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Gus Fusions - Beta-Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher-Plants
Abstract
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.
Supplementary resources (3)
... The plasmid containing pCAMBIA1301-35SN-GUS was introduced into A. senticosus plants via Agrobacterium-mediated transformation, following the method described by Fan et al. [21]. The regenerated cotyledon embryos were authenticated using the GUS histochemical assay, following the method outlined by Jefferson (1987) [22]. Subsequently, their total DNA was extracted employing the plant genomic DNA extraction kit (DP305, TianGen, Beijing, China), following the manufacturer's guidelines. ...
... The plasmid containing pCAMBIA1301-35SN-GUS was introduced into A. senticosus plants via Agrobacterium-mediated transformation, following the method described by Fan et al. [21]. The regenerated cotyledon embryos were authenticated using the GUS histochemical assay, following the method outlined by Jefferson (1987) [22]. Subsequently, their total DNA was extracted employing the plant genomic DNA extraction kit (DP305, TianGen, Beijing, China), following the manufacturer's guidelines. ...
Background: Transient and stable gene transformation systems play a crucial role in elucidating gene functions and driving genetic improvement in plants. However, their application in medicinal woody plants has been hampered by inefficient procedures for isolating protoplasts and regenerating plant in vitro.
Results: Embryogenic callus protoplast isolation and transient transformation system were successfully established, the highest yield of protoplasts was achieved at approximately 1.88×10⁶ cells per gram with a viability of 90% under the combination of 1.5% cellulase and 0.2% lysozyme, with enzymatic digestion for 6 h in darkness followed by centrifugation at 400×g for 5 min. The transient transfection rate of protoplast reached 45.56% at a PEG 4000 concentration of 40%, a transfection time of 40 min, 16 h of dark incubation, a plasmid concentration of 1.5 ng·μL⁻¹, and a heat shock of 25 min at 45°C. Additionally, 15 Agrobacterium tumefaciens-mediated GUS-positive seedlings were obtained through the somatic embryogenetic pathway under the optimized conditions.
Conclusion: This study successfully established both transient and stable genetic transformation systems, paving the way for future molecular biology research in A. senticosus.
... The DR5:GUS construct was transformed into the bta2 mutant in the Nipponbare background by crossing. After PCR screening of the transgenic plants or progeny, positive plants were identified and T 2 transgenic or F 2 lines were used for GUS staining (Jefferson et al., 1987). Images of various organs were captured with a stereoscope (Leica Application Suite 3.3, Germany). ...
Tiller angle is a key agricultural trait that establishes plant architecture, which in turn strongly affects grain yield by influencing planting density in rice. The shoot gravity response plays a crucial role in the regulation of tiller angle in rice, but the underlying molecular mechanism is largely unknown. Here, we report the identification of the BIG TILLER ANGLE2 (BTA2), which regulates tiller angle by controlling the shoot gravity response in rice. Loss‐of‐function mutation of BTA2 dramatically reduced auxin content and affected auxin distribution in rice shoot base, leading to impaired gravitropism and therefore a big tiller angle. BTA2 interacted with AUXIN RESPONSE FACTOR7 (ARF7) to modulate rice tiller angle through the gravity signaling pathway. The BTA2 protein was highly conserved during evolution. Sequence variation in the BTA2 promoter of indica cultivars harboring a less expressed BTA2 allele caused lower BTA2 expression in shoot base and thus wide tiller angle during rice domestication. Overexpression of BTA2 significantly increased grain yield in the elite rice cultivar Huanghuazhan under appropriate dense planting conditions. Our findings thus uncovered the BTA2‐ARF7 module that regulates tiller angle by mediating the shoot gravity response. Our work offers a target for genetic manipulation of plant architecture and valuable information for crop improvement by producing the ideal plant type.
... Histochemical GUS assays were carried out according to Jefferson et al. (1987). The flowers from various developmental stages as defined in Sanders et al. (1999) were submerged in a GUS substrate solution (100 mM sodium phosphate buffer pH 7.0, 1 mg ml À1 5-bromo-4-chloro-3-indolyl-b-D-glucuronide, 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide, 0.1% (v/v) triton X-100) and vacuum-infiltrated for 1 h, followed by 3-h incubation at 37°C. ...
Acyl‐CoA‐Binding Proteins (ACBPs) bind acyl‐CoA esters and function in lipid metabolism. Although acbp3‐1, the ACBP3 mutant in Arabidopsis thaliana ecotype Col‐0, displays normal floral development, the acbp3‐2 mutant from ecotype Ler‐0 characterized herein exhibits defective adaxial anther lobes and improper sporocyte formation.
To understand these differences and identify the role of ERECTA in ACBP3 function, the acbp3 mutants and acbp3‐erecta (er) lines were analyzed by microscopy for anther morphology and high‐performance liquid chromatography for lipid composition.
Defects in Landsberg anther development were related to the ERECTA‐mediated pathway because the progenies of acbp3‐2 × La‐0 and acbp3‐1 × er‐1 in Col‐0 showed normal anthers, contrasting to that of acbp3‐2 in Ler‐0. Polymorphism in the regulatory region of ACBP3 enabled its function in anther development in Ler‐0 but not Col‐0 which harbored an AT‐repeat insertion. ACBP3 expression and anther development in acbp3‐2 were restored using ACBP3pro (Ler)::ACBP3 not ACBP3pro (Col)::ACBP3. SPOROCYTELESS (SPL), a sporocyte formation regulator activated ACBP3 transcription in Ler‐0 but not Col‐0.
For anther development, the ERECTA‐related role of ACBP3 is required in Ler‐0, but not Col‐0. The disrupted promoter regulatory region for SPL binding in Col‐0 eliminates the role of ACBP3 in anther development.
... The GUS activity analysis was performed following a standard protocol (Jefferson et al., 1987). Tissues of rice at different periods were taken, cut into appropriate sizes, and immediately immersed in 90% ...
Chloroplasts play a vital role in plant growth and development, which are the main sites of photosynthesis and the production of hormones and metabolites. Despite their significance, the regulatory mechanisms governing chloroplast development remain unclear. In our investigation, we identified a rice mutant with defective chloroplasts in rice (Oryza sativa L.), named albino lethal 13 (osal13), which displayed a distinct albino phenotype in leaves, ultimately resulting in seedling lethality. Molecular cloning revealed that OsAL13 encodes a novel rice protein with no homologous gene or known conserved domain. This gene was located in the chloroplast and exhibited constitutive expression in various tissues, particularly in green tissues and regions of active cell growth. Our study's findings reveal that RNAi‐mediated knockdown of OsAL13 led to a pronounced albino phenotype, reduced chlorophyll and carotenoid contents, a vesicle chloroplast structure, and a decrease in the expression of chloroplast‐associated genes. Consequently, the pollen fertility and seed setting rate were lower compared with the wild type. In contrast, the overexpression of OsAL13 resulted in an increased photosynthetic rate, a higher total grain number per panicle, and enhanced levels of indole‐3‐acetic acid (IAA) in the roots and gibberellin A3 (GA3) in the shoot. These outcomes provide new insights on the role of OsAL13 in regulating chloroplast development in rice.
... Histochemical GUS assay was carried out as per Jefferson et al. (1987). The actively dividing transformed Hemidesmus callus cultures after three successive sub-culturing that were maintained on MS media supplemented with hygromycin ...
Hemidesmus indicus L. R. Br. is a rare and endangered plant exhibits potent medicinal properties explored for the presence of flavoring compound in the roots namely 2-hydroxy-4- methoxybenzaldehyde (HMB) having applications in pharmaceutical and nutraceutical industries. The objective of the study is to improve the regeneration potential and optimize the genetic transformation in Hemidesmus indicus. Present study exhibits direct regeneration of Hemidesmus indicus through leaf and nodal explants with subsequent plant regeneration using Murashige and Skoog (MS) medium supplemented with various plant growth regulators (auxins, cytokinins, and gibberellic acid) TRIA and with adjuvant adenine sulphate,. The caulogenic response of 78.8%, 73.3% and 71.4% was observed when the leaf explant was inoculated on MS media containing 2.3 mgL− 1 BAP + 0.2 mgL− 1 2,4-D, 0.02 mgL− 1 TRIA + 2 mgL− 1 BAP and 1 mgL− 1 KIN + 1 mgL− 1 NAA respectively with creamish yellow nodular friable callus by 4 weeks. The initiation of shoot bud was observed within three days after inoculation of nodal explant on media supplemented with 1 mgL− 1 BAP + 0.1 mgL− 1 NAA, 1 mgL− 1 BAP + 0.1 mgL− 1 NAA + 40 mgL− 1 AgNO3, 1 mgL− 1 BAP + 0.1 mgL− 1 NAA + 40 mgL− 1 AgNO3 + 40 mgL− 1 adenine sulphate respectively and incubated in the dark for 2 weeks. Shoot regeneration from the leaf explants was also observed within 4 weeks after inoculation in MS medium with 1 mgL− 1 BAP + 0.1 mgL− 1 NAA. In the present study, we also report the development of an effective and reproducible Agrobacterium tumefaciens mediated genetic transformation system in Hemidesmus indicus. Genetic transformation was achieved by inoculating in vitro calli of Hemidesmus indicus with the A. tumefaciens strain EHA105 harboring pCAMBIA1301 with 35 S CAMV promoter driving the expression of reporter β-glucuronidase gene (GUS) and hygromycin selection marker hptII. After co-cultivation, the calli were thoroughly washed and inoculated on MS basal medium with (2.3mgL− 1 BAP + 0.2 mgL− 1 2,4-D) along with cefotaxime (250 mgL− 1) and hygromycin (30 mgL− 1) and the co-cultivated callus cultures were maintained in dark at 25 ̊C ± 2 ̊C for 4 weeks. After two rounds of subculture, the putative transformed calli were assessed by performing histochemical GUS staining assay and genomic DNA PCR to detect the exogenous GUS gene sequence. The transformation efficiency was 26% with transformed calli tolerant to hygromycin (30 mg L⁻¹) and were positive to histochemical GUS staining assay and PCR. The results indicated a successful establishment of a reliable and efficient A. tumefaciens mediated genetic transformation system. The in-vitro micropropagation is a well-known technique for the mass production of the plant and thus aid in conservation of H. indicus. Through transformation, the regulatory and the biosynthetic genes can be manipulated for enhancement of nutraceutical, pharmaceutical bioactive compounds present in H. indicus and can also be applied for generating transgenic plants with desirable traits.
... After growing on the medium for 14 days, seedlings were treated with 20% PEG6000, and then placed in GUS staining solution for 12 h. The seedlings were decolorized with anhydrous ethanol the leaf (Jefferson et al. 1987). ...
Drought is one of the abiotic stresses affecting agricultural production. WRKY transcription factors have important functions in response to drought. However, functions of only a few WRKY transcription factors are understood in soybean. Here, GmWRKY17 was cloned from soybean and shown to bind the W-box sequence. GmWRKY17 was tissue-specific and induced by drought. Overexpression of GmWRKY17 was found to decrease abscisic acid (ABA) sensitivity and increase drought tolerance in Arabidopsis thaliana. GmWRKY17 improved germination rate, leaf opening and greening, root length and chlorophyll content under osmotic stress and drought. GmWRKY17 reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels under drought. GmWRKY17 promoted stomatal closure after treatment with 10% polyethylene glycol 6000 (PEG6000). Overexpression of GmWRKY17 increased superoxide dismutase (SOD) activity and catalase (CAT) activity, and GmWRKY17 promoted the expression of AtSOD1 and AtCAT1 under drought. Moreover, GmWRKY17 increased proline content and enhanced the expression of AtP5CS1, and GmWRKY17 up-regulated stress-related genes under drought stress. All results showed that overexpression of GmWRKY17 improved drought tolerance, and GmWRKY17 may regulate drought stress by enhancing antioxidant activity and upregulating stress-related genes in Arabidopsis thaliana.
... [22]. Transition 2 (circle 2, Fig 3) can be explained by the following breakthroughs in the late 1980s: better approaches to create transgenic plants and insertional mutants [23], more efficient creation of mutant plant libraries through chemical mutagenesis (e.g., [24]), and availability of gene reporter systems such as β-glucuronidase [25]. Because of these breakthroughs, molecular genetics studies shifted away from understanding the basic machinery to understanding the molecular underpinnings of specific processes, such as molecular mechanisms of flower and meristem development and the action of hormones such as auxin (topic 27, S5C Fig); this type of research was discussed as a future trend in 1988 [26] and remains prevalent to this date. ...
Scientific advances due to conceptual or technological innovations can be revealed by examining how research topics have evolved. But such topical evolution is difficult to uncover and quantify because of the large body of literature and the need for expert knowledge in a wide range of areas in a field. Using plant biology as an example, we used machine learning and language models to classify plant science citations into topics representing interconnected, evolving subfields. The changes in prevalence of topical records over the last 50 years reflect shifts in major research trends and recent radiation of new topics, as well as turnover of model species and vastly different plant science research trajectories among countries. Our approaches readily summarize the topical diversity and evolution of a scientific field with hundreds of thousands of relevant papers, and they can be applied broadly to other fields.
... transgenic lines as described 6,25 . Seedlings grown in 0.5× MS medium or soil were used for the histochemical staining 75 . Data represent more than five independent lines, which displayed similar staining patterns. ...
Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca²⁺ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3–5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca²⁺ spiking is controlled directly by water through these hypo-osmosensors—that is, Ca²⁺ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins⁶. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca²⁺-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca²⁺ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca²⁺ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.
... AtATG5 and AtATG7 native promoter space-time expression was evaluated in a representative transgenic line out of 10-12 independent homozygous lines by qualitative GUS staining (Jefferson et al., 1987) and visualized under a Leica DMi8 light microscope (Leica, Wetzlar, Germany). ...
Autophagy is a cell recycling mechanism that degrades cytoplasmic components. Although classically considered a non-selective bulk degradation mechanism, autophagy also functions selectively. Here, we investigate the impact of autophagy on seed development by studying the autophagy-related (ATG) genes AtATG5 and AtATG7 in Arabidopsis, focusing on their role in ABA responses. Seeds of atg5 and atg7 mutants germinate significantly slower than Col-0, especially in the presence of ABA. Transcriptomic analyses comparing imbibed atg7 and Col-0 seeds reveal differences in gene expression associated with lipid storage and seed maturation ontology categories. Germinating seeds of atg mutants show histochemical alterations in the organisation of lipid droplets and protein storage vacuoles (PSV) in the emerging radicle. Notably, immunolocalization of ATG8 is observed in PSV in Col-0, but not in atg mutants. In the presence of ABA, approximately 10% of the transcriptome induced in atg7 and repressed in Col-0 has been reported to be under control of the transcription factors ABI3 and ABI5, master regulators of ABA signaling in the seed. Yeast-two hybrid assays confirmed their direct interaction with the autophagy machinery through ATG8. Interestingly, the decrease in ABI5 observed in Col-0 seeds after imbibition is delayed in atg mutants, which also show altered accumulation in developing seeds of the ABI5 homolog bZIP67 that regulates reserve biosynthesis. Taken together, our data highlight the relevance of autophagy in controlling seed reserve mobilisation, its impact on seed germination, and the perception of environmental signals through ABA responses that include a transcription factor decay mechanism.
... The preliminary confirmation of the resistant buds of ZADT was positive by GUS staining, the leaves and buds of resistant buds were incubated in 100 mmol/L staining buffer overnight at 37˚C and then in 70% ethanol for 4 h to remove chlorophyll from the green tissue [37] and then these samples were observed and photographed. ...
Sunflower is one of the four major oil crops in the world. ‘Zaoaidatou’ (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.
... Transient expression of the Gus A gene was tested by histochemical staining of the tissues 48 hr after bombardment using X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronidase), procedure outline by Jefferson et al., (1987). ...
Cowpeas are important legume grown primarily in the semi-arid tropics providing great source of protein, vitamins and highly digestible energy. The demand for cowpeas is high but yield remain critically low, largely due to insect pests like other crop plants. As the germplasm of cowpea contains little or no resistance to major insect pests hence a gene technology approach for insect protection traits is now a high priority. The present work was aimed to study various factors affecting Agrobacterium tumefacience mediated transformation of Vigna unguiculata. Agrobacterium strain EHA 101 having kanamycin resistant gene as selectable marker and β-glucuronidase (GUS) as a reporter gene was used for transformation. Factors affecting transformation efficiency such as type and age of explant, effect of sucrose concentration in co-cultivation medium were studied. Histochemical analysis was performed to determine the activity of the GUS gene. Results concluded that the transgene was transmitted efficiently to the cowpea explants.
... This was repeated three times [42]. After being cultured in the dark for 24 h, the leaves were transferred to the light for 48 h before being collected and the expression level of GUS reporter gene qualitatively and quantitatively detected by the method of Jefferson et al. [43]. ...
To provide a theoretical basis for the application of ALA in pear production, the effects of exogenous 5-aminolevulinic acid (ALA) treatment on leaf photosynthetic gas exchange parameters, chlorophyll fast fluorescence properties, and relative expression of the related genes were investigated using pear (Pyrus pyrifolia Nakai cv. ‘Whasan’) as a material in the study. The results show that exogenous ALA treatment improved the photosynthetic gas exchange parameters of pear leaves, upregulated the expression of multiple key genes which are related to ALA biosynthesis, metabolism, and transformation into chlorophylls. GUS staining in tobacco leaves showed that exogenous ALA activated the promoter activity of PypHEMA and PypCHLH genes, implying that the synthesis of endogenous ALA and chlorophylls was promoted by exogenous ALA. Furthermore, ALA promoted the expression of the genes encoding photosystem II (PSII) reaction center proteins, such as core protein D1, inner light-harvesting pigment proteins CP43 and CP47, and cytochrome b559. This led to increased PSII reaction center activity. In addition, ALA alleviated the donor side oxygen-evolving complex inhibition and reduced the closure rate on the receptor side, allowing for increased photochemical electron transfer and reduced heat dissipation while improving the photosynthetic performance index PIabs and PItotal. The findings of this study contribute to a better understanding of ALA’s promotion of plant photosynthetic efficiency, providing valuable insights for further research and potential applications in pear production.
... Qualitative β-glucuronidase (GUS) histochemical assays were performed as reported elsewhere (Jefferson et al. 1987), using X-glu (5-bromo-4-chloro-3-indolyl-glucuronic acid, Inalco S.P.A., Milano, Italy) as substrate. ...
Plant argonaute (AGO) proteins—chiefly AGO1 and 2—restrict viral infections. AGO1/2 also participate in developmental processes and are tightly regulated by microRNAs. Researchers have conducted extensive studies on the regulatory loop involving miR168/AGO1 in viral infections, though comparatively less attention has been given to the miR403/AGO2 system. Here, we simultaneously studied both regulatory systems in Arabidopsis plants infected with turnip mosaic virus (TuMV). TuMV simultaneously altered both miR168 and miR403 precursors as well as their mature forms at medium to late stages of infection. While TuMV decreased miRNA precursor molecules, it induced the overaccumulation of mature miRNA forms, without evidence of concomitant transcriptional alteration. The AGO1 protein remained at basal levels, whereas the AGO2 protein overaccumulated. The application of exogenous salicylic acid (SA) in healthy plants resulted in elevated AGO2 mRNA levels. Conversely, this hormone did not induce any significant changes in either AGO1 mRNA levels or those of miRs 168 and 403. This response is coherent with previous results, which showed enhanced levels of SA under TuMV infection and the partially differential sensitivity that AGO proteins have against this defense hormone. Our results also highlight the key role of AGO2 in leaves as an antiviral molecule and demonstrate the different responsiveness of the AGO1/miR168 and AGO2/miR403 systems regarding TuMV infection and SA response. Taken together, the results presented here are in line with previous reports studying abiotic and biotic impacts on microRNA biogenesis and AGO-dependent antiviral defense and further expand the knowledge of the miR403/AGO2 regulatory system.
... Histochemical GUS assays were conducted using methods previously described [37]. Different tissues were collected and immersed in a vacuum using GUS staining solution for one hour in the dark. ...
Background
Aquaporins (AQPs) facilitate water diffusion across biological membranes and are involved in all phases of growth and development. Small and basic intrinsic proteins (SIPs) belong to the fourth subfamily of the plant AQPs. Although SIPs are widely present in higher plants, reports on SIPs are limited. Rice is one of the major food crops in the world, and water use is an important factor affecting rice growth and development; therefore, this study aimed to provide information relevant to the function and environmental response of the rice SIP gene family.
Results
The rice (Oryza sativa L. japonica) genome encodes two SIP-like genes, OsSIP1 and OsSIP2, whose products are predominantly located in the endoplasmic reticulum (ER) membrane but transient localization to the plasma membrane is not excluded. Heterologous expression in a yeast aquaglyceroporin-mutant fps1Δ showed that both OsSIP1 and OsSIP2 made the cell more sensitive to KCl, sorbitol and H2O2, indicating facilitated permeation of water and hydrogen peroxide. In addition, the yeast cells expressing OsSIP2 were unable to efflux the toxic methylamine taken up by the endogenous MEP permeases, but OsSIP1 showed subtle permeability to methylamine, suggesting that OsSIP1 may have a wider conducting pore than OsSIP2. Expression profiling in different rice tissues or organs revealed that OsSIP1 was expressed in all tissues tested, whereas OsSIP2 was preferentially expressed in anthers and weakly expressed in other tissues. Consistent with this, histochemical staining of tissues expressing the promoter-β-glucuronidase fusion genes revealed their tissue-specific expression profile. In rice seedlings, both OsSIPs were upregulated to varied levels under different stress conditions, including osmotic shock, high salinity, unfavorable temperature, redox challenge and pathogen attack, as well as by hormonal treatments such as GA, ABA, MeJA, SA. However, a reduced expression of both OsSIPs was observed under dehydration treatment.
Conclusions
Our results suggest that SIP-like aquaporins are not restricted to the ER membrane and are likely to be involved in unique membrane functions in substrate transport, growth and development, and environmental response.
... The β-glucuronidase (GUS) assay was performed as described in Ref. [32]. To confirm the presence of the target gene (AtMYB12), we extracted total DNA from nine feasible transformants, WT plant, transgenic callus, and WT callus, as outlined in Ref. [33]. ...
The study aimed to enhance quercetin production in radish by optimizing Agrobacterium tumefaciens-mediated in-planta transformation. This protocol involved infecting radish seed embryo axis with A. tumefaciens EHA105 strain carrying the 35S::AtMYB12. Radish seeds were infected with the Agrobacterium suspension (0.8 OD600) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes (AtMYB12, CHS, F3H, and FLS) and antioxidant genes (GPX, APX, CAT, and SOD) compared to WT plants. Overexpression of AtMYB12 in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In AtMYB12 overexpressed callus lines, phenolic content (357.31 mg g⁻¹ dry weight), flavonoid content (463 mg g⁻¹ dry weight), and quercetin content (48.24 mg g⁻¹ dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved in-planta transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.
... Plants were grown in the 6 d system on +Fe and -Fe, in two biological replicates, and assayed for histochemical GUS activity. Four to six seedlings of each line were incubated in 1 ml GUS staining solution containing [50 mM Na 2 HPO 4 , 50 mM NaH 2 PO 4 pH 7.2, 2 mM K 4 [Fe(CN) 6 ]Fe 2+ , 2 mM K 3 [Fe(CN) 6 ]Fe 3+ , 0.2% Triton-X, 2 mM 5-bromo-4-chloro-3-indoyl-b-D-glucuronic acid (X-Gluc)] [44] for 15 min to four hours at 37˚C in the dark, until blue staining was observed. Afterwards leaf chlorophyll was removed by incubation in 70% EtOH for 24 h (EtOH was exchanged every few hours). ...
Iron (Fe) is a crucial micronutrient needed in many metabolic processes. To balance needs and potential toxicity, plants control the amount of Fe they take up and allocate to leaves and seeds during their development. One important regulator of this process is POPEYE (PYE). PYE is a Fe deficiency-induced key bHLH transcription factor (TF) for allocation of internal Fe in plants. In the absence of PYE, there is altered Fe translocation and plants develop a leaf chlorosis. NICOTIANAMINE SYNTHASE4 ( NAS4 ), FERRIC-REDUCTION OXIDASE3 ( FRO3 ), and ZINC-INDUCED FACILITATOR1 ( ZIF1 ) genes are expressed at higher level in pye-1 indicating that PYE represses these genes. PYE activity is controlled in a yet unknown manner. Here, we show that a small Fe deficiency-induced protein OLIVIA (OLV) can interact with PYE. OLV has a conserved C-terminal motif, that we named TGIYY. Through deletion mapping, we pinpointed that OLV TGIYY and several regions of PYE can be involved in the protein interaction. An OLV overexpressing (OX) mutant line exhibited an enhanced NAS4 gene expression. This was a mild Fe deficiency response phenotype that was related to PYE function. Leaf rosettes of olv mutants remained smaller than those of wild type, indicating that OLV promotes plant growth. Taken together, our study identified a small protein OLV as a candidate that may connect aspects of Fe homeostasis with regulation of leaf growth.
... Tobacco (Nicotiana benthamiana) leaves were used to conduct transient GUS activity assay [44]. The promoter sequences from ZJU152 and ZJU163 of ClERF1 were cloned into pMDC162 vector to obtain GUS fusion expression vector using pEASY-Basic Seamless Cloning and Assembly Kit (TRANs, Beijing, China) according to the manufacturer's instructions. ...
Background
Flesh firmness is a critical factor that influences fruit storability, shelf-life and consumer’s preference as well. However, less is known about the key genetic factors that are associated with flesh firmness in fresh fruits like watermelon.
Results
In this study, through bulk segregant analysis (BSA-seq), we identified a quantitative trait locus (QTL) that influenced variations in flesh firmness among recombinant inbred lines (RIL) developed from cross between the Citrullus mucosospermus accession ZJU152 with hard-flesh and Citrullus lanatus accession ZJU163 with soft-flesh. Fine mapping and sequence variations analyses revealed that ethylene-responsive factor 1 (ClERF1) was the most likely candidate gene for watermelon flesh firmness. Furthermore, several variations existed in the promoter region between ClERF1 of two parents, and significantly higher expressions of ClERF1 were found in hard-flesh ZJU152 compared with soft-flesh ZJU163 at key developmental stages. DUAL-LUC and GUS assays suggested much stronger promoter activity in ZJU152 over ZJU163. In addition, the kompetitive allele-specific PCR (KASP) genotyping datasets of RIL populations and germplasm accessions further supported ClERF1 as a possible candidate gene for fruit flesh firmness variability and the hard-flesh genotype might only exist in wild species C. mucosospermus. Through yeast one-hybrid (Y1H) and dual luciferase assay, we found that ClERF1 could directly bind to the promoters of auxin-responsive protein (ClAux/IAA) and exostosin family protein (ClEXT) and positively regulated their expressions influencing fruit ripening and cell wall biosynthesis.
Conclusions
Our results indicate that ClERF1 encoding an ethylene-responsive factor 1 is associated with flesh firmness in watermelon and provide mechanistic insight into the regulation of flesh firmness, and the ClERF1 gene is potentially applicable to the molecular improvement of fruit-flesh firmness by design breeding.
... At 5 DAG, the seedlings were transferred to a modified Hoagland and Arnon (1950) nutrient solution containing 2 mM N-nitrate, where they remained until 14 DAG. Post germination seeds (1-3 DAG), seedlings with a welldeveloped radicle and epicotyl (4-5 DAG), and seedlings with developed roots and shoots (14 DAG) were collected and subjected to histochemical GUS staining according to Jefferson et al. (1987). The stained tissues were incubated in solutions with increasing concentrations of ethanol (70%, 80%, 90%, and 95%) and fixed in formalin-acetic acid (FAA)-70% ethanol. ...
Nitrogen (N) is an essential nutrient for plants. Nitrate is often the main inorganic N form absorbed, and amino acids are the predominant organic N form transported in most plant species. Amino acid transporters (AATs) mediate the distribution of amino acids between organs, and any disturbance in this process can cause changes in crop development. To date, the OsAAP1 amino acid transporter has been characterized in the most detail during the reproductive and ripening periods in rice plants. In this study, we identified the sites of OsAAP1 expression during the early stages of rice development and determined the implications of OsAAP1 knockout on seedling establishment, nitrate–N uptake and assimilation, and the expression of genes related to N and carbon (C) metabolism. OsAAP1 is strongly expressed in the coleorhiza, radicle, and epicotyl of seedlings and in the regions of the vascular bundles of roots and leaves. OsAAP1 knockout hindered rice seedling establishment and impaired nitrate absorption. Under low N, OsAAP1 knockout results in damage to NO3⁻ absorption, with negative effects on N assimilation and C metabolism. Under sufficient N, nitrate absorption was more strongly impaired, and downregulation of nitrate transporters and N assimilation enzymes was observed. Taken together, the data suggest that the OsAAP1 gene is essential for the early development of rice when nitrate is the predominant N source.
... C32I bacteria cell suspension. Seven dayspost-inoculation [dpi], the seedlings were subjected to GUS histochemical staining during 12 h of incubation at 37 °C in a GUS reaction buffer [0.5 mg/ml of 5-bromo-4-chloro-3indolyl-β-D-glucuronide in 100 mM sodium phosphate, pH 7] (Jefferson et al. 1987). Tissue clarification was carried out with a solution of methanol: acetone [3:1] during 2 h. ...
Plants and microorganisms establish beneficial associations that can improve their development and growth. Recently, it has been demonstrated that bacteria isolated from the skin of amphibians can contribute to plant growth and defense. However, the molecular mechanisms involved in the beneficial effect for the host are still unclear. In this work, we explored whether bacteria isolated from three tropical frogs species can contribute to plant growth. After a wide screening, we identified three bacterial strains with high biostimulant potential, capable of modifying the root structure of Arabidopsis thaliana plants. In addition, applying individual bacterial cultures to Solanum lycopersicum plants induced an increase in their growth. To understand the effect that these microorganisms have over the host plant, we analysed the transcriptomic profile of A. thaliana during the interaction with the C32I bacterium, demonstrating that the presence of the bacteria elicits a transcriptional response associated to plant hormone biosynthesis. Our results show that amphibian skin bacteria can function as biostimulants to improve agricultural crops growth and development by modifying the plant transcriptomic responses.
... Leaf discs were punched and stained with GUS staining solution. After being photographed, the leaf discs were used to assay GUS activity as described by Jefferson et al. [32]. ...
Background
Molecular mechanisms in response to drought stress are important for the genetic improvement of maize. In our previous study, nine ZmLAZ1 members were identified in the maize genome, but the function of ZmLAZ1 was largely unknown.
Results
The ZmLAZ1-3 gene was cloned from B73, and its drought-tolerant function was elucidated by expression analysis in transgenic Arabidopsis. The expression of ZmLAZ1-3 was upregulated by drought stress in different maize inbred lines. The driving activity of the ZmLAZ1-3 promoter was induced by drought stress and related to the abiotic stress-responsive elements such as MYB, MBS, and MYC. The results of subcellular localization indicated that the ZmLAZ1-3 protein localized on the plasma membrane and chloroplast. The ectopic expression of the ZmLAZ1-3 gene in Arabidopsis significantly reduced germination ratio and root length, decreased biomass, and relative water content, but increased relative electrical conductivity and malondialdehyde content under drought stress. Moreover, transcriptomics analysis showed that the differentially expressed genes between the transgenic lines and wild-type were mainly associated with response to abiotic stress and biotic stimulus, and related to pathways of hormone signal transduction, phenylpropanoid biosynthesis, mitogen-activated protein kinase signaling, and plant-pathogen interaction.
Conclusion
The study suggests that the ZmLAZ1-3 gene is a negative regulator in regulating drought tolerance and can be used to improve maize drought tolerance via its silencing or knockout.
... T2 seeds were germinated on 1/2 MS medium containing hygromycin to select for transformants, then transferred transgenic lines to a new medium adding 20 µM CdCl 2 for three days. Histochemical GUS staining was conducted with reference to the method mentioned by Jefferson et al. (1987). Sections of 4 µm thickness were obtained as described by Jia et al. (2016), and were observed with a light microscope and photographed with a CCD camera. ...
Main conclusion
SbYS1 and its upstream transcription factor SbWRKY72 were involved in Cd tolerance and accumulation and are valuable for developing sweet sorghum germplasm with high-Cd tolerance or accumulation ability through genetic manipulation.
Abstract
Cadmium (Cd) is highly toxic and can severely affect human health. Sweet sorghum, as an energy crop, shows great potential in extracting cadmium from Cd-contaminated soils. However, its molecular mechanisms of Cd-tolerance and -accumulation remain largely unknown. Here, we isolated a YSL family gene SbYS1 from the sweet sorghum genotype with high Cd accumulation ability and the expression of SbYS1 in roots was induced by cadmium. GUS staining experiment exhibited that SbYS1 was expressed in the epidermis and parenchyma tissues of roots. Further subcellular localization analysis suggested that SbYS1 was localized in the endoplasmic reticulum and plasma membrane. Yeast transformed with SbYS1 exhibited a sensitive phenotype compared to the control when exposed to Cd-NA (chelates of cadmium and nicotianamine), indicating that SbYS1 may absorb cadmium in the form of Cd-NA. Arabidopsis overexpressing SbYS1 had a longer root length and accumulated less Cd in roots and shoots. SbWRKY72 bound to the promoter of SbYS1 and negatively regulated the expression of SbYS1. Transgenic Arabidopsis of SbWRKY72 showed higher sensitivity to cadmium and increased cadmium accumulation in roots. Our results provide references for improving the phytoremediation efficiency of sweet sorghum by genetic manipulation in the future.
... Histochemical analysis of β-glucuronidase (GUS) activity was conducted as described previously (Jefferson et al., 1987). GUS enzyme activity in transgenic plants was determined by staining with 1 mg/ml X-Gluc (Duchefa, the Netherlands) as the substrate. ...
Plant genomes contain numerous genes encoding chitinase‐like (CTL) proteins, which have a similar protein structure to chitinase belonging to the glycoside hydrolase (GH) family but lack the chitinolytic activity to cleave the β ‐1,4‐glycosidic bond in chitins, polymers of N ‐acetylglucosamine. CTL1 mutations found in rice and Arabidopsis have caused pleiotropic developmental defects, including altered cell wall composition and decreased abiotic stress tolerance, likely due to reduced cellulose content. In this study, we identified suppressor of hot2 1 ( suh1 ) as a genetic suppressor of the ctl1 hot2‐1 mutation in Arabidopsis . The mutation in SUH1 restored almost all examined ctl1 hot2‐1 defects to nearly wild‐type levels or at least partially. SUH1 encodes a Golgi‐located type II membrane protein with glycosyltransferase (GT) activity, and its mutations lead to a reduction in cellulose content and hypersensitivity to cellulose biosynthesis inhibitors, although to a lesser extent than ctl1 hot2‐1 mutation. The SUH1 promoter fused with the GUS reporter gene exhibited GUS activity in interfascicular fibers and xylem in stems; meanwhile, the ctl1 hot2‐1 mutation significantly increased this activity. Our findings provide genetic and molecular evidence that the antagonistic activities of CTL1 and SUH1 play an essential role in assembling the cell wall in Arabidopsis .
... Interactions between PR1: GUS or LOX2:GUS and microorganisms and consortia were conducted as described above. After 5 days of interaction, plants were incubated with the reagent X-Gluc at 37˚C in total darkness for 24 h to promote the reaction and precipitation of the insoluble compound 5,5'dibromo-4,4'dichloro-indigo, shown in blue in the plant tissue [59]. After incubation, the plants were washed with ethanol to eliminate chlorophyll and rehydrated with 50% ethanol/ 50% glycerol. ...
Trichoderma uses different molecules to establish communication during its interactions with other organisms, such as effector proteins. Effectors modulate plant physiology to colonize plant roots or improve Trichoderma ’s mycoparasitic capacity. In the soil, these fungi can establish relationships with plant growth–promoting bacteria (PGPBs), thus affecting their overall benefits on the plant or its fungal prey, and possibly, the role of effector proteins. The aim of this study was to determine the induction of Trichoderma atroviride gene expression coding for effector proteins during the interaction with different PGPBs, Arabidopsis or the phytopathogen Fusarium brachygibbosum , and to determine whether PGPBs potentiates the beneficial effects of T . atroviride . During the interaction with F . brachygibbosum and PGPBs, the effector coding genes epl1 , tatrx2 and tacfem1 increased their expression, especially during the consortia with the bacteria. During the interaction of T . atroviride with the plant and PGPBs, the expression of epl1 and tatrx2 increased, mainly with the consortium formed with Pseudomonas fluorescens UM270, Bacillus velezensis AF12, or B . halotolerans AF23. Additionally, the consortium formed by T . atroviride and R . badensis SER3 stimulated A . thaliana PR1:GUS and LOX2:GUS for SA- and JA-mediated defence responses. Finally, the consortium of T . atroviride with SER3 was better at inhibiting pathogen growth, but the consortium of T . atroviride with UM270 was better at promoting Arabidopsis growth. These results showed that the biocontrol capacity and plant growth-promoting traits of Trichoderma spp. can be potentiated by PGPBs by stimulating its effector functions.
... The GV3101 strain of Agrobacterium tumefaciens containing the pFaMYB63::GUS recombinant plasmid was introduced into Arabidopsis using the floral dip method. Five-day-old seedlings carrying pFaMYB63::GUS were subsequently cultured on MS containing 150 mM NaCl for 48 h and were thereafter incubated overnight at 37 °C in GUS staining buffer (0.5 mM ferrocyanide, 0.1% Triton X-100, 0.1 mM EDTA, 0.5 mM ferricyanide, and 1 mM X-Gluc) (Jefferson et al. 1987). For comparison, control seedlings of the WT were grown on MS medium without NaCl. ...
Salinity is a pivotal abiotic stress factor with far-reaching consequences on global crop growth, yield, and quality and which includes strawberries. R2R3-MYB transcription factors encompass a range of roles in plant development and responses to abiotic stress. In this study, we identified that strawberry transcription factor FaMYB63 exhibited a significant upregulation in its expression under salt stress conditions. An analysis using yeast assay demonstrated that FaMYB63 exhibited the ability to activate transcriptional activity. Compared with those in the wild-type (WT) plants, the seed germination rate, root length, contents of chlorophyll and proline, and antioxidant activities (SOD, CAT, and POD) were significantly higher in FaMYB63-overexpressing Arabidopsis plants exposed to salt stress. Conversely, the levels of malondialdehyde (MDA) were considerably lower. Additionally, the FaMYB63-overexpressed Arabidopsis plants displayed a substantially improved capacity to scavenge active oxygen. Furthermore, the activation of stress-related genes by FaMYB63 bolstered the tolerance of transgenic Arabidopsis to salt stress. It was also established that FaMYB63 binds directly to the promoter of the salt overly sensitive gene SOS1, thereby activating its expression. These findings identified FaMYB63 as a possible and important regulator of salt stress tolerance in strawberries.
... GUS activity assays were conducted following the protocol outlined in a previous study (Jefferson et al. 1987). The GUS staining buffer was composed of 50 mg X-Gluc (Merck,, 0.05 M Na 2 HPO 4 , 0.1% (v/v) Triton X-100, 10 mM EDTA. ...
Artemisinin is primarily synthesized and stored in the subepidermal space of the glandular trichomes of Artemisia annua. The augmentation of trichome density has been demonstrated to enhance artemisinin yield. However, existing literature lacks insights into the correlation between the stratum corneum and trichomes. This study aims to unravel the involvement of TrichomeLess Regulator 3 (TLR3), which encodes the transcription factor, in artemisinin biosynthesis and its potential association with the stratum corneum. TLR3 was identified as a candidate gene through transcriptome analysis. The role of TLR3 in trichome development and morphology was investigated using yeast two-hybrid, pull-down analysis, and RNA electrophoresis mobility assay. Our research revealed that TLR3 negatively regulates trichome development. It modulates the morphology of Arabidopsis thaliana trichomes by inhibiting branching and inducing the formation of abnormal trichomes in Artemisia annua. Overexpression of the TLR3 gene disrupts the arrangement of the stratum corneum and reduces artemisinin content. Simultaneously, TLR3 possesses the capacity to regulate stratum corneum development and trichome follicle morphology by interacting with TRICHOME AND ARTEMISININ REGULATOR 1, and CycTL. Consequently, our findings underscore the pivotal role of TLR3 in the development of glandular trichomes and stratum corneum biosynthesis, thereby influencing the morphology of Artemisia annua trichomes.
Supplementary Information
The online version contains supplementary material available at 10.1186/s43897-024-00085-4.
... Three independent positive transgenic lines were obtained and analysed for GUS signal. The root, anther, husk and brown rice samples of the transgenic plants were incubated in an X-Gluc staining buffer at 37°C for 2 h (Jefferson et al., 1987). The stained roots were photographed using a stereomicroscope (Olympus MVX10) with a color CCD camera. ...
Copper (Cu) is an essential micronutrient for all living organisms but is also highly toxic in excess. Cellular homoeostasis of Cu is maintained by various transporters and metallochaperones. Here, we investigated the biological function of OsCOPT7, a member of the copper transporters (COPT) family, in Cu homoeostasis in rice. OsCOPT7 was mainly expressed in the roots and the expression was upregulated by Cu deficiency. OsCOPT7 was localized at the tonoplast and the endoplasmic reticulum. Knockout of OsCOPT7 increased Cu accumulation in the roots but decreased Cu concentrations in the shoots and grain. The knockout mutants contained higher concentrations of Cu in the roots cell sap but markedly lower concentrations of Cu in the xylem sap than wild‐type plants. Seed setting and grain yield were reduced significantly in the knockout mutants grown in a low Cu soil. Knockout mutants were more tolerant to Cu toxicity. Yeast two‐hybrid and bimolecular fluorescence complementation assays showed that OsCOPT7 interacts physically with the rice Cu chaperone antioxidant protein 1 (OsATX1). Taken together, our results indicate that OsCOPT7 is a specific Cu transporter functioning to export Cu from the vacuoles and the ER and plays an important role in controlling the root‐to‐shoot Cu translocation in rice.
... The positive transgenic lines (T 2 ) of pCAMBIA1304-HAK9 pro::GUS in Nip were used for GUS staining assays. Histochemical GUS staining was performed according to a previously described method (Jefferson et al., 1987), and images were collected under a dissecting microscope. ...
Soil salinity has a major impact on rice seed germination, severely limiting rice production. Herein, a rice germination defective mutant under salt stress (gdss) was identified by using chemical mutagenesis. The GDSS gene was detected via MutMap and shown to encode potassium transporter OsHAK9. Phenotypic analysis of complementation and mutant lines demonstrated that OsHAK9 was an essential regulator responsible for seed germination under salt stress. OsHAK9 is highly expressed in germinating seed embryos. Ion contents and non‐invasive micro‐test technology results showed that OsHAK9 restricted K⁺ efflux in salt‐exposed germinating seeds for the balance of K⁺/Na⁺. Disruption of OsHAK9 significantly reduced gibberellin 4 (GA4) levels, and the germination defective phenotype of oshak9a was partly rescued by exogenous GA3 treatment under salt stress. RNA sequencing (RNA‐seq) and real‐time quantitative polymerase chain reaction analysis demonstrated that the disruption of OsHAK9 improved the GA‐deactivated gene OsGA2ox7 expression in germinating seeds under salt stress, and the expression of OsGA2ox7 was significantly inhibited by salt stress. Null mutants of OsGA2ox7 created using clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR‐associated nuclease 9 approach displayed a dramatically increased seed germination ability under salt stress. Overall, our results highlight that OsHAK9 regulates seed germination performance under salt stress involving preventing GA degradation by mediating OsGA2ox7, which provides a novel clue about the relationship between GA and OsHAKs in rice.
... The GUS activity was quantified as described [64]. A plasmid carrying the luciferase gene under the control of the 35S promoter was used as an internal control to normalize the data for variations in the experiment [65]. ...
The homeodomain-leucine zipper (HD-ZIP) transcription factors, representing one of the largest plant-specific superfamilies, play important roles in the response to various abiotic stresses. However, the functional roles of HD-ZIPs in abiotic stress tolerance and the underlying mechanisms remain relatively limited in Miscanthus sinensis. In this study, we isolated an HD-ZIP TF gene, MsHDZ23, from Miscanthus and ectopically expressed it in Arabidopsis. Transcriptome and promoter analyses revealed that MsHDZ23 responded to salt, alkali, and drought treatments. The overexpression (OE) of MsHDZ23 in Arabidopsis conferred higher tolerance to salt and alkali stresses compared to wild-type (WT) plants. Moreover, MsHDZ23 was able to restore the hb7 mutant, the ortholog of MsHDZ23 in Arabidopsis, to the WT phenotype. Furthermore, MsHDZ23-OE lines exhibited significantly enhanced drought stress tolerance, as evidenced by higher survival rates and lower water loss rates compared to WT. The improved drought tolerance may be attributed to the significantly smaller stomatal aperture in MsHDZ23-OE lines compared to WT. Furthermore, the accumulation of the malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities in several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic plants. Collectively, these results demonstrate that MsHDZ23 functions as a multifunctional transcription factor in enhancing plant resistance to abiotic stresses.
Guar is a legume of industrial importance due to the presence of gum (galactomannan) in its seed endosperm. The mannose (M)/galactose (G) ratio in galactomannan molecule is critical in deciding the physico-chemical properties of guar gum. Gum with reduced galactose content has better commercial value due to better viscosity. We report down regulation of galactomannan galactosyltransferase (GMGT) which links G residues to the M backbone in galactomannan molecule. The target GMGT sequence for RNAi was directionally cloned into pENTR TM /D-TOPO® vector, followed by a LR recombination reaction with pANDA vector. The constructed gmgt RNAi cassette was delivered into guar tissue using Agrobacterium tumefaciens-mediated gene transfer. The cotyledonary node explants were pre-cultured for 72 h and co-cultivated with A. tumefaciens strain harbouring the gmgt RNAi cassette for 24 h. Maximum number of putative transformed shoots regenerated from the cotyledonary node explants cultured in vitro on Murashige and Skoog (MS) medium containing 1.5 mg/L indole-3-acetic acid (IAA), 4.0 mg/L butyric acid (BA) and 1.0 mg/L gibberellic acid (GA 3). In vitro regenerated shoots were rooted on half-strength MS medium containing 4% sucrose and 2 mg/L indole-3-butyric acid (IBA). The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis showed reduced levels of GMGT expression in the seed endosperm of 35 days after owering (DAF) stage pods. The present ndings are expected to be bene cial in taking up of gene characterization and improvement of agronomic traits in guar.
Identification of a putative cognate sensor kinase for the two-component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.
A previously identified wheat drought stress responsive Universal stress protein, TaUSP_3B‐1 has been found to work in an auxin dependent manner in the plant root tissues in the differentiation zone. We also found a novel interacting partner, TaGolS, which physically interacts with TaUSP_3B‐1 and colocalizes in the endoplasmic reticulum. TaGolS is a key enzyme in the RFO (Raffinose oligosaccharides) biosynthesis which is well reported to provide tolerance under water deficit conditions. TaUSP_3B‐1 overexpression lines showed an early flowering phenotype under drought stress which might be attributed to the increased levels of AtTPPB and AtTPS transcripts under drought stress. Moreover, at the cellular levels ER stress induced TaUSP_3B‐1 transcription and provides tolerance in both adaptive and acute ER stress via less ROS accumulation in the overexpression lines. TaUSP_3B‐1 overexpression plants had increased silique numbers and a denser root architecture as compared to the WT plants under drought stress.
MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red‐flesh phenotype due to a minisatellite insertion in its R6 promoter, but R6:MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull‐down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB‐bHLH‐WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163‐bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R6 by structural variation analysis, contains a typical W‐box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R6 background. These findings reveal a novel regulatory role of the WRKY‐containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants.
Genetic transformation is helpful in enhancing crops, utilising promoters that can be constitutive, inducible, or tissue-specific. However, the use of constitutive promoters may hinder plant growth due to energy consumption during cellular processes. To optimise transgene effects, tissue-specific promoters like root-specific ones prove valuable in addressing root-related issues and enhancing productivity. Yet, identified root-specific promoters in crop are limited. To address this gap, the expression pattern of the root-specific SlREO promoter was examined across various crops. Sequencing confirmed its identity and high homology (99%) with the NCBI database, distinct from other plants tested. Using the PLACE database, six motifs associated with root expression were identified, along with several other important elements. The 2.4 kb SlREO promoter was linked to a ß-glucuronidase (GUS) reporter gene alongside the CaMV35S promoter in pRI 201-AN-GUS vectors to study its expression. Histochemistry revealed strong root-specific expression in tomato (Solanum lycopersicum) root tissues and limited expression in stems. However, the SlREO promoter did not consistently maintain its root-specific expression in other plants. Conversely, the CaMV35S promoter exhibited constitutive expression across all tissues in various plants. This study underscores the potential of the SlREO promoter as a root-specific regulatory element, offering avenues for improving crops, particularly against environmental stresses.
Phosphite (Phi) has gained attention in agriculture due to its biostimulant effect on crops. This molecule has been found to benefit plant performance by providing protection against pathogens, improving yield and fruit quality, and improving nutrient and water use efficiency. It is still unclear how Phi enhances plant growth and protects against multiple stresses. It has been hypothesized that Phi acts by directly affecting the pathogens and interacting with the plant cellular components and molecular machinery to elicit defense responses. This study delves into elucidating the mechanisms underlying Phi’s beneficial effects on plants, revealing a complex interplay with fundamental signaling pathways. A RNA-seq study of Arabidopsis seedlings under optimal and limiting phosphate conditions helped us unveil Phi’s role in promoting plant growth by activating the expression of genes involved in the biosynthesis and signaling pathways associated with abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA). Expression of ABA-related genes, known for their involvement in stress response and development regulation, is triggered by Phi treatment, contributing to enhanced resilience and growth. Simultaneously, the activation of the SA pathway, associated with defense responses, suggests Phi’s potential in bolstering plant immunity. Moreover, Phi influences JA biosynthesis and signaling, which are crucial for defense against herbivores and pathogens, thereby strengthening plants’ defenses. Our findings reveal a multifaceted mechanism through which Phi benefits Arabidopsis development. Understanding its intricate interplay with key signaling pathways opens avenues for leveraging Phi as a strategic tool to enhance plant resilience, immunity, and growth in agricultural and ecological contexts.
Agrobacterium tumefaciens-mediated rice transformation efficiency depends on several factors such as genotype, type of explant, media composition and growth regulators, co-cultivation conditions, antibiotics, temperature, A. tumefaciens strain and their cell density, gene construct, explant competence after wounding, and control of A. tumefaciens overgrowth. Most of these issues have been addressed earlier, but only for a few elite indica rice cultivars like IR64, IR72, Pusa Basmati 1, Samba Mahsuri, etc. Besides these, there are some local cultivars having considerable consumers' preference and farmers' choice. Considering these aspects, the objective of the study was to standardize the callus transformation technique. In our earlier study, a popular rice variety, Khitish (IET 4094) from West Bengal, was found to be the most amenable genotype for indirect regeneration. Hence, this variety was selected for transformation experiments with A. tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA2301. Here, the co-cultivation-based transformation protocol was optimized by introducing several steps such as pre-culture in co-cultivation media before callus infection, washing with antibiotics dissolved in MS instead of water after co-cultivation, and pre-selection with only Agrobacterium elimination antibiotics after washing–drying step. To control the Agrobacterium, a combination of cefotaxime and timentin was used during these steps. This resulted in lowest Agrobacterium overgrowth and highest transformation frequency (28.2% in Khitish). The transient expression and stable integration of foreign gene were confirmed by GUS assay and PCR amplification of nptII selection marker gene in T0 plant genomic DNA, respectively.
Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C‐type lectin receptor‐like kinase 1 (CLRLK1) in the aspect of PD callose‐modulated symplasmic continuity. Here, we found that loss‐of‐function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine‐tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis.
Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G‐box and I‐box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost‐effective and intuitive strategy for studying the regulatory mechanism of gene expression.
Symbiotic nitrogen fixation (SNF) is crucial for legumes, providing them with the nitrogen necessary for plant growth and development. Nodulation is the first step in the establishment of SNF. However, the determinant genes in soybean nodulation and the understanding of the underlying molecular mechanisms governing nodulation are still limited. Herein, we identified a phosphatase, GmPP2C61A, which was specifically induced by rhizobia inoculation. Using transgenic hairy roots harboring GmPP2C61A::GUS, we showed that GmPP2C61A was mainly induced in epidermal cells following rhizobia inoculation. Functional analysis revealed that knockdown or knock‐out of GmPP2C61A significantly reduced the number of nodules, while overexpression of GmPP2C61A promoted nodule formation. Additionally, GmPP2C61A protein was mainly localized in the cytoplasm and exhibited conserved phosphatase activity in vitro . Our findings suggest that phosphatase GmPP2C61A serves as a critical regulator in soybean nodulation, highlighting its potential significance in enhancing symbiotic nitrogen fixation.
The regulation of seed development is critical for determining crop yield. Auxins are vital phytohormones that play roles in various aspects of plant growth and development. However, its role in amino acid biosynthesis and metabolism in seeds is not fully understood. In this study, we identified a mutant with small seeds through forward genetic screening in Medicago truncatula. The mutated gene encodes MtPIN4, an ortholog of PIN1. Using molecular approaches and integrative omics analyses, we discovered that auxin and amino acid content significantly decreased in mtpin4 seeds, highlighting the role of MtPIN4‐mediated auxin distribution in amino acid biosynthesis and metabolism. Furthermore, genetic analysis revealed that the three orthologs of PIN1 have specific and overlapping functions in various developmental processes in M. truncatula . Our findings emphasize the significance of MtPIN4 in seed development and offer insights into the molecular mechanisms governing the regulation of seed size in crops. This knowledge could be applied to enhance crop quality by targeted manipulation of seed protein regulatory pathways.
Background
Waterlogging poses a significant threat to plant growth and yield worldwide. Identifying the genes responsible for mitigating waterlogging stress is crucial. Ethylene-responsive factors (ERFs) are transcriptional regulators that respond to various biotic and abiotic stresses in plants. However, their roles and involvement in responding to waterlogging stress remain largely unexplored. Hence, this study aimed to elucidate the role of ERFs in enhancing banana plant resilience to waterlogging.
Methods
We hypothesized that introducing a group VII ERF transcription factor in Arabidopsis could enhance waterlogging stress tolerance. To test this hypothesis, we isolated MaERFVII3 from banana roots, where it exhibited a significant induction in response to waterlogging stress. The isolated MaERFVII3 was introduced into Arabidopsis plants for functional gene studies.
Results
Compared with wild-type plants, the MaERFVII3 -expressing Arabidopsis showed increased survival and biomass under waterlogging stress. Furthermore, the abundance of transcripts related to waterlogging and hypoxia response showed an elevation in transgenic plants but a decrease in wild-type and empty vector plants when exposed to waterlogging stress. Our results demonstrate the significant contribution of MaERFVII3 to waterlogging tolerance in Arabidopsis , providing baseline data for further exploration and potentially contributing to crop improvement programs.
Bud endodormancy in perennial plants is a sophisticated system that adapts to seasonal climatic changes. Growth-promoting signals such as low temperature and gibberellins (GAs) are crucial for facilitating budbreak following endodormancy release (EDR). However, the regulatory mechanisms underlying GA-mediated budbreak in tree peony (Paeonia suffruticosa) remain unclear. In tree peony, the expression of PsmiR159b among three differentially expressed miR159 members was inhibited with the prolonged chilling, and overexpression of PsMIR159b delayed budbreak, whereas silencing PsmiR159b promoted budbreak after dormancy. PsMYB65, a downstream transcription factor in the GA pathway, was induced by prolonged chilling and exogenous GA3 treatments. PsMYB65 was identified as a target of PsmiR159b, and promoted budbreak in tree peony. RNA-seq of PsMYB65-slienced buds revealed significant enrichment in the GO terms regulation of ‘cell cycle’ and ‘DNA replication’ among differentially expressed genes. Yeast one-hybrid and electrophoretic mobility shift assays demonstrated that PsMYB65 directly bound to the promoter of the type-D cyclin gene PsCYCD3;1. Dual-luciferase reporter assay indicated that PsMYB65 positively regulate PsCYCD3;1 expression, suggesting that miR159b-PsMYB65 module contributes to budbreak by influencing the cell cycle. Our findings revealed that the PsmiR159b-PsMYB65 module functioned in budbreak after dormancy by regulating cell proliferation, providing valuable insights into the endodormancy release regulation mechanism.
Zeaxanthin epoxidase (ZEP) is a key enzyme that catalyzes the conversion of zeaxanthin to violaxanthin in the carotenoid and abscisic acid (ABA) biosynthesis pathways. The rapeseed (Brassica napus) genome has 4 ZEP (BnaZEP) copies that are suspected to have undergone subfunctionalization, yet the 4 genes’ underlying regulatory mechanisms remain unknown. Here, we genetically confirmed the functional divergence of the gene pairs BnaA09.ZEP/BnaC09.ZEP and BnaA07.ZEP/BnaC07.ZEP, which encode enzymes with tissue-specific roles in carotenoid and ABA biosynthesis in flowers and leaves, respectively. Molecular and transgenic experiments demonstrated that each BnaZEP pair is transcriptionally regulated via ABA-responsive element–binding factor 3 s (BnaABF3s) and BnaMYB44s as common and specific regulators, respectively. BnaABF3s directly bound to the promoters of all 4 BnaZEPs and activated their transcription, with overexpression of individual BnaABF3s inducing BnaZEP expression and ABA accumulation under drought stress. Conversely, loss of BnaABF3s function resulted in lower expression of several genes functioning in carotenoid and ABA metabolism and compromised drought tolerance. BnaMYB44s specifically targeted and repressed the expression of BnaA09.ZEP/BnaC09.ZEP but not BnaA07.ZEP/BnaC07.ZEP. Overexpression of BnaA07.MYB44 resulted in increased carotenoid content and an altered carotenoid profile in petals. Additionally, RNA-seq analysis indicated that BnaMYB44s functions as a repressor in phenylpropanoid and flavonoid biosynthesis. These findings provide clear evidence for the subfunctionalization of duplicated genes and contribute to our understanding of the complex regulatory network involved in carotenoid and ABA biosynthesis in B. napus.
SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms. The SIZ1 gene encodes an 873-amino acid (aa) protein. Among the four SIZ1 splicing variants (SSVs), SSV1 and SSV4 encode identical 885 aa proteins; SSV2 encodes an 832 aa protein; and SSV3 encodes an 884 aa protein. SSV2 mainly localized to the plasma membrane, whereas SIZ1, SSV1/SSV4, and SSV3 localized to the nucleus. Interestingly, SIZ1 and all SSVs exhibited similar E3 SUMO ligase activities and preferred SUMO1 and SUMO2 for their E3 ligase activity. Transcript levels of SSV2 were substantially increased by heat treatment, while those of SSV1, SSV3, and SSV4 transcripts were unaffected by various abiotic stresses. SSV2 directly interacted with and sumoylated cyclic nucleotide-gated ion channel 6 (CNGC6), a positive thermotolerance regulator, enhancing the stability of CNGC6. Notably, transgenic siz1-2 mutants expressing SSV2 exhibited greater heat stress tolerance than wild-type plants, whereas those expressing SIZ1 were sensitive to heat stress. Furthermore, transgenic cngc6 plants overaccumulating a mutated mCNGC6 protein (K347R, a mutation at the sumoylation site) were sensitive to heat stress, similar to the cngc6 mutants, while transgenic cngc6 plants overaccumulating CNGC6 exhibited restored heat tolerance. Together, we propose that alternative splicing is an important mechanism that regulates the function of SSVs during development or under adverse conditions, including heat stress.
Rapid hypocotyl elongation allows buried seedlings to emerge, where light triggers de‐etiolation and inhibits hypocotyl growth mainly by photoreceptors. Phosphorylation/dephosphorylation events regulate many aspects of plant development. Only recently we have begun to uncover the earliest phospho‐signaling responders to light. Here, we reported a large‐scale phosphoproteomic analysis and identified 20 proteins that changed their phosphorylation pattern following a 20 min light pulse compared to darkness. Microtubule‐associated proteins were highly overrepresented in this group. Among them, we studied CIP7 (COP1‐INTERACTING‐PROTEIN 7), which presented microtubule (MT) localization in contrast to the previous description. An isoform of CIP7 phosphorylated at Serine ⁹¹⁵ was detected in etiolated seedlings but was undetectable after a light pulse in the presence of photoreceptors, while CIP7 transcript expression decays with long light exposure. The short hypocotyl phenotype and rearrangement of MTs in etiolated cip7 mutants are complemented by CIP7‐YFP and the phospho‐mimetic CIP7 S915D ‐YFP, but not the phospho‐null CIP7 S915A ‐YFP suggesting that the phosphorylated S ⁹¹⁵ CIP7 isoform promotes hypocotyl elongation through MT reorganization in darkness. Our evidence on Serine ⁹¹⁵ of CIP7 unveils phospho‐regulation of MT‐based processes during skotomorphogenic hypocotyl growth.
We present the DNA sequence and plant-tumor transcription pattern of some 2400 base pairs from the right border region of
pTi T37 DNA from the virulent Agrobacterium tumefaciens strain T37. This region includes the entire transcription unit encompassing the nopaline synthase gene, together with parts
of other transcription units. The strategy used to determine the sequence also produced two opposing series of defined, asymmetric
deletions across the target DNA region, some of which may serve future purposes in the exploitation of this sequence, which
is known to be expressed in a wide variety of host plant tissues.
We have developed a gene-fusion system based on the Escherichia coli beta-glucuronidase gene (uidA). The uidA gene has been cloned from E. coli K-12 and its entire nucleotide sequence has been determined. beta-Glucuronidase has been purified to homogeneity and characterized. The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates. We have constructed gene fusions of the E. coli lacZ promoter and coding region with the coding region of the uidA gene that show beta-glucuronidase activity under lac control. Plasmid vectors have been constructed to facilitate the transfer of the beta-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites. There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.
Chimaeric genes can be constructed which fuse the transit peptide of a small subunit of the chloroplast-located ribulose 1,5-bisphosphate carboxylase with a bacterial protein. The fusion protein is translocated into chloroplasts and cleaved in a similar way to the small subunit polypeptide precursor.
Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.
The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary
DNA clone of the firefly luciferase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants
produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase
was also detected in undisrupted leaves or cells in culture from transgenic plants incubated in luciferin and in whole transgenic
plants "watered" with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and
roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway
for uptake of luciferin through the vasculature of the plant.
Morphologically normal plants were regenerated from Nicotiana plumbaginifolia cells transformed with an Agrobacterium tumefaciens strain containing a tumor-inducing plasmid with a chimeric gene for kanamycin resistance. The presence of the chimeric gene in regenerated plants was demonstrated by Southern hybridization analysis, and its expression in plant tissues was confirmed by the ability of leaf segments to form callus on media containing kanamycin at concentrations that were normally inhibitory. Progeny derived from several transformed plants inherited the foreign gene in a Mendelian manner.
The endogenous small subunit of the ribulose-1,5-bisphosphate carboxylase gene rbcS and the light-harvesting chlorophyll a/b-binding protein gene (LHCP) of pea are expressed in a light-inducible manner and are active mainly in green chloroplast-containing tissue. Chimeric genes under control of the 5'-flanking sequences of the rbcS ss3.6 or LHCP AB80 genes from pea were used to study the factors relating to the issue-specific and lightinducible expression of these nuclear-encoded genes in transgenic tobacco plants. The results show that plastid development plays a crucial role in the activation of expression of these chimeric genes. Particular members of each of the above gene families respond differently to tissue-specific and environmental factors. Furthermore, the light-inducible expression directed by the 5'-flanking sequence of ss3.6 rbcSgene is not exclusively mediated by phytochrome, but probably is controlledin large part by another photoreceptor.
A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [γ32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.
A rapid and sensitive method has been developed to determine lysopine dehydrogenase (EC 1.5.1-) and nopaline dehydrogenase activities in crown gall tumour tissues. By this method, enzyme activities as low as 0.2 micrometerol octopine or nopaline per h per g fresh weight tumour tissue can still be detected. In non-infected young pea seedlings, no lysopine dehydrogenase activity was detected.
Bean golden mosaic virus, a single-stranded DNA plant virus, induced striking changes in the nuclear structure which were distinct from those known to occur in cells infected with other plant viruses. The changes, in order of occurrence, included 1) hypertrophy of the nucleolus so that it occupied up to of the nuclear volume, 2) segregation of nucleolur components into discrete granular and fibrillar regions which were similar to those induced by certain carcinogens or antibiotics in animal cells, 3) appearance of electron dense, condensed fibrillar rings in various numbers and sizes, and 4) appearance of viruslike particles either as loosely compacted aggregates or in hexagonally close-packed arrays. Cytochemical studies revealed that the segregated nucleoli were composed mainly of ribonucleoprotein while the fibrillar rings were deoxyribonucleoprotein. Virus particles occurred only in the nuclei of infected cells. Only phloem cells showed nucleopathic effects and/or contained virus particles.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The USPHS/NIOSH Membrane Filter Method is used to monitor for asbestos in occupational and mining atmospheres, and employs the phase-contrast optical microscope (PCM) that under optimum conditions has a resolution of approximately 0.25 micron. While amphibole cleavage fragments are usually visible by PCM, asbestos fibers (such as amosite and chrysotile) have finer widths that may render them invisible by PCM. In this study, personal air-monitoring filters containing chrysotile, amosite and amphibole cleavage fragments from various sources have been analyzed by PCM in accordance with the USPHS/NIOSH Method and scanning transmission electron microscopy (STEM) to assess the effectiveness of the PCM technique. Each STEM specimen was prepared using a direct-transfer technique to ensure that particle size distribution and concentration were not altered. STEM results for chrysotile samples are highly variable, with 9% to 81% of regulatory particles having widths smaller than 0.25 micron--the resolution of the optical microscope. Amosite samples have 27% to 38% of regulatory particles with widths below microscope resolution, indicating that routine particle counts by PCM on these samples would underestimate true fiber content by approximately one-third. All amphibole cleavage fragment samples had regulatory particles that would be observed by PCM. Multiplication factors have been suggested for application to routine counts by PCM to more accurately assess true particle content for mineral particulates on personal air-monitoring filters.
We have shown the expression of transformed genes in the nematode Caenorhabditis elegans using a new gene fusion system. Vectors consisting of the flanking regions of a collagen gene (col-1) or a major sperm protein gene of C. elegans fused to the Escherichia coli uidA gene, encoding beta-glucuronidase, were microinjected into worms and found to be propagated as high-copy extrachromosomal tandem arrays. We have detected beta-glucuronidase activity in transformed lines, and have shown that the activity is dependent upon the correct reading frame of the construction and on the presence of the worm sequences. The enzyme activity was shown to be encoded by the chimeric beta-glucuronidase gene by co-segregation analysis and by inactivation with specific antisera. Expression is at a very low level, and seems to be constitutive. We have used histochemical techniques to visualize the enzyme activity in embryos.
Although promoter regions for many plant nuclear genes have been sequenced, identification of the active promoter sequence has been carried out only for the octopine synthase promoter. That analysis was of callus tissue and made use of an enzyme assay. We have analysed the effects of 5' deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, including different plant tissues. We assayed the RNA transcription product which allows a more direct assessment of deletion effects. The cauliflower mosaic virus (CaMV) 35S promoter provides a model plant nuclear promoter system, as its double-strand DNA genome is transcribed by host nuclear RNA polymerase II from a CaMV minichromosome. Sequences extending to -46 were sufficient for accurate transcription initiation whereas the region between -46 and -105 increased greatly the level of transcription. The 35S promoter showed no tissue-specificity of expression.
We have cloned and sequenced a gene for the small subunit (SS) of ribulose bis-phosphate carboxylase-oxygenase from Nicotiana
tabacum. The tobacco gene is most closely related to the SS genes from the dicots soybean and pea, and less so to the monocots
wheat and Lemna; the deduced amino acid sequence of the mature protein is in all cases more closely conserved than is its
chloroplast transit sequence. Unlike the genomic sequences of the two monocots, which have one intron, and the two other dicots,
which have two introns, the tobacco gene has three introns. The third tobacco intron lies within a highly conserved region
of the protein. Its position coincides with the boundary of a 12 amino acid insertion in the SS genes of higher plants, relative
to those of blue green algae. The 5′ flanking end of the gene carries 67 bp inverted repeats, which flank a series of eight
direct repeats; the direct repeats themselves each carry inverted repeats. The 3′ untranslated end of this gene differs by
only 2 bp from that of an N. sylvestris SS gene.
A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.
A simple and rapid assay for quantitative determinations of DNA in crude homogenates is described. The method is based on the enhancement of fluorescence seen when bisbenzimidazole (Hoechst 33258) binds to DNA. Crude homogenates in which chromatin has been dissociated with high salt buffer can be assayed directly and reliably in a few minutes. The dissociation of chromatin is critical to accurate determinations of DNA in biological materials using this method. The assay can detect as little as 10 ng of DNA with rather unsophisticated instrumentation.
A model is presented according to which cauliflower mosaic virus (CaMV) DNA is replicated via an RNA intermediate. The model explains the typical S1 nuclease-sensitive sites in mature CaMV DNA, the occurrence of the large, terminally redundant transcript, the local separation of CaMV transcription and CaMV assembly, the abundance of knotted CaMV DNA forms, and the high recombination frequency. A site of perfect homology to plant tRNA was detected. Extracts from a mixture of nuclei and inclusion bodies can be separated into fast-sedimenting complexes elongating endogenous CaMV RNA, and slow-sedimenting ones elongating endogenous CaMV DNA. The CaMV DNA synthesis can be partly inhibited both by RNAase and actinomycin D, suggesting the presence of a mixture of RNA- and DNA-templates.
A simple and rapid method for preparing plasmids for restriction enzyme analysis has been developed. Bacteria are boiled for 15–40 s and an insoluble clot of genomic DNA and debris is removed by low-speed centrifugation. Plasmids are recovered from the supernatant by isopropanol precipitation and can be resuspended in buffer and immediately restricted. A 5-ml bacterial culture yields enough plasmids for many restriction enzyme digestions, but a single colony on a petri dish or a 0.5-ml miniculture will suffice for a few experiments. In addition, the procedure can be readily adapted for the preparation of plasmids from liter cultures with quantitative yields.
Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.
We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.
Four RNA transcripts encoded by cauliflower mosaic virus DNA have been detected in the polyadenylated RNA from virus-infected turnip leaves. Two of these transcripts, the major 35S and the 8S species, have the same 5' termini, at nucleotide 7435. A viral DNA fragment encompassing this region directs transcription initiation at this point in vitro. The 5' terminus of the 19S transcript is at nucleotide 5764, and a corresponding viral DNA fragment also directs transcription initiation in vitro. The major 35S RNA is a complete transcript of the circular viral genome, and is 3'-coterminal with 19S RNA at nucleotide 7615. The 8S RNA has its 3' extremity at delta 1, the single-stranded interruption in the transcribed strand of virion DNA. A minor 35S RNA has also been detected that has its 5' and 3' termini at delta 1.
The DNA sequence of the nopaline synthase gene (nos) from Agrobacterium tumefaciens Ti plasmid pTiT37 and adjacent regions up to the right border of the T-DNA was determined. The 5' and 3' termini of the polyadenylated nos mRNA, isolated from a T37 tobacco teratoma tumor line, were localized by S1 mapping. The final mRNA is unspliced, encoded by a region of about 1450 bp, and specifies an open reading frame of 413 amino acids. Potential transcriptional signals in the 5' flanking DNA, such as CATAAA ("TATA box") and GGTCACTAT ("CAT box"), bear close resemblance to other eukaryotic promoters. Two putative polyadenylation signals, AATAAA and AATAAT, are found about 135 and 50 bp from the 3' end, respectively. This study may provide information for the development of expression vectors for genes in plant cells; moreover, the structural gene can be used as an easy screenable marker.
We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase. The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5. It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids. The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping. Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site. No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence. The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene.
Promoter DNA sequences from a petunia chlorophyll a/b binding protein gene were fused to octopine synthase DNA sequences and the resulting chimaeric genes were introduced into petunia and tobacco cells. Populations of transformed regenerated petunia plants containing the chimaeric genes were examined so that the expression of any particular construction could be compared between independent transformants. Substantial variation was observed between transformants in the level of chimaeric gene expression. In general, transcriptional fusions in which a linker sequence interrupted the 5'-untranslated region gave rise to less chimaeric mRNA accumulation than a translational fusion. In the most actively expressing transformants the amount of mRNA from the introduced chimaeric genes was half that of the endogenous wild-type gene. Transcription initiated at the same place in the chimaeric and endogenous genes. Construction of the translational cab/ocs fusion caused three amino acid changes in the octopine synthase protein and functional octopine synthase enzyme was absent from plants in which mRNA for the chimaeric gene was abundantly expressed.
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