 3 to 7, becoming completely absent in all floral organs from stage 11 onward. Therefore, it seems that the two tobacco promoters have acquired similar but distinct inflorescence-, floral meristem- and floral organ-specific and development-dependent regulatory features without any leaky activity in vegetative tissues. These features are novel and have rarely been observed in other flower-specific promoters characterized to date. The potential application of these promoters for engineering sterility, increasing biomass production and modifying flower architecture, as well as their putative use in flower-specific transgene excision, will be discussed.     

Characterization of the GLP13 gene promoter in Arabidopsis thaliana

In transgenic plants, for many applications it is important that the inserted genes are expressed in a tissue-specific manner. This in turn could help better understanding their roles in plant development. Germin-like proteins (GLPs) play diverse roles in plant development and defense responses. In order to understand the functions and regulation of the GLP13 gene, its promoter (762 bp) was cloned and fused with a β-glucuronidase (GUS) reporter gene for transient expression in Arabidopsis thaliana and tobacco (Nicotiana tabacum cv. K326). Histochemical analysis of the transgenic plants showed that GUS was specifically expressed in vascular bundles predominantly in phloem tissue of all organs in Arabidopsis. Further analyses in transgenic tobacco also identified similar GUS expression in the vascular bundles.