Download Chloroplast Tubules Visualized in Transplastomic Plants Expressing

Plant CellPhysiol. 41(3): 367-371 (2000)
JSPP © 2000
Short Communication
Chloroplast Tubules Visualized in Transplastomic Plants Expressing Green
Fluorescent Protein
Takashi Shiina1, Keiko Hayashi2, Nao Ishii, Kazuya Morikawa and Yoshinori Toyoshima3
Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto, 606-8501 Japan
A fusion between the plastid psbA promoter and the
greenfluorescentprotein gene igfp) was introduced into the
tobacco chloroplast genome by stable plastid transformation. GFP was synthesized actively and exclusively in
the chloroplasts. Tubular projections filled with GFP but
containing no chlorophyll were visualized for the first time
in chloroplasts of these transplastomic plants. Occasionally, the tubules connect chloroplasts with each other,
suggesting the possibility of the exchange of endogenous
proteins. However, the fusion of protoplasts between the
transplastomic and wild-type plants showed that such
chloroplast connections might be rare in mesophyll protoplasts.
tween chloroplasts were reported in higher plant cells decades ago (Weier and Thomson 1962, Wildman et al. 1962,
Vesk et al. 1965). However, chloroplast interconnections
were not recognized in general and neither their function
nor their origin has been clarified.
Green fluorescent protein (GFP) is a powerful tool to
visualize subcellular structures in living cells (Rizzuto et
al. 1995, Kohler et al. 1997a, b). Recently, tubular connections between chloroplasts were visualized in transgenic
plants in which translational fusion of the chloroplast
transit signal and GFP was expressed in cytoplasm and
targeted into the stroma of chloroplasts (Kohler et al.
1997b). Similar connections were also observed in etioplasts (Tirlapur et al. 1999). These tubular connections are
likely to be involved in the exchange of endogenous proteins between chloroplasts. When GFP was selectively
photobleached in one of the two connected chloroplasts, a
rapid recovery of GFP fluorescence in the bleached chloroplast was observed, indicating the flow of GFP through
the tubules (Kohler et al. 1997b). The tubules may also
have an important role as a route for the flow of genomic
information between chloroplasts to establish genetic homoplasmicity. However, it is difficult to estimate the exchange rates of macromolecules between chloroplasts by
using nuclear transformants.
As mentioned above, the chloroplast tubular connections have been so far visualized in nuclear transgenic
plants (Kohler et al. 1997b, Tirlapur et al. 1999). In this
paper, we visualized chloroplast tubules for the first time in
stable transplastomic plants expressing GFP via the plastid
genome. The psbA promoter is recognized by eubacterial
multi-subunit plastid RNA polymerase (PEP). It comprises
the well conserved —35 and —10 elements spaced 18
nucleotides apart and exhibits the highest transcription
rates in chloroplasts (Rapp et al. 1992). Previously, we
reported that the —35 element was required for transcription initiation from the psbA promoter in younger leaves,
but not in developed leaves in wheat (Satoh et al. 1999). In
order to examine the role of the —35 element of the psbA
promoter in dicots, we constructed tranplastomic tobacco
plants with the green fluorescent protein gene (gfp) expressed from psbA promoter derivatives (Hayashi et al.,
in preparation). By using the transplastomic plant that
expresses GFP from the wild-type psbA promoter core
Key words: Chloroplast — Chloroplast transformation —
Chloroplast tubule — Green fluorescent protein —
Nicotiana tobaccum — psbA promoter.
Plastids are semiautonomous organelles that contain
their own genome and are reproduced by fission. The surface of plastids is bounded by an envelope consisting of
two membranes. Most macromolecules, such as proteins
and nucleic acids are not able to penetrate the inner membrane of the envelope. Thus, chloroplasts in general have
been considered as discrete organelles. On the other hand,
fusions and/or connections between chloroplasts have
been observed in some algae. It has been reported that
Euglena chloroplasts connect to each other by many
bridges prior to chloroplast division (Ehara et al. 1990). In
Acetabularia, membrane tubules emanating from chloroplasts connect the chloroplasts with each other (Menzel
1994). In addition, similar images of interconnections beAbbreviations: GFP, Green fluorescent protein; MES, 2-(NMorpholino)ethanesulfonic Acid; MS medium, Murashige and
Skoog medium; SD signal, Shine-Dalgarno signal.
1
Present address: Faculty of Human Environment, Kyoto Prefectural University Hangi-cho, Shimogamo, Sakyo-ku, Kyoto,
606-8522 Japan.
2
Present address: Hokuriku National Agricultural Experiment
Station, Inada 1-2-1, Joetu, Niigata, 943-0193 Japan.
3
Corresponding author: Fax: +81-75-753-6577, E-mail: ytoyoshi
@ip. media. kyoto-u. ac .j p.
367
Tubular connections between chloroplasts
368
(KH6), we could visualize tubular projections emanating
from chloroplasts. This transgenic line offers an opportunity for estimating rates of protein transport among
chloroplasts via tubular connections by examining the distribution of GFP among chloroplasts following protoplast
fusion.
In wheat chloroplasts, the psbA promoter core is
responsible for maintaining the high transcription rate
(Satoh et al. 1999). Thus, the tobacco psbA promoter core
was cloned in upstream of the gfp cassette consisting of a
SD signal; gfp and Trpsl6 (Fig. 1). The gfp gene was
modified by introducing mutations into a chromophore
region (Phe^Ser65 to Leu^Thr65). The chimeric gfp gene
was cloned into a pKH5 plastid transformation vector
designed following pRV112A (Zoubenko et al. 1994). The
pKH5 contains the aadA cassette (P16SrDNA::aadA::
TpsbA) excised from a plasmid pCT08 (Shikanai et al.
1998). Details of the plasmid construction will be described
elsewhere (Hayashi et al., in preparation). Transformation
of tobacco (Nicotiana tabacum cv. Xanthi) plastids was
carried out using a Biorad PDSlOOOHe Biolistic gun at
1100 PSI following the method described by Svab and
Maliga (1993). Transgenic shoots were selected on agarsolidified MS medium (Murashige and Skoog 1962) containing sucrose (3%), 1-naphthaleneacetic acid (0.1 mg
liter"1), A^-benzyladenine (1 mg liter" 1 ), thiamine (1 mg
liter" 1 ), inositol (100 mg liter"1) and spectinomycin dihydrochloride (500 mg liter"1) as described by Svab et al.
(1990). Transgenic plants rooted on MS agar medium
containing sucrose (3%) or soil were used for observation
with microscopes.
Confocal imaging of cells was performed using a
confocal laser-scanning microscope (CLSM) (MRC600 Z
or /^Radiance BioRad U.S.A.). The GFP was excited with
the 488 nm of blue line and the chlorophyll was excited
with the 568 nm line. These fluorescence images were col-
TpsbA
I
16SrDNA
| |"|
tmV
lected in green or red channels, respectively. Serial optical
sections were obtained at 0.5 //m intervals and processed
using NIH image and PhotoShop (Adobe Systems Inc).
GFP fluorescence was detected in transplastomic
plants, while control wild-type plants did not show GFP
fluorescence (data not shown). Fig. 2 shows fluorescence
images of mesophyll and guard cells in a transplastomic
tobacco plant. As expected, GFP fluorescence was localized in chloroplasts, indicating that GFP was synthesized
and accumulated in chloroplasts and did not move to other
organelles. Thin tubular projections emanating from chloroplasts were detected, but their avandance varied from
cell to cell. The diameter of tubules was around 0.5 /um and
their length was up to 10 /um. Sometimes a branching of
tubules could also be observed. No chlorophyll autofluorescence was detected from the tubules, indicating that
thylakoid membranes do not extend into them. The shape
Prm PpsbA Trps16
iiinili
70B rps7/12
—*•
1 2kb
5bp
Fig. 1 A gfp cassette driven by psbA promoter. PpsbA indicates
the position of the psbA promoter; gfp encodes the modified
green fluorescent protein; Trpsl6 refers to the 3-UTR of the rpsl6
ribosomal protein gene; Prrn indicates the position of the
16SrRNA promoter; aadA encodes aminoglycocide 3 "-adenylyltransferase; TpsbA refers to the 3'-UTR of the psbA gene. The
psbA promoter core was connected with a gfp gene via a linker
containing a synthesized SD signal.
Fig. 2 Images of GFP (a, d) and chlorophyll (c, f) fluorescence
taken with a laser confocal microscope (CLSM). (a-c) are projections of 85 optical sections of the same mesophyll cells; (d-f)
are projections of 34 optical sections of a guard cell. Panels b and
e show merged images of both GFP and chlorophyll fluorescence.
All optical sections were taken at 0.5 ^m intervals by CLSM.
Chloroplast tubules are indicated by arrows. Tubules connecting
chloroplasts are indicated by red arrows. Scale bars, 10 ^m.
Tubular connections between chloroplasts
and length of the tubules changed dynamically in the living
cell. They wave, shrink and extend continuously. In some
cells, tubules filled with GFP connect two or more chloroplasts (Fig. 2b).
It is expected that such connections between chloroplasts would contribute to keep the concentration of inorganic ions, metabolic intermediates, proteins and enzymes uniform among several chloroplasts in the same cell.
Transplastomic plants in which GFP was exclusively synthesized in chloroplasts enable us to estimate the rate of
GFP transfer among chloroplasts by fusioning protoplasts
between the transplastomic and wild-type plants. We isolated protoplasts from mesophyll cells of both plants
by treatment with a solution containing 0.5 M mannitol,
2.5 mM MES (pH 5.7), 1% Cellulase Onozuka and 0.5%
Macerozyme at 25°C in the dark for I d . Wild-type protoplasts were mixed with protoplasts from the transplastomic plants and polyethylene glycol-induced fusion
was carried out. The fused cells were incubated at 25°C in
the light. At first, the two types of chloroplast with and
without GFP fluorescence coexisted at the ratio of one to
one in most of the fused cells. A rapid flow of GFP through
the tubular connections between chloroplasts has been
demonstrated by selective photobleaching of individual
chloroplasts (Kohler et al. 1997b, Tirlapur et al. 1999). If
tubular connections between chloroplasts were so frequent
that all chloroplasts connected with each other via tubules,
GFP fluorescence would distribute in all fused cell chloroplasts. Contrary to this, we found that the fraction of
chloroplasts exhibiting GFP fluorescence in the fused
cells did not increase significantly up to 3 d (Fig. 3). This
strongly suggests that chloroplast connections are rare in
most mesophyll protoplasts and exchange of endogenous
proteins between chloroplasts does not occur frequently.
One may assume that the tubular connections observed in
mesophyll cells of leaves disappeared during protoplast
Green channel
369
Merged image
Red channel
Fig. 3 Fluorescent image of fused protoplasts with a conventional fluorescent microscope, (a) GFP fluorescence; (c) chlorophyll fluorescence; (b) merged image of both GFP and chlorophyll
fluorescence. Images were taken 2 d after the fusion of protoplasts.
preparation due to a loss of turgor pressure or change of
light or hormone conditions. However, the following observations provided further support to the above conclusion. When transient expression of GFP in chloroplasts
was examined two days after the introduction of the gfp
gene by bombardment, GFP fluorescence was usually detected in only a single chloroplast per cell (data not shown).
The similar observation has been reported by Hibberd et
al. (1998). GFP did not distribute to all chloroplasts in a
few days following microinjection of the gfp gene into a
single chloroplast in a living mesophyll cell (Knoblauch et
al. 1999). Furthermore, Khan and Maliga (1999) reported
that some plastids express GFP whereas others do not in
the same cell in chimeric transplastomic sectors. From
these observations, it may be concluded that exchange of
endogenous proteins between chloroplasts is a rare event in
most leaf cells.
However, it may occur actively at some specific cell
stages or in some special cells. In facts, the tubules were
more frequently observed in mesophyll cells of the matured
leaves than those of the etiolated cotyledons in tobacco
(Fig. 4). Furthermore, we could observe well-developed
Fig. 4 Images of GFP fluorescence of mesophyll cells of the matured leaves (a) and those of the etiolated cotyledons (b) taken with
CLSM. All images are single optical sections taken at 0.5 ^m intervals by CLSM and merged images of both GFP and chlorophyll
fluorescence are shown. In etioplasts, GFP fluorescence overwhelms red fluorescence of chlorophyll. Chloroplast tubules are indicated
by arrowheads. Scale bar, 10 fxm.
Tubular connections between chloroplasts
370
Green channel
Red channel
Fig. 5 Images of GFPfluorescenceof a trichome cell taken with
CLSM. (a) GFPfluorescence;(b) chlorophyllfluorescence.All
images are projections of 6 optical sections taken at 0.5 /xm intervals by CLSM. The cell margin is indicated by dot lines. Scale
bar,
chloroplast tubules in mesophyll cells adjacent to the epidermal cells in some leaves (Fig. 4a). These observations
suggest that chloroplast tubules may be regulated developmentally and/or tissue-specifically and have functions related to photosynthesis and the intracellular signal transduction. Previously, the presence of tubular connections
between etioplasts has been demonstrated in stomatal
guard cells of Arabidopsis (Tirlapur et al. 1999). The chloroplast tubules may be regulated in a different manner
during chloroplast development between tobacco and Arabisopsis. Recently, Kohler and Hanson (2000) reported
that the appearance of the plastid tubules (stromules) are
abundant in root cells. However, we could not observe
them due to the low level GFP expression from the leafspecific psbA promoter in root cells. Further study is
required to understand the developmental regulation of
chloroplast tubules. In addition, we found that chloroplast
tubules were most abundant in trichome cells of tobacco
leaves (Fig. 5). Several tubules emanate from single chloroplasts and many of them end at a near cell membrane.
They might be involved in transducing membrane signals
to the chloroplasts or acquiring molecules which are localized in some special compartments in the cell (Tilapur et al.
1999). Further study is needed to characterize the protein
transport through the tubular connections between chloroplasts.
It would be interesting to know whether chloroplast
genome or genetic information is exchangeable through the
tubules. There are more than 10 plastids in a leaf cell. As
described above, in transient expression of GFP in chloroplasts, GFP fluorescence was usually detected in only a
single chloroplast in a cell after bombardment. However,
in stable plastid transformation all the plastid genome copies are uniformly altered. Transformed and non-transformed plastids may sort out during cultivation of the cells
on a selective medium. Alternatively, it is expected that the
introduced gfp gene may pass to other plastids through
chloroplast tubules during this process. In addition, intergenomic recombination between chloroplast genomes has
also been reported in some somatic hybrids in higher plants
(Medgyesy et al. 1985, Fejes et al. 1990, Baldev et al. 1998).
The chloroplast tubules may have an important role in
recombination of the chloroplast genome and establishing
homoplasmicity.
The chloroplast tubules are reminiscent of bacterial
pilli. It is interesting to note that the appearance of the
tubular connections is coordinated with a cycle of plastid
division in Euglena (Ehara 1990). Interconnections of chloroplasts have been observed in Euglena (Ehara 1990),
Acetabularia (Menzel 1994) and several higher plants
(Koler et al. 1997b, Tirlapur et al. 1999). The evolutional
diversity of the chloroplast tubules also supports the
physiological importance of the interconnection between
chloroplasts.
We thank Dr. T. Shikanai (Nara Institute of Science and
Technology, Japan) for providing the plasmid pCT08, and Drs. S.
Tsukita and A. Nagafuchi (Graduate School of Medicine, Kyoto
University, Japan) for access to a confocal laser microscope.
Thanks are also due to Dr. E.O. Hideg (Biological Reserach
Center of the Hungarian Academy of Sciences, Hungary) for
critical reading of the manuscript. This work was supported partly
by a Grant-in-Aid for Scientific Research in Priority Areas (no.
11151218) to YT and Grants-in-Aid for Scientific research (no.
10640629 and 10309008) to TS from the Ministry of Education,
Science and Culture of Japan; by a grant from the Research for
the Future program, Japan Society for the Promotion of Science
(JSPS-RFTF96L00604) to TS. KM is a Research Fellow of the
Japan Society for the promotion of Science.
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(Received September 27, 1999; Accepted December 21, 1999)