Planta (1987)171:82-87
P l a n t a 9 Springer-Verlag 1987
Quality of a stress-sensitive Cucurbita pepo L. pollen G. Gay*, C. Kerhoas and C. Dumas Laboratoire reconnaissance cellulaire et amtlioration des plantes, U M CNRS 380024,Bfitiment 741, 43 bd du 11 novembre 1918, F-69622 Villeurbanne, France
Abstract. The quality of Cucurbita pepo L. pollen was studied using field pollinations and the fluorochromatic-reaction test. The extreme sensitivity of this pollen to dehydration and ageing is demonstrated. Controlled stress applied to mature pollen leads to the development of seedless fruits. Molecular signals seem to be involved in the induction of this parthenocarpy. These results indicate the existence of distinct sequences involved in the completion of the fertilization program of pollen. With pollen altered by stress, the fertilization process may be stopped at different stages of its completion. We bring evidence that Cucurbita pepo plants have developed special adaptations in order to compensate for the poor viability of their pollen.
(1985). One of the simplest and best tests remains the fluorochromatic-reaction (FCR) test (HeslopHarrison and Helsop-Harrison 1970), which is essentially based on the permeability of the plasma membrane of the vegetative cell. In this paper, we have studied Cucurbita pepo L. pollen quality using the FCR test and have also used seed set after field pollination as the reference test. Pollen samples were submitted to controlled dehydration and ageing. In contrast to pollen from many species, Cucurbita pepo pollen appears to be a stress-sensitive pollen. We suggest that the decrease of pollen viability may be proceeding in distinct steps.
Key words: Ageing (pollen) - Cucurbita- Hydration (pollen) - Parthenocarpy - Pollen (hydration; viability) - Water stress (pollen).
Material and methods
Introduction
Pollen quality is a parameter which is rather difficult to estimate. In part, this difficulty arises from the fact that the word pollen covers a large population of individual complex organisms, and within each population complex interactions occur in individuals. These interactions have been described by several terminologies such as 'pioneer effect' (Visser 1980), 'mentor effect' (Gaget et al. 1984), and 'mass effect' (Knox et al. 1986). These kinds of interactions can only be visualized by in-vivo studies. The concept of pollen quality has been re-examined by Heslop-Harrison and Heslop-Harrison * To whom correspondence should be addressed Abbreviation: F C R = fluorochromatic reaction
Plant material. Flowering squash (Cucurbita pepo L., cv. Seneca) plants were supplied by Tezier (Valence, France) and grown in the open field (except where otherwise stated). Staminate flower buds were closed in the evening in order to harvest the pollen the following morning. In-vitro germination. Pollen was placed on a semi-solid medium (adapted from that described by Brewbaker and Kwack 1963) containing 12% sucrose and covered by a thin film of paraffin oil to prevent pollen grains bursting. After an incubation of l h at room temperature, pollen grains were stained with Toluidine blue (0,06% Toluidine blue in acetate buffer 0.1 M, pH 4.5, containing 60% sucrose). In vivo germination. Pistillate flower buds were closed in the evening, for pollination occurring the following morning. After pollination the flowers were enclosed in paper bags to prevent insects entering. Pollen germination was examined after 2h of delay using the aniline-blue-fluorescence technique (Dumas and Knox 1983). Fruit development and seed set were observed two months after pollination. Pollen ageing. Freshly harvested pollen was divided into samples weighing 80 mg and placed at 5~ in sealed Eppendorf tubes. Each day one sample was assayed by the FCR test. At 0, 5 and 8 d of ageing, pollations of female flowers of Seneca plants were performed in duplicate.
G. Gay et al. : Quality of stress sensitive Cucurbita pollen
83
Pollen dehydration. Freshly harvested pollen was divided into samples, weighing 100 mg, in aluminium cuplets and placed at 25 ~ C, 40% relative humidity. At various times one sample was assayed by the F C R test, for water content (85 ~ C, 5 min in an infrared Sartorius desiccator [Sartorius, G6ttingen, FRG]), and was used for field pollinations in duplicate.
80
Thefluorochromatic reaction9 The fluorochromatic reaction was performed according to Heslop-Harrison and Heslop-Harrison (1985). The pollen grains were assessed visually into three groups: (i) Grade 2: pollen grains showing an acceptable fluorescence intensity by analogy with pollen samples of high germinative capacity. (ii) Grade 1: pollen grains showing a low fluorescence intensity and considered as intermediates of uncertain state. (iii) Grade 0: pollen grains with poor fluorescence and showing continuous leakage of fluorescein. Around 400 pollen grains were observed for each pollen sample.
x40
Flower study. In these experiments, plants were grown in a greenhouse. A hygrometric probe (Hygrocor, Bioblock, Strasbourg, France) was placed inside a pistillate flower 2 mm from the stigma, and another hygrometric probe was placed outside for flower. Relative humidities were recorded from 08.00 to 16.00 h. The distance between two opposite petals was measured every 30 rain on the same flower. On staminate flowers, the corollas were excised at 08.00 h. Pollen water content was measured at 12.00 h using pollen from untouched flowers and from excised flowers. Scanning electron microscopy. Thirty minutes after pollination, stigmas were fixed with 3 % glutaraldehyde in cacodylate buffer 0.1 M with 3% sucrose (v/w) for 2 h, washed three times for 30 min in the buffer and then washed for 10 rain in distilled water. Then they were gently dehydrated in an acetone series, further prepared by critical-point drying, and coated with goldpalladium. Observations were carried out using a JOEL CF 35 scanning-electron microscope (JOEL, Tokyo, Japan) belonging to the Centre of Electron Microscopy of Lyon (CMEABG, Lyon, France).
Results
Characterization of Cucurbita pollen. Squash pollen has certain exceptional features: (i) its water content at anthesis is high and extremely stable as long as it is enclosed in the intact flower (46 + 3 % of the fresh weight, independent of atmospheric conditions); (ii) this pollen is also characterized by its large size (200 pm diameter in hydrated state). Flower mechanisms. Pistillate and staminate flowers open their corollas at the end of the night, and close them gradually when the sun rises. During hot summer days, the corollas are fully closed before midday 9 We studied the mechanism of enclosure of the corolla. The corolla maintains a micro-environment around the stigma or the pollen. Transpiration and the progressive closure of the flower ensure a high relative humidity inside the corolla (Fig. 1) and may also decrease the temperature (not verified). The importance of this regula-
9
./(I
60
2O
14 10 ~m o
"~
20
8 6
q []
4
X ,
0
,
, , , ,
11
12
, , , ,
,,m,
12; 14 15 16 TIME (hours)
17
2
18
Fig. 1. Closing of a pistillate flower of Cucurbita pepo. The flower maintains a high relative humidity (RH) around the stigma, n - - n , Corolla diameter; ~ a , RH inside corolla; zx--zx, R H outside corolla Table 1. Control of pollen water content and pollen viability by the corolla of the staminate flower in Cucurbita pepo (each value is the mean of three measurements)
Intact corolla 08.00 h 12.00 h Excised corolla 12.00 h
Water content (% fresh weight)
Viability (FCR) (% grade 2)
46.8
100
45.0
100
15.4
0
tion is demonstrated if the corolla is excised. The pollen water content remains constant while the flower closes slowly but when the corolla is excised, pollen water content falls dramatically (Table 1).
In-vivo pollen germination. Pollen germination on the stigma surface was studied using scanning electron microscopy. Thirty minutes after pollination three kinds of pollen grains were observed: (i) grains which had failed to swell though they were retained on the stigma surface by their spiculas (Fig. 2 a); (ii) hydrated pollen grains which showed strong interactions with the stigma surface indicated by the establishment of a physical linkage with a glue-like substance (Fig. 2 b), a prominence that we term the pregerm had developed at each aperture; (iii) germinated pollen grains (Fig. 2c). Pollen-tube formation and elongation occurred after the pregerm formation. The structural difference between the pregerm wall and the pollen-tube wall can be clearly seen in Fig. 2 c. In-vitro germination. In contrast to the results obtained for in-vivo germination, in vitro all the pol-
84
G. Gay et al. : Quality of stress sensitive Cucurbita pollen I00"
300
13
2
j.f "2"
~.e 80"
-g 'I0
I,,U
.I
P l a n t a 9 Springer-Verlag 1987
Quality of a stress-sensitive Cucurbita pepo L. pollen G. Gay*, C. Kerhoas and C. Dumas Laboratoire reconnaissance cellulaire et amtlioration des plantes, U M CNRS 380024,Bfitiment 741, 43 bd du 11 novembre 1918, F-69622 Villeurbanne, France
Abstract. The quality of Cucurbita pepo L. pollen was studied using field pollinations and the fluorochromatic-reaction test. The extreme sensitivity of this pollen to dehydration and ageing is demonstrated. Controlled stress applied to mature pollen leads to the development of seedless fruits. Molecular signals seem to be involved in the induction of this parthenocarpy. These results indicate the existence of distinct sequences involved in the completion of the fertilization program of pollen. With pollen altered by stress, the fertilization process may be stopped at different stages of its completion. We bring evidence that Cucurbita pepo plants have developed special adaptations in order to compensate for the poor viability of their pollen.
(1985). One of the simplest and best tests remains the fluorochromatic-reaction (FCR) test (HeslopHarrison and Helsop-Harrison 1970), which is essentially based on the permeability of the plasma membrane of the vegetative cell. In this paper, we have studied Cucurbita pepo L. pollen quality using the FCR test and have also used seed set after field pollination as the reference test. Pollen samples were submitted to controlled dehydration and ageing. In contrast to pollen from many species, Cucurbita pepo pollen appears to be a stress-sensitive pollen. We suggest that the decrease of pollen viability may be proceeding in distinct steps.
Key words: Ageing (pollen) - Cucurbita- Hydration (pollen) - Parthenocarpy - Pollen (hydration; viability) - Water stress (pollen).
Material and methods
Introduction
Pollen quality is a parameter which is rather difficult to estimate. In part, this difficulty arises from the fact that the word pollen covers a large population of individual complex organisms, and within each population complex interactions occur in individuals. These interactions have been described by several terminologies such as 'pioneer effect' (Visser 1980), 'mentor effect' (Gaget et al. 1984), and 'mass effect' (Knox et al. 1986). These kinds of interactions can only be visualized by in-vivo studies. The concept of pollen quality has been re-examined by Heslop-Harrison and Heslop-Harrison * To whom correspondence should be addressed Abbreviation: F C R = fluorochromatic reaction
Plant material. Flowering squash (Cucurbita pepo L., cv. Seneca) plants were supplied by Tezier (Valence, France) and grown in the open field (except where otherwise stated). Staminate flower buds were closed in the evening in order to harvest the pollen the following morning. In-vitro germination. Pollen was placed on a semi-solid medium (adapted from that described by Brewbaker and Kwack 1963) containing 12% sucrose and covered by a thin film of paraffin oil to prevent pollen grains bursting. After an incubation of l h at room temperature, pollen grains were stained with Toluidine blue (0,06% Toluidine blue in acetate buffer 0.1 M, pH 4.5, containing 60% sucrose). In vivo germination. Pistillate flower buds were closed in the evening, for pollination occurring the following morning. After pollination the flowers were enclosed in paper bags to prevent insects entering. Pollen germination was examined after 2h of delay using the aniline-blue-fluorescence technique (Dumas and Knox 1983). Fruit development and seed set were observed two months after pollination. Pollen ageing. Freshly harvested pollen was divided into samples weighing 80 mg and placed at 5~ in sealed Eppendorf tubes. Each day one sample was assayed by the FCR test. At 0, 5 and 8 d of ageing, pollations of female flowers of Seneca plants were performed in duplicate.
G. Gay et al. : Quality of stress sensitive Cucurbita pollen
83
Pollen dehydration. Freshly harvested pollen was divided into samples, weighing 100 mg, in aluminium cuplets and placed at 25 ~ C, 40% relative humidity. At various times one sample was assayed by the F C R test, for water content (85 ~ C, 5 min in an infrared Sartorius desiccator [Sartorius, G6ttingen, FRG]), and was used for field pollinations in duplicate.
80
Thefluorochromatic reaction9 The fluorochromatic reaction was performed according to Heslop-Harrison and Heslop-Harrison (1985). The pollen grains were assessed visually into three groups: (i) Grade 2: pollen grains showing an acceptable fluorescence intensity by analogy with pollen samples of high germinative capacity. (ii) Grade 1: pollen grains showing a low fluorescence intensity and considered as intermediates of uncertain state. (iii) Grade 0: pollen grains with poor fluorescence and showing continuous leakage of fluorescein. Around 400 pollen grains were observed for each pollen sample.
x40
Flower study. In these experiments, plants were grown in a greenhouse. A hygrometric probe (Hygrocor, Bioblock, Strasbourg, France) was placed inside a pistillate flower 2 mm from the stigma, and another hygrometric probe was placed outside for flower. Relative humidities were recorded from 08.00 to 16.00 h. The distance between two opposite petals was measured every 30 rain on the same flower. On staminate flowers, the corollas were excised at 08.00 h. Pollen water content was measured at 12.00 h using pollen from untouched flowers and from excised flowers. Scanning electron microscopy. Thirty minutes after pollination, stigmas were fixed with 3 % glutaraldehyde in cacodylate buffer 0.1 M with 3% sucrose (v/w) for 2 h, washed three times for 30 min in the buffer and then washed for 10 rain in distilled water. Then they were gently dehydrated in an acetone series, further prepared by critical-point drying, and coated with goldpalladium. Observations were carried out using a JOEL CF 35 scanning-electron microscope (JOEL, Tokyo, Japan) belonging to the Centre of Electron Microscopy of Lyon (CMEABG, Lyon, France).
Results
Characterization of Cucurbita pollen. Squash pollen has certain exceptional features: (i) its water content at anthesis is high and extremely stable as long as it is enclosed in the intact flower (46 + 3 % of the fresh weight, independent of atmospheric conditions); (ii) this pollen is also characterized by its large size (200 pm diameter in hydrated state). Flower mechanisms. Pistillate and staminate flowers open their corollas at the end of the night, and close them gradually when the sun rises. During hot summer days, the corollas are fully closed before midday 9 We studied the mechanism of enclosure of the corolla. The corolla maintains a micro-environment around the stigma or the pollen. Transpiration and the progressive closure of the flower ensure a high relative humidity inside the corolla (Fig. 1) and may also decrease the temperature (not verified). The importance of this regula-
9
./(I
60
2O
14 10 ~m o
"~
20
8 6
q []
4
X ,
0
,
, , , ,
11
12
, , , ,
,,m,
12; 14 15 16 TIME (hours)
17
2
18
Fig. 1. Closing of a pistillate flower of Cucurbita pepo. The flower maintains a high relative humidity (RH) around the stigma, n - - n , Corolla diameter; ~ a , RH inside corolla; zx--zx, R H outside corolla Table 1. Control of pollen water content and pollen viability by the corolla of the staminate flower in Cucurbita pepo (each value is the mean of three measurements)
Intact corolla 08.00 h 12.00 h Excised corolla 12.00 h
Water content (% fresh weight)
Viability (FCR) (% grade 2)
46.8
100
45.0
100
15.4
0
tion is demonstrated if the corolla is excised. The pollen water content remains constant while the flower closes slowly but when the corolla is excised, pollen water content falls dramatically (Table 1).
In-vivo pollen germination. Pollen germination on the stigma surface was studied using scanning electron microscopy. Thirty minutes after pollination three kinds of pollen grains were observed: (i) grains which had failed to swell though they were retained on the stigma surface by their spiculas (Fig. 2 a); (ii) hydrated pollen grains which showed strong interactions with the stigma surface indicated by the establishment of a physical linkage with a glue-like substance (Fig. 2 b), a prominence that we term the pregerm had developed at each aperture; (iii) germinated pollen grains (Fig. 2c). Pollen-tube formation and elongation occurred after the pregerm formation. The structural difference between the pregerm wall and the pollen-tube wall can be clearly seen in Fig. 2 c. In-vitro germination. In contrast to the results obtained for in-vivo germination, in vitro all the pol-
84
G. Gay et al. : Quality of stress sensitive Cucurbita pollen I00"
300
13
2
j.f "2"
~.e 80"
-g 'I0
I,,U
.I
