Rapid Isolation of Arabidopsis Chloroplasts and Their Use for In Vitro Protein Import Assays

Article (PDF Available)inMethods in molecular biology (Clifton, N.J.) 774:281-305 · January 2011with71 Reads
DOI: 10.1007/978-1-61779-234-2_17 · Source: PubMed
Abstract
In vitro chloroplast protein import assays have been performed since the late 1970s, initially with plant species (e.g., pea and spinach) that readily provide an abundant source of starting material and also, subsequently, a good yield of chloroplasts for import assays. However, the sequencing of the Arabidopsis genome paved the way for an additional model system that is more amenable to genetic analysis, as a complement to the more biochemically orientated models such as pea and spinach. A prerequisite for this change was an efficient and reliable protocol for the isolation of chloroplasts for use in protein import assays, enabling biochemical approaches to be combined with the genetic potential of the plant. The method described here was developed as a rapid and low-cost procedure that can be accessed by everyone due to its simplicity. Despite its rapidity and simplicity, the method yields highly pure chloroplasts, and in addition works well with mutant plants that exhibit pale or chlorotic phenotypes. The protocol is also optimized for work with material from young plants (10-14 days old), when protein import is believed to be at its peak, and so plant growth can be conducted in vitro on Murashige and Skoog medium. The isolation method has been used not only for protein import assays, but also for proteomic analysis and further subfractionation studies.
A simple method for isolating import-competent Arabidopsis chloroplasts
Henrik Aronsson, Paul Jarvis
Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
Received 15 August 2002; accepted 23 August 2002
First published online 17 September 2002
Edited by Ulf-Ingo Flu
«
gge
Abstract We present a simple, rapid and low-cost method for
isolating a high yield of Arabidopsis chloroplasts that can be
used to study chloroplast protein import. E⁄ciency of chloro-
plast isolation was dependent upon the ratio between amount of
plant tissue and the bu¡er volume, the size and speed of the
homogenisation equipment, and the size of the homogenisation
beaker. The import method proved useful when characterising
di¡erent precursor proteins, developmental stages and import-
defective mutants. Time-course experiments enabled the mea-
surement of import rates in the linear range. Compared to pro-
toplastation, this isolation method has signi¢cant time and cost
savings (VV80% and VV95%, respectively), and yields chloro-
plasts with a higher capacity to import proteins.
 2002 Federation of European Biochemical Societies. Pub-
lished by Elsevier Science B.V. All rights reserved.
Key words: Chloroplast ; Protein import; Precursor protein;
Arabidopsis thaliana
1. Introduction
Most chloroplast proteins are encoded in the nucleus, trans-
lated in precursor form in the cytosol, and transported across
the chloroplast envelope post-translationally [1,2]. Precursor
proteins (preproteins) each have an amino-terminal targeting
signal called the transit peptide that directs their translocation
through a translocon complex in the envelope membranes,
and is ¢nally cleaved by a stromal processing peptidase to
yield mature protein [3,4]. Imported proteins may subse-
quently be localised to di¡erent intrachloroplastic compart-
ments [1].
The chloroplast protein import ¢eld has, during the last
decade, undergone rapid progress concerning the discovery
of translocon components [1,2]. Pea has been the model sys-
tem of choice for studies of chloroplast protein import, but
Arabidopsis is now emerging as an alternative model system
[5^9]. Advanced genetic and molecular techniques and the
complete genome sequence are enabling the import mecha-
nism to be explored in greater detail, in vitro and in vivo.
The exploitation of Arabidopsis in chloroplast import re-
search, however, has been retarded by the inconsistency and
low yield of Arabidopsis chloroplast isolation procedures. This
was partly resolved by a method for isolating Arabidopsis
chloroplasts from protoplasts [10]. However, because this
method relies on protoplastation prior to chloroplast isola-
tion, it is time-consuming and expensive. We present a simple,
low-cost method that can be used to isolate large numbers of
intact chloroplasts from Arabidopsis seedlings in V1 h, and
describe how the isolated chloroplasts can be used to study
chloroplast protein import. The simplicity of the protocol,
and the functional integrity of the isolated chloroplasts, indi-
cate that the isolation method may prove useful in studies of
many di¡erent aspects of chloroplast biology in addition to
protein import.
2. Materials and methods
2.1. Plant growth conditions
Seeds of Arabidopsis thaliana (ecotype Columbia-0) were sterilised
in 70% (v/v) ethanol, 0.05% (v/v) Triton X-100 for 5 min, followed by
10 min in 100% ethanol, and allowed to air-dry in a laminar £ow
hood. Seeds were sown on petri plates (9 cm in diameter; 100^120
seeds/plate) containing Murashige and Skoog salt and vitamin mix-
ture (Sigma), 0.5% (w/v) sucrose, and 0.6% (w/v) bactoagar. Each
plate was sealed with Leukopor tape (Beiersdorf), incubated at 4‡C
for 48 h to break seed dormancy, and thereafter grown at 20‡C in
120 Wmol/m
2
/s white light with a long day cycle (16-h light/8-h dark)
for exactly 10, 14 or 28 d in a tissue culture chamber (Percival).
2.2. Isolation of Arabidopsis chloroplasts
For each isolation procedure, 25^40 petri plates of 10-, 14- or 28-d-
old plants were used; with 10-d-old plants, this is equivalent to
V2500^4000 individuals or V7.5^12 g tissue. During the isolation
procedure the plant material was kept at 4‡C. Plants were homoge-
nised for 3^4 s using a polytron (Kinematica PT20) with a small rotor
(13-mm diameter, V40% max speed) in 20 ml isolation bu¡er (0.3 M
sorbitol, 5 mM MgCl
2
, 5 mM EGTA, 5 mM EDTA, 20 mM HEPES/
KOH, pH 8.0, 10 mM NaHCO
3
) in a 50-ml beaker. The homogenate
was ¢ltered through a double layer of Miracloth (Calbiochem). The
debris retained in the Miracloth was returned to the beaker with 20 ml
fresh isolation bu¡er and the homogenisation was repeated. This ho-
mogenisation procedure was carried out ¢ve times in total. The com-
bined homogenate was centrifuged at 1000Ug
max
for 5 min (brake on)
and the pellet was resuspended in V500 Wl isolation bu¡er.
The resuspended chloroplasts were loaded onto a two-step Percoll
gradient or a linear Percoll gradient. Two-step gradients were pre-
pared in 20-ml Corex tubes (DuPont), and linear gradients were pre-
pared in 30-ml Nalgene tubes (Fisher). Two-step gradients consisted
of a bottom layer (3 ml) comprising 2.55 ml Percoll solution (95%
(w/v) Percoll, 3% (w/v) PEG 6000, 1% (w/v) Ficoll, 1% (w/v) BSA)
plus 0.45 ml gradient mixture (25 mM HEPES^NaOH, pH 8.0, 10 mM
EDTA, 5% (w/v) sorbitol) and a top layer (7 ml) comprising 2.94 ml
Percoll solution plus 4.06 ml gradient mixture. Two-step gradients
were centrifuged in a swing-out rotor at 1500Ug
max
for 10 min (brake
o¡). Linear gradients (26 ml) consisted of 13 ml Percoll, 13 ml 2U
isolation bu¡er and 5 mg glutathione, and were pre-centrifuged in a
¢xed angle rotor at 43 000Ug
max
for 30 min (brake o¡). After adding
the chloroplasts, linear gradients were centrifuged in a swing-out rotor
at 7800Ug
max
for 10 min (brake o¡).
The band that appeared between the phases when using two-step
gradients, and the lower band that appeared when using linear gra-
dients, contained intact chloroplasts. The upper band in each case
contained broken chloroplasts. Broken chloroplasts were removed
and discarded, and then the intact chloroplasts were recovered using
0014-5793 / 02 / $22.00 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S0014-5793(02)03342-2
*Corresponding author. Fax: (44)-116-252 3330.
E-mail address: rpj3@le.ac.uk (P. Jarvis).
FEBS 26550 30-9-02
FEBS 26550 FEBS Letters 529 (2002) 215^220
a 1-ml Gilson pipette tip, cut at the end. Next, 30 ml HEPES^
MgSO
4
^sorbitol (HMS) bu¡er (50 mM HEPES, 3 mM MgSO
4
,
0.3 M sorbitol), or, where speci¢cally stated, 30 ml HEPES^sorbitol
(HS) bu¡er (50 mM HEPES, 0.3 M sorbitol), was added to the chlo-
roplasts and the tube was inverted carefully one time to wash the
Percoll. The chloroplasts were centrifuged in a swing-out rotor at
1000Ug
max
for 5 min (brake on). The supernatant was decanted
and discarded, and the pellet was resuspended in the residual HMS
(or HS) bu¡er. Determination of the yield of chloroplasts was carried
out using a haemocytometer, and the intactness of the chloroplasts
was veri¢ed by phase-contrast microscopy (Zeiss) and transmission
electron microscopy [11].
Isolation of chloroplasts from protoplasts was carried out as de-
scribed previously [10], with the following minor changes. The growth
conditions of the plants were as described above. The cellulase con-
centration was reduced two-fold to 2%; this lower cellulase concen-
tration was found to be e¡ective for isolating Arabidopsis chloroplasts
for photosynthetic electron transport studies [12], and did not signi¢-
cantly a¡ect chloroplast yield (Aronsson and Jarvis, unpublished ob-
servation). A two-step Percoll gradient was used in place of the linear
Percoll gradient in some cases.
2.3. Import into Arabidopsis chloroplasts
Template DNA for the in vitro transcription/translation of prepro-
teins was ampli¢ed by PCR from cDNA clones using M13 primers.
Transcription/translation was performed using a wheat germ system
(Promega) containing [
35
S]-methionine and either T7 RNA polymer-
ase (Arabidopsis pSS and pL11) or SP6 RNA polymerase (pea pSS)
according to the manufacturer’s instructions.
Import reactions were carried out in HS bu¡er [13], or HMS bu¡er
containing 20 mM gluconic acid (potassium salt), 10 mM NaHCO
3
and 0.2% (w/v) bovine serum albumin [14]. Unless stated otherwise,
each 150 Wl import assay contained 10
7
chloroplasts (V20 Wg chlo-
rophyll when using wild-type) from 10-d-old plants, 5 mM MgATP,
10 mM methionine, and translation mixture not exceeding 10% of the
total volume.
Import was performed in white light (100 Wmol/m
2
/s) at 25‡C for 5,
10, 15 or 20 min exactly. When necessary, import reactions were di-
vided in two for protease treatment: one half was treated with 100 Wg/
ml thermolysin on ice for 30 min, and the other half was left un-
treated. Thermolysin reactions, and all import reactions that were
not subjected to thermolysin treatment, were stopped by adding an
equal volume of ice-cold HS bu¡er containing 50 mM EDTA, fol-
lowed by a short centrifugation burst (V4 s) in a microfuge. Pellets
were resuspended in 2U denaturation bu¡er [15]. After heating at
95‡C for 1.5 min, samples were resolved on 12.5% or 15% SDS^
PAGE gels [15]; translation mixture equivalent to 10% of that added
to each import reaction was run on each gel for quanti¢cation pur-
poses. Gels were ¢xed in 7% (v/v) acetic acid, coated with NAMP100
£uorescent signal ampli¢er (Amersham), dried, and exposed to X-ray
¢lm or a phosphorimager screen. Quanti¢cation was performed using
ImageQuant software (Molecular Dynamics).
To deplete endogenous ATP, chloroplasts were kept in the dark at
room temperature for 10 min [9,16], or in the dark on ice in the
presence of 6 WM nigericin (Sigma) for 10 min [17]. Small molecules,
including ATP, were removed from the translation products by Se-
phadex G-25 ¢ltration (Pharmacia). MgATP was added to import
reactions, containing dark-adapted chloroplasts and ATP-depleted
preprotein at di¡erent concentrations (50^5000 WM), or was omitted
completely. Reactions were incubated in the dark at 25‡C for 15 min,
and then analysed as described above.
3. Results and discussion
3.1. Isolation of intact chloroplasts from Arabidopsis plants
To minimise the time needed for plant growth, and to allow
the analysis of import-defective mutants during early develop-
ment when their phenotypes are often more severe [5],we
developed a chloroplast isolation protocol that can be used
with 10-d-old Arabidopsis plants. To reduce the time and cost
of the isolation procedure, we based our protocol on estab-
lished methods that do not involve protoplastation [13,14].
During the development of the isolation method, we found
the ratio between the amount of tissue and the volume of
isolation bu¡er to be of great importance. The type of homog-
enisation equipment used was also found to be important, as
was the size and speed of the polytron rotor, and the size of
the homogenisation beaker. The isolation conditions pre-
sented here were found to be optimal. Increasing the size or
speed of the polytron rotor, or using a kitchen blender instead
of the polytron, resulted in a higher proportion of broken
chloroplasts (data not shown). A key feature of the isolation
procedure is the repetition of the homogenisation step, since
this ensures a high yield of intact chloroplasts.
After homogenisation, intact chloroplasts were separated
from damaged chloroplasts by Percoll density gradient centri-
fugation (two-step gradients were used for most experiments).
A typical Percoll gradient showing the distribution of chloro-
plasts between two discrete bands is shown (Fig. 1A). Chloro-
plasts from the lower band were washed and estimated to be
s 85% intact by phase-contrast microscopy. The integrity of
the isolated chloroplasts was also con¢rmed by transmission
electron microscopy, and a typical micrograph is shown
(Fig. 1B). A typical isolation procedure using V2500 plants
(V7.5 g tissue) yielded s 15U10
7
intact chloroplasts (V40 Wg
chlorophyll/g tissue), which was su⁄cient for at least 15 im-
port assays each containing 10
7
chloroplasts; import using
2U10
7
chloroplasts per assay did not signi¢cantly increase
import e⁄ciency (data not shown). The yield of V40 Wg chlo-
Fig. 1. Isolation of intact Arabidopsis chloroplasts. A: Typical two-
step Percoll gradient showing the distribution of chloroplasts be-
tween two bands. The upper band contains broken chloroplasts,
and the lower band contains intact chloroplasts. B : Electron micro-
graph of an intact chloroplast from the lower band of the Percoll
gradient. Size bar indicates 1.0 Wm. C: Import of at-pSS into intact
chloroplasts. After 15 min import reactions, samples were either
treated with thermolysin (+th) or left untreated (3th). TM, transla-
tion mixture; p, preprotein; m, mature.
FEBS 26550 30-9-02
H. Aronsson, P. Jarvis/FEBS Letters 529 (2002) 215^220216
rophyll/g tissue is approximately eight-fold higher than previ-
ously reported yields for direct isolation from Arabidopsis
plants [7,10], but slightly less than the yield reported for iso-
lation from protoplasts [10].
To assess the import competence of the chloroplasts, and to
further con¢rm their integrity, an import assay using Arabi-
dopsis Rubisco small subunit preprotein (at-pSS) was con-
ducted (Fig. 1C). Radiolabelled pSS was imported with high
e⁄ciency (V10% of added preprotein was imported after
15 min), and the imported protein was found to be insensitive
to exogenously applied thermolysin (Fig. 1C). These data
demonstrate that the chloroplasts are highly import-compe-
tent and that their envelope membranes are intact.
3.2. Import using di¡erent preproteins
Several previous studies of Arabidopsis chloroplast protein
import utilised heterologous import systems in which the pre-
protein and the chloroplasts were from di¡erent species
[6,7,9,10]. To determine if the use of heterologous systems
a¡ects import e⁄ciency, we compared the import e⁄ciencies
of at-pSS and pea pSS (ps-pSS) using chloroplasts from 10-d-
old Arabidopsis plants (Fig. 2A). ps-pSS import was at least as
e⁄cient as at-pSS import, suggesting that, as with other spe-
cies [18], there is no strong disadvantage in using a heterolo-
gous import system, at least in the case of pSS. Nevertheless,
the use of homologous import systems is preferable, since
doing so reduces the risk of obtaining spurious results. The
Arabidopsis genome contains multiple genes for many import
apparatus components [19], hinting at the existence of multi-
ple import pathways in plastids, perhaps with di¡erent
precursor recognition speci¢cities [5,8]. In light of this remark-
able complexity, it will be important to work with homolo-
gous import systems to ensure that the results obtained accu-
rately re£ect the in vivo situation.
In the past, import studies utilised pSS more than other
preproteins since it is imported with high e⁄ciency in vitro.
However, the recently discovered complexity of the Arabidop-
sis import apparatus [19] means that it will be important to
conduct future import studies using multiple precursors. We
therefore investigated the competence of isolated Arabidopsis
chloroplasts to import a second Arabidopsis preprotein : the
50S ribosomal subunit precursor, pL11 (Fig. 2B). This pre-
protein was selected since it is non-photosynthetic, whereas
pSS is photosynthetic; comparisons of the import of function-
ally distinct proteins may be important, since photosynthetic
and non-photosynthetic preproteins may follow di¡erent im-
port pathways [8,20]. Import of pL11 was at least as e⁄cient
as pSS import (Fig. 2B, lane 2; V10% of added pL11 was
imported after 15 min), indicating that the method is useful
for analysing precursors other than pSS. Several other Arabi-
dopsis preproteins were also imported with high e⁄ciency
(Aronsson, Kubis and Jarvis, unpublished).
Fig. 2. Import using di¡erent preproteins and at di¡erent develop-
mental stages. A: Comparison of homologous and heterologous im-
port systems. at-pSS (lanes 1^2) and ps-pSS (lanes 3^4) were each
imported (Imp) into Arabidopsis chloroplasts for 20 min. B: Import
at di¡erent developmental stages. Chloroplasts isolated from 10-,
14- and 28-d-old plants were used in import assays with at-pL11;
import was carried out for 15 min in each case.
Fig. 3. Energetic requirements for binding and import. A: ATP-de-
pleted chloroplasts and at-pSS were incubated together for 15 min
in darkness. Reactions were carried out at di¡erent ATP concentra-
tions as indicated. B: As A, except that the chloroplasts were de-
pleted of ATP in the presence of nigericin. C: Bound preprotein
cannot be removed by washing. Chloroplasts were incubated with
at-pSS in the presence of 5 mM ATP in the light for 15 min, and
were either analysed immediately (0) or washed once (1U) or twice
(2U) in HS bu¡er prior to analysis.
FEBS 26550 30-9-02
H. Aronsson, P. Jarvis/FEBS Letters 529 (2002) 215^220 217
3.3. Import at di¡erent developmental stages
The isolation method was optimised for use with 10-d-old
plants for reasons given earlier. Because it has been demon-
strated that the import capabilities of pea and wheat chloro-
plasts can change signi¢cantly during development [21], and
that the e¡ects of Arabidopsis translocon mutations can
change through development [5], it will be important to be
able to study import at di¡erent developmental stages. To
assess the suitability of the method for studying import at
later stages, we isolated chloroplasts from 10-, 14- and 28-d-
old plants and compared their import capabilities using pL11
(Fig. 2B). In each case, V10% of the added pL11 was im-
ported after 15 min, demonstrating that the protocol is indeed
suitable for use with older plants. Interestingly, the import
capabilities of chloroplasts from plants of di¡erent ages did
not appear to di¡er signi¢cantly (Fig. 2B). A similar observa-
tion was made previously after comparing pSS import into
chloroplasts isolated from 2-, 3-, 4- and 5-week-old Arabidop-
sis plants [10]. These results may indicate fundamental regu-
latory di¡erences between the import systems of Arabidopsis
and pea or wheat, or re£ect the fact that whole plants were
used to make the Arabidopsis import capacity comparisons,
whereas developmentally di¡erent tissues were used in the pea
and wheat experiments.
3.4. Energetic requirements for import
Protein import into pea chloroplasts can be divided into
three distinct stages based on energetic requirements: en-
ergy-independent binding (ATP not required), import inter-
mediate formation ( 6 100 WM ATP) and complete transloca-
tion ( s 100 WM ATP) [16,22]. To assess the energetic
requirements for protein import into Arabidopsis chloroplasts
isolated using our method, import experiments containing dif-
ferent concentrations of ATP were conducted. As in pea, pSS
bound to chloroplasts in the absence of ATP (Fig. 3A, lane 2),
complete translocation only occurred at ATP concentrations
exceeding 50 WM(Fig. 3A, lanes 4^6), and translocation was
most e⁄cient at mM ATP concentrations (Fig. 3A, lane 6).
However, import was observed to di¡er from pea import in
two ways: (1) low ATP concentrations (50 WM) did not sig-
ni¢cantly stimulate preprotein binding (Fig. 3A, lane 3); (2)
relatively high levels of preprotein remained bound to the
chloroplast surface at all ATP concentrations (Fig. 3A). To
determine if the former discrepancy was due to residual ATP
within the chloroplasts, we repeated the experiment using
chloroplasts depleted of ATP in the presence of the iono-
phore, nigericin [17]. This time, stimulated preprotein binding
Fig. 4. Comparing import in di¡erent genotypes and using di¡erent
import bu¡ers. A: at-pSS was imported into chloroplasts from dgd1
(d) and wild-type (wt) Arabidopsis plants for 5 and 15 min as indi-
cated. B: at-pSS was imported into chloroplasts using HMS bu¡er
or HS bu¡er for 5, 10 and 20 min as indicated. C: Radioactivity as-
sociated with each mature band in B was quanti¢ed and expressed
as a percentage of the total pSS-associated radioactivity added to
each reaction.
Fig. 5. Comparison of chloroplasts isolated using the direct and
protoplast methods. Arabidopsis pSS (A) and pL11 (B) preproteins
were imported into chloroplasts isolated from 14-d-old Arabidopsis
plants by either the direct method (D) or the protoplast method (P)
as indicated. Chloroplasts were puri¢ed using either a linear or a
two-step Percoll gradient. Import was carried out for 15 min.
FEBS 26550 30-9-02
H. Aronsson, P. Jarvis/FEBS Letters 529 (2002) 215^220218
at low ATP concentrations was clearly observed (Fig. 3B,
lanes 3^4), indicating that more stringent ATP-depletion con-
ditions are required for Arabidopsis than pea [16]. High levels
of preprotein binding have been observed previously [6,10],
and this therefore appears to be a general feature of Arabi-
dopsis chloroplasts. To assess the strength of association be-
tween the bound preprotein and the chloroplast surface, we
attempted to remove the preprotein by persistent washing in
HS bu¡er (Fig. 3C). Even after two washes, none of the
bound preprotein could be removed, indicating that it is
tightly bound to the chloroplast surface. High levels of pre-
protein binding may therefore re£ect some speci¢c property of
the Arabidopsis import apparatus.
3.5. Studying import-defective mutants and quantifying import
rates
The ability to characterise import in a range of di¡erent
genotypes is an essential requirement for chloroplast import
research using the Arabidopsis model system. We therefore
tested our protocol using several di¡erent chloroplast biogen-
esis mutants, including ppi1, dgd1 and mgd1 [5,6,23], and
found that it works e⁄ciently in each case. Data generated
using the mutants are intended for other papers, and so only
data on the previously characterised mutant, dgd1, are shown
here (Fig. 4A). The data are consistent with previous results
[6], and con¢rm that dgd1, a mutant de¢cient in the plastidic
galactolipid, digalactosyldiacylglycerol, is an import-defective
mutant, clearly demonstrating the usefulness of the procedure
for studying import defects in Arabidopsis mutants.
When characterising import-defective mutants, or when
studying import at di¡erent developmental stages or under
di¡erent environmental conditions, it may be necessary to
make comparisons of import e⁄ciency between samples.
The best way to do this is to measure the import rate in
each sample. Import rates decline during extended import re-
actions (data not shown), and so time-course experiments
must be conducted during the ¢rst 20 min. We used time-
course experiments to compare two di¡erent import bu¡ers:
HMS bu¡er [14] and HS bu¡er [13] (Fig. 4B). Quanti¢cation
of the data demonstrates that import using HMS bu¡er is
more e⁄cient than import using HS bu¡er (Fig. 4C), and so
HMS bu¡er was used for all our import experiments.
3.6. Comparison with chloroplasts isolated from protoplasts
The isolation of Arabidopsis chloroplasts from protoplasts
has been described [12]. Recently, a modi¢cation of this pro-
tocol was used to isolate Arabidopsis chloroplasts for import
studies (referred to as the protoplast method) [10]. Direct
homogenisation methods have been used more commonly
for chloroplast isolation, have been in use for many years
[3,24], and have been shown to work well with several di¡er-
ent species [20,21,25]. There are two major di¡erences between
the protoplast method and the direct homogenisation method
described here (referred to as the direct method). First, the
time between tissue harvesting and the beginning of the chlo-
roplast isolation procedure is 4^5 h with the protoplast meth-
od [10] ; with the direct method, chloroplast isolation starts
immediately after tissue harvesting. Second, the use of the cell
wall-degrading enzymes (cellulase and pectinase) for proto-
plastation adds an extra cost to each isolation, making the
protoplast method V20 times more expensive than the direct
method. To determine if these time and cost disadvantages are
compensated for by higher chloroplast yields or improved
import-competence, we compared the two isolation proce-
dures directly. To make the results directly comparable with
those described previously [10], 14-d-old plants were used.
We used two di¡erent types of Percoll gradient to isolate
intact chloroplasts from homogenates prepared using each
method: a two-step gradient and a linear gradient. Using
both types of gradient, we obtained a lower band containing
intact chloroplasts with each method (data not shown). The
di¡erent Percoll gradients used did not produce signi¢cantly
di¡erent results. Chloroplast yield was found to be similar,
although in some cases it was slightly higher using the proto-
plast method; in our hands, maximal yields using the proto-
plast method were 50^60 Wg chlorophyll/g tissue. The import
capabilities of chloroplasts from the direct and protoplast
methods, isolated using linear and two-step gradients, were
compared in import assays using pSS and pL11 (Fig. 5).
The results demonstrate that the import of both preproteins
is signi¢cantly more e⁄cient with chloroplasts isolated using
the direct method. In a previous study using the protoplast
method, import e⁄ciencies were also relatively low: only
V3% of the added pSS was imported after 30 min [9]. The
very high import-competence of chloroplasts isolated using
the direct method presumably re£ects their integrity and nor-
mal functionality, indicating that the protocol may be useful
for studying many di¡erent aspects of chloroplast biology in
addition to protein import.
Acknowledgements: We thank Ramesh Patel and Anthony Wardle for
excellent technical assistance, Stefan Hyman for transmission electron
microscopy, and Amy Baldwin, Jocelyn Be
¤
dard, Penelope Dudley and
Sybille Kubis for verifying the usefulness of the method. cDNAs were
kindly provided by Dario Leister (pL11), Bernd Reiss (pea pSS) and
the Arabidopsis Biological Resource Center (pSS ; EST 188D4T7). We
thank Sabina Kovacheva, Sybille Kubis and David Stevenson for
their comments on the manuscript. This work was supported by a
Wenner^Gren Foundation Fellowship (H.A.) and by the Royal Soci-
ety Rosenheim Fellowship and BBSRC grants 91/C12976 and 91/
P12928 (P.J.).
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    • "It has been shown that pea roots contain a higher phenol oxidase activity than do pea leaves (Henry et al., 1979) and it is also possible that isolated leucoplasts are more sensitive to oxidative damages than do isolated chloroplasts. We therefore added reducing agents (2 mM ascorbic acid, 0.1 mM DTT, and 1.2 mM glutathione, designated as ADG inFigure 1B) to the homogenization buffer (Schulz et al., 2004; Aronsson and Jarvis, 2011). Indeed after adding the reducing agents, we no longer observed dark-brown leucoplast pellets and the import efficiency of prTic40 further increased (Figure 1B, lanes 5–6). "
    [Show abstract] [Hide abstract] ABSTRACT: Leucoplasts are important organelles for the synthesis and storage of starch, lipids and proteins. However, molecular mechanism of protein import into leucoplasts and how it differs from that of import into chloroplasts remain unknown. We used pea seedlings for both chloroplast and leucoplast isolations to compare within the same species. We further optimized the isolation and import conditions to improve import efficiency and to permit a quantitative comparison between the two plastid types. The authenticity of the import was verified using a mitochondrial precursor protein. Our results show that, when normalized to Toc75, most translocon proteins are less abundant in leucoplasts than in chloroplasts. A precursor shown to prefer the receptor Toc132 indeed had relatively more similar import efficiencies between chloroplasts and leucoplasts compared to precursors that prefer Toc159. Furthermore we found two precursors that exhibited very high import efficiency into leucoplasts. Their transit peptides may be candidates for delivering transgenic proteins into leucoplasts and for analyzing motifs important for leucoplast import.
    Full-text · Article · Sep 2015
    • "In vitro transcription/translation was performed using a coupled TNT system (Promega, Madison, WI, USA) based on rabbit reticulocyte lysate containing [ 35 S]-methionine and T7 RNA polymerase, according to the manufacturer's instructions (Promega ). Using M13 primers, the template DNA for the transcription/translation reactions was amplified by PCR from Arabidopsis cDNA clones for the precursors of Rubisco small subunit 1A and atTic22-III, according to Aronsson and Jarvis [56]. Chloroplast incubations with thermolysin contained 50 mg/ml thermolysin and 300 mM CaCl 2 , and reactions were conducted for 5 min on ice [24]. "
    [Show abstract] [Hide abstract] ABSTRACT: The Tic22 protein was previously identified in pea as a putative component of the chloroplast protein import apparatus. It is a peripheral protein of the inner envelope membrane, residing in the intermembrane space. In Arabidopsis, there are two Tic22 homologues, termed atTic22-III and atTic22-IV, both of which are predicted to localize in chloroplasts. These two proteins defined clades that are conserved in all land plants, which appear to have evolved at a similar rates since their separation .400 million years ago, suggesting functional conservation. The atTIC22-IV gene was expressed several-fold more highly than atTIC22-III, but the genes exhibited similar expression profiles and were expressed throughout development. Knockout mutants lacking atTic22-IV were visibly normal, whereas those lacking atTic22-III exhibited moderate chlorosis. Double mutants lacking both isoforms were more strongly chlorotic, particularly during early development, but were viable and fertile. Double-mutant chloroplasts were small and under-developed relative to those in wild type, and displayed inefficient import of precursor proteins. The data indicate that the two Tic22 isoforms act redundantly in chloroplast protein import, and that their function is non-essential but nonetheless required for normal chloroplast biogenesis, particularly during early plant development. Copyright: ß 2013 Kasmati et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    Full-text · Dataset · May 2013 · PLoS ONE
    • "In vitro transcription/translation was performed using a coupled TNT system (Promega, Madison, WI, USA) based on rabbit reticulocyte lysate containing [ 35 S]-methionine and T7 RNA polymerase, according to the manufacturer's instructions (Promega ). Using M13 primers, the template DNA for the transcription/translation reactions was amplified by PCR from Arabidopsis cDNA clones for the precursors of Rubisco small subunit 1A and atTic22-III, according to Aronsson and Jarvis [56]. Chloroplast incubations with thermolysin contained 50 mg/ml thermolysin and 300 mM CaCl 2 , and reactions were conducted for 5 min on ice [24]. "
    [Show abstract] [Hide abstract] ABSTRACT: The Tic22 protein was previously identified in pea as a putative component of the chloroplast protein import apparatus. It is a peripheral protein of the inner envelope membrane, residing in the intermembrane space. In Arabidopsis, there are two Tic22 homologues, termed atTic22-III and atTic22-IV, both of which are predicted to localize in chloroplasts. These two proteins defined clades that are conserved in all land plants, which appear to have evolved at a similar rates since their separation >400 million years ago, suggesting functional conservation. The atTIC22-IV gene was expressed several-fold more highly than atTIC22-III, but the genes exhibited similar expression profiles and were expressed throughout development. Knockout mutants lacking atTic22-IV were visibly normal, whereas those lacking atTic22-III exhibited moderate chlorosis. Double mutants lacking both isoforms were more strongly chlorotic, particularly during early development, but were viable and fertile. Double-mutant chloroplasts were small and under-developed relative to those in wild type, and displayed inefficient import of precursor proteins. The data indicate that the two Tic22 isoforms act redundantly in chloroplast protein import, and that their function is non-essential but nonetheless required for normal chloroplast biogenesis, particularly during early plant development.
    Full-text · Article · May 2013
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