SLIDE 4 Cytoplasmic carbohydrate content during Helleborus pollen presentation 17
Each suspension was then transferred to a 1.8 ml Eppendorf tube,
composition and viability of Helleborus foetidus L. and H.
dried under vacuum (Speedvac, Savant Instruments) and stored
bocconei Ten. pollen grains change in experimental conditions
at Õ80ß.
similar to those that occur naturally. The two species ower
Frozen samples were resuspended in 0.5 ml de-ionized water,
from January to March, when minimum temperature is
placed in a vortex apparatus and centrifuged in a ALC 4224
around 0ßC and maximum temperature can reach 18ßC for
centrifuge at 11,000 r.p.m. for 8 minutes. One hundred ml of the
a few hours (Vesprini & Pacini 2000). Their owers have
supernatant were withdrawn for sugar analysis by enzymatic test (Boehringer Mannheim Sucrose/D-Glucose/D-Fructose enzymatic
many anthers that do not open simultaneously and each
test, Cat. No. 716 260), while the remaining liquid and the pellet
anther exposes its pollen for about three days. Pollen is
were pretreated for starch testing. Pretreatment was necessary to
gathered by insects of the Bombus genus, which also feed on
convert starch in a soluble form and was carried out by adding
the nectar (Vesprini et al. 1999). Although both species are
200 ml of DMSO (dimethylsulphoxide) and 50 ml of HCl 8 M. Samples
partially self compatible, spontaneous self pollination does
for starch analysis were incubated for 30 minutes at 60ßC; 50 ml of
not occur and the species rely on Bombus visits for seed
NaOH 8 M was then added and a nal dilution to 1 ml was made with citrate buVer (pH=4). Following a second centrifuge cycle
production (Vesprini & Pacini 2000).
at 11,000 r.p.m. for 8 minutes, a 100 ml aliquot of supernatant
This study is part of a research on the reproductive
was withdrawn for starch analysis using another enzymatic test
ecophysiology of these two species as well as research on the
(Boehringer Mannheim enzymatic test Cat. No. 207 748).
general physiology of pollen and anther carbohydrates.
All the analyses were performed in triplicate with a Varian (Cary)
Carbohydrates are known to play a role in drought and low
UV-visible spectrophotometer at 365 nm wave length.
temperature tolerance in vegetative parts of the plants (Akazawa & Okamoto 1980). Sucrose is responsible for Pollen viability desiccation tolerance during pollen development (Hoekstra & van Roekel 1988) but the function of carbohydrates during
After experiments, pollen viability was determined by uorochro-
pollen presentation and dispersal is lacking.
matic reaction (Heslop-Harrison et al. 1984) in which a 30% sucrose solution was used. Pollen grains were rehydrated for one hour at room temperature and 100% RH before performing the viability
MATERIALS AND METHODS
- test. For each sample at least 300 pollen grains were scored and only
the fully stained pollen grains were considered viable.
Pollen collection Cytological detection of total insoluble polysaccharides
Helleborus foetidus and H. bocconei pollen was collected from the
- wers of diVerent plants growing in their natural habitat located
Mature Helleborus foetidus and Helleborus bocconei anthers were in oak forests of Central Tuscany, Italy (Monteriggioni, Siena). collected and xed in 5% glutaraldehyde in phosphate buVer at Flowers with some dehisced anthers were cut from the plants. The pH 6.9, dehydrated in an ethyl alcohol series and embedded in already exposed pollen was tapped from opened anthers and dis- Technovit 7100 (Heraeus Kulzer GmbH).
- carded. The owers were placed in water overnight. At 8:00 a.m.
To detect the presence of total insoluble polysaccharides, the the following morning, recently exposed pollen was tapped out of embedded material was cut into semi-thin sections (2±5 micrometers) newly opened anthers from about 100 owers per species. and stained with: a) PAS (Periodic acid-ShiV reaction) for total insoluble polysaccha-
Sample treatment
rides after free aldehyde groups blockage (O’Brien & McCully 1981); After collection, pollen was divided into aliquot of 30 mg, which in b) IKI (iodine-potassium iodide) for starch (Johansen 1940). total took one hour to complete, pollen treatment therefore began at 9:00 a.m.. Each aliquot of pollen was spread out into thin layers
- n paralm (American National Can) inside Petri dishes, which
RESULTS
were stored in a refrigerator with controlled temperature (0ßC) and RH (25%). Sugars and starch were determined as follows:
Carbohydrates in pollen grains at the beginning of experiment
1) immediately after pollen collection 2) after 24 hours at 0ßC
Cytological analysis
total insoluble polysaccharides
3) after 36 hours at 0ßC
showed that both species contain uniformly distributed cyto-
4) after 72 hours at 0ßC 5) after 32 hours at 0ßC, plus 4 more hours at 18ßC, plus 36 more
plasmic polysaccharides. Glucose, fructose and sucrose con-
hours at 0ßC
tents reached similar values in both species. Starch is present
6) after 24 hours at 0ßC, plus 4 more hours at 18ßC, 20 more hours
in H. foetidus only. Biochemical assay of carbohydrates is
at 0ßC, 4 more hours at 18ßC and 20 more hours at 0ßC
shown in Table I.
Treatments 5 and 6 were carried out to test whether thermal cycles, which occur during the owering period, have further eVects other than low temperatures.
Table I. Carbohydrate type and quantity expressed in mg/mg
- f shedding pollen. Values for glucose, fructose and sucrose
Sugar and starch analysis were similar in the two species.
For each treatment, 30 mg of pollen was placed in an 80% methanol Glucose Fructose Sucrose Starch solution (v/v) and homogenized for 3 minutes in a potter homogen- izer, to completely break down the pollen grains. Samples were
1.60Ô0.2 0.46Ô0.08 70.25Ô11.3 not detectable
- bserved microscopically to check that pollen was completely
- H. foetidus
1.23Ô0.18 0.32Ô0.06 68.54Ô 9.7 9.79Ô1.6 ruptured.
Grana 41 (2002)
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