performed in a rapid way using protoplasts transient expression [7–
11]. Dose-dependent analysis through co-expression in protoplasts
permitted the evaluation of individual sorting machinery effects on
cargo molecule transport in a quantitative way [12, 13]. Likewise,
our knowledge on plant signaling pathway is growing with the
application of drug treatments and other biochemical assay in the
protoplasts transient expression system [14].
Confocal microscopy is widely used for studying protein subcellular localization with the introduction of fluorescence protein as
fusion tags or reporters. Nevertheless, the spatial resolution of
conventional confocal microscopy is restricted due to the diffraction limit of light and can normally reach a resolution of only
200 nm in biological specimens. Thus, this limited resolution leaves
a gap between light microscopy and electron microscopy. Recently,
to conquer this barrier and to bridge the resolution gap between
confocal and electron microscopy, super-resolution techniques
such as SIM (Structured Illumination Microscopy) were developed
for analyzing structures and molecules beyond the diffraction limit
of light. There is an increasing application of super-resolution
imaging in plant cell biology research [14, 15].
A recent study using SIM in combination with protoplast
transient expression system to study the spatial relationship
between ERESs (Endoplasmic Reticulum Exit Sites) and Golgi
apparatus unraveled a distinct population of ERESs [14]. Arabidopsis COPII machinery or ERESs marker, the small GTPase Sar1
homologs AtSar1a and AtSar1c, exhibited distinct spatial relationship with Golgi markers such as EMP12 (a cis-Golgi-localized
integral membrane protein) under super-resolution imaging using
protoplast transient expression (Fig. 2) [7, 14, 16]. This study
paraphrased the current model of ER-Golgi interface in higher
plants and demonstrated the usefulness of protoplast transient
expression combining with super-resolution imaging in future
plant cell biology research [17, 18].
2 Materials
2.1 Arabidopsis
PSBD Cell Culture
1. 3–5-day-old suspension cell cultures (Arabidopsis plant system
biology dark-type culture PSB-D from Landsberg erecta root
cells).
2. Arabidopsis suspension culture medium: 4.3 g/L Murashige
and Skoog Basal Salt Mixture (MS), 30 g/L sucrose, 0.1 g/L
myo-Inositol, 0.05 g/L kinetin, 0.8 g/L 1-naphthaleneacetic
acid, and 0.4 mg/L Thiamine hydrochloride, pH 5.7 (with
KOH). Sterilize by autoclave.
3. Orbital rotary shaker (New Brunswick™ Innova
® 40/40R
Shakers).
158
Yonglun Zeng et al.
11]. Dose-dependent analysis through co-expression in protoplasts
permitted the evaluation of individual sorting machinery effects on
cargo molecule transport in a quantitative way [12, 13]. Likewise,
our knowledge on plant signaling pathway is growing with the
application of drug treatments and other biochemical assay in the
protoplasts transient expression system [14].
Confocal microscopy is widely used for studying protein subcellular localization with the introduction of fluorescence protein as
fusion tags or reporters. Nevertheless, the spatial resolution of
conventional confocal microscopy is restricted due to the diffraction limit of light and can normally reach a resolution of only
200 nm in biological specimens. Thus, this limited resolution leaves
a gap between light microscopy and electron microscopy. Recently,
to conquer this barrier and to bridge the resolution gap between
confocal and electron microscopy, super-resolution techniques
such as SIM (Structured Illumination Microscopy) were developed
for analyzing structures and molecules beyond the diffraction limit
of light. There is an increasing application of super-resolution
imaging in plant cell biology research [14, 15].
A recent study using SIM in combination with protoplast
transient expression system to study the spatial relationship
between ERESs (Endoplasmic Reticulum Exit Sites) and Golgi
apparatus unraveled a distinct population of ERESs [14]. Arabidopsis COPII machinery or ERESs marker, the small GTPase Sar1
homologs AtSar1a and AtSar1c, exhibited distinct spatial relationship with Golgi markers such as EMP12 (a cis-Golgi-localized
integral membrane protein) under super-resolution imaging using
protoplast transient expression (Fig. 2) [7, 14, 16]. This study
paraphrased the current model of ER-Golgi interface in higher
plants and demonstrated the usefulness of protoplast transient
expression combining with super-resolution imaging in future
plant cell biology research [17, 18].
2 Materials
2.1 Arabidopsis
PSBD Cell Culture
1. 3–5-day-old suspension cell cultures (Arabidopsis plant system
biology dark-type culture PSB-D from Landsberg erecta root
cells).
2. Arabidopsis suspension culture medium: 4.3 g/L Murashige
and Skoog Basal Salt Mixture (MS), 30 g/L sucrose, 0.1 g/L
myo-Inositol, 0.05 g/L kinetin, 0.8 g/L 1-naphthaleneacetic
acid, and 0.4 mg/L Thiamine hydrochloride, pH 5.7 (with
KOH). Sterilize by autoclave.
3. Orbital rotary shaker (New Brunswick™ Innova
® 40/40R
Shakers).
158
Yonglun Zeng et al.