Journal of Experimental Botany, Vol. 54, No. 381, pp. 345-348,
January 2, 2003
© 2003 Oxford University Press
Vascular connections between the receptacle and empty achenes in sunflower (Helianthus annuus L.)
Received 13 June 2002; Accepted 30 August 2002
Martin-Luther-Universität Halle-Wittenberg, Landwirtschaftliche Fakultät, Institut für Acker- und Pflanzenbau, Ludwig-Wucherer-Str. 2, D-06099 Halle (Saale), Germany
1 To whom correspondence should be addressed. Fax: +49 345 5527129. E-mail: grimm{at}landw.uni-halle.de
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Abstract |
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Empty achenes in sunflower, particularly in the centre of the capitulum, may be caused by poor vascularization. This hypothesis was tested by microscopic examination and translocation experiments. Phloem and xylem were identified by fluorescence of aniline-blue-stained callose and autofluorescence, respectively. Vascular strands that extended from the receptacle into empty achenes were regularly found in longitudinal sections. The phloem-mobile probe, carboxyfluorescein, was translocated from the receptacle to the pericarp and the testa of empty achenes. Similarly, 14CO2-derived 14C-photoassimilates moved into empty achenes. The observations suggest that empty achenes are both structurally and functionally connected with the vascular system of the receptacle. Hence, deficient vascular connections do not prevent seed filling in sunflower.
Key words: 14C-photoassimilates, carboxyfluorescein, phloem transport, seed filling.
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Introduction |
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The head-like inflorescence of sunflower (capitulum) contains varying numbers of empty grains (achenes) at maturity. Poor seed filling may reduce yield considerably and is thus the most common problem in sunflower cultivation. Empty achenes do not contain an embryo, or the embryo does not incorporate significant amounts of storage compounds. Empty ovules result from any kind of fertilization failure. Further, the embryo can be aborted at different stages of seed development due to genotypic and environmental reasons (e.g. non-optimal temperature, radiation, nitrogen, and water supply; for a review see Connor and Hall, 1997).
Generally, the occurrence of empty achenes is highest in the centre of the capitulum. Poor seed filling is frequently thought to be related to poor vascularization of the receptacle (Beltrano et al., 1994; Chone, 1983; Durrieu et al., 1985; Goffner et al., 1988; Morozov, 1958; Yegappan et al., 1982). Macroscopically, vascular bundles originating from the stem were identified that run radially towards the periphery of the capitulum, and from there towards the centre of the capitulum (Morozov, 1958; Durrieu et al., 1985; Goffner et al., 1988). According to Durrieu et al. (1985) and Goffner et al. (1988) the central part of the flattened inflorescence axis (receptacle) appears nearly deprived of vascular bundles. However, no information on the microscopic structure of the region between the receptacle and the achenes is available. The objectives of this study were, therefore, (1) to investigate, using fluorescence microscopy, whether vascular connections between the receptacle and empty achenes are present, and (2) if present, to establish whether these connections are functional, as indicated by the translocation of carboxyfluorescein and 14C-photoassimilates.
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Materials and methods |
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Plants
Sunflower (Helianthus annuus L. cv. Rigasol) plants were raised in a greenhouse at 22/16 ±1 °C day/night temperature. Daylight was supplemented from 06.00 h to 22.00 h with 400 W HPS lamps (SON-T AGRO; Philips, Belgium) providing a minimum photosynthetic photon flux density of 100 µmol m–2 s–1. Plants were cross-pollinated by hand. Capitula were excised and analysed in the late seed-filling stages (translocation experiments) or at full maturity (microscopy).
Microscopy
The vascular structures between empty seeds and receptacle were analysed in hand sections of the periphery (20 achenes) and the centre (70 achenes) of the capitulum. The sections were cleared in 90% lactic acid at room temperature overnight, subsequently neutralized with 0.1 M NaOH and rinsed with water. To detect callose, sections were stained with 0.01% aniline blue in 0.2 M phosphate buffer (pH 7.2) and then examined under a fluorescence microscope (BX60 with filterset BP 330–385, DM 400, BA 420; Olympus, Tokyo, Japan). Under these conditions, sieve tubes were visible due to fluorescent callose deposits, and xylem vessels were visible due to autofluorescence.
Phloem transport into the empty achenes was investigated in six plants using the phloem-mobile probe 4(5)-carboxyfluoresceindiacetate (CF; Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). CF was applied either to the stem (method A) or to an excised block of the receptacle (method B; Wolswinkel, 1987). Method A: the capitulum was excised leaving a stump of the stem about 3 cm in length. The cut surface was placed in 0.03% CF-solution for 24–72 h. Method B: Blocks of approximately 30–40 achenes were excised from the centre of the capitulum. The receptacle was partly removed, leaving a 1.5 cm thick layer below the achenes. Blocks were positioned on a 0.03% CF-solution for 24–72 h such that the bottom of the block was in contact with the dye solution. During the CF application the explants were maintained at high humidity. Subsequently, hand sections of empty achenes (n=50) were examined under a fluorescence microscope (Axioskop 20; Carl Zeiss, Jena, Germany) equipped with the filterset BP 450–490, FT 510, LP 515. In addition, hand sections of filled achenes with and without CF application served as positive and negative controls, respectively (n=20 each).
Translocation of 14C-photoassimilates
A 14CO2-pulse of 5 min was applied to an upper source leaf blade (n=9). Following a chase period of 3 h the capitulum was excised, photographed and freeze-dried. All achenes were numbered, removed from the capitulum and cut into half. To detect 14C-photoassimilates half-achenes were placed on a storage phosphor screen (BAS MP 2040; Fuji, Tokyo, Japan) for 10–20 h. The exposed screen was scanned using a phosphorimager (BAS 1000; Fuji, Tokyo, Japan).
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Results and discussion |
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Light microscopy revealed that the uppermost 2–3 mm layer of the receptacle was rich in vascular tissue in the periphery as well as in the centre of the capitulum. Empty achenes were always connected with the vascular network of the receptacle, each by three separate strands (Fig. 1A–C). The connecting vascular strands bent nearly perpendicularly about 1–2 mm below the achenes in the receptacle (Fig. 1A, B). Furthermore, individual sieve tubes and xylem vessels extended from the receptacle into the empty achenes (Fig. 1C–E). These findings are not in agreement with the conclusions of Durrieu et al. (1985) and Goffner et al. (1988) who reported a lack of vascular bundles in the centre of the sunflower capitulum and stated this to be a cause for poor seed filling. However, these authors did not investigate the interface between the receptacle and the achenes. Also, the staining procedure employed (safranin/fast green) allows the identification of xylem, but does not specifically distinguish phloem elements.
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Although sink activity of the empty achenes is expected to be low and hence may limit translocation and accumulation of phloem-mobile tracers, in several experiments they had been transported into empty achenes. CF, frequently used as an indicator for assimilate transport, moved from the receptacle into empty achenes (Fig. 1E–H). In four out of six plants, CF was detected in the phloem of the receptacle, the fruit wall (pericarp; Fig. 1E, G) and the seed coat (testa; Fig. 1F, H) of empty achenes. In preliminary experiments, following CF application to one of the upper leaves (method adapted from Pradel et al., 1999), CF was not detected in empty achenes, and only rarely in the receptacle or in the filled achenes (in two out of nine plants). This observation may be accounted for by dilution along the translocation pathway, thus preventing the movement of detectable amounts of CF. In addition, CF does not move into the embryo due to the absence of a symplasmic pathway between the seed coat and embryo (Thorne, 1985).
When leaves were exposed to 14CO2, the pericarp of the empty achenes rarely incorporated detectable quantities of 14C-assimilates (Fig. 1M). In one out of nine plants, empty achenes were found to be labelled above background.
The present experiments established the existence of functioning phloem pathways into empty achenes. Con siderations of the ontogeny of the achenes corroborate these findings. Principally, each floral organ is connected with the vascular tissue in the receptacle (Esau, 1969). In sunflower, the anatomic peculiarities may be interpreted in the following way: Three vascular strands connect the receptacle with all floral organs during anthesis, and with achenes during ripening. Newcomb (1972) studied the cellular development of the ovary. He found vascular tissue entering the ovule near the micropyle, extending along the periphery and terminating at the chalaza. Most likely this vascular tissue corresponds to the central vascular strand shown in Fig. 1A, C. The two vascular strands running into the pericarp of the achene (peripheral strands shown in Fig. 1A, C) are identical with the ones, which entered the ovary wall during anthesis. In Asteraceae, the ovary wall of the epigynous flower is regarded as a floral tube, i.e. the adnate bases of the floral organs (Esau, 1965). The ovary wall transforms into the pericarp during seed development. In the ovary wall the vascular strands fork, and run as several parallel bundles to the apical plate, from where branches extend to the sepals, corolla, stamens, and the style. Before fertilization and seed filling, assimilates and nutrients are required for floret development and flowering. Following anthesis, if no fertilization happens, or the embryo is aborted, the assimilate demand is reduced. It may be speculated whether the reduced assimilate demand might cause the vascular tissue to degenerate. Such degeneration was not observed. Moreover, despite the defects in the embryogenesis, empty achenes often develop pericarps of normal size and colour, indicating that assimilate transport takes place. Steer et al. (1988) showed that the central florets developed into well-filled achenes when the competition of the peripheral florets was eliminated.
Beside the poor vascularization, several other factors are discussed to explain the phenomenon of empty achenes (Connor and Hall, 1997). Morozov (1958) distinguished between the empty achenes in the periphery and those in the centre of the capitulum. This classification is useful because different reasons may cause the lack of seed filling in the different positions. In the periphery, empty achenes are usually surrounded by well-developed ones, indicating that intrinsic factors of the individual seed (e.g. defective pollination, disturbed embryogenesis) may cause the lack of filling. On the contrary, the ‘empty centre syndrome’ may preferentially depend on environmental conditions and overall regulation in the plant (e.g. shortage of water, nutrients and space, hormonal regulation). The presence of functioning vascular connections between the empty achenes and the receptacle, particularly in the centre of sunflower capitulum, conflicts with the view that empty seeds are a consequence of poor vascularization.
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Acknowledgements |
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We thank Alexander Schulz and Moritz Knoche for helpful discussions. The research was supported by the Deutsche Forschungsgemeinschaft (DFG).
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References |
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