An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora
© Bu et al.; licensee BioMed Central Ltd. 2014
Received: 12 January 2014
Accepted: 26 May 2014
Published: 5 June 2014
An efficient transformation method is lacking for most non-model plant species to test gene function. Therefore, subcellular localization of proteins of interest from non-model plants is mainly carried out through transient transformation in homologous cells or in heterologous cells from model species such as Arabidopsis. Although analysis of expression patterns in model organisms like yeast and Arabidopsis can provide important clues about protein localization, these heterologous systems may not always faithfully reflect the native subcellular distribution in other species. On the other hand, transient expression in protoplasts from species of interest has limited ability for detailed sub-cellular localization analysis (e.g., those involving subcellular fractionation or sectioning and immunodetection), as it results in heterogeneous populations comprised of both transformed and untransformed cells.
We have developed a simple and reliable method for stable transformation of plant cell suspensions that are suitable for protein subcellular localization analyses in the non-model monocotyledonous plant Puccinellia tenuiflora. Optimization of protocols for obtaining suspension-cultured cells followed by Agrobacterium-mediated genetic transformation allowed us to establish stably transformed cell lines, which could be maintained indefinitely in axenic culture supplied with the proper antibiotic. As a case study, protoplasts of transgenic cell lines stably transformed with an ammonium transporter-green fluorescent protein (PutAMT1;1-GFP) fusion were successfully used for subcellular localization analyses in P. tenuiflora.
We present a reliable method for the generation of stably transformed P. tenuiflora cell lines, which, being available in virtually unlimited amounts, can be conveniently used for any type of protein subcellular localization analysis required. Given its simplicity, the method can be used as reference for other non-model plant species lacking efficient regeneration protocols.
KeywordsNon-model plant Suspension-cultured cells Green fluorescent protein (GFP) Agrobacterium Subcellular localization
The subcellular localization of plant proteins is highly correlated with their functions, and as such, is a particularly relevant aspect of functional studies. In many cases, subcellular localization can be predicted in silico based on the primary protein sequence by exploiting the conservation of signal peptides and motifs responsible for protein targeting to different cell compartments . Despite recent advances in improving the accuracy of algorithms for prediction [2, 3], experimental validation of predicted localization is still the golden standard to obtain reliable functional information.
Transient gene expression assays allow for rapid and high-throughput analyses of plant genes, and thus have become widely used for characterization studies of gene function. Accordingly, several methods for transient gene expression have been developed, such as polyethylene glycol-mediated protoplast transfection , biolistic bombardment , and Agrobacterium-mediated transient assays . In addition, stable plant transgenic lines (particularly Arabidopsis) expressing epitope-tagged or otherwise modified genes offer advantages in terms of ensuring a sustainable supply of plant material with homologous protein expression, the potential for mutant complementation, as well as the ability to conduct a global-scale examination throughout all tissues and cell types. Although the commonly used floral dip procedure can also be used toward this end, this procedure is more time consuming and laborious as it requires the maintenance and growth of the transgenic plants to maturity, which can take up to several weeks or longer.
Materials and preparations
Wild P. tenuiflora seeds and Agrobacterium tumefaciens harboring the plasmid pBI121-ER-GFP were provided from the Alkali Soil Natural Environment Science Center of Northeast Forestry University, Anda practice base, Harbin, China. This plasmid contains 499 amino acids from the PutAMT1;1 ammonium transporter from P. tenuiflora, which has been previously demonstrated to be localized at the nucleus periphery in correspondence with the ER and the plasma membrane . P. tenuiflora plants were collected from an area of alkaline soil in Northeast China (Heilongjiang Province). No specific permissions were required for these activities, as sampling did not involve any endangered or protected species.
Callus induction and subculture training
P. tenuiflora seeds were soaked in distilled water at 4°C for 1–2 d. After the seeds were dried and soaked in 75% alcohol for 1 min, they were rinsed 3–4 times with sterile water, then with 10% sodium hypochlorite for 30 min, and finally with sterile water 3–5 times. The calli were induced on the disinfected dry seeds after inoculation in the induction medium, which comprised Murashige and Skoog (MS) medium containing 0.5 g/L proline, 0.5 mg/L glutamic acid, 30 g/L sucrose, 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.8% agar (pH 5.8) at 25°C under illumination of 80 μmol/(m2 · s) for 12 h/d. Calli were obtained after 2 weeks. Weakly yellow, loose calli were selected for subculture training.
Establishment of the system of P. tenuiflorasuspension-cultured cell system
The selected calli were cultured at 22°C with shaking at 120 rpm in the dark. After culturing for 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 d, the evenly suspended cells were harvested. Simultaneously, for subculture training, 10 mL of suspension-cultured cells (dry weight approximately 40 mg) were inoculated in liquid MS medium with different nutrient conditions: 1) different types of nitrogen nutrients, including 1/2 MS (i.e., half concentration of regular MS), MSN (i.e., half the ammonium nitrate amount of MS), MSK (i.e., half the potassium nitrate amount), MSE (i.e., additional 0.5 mg/L glutamic acid), MSP (i.e., additional 0.5 g/L proline), MSPE (i.e., 0.5 mg/L glutamic acid and 0.5 g/L proline), MSCH (i.e., 0.6 g/L casein acid hydrolysate), and MSPECH (i.e., 0.5 mg/L glutamic acid, 0.5 g/L proline, and 0.6 g/L casein acid hydrolysate); all the different media contained 2.0 mg/L 2,4-D, and 30 g/L sucrose (pH = 5.8); 2) different concentrations of sucrose, including 2.0 mg/L 2,4-D, and 0, 10, 20, 25, 30, 35, 40, or 50 g/L sucrose at pH = 5.8; 3) the same hormone at different concentrations: MSPECH + 0, 1.0, 2.0, 3.0, or 4.0 mg/L 2, 4-D and 30 g/L sucrose at pH = 5.8; 4) different pH: MSPECH + 2.0 mg/L 2, 4-D and 30 g/L sucrose at pH = 3.8, 4.8, 5.8, 6.8, 7.8, 8.8, 9.8, or 10.8. All media were tested at 22 ± 1°C with shaking at 120 rpm in the dark. After culturing for 15 d, the suspension cultures (50 mL) were centrifuged at 2000 rpm for 5 min, then dried at 70°C for 24 h and weighed; this process was repeated three times.
Effect of acetosyringone (AS) on transformation
Three-day-old P. tenuiflora suspension-cultured cells were used to test the effects of AS on the transformation process. AS (100 mg/L) was added to the culture medium to create our subculture, while the cells in the culture medium without AS were used as the control.
Isolation of protoplasts from P. tenuiflorasuspension-cultured cells
Five-day-old P. tenuiflora suspension-cultured cells (5 mL) were centrifuged at 800 rpm for 5 min, and the precipitated cells were washed and centrifuged again at 800 rpm for 5 min. The newly precipitated cells were weighed and incubated in 0.05 mM MES (pH 5.8) containing 2% cellulose enzyme, 1% pectinase, 0.2 M CaCl2 · 2H2O, and 0.6 M mannitol for 3–4 h at 28°C with gentle shaking. After the enzymatic digestion, the cells were centrifuged at 100 rpm for 5 min and the supernatant was discarded. The cells were resuspended in 2 mM MES (pH 5.7) containing 154 mM NaCl, 125 mM CaCl2, and 5 mM KCl for 30 min. Protoplasts were harvested by centrifugation at 1000 rpm for 5 min, washed three times using the resuspension solution, and then solubilized in the solution containing 0.16 M mannitol and 0.02 M CaCl2 · 2H2O.
Transformation of P. tenuiflorasuspension-cultured cells
Two-week-old suspension-cultured P. tenuiflora cells (5 mL) were inoculated into 20 mL of freshly modified MS medium containing 100 mg/L AS. After 3 d, the cells were co-cultured with Agrobacterium at optical density at 600 nm (OD600) of 0.5, 1.0, 1.5, or 2.0 with shaking at 120 rpm and maintained at 22°C in the dark for 3 d. Then, the cells were filtered over nylon nets and washed with 200 mg/L cefotaxime (Cef) in sterile water. After being transferred to the liquid medium for 24 h, 1 mL suspension-cultured cells was inoculated into the modified MS medium containing 2.0 mg/L 2,4-D, 30 g/L sucrose (pH = 5.8), additional 0, 25, 50, 75, or 150 mg/L kanamycin (Kan), and 0, 100, 200, 300, 400, or 500 mg/L Cef at 25°C in the dark. Subsequently, the cluster of cells was transferred into 50 mL of liquid medium containing Kan and Cef and incubated at 22°C with shaking at 120 rpm in the dark for 9–14 d to obtain the transgenic suspension-cultured cells. Finally, the cells were harvested and dried at 70°C for 24 h. The dry weight was measured three times.
GFP fluorescence observation
Green fluorescence of P. tenuiflora transgenic cells was observed using a stereomicroscope (Olympus, SZX9, Japan). The detection of the GFP signals was carried out using a laser-scanning confocal imaging system (Olympus Fluoview, FV500, Japan).
Results and discussion
Most of the currently available methods for protein subcellular localization can be readily applied to the majority of model plant species such as Arabidopsis and tobacco, which could, in principle, lend themselves as heterologous recipients for localization studies of proteins from non-model plant species [18, 19]. However, the relatively high failure rates reported in studies adopting such heterologous approaches (e.g., for Arabidopsis proteins expressed in tobacco) indicate that homologous expression systems are preferable whenever possible [10, 20]. The lack of established regeneration protocols for the large majority of non-model plant species hinders the application of whole-plant stable transformation methods for protein subcellular localization studies, which rely mainly on transient transformation approaches. The latter methods, however, are limited with respect to the level of detail to which localization can be assessed, as subcellular fractionation or sectioning and immunodetection methods cannot be applied, thus seriously preventing analyses of functional characterization of genes in non-model species. Therefore, adaptation of methods for the stable transformation of cell cultures that are currently applied mainly for functional studies [21, 22] to protein subcellullar localization analyses would provide two main advantages for cases dealing with non-model species: (1) the faithful representation of native localization patterns that are typical of homologous systems, and (2) the versatility, robustness, and virtually unlimited amount of material available for characterizing stable transformation methods. As a case study for the development of such an approach, we chose to use P. tenuiflora, a non-model halophite grass belonging to the Poaceae family that has been the subject of several previous studies owing to its ability to grow in soils with extremely high salinity and pH [23–26]. Given that P. tenuiflora is recalcitrant to regeneration, it can be conveniently used as a representative case for the proof of concept of the novel method.
Establishment of suspension cell lines of P. tenuiflora
Optimization of the liquid medium
Growth performance of P. tenuiflora suspension-cultured cells
P. tenuiflorasuspension-cultured cells are a stable genetic transformation system
The Agrobacterium-mediated transformation was used to establish the genetic transformation system of the P. tenuiflora suspension-cultured cells. The pBI121-ER-GFP plasmid was used to transform the P. tenuiflora suspension-cultured cells. The transgenic cells were screened on a solid medium containing the appropriate marker. To ensure high transformation efficiency, the Agrobacterium concentration, AS effect, co-culture time during transformation, and working concentrations of Kan and Cef were optimized as described below.
Considering that Agrobacterium concentration can affect the transformation efficiency, four different concentrations of Agrobacterium were tested: OD600 = 0.5, 1.0, 1.5, and 2.0. Although Kan-resistant cell clusters grew at Agrobacterium concentrations of an OD600 of 0.5, 1.0, and 1.5, the growth of the cell clusters was negatively affected at higher concentration (OD600 = 2.0). Therefore, we suggest that Agrobacterium concentration for genetic transformation should be kept as low as possible (an Agrobacterium concentration corresponding to OD600 = 0.3 was suitable for genetic transformation; data not shown). In addition, the length of co-culture during the transformation was also important for obtaining high transformation efficiency, as the bacteria were observed to be completely removed from the suspension cells after 3 d of co-culture (data not shown). Therefore, 3 d of co-culture was chosen as the optimum culturing time in order to obtain high transformation efficiency. Pandey et al. reported that optimal β-glucuronidase expression was observed in cumin embryos co-cultivated with an Agrobacterium cell suspension at an OD600 of 0.6 for 72 h , while the optimal transformation efficiency of suspension-cultured Glycyrrhiza inflata Batalin cells was achieved using an Agrobacterium suspension of an OD600 of 0.4 over 24 h of co-cultivation . These results suggested that co-cultivating the lowest concentration of Agrobacterium possible with plant suspension-cultured cells for approximately 3 d could provide optimal conditions for achieving highly efficient transformation.
Optimization of the concentrations of Kan, Cef, and AS
AS is a commonly used agent affecting Agrobacterium-mediated plant genetic transformation. To test the AS-mediated effects on the Agrobacterium-mediated genetic transformation of P. tenuiflora suspension-cultured cells, 3-d-old AS-pretreated suspension cells were co-cultured with Agrobacterium at OD600 = 0.5 for 3 d; non-pretreated cells were used as the control. A slight increase in the number of cell clusters from the AS-treated suspension cells on the solid medium was observed compared to that in the control (data not shown), suggesting that AS pre-treatment could improve the efficiency of infection of P. tenuiflora suspension-cultured cells.
Subcellular localization of proteins
Taken together, the results presented herein indicate that the P. tenuiflora suspension-cultured cell system could be successfully established and employed for protein subcellular localization analysis. Although previous studies reported that this approach may not be suitable for subcellular localization and other fluorescence-based analyses [31, 32], we demonstrate that this optimized expression system based on P. tenuiflora suspension-cultured cells proved to be simple, reliable, and stable. Nevertheless, like all transformation methods relying on cells isolated from single organs without intervening regeneration of whole plants, this method can only be applied to examine the localization of proteins in cell compartments/structures that are present in the recipient cells. Therefore, the method is not suitable for studies requiring localization to the cell walls or plasmodesmata, for example, or to track protein movements in the context of organized tissues or organs. We note, however, that application of this method to other cell types deriving from different tissues such as the leaf, root, and inflorescence requires further validation to best ascertain the scope of applications of the method. In comparison to transient assays, our method is more labor-intensive and time-consuming, and thus does not lend itself readily to application in high-throughput studies . However, the method does have an important advantage of providing homogeneous populations of transformed cells in virtually unlimited amounts for extended periods of time that can be used in all localization assays, which cannot otherwise be carried out on transiently transformed protoplasts owing to their complexity or particular technical requirements (e.g., cell fractionation, embedding and sectioning, immunolocalization [33, 34]). Furthermore, we expect that our stable transformation method could be suitable for dynamically documenting protein re-localization through the different phases of the cell cycle or in response to environmental cues, although this was not directly tested in the present study. Most importantly, the fact that this method does not rely on whole plant regeneration makes it applicable to any non-model plant species lacking suitable regeneration protocols, which is a substantial advantage. As the method is based on simple, species-specific optimization steps of protocols that were previously developed for functional analyses in a relatively distantly related monocot species (rice), we expect that, with minor modifications, the method could be applied to several other non-model species from the Poaceae family.
We have developed a rapid and stable suspension-cultured cell system for non-model plants (i.e., those lacking suitable regeneration protocols) based on an Agrobacterium-mediated approach. This method was successfully used for determining ER-GFP fusion protein subcellular localization in P. tenuiflora. Our method is simple and rapid, and is applicable to evaluating the localization of proteins only in cell compartments/structures that are present in the recipient cells.
Green fluorescent protein
Murashige and Skoog
4-days: 2,4-dichlorophenoxyacetic acid
- 1/2 MS:
Half concentration of regular MS
Contains 1/2 amount of ammonium nitrate of MS
Contains 1/2 amount of potassium nitrate
Contains additional 0.5 mg/L glutamic acid
Contains additional 0.5 g/L proline
Contains 0.5 mg/L glutamic acid and 0.5 g/L proline
Contains 0.6 g/L casein acid hydrolysate
Contains 0.5 mg/L glutamic acid, 0.5 g/L proline, and 0.6 g/L casein acid hydrolysate
This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University of China (PCSIRT), State Forestry Administration 948 Program of PR China (No. 2008429) and the Heilongjiang Provincial Program for Distinguished Young Scholars (JC200609) awarded to Shenkui Liu. Further support was provided by the Fundamental Research Funds for the Central Universities (No. DL13BAX13) awarded to Yuan-yuan Bu. The funders had no role in study design. We thank the Editage Corporation for providing the English editing service.
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