Journal of Experimental Botany, Vol. 54, No. 381, pp. 345±348, January 2003
DOI: 10.1093/jxb/erg019
RESEARCH PAPER
Vascular connections between the receptacle and empty
achenes in sun¯ower (Helianthus annuus L.)
Merianne Alkio and Eckhard Grimm1
Martin-Luther-UniversitaÈt Halle-Wittenberg, Landwirtschaftliche FakultaÈt, Institut fuÈr Acker- und P¯anzenbau,
Ludwig-Wucherer-Str. 2, D-06099 Halle (Saale), Germany
Received 13 June 2002; Accepted 30 August 2002
Abstract
Empty achenes in sun¯ower, 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 identi®ed by ¯uorescence of
aniline-blue-stained callose and auto¯uorescence,
respectively. Vascular strands that extended from the
receptacle into empty achenes were regularly found
in longitudinal sections. The phloem-mobile probe,
carboxy¯uorescein, 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, de®cient vascular connections do not prevent
seed ®lling in sun¯ower.
14
Key
words:
C-photoassimilates,
phloem transport, seed ®lling.
carboxy¯uorescein,
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 ®lling 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 identi®ed 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 ¯attened in¯orescence 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
¯uorescence 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 carboxy¯uorescein and 14C-photoassimilates.
Introduction
Materials and methods
The head-like in¯orescence of sun¯ower (capitulum)
contains varying numbers of empty grains (achenes) at
maturity. Poor seed ®lling may reduce yield considerably
and is thus the most common problem in sun¯ower
cultivation. Empty achenes do not contain an embryo, or
the embryo does not incorporate signi®cant 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,
Plants
Sun¯ower (Helianthus annuus L. cv. Rigasol) plants were raised in a
greenhouse at 22/16 61 °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 ¯ux density of 100 mmol m±2 s±1. Plants were cross-pollinated
by hand. Capitula were excised and analysed in the late seed-®lling
stages (translocation experiments) or at full maturity (microscopy).
1
Microscopy
The vascular structures between empty seeds and receptacle were
analysed in hand sections of the periphery (20 achenes) and the
To whom correspondence should be addressed. Fax: +49 345 5527129. E-mail: [email protected]
ã Society for Experimental Biology 2003
346 Alkio and Grimm
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 ¯uorescence
microscope (BX60 with ®lterset BP 330±385, DM 400, BA 420;
Olympus, Tokyo, Japan). Under these conditions, sieve tubes were
visible due to ¯uorescent callose deposits, and xylem vessels were
visible due to auto¯uorescence.
Phloem transport into the empty achenes was investigated in six
plants using the phloem-mobile probe 4(5)-carboxy¯uoresceindiacetate (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% CFsolution 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 ¯uorescence microscope (Axioskop 20; Carl
Zeiss, Jena, Germany) equipped with the ®lterset BP 450±490, FT
510, LP 515. In addition, hand sections of ®lled 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 14Cphotoassimilates 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).
Results and discussion
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 ®ndings 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 sun¯ower capitulum and stated
this to be a cause for poor seed ®lling. However, these
authors did not investigate the interface between the
receptacle and the achenes. Also, the staining procedure
employed (safranin/fast green) allows the identi®cation of
xylem, but does not speci®cally distinguish phloem
elements.
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 ®lled 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
14
C-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. Considerations of the ontogeny of the achenes corroborate
these ®ndings. Principally, each ¯oral organ is connected
with the vascular tissue in the receptacle (Esau, 1969). In
sun¯ower, the anatomic peculiarities may be interpreted in
the following way: Three vascular strands connect the
receptacle with all ¯oral 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 ¯ower is
regarded as a ¯oral tube, i.e. the adnate bases of the ¯oral
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 ®lling, assimilates and nutrients are required for ¯oret
development and ¯owering. 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
Vascular connections of empty achenes 347
Fig. 1. Vascular connections between the receptacle and empty achenes in sun¯ower. (A) Light micrograph and (B) explanatory drawing:
longitudinal section through the insertion site of an empty achene on the receptacle. Three main vascular strands connect the achene with the
receptacle. Boxes in (B) indicate the positions of ¯uorescent micrographs (C±L). In (C), (D), (K), and (L) sieve tubes are indicated by
¯uorescence of callose in sieve plates after staining with aniline blue. (C) Vascular strands between the receptacle and an empty achene. (D)
Detail of the insertion site of an empty achene with vascular connection to the receptacle. (E) Same region as in (D) but after CF application,
yellow ¯uorescence indicates CF being transported into the empty achene. (F) Testa of an empty achene, CF has been transported in the phloem
to the branching point of the central incoming vascular strand. (G) Pericarp and (H) testa of an empty achene, CF is visible in sieve tubes.
(I) Positive control: Testa of a ®lled achene after CF application, CF in sieve tubes. (J, K, L) Negative controls: Testa of a ®lled achene without
CF application, the xylem exhibits auto¯uorescence. (M) 14C-photoassimilate translocation into developing achenes after 14CO2-application to a
leaf. Incorporated 14C-photoassimilates were detected in longitudinally split achenes using a phosphorimager. The ®rst three parts of (M) show
top-views of the position and arrangement of the achenes 1±7 in the capitulum. One empty achene (4) in the periphery of the capitulum was
surrounded by six ®lled ones (1±3, 5±7). Autoradiographs show low and high radioactivity in the empty achene (4) and in the ®lled achenes (1±3,
5±7), respectively. ea, empty achene; p, phloem; pe, pericarp; r, receptacle; vp, vascular strand in the pericarp; vt, vascular strand in the testa; vc,
central vascular strand; vr, vascular strand in the receptacle; x, xylem; darts indicate sieve-plates. The bars indicate 100 mm (A±L) and 5 mm (M).
348 Alkio and Grimm
size and colour, indicating that assimilate transport takes
place. Steer et al. (1988) showed that the central ¯orets
developed into well-®lled achenes when the competition of
the peripheral ¯orets 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 classi®cation is useful
because different reasons may cause the lack of seed ®lling
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 ®lling. 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
sun¯ower capitulum, con¯icts with the view that empty
seeds are a consequence of poor vascularization.
Acknowledgements
We thank Alexander Schulz and Moritz Knoche for helpful
discussions. The research was supported by the Deutsche
Forschungsgemeinschaft (DFG).
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