Photoassimilate transport from upper source leaves to the capitulum
The present investigations show that the upper 10 leaves export photoassimilates to the sunflower capitulum. A transition to a basipetal assimilate movement can be assumed to take place between leaves 10 and 15. Since the plants investigated had a total number of 24 leaves, we conclude that the upper 40–60% of leaves export photoassimilates to the capitulum.
Each source leaf exported photoassimilates to a well defined area (sector) of the capitulum. There was a clear spatial relationship between the supplied sector and the supplying leaf: The orientation of the sector in the capitulum was identical with the orientation of the insertion site of the leaf at the stem (Figs 2a–d and 3). The size of labelled sector varied slightly, which can be attributed to physiological and anatomical reasons.
Physiologically, the initial level of 13C-enrichment and the specific 14C-radioactivity after pulse application of 13/14CO2 in the leaf depend on actual photosynthesis. The initial level of 13/14C-label and the loss of labelled compounds along the pathway determine the precision of detection of the distribution patterns. This may explain minor differences in sector boundaries.
Anatomically, three main leaf traces connect the vascular network of a sunflower leaf blade with that of the stem (Esau, 1945). Leaf traces are described to enter the stem and run down to the next lower leaf of the same orthostichy. Near the site where the petiole inserts leaf traces join the vascular bundles of the next higher leaf of the same orthostichy (Priestley & Scott, 1936; Esau, 1945; Thompson et al., 1979). In our experiments photoassimilates exported via the midvein primarily terminated in the 1/8 sector of the capitulum, which exactly aligned with the insertion site of the leaf (Table 2; Figs 2a–d and 3), whereas photoassimilates exported via the two main lateral veins were mostly delivered to the two adjacent 1/8 sectors (Table 2). Thus, we suggest that a single floret or achene is typically connected with the leaves of three neighbouring ortostichies. This view was confirmed by the 14C-labelling experiments. Since the leaves of plants grown in a glasshouse were considerably smaller than those from field-grown ones, the leaf chamber for 14C-application enclosed the three main leaf traces. Correspondingly, 14C-photoassimilates were found in a 2/8–3/8 sector of the capitulum. The maximum radioactivity, however, aligned with the insertion site indicating bulk export via the midvein.
Previous findings support this view: Asymmetrical growth of the sunflower capitulum as a result of one-sided defoliation (Caldwell, 1930; Prokofiev et al., 1957), may indicate limited lateral branching of the phloem pathway. Moreover, eosin, applied to a petiole, moved from leaf to leaf within one and the same orthostichy (Caldwell, 1930).
Following foliar application of [14C]acetate and [14C]phosphate, Prokofiev et al. (1957) found radiolabel in sectors of the receptacle. They supposed that the insertion height of the leaves correlates with the width of the labelled sector. However, present results showed that photoassimilates leaving a source leaf via one of the three main veins mostly move to a 1/8-sector of the capitulum, independent of the insertion height of the leaf. Further, Prokofiev et al. (1957) detected radiolabel in achenes of 3–4 single parastichies, located above the treated leaf. Labelled parastichies were separated by 4–5 unlabelled ones. The pattern most likely resulted from a selective mode of sampling. We never found labelled photoassimilates predominantly enriched in parastichies. The most prominent 13/14C-label was always concentrated in a narrow sector (Fig. 3). The maximum amount of label was found in radially arranged achenes, indicating that the main vascular bundles and their primary branches should also be radially orientated in the receptacle. Notably, chase periods in the experiments of Prokofiev et al. (1957) were 5–15 d. Therefore, it is difficult to decide whether phloem transport or sink metabolism caused the pattern. Pulse-chase experiments used here allow the study of photoassimilate movement almost free of secondary processes (e.g. long-term sink metabolism, short distance transport of metabolites in the receptacle). The differences in experimental design most likely explain the differing results.
The discovery of sectorial photoassimilate supply in sunflower contributes to understanding assimilate distribution on the whole plant level. Moreover, the finding can directly be put to use in exploring transport physiology and seed development in sunflower, as it allows one side of the plant to be treated (e.g. foliar application of plant growth regulators) and the opposite side to be regarded as the untreated control. Spatially limited photoassimilate distribution was also found in sinks of other species, for instance in sugar beet (Schilling et al., 1986), tomato (Li et al., 2000) and grape vine (Motomura, 1993).
Photoassimilate distribution in the capitulum during anthesis and seed filling
Development of the reproductive sinks of the sunflower starts with the formation of the involucral bracts, which are the main sinks until the appearance of the first floret primordia (Hernandez & Palmer, 1992). During floret differentiation, photoassimilates move preferably to these primordia and the receptacle (Hernandez & Palmer, 1992). Generally, peripheral floret primordia start to import earlier, and they incorporate higher amounts of carbohydrates than the central ones (Hernandez & Palmer, 1992). Present investigations revealed that during the first days of anthesis florets at the staminate stage are strong sinks attracting considerable quantities of 13C-photoassimilates (Table 3, plants 1–3). More precisely, the high sink activity occurred when florets converted from buds to staminate florets or when they persisted in the staminate stage. Different events may account for the high sink activity (for summary, see Putt, 1940; Miller & Fick, 1997): opening of the bud and rapid elongation of filaments, exerting the anther tube through the corolla; pollen production; discharging of pollen inside the anther tube after full extension of pollen sacs; and nectar production. During late stages of anthesis, photoassimilate import maximum shifted from the staminate florets to the youngest developing achenes.
13C-translocation following the pulse incubation of source leaves does not allow quantification of the total flux of photoassimilates into the capitulum. Nevertheless, it describes, spatially resolved, the import of newly fixed photoassimilates. Sunflower capitula develop individual patterns of competition for photoassimilates across the zones during seed filling (Table 4). 13C-distribution patterns varied considerably, notwithstanding the facts that plants had been grown under identical conditions and analyses were done at the same developmental stage, referred to as days after first flowering. In the outer whorls (zones 1–3) competition for photoassimilates seems to play a secondary role. Records over 2 wk (12–26 DAF) showed that the relative 13C-enrichment was very similar in these zones (Fig. 4). Apart from import peaks during anthesis, another maximum of photoassimilate import appeared in zone 4, especially between 18 and 26 DAF. In contrast, the centre of capitulum was generally deprived concerning the photoassimilate import during the stages examined. Filled achenes in zone 6 exhibited only low 13C-enrichments. Empty achenes, frequently occurring in the centre of the capitulum, increase this tendency: If at all, they import very low amounts of photoassimilates into husks (Alkio & Grimm, 2003).
Phloem transport sugars
Present investigations on phloem transport sugars in sunflower were based on the analysis of 13/14C-labelled carbohydrates exported from the leaf blade to the petiole and stem. Depending on the length of pulse and chase, extracts of the petiole and stem include both carbohydrates translocated in sieve tubes and those unloaded and subsequently metabolized in accompanying tissues.
Among 14CO2-derived photoassimilates up to 98% of the radioactivity in petiole and stem extracts was found in sucrose 1 h after label application (Table 5; Fig. 5). Even after the long chase period of 24 h sucrose was the only labelled sugar detected in the petiole of the 13CO2-treated leaf (Table 6). So far, the results are in accordance with data reported by Ito & Mitsumori (1992) who concluded from 13C-NMR analyses that sucrose is the main transport sugar in the sunflower phloem. However, the sunflower extracts additionally contained raffinose and myo-inositol (Table 5), which are further candidates for phloem transport.
Raffinose was present in petiole and stem extracts (Table 5; Fig. 5). It has been reported as a constituent of sunflower achenes (Kuo et al., 1988) and of the phloem sap in other Asteraceae species (Artemisia canarensis and Euryps spec.; Zimmermann & Ziegler, 1975). Small amounts of raffinose are translocated in the phloem of some species as a supplement to sucrose (Hoffmann-Thoma et al., 2001). The role of raffinose in sunflower was not discussed in previous studies (Chopowick & Forward, 1974; Shiroya, 1977). Chopowick & Forward (1974) found [14C]raffinose (2% of total radioactivity) in the petiole 1 h after pulse application of 14CO2. The results here suggest that raffinose is not transported in sunflower phloem: (i) Labelled raffinose was absent in the petiole 1 h after pulse application of 14CO2. If raffinose is a phloem transport sugar it should be present in this organ. (ii) Percentages of [14C]raffinose in the stem extracts did not exceed those of [14C]glucose and [14C]fructose. Both monosaccharides most likely arise from unloading of sucrose and metabolism in cells near to the sieve tubes; phloem transport of hexoses usually does not occur. Considering the low percentages of raffinose an analogous origin may be assumed.
Myo-inositol was found in the sieve tube saps of various plants, among them also species of Asteraceae (Zimmermann & Ziegler, 1975). The lack of radioactive myo-inositol outside of the treated leaf blade (Table 5) suggests that the compound is not transported in the phloem of sunflower.
Finally, we identified trehalose in stem extracts (Table 5; Fig. 5). Higher plants do not commonly contain notable amounts of this sugar. Trehalose in plants is discussed in the context of drought, freezing or heat tolerance and, most recently, of regulation in carbohydrate partitioning (Müller et al., 2001). Under sterile growth conditions a trehalose : sucrose ratio of 1/70 was found (Müller et al., 2001) in Arabidopsis. In sunflower, grown in an environment not free from microorganisms, the ratio was much higher (1/4; Table 5). We cannot exclude the possibility that considerable amounts of trehalose in our experiments originated from microbial contamination (see Discussion in Müller et al., 2001). However, due to the lack of radioactivity in trehalose it was obviously not synthesized and transported in sunflower.
On the basis of the 13C- and 14C-sugar analyses presented here sucrose can be considered as the only carbohydrate translocated in sunflower phloem. However, the analysis of the sieve tube sap would complete the proof. To our knowledge, this has not been done for sunflower.