Plant growth and evaluation of sexuality
Seeds of monoecious RS-M (MMff) and gynoecious RS-G (MMFF) cucumbers(Cucumis sativus L.) were provided by the Tohoku Seed Co., Ltd.(Utsunomiya, Japan). These two cucumbers are near isogenic lineswith the exception of the F gene. Cucumber seeds were germinatedon wet filter paper in a Petri dish at 28 °C in the darkovernight. The resulting seedlings were transferred to plasticpots (12 cm diameter) containing the soil composite Kureha–Engei–Baido(0.4 g N, 1.9 g P, 0.6 g K kg–1; Kureha Chemical Co.,Tokyo). When the leaf blade of the fifth leaf was approximately2 cm long (defined as the 5-leaf stage), plants were transplantedinto larger pots (27 cm diameter) filled with Kureha–Engei–Baidosoil composite. Plants were adequately watered and suppliedwith fertilizer (0.002% (v/v) Hyponex (Hyponex-Japan, Osaka))during the experiments. Plants were grown in a greenhouse untilanthesis of the flowers on node 25 of the main stems, and thesex of flowers on each node was recorded. Cucumber plants weregrown in two different seasons, mid-September to the end ofNovember in 2003 and mid-August to the end of October in 2004,and they were designated as Experiment I and Experiment II,respectively. There were differences in temperature and day-lengthconditions in the two experiments (Table 1). Flower sex wasdetermined until node 25 or an even higher node at 7 weeks aftergermination. Day-length became shorter from approximately 14h to 12 h during the experiments, and it was approximately 70–80min longer in Experiment II than Experiment I. The average daytemperature and the minimum day temperature in Experiment IIwere 2–9 °C higher than in Experiment I. The differenceswere greater than 4 °C during weeks 2 to 7 in particular,when cucumber plants differentiated flower sex until node 25or higher on the main stems. Because short-day and low nighttemperature conditions are favourable for cucumber plants toproduce female flowers (Nitsch et al., 1952; Ito and Saito, 1960;reviewed in Frankel and Galun, 1977), plants grown in ExperimentI could produce a greater number of female flowers comparedwith those grown in Experiment II as discussed below.
Expression analysis of CS-ACS2 by RT-PCR
Total RNA was isolated from shoot apices, stems, leaves, androots when plants were at the 5-leaf stage. Shoot apices ofapproximately 1 cm long included immature leaves. Stems andleaves were obtained from the third internodes and the fourthleaves, respectively. These samples were excised with a razorblade and immediately frozen in liquid nitrogen and stored at–80 °C prior to the extraction of nucleic acids.
Total RNA was extracted from the cucumber organs with the TRIreagent (Sigma Chemical Co., St Louis). cDNAs synthesis wasperformed with 0.5 µg of total RNA using ReverTra Ace (Toyobo, Osaka) and random hexamers (Takara Bio Inc., Shiga,Japan) in a 20 µl volume. PCR was performed with TaKaRaEx TaqTM (Takara Bio Inc.) using 1 µl of the cDNA solutionand specific primer sets for CS-ACS2 and Cs-actin. The primerswere as follows: CS-ACS2 forward primer, 5'-TTC AAG CGT TGAACT TTC GCG-3', and CS-ACS2 reverse primer, 5'-GAA TGT CTT CGATTG TGG ACC G-3', for CS-ACS2; Cs-actin forward primer, 5'-GACATT CAA TGT GCC TGC TAT-3', and Cs-actin reverse primer, 5'-CATACC GAT GAG AGA TGG CTG-3', for Cs-actin (Yamasaki et al., 2001).The PCR conditions were 94 °C for 30 s, 55 °C for 30s, and 72 °C for 1 min. After 24 cycles for CS-ACS2 and20 cycles for Cs-actin, the PCR products were analysed on a1.0% agarose gel by electrophoresis and visualized by ethidiumbromide staining.
Expression analysis of the CS-ACS2 gene by in situ hybridization
The CS-ACS2 fragment was isolated as described previously (Yamasaki et al., 2001),labelled according to the instructions provided with the DIG(digoxigenin) RNA Labelling and Detection Kit (Roche Diagnostics,Basel), and used as a probe for in situ hybridization.
Shoot apices, approximately 1 cm long, were harvested from cucumberplants when the leaf blade of the fourth or the sixth leaf wasapproximately 2 cm long (defined as the 4-leaf stage and the6-leaf stage, respectively). All flower buds were collectedfrom shoot apices of five plants in Experiment I and three plantsin Experiment II using a stereomicroscope. Each excised flowerbud was put into a vial and fixed with 0.05 M sodium phosphatebuffer (pH 7.2) containing 4% paraformaldehyde and 0.25% glutaraldehyde.Samples were incubated twice with fresh fixative for 5 min eachtime, and subsequently underwent a secondary fixation for 90min. After fixation, the samples were dehydrated with an ethanolseries, which was then replaced by butanol, and finally embeddedin Paraplast Plus (Oxford, Labware, St Louis).
For in situ
hybridization, a previously described method bySaito et al. (2004)
was modified according to Sugiyama et al. (2006)
as follows. Sections (9 µm thick) were placed on silicon-coated
glass slides (Matsunami Glass Ind., Osaka) and incubated at
47 °C overnight. The paraffin was removed from the slides
by immersion in xylene. Sections were rehydrated and incubated
for 10 min in 0.2 N HCl, 100 mM TRIS-HCl (pH 7.5), and 50 mM
EDTA containing 7.5 µg ml–1
proteinase K (Roche
Diagnostics) at 25 °C, and for 20 min in 100 mM triethanolamine
(pH 8.0) and 0.25% acetic anhydrate. After dehydration in an
ethanol series, slides were air-dried before application of
the hybridization solution. Hybridization solution (100 µl)
containing 50% formamide, 5x SSC, 5x Denhardt's solution, 100
µg ml–1Escherichia coli
tRNA, and 500 µg
poly(A) was applied to each slide. After 2 h of prehybridization,
50 µl of the hybridization solution was discarded, and
100 µl hybridization solution containing 3 µg ml–1
of digoxigenin (DIG)-labelled RNA probe was added to each slide.
These slides were incubated in a humid chamber at 65 °C
overnight, and unhybridized probe was removed by immersing the
slides in 2x SSC for 2 h at 65 °C. Slides were immersed
in NT buffer (150 mM NaCl, 100 mM TRIS-HCl, pH 7.5), and incubated
for 60 min with 100 µl of 1.5% (w/v) blocking reagent
(Roche Diagnostics) at room temperature. Subsequently, 100 µl
of anti-DIG-alkaline-phosphatase-conjugated antibody (Roche
Diagnostics) diluted into 1:1000 in NT buffer was applied to
each slide. The slides were washed with NT buffer and TNM buffer
(100 mM TRIS-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2
sections were stained for 2 h in 200 µl of 175 µg
nitroblue tetrazolium (Roche Diagnostics) and 450
Diagnostics) in TNM buffer. The staining reaction was stopped
by immersion in 10 mM TRIS-HCl (pH 7.5) and 100 mM EDTA. The
slides were dehydrated with an ethanol series, which was then
replaced by xylene, sealed with MP500 (Matsunami Glass Ind.),
and covered with a glass cover slip. The sections were observed
under a microscope (BX50, Olympus, Tokyo), and photographs were
taken with a digital camera (Camedia E-10, Olympus). All sections
obtained from three to five plants were observed, but only those
of one plant are represented in each figure.