Sub-cellular markers highlight intracellular dynamics of membrane proteins in response to abiotic treatments in rice
Cell biology approach using membrane protein markers tagged with fluorescent proteins highlights the dynamic behaviour of plant cell membranes, not only in the standard but also in changing environmental conditions. In the past, this strategy has been extensively developed in plant models such as Arabidopsis.
Here, we generated a set of transgenic lines expressing membrane protein markers to extend this approach to rice, one of the most cultivated crop in the world and an emerging plant model. Lines expressing individually eight membrane protein markers including five aquaporins (OsPIP1;1, OsPIP2;4, OsPIP2;5, OsTIP1;1, OsTIP2;2) and three endosomal trafficking proteins (OsRab5a, OsGAP1, OsSCAMP1) were obtained. Importantly, we challenged in roots the aquaporin-expressing transgenic lines upon salt and osmotic stress and uncovered a highly dynamic behaviour of cell membrane.
We have uncovered the relocalization and dynamics of plasma membrane aquaporins upon salt and osmotic stresses in rice. Importantly, our data support a model where relocalization of OsPIPs is concomitant with their high cycling dynamics.
KeywordsOryza sativa Subcellular markers Intracellular dynamics Abiotic stress
In plants, popularization of cell biology approaches, such as laser scanning confocal microscopy, was promoted by the use in particular of shared sets of transgenic lines expressing fluorescent-protein fusions to subcellular markers. This approach has been mostly developed in the plant model Arabidopsis (Cutler et al. 2000). New sets of transgenic lines have also been developed in leading crop models such as maize (Krishnakumar et al. 2015), and rice (Wu et al. 2016). These sets of markers are interesting tools for highlighting subcellular compartments. Most interestingly, the use of protein markers with known biological functions allows one to uncover novel subcellular regulations. For instance, a set of multicolour markers of membrane compartments was used to study the intracellular dynamics of aquaporins in Arabidopsis (Wudick et al. 2015). Here, we present a new set of transgenic rice lines stably-expressing individually fluorescent protein fusions with subcellular protein-markers. These include (i) plasma membrane (PM) and tonoplast aquaporins (Sakurai et al. 2005), (ii) PM-marker low-temperature inducible protein 6A (LTi6a; (Cutler et al. 2000)), (iii) OsRab5a known to be localized in a pre-vacuolar compartment (Shen et al. 2011), (iv) OsGAP1 which has a putative function for Golgi apparatus to PM and trans-Golgi network (TGN) trafficking and potentially localizes in endosomal compartments (Heo et al. 2005), and (v) a rice secretory carrier membrane protein (OsSCAMP1) which is localized in an early endosome compartment and may have a function in the early stage of membrane trafficking from the PM (Cai et al. 2011). Apart from aquaporins and LTi6a, all of these proteins are identified components of key compartments en route towards the vacuole. Importantly, we challenged the aquaporin-expressing transgenic lines upon salt and osmotic stress to uncover their dynamic behaviour in rice roots.
Results and Discussion
Rice Transgenic Line Creation and Subcellular Localization Visualization
Redistribution of PM Aquaporins upon Salt and Drought Stress
Dynamic of Endocytosis of PM Aquaporins upon Salt Stress
In conclusion, we have uncovered the relocalization and dynamics of PM aquaporins upon salt and osmotic stresses in rice. Importantly, our data support a model where relocalization of OsPIPs is concomitant with their high cycling dynamics. Altogether these data indicate that the rice research community has at its disposal a new set of subcellular markers amenable for cell biology approaches on a large array of topics.
Molecular Cloning of Membrane Protein Markers and Plant Transformation
Molecular cloning information is summarized in Additional file 2: Table S1. OsPIP1;1 was subcloned into pDONR207 and transferred into the destination vector pGWB5 (Nakagawa et al. 2007) using Gateway® Gene Cloning technology (Invitrogen, USA), according to the manufacturer’s instruction. The whole set of the other protein markers were subcloned into pBluescript SK vector (Stratagene, USA) or pUC57 (see Additional file 2: Table S1), and then cloned into the pGreenII 0179 binary vector (Hellens et al. 2000) under the transcriptional and translational control of a double enhanced cauliflower mosaic virus 35S promoter and the 3′ end of the pea ribulose-1,5-bisphosphate carboxylase small subunit rbcS gene. A molecular construct consisting of the maize ubiquitin-1 promoter controlling the expression of a fluorescent plasma-membrane-localized fusion protein (ECFP-LTI6a) (Cutler et al. 2000) was obtained by synthesis (Genscript) and cloned into the plasmid pUC57. The insert was released by a double digestion with EcoRI et KpnI and cloned into the binary vector pCAMBIA2300 linearized by EcoRI and KpnI. All constructs were confirmed by DNA sequencing by Eurofins Genomics (Germany). The recombinant DNA plasmids were electroporated into Agrobacterium tumefaciens strain EHA105 or GV3101 for rice or Arabidopsis transformation, respectively. Japonica rice Nipponbare cultivar was transformed according to a modified seed-embryo callus transformation procedure (Sallaud et al. 2003). Arabidopsis transformation was performed according to flower dip protocol (Clough and Bent 1998). Selection of transgenic plants was performed with medium supplemented with hygromycin.
Plant Materials and Growth Conditions
Rice Oryza sativa L. cv. Nipponbare and Arabidopsis thaliana L. (Heyn.) accession Columbia 0 were used in this study. Rice seeds were dehusked, then sterilized by dipping in 70% ethanol for 2 min, soaking in 3.6% (w/v) sodium hypochlorite solution for 30 min and rinsing several times with distilled water. After sterilization, the seeds were put in the petri dish containing moist filter paper. After emergence of the coleoptile and germination of the radicle, seeds were transferred onto a raft floating on deionised water. Conditions of the growth chamber were 14 h of day cycle (~ 200 μE m− 2 s − 1) and 10 h of night at 28/25 °C and 70% relative humidity. Seven to eight days after germination (DAG) rice seedlings were used for cell biology approaches. Arabidopsis seeds were surface sterilized in a solution (50% ethanol, 4 g L− 1 Bayrochlore and 0.02% (w/v) Clean N for 10 min, thoroughly washed with 70% ethanol and air-dried under the sterile hood for 2 h. Sterilized seeds were sown on sucrose-added (10 g L− 1) half-strength Murashige and Skoog (MS) medium (Murashige and Skoog 1962) in clear polystyrene plates (12 × 12 cm) sealed with an air-permeable tape. After 48 h of stratification in 4 °C dark room, plates were transferred vertically into a growth chamber with cycles of 16 h of light (~ 150 μE m− 2 s − 1) and 8 h of dark at 21 °C and 65% relative humidity. Seven days after sowing (DAS), Arabidopsis seedlings were used for cell biology approaches.
Confocal Microscopy Visualization
A laser scanning confocal microscope (Leica TSC SP8 system, Germany) was used with the excitation wavelengths 405 nm, 488 nm and 561 nm for CFP, GFP and mCherry, respectively. The detection wavelengths were in the range of 450–500 nm for CFP, 500–535 nm for GFP and 580–630 nm for mCherry. For ER-Tracker Blue-White DPX staining, observations were made with excitation wavelength 405 nm and detection wavelengths in the range of 420–500 nm. For FM4–64 and propidium iodide staining, observations were made with excitation wavelength 561 nm and detection wavelengths in the range of 580–630 nm. Images were taken at a region ~ 0.5–1 cm from the root tips. Images were captured in a z-stack of 0.5 μm intervals for subcellular localization and a time lapse for mobility of protein observation.
Multiphoton microscope (Zeiss LSM 7MP OPO, Germany) was used to observe ClearSee-prepared tissue with the excitation wavelength 836 nm and signals were collected in detection range of 500–550 nm.
ClearSee Tissue Preparation
ClearSee solution was prepared by mixing xylitol (10% w/v), sodium deoxycholate (15% w/v) and urea (25% w/v) in water (Kurihara et al. 2015). Briefly, rice seeds were dehusked and surface sterilized in 3.6% (w/v) sodium hypochlorite solution for 30 min, then washed carefully with sterile water. Next, seeds were sown on half-strength MS medium in clear polystyrene plates (24.5 × 24.5 cm), 25 seeds for 1 line at the middle per plate. Plates were kept vertically in culture room at 29 °C for 12 h of day, 25 °C for 12 h of night and relative humidity at 66%. Seven DAG, rice roots were collected and immediately immersed in a 4% (w/v) paraformaldehyde solution prepared with 1X PBS, subjected to vacuum for 30 min, washed again with 1X PBS, and then immersed in ClearSee solution under vacuum for 2 h, followed by 1 week at ambient conditions. When rice root became transparent, each root type was mounted in a 1% (w/v) agarose solution and visualized by means of a multiphoton microscope.
Fluorescent Dye Staining
We used 1 μM ER-Tracker Blue-White DPX (1 mM stock solution in dimethylsulfoxide), 10 μM FM4–64 (10 mM stock solution in H2O), and propidium iodide 1 μg/mL (1 mg/mL stock solution in water). Dyes were from Molecular Probes (Eugene, USA) and diluted into water before root staining. Roots were immersed into FM4–64 for 2–5 min before observation. For propidium iodide staining, roots were stained for 5 min, and then transferred into a 500 mM mannitol solution for plasmolysis before observation. Staining with ER-Tracker Blue-White DPX was carried out for 30–60 min before observation.
Stress Application and Pharmacological Approach
Plants were stress challenged by incubating the roots in solutions of 100 mM NaCl or 20% (w/v) PEG6000. Brefeldine A was used at a concentration of 50 μM, dissolved into water supplemented or not with 100 mM NaCl. Importantly, cycloheximide was added at a concentration of 50 μM prior to and during BFA treatments to prevent new protein biosynthesis. All chemicals listed in this section were from Sigma-Aldrich (USA).
We thank Xavier Dumont for assistance in Arabidopsis transformations, Cecile Fizames for assistance in bioinformatics sequence analysis and other staff members of the Institut de Biologie Intégrative des Plantes for technical assistance in biological material culture. We thank Nguyen Thi Hue and Dinh Van Lam from National key laboratory for Plant Cell Biotechnology for assistance in rice transformation. We acknowledge the imaging facility MRI, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future») for confocal observations. TTHC was supported by a PhD scholarship from the Ministry of Education and Training of the Socialist Republic of Vietnam and by the Embassy of France in Vietnam.
DCT was supported by a Master scholarship from the University of Science and Technology of Hanoi. Funding of DTL was supported by the EU Marie Curie International Outgoing Fellowship “ORYZAQUA – Cell biology of rice aquaporins” (PIOF-GA-2011-300150) which we kindly acknowledge. This work was also supported by the French Ministry of Foreign Affairs and International Development, the French Ministry of Higher Education and Scientific Research and the Ministry of Science and Technology of the Socialist Republic of Vietnam in the frame of a Hoa Sen – Lotus Hubert Curien partnership N° 30598PC “Application of functional genomics and association genetics to characterize genes involved in abiotic stresses tolerance in rice”.
Availability of Data and Materials
The datasets supporting the conclusions of this article are included within the article (and its additional files).
TTHC, DCT carried out the experiments, interpreted the results and wrote the paper. HTG and DTL designed experiments carried out the experiments, interpreted the results and wrote the paper. CB, DM, AV, MI carried out the experiments. NVD, CP, EM, PG, CM designed experiments, interpreted the results and wrote the paper. All authors read and approved the final manuscript.
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