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An improved assay for ??-glucuronidase in transformed cells: Methanol almost completely suppresses a putative endogenous ??-glucuronidase activity
Abstract
The bacterial β-glucuronidase (GUS) gene is often introduced into plants as a reporter gene fused to a promoter because of advantages over other reporter genes. However, many plants have non-neglegible levels of endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene, especially if this level is a low one. To eliminate the endogenous GUS activity, we studied the effect of some water-soluble organic solvents on decreasing it and found that addition of methanol at 20% volume to a GUS reaction mixture was very effective, lowering the endogenous activity to 0–15% of the origin in all plant extracts tested. Moreover, methanol at this concentration enhanced by 1.4-fold the activity originating from the introduced GUS gene or GUS purified from E. coli. This method enabled us to determine GUS gene expression more reliably, after introduction of GUS gene fusion into rice protoplasts. In histochemical GUS analysis of transgenic tobacco, this method was also effective for suppressing the endogenous GUS activity.
... According to our qRT-PCR results, both genes were expressed in the ovaries before and after the anthesis stage (Fig. 1), followed by no expression beginning 6 DAA (Solyc03g007780) or 2 DAA (Solyc02g067760). However, in the seeds and seed-surrounding tissues of fruits in the WT as well as all promoter-GUS plants, we observed a pale blue color, despite the use of modified methods to reduce intrinsic GUS-like activity according to previous reports 35,36 . The reproductive organs of tomatoes, such as the pericarp, seed coat and embryo, have unexpected GUS-like activity 37 . ...
... GUS and histological assay. Histochemical ß-glucuronidase (GUS) analysis was performed with 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-Gluc) using a previously described method, with a slight modification: phosphate buffer (pH of 8.0) and 20% methanol 35,36,38 . The tissues were incubated in the GUS staining solution at room temperature for 16 h, followed by washing with 70% ethanol to eliminate chlorophyll. ...
Parthenocarpy is the development of an ovary into a seedless fruit without pollination. The ubiquitous downregulation of SlIAA9 induces not only parthenocarpic fruit formation but also an abnormal vegetative phenotype. To make parthenocarpic transgenic tomato plants without unwanted phenotypes, we found two genes, namely, Solyc03g007780 and Solyc02g067760, expressed in ovary tissue but not in vegetative tissues. Solyc03g007780 was expressed in developing ovaries and anthers. Solyc02g067760 mRNA was detected in whole-flower tissues. The promoters of Solyc03g007780 (Psol80) and Solyc02g067760 (Psol60) predominantly induced the expression of genes in the ovule, placenta, endocarp and pollen and in whole-flower tissues, respectively. Psol80/60-SlIAA9i lines, created for SlIAA9-RNA interference controlled by two promoters, successfully formed parthenocarpic fruits without pleiotropic effects in vegetative tissues. Downregulation of SlIAA9, responsible for parthenocarpic fruit formation, was observed in ovules rather than ovaries in the Psol80/60-SlIAA9i lines. Although the weight of parthenocarpic fruits of the Psol80/60-SlIAA9i lines was lower than the weight of pollinated fruits of the wild type (WT), the parthenocarpic fruits presented redder and more saturated colors and higher levels of total soluble solids and titratable acidity than the WT fruits.
... 9. GUS histochemical assay [19,20]: Place plant tissue in a test tube containing GUS reaction buffer [GUS substrate solution with 20% (v/v) methanol] (see Note 7), and incubate at 37 C for 30 min to 24 h. GUS-positive cell aggregates will be staining blue (Fig. 2c, d) 11. ...
... 14. The addition of methanol to the GUS reaction has been shown to increase the activity of the bacterial enzyme and inhibit endogenous GUS-like activities in plant tissues [20]. ...
Genetic transformation of cotton (Gossypium hirsutum L.) is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependent. However, once embryogenic cell cultures are available, they can be effectively used for transformation by Agrobacterium or biolistic bombardment methods. Here I describe a detailed procedure to transform cotton embryogenic cell suspension cultures by biolistic bombardment. A commercially available, helium-driven biolistic device (Bio-Rad PDS1000/He) was used to bombard gold particles coated with plasmid DNA (for visual identification of transformed cells and/or selection) into embryogenic cells. Stable transformation at a high frequency (up to 4% of the transiently expressing cells) is possible. Regeneration of fertile transgenic plants from embryogenic cells takes only about 2 months. Another advantage of the embryogenic cell suspension cultures is that they are amenable for cryopreservation and long-term storage. It is highly preferable to transform commercial varieties of choice than obsolete varieties to avoid genetic drug due to backcrossing.
... 7. GUS histochemical assay [16,17]: Place plant tissue in a test tube containing GUS reaction buffer [GUS substrate solution with 20% (v/v) methanol] (see Note 7) and incubate at 37 C for 30 min. to 24 h. ...
... 7. The addition of methanol to the GUS reaction has been shown to increase the activity of the bacterial enzyme and inhibit endogenous GUS-like activities in plant tissues [17]. ...
Biolistic transformation of cotton (Gossypium hirsutum L.) meristems, isolated from mature seed, is detailed in this report. A commercially available, helium-driven biolistic device (Bio-Rad PDS1000/He) was used to bombard gold particles coated with a marker gene (uidA or “GUS”) into the shoot meristem. The penetration of gold particles was dependent on bombardment parameters, and it was mostly one- to two-cell layers deep. Stable transformation of epidermal L1 layer was consistently observed in approximately 5% of the seedlings. Germ line transformation was observed in up to 0.71% of bombarded meristems by several laboratories. Using this method identification of germ line transformation is laborious and time-consuming. However, the protocol described here represents a simple and efficient method for generating germ line transformation events. In addition, this procedure offers a quick method to evaluate gene constructs in cotton tissues (embryos, cotyledons, leaf) especially fibers which originate as single cells from the maternal epidermis layer.
... After staining, calli and regenerated shoots were decolorized with 99% ethanol for the extraction of chlorophyll. GUS activity in transgenic callus was analyzed by the fluorometric method reported by Kosugi et al. (1990) with 4-methyl-umbelliferyl glucuronide as the substrate. Twelve leaf segments were used in the GUS assay (number of blue spots) and 10 replications were performed. ...
The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium. Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L−1 and 6-benzylaminopurine (BAP) 2 mg L−1, regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L−1 and GA3 0.2 mg L−1, and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L−1 and BAP 0.5 mg L−1. To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression of the nptII resistance gene were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L−1 to select transformed cells. Using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA. This technique will be useful for creating genetically modified T. cinerariifolium, particularly for elucidating the mechanism of pyrethrin biosynthesis toward the creation of pyrethrin-rich traits.
... To remove the chlorophyll and other pigments, a mixture of acetone and methanol (1:3) was added and explants were incubated at 4 • C for 1 h before being visually inspected. After the tissue became transparent, the explants were washed with distilled water twice and stored in 50% glycerol [60,61]. Green fluorescent protein expression in leaves, hypocotyl segments, calli, and developing embryos was visualized using an Olympus SZX12 Stereo Fluorescence Mi-croscope equipped with an HBO 100 W mercury bulb light mounted with a long pass GFP filter and DP74 Cooled Color camera (Olympus America Inc., Melville, NY, USA). ...
Industrial hemp Cannabis sativa L. is an economically important crop mostly grown for its fiber, oil, and seeds. Due to its increasing applications in the pharmaceutical industry and a lack of knowledge of gene functions in cannabinoid biosynthesis pathways, developing an efficient transformation platform for the genetic engineering of industrial hemp has become necessary to enable functional genomic and industrial application studies. A critical step in the development of Agrobacterium tumefaciens-mediated transformation in the hemp genus is the establishment of optimal conditions for T-DNA gene delivery into different explants from which whole plantlets can be regenerated. As a first step in the development of a successful Agrobacterium tumefaciens-mediated transformation method for hemp gene editing, the factors influencing the successful T-DNA integration and expression (as measured by transient β-glucuronidase (GUS) and Green Florescent Protein (GFP) expression) were investigated. In this study, the parameters for an agroinfiltration system in hemp, which applies to the stable transformation method, were optimized. In the present study, we tested different explants, such as 1- to 3-week-old leaves, cotyledons, hypocotyls, root segments, nodal parts, and 2- to 3-week-old leaf-derived calli. We observed that the 3-week-old leaves were the best explant for transient gene expression. Fully expanded 2- to 3-week-old leaf explants, in combination with 30 min of immersion time, 60 µM silver nitrate, 0.5 µM calcium chloride, 150 µM natural phenolic compound acetosyringone, and a bacterial density of OD600nm = 0.4 resulted in the highest GUS and GFP expression. The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest, using the latest technologies such as genome editing, and studying gene functions that regulate secondary metabolites in hemp.
... The binary vector pCAMBIA1305 containing GUSPlus with a CaMV 35S promoter was used in these experiments. GUS activity in transformed tissue was visualized using histochemical staining with 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc) [60]. For Agrobacterium-mediated transformation, individual colonies of A. tumefaciens EHA105 containing pCAMBIA1305, pYLCRISPR/Cas9P 35S -sgRNA(GUS), pISV-CRISPR/Cas9P 35S -sgRNA(GUS), and pISV-CRISPR/Cas9P GmUbi -sgRNA(GUS) were grown in 5 mL of LB medium (supplemented with 50 mg L −1 kanamycin and 25 mg L −1 rifampicin) overnight at 27 • C. The cultures were then harvested using centrifugation (6000 rpm for 5 min), washed with 10 mM MgCl 2 , and then resuspended in the infiltration solution (10 mM MgCl 2 supplemented with 200 µM acetosyringone) to obtain an OD 600 ≈ 0.1. ...
Simple Summary
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has become an important research tool for targeted mutagenesis in plants. However, the construction of mutants in the model legume Medicago truncatula remains challenging as it requires the combination of an efficient CRISPR/Cas9-mediated gene editing system for explants and specific tissue culture techniques for regeneration to whole plants. Here, we describe the construction of M. truncatula mutants using the Agrobacterium tumefaciens-mediated transformation of leaf explants. Regeneration to whole plants was performed at high efficiency (80%), and a gene editing efficiency of up to 70% was reached in the obtained plants. CRISPR/Cas9-mediated gene editing was achieved for three M. truncatula genes. Three mutant lines with knockout mutations in the LysM receptor kinase gene MtLYK10 were further characterized. Inoculation experiments with nitrogen-fixing Sinorhizobium meliloti bacteria indicated that root nodule formation was impaired in the constructed MtLYK10 mutants. In conclusion, we show that the described CRISPR/Cas9 system is suitable for efficient gene editing in M. truncatula and that MtLYK10 plays a role in nodule symbiosis.
Abstract
CRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in Medicago truncatula remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of M. truncatula mutants. Gene editing was performed for the LysM receptor kinase gene MtLYK10 and two major facilitator superfamily transporter genes. The functionality of CRISPR/Cas9 vectors was tested in Nicotiana benthamiana leaves by editing a co-transformed GUSPlus gene. Transformed M. truncatula leaf explants were regenerated to whole plants at high efficiency (80%). An editing efficiency (frequency of mutations at a given target site) of up to 70% was reached in the regenerated plants. Plants with MtLYK10 knockout mutations were propagated, and three independent homozygous mutant lines were further characterized. No off-target mutations were identified in these lyk10 mutants. Finally, the lyk10 mutants and wild-type plants were compared with respect to the formation of root nodules induced by nitrogen-fixing Sinorhizobium meliloti bacteria. Nodule formation was considerably delayed in the three lyk10 mutant lines. Surprisingly, the size of the rare nodules in mutant plants was higher than in wild-type plants. In conclusion, the symbiotic characterization of lyk10 mutants generated with the developed CRISPR/Cas9 protocol indicated a role of MtLYK10 in nodule formation.
... After staining, calli and regenerated shoots were decolorized with 99% ethanol for the extraction of chlorophyll. GUS activity in transgenic callus was analyzed by the uorometric method reported by Kosugi et al. (1990) with 4-methyl-umbelliferyl glucuronide as the substrate. Twelve leaf segments were used in the GUS assay (number of blue spots) and 10 replications were performed. ...
The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium . Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L ⁻¹ and 6-benzylaminopurine (BAP) 2 mg L ⁻¹ , regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L ⁻¹ and GA 3 0.2 mg L ⁻¹ , and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L ⁻¹ and BAP 0.5 mg L ⁻¹ . To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L ⁻¹ to select transformed cells. By using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA . This technique allowed us to elucidate the mechanisms underlying pyrethrum biosynthesis in T . cinerariifolium and was useful for creating transgenic T . cinerariifolium with increased pyrethrum biosynthesis.
... The bacterial β-glucuronidase (GUS) gene is often introduced into plants as a reporter gene fused to a promoter because of its advantages over other reporter genes [37]. The promoter fragments of FvTrihelix6 were amplified using specific primers P-FvTrihelix6-GUS-F and P-FvTrihelix6-GUS-R containing EcoR I and Pst I restriction sites, and the product was incorporated into the GUS reporter plasmid to create the pFvTrihelix6::GUS vector. ...
The Trihelix is a plant-specific transcription factor family and has critical roles in plant growth and development and stress resistance. There is less information about Trihelix transcription factor genes and their potential functions in strawberries (Fragaria vesca). In the present study, we performed a detailed bioinformatics analysis of the Trihelix family in strawberries including physicochemical properties, chromosomal location, exon–intron distribution, domain arrangement, and subcellular localization. Thirty Trihelix family members were identified and divided into five subfamilies. The expression of FvTrihelix genes in different tissues/organs, i.e., root, stolon, leaf, flower, and fruit, was measured in strawberries after infection with Colletotrichum. gloeosporioides and foliar applications of salicylic acid (SA) and jasmonic acid (JA). Most of the genes showed differential expression responses following C. gloeosporioides infection and hormone treatments (SA and JA), suggesting critical roles in disease resistance and hormonal signaling pathways. As anticipated, the ectopic expression of FvTrihelix6 in Arabidopsis thaliana increased resistance against Colletotrichum. higginsianum infection. FvTrihelix6 protein was localized in the nucleus. We surmise that FvTrihelix6 enhances resistance against pathogens through the SA and JA signaling pathways. This study provides novel insights into the strawberry Trihelix transcription factor genes and new candidates for disease-resistance breeding of strawberries.
... The histochemical assay for GUS gene expression (β-D-Glucuronidase) was performed in sorghum shoot tip calli with 5-Bromo-4-Chloro-3-Indolyl Glucuronide (X-Gluc) as a substrate by established methods [26,27]. Following cocultivation, tissues were harvested for GUS staining. ...
... The positive and negative controls used in the experiment included the CaMV35S-GUS vector and the water treatment, respectively. GUS activity experiments were performed as previously described 46 . ...
Fruit shape is an essential agronomic feature in many crops. We identified and functionally characterized an auxin pathway-related gene VvSUN. VvSUN, which belongs to the SUN/IQ67-DOMAIN (IQD) family, localizes to the plasma membrane and chloroplast and may be involved in controlling fruit shape through auxin. It is highly expressed in the ovary, and the expression level at one week before the anthesis stage is positively correlated with the fruit shape index. Functional analyses illustrated that VvSUN gene overexpression in tomato and tobacco plants changed fruit/pod shape. VvSUN promoter directly bound to VvARF6 in yeast and activated the GUS activity by IAA treatments in grapevine leaves, indicating VvSUN functions are in coordination with auxin. Further analysis of 35S::VvSUN transgenic tomato ovaries showed that the fruit shape changes caused by VvSUN were predominantly caused by variations in cell number along longitudinal directions by regulating endogenous auxin levels via polar transport and/or auxin signal transduction process variations. Moreover, an enrichment of the 35S::VvSUN transgenic tomato differentially expressed genes was found in a variety of biological processes, including primary metabolic process, transmembrane transport, calcium ion binding, cytoskeletal protein binding, tubulin binding, microtubule−based movement and so on. Using weighted gene co-expression network analysis (WGCNA), we confirmed that the plant hormone signal transduction may play a crucial role in controlling fruit shape. As a consequence, it is possible that VvSUN acts as a hub gene, altering cellular auxin levels and plant hormone signal transduction pathway, which plays a role in cell division patterns, leading to anisotropic growth of the ovary and, ultimately, an elongated fruit shape.
... The GUS activity assay was performed essentially as described previously (Kosugi et al. 1990). Briefly, proteins were extracted with lysis buffer containing 50 mM sodium phosphate buffer, pH 7, 10 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100 and 20% methanol. ...
Salicylic acid (SA) plays a key role in plant resistance to pathogens. In Arabidopsis, the isochorismate synthase pathway mainly contributes to pathogen-induced SA synthesis, and expression of SA synthesis genes is activated by two CBP60-type transcription factors, CBP60g and SARD1. In tobacco, the mechanisms underlying SA synthesis remain largely unknown. SA production is induced by wounding in tobacco plants in which the expression of two stress-related mitogen-activated protein kinases is suppressed. Using this phenomenon, we identified genes whose expression is associated with SA synthesis. One of the genes, NtCBP60g, showed 23% amino acid sequence identity with CBP60g. Transient overexpression of NtCBP60g as well as NtSARD1, a tobacco homolog of SARD1, induced SA accumulation in Nicotiana benthamiana leaves. NtCBP60g and NtSARD1 bound calmodulin (CaM), and CaM enhanced SA accumulation induced by NtCBP60g and NtSARD1. Conversely, mutations in NtCBP60g and NtSARD1 that abolished CaM binding reduced their ability to induce SA. Expression profiling and promoter analysis identified hypersensitivity-related genes HSR201 and HSR203J as the targets of NtCBP60g and NtSARD1. Virus-induced gene silencing of both NtCBP60g and NtSARD1 homologs compromised SA accumulation and the expression of HSR201 and HSR203J homologs, which were induced by a pathogen-derived elicitor in N. benthamiana leaves. Moreover, elicitor-induced SA accumulation was compromised by silencing of the HSR201 homolog and the HSR203J homolog. These results suggested that HSR201 and HSR203J are regulated by NtCBP60g and NtSARD1 and are required for elicitor-induced SA synthesis.
... Bacteria (logarithmic growth phase) were collected by centrifugation, washed twice with double deionized water (ddH 2 O), and resuspended in ddH 2 O (OD 600 = 1.0). One hundred microlitres of the bacterial suspension was mixed with 200 μl of GUS staining solution (50 mM NaH 2 PO 4 , 50 mM Na 2 HPO 4 , 10 mM Na 2 EDTA, 0.1% Triton X-100, 0.5 mM K 3 [Fe(CN) 6 ], 0.5 mM K 4 [Fe(CN) 6 ], 20 mM X-Gluc), incubated at 37°C for 4 h, and the colour change was observed (Kosugi et al., 1990). ...
Xanthomonas campestris pv. campestris (Xcc) can cause black rot in cruciferous plants worldwide. Two‐component systems (TCSs) are key for bacterial adaptation to various environments, including hosts. VemR is a TCS response regulator and crucial for Xcc motility and virulence. Here, we report that RavA is the cognate histidine kinase (HK) of VemR and elucidate the signalling pathway by which VemR regulates Xcc motility and virulence. Genetic analysis showed that VemR is epistatic to RavA. Using bacterial two‐hybrid experiments and pull‐down and phosphorylation assays, we found that RavA can interact with and phosphorylate VemR, suggesting that RavA is the cognate HK of VemR. In addition, we found that RpoN2 and FleQ are epistatic to VemR in regulating bacterial motility and virulence. In vivo and in vitro experiments demonstrated that VemR interacts with FleQ but not with RpoN2. RavA/VemR regulates the expression of the flagellin‐encoding gene fliC by activating the transcription of the rpoN2‐vemR‐fleQ and flhF‐fleN‐fliA operons. In summary, our data show that the RavA/VemR TCS regulates FleQ activity and thus influences the expression of motility‐related genes, thereby affecting Xcc motility and virulence. The identification of this novel signalling pathway will deepen our understanding of Xcc–plant interactions. RavA phosphorylates VemR and might affect the interaction of VemR with FleQ, which regulates Xanthomonas campestris pv. campestris motility and virulence via FliA and Clp.
... Para determinar la expresión de los genes GusPlus y gusA, se utilizó el kit β-Glucuronidase Reporter Gene Staining (Sigma, GUS) y se les realizó una aplicación de metanol al 20 %; todas las coloraciones azules que se observaron en los callos se consideraron como expresiones transitorias del gen (Abdollahi et al., 2011;Kosugi et al., 1990;Tripathi et al., 2010). Posterior a la tinción, los callos embriogénicos transformados y los controles se almacenaron en tubos de 1,5 ml con alcohol al 75 %. ...
Introducción. La transformación del arroz (Oryza sativa L. ssp indica) mediada por Agrobacterium, representa una oportunidad para la investigación científica y el mejoramiento genético. Es necesaria la optimización del protocolo para obtener la mayor eficiencia de transformación. Objetivo. Evaluar diferentes factores que afectan la transformación genética en callos embriogénicos de arroz de la subespecie indica vía Agrobacterium tumefaciens. Materiales y métodos. Este estudio se realizó en San José, Costa Rica, entre 2012 y 2014. En seis tratamientos se evaluaron: el efecto de la edad del callo, la concentración de acetosiringona, condición luminosa, la presencia o ausencia de radícula y la cepa de Agrobacterium tumefaciens en la transformación genética de callos embriogénicos de arroz de la variedad CR5272 con el gen reportero gus. Se compararon la cepa de Agrobacterium LBA4404 con el plásmido pCAMBIA1305.2 y las cepas ATHV, GV3101 y LBA4404, con el plásmido pCAMBIA1303; mediante pruebas histoquímicas para la detección de la expresión transitoria del gen marcador que codifica para la β-glucuronidasa. Resultados. La evaluación de los seis tratamientos con la cepa LBA4404::pCAMBIA1305.2 resultó en expresión transitoria del gen GusPlus de 1,33-7,00 % para la variedad CR5272 y de 8,00 % para el control con la variedad Nipponbare (ssp. japonica). Las cepas con el plásmido pCAMBIA1303 presentaron una expresión transitoria del gen gusA entre el 100-65 % con un área promedio de 14,23 mm2 (ATHV), 8,81 mm2 (GV3101), y 8,83 mm2 (LBA4404), sin diferencias significativas entre ellas; sin embargo, sí hubo diferencias al compararlas con la cepa LBA4404::pCAMBIA1305.2 (85 %, 4,39 mm2). Conclusiones. La utilización de las condiciones: callos de seis días, concentración de acetosiringona de 76 µM, luz antes y después del cocultivo, presencia de radícula y la cepa ATHV::pCAMBIA 1303, mejoraron la eficiencia de transformación con Agrobacterium tumefaciens en la variedad de arroz CR5272.
... However, the wild-type control plants also showed weak blue staining in the flowers under similar experimental conditions (data not shown). These background GUS activities have previously been detected when using the histochemical stain with tobacco (Kosugi et al. 1990;Sudan et al. 2006). To eliminate the effect of endogenous GUS activity, we performed a fluorometric assay by calculating the mean activity value of the lines of the transgenic tobacco plants. ...
Key message
The promoter of the Arabidopsis thaliana β-glucosidase 19 gene directs GUS expression in a seed-specific manner in transgenic Arabidopsis and tobacco.
Abstract
In the present study, an 898-bp putative promoter of the Arabidopsis β-glucosidase 19 (AtBGLU19) gene was cloned. The bioinformatics analysis of the cis-acting elements indicated that this putative promoter contains many seed-specific elements, such as RY elements. The features of this promoter fragment were evaluated for the capacity to direct the β-glucuronidase (GUS) reporter gene in transgenic Arabidopsis and tobacco. Histochemical and fluorometric GUS analyses of transgenic Arabidopsis plants revealed that the AtBGLU19 promoter directed strong GUS activity in late-maturing seeds and dry seeds, whereas no GUS expression was observed in other organs. The results indicated that the AtBGLU19 promoter was able to direct GUS expression in a seed-specific manner in transgenic Arabidopsis. In tobacco, the intensity of the staining and the level of GUS activity were considerably higher in the seeds than in the other tissues. These results further confirmed that the AtBGLU19 promoter is seed specific and can be used to control transgene expression in a heterologous plant system.
... GUS staining of 5-day-old seedlings was performed following Kosugi et al. (1990) following 9 h of exposure to hydroponic solution containing 10 µM AlCl 3 at pH 5.0. Expression level analysis of GUS was conducted as described above using UBQ1 (AT3G52590) as an internal control (three technical replicates in three individual transgenic lines for each construct). ...
Under acid soil conditions, Al stress and proton stress can occur, reducing root growth and function. However, these stressors are distinct, and tolerance to each is governed by multiple physiological processes. To better understand the genes that underlie these coincidental but experimentally separable stresses, a genome-wide association study (GWAS) and genomic prediction (GP) models were created for approximately 200 diverse Arabidopsis thaliana accessions. GWAS and genomic prediction identified 140/160 SNPs associated with Al and proton tolerance, respectively, which explained approximately 70% of the variance observed. Reverse genetics of the genes in loci identified novel Al and proton tolerance genes, including TON1-RECRUITING MOTIF 28 (AtTRM28) and THIOREDOXIN H-TYPE 1 (AtTRX1), as well as genes known to be associated with tolerance, such as the Al-activated malate transporter, AtALMT1. Additionally, variation in Al tolerance was partially explained by expression level polymorphisms of AtALMT1 and AtTRX1 caused by cis-regulatory allelic variation. These results suggest that we successfully identified the loci that regulate Al and proton tolerance. Furthermore, very small numbers of loci were shared by Al and proton tolerance as determined by the GWAS. There were substantial differences between the phenotype predicted by genomic prediction and the observed phenotype for Al tolerance. This suggested that the GWAS-undetectable genetic factors (e.g., rare-allele mutations) contributing to the variation of tolerance were more important for Al tolerance than for proton tolerance. This study provides important new insights into the genetic architecture that produces variation in the tolerance of acid soil.
... GUS detection and quantification were performed using histochemical (Kosugi et al. 1990) and spectrophotometric assays (Myronovskyi et al. 2011), respectively, at each treatment time point. For the quantitative spectrophotometric GUS assay, 4-nitrophenyl β-d-glucuronide (PNPG; #N1627, Sigma-Aldrich, MO, USA) reaction was measured at 415 nm with a spectrophotometer (NanoDrop ® spectrophotometer 1000, Thermo fisher scientific Inc., TX, USA). ...
Homology-dependent gene silencing (HDGS) is a resistance mechanism against homologous transgene introduction or virus infection in various organisms. In plants in particular, HDGS phenomena in homologous DNA or RNA sequences can cause gene silencing. In this study, GUS-silenced transgenic lines were developed by agroinfiltration with the i35S binary vector for inducing targeted de novo CaMV 35S promoter methylation. Agroinfiltration with i35S triggered inheritable de novo methylation of the CaMV 35S promoter and CAAT box region by HDGS. Moreover, targeted de novo CaMV 35S promoter methylation appeared to be controlled by the expression of SUVH9, which is involved in endogenous DNA methylation and hyper-methylation of the introduced promoter. In summary, this study demonstrated that targeted de novo CaMV 35S promoter methylation may be subjected to an HDGS mechanism that can downregulate gene expression and maintain the heritable stability of the methylated promoter.
... Control plants were treated with corresponding amounts of sterile water. Finally, harvested roots were subjected to histochemical GUS staining using 5-bromo-4-chloro-3-indol-glucuronide cyclohexylamine salt [39]. Briefly, L. japonicus roots were transferred to 10-mL tubes, then soaked with GUS staining solution and exposed to a vacuum for 20 min. ...
Background:
Plant receptors with lysin motifs (LsyM) recognize microbial signals such as fungal chitin and lipo-chitooligosaccharidic Nod factors of nitrogen-fixing rhizobia. It is generally assumed that ligand-induced dimerization of LysM receptors is an essential step in activation of intracellular kinase domains and downstream signaling. Consequently, genes required for plant defense and establishment of symbiosis are expressed. We recently found that three LysM receptor proteins (namely LYK1, LYK4 and LYK5) of Arabidopsis thaliana form a tripartite receptor complex to perceive chitin. However, constitutive and ligand-induced interactions of LysM receptors generally remain difficult to be characterized.
Results:
Interactions between ectodomains of LYK1, LYK4 and LYK5 were investigated by a chimeric receptor approach using hairy roots of the legume Lotus japonicus. Synthetic receptor pairs consisting of a LYK ectodomain and the intracellular domain of a L. japonicus Nod factor receptor (NFR1 and NFR5, respectively) were tested for their capacity to activate expression of the symbiotic NIN (nodule inception) gene. The results indicated constitutive (LYK4ED-LYK4ED, LYK4ED-LYK5ED) and chitin-induced interactions (LYK1ED-LYK1ED, LYK1ED-LYK5ED) of the examined ectodomains.
Conclusion:
We present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.
... Finally, the cross-sections were analyzed, and images taken with a Carl Zeiss Axiopot equipped with Axio Cam ICc5. Fluorometric quantification of the GUS activity (MUG assay) was performed according to the protocols of Jefferson et al. (1987) and Kosugi et al. (1990). Activity of GUS expression was measured with the help of a plate reader (Synergy/HTX-multi mode reader, BioTek, Vermont, USA). ...
C4 photosynthetic plants have evolved from C3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C4 enzymes either to mesophyll (M) or bundle sheath (BS) cells is considered a crucial step towards the evolution of C4 photosynthesis. In this study we demonstrate that the 5´‐flanking sequences of the C4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C4 grass species could drive M cell‐specific expression of a reporter gene in rice. In addition to that, we identified around 450 bp (upstream of their transcription start site) of the analyzed C4Ppc promoters contain all the essential regulatory elements for driving M cell‐specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNS)s were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M cell‐specific expression. This work identifies the evolutionary conservation of C4Ppc regulatory mechanisms of multiple closely related C4 grass species.
... GUS activity was analyzed both histochemically and fluorometrically according to the method of Kosugi et al. (1990), with the following modifications. For histochemical semi-quantitative GUS staining, organs were detached from greenhouse-grown plants, immediately transferred to GUS reaction mixture (containing 1 mM 5-bromo-4-chloro-3indolyl-β-D-glucuronide, 50 mM potassium phosphate buffer [pH 7.0], 10%-20% [v/v] methanol and 1 mM dithiothreitol [DTT]), vacuum infiltrated for 30 min at room temperature, and incubated for approximately 16-24 h at 37°C. ...
The chrysanthemum (Chrysanthemum morifolium) is one of the most popular ornamental plants in the world. Genetic transformation is a promising tool for improving traits, editing genomes, and studying plant physiology. Promoters are vital components for efficient transformation, determining the level, location, and timing of transgene expression. The cauliflower mosaic virus (CaMV) 35S promoter is most frequently used in dicotyledonous plants but is less efficient in chrysanthemums than in tobacco or torenia plants. Previously, we used the parsley ubiquitin (PcUbi) promoter in chrysanthemums for the first time and analyzed its activity in transgenic calli. To expand the variety of constitutive promoters in chrysanthemums, we cloned the upstream region of the actin 2 (CmACT2) gene and compared its promoter activity with the 35S and PcUbi promoters in several organs, as well as its durability for long-term cultivation. The CmACT2 promoter has higher activity than the 35S promoter in calli but is less durable. The PcUbi promoter has the highest activity not only in calli but also in leaves, ray florets, and disk florets, and retains its activity after long-term cultivation. In conclusion, we have provided useful information and an additional type of promoter available for transgene expression in chrysanthemums.
... There are two major differences between E. coli and endogenous GUSB activities. Optimal pH for E. coli GUSB is 7-8 while that for endogenous GUSB is 4-5, and the other difference is the sensitivity to the saccharic acid 1,4-lactone inhibitor present in the enzyme assay mixture, which significantly decreases the endogenous GUSB activity (Kosugi et al., 1990;Abdollahi et al., 2011). An improved protocol for eliminating the endogenous GUSB activity interfering with the transformed plants has been developed (Alwen et al., 1992;Hänsch et al., 1995). ...
... Subsequently YFP was excited at 514 nm and light emission collected at 518-565 nm. Jefferson et al., 1987;Kosugi et al., 1990). To visualize the blue GUS staining, the leaf 508 chlorophyll was extracted using a series of ethanol dilutions spanning from 40-100% (v/v) 509 ethanol. ...
Photosynthesis is limited by the slow relaxation of nonphotochemical quenching, which primarily dissipates excess absorbed light energy as heat. Because the heat dissipation process is proportional to light-driven thylakoid lumen acidification, manipulating thylakoid ion and proton flux via transport proteins could improve photosynthesis. However, an important aspect of the current understanding of the thylakoid ion transportome is inaccurate. Using fluorescent protein fusions, we show that the Arabidopsis (Arabidopsis thaliana) two-pore K+ channel TPK3, which had been reported to mediate thylakoid K+ flux, localizes to the tonoplast, not the thylakoid. The localization of TPK3 outside of the thylakoids is further supported by the absence of TPK3 in isolated thylakoids as well as the inability of isolated chloroplasts to import TPK3 protein. In line with the subcellular localization of TPK3 in the vacuole, we observed that photosynthesis in the Arabidopsis null mutant tpk3-1, which carries a transfer DNA insertion in the first exon, remains unaffected. To gain a comprehensive understanding of how thylakoid ion flux impacts photosynthetic efficiency under dynamic growth light regimes, we performed long-term photosynthesis imaging of established and newly isolated transthylakoid K+- and Cl--flux mutants. Our results underpin the importance of the thylakoid ion transport proteins potassium cation efflux antiporter KEA3 and voltage-dependent chloride channel VCCN1 and suggest that the activity of yet unknown K+ channel(s), but not TPK3, is critical for optimal photosynthesis in dynamic light environments.
... GUS staining was performed as described by Kosugi et al. (1990). Images of stained grains were captured by using a Discovery V20 stereomicroscope (Carl Zeiss, Germany). ...
BRITTLE1 (BT1), responsible for unidirectional transmembrane transport of ADP-glucose, plays a pivotal role in starch synthesis of cereal grain. In this study, we isolated three TaBT1 homoeologous genes located on chromosomes 6A, 6B, and 6D in common wheat. TaBT1 was mainly expressed in developing grains, and knockdown of TaBT1 in common wheat produced a decrease in grain size, thousand kernel weight (TKW), and grain total starch content. High diversity was detected at the TaBT1-6B locus, with 24 polymorphic sites forming three haplotypes (Hap1, Hap2, and Hap3). Association analysis revealed that Hap1 and Hap2 were preferred haplotypes in modern breeding, for their significant correlations with higher TKW. Furthermore, β-glucuronidase (GUS) staining and enzyme activity assays in developing grains of transgenic rice with exogenous promoters indicated that the promoters of Hap1 and Hap2 showed stronger driving activity than that of Hap3. Evolutionary analysis revealed that BT1 underwent strong selection during wheat polyploidization. In addition, the frequency distribution of TaBT1-6B haplotypes revealed that Hap1 and Hap2 were preferred in global modern wheat cultivars. Our findings suggest that TaBT1 has an important effect on starch synthesis and TKW, and provide two valuable molecular markers for marker assisted selection (MAS) in wheat high-yield breeding.
... Histochemical GUS assays were carried out as described by Jefferson et al. (1987) with minor modifications. Leaves and sectioned seeds were incubated at 37°C overnight (12 h) in the dark in 1 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-gluc) in 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.3% (v/v) Triton X-100 and 20% (v/v) methanol to eliminate endogenous GUS activity (Kosugi et al., 1990). After staining, tissues were destained in an ethanol series (50%, 70%, 80% and 95%) to remove Chl, and then stored in 70% (v/v) ethanol, and photographed with a digital camera. ...
The maize (Zea mays) enzyme β‐carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of β‐carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm‐preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown.
To gain insight into the regulation of ZmBCH2, we isolated 2036 bp of the 5′‐flanking region containing the 263 bp 5′‐untranslated region (5′‐UTR) including the first intron. We linked this to the β‐glucuronidase reporter gene gusA.
We found that high‐level expression of gusA in rice seeds requires the 5′‐UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed‐preferred expression profile as long as a prolamin box and AACA motif were present.
We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants.
... Histochemical GUS staining and fluorometric quantitative GUS assays were done according to Kosugi et al. (1990). Crude protein concentrations were determined with a Quick Start Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using bovine serum albumin as a standard. ...
The Cauliflower mosaic virus 35S promoter (P35S) induces transgene expression with insufficient activity and stability in some plant species, including lettuce. To develop a system to provide sufficient gene expression, a polyubiquitin promoter (PLsUbi) and terminator (TLsUbi) were isolated from lettuce, and this system was functionally compared with the conventional P35S-NOS terminator (P35S-Tnos) system by using a β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis, PLsUbi induced higher GUS activity than P35S, and the PLsUbi-TLsUbi combination induced higher GUS activity compared with the PLsUbi-Tnos combination, suggesting that the polyubiquitin terminator promotes transgene expression in concert with PLsUbi. The PLsUbi-TLsUbi combination induced less accumulation of GUS mRNA but > 10-fold higher GUS enzyme activity than the P35S-Tnos combination, suggesting that the PLsUbi-TLsUbi combination translationally promoted GUS expression in Arabidopsis. In transgenic lettuce, PLsUbi-TLsUbi transcriptionally and translationally promoted GUS expression, inducing approximately 16-fold-higher accumulation of GUS mRNA and > 800-fold-higher GUS enzyme activity compared with those induced by P35S-Tnos. Bisulfite sequencing methylation analysis of the introduced promoter sequences indicated that, for PLsUbi, the mean percentage of methylated cytosines in lettuce was 3.5 times that in Arabidopsis. For P35S, the mean percentage of methylated cytosines in lettuce was > 10 times that in Arabidopsis, and this methylation may be a major reason underlying the transcriptional inactivation of P35S in lettuce. Together, our results indicate that PLsUbi-TLsUbi promotes transgene expression in lettuce and Arabidopsis and may have broad applications in genetic engineering of additional plant species.
... Histochemical GUS assay: GUS assay was performed following Jefferson et al. (1987). Methanol was added to the buffer solution at a final concentration of 20% to suppress endogenous β-glucuronidase activity following Kosugi et al. (1990). Transient GUS expression was observed 24-48 h after bombardment of explants by overnight incubation in the GUS staining solution (88.9 mg X-Gluc, 10.0 mg Chloramphenicol, 1.0 ml NaH2PO4 (0.2 M stock), 1.0 ml Triton X-100, 20.0 % Methanol, pH 7.0-8.0) ...
Many factors which affect bombardment efficiency were studied and optimized to develop a simple and reproducible system for transformation of rice through particle-bombardment. Conditions were optimized by varying particle size, helium pressure, target distance and DNA/particle loads. Effect of pre- and post-bombardment osmotic treatment and effective concentration of hygromycin B for selection of transformed cells were also investigated. Both types of explants gave an equally good response in transient GUS expression, however, mature embryos were found to be more suitable giving higher selection efficiency. Gold particles of 1 µm size, helium pressure of 1,100 psi, target distance of 9 cm and a DNA to particle ratio of 3:1 produced the highest transient GUS expression in embryos. Osmotic treatment resulted in 2.7 to 2.9 fold increase in transient GUS expression. Hygromycin B at 50 mg L⁻¹ was found to be optimum for effective selection of transformed cells. Transgenic plants were recovered at an average transformation efficiency of 1.5%. Analysis of putative transgenic plants revealed that 96% of the recovered plants were positive for both GUS and hpt genes. All the established plants were phenotypically normal and fertile.
... A similar procedure was used in M. truncatula, although for entire seeds and their dissected compartments, 20% methanol (v/v) was added to the staining solution to suppress endogenous GUS-like activities, as previously suggested [53]. ...
Phytic acid (InsP6) is the main storage form of phosphate in seeds. In the plant it plays an important role in response to environmental stress and hormonal changes. InsP6 is a strong chelator of cations, reducing the bioavailability of essential minerals in the diet. Only a common bean low phytic acid (lpa1) mutant, affected in the PvMRP1 gene, coding for a putative tonoplastic phytic acid transporter, was described so far. This mutant is devoid of negative pleiotropic effects normally characterising lpa mutants. With the aim of isolating new common bean lpa mutants, an ethyl methane sulfonate mutagenized population was screened, resulting in the identification of an additional lpa1 allele. Other putative lpa lines were also isolated. The PvMRP2 gene is probably able to complement the phenotype of mutants affected in the PvMRP1 gene in tissues other than the seed. Only the PvMRP1 gene is expressed at appreciable levels in cotyledons. Arabidopsis thaliana and Medicago truncatula transgenic plants harbouring 1.5 kb portions of the intergenic 5′ sequences of both PvMRP genes, fused upstream of the GUS reporter, were generated. GUS activity in different organs suggests a refined, species-specific mechanisms of regulation of gene expression for these two PvMRP genes.
... Transgenic tobacco plants were obtained by agrobacterial transformation of leaf explants (Horsh et al., 1985). Histochemical analysis was assayed as described (Jefferson, 1987) with some modification (Kosugi et al., 1990). ...
... Histochemical staining for GUS activity was performed as described by Jefferson et al. (1987) and modified by Kogushi et al. (1990) to avoid background (i.e., with 20% methanol in the 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid (X-Gluc) solution). Fragments of stems, roots, and leaves (plants grown in vitro) as well as bolls and seeds (greenhouse plants) were hand-cut and subsequently incubated with 1-mg/l X-Gluc (Duchefa). ...
This study provides new insights into the biosynthesis regulation and in planta function of the lignan yatein in flax leaves.
Pinoresinol-lariciresinol reductases (PLR) catalyze the conversion of pinoresinol into secoisolariciresinol (SECO) in lignan biosynthesis. Several lignans are accumulated in high concentrations, such as SECO accumulated as secoisolariciresinol diglucoside (SDG) in seeds and yatein in aerial parts, in the flax plant (Linum usitatissimum L.) from which two PLR enzymes of opposite enantioselectivity have been isolated. While LuPLR1 catalyzes the biosynthesis of (+)-SECO leading to (+)-SDG in seeds, the role(s) of the second PLR (LuPLR2) is not completely elucidated. This study provides new insights into the in planta regulation and function of the lignan yatein in flax leaves: its biosynthesis relies on a different PLR with opposite stereospecificity but also on a distinct expression regulation. RNAi technology provided evidence for the in vivo involvement of the LuPLR2 gene in the biosynthesis of (-)-yatein accumulated in flax leaves. LuPLR2 expression in different tissues and in response to stress was studied by RT-qPCR and promoter-reporter transgenesis showing that the spatio-temporal expression of the LuPLR2 gene in leaves perfectly matches the (-)-yatein accumulation and that LuPLR2 expression and yatein production are increased by methyl jasmonate and wounding. A promoter deletion approach yielded putative regulatory elements. This expression pattern in relation to a possible role for this lignan in flax defense is discussed.
... The transformed hairy roots were stained in GUS reaction solution, which contained 1 mM X-Gluc, 100 mM NaH 2 PO 4 , 10 mM Na 2 EDTA, 0.5 mM K 3 Fe(CN) 6 , 0.5 mM K 4 Fe(CN) 6 , and 0.1% Triton X-100. 20% methanol was used to eliminate endogenous GUS activity (Kosugi et al., 1990) and were then photographed with a digital camera after overnight incubation at 37°C. ...
... GUS staining was performed following the protocol of Kosugi et al. (1990). The images of stained samples were captured using a Discovery V20 stereomicroscope (Carl Zeiss, Germany). ...
ADP-glucose pyrophosphorylase (AGPase), comprising two small subunits and two large subunits, is considered a key enzyme in the endosperm starch synthesis pathway in wheat (Triticum aestivum L.). Two genes, TaAGP-S1-7A and TaAGP-L-1B, were investigated in this study. Haplotypes of these genes were associated with thousand kernel weight (TKW) in different populations. Mean TKWs of favored haplotypes were significantly higher than those of non-favored ones. Two molecular markers developed to distinguish these haplotypes could be used in molecular breeding. Frequencies of favored haplotypes were dramatically increased in cultivars released in China after the 1940s. These favored haplotypes were also positively selected in six major wheat production regions globally. Selection of AGP-S1 and AGP-L-1B in wheat mainly occurred during and after hexaploidization. Strong additive effects of the favored haplotypes of with other genes for starch synthesis were also detected in different populations. This article is protected by copyright. All rights reserved.
... GUS activity in various tissues of transgenic (proZmSTK2_USP-GUS) and non-genetic modified maize plants (McC) at different developmental stages was detected via histochemical staining using 5-bromo-4-chloro-3indolyl--D-glucuronide (X-Gluc). Specifically, all tissues were placed in the GUS staining solution (50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.5 mg/mL X-Gluc, 0.1% Triton X-100, and 20% methanol) (Kosugi et al. 1990) and incubated at 37°C overnight. After staining, chlorophyll was removed from the samples by 70% (v/v) ethanol treatment. ...
i>ZmSTK2_USP , located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2,100 bp upstream from the start codon ATG has shown that it contains cis -element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In pro ZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetable tissues. These results show that pro ZmSTK2_USP-GUS is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.
... In situ detection of β-glucuronidase and fluorimetric activity measurements Fluorimetric measurements of GUS activity were performed according to Jefferson et al. (1987) and Kosugi et al. (1990). The statistical significance of the difference between two data sets was analysed using the Mann-Whitney U test (Mann and Whitney, 1947). ...
The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4 Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter–reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4 Flaveria ca3 and ppcA1 genes specifically in M cells.
... Seeds (harvested from transgenic plants grown in the greenhouse) at different stages of development (S1, S2, S3, and S5) were cut transversally and subjected to histochemical staining for GUS activity as described by Jefferson et al. (1987) and modified (Kosugi et al., 1990) to avoid background that could be due to non-specific endogenous GUS activity (i.e., with 20% MeOH in 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) solution). A K + ferricyanide/ferrocyanide mixture (2 mM of each) was also added to the incubation buffer to prevent the diffusion of the indoxyl derivative before its oxidative dimerization. ...
The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.
... PCR-positive plants were analyzed by GUS method. Soybean explants were placed in GUS assay solution containing 80 mM Na 2 HPO, 8 mM Na 2 EDTA, 0.8 % Triton-X, 1.6 % dimethyl sulfoxide, 20 % methanol, 0.38 mM K 4 Fe(CN) 6 , and 1 mM Xglucuro, pH 8.0 for 1 day at 37°C to detect the stable GUS expression on the transgenic plants, after which the explants were washed in 70 % ethanol and in 95 % ethanol [22,23]. The transformation frequency was calculated as the percentage of GUS-positive plants (%) = (GUS-positive explants / tolal number of explants) × %. ...
In the present study, a chi gene from Trichoderma asperellum, designated Tachi, was cloned and functionally characterized in soybean. Firstly, the effects of sodium thiosulfate on soybean Agrobacterium-mediated genetic transformation with embryonic tip regeneration system were investigated. The transformation frequency was improved by adding sodium thiosulfate in co-culture medium for three soybean genotypes. Transgenic soybean plants with constitutive expression of Tachi showed increased resistance to Sclerotinia sclerotiorum compared to WT plants. Meanwhile, overexpression of Tachi in soybean exhibited increased reactive oxygen species (ROS) level as well as peroxidase (POD) and catalase (SOD) activities, decreased malondialdehyde (MDA) content, along with diminished electrolytic leakage rate after S. sclerotiorum inoculation. These results suggest that Tachi can improve disease resistance in plants by enhancing ROS accumulation and activities of ROS scavenging enzymes and then diminishing cell death. Therefore, Tachi represents a candidate gene with potential application for increasing disease resistance in plants.
... The most favoured marker gene is the GUS gene (uidA from Escherichia coli K-12). Such a discrepancy between the results/observations of GUS expression in bombarded pollen of lily has been attributed to the presence (Nishihara et al. 1993) or absence van der Leede-Plegt et al. 1992) of 20 % methanol in the X-Gluc solution used for the assay (Jefferson et al. 1987;Kosugi et al. 1990). Discrepancies such as 'background stain' have also been reported in vegetative tissues as well as pollen (Hu et al. 1990;Shi et al. 1995). ...
In present times, when genetically modified (GM) crops are creating a niche for themselves in the agricultural arena, germline transformation is likely to reduce the time and effort to produce transgenics. In the last three decades of transgenic research, phenomenal success has been achieved but has remained limited to species that lent themselves easily to genetic modification. More than 15 dicot and 11 monocot taxa have been tested for male germline transformation. On the other hand at least 23 three dicot and four monocot taxa have been tested for genetic modification through female germline. Amongst the male germ cells, cellular systems ranging from microspore, immature and mature pollen, pollinaria, pollen protoplasts, pollen tubes, exine detached pollen (EDP) and pollen derived embryos have been tested for transient or stable integration of foreign genes. A variety of methods and variants and combinations of methods such as agrolistics that combines Agrobacterium mediated transformation and biolistics, are available for the introduction of genes into the male germline, as it is accessible to treatments under a variety of conditions. Amongst the methods tested in male germline transformation, particle bombardment remains the most preferred method. The female germline, being largely inaccessible has lent itself to modification mostly via Agrobacterium-mediated methods. The success of in planta vacuum infiltration and floral dip exercise seems to be confined to Crucifers with ovule as the prime target. Applicability of germline transformation methods is being tested on a wider range of crop plants.
The β-glucuronidase (GUS) reporter gene system is an important technique with versatile uses in the study of flower development in a broad range of species. Transcriptional and translational GUS fusions are used to characterize gene and protein expression patterns, respectively, during reproductive development. Additionally, GUS reporters can be used to map cis-regulatory elements within promoter sequences and to investigate whether genes are regulated post-transcriptionally. Gene trap/enhancer trap GUS constructs can be used to identify novel genes involved in flower development and marker lines useful in mutant characterization. Flower development studies primarily have used the histochemical assay in which inflorescence tissue from transgenic plants containing GUS reporter genes are stained for GUS activity and examined as whole-mounts or subsequently embedded into wax and examined as tissue sections. In addition, quantitative GUS activity assays can be performed on either floral extracts or intact flowers using a fluorogenic GUS substrate. Another use of GUS reporters is as a screenable marker for plant transformation. A simplified histochemical GUS assay can be used to quickly identify transgenic tissues.
The phytohormone salicylic acid (SA) regulates plant defense responses against pathogens. Previous studies have suggested that SA is mainly produced from trans-cinnamic acid (CA) in tobacco, but the underlying mechanisms remain largely unknown. SA synthesis is activated by wounding in tobacco plants in which the expression of WIPK and SIPK, two mitogen-activated protein kinases, is suppressed. Using this phenomenon, we previously revealed that HSR201 encoding benzyl alcohol O-benzoyltransferase is required for pathogen signal-induced SA synthesis. In this study, we further analyzed the transcriptomes of wounded WIPK/SIPK-suppressed plants and found that the expression of NtCNL, NtCHD and NtKAT1, homologous to cinnamate-coenzyme A (CoA) ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD) and 3-ketoacyl-CoA thiolase (KAT), respectively, is associated with SA biosynthesis. CNL, CHD and KAT constitute a β-oxidative pathway in the peroxisomes and produce benzoyl-CoA, a precursor of benzenoid compounds in petunia flowers. Subcellular localization analysis showed that NtCNL, NtCHD and NtKAT1 localize in the peroxisomes. Recombinant NtCNL catalyzed the formation of CoA esters of CA, whereas recombinant NtCHD and NtKAT1 proteins converted cinnamoyl-CoA to benzoyl-CoA, a substrate of HSR201. Virus-induced gene silencing of any one of NtCNL, NtCHD and NtKAT1 homologs compromised SA accumulation induced by a pathogen-derived elicitor in Nicotiana benthamiana leaves. Transient overexpression of NtCNL in N. benthamiana leaves resulted in SA accumulation, which was enhanced by co-expression of HSR201, although overexpression of HSR201 alone did not cause SA accumulation. These results suggested that the peroxisomal β-oxidative pathway and HSR201 cooperatively contribute to SA biosynthesis in tobacco and N. benthamiana.
Numerous studies suggest a relevant role of cytokinin (CK) distribution in shaping of plant morphology upon changing environment. Nonetheless, our knowledge about an involvement of short-distance CK translocation in root mineral nutrition is still scarce and specific role of CK transporters in root morphology has yet to be established. Revealing the molecular identity of CK transporters is thereby crucial for better understanding of root plasticity during soil fertility, as well as more frequently encountered plant nutrients deficiencies. In this work, we identified and characterized the Medicago truncatula full-size ATP-binding cassette (ABC) transporter belonging to the G subfamily, namely MtABCG40 as a CK importer. Its expression is root-specific and is induced by nitrogen deprivation and CKs. Our analyses indicate that MtABCG40 has a negative impact on lateral root density through decreased lateral root initiation and enhancement of primary root elongation. Moreover, in line with postulated resemblance to lateral roots, we also observed an inhibitory influence of this transporter on nodule number. Our results suggest that MtABCG40 action affects CK signaling which impacts on the cellular response to auxin. We present data that demonstrate a full-size ABCG transporter with a novel function in legumes and CK transport.
Seed-specific expression using appropriate promoters is a recommended strategy for the efficiently producing valuable metabolites in transgenic plants. In the present study, we investigated the sequence of sucrose binding protein (SBP) as a seed-specific promoter to find the cis -acting elements specific to gene expression in seeds. The 1860 bp SBP sequence was analyzed using Plant Care and PLACE databases to find cis -acting elements, which resulted in a finding of 22 cis -acting elements required for seed expression. In addition, we have discovered cis - acting elements that are indirectly involved in triacylglycerol synthesis (GATABOX, DOFCOREZM, CACGTGMOTIF). The seed specificity of SBP was analyzed by generating a stable transgenic tobacco plant harboring β-glucuronidase (GUS) reporter gene under the control of the SBP promoter. Histochemical analysis of these transgenic tobacco plants indicated decreasing GUS activity in the leaves during the vegetative stage. However, the mature seeds of transgenic plants showed GUS activity. Moreover, the SBP promoter function in the seed oil content was evaluated by the expression of DGAT1 . The expression analysis of DGAT1 in SBP-DGAT1 transgenic tobacco seeds using quantitative real-time PCR revealed a 7.8-fold increase in DGAT1 than in non-transgenic plants. Moreover, oil content increased up to 2.19 times more than in non-transgenic plants. And the oil content of the SBP-DGAT1 transgenic tobacco leaves did not change compared to the control plant. Therefore, we suggested that the SBP promoter could be used as a seed-specific promoter for targeted expression of desired genes in the metabolite engineering of oilseed crops.
Main conclusion
A novel Torenia phenotype having separate petals was obtained by the combination of NF-YA6-VP16 with a floral organ-specific promoter.
Abstract
Genetic engineering techniques helped in obtaining novel flower colors and shapes, in particular, by introducing functionally modified transcription factors (TFs) to ornamental flower species. Herein, we used functionally modified Arabidopsis TFs fused with the repression domain SRDX and the activation domain VP16 to screen for novel floral traits in Torenia fournieri Lind (torenia). We avoided undesired phenotypes unrelated to flowers by expressing these TFs through a floral organ-specific promoter belonging to the class-B genes, GLOBOSA (TfGLO). Fourteen constructs were produced to express functionally modified Arabidopsis TFs in which each of SRDX and VP16 was fused into 7 TFs that were used for the collective transformation of Torenia plants. Among the obtained transgenic plants, phenotypes with novel floral traits reflected in separate petals within normally gamopetalous flower lines. Sequencing analysis revealed that the transgenic plants contained nuclear factor-YA6 (NF-YA6) fused with the VP16. In the margin between the lips of the petals and tube in the TfGLOp:NF-YA6-VP16 plants, staminoid organs have been developed to separate petals. In the petals of the TfGLOp:NF-YA6-VP16 plants, the expression of a Torenia class C gene, PLENA (TfPLE), was found to be ectopically increased. Moreover, expression of TfPLE-VP16 under the control of the TfGLO promoter brought a similar staminoid phenotype observed in the TfGLOp:NF-YA6-VP16 plants. These results suggest that the introduction of the TfGLOp:NF-YA6-VP16 induced TfPLE expression, resulting in the formation of staminoid petals and separation of them.
The β‐glucuronidase gene, uidA (GUS), has remained a favorite reporter gene in plants since its introduction in 1987 for its stability and versatility in a variety of fluorometric, spectrophotometric, and histochemical techniques. One of the most popular uses is as a reporter gene for visualizing endogenous promoter activities within plant tissues. Despite this popularity, specific protocols for minimizing nonrepresentative staining patterns, including false negatives, in challenging tissue types are not common. This became a large issue during our work on dark‐grown Arabidopsis hypocotyls, and we set out to develop a protocol that would ensure accurate staining in a tissue that is biologically resistant to reagent penetration. Through extensive testing using a variety of constitutive and endogenous promoter::GUS fusion lines, we have developed an optimized GUS staining protocol that combines the use of acetone as a fixative, deliberate physical damage, and proper positive and negative controls to help ensure accurate staining along the hypocotyl while minimizing false negatives. Hopefully, our recommendations will allow for improved staining that more accurately reflects the true activity of cloned endogenous promoters and thus facilitate a more accurate understanding of promoter activity in Arabidopsis hypocotyls and other hard‐to‐stain tissues.
Main conclusion
Functional suppression of two types of class-C genes caused transformation of pistils and stamens into petaloid organs that exhibit novel phenotypes, which gives a distinct gorgeous impression in the florets of chrysanthemum.
AbstractThe multiple-petal trait is a breeding objective for many horticultural plants. The loss of function of class-C genes causes the multiple-petal trait in several plant species. However, mechanisms involved in the generation of the multiple-petal trait are unknown in Chrysanthemum morifolium (chrysanthemum). Here, we isolated 14 class-C AGAMOUS (AG) genes, which were classified into two types of class-C genes, in chrysanthemum. Seven of these were categorized into CAG type 1 genes (CAG1s) and seven into CAG type 2 genes (CAG2s). Functions of class-C genes were co-suppressed by chimeric repressors and simultaneously knocked-down by RNAi to produce the multiple-petal phenotype in chrysanthemum. The expression of chimeric repressors of CAG1s and CAG2s caused morphological alteration of the pistils and stamens into petaloid organs in the ray and disk florets. Interestingly, the reproductive organs of the disk florets were transformed into petaloid organs similar to the petals of the disk florets, and those of the ray florets were transformed into petaloid organs such as the petals of the ray florets. Simultaneous knockdown of CAG1s and CAG2s expression by RNAi also exhibited a petaloid phenotype as observed in transgenic plants obtained by chimeric repressors. These results showed that CAG1s and CAG2s play important roles in the development of pistils and stamens, and the simultaneous repression of CAG1s and CAG2s resulted in a multiple-petal phenotype in chrysanthemum.
Characterization of green tissue-specific promoters helps facilitate genetic improvement in crops. Here, we isolated a novel green tissue-specific expression gene Psak encoding photosystem I protein in rice through RT-PCR analysis. The 5′ flanking region from −635 to +60 (transcription start site of Psak as +1) covering parts of Psak gene and its promoter Ppsak was identified as be critical for green tissue-specific expression (including leaves and stems) in rice. Further promoter deletion analyses demonstrated that the promoter region from −559 to −232 was necessary and sufficient for green tissue-specific expression of Psak gene. Histochemical assays showed that the GUS expression of this 267-bp region was highly presented in leaves and stems, but reduced or absent in other tissues examined. The GUS expression level of this core promoter region could be 14.6- to 21.6-fold higher than that of the full-length region of Ppsak promoter in leaves and stems. These results combined with cis-acting elements prediction in Ppsak promoter suggested that the TCT motif located in this promoter played a key role in strong green tissue-specific expression.
The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9 We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing.
The objective of this study was to develop an efficient. reproducible and genotype independent Agrobacterium tumefaclens transforrnation procedure for Egyptian and Canadian flax. Priliminarily experiments showed that the concentrations of 250 ppm cefolaxime and 500 ppm carbencillin were effective to suppress Agrobacterium growth and safe for use since their effects on regeneration was found negligible. Two Egyptian (Giza-7 and Giza-8) and one Canadian (NorLin) cultivars were used. The binary plasmid P35GUSINT in the octopine-type Agrobacterium tumefaciens strain C58ClRif (l.cGV2260) was used as the vector system. An intron-containing B-glucuronidase (GUS) gene driven by Ihe CaMV 355 promoter served as the repoter in the histochemical assay. The neomycin phosphotransferase II (NPTII) gene (driven by the nos promoter) in T·DNA providing the resistance to kanamycin was used as the selectable marker. The flax explants used were the hypocotyls exised from 5-days old seedlings. The GUS assay was done on hypocotyls underwent 3 different duration of pre-peeling (I, 2, 3 or 4 days), post peeling (I, 2, 3 or 4 days) and co-cuItivation (2,4,6 or 8 days) with a Iotal of 64 treatments. Co-cultivation of hypocotyl for 4 days or more and or pre-peeling them for 2 days or more had resulted in 100% transformation frequency (TE). There was a gradual increase in area of GUS transformation tissues (PATT), transformation intensity (TI). number of regenerated shoots (RS/H), number of putative transgenic shoots / hypocol (PTS/H), transformation efficiency (TE) and transformation rate (TR) as duration of pre-peeling, post-peeling and co-cullivalion was increased. Maximum values of PATT, TI and RS/H for all cultivars were obtained from 4 days pre-peeling, 4 days post-peeling and 8 days co-cullivalion. The highest PTSI/H, TE and TR values were obtained from 4 days pre·peeling, 3 days post-peeling and 8 days co-culture for Giza-7 and Giza-8 and from 8 days pre-culture and 8 days co-culture for Norlin. The maximum TR values (10% for Giza 7 and Norlin and 11.67% for Gizas-8) are still below the theoretical maximum, though they are high enough to render the production of transgenic flax plants feasible. A total of 68 putative transgenic Ro plants were recovered; all of them produced R1 seed. GUS assay of Ro plants gave positive results, indicating that the transfomed plants were not chimeric for the lransferred trails. Inheritance studies indicated that kanamycin resistance was inherited as a single dominant allele and transmitted through meiosis in a Mendelian fashion GUS and NPTII genes were expressed and transmitted to the seed progeny and appear to be inherited as a single unit. This gene transfer method could be considered as genotype -apid, efficient and does not involve a distinct callus phase. independent,
The Lupme3 flax (Linum usitatissimum) gene encodes a putative pectin methylesterase (PME). A cDNA was generated by RACE-PCR using two consensus sequences to generate primers. PMEs are encoded by numerous genes (more than 60 ORFs in Arabidopsis contain PME consensus sequences) and little information is available on their expression regulation, and the role of the different isoforms in the plant, except for those that are active in fruits.
A construct using a partial sequence of this gene inserted in antisense orientation downstream of the CaMV 35S promoter was inserted in the flax genome via Agrobacterium tumefaciens. Transgenic lines were analysed for gene expression (northern blots) and PME activity in vitro. PME activity in calli was decreased at basic and neutral experimental pH (6.5, 7.5, 8.5) but increased at pH 5.5. This slight increase at pH 5.5 could be due to compensation by other isoforms, a similar (although opposite) phenomenon has been encountered by other workers who performed sense transgenesis with PME in potato (Pilling et al., 2000). For all transgenic lines but one, PME activity at high pH (pH 8.0) was reduced in calli. On the contrary, activity in mature shoots of plants grown in greenhouse was unchanged as well as the overall growth and aspect of the plants. Similar experiments were conducted with tobacco in which the transgenic plants growth was delayed as compared to the standard plants.
Another type of experiment was performed using a “Lupme3 promoter-reporter gus” fusion construct, in order to analyse the expression of Lupme3 gene in planta: this construct was introduced in an heterologous system i.e tobacco, and in the homologous system i.e. flax. Expression in transgenic tobacco was observed in small leaves veins and in the root central cylinder as well as in calli, but not in mature shoots. In flax also, expression was detected in calli but not in mature shoots.
These results correlate well with those of the PME activity in transgenic antisense flax plants and calli and indicate that this PME isoform is active in young vascular tissues and in undifferentiated cells but is not produced in mature stems.
The genus Rhododendron (Ericaceae) has several hundred species. Horticulturally, it is divided into either the evergreen and deciduous azaleas or the lepidote and elepidote rhododendrons. Esthetically, all of them are esteemed highly, but the elepidote rhododendrons, which are the derivatives of interspecific hybridization among R. arboreum, R, catawbiense, R. ponticum, R. wardii, R. yakushimanum, etc., have been the most highly evaluated because of their outstanding ornamental beauty. They have a marvelous range of flower colors from white to deepest reds and violets through various shades of yellow, orange, pink, mauve, and purple, together with various tree shapes from dwarf creeping shrubs to very high trees, adorned with many beautiful flower trusses (Arisumi 1989). Thus, together with evergreen azaleas, the elepidote rhododendrons constitute one of the most important groups in ornamental woody plants.
The principle of using reporter genes in studying molecular processes in a living cell means that in the natural gene, a synthetic modification is introduced (or the protein coding sequence is deleted and replaced by another gene) in order either to simplify the detection of the gene product or to distinguish it from similar or identical genes in the genome. The use of reporter genes requires a method of gene transfer — either transient or stable. Reporter gene technology can take very different shapes according to how and how much of the gene is tagged, and how the tag is detected or assayed. If one considers a schematic representation of an eukaryotic gene, with its cis-regulating regions, core promoter, transcript leader, translation initiation site, coding sequence, and 3′ control regions, a reporter tag can replace the gene to nearly any desired extent. Figure 1 shows that by replacing the gene to various extents by reporter sequences, gene fusions that can be used to analyze various levels of control of gene expression, as well as protein trafficking, can be generated.
In this study, we attempted to develop a new biotechnological method for the efficient modification of floral traits. Because
transcription factors play an important role in determining floral traits, chimeric repressors, which are generated by attaching
a short transcriptional repressor domain to transcription factors, have been widely used as effective tools for modifying
floral traits in many plant species. However, the overexpression of these chimeric repressors by the Cauliflower mosaic virus 35S promoter sometimes causes undesirable morphological alterations to other organs. We attempted simultaneously to generate
new floral traits and avoid such quality loss by examining five additional floral organ-specific promoters, one Arabidopsis thaliana promoter and four Torenia fournieri promoters, for the expression of the chimeric repressor of Arabidopsis TCP3 (AtTCP3), whose overexpression drastically alters floral traits but also generates dwarf phenotypes and deformed leaves. We found
that the four torenia promoters exhibited particularly strong activity in the petals but not in the leaves, and that the combination
of these floral organ-specific promoters with the chimeric repressor of AtTCP3 caused changes in the color, color patterns and cell shapes of petals, whilst avoiding other unfavorable phenotypes. Interestingly,
each promoter that we used in this study generated characteristic and distinguishable floral traits. Thus, the use of different
floral organ-specific promoters with different properties enables us to generate diverse floral traits using a single chimeric
repressor without changing the phenotypes of other organs.
Genetic tranformation technologies are essential to programs of molecular biology and genetic engineering. Although significant efforts in conifer molecular biology have been initiated since the late 1980s, so far only a limited number of genes have been cloned [1]. Conifers are by far the most difficult plant group for this type of study because of their large genomes and lengthy life cycles. Furthermore, progress has been hindered by the present inefficiencies in gene transfer methods and tissue culture protocols for certain species such as pines. Nevertheless, there is a significant body of literature on gene transfer in several conifer species using Agrobacterium-mediated transformation and other protocols of direct DNA transfer.
The cis-acting elements for regulating gene expression of the tobacco pathogenesis-related la protein gene were analyzed in transgenic plants. The 5'-flanking 2.4-kilobase fragment from the pathogenesis-related Ia protein gene was joined to the bacterial 8-glucuronidase gene and introduced into tobacco cells by Agrobacterium-mediated gene transfer. Promoter activity was monitored by quantitative and histochemical assay of 8-glucuronidase activity in leaves of regenerated transgenic plants. The level of p-glucuronidase activity was clearly increased by treatment with salicylic acid, by cutting stress, and by local lesion formation caused by tobacco mosaic virus infection. Cytochemical studies of the induced 0-glucuronidase activity revealed tissue-specific and developmentally regu- lated expression of the pathogenesis-related la gene after stress or chemical treatment and after pathogen attack. To identify the cis-acting element more precisely, a series of 5'-deleted chimeric genes was constructed and transformed into tobacco plants. Transgenic plants with a 0.3-kilobase fragment of the 5'-flanking region of the pathogenesis-related Ia gene had the same qualitative response as those with the 2.4-kilobase fragment upon treatment with salicylic acid or infection with TMV. Thus, the 0.3-kilobase DNA sequence fragment was sufficient to allow the regulated expression of the pathogenesis-related la gene.
The reporter gene for chloramphenicol acetyltransferase (CAT) was introduced into white spruce (Picea glauca (Moench) Voss.) protoplasts by electroporation. CAT transient gene expression was increased by increasing the concentration of pCaMVCN plasmid and was affected by the level of the applied voltage. Highest CAT activities were obtained after electroporation with a pulse of 350V.cm(-1) having an exponential decay constant of approximately 105ms. Linearized plasmid constructs gave much higher levels of CAT activity than circular plasmid. Attempts to use the Escherichia coli β-glucuronidase gene (β-GUS) as a marker gene revealed very high levels of β-GUS-like activity in electroporated protoplasts. This activity was mainly due to a small molecule and may mask successful transformation since β-GUS-like activity increased when plasmid DNA was present during electroporation.
Upon discovery that Brassica campestris leaf extracts harbour some chloramphenicol acetyl transferase (CAT) activity, a systematic screening of plant tissue for this activity, so far only reported for prokaryotic microorganism, has been conducted. Results were negative for three solanaceous plants as well as for the Cruciferae Arabidopsis thaliana and Orychophragmus violaceus. By contrast, the three tested species of the Cruciferae genus Brassica exhibit significant CAT activity. The Brassica CAT activity is much more heat labile than the enzyme encoded by the bacterial transposon, Tn9, that is commonly used as a reporter in gene fusion experiments.
α-Galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) and β-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) were extracted from spinach leaves and separated from each other by chromatography on DEAE-cellulose and by Sephadex gel filtration. The α-galactosidase was purified 15–30-fold and β-galactosidase over 1000-fold. Using galactosides, α-galactosidase had an optimal pH at 5.3 and a of 0.3 mM; it hydrolyzed melibiose at 1.5% of the rate of hydrolysis of the α-galactoside. This enzyme still had phosphatase, sulfatase and esterase activities. β-Galactosidases had an optimal pH at 4.2 and a of 0.4 mM. It hydrolyzed lactose at 2% of the rate of hydrolysis of β-galactoside. The enzyme was free of α- or β-glucosidase, phosphatase or esterase; it still had considerable sulfatase activity.Monogalactosyldiglyceride, adsorbed onto celite was hydrolyzed by the β-galactosidase at a 250-fold lesser rate than that of β-galactoside. Neither enzyme hydrolyzed digalactosyldiglyceride from Anacystis nidulans.
Chlorate-resistant cell lines were established from survivors after plating allodihaploid cells of Nicotiana tabacum into solid medium containing 20 mM chlorate and amino acids as sole nitrogen source. Data characterizing 9 of the most resistant lines are presented. The mutational origin of these lines was inferred on the basis of the enhancement of the variant frequency by mutagen treatment, and of the persistance of the variant phenotype in cell progeny during growth in the absence of selection for more than 3 years and in plants regenerated from two of the lines.
Seven lines completely lacked in vivo nitrate reductase (NR) activity and two lines exhibited low (less than 5% of the wild type) NR activity. The abolition of NR activity was found to be not due to an impaired induction by nitrate. Data reported elsewhere show that one of the NR-negative mutants simultaneously lacks xanthine dehydrogenase activity. This pleiotropic mutation is interpreted to affect the synthesis of a molybdenum-containing cofactor, whereas the 8 other lines carry mutations specifically affecting the synthesis of the NR. Both types of NR-negative mutants were unable to grow on minimal medium containing nitrate as sole nitrogen source, but grew well on amino acids. They proved extremely sensitive to the standard medium containing nitrate and ammonium. Differences between the NR-negative mutants with respect to chlorate resistance suggest that chlorate inhibits cultured N tabacum cells not only via its NR-catalysed conversion to chlorite, but also by NR-independent mechanisms.
Beta-glucuronidase (GUS) was histochemically analyzed in anthers and pollen of potato, tobacco and tomato. GUS activity was determined in transgenic plants containing a chimaeric GUS gene and in untransformed plants. In anthers of transgenic plants at premeiotic and meiotic stages of sporogenous development, indigogenic precipitation indicative of GUS activity was consistently manifest in cells of the vascular cylinder, the connectivum and the stomium while no activity was found in the tapetal and sporogenous tissues. At similar stages, anther sections of untransformed plants did not show any indigo blue staining. At later stages of microspore and pollen development, anthers of both transgenic and untransformed plants demonstrated consistently high levels of GUS activity in tapetal and sporogenic cells. In anthers of transgenic plants, GUS was also present in the vascular cylinder, the connectivum and the stomium. These results indicate that in anthers of transgenic potato, tobacco and tomato the chimaeric GUS gene product was localized tissue specifically. They also show that an endogenous GUS gene was expressed in a temporal- and spatial-specific manner in the tapetum and pollen of both transformed and untransformed plants.
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.
The nutrient requirements of suspension cultures from soybean root have been investigated, and a simple medium consisting of mineral salts, sucrose, vitamins and 2,4-dichlorophenoxyacetic acid (2,4-d) has been designed.The cells required thiamine, 2,4-d and ammonium in addition to the usual mineral salts and sucrose.Optimum concentrations of nitrate and ammonium were 25 and 2 mM respectively. The highest yield of cells was achieved at an initial pH of 4.5–5.5. During the growth cycle the pH gradually increased to 6.0–6.2.
Analysis of gene organization and expression in plants
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