Ethanol vapor is an efficient inducer of the alc gene expression system in model and crop plant species

We have demonstrated that low concentrations of ethanol vapor efficiently induce the alc gene expression system in tobacco (Nicotiana tabacum cv Samsun NN), potato (Solanum tuberosum cv Solara), and oilseed rape (Brassica napus cv Westar). For many situations, this may be the preferred method of induction because it avoids direct application of comparatively high concentrations of an ethanol solution. Although induction was seen with less than 0.4 microM ethanol vapor, maximal induction of the chloramphenicol acetyl transferase gene was achieved after 48 h in leaves of tobacco plants enclosed with 4.5 microM ethanol vapor. In the absence of ethanol, there is no detectable gene expression. Treatment of potato tubers with ethanol vapor results in uniform beta-glucoronidase (GUS) expression. Vapor treatment of a single oilseed rape leaf resulted in induction of GUS in the treated leaf only and (14)C-ethanol labeling in tobacco confirmed that the inducer was not translocated. In contrast, enclosure of the roots, aerial parts, or whole plant with ethanol vapor resulted in induction of GUS activity in leaves and roots. The data reported here broaden the utility of the alc system for research and crop biotechnology.     

Local induction of the alc gene switch in transgenic tobacco plants by acetaldehyde

The alc promoter system, derived from the filamentous fungi Aspergillus nidulans, allows chemically regulated gene expression in plants and thereby the study of gene function as well as metabolic and developmental processes. In addition to ethanol, this system can be activated by acetaldehyde, described as the physiological inducer in A. nidulans. Here, we show that in contrast to ethanol, acetaldehyde allows tissue-specific activation of the alc promoter in transgenic tobacco plants. Soil drenching with aqueous acetaldehyde solutions at a concentration of 0.05% (v/v) resulted in the rapid and temporary induction of the alc gene expression system exclusively in roots. In addition, the split root system allows activation to be restricted to the treated part of the root. The temporary activation of the alc system by soil drenching with acetaldehyde could be prolonged over several weeks by subsequent applications at intervals of 7 d. This effect was demonstrated for the root-specific induction of a yeast-derived apoplast-located invertase under the control of the alcohol-inducible promoter system. In leaves, which exhibit a lower responsiveness to acetaldehyde than roots, the alc system was induced in the directly treated tissue only. Thus, acetaldehyde can be used as a local inducer of the alc gene expression system in tobacco plants.     

Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.     

Auxin Levels Regulate the Expression of a Wound-Inducible Proteinase Inhibitor II-Chloramphenicol Acetyl Transferase Gene Fusion in Vitro and in Vivo

Proteinase inhibitor genes are expressed in solanaceous and leguminous plants following wounding of the foliage by mechanical methods. Previous studies have shown that a cloned proteinase inhibitor II-chloramphenicol acetyl transferase (pin2-CAT) chimeric gene is regulated in a wound-inducible manner in transgenic plants. In this study, we analyzed transgenic plant tissues for expression of the pin2-CAT gene in response to various plant hormones. We found that CAT activity was induced in tobacco (Nicotiana tabacum) callus incubated in the absence of any plant growth regulators. Addition of growth regulators to the medium thus permitted us to measure the effects of these substances on the activity of the pin2-CAT gene construction. Cytokinin (BAP) and ethylene (ethophon) even at low concentrations stimulated the expression of CAT activity by 25 to 50%. Abscisic acid at concentrations up to 4.4 × 10⁻⁵ molar had no effect upon CAT activity, but increasing auxin (naphthalene acetic acid) levels completely inhibited the synthesis of CAT protein. Gibberellic acid had little effect except at very high concentration (2.9 × 10⁵ molar). The kinetics of activation of the pin2-CAT gene were quite long (5 to 7 days) when unwounded calli were plated on media lacking auxin. This effect was documented for calli derived from several transformed plants, containing the full, chimeric pin2-CAT (pRT45) gene. In addition, calli from tissues transformed with wild-type vectors or from several plants transformed with pRT50 (a noninducible derivative of pRT45) were not induced by plating on media lacking auxin. Other naturally occurring and synthetic auxins had similar effects to naphthalene acetic acid in inhibiting the induction of the chimeric gene fusion. Finally, leaf discs from transformed plants were induced by incubation in MS liquid medium in the presence and absence of naphthalene acetic acid. NAA was also effective in down regulating the chimeric gene in whole plant tissues.     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

Characterization of the ethanol-inducible alc gene expression system in tomato

The efficacy of the ethanol-inducible alc transgene expression system, derived from the filamentous fungus Aspergillus nidulans, has been demonstrated in transgenic tomato. Two direct comparisons have been made. First, this study has utilized two transgenic lines carrying distinct reporter genes (chloramphenicol acetyltransferase and beta-glucuronidase) to distinguish aspects of induction determined by the nature of the gene/gene product rather than that of the plant. Second, comparisons have been made to data generated in other species in order to identify any species-specific effects. The induction profiles for different genes in different species have shown remarkable similarity indicating the broad applicability of this gene switch. While there are minor differences observed between species, these probably arise from diversity in their metabolism. A series of potential alternative inducers have also been tested, revealing that ethanol (through metabolism to acetaldehyde) is better than other alcohols and ketones included in this study. Expression driven by alc was demonstrated to vary spatially, the upper younger leaves having higher activity than the lower older leaves; this will be important for some applications, and for experimental design. The highest levels of activity from ethanol-inducible transgene expression were determined to be the equivalent of those from the constitutive Cauliflower Mosaic Virus 35S promoter. This suggests that the alc system could be an important tool for plant functional genomics.     

Alcohol-inducible gene expression in transgenic Populus

We tested the efficiency and optimized the conditions for controlled alcohol-inducible transgene expression in Populus using gus as a reporter gene. Specificity of induction, efficiency in different organs, effect of three chemical inducers, and induction methods were tested using up to 10 independent transgenic events generated in two different Populus genotypes. The optimal inducer concentration and the duration of induction period were determined in dose–response and in time–course experiments. Under in vitro conditions, β-glucuronidase (GUS) induction was efficient both in the aerial parts and in the roots of regenerated plantlets. Among the chemical inducers tested, ethanol was the most effective activator with no apparent phytotoxicity when concentrations were at or below 2%. After 5 days of treatment, fluorometrically-determined the GUS activity could be detected when inducing with ethanol at concentrations as low as 0.5%. Prolonged induction by ethanol vapors significantly increased the GUS activity in leaves from both the tissue culture plants and greenhouse-grown plants.Electronic Supplementary Material Supplementary material is available for this article at     

The yeast polyadenylate-binding protein (PAB1) gene acts as a disease lesion mimic gene when expressed in plants

We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco. Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth. The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants. In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control. Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene. These two properties – impairment of growth and induction of defense responses – indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants.     

AtZFP1, encoding Arabidopsis thaliana C2H2 zinc-finger protein 1, is expressed downstream of photomorphogenic activation

C₂H₂ zinc-finger proteins play important roles in plant development including floral organogenesis, leaf initiation, lateral shoot initiation, gametogenesis and seed development. The gene for one such protein from Arabidopsis, AtZFP1 (Arabidopsis thalianazinc-finger protein 1), is expressed at high levels in the shoot apex, including the apical meristem, developing leaves and the developing vascular system. In light-grown seedlings, AtZFP1 expression is induced about three days after germination, before the expansion of the true leaves. Dark-grown plants, in which photomorphogenesis is repressed, have no detectable AtZFP1 expression in the shoot apex. Under conditions which induce or mimic photomorphogenic development including growth in the light, shifting dark-grown plants to continuous light or growth on cytokinin in the dark, high levels of AtZFP1 expression are detected. Furthermore, AtZFP1 expression does not depend on active photosynthesis as shown by analysis of plants grown on the carotenoid biosynthetic inhibitor norflurazon. These results are discussed in relation to a possible role for AtZFP1 in shoot development, downstream of photomorphogenic activation.     

The Arabidopsis HSP18.2 promoter/GUS gene fusion in transgenic Arabidopsis plants: a powerful tool for the isolation of regulatory mutants of the heat-shock response

A detailed study of the expression of the promoter of the HSP18.2 gene from Arabidopsis fused to the bacterial gene for β-glucuronidase (GUS) in transgenic Arabidopsis plants is described. High levels of GUS activity were induced in all organs of transformants except for seeds during heat shock. The optimum temperature for expression of GUS in Arabidopsis was 35°C regardless of the plant growth temperature. Heat shock of 40°C did not induce any detectable levels of GUS activity. Pre-incubation at 35°C was found to have a protective effect on the induction of GUS activity at 40°C. GUS activity was also increased in response to a gradual increase in temperature. Histochemical analysis revealed that basal levels of GUS activity were induced in the vascular tissue of leaves and sepals, as well as at the tips of carpels, at the normal growth temperature. Heat treatment of a limited part of the plant tissue did not appear to cause systemic induction of GUS activity. To extend the analysis of the plant heat-shock response, we attempted to screen mutations in genes involved in the regulation of the induction of heat-shock protein (HSP) genes, using the GUS gene as a selection marker in transgenic Arabidopsis plants, and the results of this analysis are described.     

Analysis of catalase mutants underscores the essential role of CATALASE2 for plant growth and day length‐dependent oxidative signalling

Three genes encode catalase in Arabidopsis. Although the role of CAT2 in photorespiration is well established, the importance of the different catalases in other processes is less clear. Analysis of cat1, cat2, cat3, cat1 cat2, and cat2 cat3 T‐DNA mutants revealed that cat2 had the largest effect on activity in both roots and leaves. Root growth was inhibited in all cat2‐containing lines, but this inhibition was prevented by growing plants at high CO₂, suggesting that it is mainly an indirect effect of stress at the leaf level. Analysis of double mutants suggested some overlap between CAT2 and CAT3 functions in leaves and CAT1 and CAT2 in seeds. When plants had been grown to a similar developmental stage in short days or long days, equal‐time exposure to oxidative stress caused by genetic or pharmacological inhibition of catalase produced a much stronger induction of H₂O₂ marker genes in short day plants. Together, our data (a) underline the importance of CAT2 in basal H₂O₂ processing in Arabidopsis; (b) suggest that CAT1 and CAT3 are mainly “backup” or stress‐specific enzymes; and (c) establish that day length‐dependent responses to catalase deficiency are independent of the duration of oxidative stress.     

Functional comparison of catalase genes in the elimination of photorespiratory H2O2 using promoter‐ and 3′‐untranslated region exchange experiments in the Arabidopsis cat2 photorespiratory mutant

Photorespiration‐associated production of H₂O₂ accounts for the majority of total H₂O₂ in leaves of C₃ plants and is mainly eliminated by catalases. In Arabidopsis, lack of CAT2, but not CAT1 or CAT3, results in growth suppression and a marked accumulation of H₂O₂ in leaves. To evaluate the contribution of individual catalase genes and their promoters to catalase function, we investigated the growth suppression and H₂O₂ accumulation phenotypes of Arabidopsis derivatives expressing catalase genes from heterologous CAT promoters in a cat2 mutant background. The expression of CAT2 from the CAT2 promoter restored the wild‐type phenotype in a cat2‐1 mutant, while CAT1 and CAT3 promoter‐driven expression of CAT2 did not. Ectopic expression of CAT3 from the CAT2 promoter also restored the normal phenotype, unlike that of CAT1 which required replacement of the CAT1 3′‐untranslated region (UTR) with that of CAT2. These results demonstrated that the photorespiratory role of CAT2 is determined mainly by the regulation of its promoter activity. The 3′‐UTR of CAT2 was vital for controlling CAT2 protein levels under photorespiratory conditions. Identification of component of heterotetramers catalase isoforms suggested that there is some functional redundancy between CAT2 and CAT1 and CAT3.     

Expression of a plant defensin in rice confers resistance to fungal phytopathogens

Transgenic rice (Oryza sativa L. cv. Pusa basmati 1), overexpressing the Rs-AFP2 defensin gene from the Raphanus sativus was generated by Agrobacterium tumefaciens-mediated transformation. Expression levels of Rs-AFP2 ranged from 0.45 to 0.53% of total soluble protein in transgenic plants. It was observed that constitutive expression of Rs-AFP2 suppresses the growth of Magnaporthe oryzae and Rhizoctonia solani by 77 and 45%, respectively. No effect on plant morphology was observed in the Rs-AFP2 expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of Rs-AFP2 plants on the in vitro growth of M. oryzae indicated that the Rs-AFP2 protein produced by transgenic rice plants was biologically active. Transgene expression of Rs-AFP2 was not accompanied by an induction of pathogenesis-related (PR) gene expression, suggesting that the expression of Rs-AFP2 directly inhibits the pathogens. Here, we demonstrate that transgenic rice plants expressing the Rs-AFP2 gene show enhanced resistance to M. oryzae and R. solani, two of the most important pathogens of rice.     

Chloramphenicol Acetyl Transferase (CAT) Protein Is Expressed in Transgenic Tobacco in Field Tests following Attack by Insects

The expression of chloramphenical acetyl transferase (CAT) protein driven by the wound-inducible promoter from the proteinase inhibitor II K (pin2) gene was examined in whole tobacco (Nicotiana tabacum L.) plants under field conditions. Mechanical wounding of the field-grown leaves caused an accumulation of CAT protein in these leaves which begins several hours after wounding and continues to accumulate for about 36 hours. When sections of leaves were assayed for accumulation of CAT protein following wounding, the CAT protein was found to accumulate in the apical portions of the leaves. When endogenous insects attacked the leaves of transgenic plants grown in the field, the plants responded by inducing CAT protein. The mesophyll cells of the leaf were the site of expression of the CAT protein rather than the mid-vein or major veins within the leaf blade, indicating that the wound-inducible pin2 promoter specifically directs the synthesis of novel genes in tissues preferentially consumed by larval insects.     

Low level expression of prokaryotic tzs gene enhances growth performance of transgenic poplars

We modulated the level of a hormone gene expression in poplars using either 35S promoter (p35S) of cauliflower mosaic virus (CaMV) or aux promoter (pAUX) of A. rhizogenes. The transgenic poplars (Populus alba × P. tremula var. glandulosa), in which the bacterial trans-zeatin secretion (tzs) gene was attached either to the 35S promoter or to the aux promoter, were compared for their performance in tissue culture as well as in nursery. Northern blot analysis of total RNA probed with tzs coding region showed that the total tzs mRNA expression by p35S was approximately 200–300-fold higher than that driven by pAUX. In contrast, the cellular zeatin content of p35S-tzs transgenic poplars was merely 13-fold of those found in pAUX-tzs plants. Due to different levels of cellular zeatin levels, the two types of transgenic poplars showed different morphogenetic as well as growth responses. The p35S-tzs transgenic plants showed morphological characteristics typical of those treated with cytokinin in culture. These include multiple axillary shoot formation, thick stems, narrow leaves and absence of roots. In contrast, the pAUX-tzs plants had slightly higher cellular cytokinin levels than did control plants and showed a lower degree of cytokinin-related phenotypes, including a few axillary shoots in root-inducing media. Since p35S-tzs did not develop roots, only pAUX-tzs transgenic poplars could be transplanted to the nursery where they resumed a close-to-normal growth. Nevertheless, pAUX-tzs plants transferred to the nursery developed cytokinin-related phenotypes, including greater number of shoots, smaller leaves and slightly retarded growth in height, but with a high total biomass.     

Transgenic Arabidopsis plants expressing Escherichia coli pyrophosphatase display both altered carbon partitioning in their source leaves and reduced photosynthetic activity

The effects of the cytosolic expression of Escherichia coli pyrophosphatase (ppa) were investigated in the rosette leaves of transgenic Arabidopsis plants. During the daytime, glucose and fructose were found to accumulate at levels that were approximately two- to threefold higher in these plants than in the wild type. Interestingly, however, neither sucrose nor starch levels showed any distinctive build up in transgenic plants except under continuous white light growth conditions, during which they accumulated at high levels. Additionally, the leaves of transgenic Arabidopsis plants contain two- to threefold higher levels of inorganic phosphate (Pi) and two- to sixfold higher levels of uridine diphosphate-glucose than wild type plants during the diurnal cycle. In contrast, triose phosphate contents in the leaves of E. coli ppa transformants were either similar or slightly decreased when compared with wild type leaves. Furthermore, the photosynthetic activity of these transgenic plants was found to be reduced by 20–40% compared to normal levels. These results indicate that induction of ppa activity in the cytosol affects carbon partitioning between source and sink organs and also that the concomitant increase in Pi caused the accumulation of carbon metabolites and reduced photosynthetic activity.     

Identification and suppression of the p‐coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

Grasses, such as Zea mays L. (maize), contain relatively high levels of p ‐ coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p ‐ coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full‐length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls.