An approach using leaf tissue that has naturally lost the ability to photosynthesize is ideal for
studying the expression and regulation of genes related to photosynthesis [6,19]. Genes that di-
rectly or secondarily affect chloroplast form and function have been identified in variegated mu-
tants [2,3,8,10,16]. Therefore, the variegated areas of mutants are extremely useful for studying alter-
ations of photosynthetic genes [7] and they are an excellent starting point to dissect the poorly under-
stood pathways of communication between the nuclear-cytoplasmic, chloroplast, and mitochon-
drial genetic systems [20,21]. In this study, we present the genetic, histological, and molecular
characteristics of a variegated tobacco phenotype, which is a periclinal chimera that has undeveloped
chloroplasts in some of the leaf mesophyll cell layers.
2. Materials and methods
2
.
1
. Plant materials The ovaries of tobacco Nicotiana tabacum L.
cv. BY-4 containing embryos 24 – 96 h after self- pollination were irradiated with a
14
N beam 135A MeV accelerated by the Riken Ring Cyclotron
with a dose range of 5 – 200 gray. The linear energy transfer of the nitrogen ions corresponded
to 28.5 keVmm [22]. One month after irradiation, seeds of the M
1
generation were harvested. A variegated plant was found in an M
2
population derived from self-pollinated M
1
plants that had been irradiated with 50 gray of a N-ion beam 84 h
after pollination. The M
2
seeds were germinated in half-strength Murashige and Skoog MS [23]
medium containing 2.0 sucrose and 0.8 agar at 25°C under continuous light 80 mmol quanta m
− 2
s
− 1
. Two weeks later, the variegated plant was transferred into a plant box to be grown under the
same condition. After 2 months, the plant was planted in a plastic pot containing soil, and was
placed in a greenhouse under 18-h photoperiod 100 mmol quanta m
− 2
s
− 1
at 25°C. A fully expanded upper leaf of the plant was harvested
monthly to be used in biochemical experiments. The variegated plant was permitted to carry flower
at a 6 month-old stage to achieve for self-pollina- tion analysis.
2
.
2
. Chlorophyll estimation The chlorophyll content was determined using a
modified version of the procedure of Aron [24]. To determine the chlorophyll content, 0.3 g fresh
weight of leaves from white and green areas of a variegated plant were homogenized in 80 ace-
tone and centrifuged at 9000 × g for 10 min. The clear supernatant was then diluted to 1:80 in 80
vv acetone, and the absorption spectra were determined at 645 and 663 nm.
2
.
3
. Genetic analysis The M
2
variegated plant was derived from M
1
line S201 [25]. In M
2
progeny of the S201 self-pol- lination, the segregation ratio among green, white
and variegated was 2588:587:1 and reciprocal crosses of the S201 line with wild-type resulted in
all F
1
progeny having the green plants. The mode of inheritance of the variegated phenotype was
determined by self-pollination of the variegated mutant, and by reciprocal crosses between the
mutant and wild-type plants. Cross-pollination be- tween the mutant line and wild-type plants was
achieved with emasculated flowers, which were covered with paper bags to prevent contamination
with foreign pollen. F
1
seeds were harvested and planted as described above. One month after sow-
ing, the number of white and green seedlings was recorded. F
1
-green plants were grown to maturity in the greenhouse to obtain F
2
seeds.
2
.
4
. Microscopic obser6ation Pieces of leaves were fixed in 2 glutaraldehyde
dissolved in 20 mM sodium cacodylate buffer pH 7.2 for 16 h at 4°C, dehydrated though an ethanol
series, and then embedded in Technovit 7100 resin Kulzer and Co., GmbH, Wehrheim, Germany as
described previously [26]. The samples were sec- tioned 0.6 mm thick with a glass knife on a
Sorvall MT-2B Ultra Microtome DuPont, Dela- ware, USA and dried on cover slips. They were
stained with 100 mMml DiOC6 3,3-dihexylox- acarbocyanine iodide in ethanol for 30 s at room
temperature, and washed in 50 ethanol for 10 s and then in distilled water for 10 s. The samples
were then observed with an Olympus IX70 fluores- cence microscope. Photographs were taken on 35
mm Ektachrome DYNA EX 400 color film Ko- dak, Rochester, USA.
2
.
5
. Electron microscopy The samples were fixed in 3 glutaraldehyde,
which was buffered with 20 mM sodium cacody- late at pH 7.0 for 6 h at 4°C, and washed with the
same buffer for 16 h at 4°C. Then they were post-fixed with 2 osmium tetroxide in 20 mM
cacodylate buffer pH 7.0 for 6 h at 4°C. The fixed samples were run through an alcohol series
and embedded in Spurr’s resin. Ultrathin sections were cut with a diamond knife on an ULTRA-
CUT UCT ultramicrotome Leica, Wien, Austria, transferred to Formvar-coated grids. They were
double-stained with 1 uranyl acetate for 15 min at 37°C and with lead citrate solution for 10 min
at room temperature. After washing with distilled water, the samples were observed with a JEM-
2000 FX II electron microscope Jeol, Tokyo, Japan.
2
.
6
. RNA gel blot analysis Young leaves were frozen in liquid nitrogen and
total RNA was prepared according to the method of Sambrook et al. [27] with minor modifications.
Total RNA 3 mg was electrophoresed on a formaldehyde denaturing 1.5 agarose gel in 1 ×
MOPS buffer 20 mM MOPS-KOH, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA and then
blotted onto a GeneScreen Plus membrane Du- Pont, using standard protocols [27].
The clones of rbcL which encoded the large subunit of ribulose-1,5-bisphosphate carboxylase;
and 16S rDNA which encoded the plastid 16S rRNA were gift of M. Sugiura and M. Sugita in
Nagoya Univ. Tobacco psbA which encoded the D1 subunit of the photosystem II reaction center
fragments of tobacco GeneBank Accession No. S54304, cab which encoded a major chlorophyll
ab binding protein Cab21 of tobacco GeneBank Accession No. X52743, rbcS which encoded the
small subunit of ribulose-1,5-bisphosphate car- boxylase of tobacco GeneBank Accession No.
M36685 and cytoplasmic 25S rRNA of tobacco GeneBank Accession No. X76056 were amplified
by polymerase chain reaction PCR using Expand High
Fidelity PCR
system Boehringer
Mannheim from purified tobacco chloroplast DNA and nuclear genomic DNA using two sets of
primer, 5-TTATCCATTTGTAGATGGAGCTT- CGATC-3 and 5-ATGACTGCAATTTTAGA-
GAGACGCGAA-3 for
psbA, and
5-ATG- GTACGGCCCAGACCGTGTTAAGTAC-3 and
5-TCACTTTCCGGGGACAAAGTTTGTGGC- G-3 for cab, and 5-ATGGCTTCCTCAGTT-
CTTTCCTCTGCAG-3 and
5-TTAGTAGC- CTTCTGGCTTGTAGGCAATGAA-3 for rbcS,
and 5-GAATTCACCAAGTGTTGGATTGT-3
and 5-ACGAATCGGAGCGACAAAGGG-3
for 25S rRNA. The fragment was cloned into pCR2.1 vector Invitrogen and sequenced by
DNA sequencer model PRISM310, ABI with Dye Terminator Cycle Sequencing Kit ABI. The
clones were labeled with
32
P 6000 CimM by Ready To Go-DNA labeling beads Amersham
and used as hybridization probes. After hybridization for 24 h at 68°C, the filters
were washed in 1 × SSPE [0.15 M NaCl, 0.015 M sodium citrate and 0.1 wv SDS] twice at room
temperature and three times at 68°C, and exposed to X-ray films for autoradiography. The radioac-
tivity of the bands was measured using the BAS 2000 system Fuji Film Co. Ltd., Tokyo, Japan.
Experiments were repeated three times.
3. Results
