Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1

Abstract

Background: Magnolia is a woody ornamental plant, which is widely used in urban landscaping. However, its lengthy juvenile period and recalcitrance to regeneration impedes functional characterization of its genes.

Results: We developed an efficient protoplast isolation and transient expression system for Magnolia denudata × Magnolia acuminata 'Yellow River'. The highest yield of protoplasts was obtained from young leaves digested in 3% Cellulase R10, 0.8% Macerozyme R10, 0.04% pectinase and 0.4 M mannitol enzymolysis solution for 6 h. For transfection of protoplasts, 20% PEG4000 for 5 min was optimal. To verify the protoplast system and begin to understand heat tolerance in Magnolia, a heat shock transcription factor MdeHSF1 was cloned from 'Yellow River', which belongs to the HSF subfamily A and has significant homology with AtHSFA1A. Subcellular localization analysis indicated that MdeHSF1 was expressed in the cell nucleus. Furthermore, qPCR analysis of the MdeHSF1 transcript level in response to high temperature stress suggested that MdeHSF1 might be involved in regulating heat stress tolerance in 'Yellow River'.

Conclusion: The described protocol provides a simple and straightforward method for isolating protoplast and exploring gene subcellular localization of MdeHSF1 in Magnolia. This expands the new research of protoplast isolation and transfection in Magnolia.

Keywords: Heat shock transcription factor; Heat stress; Magnolia; Protoplast isolation; Subcellular localization.

Fig. 1

Different tissues for protoplast isolation in ‘Yellow River’. The shape of ‘Yellow River’ (a), mature leaves of ‘Yellow River’ (b), young leaves of ‘Yellow River’ (c), ‘cut-off’ leaves from ‘Yellow River’ (d, e), Protoplasts isolated from the young leaves (f, g), bar 50 μm

Fig. 2

The protoplast yield under different treatments. In response to a range (0.2, 0.4, 0.6 and 0.8 M) of mannitol and sorbitol concentrations in digestion solution with 3% Cellulase R10, 0.8% Macerozyme R10, and 0.4% Pectinase (a), protoplast yield as affected by different concentrations of digestion enzymes (b): (a) 1% (w/v) Cellulase R10, 0.4% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (b) 1.5% (w/v) Cellulase R10, 0.5% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (c) 2% (w/v) Cellulase R10, 0.6% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (d) 2.5% (w/v) Cellulase R10, 0.7% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (e) 3% (w/v) Cellulase R10, 0.8% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (f) 3.5% (w/v) Cellulase R10, 0.9% (w/v) Macerozyme R10, and 0.04% (w/v) Pectinase, protoplast yield as affected by digestion time (2, 4, 6, 8, 10 or 12 h) (c), protoplast yield as affected by tissue type (buds, young leaves and mature leaves) (d)

Fig. 3

The protoplast transfection ratio and transient expression. The ratio of transfection at 10, 15, 20, 25 and 30% (w/v) PEG4000 concentration (a), the ratio of transfection after 5, 10, 15 and 20 min (b), images of GFP and chlorophyll autofluorescence (Chl) as well as bright field images of protoplasts captured 12 h after transfection (c, d). Bar in c, 50 μm. Bar in d, 10 μm

Fig. 4

The protein of MdeHSF1 and three AtHSF proteins were compared using DNAman6.0 software. AtHSFA1A is At4g17750; AtHSFB1 is At4g36990; AtHSFC1 is At3g24520

Fig. 5

The HSF DNA binding domain is underlined (a). Phylogenetic relationship between MdeHSF1 and AtHSF proteins drawn using MEGA6.0 (b), the conserved domain analysis of the MdeHSF1 and AtHSF proteins using the MEME website (c)

Fig. 6

qPCR analysis of the MdeHSF1 expression level under high temperature (42 °C) from 0 to 5 h in ‘Yellow River’ and M. × soulangeana leaves

Fig. 7

Plasmids can be transferred to protoplasts for transient expression of MdeHSF1. The protected picture of the subcellular location of MdeHSF1 (a), the subcellular location of MdeHSF1 in ‘Yellow River’ protoplast (b) Bar 10 μm