An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1
© The Author(s) 2017
Received: 8 May 2017
Accepted: 25 May 2017
Published: 5 June 2017
Perennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system.
In this report, a highly efficient protoplast isolation (5.6 × 107 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein–protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time.
This efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein–protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.
Perennial ryegrass (Lolium perenne L.) is the most widely distributed and cultivated turf and forage grass in temperate zones. World-wide consorted programs are working on the molecular genetics of this grass species. The draft genome of this perennial ryegrass was recently published  and the assembly of the genome dataset is undergoing. The accumulation of genomic and transcriptomic datasets provided unprecedented opportunity to conduct functional genomic studies in perennial ryegrass. Stable genetic transformation systems have been established in perennial ryegrass, yet the whole transformation takes months to obtain rooted transgenic plants [2, 3].
The plant protoplast system provides a complementary or, sometimes, an alternative way to the stable genetic transformation system for gene functional analysis in many cases, such as protein subcellular localization, in vivo protein–protein and protein-DNA interactions, protein trafficking and signal transduction, etc. Currently, mesophyll protoplast-based transient expression assays are routinely used in biological studies in Arabidopsis (Arabidopsis thaliana) [4, 5], maize (Zea mays) , tabacco (Nicotiana tabacum) , rice (Oryza sativa) [7, 8], Populus [9, 10], and cucumber (Cucumis sativus) . Successful ryegrass protoplast isolation from mesophyll cells has been reported previously and was used in the study on chloroplast photosynthetic and photorespiratory carbon metabolism . However, its efficiency remains low and not sufficient engouth for protoplast transformation studies according to our preliminary experimental results following the previously published one (data not shown). An efficient transient expression system based on ryegrass mesophyll protoplast transformation was not reported yet.
In this study, a highly repeatable and efficient protocol for mesophyll protoplast isolation and gene transient expression was developed using ryegrass leaves as starting materials. This protocol provides a facile tool for protein subcellular localization and bimolecular fluorescence complementation (BIFC) assays as shown in this study as well as the other in vivo molecular studies where this system is applicable.
Plant material and growth conditions
Perennial ryegrass (cv. Buena vista) was grown in vermiculite: perlite: peat moss (1:3:9) in a growth chamber with temperature set at 25/20 °C (day/night), photosynthetically active radiation (PAR) of 750 µmol photons m−2 s−1 and 14 h of light per day. The potted plants were watered at about three-day intervals and fertilized with ½ MS minerals (Murashige and Skoog 1962) once a week. It is CRITICAL to water and fertilizer ryegrass plants regularly to obtain fine starting plant material.
Reagents and solutions
Solution recipes for protoplast isolation and transformation
Enzyme solution (resuspension solution)
10 mM MES, 1.5% (wt/vol) cellulase R10, 0.75% (wt/vol) macerozyme R10, and 20 mM KCl. 10 mM CaCl2, 0.1% BSA, 1–5 mM β-mercaptoe- thanol (optional) and mannitol (0.6 M), pH 5.7
Room temp. (freshly prepared)
Leaf strips lysis
2 mM MES, 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl, pH 5.7
Release and wash protoplasts
4 mM MES, 0.4 M mannitol and 15 mM MgCl2, pH 5.7
Resuspend protoplast pellet
PEG-Ca2+ solution (resuspension solution)
20% (wt/vol) PEG4000, 100 mM CaCl2 and mannitol (0.3 M)
Room temp. (freshly prepared)
Transform plasmids (10ug is used in this study) into protoplasts
PEG-4000 (Cat. No. 25322-68-3), D-Mannitol (Cat. No. DH190-2), and Bovine Serum Albumin (BSA) (Cat. No. 9048-46-8) were purchased from Bei Jing Ding Guo Chang Sheng Biotech Co., Ltd., China; MES (Cat. No. E169) was from Ameresco LLC, USA; CaCl2·2H2O (Cat. No. 10035-04-8) was from Xi long Chemical Co., Ltd., China; and KCl (Cat. No. 7447-40-7), NaCl (Cat. No. 7647-14-5), and MgCl2·6H2O (Cat. No. 7791-18-6) were from Sinopharm Chemical Reagent Co., Ltd., China.
Fully expanded leaves (about 10–12 days after leaf emergence) were collected from healthy ryegrass plants and only the middle sectioned leaves were used as the starting material (the tips and bases of leaf blades were removed). A total of 0.4–0.6 g of leave sections were cut into 0.5 mm strips transversely with sharp razors wetted with the enzyme solution, and the leaf strips, once cut off, were immediately emerged into the 6 ml enzyme solution (Table 1). The leaf strips were then vacuumed (0.1 MPa) for 1 h under dark at room temperature. The enzymatic digestion was carried out in a thermal-stable shaker at 30 rpm, 28 °C in dark for about 6 h to the extent that the leaf strips were readily dissembled upon gentle touches with a pipette tip.
The suspended protoplasts were firstly filtered through a layer of cheese clothes and then through a nylon mesh (75 μm), and the residue was washed twice with 10 ml pre-chilled modified W5 solution (Table 1). The filtrate was centrifuged at 100×g for 2 min with low acceleration and deceleration speed (BECKMAN, Model Allegra 64R, Hamburg, Germany). The pelleted protoplasts were re-suspended in 10 ml pre-chilled modified W5 solution, and set still for about one hr on ice (or overnight at 4 °C) to allow protoplast to sediment. The precipitated protoplasts were re-suspended in 3 ml MMg solution (Table 1). According to the counted number of viable protoplasts, the suspension was centrifuged down at 100×g for 2 min and re-suspended in the MMg solution to a final concentration of ~5 × 105 cells ml−1 (depending on experimental purposes, the concentration can be readily achieved up to 7.3 × 106 cells ml−1).
Noting that the pipette tips used in transferring protoplasts should be cut with scissors to minimize mechanical damage.
Vector construction and plasmid preparation
Primers used in gene cloning and vector construction
PEG-mediated protoplast transformation
The transient gene expression system using ryegrass protoplasts was modified from a protocol reported by Yoo et al. . The optimal amount of plasmid (from 1 to 10 µg) and concentration of mannitol (from 0.1 to 0.3 M) were tested. In brief, plasmids were mixed with 200 µl protoplast stock at RT, adding equal volume of PEG-Ca2+ medium (Table 1), gently mixed and setting still for 5 min at RT for plasmid transformation. Then, three ml W5 solution was added slowly and gently mixed with the protoplast suspension, followed by a centrifugation at 100×g for 2 min and the pellet was re-suspended gently in 0.5 ml of W5 solution. Finally, the protoplast suspension was transferred into 1.5 ml tubes that were pre-coated with 1% BSA to avoid protoplast attachment to the tube surface, and incubated for 10–24 h in dark at RT.
The density and viability of isolated protoplasts were counted by using the standard hemocytometer and FDA staining assay  and observed under a light microscope (OLYMPUS Model BX53, Tokyo, Japan).
Transformed protoplasts were observed with a confocal laser scanning microscope (Zeiss LSM780 Exciter) for GFP, CFP (citrine), and chloroplast auto-fluorescence with the excitation wavelengths and emission filters set at 488 nm/band-pass 505–530 nm for GFP, 458 nm/band-pass 465–530 nm for CFP, and 488 nm/band-pass 650–710 nm for chloroplast auto-fluorescence. Image processing was performed using the Volocity software (Zeiss).
Results and discussion
Protoplast isolation from mesophyll cells of perennial ryegrass
PEG-mediated protoplast transformation
The isolated protoplasts were subjected to PEG-mediated transformation. After adjusting variable parameters, we achieved up to 55.2% transformation efficiency with 10 µg plasmid DNA (plasmid size ~10 kb) prepared with a regular plasmid midi-prep kit. Higher protoplast transformation efficiency could be achieved using higher amount or purity (e.g. with CsCl purified plasmid DNA) of plasmid DNA . The mannitol concentration in the PEG-Ca2+ solution was also critical for the transformation efficiency that the highest efficiency was achieved at 0.3 M mannitol (data not shown).
LpNOL and LpNYC1 interact in vivo using BIFC
GY, QC and ZX performed vector construction, ryegrass protoplast isolation and transformation experiments, and participated in manuscript preparation; QC and BZ designed and constructed the pair of BiFC vectors; BX and BH conceived and designed the experiments, analyzed data and prepared the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions and description of a complete protocol are included within the article.
Consent for publication
All authors agreed to publish this manuscript.
Ethics approval and consent to participate
All authors read and approved the manuscript.
This project was supported by a Grant from 31572455 from the National Science Foundation of China, by grant BK20140693 from the Natural Science Foundation of Jiangsu Province, China, and by Grant KYZ201552 from the Fundamental Research Funds for the Central Universities.
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