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

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Abstract

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 β-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.

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... This system has been successfully used to control different genes in tobacco, Arabidopsis, potato, oilseed rape, tomato, and rice (Caddick et al. 1998, Roslan et al. 2001, Runzhi et al. 2005. As ethanol is less expensive, readily available, non-toxic in moderate amounts and can be easily supplied to the plants, this system is considered to have a great potential for field application (Corrado and Karali 2009). ...
... In this way, the manipulation of NCED gene expression using the alcohol-inducible promoter (ALC) (Caddick et al. 1998, Roslan et al. 2001, Tomsett et al. 2004, Randall 2021) could be used to stimulate ABA biosynthesis and responses in plants before drought stress occurs in a commercial setting. This approach would allow the control NCED expression and subsequent ABA generation during an effective timeframe, triggering preemptive xylem development and functionality, decreasing BER incidence. ...
... This study was carried out with two ALC::NCED transgenic lines and wild type 'New Yorker' tomato plants. Transgenic lines were obtained and selected for this experiment using the ALC::NCED construct that contains the alcohol (ALC) responsive promoter (Roslan et al. 2001), controlling NCED gene expression. This gene codes for 9cis-epoxycarotenoid dioxygenase (NCED), which is a key enzyme responsible for ABA biosynthesis (Estrada-Melo et al. 2015) Tomato plant transformation was accomplished according to the protocol described by Liang et al. (2014). ...
Article
Fruit susceptibility to the physiological disorder known as blossom-end rot (BER) is an important limitation in tomato production. Abscisic acid (ABA) is known to reduce leaf transpiration, which can enhance plant water use efficiency (WUE), as well as increase fruit xylem functionality, Ca2+ uptake and oxidative stress defenses, which has been suggested to reduce BER incidence. However, the role of ABA on most of these factors determining fruit susceptibility to BER remains poorly understood. ABA production is mainly regulated by the expression of 9-cis-epoxycarotenoid dioxygenase (NCED) genes. Manipulation of NCED gene expression by the alcohol inducible promoter (ALC) could be an alternative approach to stimulate ABA production and its beneficial effects on inhibiting BER incidence. The objectives of this study were to use ALC::NCED transgenic tomato plants to decrease BER incidence and investigate which mechanisms were involved in BER. In this study, two transgenic tomato lines (1 and 2) were developed with the ALC::NCED construct. This construct allows the inducible activation of the ALC promoter by treating the plants with ethanol vapor that drives NCED expression and ABA synthesis. According to the results, after full bloom, weekly spraying transgenic plants with ethanol (2%) decreased BER incidence in both transgenic lines, compared to the wild type ‘New Yorker’ plants. The transgenic line 1 had higher NCED expression in response to ethanol than the transgenic line 2 and wild type ‘New Yorker’. At 15 and 30 days after pollination, transgenic lines 1 and 2 had higher number of functional xylem vessels which helped to increase Ca2+ concentration in the distal end of the fruit, compared to the wild type fruit. In response to higher NCED expression, WUE and antioxidant content in leaves and fruit were higher in both transgenic lines, compared to the wild type, helping to explain the lower BER incidence. Therefore, our study shows that stimulating NCED expression with an inducible system increases the number of functional xylems and Ca2+ uptake into the fruit, improving plant WUE and reducing BER incidence.
... Over the past two decades or so, a number of chemicalinducible promoters have been developed from heterologous elements from animal and microorganisms that direct highly inducible expression of transgenes in plants [60] (Figure 2). The ethanol inducible gene expression system, for example, is based on the AlcR transcription factor and its target promoter AlcA in the fungus Aspergillus nidulans with a great potential for large-scale application because ethanol is cheap with relatively low toxicity and can be easily applied to plants [61][62][63][64] (Figure 3). ...
... Over the past two decades or so, a number of chemical-inducible promoters have been developed from heterologous elements from animal and microorganisms that direct highly inducible expression of transgenes in plants [60] ( Figure 2). The ethanol inducible gene expression system, for example, is based on the AlcR transcription factor and its target promoter AlcA in the fungus Aspergillus nidulans with a great potential for large-scale application because ethanol is cheap with relatively low toxicity and can be easily applied to plants [61][62][63][64] (Figure 3). ...
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The production of therapeutic and industrial recombinant proteins in plants has advantages over established bacterial and mammalian systems in terms of cost, scalability, growth conditions, and product safety. In order to compete with these conventional expression systems, however, plant expression platforms must have additional economic advantages by demonstrating a high protein production yield with consistent quality. Over the past decades, important progress has been made in developing strategies to increase the yield of recombinant proteins in plants by enhancing their expression and reducing their degradation. Unlike bacterial and animal systems, plant expression systems can utilize not only cell cultures but also whole plants for the production of recombinant proteins. The development of viral vectors and chloroplast transformation has opened new strategies to drastically increase the yield of recombinant proteins from plants. The identification of promoters for strong, constitutive, and inducible promoters or the tissue-specific expression of transgenes allows for the production of recombinant proteins at high levels and for special purposes. Advances in the understanding of RNAi have led to effective strategies for reducing gene silencing and increasing recombinant protein production. An increased understanding of protein translation, quality control, trafficking, and degradation has also helped with the development of approaches to enhance the synthesis and stability of recombinant proteins in plants. In this review, we discuss the progress in understanding the processes that control the synthesis and degradation of gene transcripts and proteins, which underlie a variety of developed strategies aimed at maximizing recombinant protein production in plants.
... Earlier, it was shown that in the filamentous fungus Aspergillus nidulans, the transcription factor AlcR induces transcription of the AlcA gene upon ethanol addition [114]. The ethanol-inducible system consists of the transcription factor AlcR under the control of the constitutive viral promoter CaMV35S and a target gene driven by the chimeric promoter containing the CaMV35S minimal promoter fused at the TATA box to the upstream promoter sequences of the AlcA gene [115]. This system was used for ethanol-induced expression of the β-glucuronidase, GFP and luciferase reporter genes in transgenic Arabidopsis plants ( Figure 6K) [115]. ...
... The ethanol-inducible system consists of the transcription factor AlcR under the control of the constitutive viral promoter CaMV35S and a target gene driven by the chimeric promoter containing the CaMV35S minimal promoter fused at the TATA box to the upstream promoter sequences of the AlcA gene [115]. This system was used for ethanol-induced expression of the β-glucuronidase, GFP and luciferase reporter genes in transgenic Arabidopsis plants ( Figure 6K) [115]. The ethanol-inducible system was combined with the Gal4/UAS system for the cell type-specific AlcR gene expression in Arabidopsis [116]. ...
Article
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Continuous and ubiquitous expression of foreign genes sometimes results in harmful effects on the growth, development and metabolic activities of plants. Tissue-specific promoters help to overcome this disadvantage, but do not allow one to precisely control transgene expression over time. Thus, inducible transgene expression systems have obvious benefits. In plants, transcriptional regulation is usually driven by chemical agents under the control of chemically-inducible promoters. These systems are diverse, but usually contain two elements, the chimeric transcription factor and the reporter gene. The commonly used chemically-induced expression systems are tetracycline-, steroid-, insecticide-, copper-, and ethanol-regulated. Unlike chemical-inducible systems, optogenetic tools enable spatiotemporal, quantitative and reversible control over transgene expression with light, overcoming limitations of chemically-inducible systems. This review updates and summarizes optogenetic and chemical induction methods of transgene expression used in basic plant research and discusses their potential in field applications.
... However, in spi lip5 double mutants we found a severe reduction of cells displaying vacuolar CPY and AALP (Figures 5A-D). The vacuolar sorting defect was even more obvious after controlled induction of CPY-mCHERRY expression using an EtOH-inducible vector system (Roslan et al., 2001) (Figures 5E,F and Supplementary Figure S4). ...
... CLSM was performed as described (Steffens et al., 2014). Expression of alcR pro -fusion proteins (pCAMPARI-vector) was induced by fumigation of transfected leaves with 2% ethanol (v/v) for 4 h (Roslan et al., 2001). Before induction of protein expression, transfected cells were identified by the presence of constantly co-expressed free YFP. ...
Article
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Beige and Chediak Higashi (BEACH) domain-containing proteins (BDCPs) are facilitators of membrane-dependent cellular processes in eukaryotes. Mutations in BDCPs cause malfunctions of endosomal compartments in various cell types. Recently, the molecular analysis of the BDCP homolog gene SPIRRIG (SPI) has revealed a molecular function in P-bodies and the regulation of RNA stability. We therefore aimed to analyze, whether SPI has also a role in membrane-dependent processes. In this study, we show that SPI physically interacts with endosomal sorting complex required for transport associated ATPase Suppressor of K⁺-transport growth defect1 (SKD1) and its positive regulator, LYST Interacting Protein 5 (LIP5) and report genetic interactions between SPI and SKD1 and LIP5. We further show that the endosomal transport route of soluble proteins to the lytic vacuole is disturbed in spi lip5 double mutants but not in the single mutants. These vacuolar transport defects were suppressed by additional expression of SKD1. Our results indicate that the BEACH domain protein SPI has in addition to a role in P-bodies a function in endosomal transport routes.
... Cas9 mRNA, the promoter utilized to drive sgRNA, and the quantity of sgRNA:Cas9 RNP complex in plant cell, further influence the heat-shock IGE systems. Chemicals, such as estradiol, ethanol, and glucocorticoid, have been used to develop inducible systems for plants; however, some of these inducers require high concentrations to be effective, which may lead to unintended growth defects (Amirsadeghi et al., 2007;Kang et al., 1999;Padidam, 2003;Roslan et al., 2001). The heat shock-inducible system is flexible and tractable to use compared to other induction systems, making it suitable for a wide range of applications. ...
Article
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Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated nuclease 9 (Cas9) has emerged as a powerful tool to generate targeted loss‐of‐function mutations for functional genomic studies. As a next step, tools to generate genome modifications in a spatially and temporally precise manner will enable researchers to further dissect gene function. Here, we present two heat shock–inducible genome‐editing (IGE) systems that efficiently edit target genes when the system is induced, thus allowing us to target specific developmental stages. For this conditional editing system, we chose the natural heat‐inducible promoter from heat‐shock protein 18.2 (HSP18.2) from Arabidopsis thaliana and the synthetic heat–inducible promoter heat shock–response element HSE‐COR15A to drive the expression of Cas9. We tested these two IGE systems in Arabidopsis using cyclic or continuous heat‐shock treatments at the seedling and bolting stages. A real‐time quantitative polymerase chain reaction analysis revealed that the HSP18.2 IGE system exhibited higher Cas9 expression levels than the HSE‐COR15A IGE system upon both cyclic and continuous treatments. By targeting brassinosteroid‐insensitive 1 (BRI1) and phytoene desaturase (PDS), we demonstrate that both cyclic and continuous heat inductions successfully activated the HSP18.2 IGE system at the two developmental stages, resulting in highly efficient targeted mutagenesis and clear phenotypic outcomes. By contrast, the HSE‐COR15A IGE system was only induced at the seedling stage and was less effective than the HSP18.2 IGE system in terms of mutagenesis frequencies. The presented heat shock–IGE systems can be conditionally induced to efficiently inactivate genes at any developmental stage and are uniquely suited for the dissection and systematic characterization of essential genes.
... A tetracycline-inducible system was developed for tobacco BY-2 suspension cells [74]. Alcohol-inducible gene expression system (AlcR-PalcA) has been successfully used in various plants such as Arabidopsis thaliana [75], Lycopersicon esculentum (tomato), and Populus sp. [76,77]. ...
Article
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Native/endogenous promoters have several fundamental limitations in terms of their size,Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in theirinsufficient transcriptional activity. Several customized synthetic promoters were designed andtested in plants during the past decade to circumvent such constraints. Such synthetic promotershave a built-in capacity to drive the expression of the foreign genes at their maximum amplitude inplant orthologous systems. The basic structure and function of the promoter has been discussed inthis review, with emphasis on the role of the Cis-element in regulating gene expression. In additionto this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. Thisreview also provides explicit information on the two major approaches for developing plant-basedsynthetic promoters: the conventional approach (by utilizing the basic knowledge of promoterstructure and Cis-trans interaction) and the advancement in gene editing technology. The success ofplant genetic manipulation relies on the promoter efficiency and the expression level of thetransgene. Therefore, advancements in the field of synthetic promoters has enormous potential ingenetic engineering-mediated crop improvement
... A tetracycline-inducible system was developed for tobacco BY-2 suspension cells [74]. Alcohol-inducible gene expression system (AlcR-PalcA) has been successfully used in various plants such as Arabidopsis thaliana [75], Lycopersicon esculentum (tomato), and Populus sp. [76,77]. ...
Article
Full-text available
Native/endogenous promoters have several fundamental limitations in terms of their size, Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in their insufficient transcriptional activity. Several customized synthetic promoters were designed and tested in plants during the past decade to circumvent such constraints. Such synthetic promoters have a built-in capacity to drive the expression of the foreign genes at their maximum amplitude in plant orthologous systems. The basic structure and function of the promoter has been discussed in this review, with emphasis on the role of the Cis-element in regulating gene expression. In addition to this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. This review also provides explicit information on the two major approaches for developing plant-based synthetic promoters: the conventional approach (by utilizing the basic knowledge of promoter structure and Cis-trans interaction) and the advancement in gene editing technology. The success of plant genetic manipulation relies on the promoter efficiency and the expression level of the transgene. Therefore, advancements in the field of synthetic promoters has enormous potential in genetic engineering-mediated crop improvement.
... Eine weitere Variante eines induzierbaren Systems ist das Ethanol-induzierbare alc-Genexpressionssystem, bei dem in Arabidopsis thaliana Expressionen von Genen mittels Ethanol induziert werden können (Roslan et al., 2001;Deveaux et al., 2003). AtSUC2 ist für die Beladung des Phloems in den Hauptvenen von Source-Blättern verantwortlich (Sauer & Stolz, 1994;Truernit & Sauer, 1995;Stadler & Sauer, 1996). ...
Thesis
Pollenschläuche gehören zu den am schnellsten wachsenden Zellen im Pflanzenreich. Pollen benötigen während der Keimung und Pollenschläuche während des Wachstums viel Energie. Eine mögliche Energiequelle können das Disaccharid Saccharose, aber auch die Spaltprodukte Glukose oder Fruktose sein. Expressionsanalysen konnten zeigen, dass zwei Gene für Zellwandinvertasen, sieben Gene für alkaline/neutrale Invertasen und ein Gen für eine vakuoläre Invertase in Pollen und/oder Pollenschläuchen exprimiert werden. Die Expression von Saccharose-Synthase-Genen war nicht nachweisbar. Pollenkeimungstests mit knockout(k.o.)-Linien ergaben, dass nur die vakuoläre Invertase AtVI2 in vitro für die Pollenkeimung wichtig ist. In reproduktiven Organen wie Funiculi, Durchlassgewebe, Stigma, Pollen, Pollenschläuchen, Antheren etc. konnte die Expression von Zuckertransportergenen der AtSUC- und AtSWEET-Genfamilie nachgewiesen werden. Es stellte sich heraus, dass sieben AtSUC-Gene in Pollen und/oder Pollenschläuchen exprimiert werden, aber AtSUC1 für die Funktionalität der Pollen der wichtigste Saccharosetransporter ist. Ionenchromatographische Messungen des Saccharosegehaltes von Atsuc1- und Atsuc1Atsuc3Atsuc8Atsuc9-Pollen zeigten, dass der Verlust von AtSUC1 eine Reduktion des Saccharosegehaltes im reifen Pollen um ca. 50 % zur Folge hatte. Dies belegt, dass AtSUC1 bereits bei der Beladung des Pollen während der Reifung in der Anthere eine Rolle spielt. Keimungstests zeigten, dass Pollen am besten bei 250 mM Saccharose keimen. Das lässt vermuten, dass in vivo möglicherweise hohe apoplastische Saccharosekonzentrationen die Keimung fördern. Auch das Pollenschlauchwachstum wurde in vitro durch Saccharose gefördert. Ab 100 mM Saccharose im Pollenkeimungsmedium stieg die Pollenschlauchlänge mit zunehmender Saccharosekonzentration bis zu einem Optimum bei 250 mM Saccharose an. Es konnte gezeigt werden, dass Pollen viel Saccharose, aber keine nachweisbare Stärke enthalten. Saccharose könnte deshalb vor allem während der Keimung als Energiequelle dienen oder womöglich als Signalmolekül, da sie die Keimung in niedrigen Konzentrationen hemmt und in hohen Konzentrationen fördert. Maltose, Prolin und andere Zucker neben Saccharose konnten Saccharose im Medium nicht ersetzen. Aufgrund dieser Daten wird vorgeschlagen, Saccharose für in vitro-Analysen mit Pollenschläuchen als Energiequelle im Medium zu verwenden. Das Enzym der Prolinsynthese, AtP5CR, ist für die Pollenkeimung und das Pollenschlauchwachstum in vitro wichtig, da AtP5CR/Atp5cr-Pollen eine signifikant niedrigere Keimungsrate und kürzere Pollenschläuche zeigten. Das Nebenprojekt zur Analyse der Regulation der AtSUC2-Expression ergab, dass der AtSUC2-Promotor in weißen, chlorophyllfreien Bereichen der grün-weiß gefleckten Blätter von immutans-Mutanten nicht aktiv war. Da bekannt ist, dass AtSUC2 in Sink-Blättern nicht exprimiert wird, deutet dies an, dass die weißen Bereiche der immutans-Blätter in einem Sink-Status bleiben. Eine Zugabe von externer Saccharose konnte den AtSUC2-Promotor in weißen Bereichen induzieren, was belegt, dass die Expression von AtSUC2 durch den Saccharosegehalt im Apoplasten induzierbar ist. Im zweiten Nebenprojekt konnte gezeigt werden, dass BvSUT3 Esculin transportieren kann. Dies ist bereits von Transportern bekannt, welche Saccharose als Substrat erkennen. Die Daten deuten an, dass BvSUT3 sehr wahrscheinlich in vivo die Aufnahme von Saccharose katalysiert. BvSUT3 lokalisierte möglicherweise in Blättern und Wurzeln von Beta vulgaris in Zellen des Phloems.
... An ethanol-inducible operon, including the alcA promoter and transcription factor for the alcohol dehydrogenase regulon (ALCR) in Aspergillus nidulans, was combinated with T7 expression system for ethanol induction. [97][98][99] After treating with 5% ethanol, PHB synthesis was induced and reached 1383 ppm in dry weight while there was only 171 ppm in uninduced tissues. Moreover, the seedling germination, growth, flower, and fertile seeds were possible when the seedling grew under the condition of ethanol non-induction. ...
Article
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The T7 system is an orthogonal transcription‐system, which is characterized by simplicity, higher efficiency, and higher processivity, and it is used for protein or mRNA synthesis in various biological‐systems. In comparison with prokaryotes, the construction of the T7 expression system is still on‐going in eukaryotes, but it shows greatly applicable prospects. In the present paper, development of T7 expression system construction in eukaryotes is reviewed, including its construction in animal (mammalian cells, trypanosomatid protozoa, Xenopus oocytes, zebrafish), plant, and microorganism and its application in vaccine production and gene therapy. In addition, the innate challenges of T7 expression system construction in eukaryote and its potential application in vaccine production and gene therapy are discussed.
... Several chemicals such as estradiol, ethanol, ecdysone, glucocorticoid, etc., have been used for developing inducible systems in plants. However, some of these can lead to untended growth defects (Kang et al., 1999;Roslan et al., 2001;Amirsadeghi et al., 2007). The estrogen-inducible chimeric transcription activator (XVE) system has been successfully used in several plant species without any undesirable impact on plant growth and morphology. ...
Article
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CRISPR/Cas-mediated editing has revolutionized crop engineering. Due to the broad scope and potential of this technology, many studies have been carried out in the past decade towards optimizing genome editing constructs. Clearly, the choice of the promoter used to drive gRNA and Cas9 expression is critical to achieving high editing efficiency, precision, and heritability. While some important considerations for choosing a promoter include the number and nature of targets, host organism, mode of transformation and goal of the experiment, spatiotemporal regulation of Cas9 expression using tissue-specific or inducible promoters enables higher heritability and efficiency of targeted mutagenesis with reduced off-target effects. In this review, we discuss specific studies that highlight the prospects and trade-offs associated with the choice of promoters on genome editing and emphasize the need for inductive exploration and discovery to further advance this area of research in crop plants.
... To evaluate the regulation capability over the pol-II-synthesized gRNAs, the synthetic CBS:minDFR promoter was used for copper-regulation. In parallel, ethanol regulation was attempted as well, using AlcR as the ethanol-responsive transcriptional factor that binds to AlcA operator in presence of ethanol [28]. All the constructs for Pol-II-synthesized gRNAs contained, like in previous experiments, a protospacer targeting a DFR promoter upstream of the reporter FLuc gene. ...
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Background CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). Results In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger. Conclusions The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.
... Methods to analyze the spatiotemporal activity of regulatory elements in plants are well-established, allowing the most appropriate promoters to be selected (Xiong et al. 2016). For example, the ethanolinducible alc promoter has been used to induce the expression of recombinant enzymes in tobacco (Salter et al. 1998) and other plants (Roslan et al. 2001), and can even be used to restrict expression to specific plant tissues (Schaarschmidt et al. 2004). The method is compatible with large-scale applications because ethanol vapor can be used for induction (Sweetman et al. 2002). ...
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Plants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as “plant molecular farming” (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose “chassis” for PMF.
... One such system is an ethanol-inducible system, where a positively regulated alcA promoter in the nucleus regulates the downstream gene encoding an RNA polymerase derived from T7 bacteriophage. The ethanol-inducible alcA promoter controls the expression of T7-RNA polymerase by the transcription factor AlcR derived from the alcohol dehydrogenase regulon of Aspergillus nidulans (Salter et al., 1998;Roslan et al., 2001) and targeted to chloroplasts via the N-terminally fused transit peptide of the Rubisco small subunit from Pisum sativum (Dasgupta et al., 1998). The T7-RNA polymerase controls the expression from a T7 promoter stably transformed into the plastids (McBride et al., 1994) and the expression of the gene of interest under the control of the T7 promoter takes place (Lössl et al., 2005). ...
Article
Mycobacterium tuberculosis causes tuberculosis in humans. The major disease burden of tuberculosis lies in developing countries. Lack of an effective vaccine for adults is one of the major hurdles for controlling this deadly disease. In the present study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was inducibly expressed in chloroplasts of Nicotiana tabacum. The expression of ESAT-6 in chloroplasts was controlled by T7 promoter that was activated by nuclear-generated signal peptide. Tobacco plants, containing nuclear component, were transformed via biolistic bombardment with pEXP-T7-ESAT-6 obtained by Gateway® cloning. Transformation and homoplasmic status of transplastomic plants was confirmed by polymerase chain reaction and Southern blotting. Plants were induced for protein expression by spraying with 5% ethanol for 1 day, 3 days, 7 days and 10 days. ESAT-6 protein was detected by immunoblot analysis and maximum protein was obtained for 10 days induced plants that was estimated to accumulate up to 1.2% of total soluble fraction of protein. Transplastomic plants showed completely normal morphology. Transplastomic and untransformed plants became slightly chlorotic upon prolonged exposure to ethanol until 10 days. Taken together, this data could help in the development of an antigen-based subunit vaccine against tuberculosis.
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Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species interact with each other and plants using a large chemical language that is interpreted by complex regulatory networks. In this work, we develop modular interkingdom communication channels, enabling bacteria to convey environmental stimuli to plants. We introduce a “sender device” in Pseudomonas putida and Klebsiella pneumoniae, that produces the small molecule p-coumaroyl-homoserine lactone (pC-HSL) when the output of a sensor or circuit turns on. This molecule triggers a “receiver device” in the plant to activate gene expression. We validate this system in Arabidopsis thaliana and Solanum tuberosum (potato) grown hydroponically and in soil, demonstrating its modularity by swapping bacteria that process different stimuli, including IPTG, aTc and arsenic. Programmable communication channels between bacteria and plants will enable microbial sentinels to transmit information to crops and provide the building blocks for designing artificial consortia.
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Chlorophyll is the major light‐absorbing pigment for plant photosynthesis. While evolution has been selected for high chlorophyll content in leaves, previous work suggests that domesticated crops grown in modern high‐density agricultural environments overinvest in chlorophyll production, thereby lowering light use and nitrogen use efficiency. To investigate the potential benefits of reducing chlorophyll levels, we created ethanol‐inducible RNAi tobacco mutants that suppress Mg‐chelatase subunit I ( CHLI ) with small RNA within 3 h of induction and reduce chlorophyll within 5 days in field conditions. We initiated chlorophyll reduction later in plant development to avoid the highly sensitive seedling stage and to allow young plants to have full green leaves to maximise light interception before canopy formation. This study demonstrated that leaf chlorophyll reduction >60% during seed‐filling stages increased tobacco seed nitrogen concentration by as much as 17% while canopy photosynthesis, biomass and seed yields were maintained. These results indicate that time‐specific reduction of chlorophyll could be a novel strategy that decouples the inverse relationship between yield and seed nitrogen by utilising saved nitrogen from the reduction of chlorophyll while maintaining full carbon assimilation capacity.
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The generation of dominant gain-of-function mutants through activation tagging is a forward genetic approach that can be applied to study the mechanisms of flower development, complementing the screening of loss-of-function mutants. In addition, the functions of genes of interest can be further analyzed through reverse genetics. A commonly used method is gene overexpression, where ectopic expression can result in an opposite phenotype to that caused by a loss-of-function mutation. When overexpression is detrimental, the misexpression of a gene using tissue-specific promoters can be useful to study spatial-specific function. As flower development is a multistep process, it can be advantageous to control gene expression, or its protein product activity, in a temporal and/or spatial manner. This has been made possible through several inducible promoter systems as well as inducible proteins by constructing chimeric fusions between the ligand-binding domain of the glucocorticoid receptor (GR) and the protein of interest. The recently introduced CRISPR-Cas9-based platform provides a new way of bioengineering transcriptional regulators in plants. By fusing a catalytically inactive dCas9 with functional activation or repression domains, the CRISPR-Cas9 module can achieve transcriptional activation or repression of endogenous genes. All these methods allow us to genetically manipulate gene expression during flower development. In this chapter, we describe methods to produce the expression constructs, method of screening, and more general applications of the techniques.
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Background Elucidating the genetic and molecular control of plant reproduction often requires the deployment of functional approaches based on reverse or forward genetic screens. The loss-of-function of essential genes, however, may lead to plant lethality prior to reproductive development or to the formation of sterile structures before the organ-of-interest can be analyzed. In these cases, inducible approaches that enable a spatial and temporal control of the genetic perturbation are extremely valuable. Genetic induction in reproductive organs, such as the ovule, deeply embedded in the flower, is a delicate procedure that requires both optimization and validation. Results Here we report on a streamlined procedure enabling reliable induction of gene expression in Arabidopsis ovule and anther tissues using the popular pOP/LhGR Dex-inducible system. We demonstrate its efficiency and reliability using fluorescent reporter proteins and histochemical detection of the GUS reporter gene. Conclusion The pOP/LhGR system allows for a rapid, efficient, and reliable induction of transgenes in developing ovules without compromising developmental progression. This approach opens new possibilities for the functional analysis of candidate regulators in sporogenesis and gametogenesis, which is otherwise affected by early lethality in conventional, stable mutants.
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The author describes her life's pathway from her beginnings at a time when women were not well represented in the sciences. Her grandparents were immigrants to the United States. Although her parents were not able to go to college because of the Great Depression, they supported her education and other adventures. In addition to her interest in science, she describes her interest and involvement in politics. Her education at Oberlin, Stanford, and Harvard prepared her for her independent career at the University of California, Los Angeles, where she was an affirmative action appointment. Her research initially centered on the plant photoreceptor phytochrome, but later in her career she investigated circadian rhythms in plants, discovering and characterizing one of the members of the central oscillator. Expected final online publication date for the Annual Review of Plant Biology, Volume 73 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Inducible systems for transgene expression activated by a chemical inducer or an inducer of non-plant origin are desirable tools for both basic plant research and biotechnology. Although, the technology has been widely exploited in dicotyledonous model plants such as Arabidopsis, it has not been optimised for use with the monocotyledonous model species, namely rice. We have adapted the dexamethasone-inducible pOp6/LhGR system for rice and the results indicated that it is fast, sensitive and tightly regulated, with high levels of induction that remain stable over several generations. Most importantly, we have shown that the system does not cause negative growth defects in vitro or in soil grown plants. Interestingly in the process of testing, we found that another steroid, triamcinolone acetonide, is a more potent inducer in rice than dexamethasone. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.
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Synthetic biology has been developing rapidly in the last decade and is increasingly attracting the attention of many plant biologists. The production of high-value plant specific secondary metabolites is however, mostly limited to microbes. This is potentially problematic, due to incorrect post-translational modification of proteins, different micro-compartmentalization of proteins, substrate availability, chaperone availability, product toxicity and cytochromes p450 enzyme reductase. Unlike other heterologous systems, plant cells could be a promising alternative source for the production of high-value metabolites. Several commercial plant suspension cell cultures from different plant species have been successfully used to produce valuable metabolites with low cost, safety and by environmentally friendly means. However, few metabolites are currently being biosynthesized using plant platforms, with the exception of the natural pigment, anthocyanin. Both Arabidopsis thaliana and Nicotiana tabacum cell cultures can be developed by multiple gene transformations and genome editing by CRISPR-Cas9. Given that the introduction of heterologous biosynthesis pathways into Arabidopsis and N. tabacum metabolites is not widely used, the biosynthesis of foreign metabolites is currently limited; however, therein lies great potential. Here, we discuss the exemplary use of plant cell cultures as well as the prospective of using A. thaliana and N. tabacum cell cultures to produce valuable plant specific metabolites.
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In the scenario of global climate change, abiotic stresses such as rising temperature, water deficit and stress combinations are posing serious threats to sustainable agricultural production, and these factors account for more yield losses than any other factor. Developing crop plants with enhanced abiotic stress tolerance has become a priority now-a-days. Agricultural biotechnology including genomics-assisted breeding and genetic engineering help in studying and understanding the complex nature of abiotic stress responses and provide measures for enhancing crop productivity under adverse environmental conditions. Plants respond and adapt to adverse environmental factors by activating molecular network cascades that are implicated in stress perception, signal transduction, genes expression and accumulation of certain metabolites. Various functional genomics approaches have helped to identify numerous genes involved in stress-associated molecular regulatory networks in both model plants and non-model crop species. Thus engineering genes that activate the transcription of other stress responsive genes or play important roles in protection and maintenance of cellular components and macromolecules might expedite crop improvement programs. Several of these candidate genes have been transformed in agriculturally important crops to improve their abiotic stress tolerance. Nonetheless, genetic engineering for stress tolerance not only encompasses attempts to engineer “single action genes” and “gene pyramiding” but also introgressions of regulatory machinery involving transcription factors. Further, development of transgenic crops not only depends upon the success rate of genetic transformation but also upon their proper integration and evaluation of stress tolerance. This review thus, summarizes the recent progress in the application of genetic engineering and/or transgenic technology for crop improvement in order to realize global food security by developing varieties superior in abiotic stress tolerance.
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Flavonols are a subclass of flavonoids that exhibit a wide array of physiological functions in plants. Commonly found flavonols are synthesized from dihydroflavonols by flavonol synthase (FLS). The genome of Arabidopsis thaliana contains six FLS genes, among which, FLS1, encodes a functional enzyme. Previous work has demonstrated that R2R3-MYB subgroup 7 transcription factors MYB11, MYB12 and MYB111 redundantly regulate flavonol biosynthesis. However, flavonol accumulation in pollen grains was unaffected in the myb11myb12myb111 triple mutant. Here we show that MYB21 and its homologs MYB24 and MYB57, which belong to subgroup 19, promote flavonol biosynthesis through regulation of FLS1 gene expression. A combination of genetic and metabolite analysis is used to identify the role of MYB21 in flavonol biosynthesis regulation through direct binding to the GARE cis-element in the FLS1 promoter. Interestingly, treatment with kaempferol or over-expression of FLS1 rescue stamen defects in the myb21 mutant. We also observed that excess ROS accumulated in myb21 stamen, and that treatment with dipenyleneiodonium chloride (DPI, a ROS inhibitor) can partly rescue the reduced-fertility of a myb21 mutant. Furthermore, drought stress increased ROS abundance and impaired fertility in myb21, myb21myb24myb57 and chs, but not in wild type or myb11myb12myb111. This suggested that pollen-specific flavonol accumulation contributes to drought-induced male fertility by ROS scavenging in Arabidopsis. These are previously unreported insights into the biological function of flavonols in Arabidopsis.
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During the transformation of wild-type (WT)Arabidopsis thaliana, a T-DNA containing OLEOSIN-GFP (OLE1-GFP) was inserted by happenstance within the GBSS1 gene, resulting in significant reduction in amylose and increase in leaf oil content in the transgenic line (OG). The synergistic effect on oil accumulation of combining gbss1 with the expression of OLE1-GFP was confirmed by transforming an independent gbss1 mutant (GABI_914G01) with OLE1-GFP. The resulting OLE1-GFP/gbss1 transgenic lines showed higher leaf oil content than the individual OLE1-GFP/WT or single gbss1 mutant lines. Further stacking of the lipogenic factors WRINKLED1, Diacylglycerol O-Acyltransferase (DGAT1), and Cys-OLEOSIN1 (an engineered sesame OLEOSIN1) in OG significantly elevated its oil content in mature leaves to 2.3% of dry weight, which is 15 times higher than that in WT Arabidopsis. Inducible expression of the same lipogenic factors was shown to be an effective strategy for triacylglycerol (TAG) accumulation without incurring growth, development, and yield penalties.
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Human papillomavirus type 16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction (PCR) and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for seven consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3 μg/gm of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. This data confirms the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology. This article is protected by copyright. All rights reserved.
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The sections in this article are Introduction How Plant Circadian Biology got to Where it is Today How the Components Were Placed in the Plant Clock Current Framework for Understanding the Arabidopsis Clock What may Pave the Way to Greater Understanding of the Clock Conclusion
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The sections in this article are Introduction Conditional Expression of Transgenes Applications to Plant Functional Genomics Potential Applications to Plant Biotechnology Conclusions
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Background: Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana, they have not yet been implemented in grasses. Results: Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). Conclusions: This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.
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Key message Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Abstract Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus, their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus. Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes-mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus, respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or β-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5 g/L gelrite, and 0.2 mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100 mg/L CaCl2. These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.
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Synthetic biology has advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits to provide organisms with new functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to the lack of reliable DNA parts and devices that enable precise control over these new synthetic functions. In particular, memory switches based on DNA site-specific recombination have been the tool of choice to build long-term and stable synthetic memory in other organisms, because they enable a shift between two alternative states registering the information at the DNA level. Here we report a memory switch for whole plants based on the bacteriophage ϕC31 site-specific integrase. The switch was built as a modular device made of standard DNA parts, designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally operated by action of the ϕC31 integrase (Int), and its recombination directionality factor (RDF). The kinetics, memory, and reversibility of the switch were extensively characterized in Nicotiana benthamiana plants.
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To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness. Spatial control of auxin signaling maintains a balance between stem-cell self-renewal and differentiation at the plant shoot apex. Here Ma et al. show that rheostatic control of auxin response by the WUSCHEL transcription factor maintains stem cells by conferring resistance to auxin mediated differentiation.
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Recent advances in the study of plant developmental and physiological responses have benefited from tissue‐specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes. However, both tissue‐specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5‐fluorocytosine (5‐FC) into 5‐fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY‐UPP gene construct in specific cell types using enhancer trap lines and promoters demonstrating that this marker acts cell autonomously. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5‐FC precursor acts systemically as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell specific promoter. Finally, we demonstrate that the tissular inactivation is reversible and can therefore be used to synchronize plant responses or determine cell type specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.
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Main conclusion Bioethanol from lignocellulosic biomass is a promising step for the future energy requirements. Grass is a potential lignocellulosic biomass which can be utilised for biorefinery-based bioethanol production. Grass biomass is a suitable feedstock for bioethanol production due to its all the year around production, requirement of less fertile land and noninterference with food system. However, the processes involved, i.e. pretreatment, enzymatic hydrolysis and fermentation for bioethanol production from grass biomass, are both time consuming and costly. Developing the grass biomass in planta for enhanced bioethanol production is a promising step for maximum utilisation of this valuable feedstock and, thus, is the focus of the present review. Modern breeding techniques and transgenic processes are attractive methods which can be utilised for development of the feedstock. However, the outcomes are not always predictable and the time period required for obtaining a robust variety is generation dependent. Sophisticated genome editing technologies such as synthetic genetic circuits (SGC) or clustered regularly interspaced short palindromic repeats (CRISPR) systems are advantageous for induction of desired traits/heritable mutations in a foreseeable genome location in the 1st mutant generation. Although, its application in grass biomass for bioethanol is limited, these sophisticated techniques are anticipated to exhibit more flexibility in engineering the expression pattern for qualitative and qualitative traits. Nevertheless, the fundamentals rendered by the genetics of the transgenic crops will remain the basis of such developments for obtaining biorefinery-based bioethanol concepts from grass biomass. Abstract Grasses which are abundant and widespread in nature epitomise attractive lignocellulosic feedstocks for bioethanol production. The complexity offered by the grass cell wall in terms of lignin recalcitrance and its binding to polysaccharides forms a barricade for its commercialization as a biofuel feedstock. Inspired by the possibilities for rewiring the genetic makeup of grass biomass for reduced lignin and lignin–polysaccharide linkages along with increase in carbohydrates, innovative approaches for in planta modifications are forging ahead. In this review, we highlight the progress made in the field of transgenic grasses for bioethanol production and focus our understanding on improvements of simple breeding techniques and post-harvest techniques for development in shortening of lignin–carbohydrate and carbohydrate–carbohydrate linkages. Further, we discuss about the designer lignins which are aimed for qualitable lignins and also emphasise on remodelling of polysaccharides and mixed-linkage glucans for enhancing carbohydrate content and in planta saccharification efficiency. As a final point, we discuss the role of synthetic genetic circuits and CRISPR systems in targeted improvement of cell wall components without compromising the plant growth and health. It is anticipated that this review can provide a rational approach towards a better understanding of application of in planta genetic engineering aspects for designing synthetic genetic circuits which can promote grass feedstocks for biorefinery-based bioethanol concepts. Graphic abstract Open image in new window
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The engineering of plant genomes presents exciting opportunities to modify agronomic traits and to produce high-value products in plants. Expression of foreign proteins from transgenes in the chloroplast genome offers advantages that include the capacity for prodigious protein output, the lack of transgene silencing and the ability to express multicomponent pathways from polycistronic mRNA. However, there remains a need for robust methods to regulate plastid transgene expression. We designed orthogonal activators that boost the expression of chloroplast transgenes harbouring cognate cis-elements. Our system exploits the programmable RNA sequence specificity of pentatricopeptide repeat proteins and their native functions as activators of chloroplast gene expression. When expressed from nuclear transgenes, the engineered proteins stimulate the expression of plastid transgenes by up to ~40-fold, with maximal protein abundance approaching that of Rubisco. This strategy provides a means to regulate and optimize the expression of foreign genes in chloroplasts and to avoid deleterious effects of their products on plant growth.
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Ethanol fermentation is considered as one of the main metabolic adaptations to ensure energy production in higher plants under anaerobic conditions. Following this pathway, pyruvate is decarboxylated and reduced to ethanol with the concomitant oxidation of NADH to NAD +. Despite its acknowledgement as an essential metabolic strategy, the conservation of this pathway and its regulation throughout plant evolution have not been assessed so far. To address this question, we compared ethanol fermentation in species representing subsequent steps in plant evolution and related it to the structural features and transcriptional regulation of the two enzymes involved: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). We observed that, despite the conserved ability to produce ethanol upon hypoxia in distant phyla, transcriptional regulation of the enzymes involved is not conserved in ancient plant lineages, whose ADH homologues do not share structural features distinctive for acetaldehyde/ethanol-processing enzymes. Moreover, Arabidopsis mutants devoid of ADH expression exhibited enhanced PDC activity and retained substantial ethanol production under hypoxic conditions. Therefore, we concluded that, whereas ethanol production is a highly conserved adaptation to low oxygen, its catalysis and regulation in land plants probably involve components that will be identified in the future .
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Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical- and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi- and fully synthetic open- and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals. © 2019 American Society of Plant Biologists. All rights reserved.
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Transcription factors play key regulatory roles in all the life processes across kingdoms. In plants, the genome of a typical model species such as Arabidopsis thaliana encodes over 1500 transcription factors that regulate the expression dynamics of all the genes in time and space. Therefore, studying their function by analyzing the loss and gain-of-function lines is of prime importance in basic plant biology and its agricultural application. However, the current approach of knocking out genes often causes embryonic lethal phenotype, while inactivating one or two members of a redundant gene family yields little phenotypic changes, thereby making the functional analysis a technically challenging task. In such cases, inducible knock-down or overexpression of transcription factors appears to be a more effective approach. Restricting the transcription factors in the cytoplasm by fusing them with animal glucocorticoid/estrogen receptors (GR/ER) and then re-localizing them to the nucleus by external application of animal hormone analogues has been a useful method of gene function analysis in the model plants. In this chapter, we describe the recent advancements in the GR and ER expression systems and their use in analyzing the function of transcription factors in Arabidopsis.
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Transcription factors are pivotal for the control of development and the response of organisms to changes in the environment. Therefore, a detailed understanding of their functions is of central importance for biology. Over the years, different experimental methods have been developed to study the activities of transcription factors in plants. These methods include perturbation assays, where the activity of a given transcription factor is disrupted and subsequently, the resulting effects are monitored using molecular, genomic, or physiological approaches. Perturbation assays can also be used to distinguish primary roles of transcription factors of interest from secondary effects. Thus, molecular genetic experiments after perturbation can be advantageous or even necessary for the precise understanding of transcription factor function at a certain stage of plant development or in a single tissue or organ type. In this chapter, we describe several commonly used techniques to knock down transcription factor activities and provide detailed information on how those techniques are employed in the model plant Arabidopsis thaliana.
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Microalgae have been widely considered for the production of valuable products, such as lipid-based biofuel, value-added pigments, and anti-photo aging reagents. More recently, microalgae have been considered an alternative host for recombinant protein production because of their economic benefits and ecofriendly characteristics. Additionally, many microalgal strains identified to date are generally recognized as safe (GRAS); therefore, the use of microalgae-based technology is promising. However, basic studies on the genetic engineering of microalgae are rare, despite their importance. Particularly, inducible promoter systems that can be applied for strain engineering or recombinant protein production are rarely studied; hence, a number of challenging issues remain unsolved. Therefore, in this study, we focused on the development of a convenient and compact-inducible promoter system that can be used in microalgae. Based on previous success with plant systems, we employed the alcohol-inducible AlcR-PalcA system, which originates from the filamentous fungus, Aspergillus nidulans. This system comprises only two components, a regulatory protein, AlcR, and an inducible promoter, PalcA. Therefore, construction and transformation of the gene cassettes can be easily performed. Ethanol-dependent gene expression was observed in the transformants with no significant growth retardation or inducer consumption observed in the cells cultivated under optimized conditions. Electronic supplementary material The online version of this article (10.1007/s10811-018-1480-8) contains supplementary material, which is available to authorized users.
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Development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables a tight control of symplasmic cell‐to‐cell transport. Here we report on an elegant, non‐invasive method to quantify passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA‐s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA‐s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA‐s fluorescence in selected cells of the root meristem. This enabled us to compare DRONPA‐s movement from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the least efflux of DRONPA‐s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed a software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA‐s system generates reproducible data and is a valuable tool to study symplasmic connectivity. This article is protected by copyright. All rights reserved.
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Plant synthetic biology is a rapidly emerging field that aims to engineer genetic circuits to function in plants with the same reliability and precision as electronic circuits. These circuits can be used to program predictable plant behavior, producing novel traits to improve crop plant productivity, enable biosensors, and serve as platforms to synthesize chemicals and complex biomolecules. Herein we introduce the importance of developing orthogonal plant parts and the need for quantitative part characterization for mathematical modeling of complex circuits. In particular, transfer functions are important when designing electronic-like genetic controls such as toggle switches, positive/negative feedback loops, and Boolean logic gates. We then discuss potential constraints and challenges in synthetic regulatory circuit design and integration when using plants. Finally, we highlight current and potential plant synthetic regulatory circuit applications.
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Eukaryotic 14-3-3 proteins have been implicated in the regulation of diverse biological processes by phosphorylation-dependent protein-protein interactions. The Arabidopsis genome encodes two groups of 14-3-3s, one of which – epsilon – is thought to fulfill conserved cellular functions. Here, we assessed the in vivo role of the ancestral 14-3-3 epsilon group members. Their simultaneous and conditional repression by RNA interference and artificial microRNA in seedlings led to altered distribution patterns of the phytohormone auxin and associated auxin transport-related phenotypes, such as agravitropic growth. Moreover, 14-3-3 epsilon members were required for pronounced polar distribution of PIN-FORMED auxin efflux carriers within the plasma membrane. Defects in defined post-Golgi trafficking processes proved causal for this phenotype and might be due to lack of direct 14-3-3 interactions with factors crucial for membrane trafficking. Taken together, our data demonstrate a fundamental role for the ancient 14-3-3 epsilon group members in regulating PIN polarity and plant development. DOI: http://dx.doi.org/10.7554/eLife.24336.001
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Obtaining transgenic plants is a common method for analyzing gene function. Unfortunately, stable genetic transformation is difficult to achieve, especially for plants (e.g., soybean), which are recalcitrant to genetic transformation. Transient expression systems, such as Arabidopsis protoplast, Nicotiana leaves, and onion bulb leaves are widely used for gene functional studies. A simple method for obtaining transgenic soybean callus tissues was reported recently. We extend this system with simplified culture conditions to gene functional studies, including promoter analysis, expression and subcellular localization of the target protein, and protein-protein interaction. We also evaluate the plasticity of this system with soybean varieties, different vector constructs, and various Agrobacterium strains. The results indicated that the callus transformation system is efficient and adaptable for gene functional investigation in soybean genotype-, vector-, and Agrobacterium strain-independent modes. We demonstrated an easy set-up and practical homologous strategy for soybean gene functional studies.
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In plants the dorsoventral boundary of leaves defines an axis of symmetry through the centre of the organ separating the top (dorsal) and bottom (ventral) tissues. Although the positioning of this boundary is critical for leaf morphogenesis, how the boundary is established and how it influences development remains unclear. Using live-imaging and perturbation experiments we show that leaf orientation, morphology and position are pre-patterned by HD-ZIPIII and KAN gene expression in the shoot, leading to a model in which dorsoventral genes coordinate to regulate plant development by localizing auxin response between their expression domains. However we also find that auxin levels feedback on dorsoventral patterning by spatially organizing HD-ZIPIII and KAN expression in the shoot periphery. By demonstrating that the regulation of these genes by auxin also governs their response to wounds, our results also provide a parsimonious explanation for the influence of wounds on leaf dorsoventrality.
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Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding.
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Assessing molecular changes that occur through altering a gene’s activity is often hampered by difficulties that arise due to the typically static nature of the introduced perturbation. This is especially problematic when investigating molecular events at specific stages and/or in certain tissues or organs during Arabidopsis development. To circumvent these issues, we have employed chemically inducible artificial microRNAs (amiRNAs) for the specific knockdown of developmental regulators. For our own research, we have combined this gene perturbation approach with a floral induction system, which allows the simultaneous induction of a large number of flowers on the inflorescence of a single plant, and the ability to knock down a gene’s activity at any given stage of development. To enable the plant community to avail of the full benefits of these systems, we describe, in this chapter, strategies for amiRNA-mediated gene perturbations and address some common problems that can be encountered when generating inducible amiRNA constructs, growing these plants, and collecting floral buds for analysis.
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Background: A mechanism of innate antiviral immunity operating against viruses infecting mammalian cells has been described during the last decade. Host cytidine deaminases ( e.g., APOBEC3 proteins) edit viral genomes, giving rise to hypermutated nonfunctional viruses; consequently, viral fitness is reduced through lethal mutagenesis. By contrast, sub-lethal hypermutagenesis may contribute to virus evolvability by increasing population diversity. To prevent genome editing, some viruses have evolved proteins that mediate APOBEC3 degradation. The model plant Arabidopsis thaliana genome encodes nine cytidine deaminases ( AtCDAs), raising the question of whether deamination is an antiviral mechanism in plants as well. Methods: Here we tested the effects of expression of AtCDAs on the pararetrovirus Cauliflower mosaic virus (CaMV). Two different experiments were carried out. First, we transiently overexpressed each one of the nine A. thalianaAtCDA genes in Nicotianabigelovii plants infected with CaMV, and characterized the resulting mutational spectra, comparing them with those generated under normal conditions. Secondly, we created A. thaliana transgenic plants expressing an artificial microRNA designed to knock-out the expression of up to six AtCDA genes. This and control plants were then infected with CaMV. Virus accumulation and mutational spectra where characterized in both types of plants. Results: We have shown that the A. thalianaAtCDA1 gene product exerts a mutagenic activity, significantly increasing the number of G to A mutations in vivo, with a concomitant reduction in the amount of CaMV genomes accumulated. Furthermore, the magnitude of this mutagenic effect on CaMV accumulation is positively correlated with the level of AtCDA1 mRNA expression in the plant. Conclusions: Our results suggest that deamination of viral genomes may also work as an antiviral mechanism in plants.
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The chimeric transcriptional activator tTA, a fusion between the Tn 10 encoded Tet repressor and the activation domain of the Herpes simplex virion protein VP16, was stably expressed in transgenic tobacco plants. It stimulates transcription of the β-glucuronidase (gus) gene from an artificial promoter consisting of 7 tet operators and a TATA-box. Tetracycline, which interferes with binding of tTA to operator DNA, reduces gus expression over several orders of magnitude. This stringency of regulation suggests that the system can be used to construct transgenic plants encoding a potentially lethal gene product. Furthermore, the specific and fast inactivation of tTA allows study of the stability of RNAs and proteins.
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In Aspergillus nidulans there are two alcohol dehydrogenases. In the presence of ethanol, alcohol dehydrogenase I (AHH I) is induced and alcohol dehydrogenase II (ADH II) is repressed. ADH I and ADH II have molecular weights of 39,000 and 36,000 respectively. At least ADH I is under the control of alcR, a transacting regulatory gene that is adjacent to alcA (the structural gene for ADH I, Pateman et al. 1983). Mutations in the alcR regulatory gene result in non inducibility of ADH I specific mRNA. Extreme alcA and alcR mutations result in derepressed levels of ADH II, and it is not clear whether alcR controls ADH II directly or through its control of ADH I synthesis. Both enzymes are subject to carbon catabolite repression. Induction of ADH I and ADH II operates at the level of synthesis or processing of mRNA.
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Summary We describe a chemically induced gene control mechanism for plants based on the ALCR transcription factor andalcApromoter ofAspergillus nidulans, which we have called thealcsystem. Ethanol, the chemical inducer, is not toxic at levels required for induction, and can be applied to the plants by spraying, root drenching and addition to liquid growth media. Thealcsystem is very sensitive to ethanol and the induction is rapid; 0.01% (1.7 mM) ethanol in liquid growth media initiates chloramphenicol acetyl transferase (CAT) reporter gene expression within 4 h, with maximal expression occurring after 4 days. In the complete absence of ethanol, there is no detectable expression of CAT, nor do we observe induction in plants subjected to wound, cold or drought stress, or following treatment with either salicylic acid or methyl jasmonate. However, extreme anoxia resulting in elevated levels of alcohol dehydrogenase activity in both roots and leaves gave substantial induction of CAT in leaves but not in roots. We believe that thealcsystem will have broad utility in the exogenous control of plant gene expression in pure science and that it also has considerable potential in agriculture.
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Summary A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven PTop10 reach from undetectable levels of the reporter enzyme -glucuronidase in the absence of dexa- methasone to induced levels reaching 15–20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.
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SummaryA novel chemical induction system for transcription in plants has been developed, taking advantage of the regulatory mechanism of vertebrate steroid hormone receptors. A chimeric transcription factor, designated GVG was constructed, consisting of the DNA-binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor (GR). The GVG gene was introduced into transgenic tobacco and Arabidopsis together with a luciferase (Luc) gene which was transcribed from a promoter containing six tandem copies of the GAL4 upstream activating sequence. Induction of luciferase activity was observed when the transgenic tobacco plants were grown on an agar medium containing dexamethasone (DEX), a strong synthetic glucocorticoid. Induction levels of the luciferase activity were well correlated with DEX concentrations in the range from 0.1 to 10 µM and the maximum expression level was over 100 times that of the basal level. Analysis of the induction kinetics by Northern blot analysis showed that the Luc mRNA was first detected 1 h after DEX treatment and increased to the maximum level in 4 h. The stationary induction level and the duration of the induction varied with the glucocorticoid derivative used. The GVG gene activity can also be regulated by DEX in transgenic Arabidopsis plants. The results indicate that a stringent chemical control of transcription can be achieved in plants with the GVG system. Advantages and potential uses of this system are also discussed.
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We have developed an estrogen receptor-based chemical-inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol-activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the −46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating transgene expression in plants.
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Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.
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The properties of the firefly luciferase (LUC) make it a very good non-destructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measured in planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed in in planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegated in planta luciferase activity, and we show when in planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on the in vivo and in vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially for in vivo gene expression studies in plant research.
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme beta-glucuronidase (GUS) increased up to 500-fold in tissue culture as well as under greenhouse conditions. Efficient de-repression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de-repressing its activity by a specific inducer at any time point of further development.
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Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and beta-glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.
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In Aspergillus nidulans alcohol dehydrogenase (ADH) I and aldehyde dehydrogenase (AldDH) are co-inducible by acetaldehyde (Pateman et al., 1983; Sealy-Lewis and Lockington, 1984) and subject to carbon catabolite repression. The structural genes alcA and aldA are unlinked, but alcA is closely linked to the positive control gene alcR. We have obtained cDNA clones of alcA and aldA and genomic clones comprising alcA and alcR. The location of these genes in a genomic clone carrying a 13-kb insert was determined by subcloning and subsequent transformation of previously characterised point mutants. We have characterised at the physical level some large deletions encompassing both linked genes. We have shown that induction affects the level of RNA hybridisible with alcA and aldA probes. Mutations in the regulatory gene alcR, result in non-inducibility of RNA hybridisible with either probe. Thus the induction process is possibly at the level of transcription. Analogous experiments suggest that carbon catabolite repression of alcohol dehydrogenase I is equally at the level of transcription.
Article
The alcA and aldA genes of Aspergillus nidulans are regulated in exactly the same manner, being subject to positive control by the product of the alcR gene. We report the complete nucleotide sequence of the alcA gene and its 5' non-coding region, preliminary localization of the region involved in the regulation of alcA expression, and a detailed comparison of this region to the 5' non-coding region of aldA (Pickett et al., 1987). The 5' flanking regions of the genes contain six similar sequence elements. Three of these elements are located upstream from the messenger RNA start points and one is related to a sequence element found in the region responsible for ethanol induction of the yeast ADH2 gene (Beier et al., 1985). The other homologous elements are located within the messenger RNA leader and may be associated with selection of messenger RNA start points. The amino acid sequence of alcohol dehydrogenase I (348 residues) shows a significant level of homology with analogous sequences in other organisms. Gene alcA contains introns which are similar in size and structure to other fungal introns. We discuss the positions of the introns in alcA of A. nidulans with particular reference to the conservation of intron position in and the evolutionary assembly of enzymes which possess NAD-binding domains.
Article
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Article
Although promoter regions for many plant nuclear genes have been sequenced, identification of the active promoter sequence has been carried out only for the octopine synthase promoter. That analysis was of callus tissue and made use of an enzyme assay. We have analysed the effects of 5' deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, including different plant tissues. We assayed the RNA transcription product which allows a more direct assessment of deletion effects. The cauliflower mosaic virus (CaMV) 35S promoter provides a model plant nuclear promoter system, as its double-strand DNA genome is transcribed by host nuclear RNA polymerase II from a CaMV minichromosome. Sequences extending to -46 were sufficient for accurate transcription initiation whereas the region between -46 and -105 increased greatly the level of transcription. The 35S promoter showed no tissue-specificity of expression.
Article
Anaerobic treatment drastically alters the pattern of protein synthesized by maize primary roots. During the first hour of anaerobiosis, aerobic protein synthesis is halted and there is an increase in the synthesis of a class of polypeptides with approximate molecular weights of 33,000 daltons. During the second hour of anaerobic treatment, the synthesis of another small group of polypeptides is initated. This group, the anerboic polypeptides (ANPs), accounts for > 70% of total protein synthesis after 5 hr of anaerobiosis, and is synthesized in basically the same ratio until root death (approximately 70 hr). The alcohol dehydrogenase polypeptides are major ANPs. RNA isolated from roots treated anaerobically for at least 24 hr directs the translation of only the anaerobic polypeptides. However, RNA from roots treated anaerobically for only 5 hr directs translation of both anaerobic and aerobic polypeptides. Thus an early response to anaerobic treatment is the suppression of aerobic message translation. Although the anaerobic polypeptides share a formal similarity to heat-shock proteins in animals, it is probable that the anaerobic genes are an adaptation to flooding.
Article
Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.
Article
The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.
Article
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Article
A novel chemical-induced gene regulatory system for plants consisting of two molecular components is described. The first, or regulatory, cassette comprises a chimeric receptor composed of the hinge and ligand binding domains of the Heliothis virescens ecdysone receptor and the transactivation domain of the Herpes simplex VP16 protein fused to the DNA binding domain and transactivation of a mammalian glucocorticoid receptor. The second component, a reporter cassette, contains six copies of the glucocorticoid response element (GRE) fused to the minimal 35SCaMV promoter and beta-glucuronidase. The system uses a commercially available non-steroidal ecdysone agonist, RH5992 (tebufenozide), as an inducer. Activation of gene expression is shown in both tobacco transient protoplasts and transgenic plants. The response is ligand dependent and is modulated by the change in minimal promoter context. The system is capable of inducing transgene activity up to 420-fold corresponding to 150% of the activity observed with positive controls (35SCaMV:GUS).
Article
A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven PTop10 reach from undetectable levels of the reporter enzyme beta-glucuronidase in the absence of dexa- methasone to induced levels reaching 15-20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.
Article
It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.
Article
We have developed an estrogen receptor-based chemical-inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10-90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol-activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the -46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical-inducible system for regulating transgene expression in plants.
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