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Keywords:

  • 13C;
  • 14C pulse-chase experiment;
  • distribution pattern;
  • Helianthus annuus (sunflower);
  • phloem transport;
  • phyllotaxy;
  • source–sink relationship

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • •  
    Photoassimilate transport from source leaves to the capitulum was investigated in sunflower (Helianthus annuus) during anthesis and seed filling.
  • •  
    Following foliar application of a 13/14CO2-pulse, labelled photoassimilates were detected using mass spectrometry, phosphorimaging, HPTLC and HPLC.
  • •  
    The upper 10 (to 15) leaves exported photoassimilates into the capitulum. Photoassimilate distribution patterns were sectorial: each leaf supplied a defined 2/8–3/8 sector of the capitulum. Photoassimilates exported via the midvein accumulated in a 1/8 sector, which aligned exactly with the insertion site of the leaf. The two main lateral veins of the leaf exported photoassimilates into the two adjacent 1/8 sectors of the capitulum. During early and late stages of anthesis, strong sinks were staminate florets and young achenes, respectively. During seed filling, an import maximum and minimum appeared in the intermediate and central whorls, respectively. Sucrose was established as the only phloem transport sugar. Raffinose, although also 14C-labelled in the path, is not transported in sunflower.
  • •  
    It is concluded that a single floret is typically connected with the leaves of three neighbouring ortostichies in sunflower. Photoassimilate distribution patterns demonstrated here generally may reflect the functional relationships between the phyllotaxy of source leaves and the position of sinks in developing inflorescences like those of Asteraceae.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In sunflowers, 73–85% of carbon accumulated in seeds (achenes) derives from carbohydrates of the current photosynthesis (Hall et al., 1990) referred to as photoassimilates. The complementary C-proportion is allocated from storage sinks, which had been filled during the preanthesis period. The largest upper leaves, exhibiting the highest photosynthetic capacity (English et al., 1979), export 80% of the dry matter incorporated in achenes (McWilliam et al., 1974). Thus, growth of achenes largely depends on phloem transport from upper, fully expanded, green leaves to the capitulum. Improved assimilate supply to growing achenes is regarded as the main factor, which may increase the yield potential of modern sunflower hybrids (Lopez Pereira et al., 1999). Several investigations have dealt with source–sink relationships and biomass partitioning in sunflowers (Connor & Hall, 1997). For instance, leaf removal on one half of the stem caused asymmetric development of the head (Caldwell, 1930; Prokofiev et al., 1957). Compared with the control, floret development lagged behind at the defoliated side. Further, the treated half produced smaller achenes. Moreover, foliar application of [14C]acetate resulted in a sectorial 14C-labelling of the receptacle (Prokofiev et al., 1957). These results suggest that photoassimilate distribution is laterally restricted in the stem and the capitulum. However, the phloem pathway was not directly investigated. Therefore, the first objective of the present paper was to analyse photoassimilate transport from source leaves to the capitulum, in particular to describe photoassimilate distribution in the capitulum in dependence on export from leaves of defined insertion sites.

The sunflower capitulum contains hundreds of florets or achenes. Each of them represents an individual sink. Photoassimilate transport depends on the physiological activity and the competition of these sinks, both changing in the course of ontogeny. Photoassimilate distribution in the capitulum was studied during the preanthesis (Hernandez & Palmer, 1992) and postanthesis stages (Goffner et al., 1988; Luthra et al., 1991), whereas the period between floret opening of outer whorls and seed filling has not been as intensively investigated. Therefore, the second objective of the present study was to analyse photoassimilate distribution in the capitulum during anthesis and seed filling.

Regarding the recent progress in research on phloem transport of macromolecules (Oparka & Cruz, 2000), hormones (Baker, 2000) and even lipids (Madey et al., 2002), one might expect only a few open questions to be left in the field of carbohydrate transport. However, in the large family of Asteraceae sieve tube carbohydrates have been scarcely analysed. For sunflower, although a widely used object in botany, no comprehensive report on the phloem transport sugars exists. Sucrose is evidently the main transport sugar (e.g. Ito & Mitsumori, 1992). However, in the present study raffinose and myo-inositol were also found in petiole and stem. Both substances are constituents of sieve tube saps in other species (Zimmermann & Ziegler, 1975). Motivated through these facts we evaluated sunflower carbohydrates concerning their role in photoassimilate transport.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plants

For 13C-labelling experiments sunflowers (Helianthus annuus L. cvs. Eurosol and Rigasol; Deutsche Cargill GmbH, Salzgitter, Germany) were grown at the experimental station Julius-Kühn-Feld (Martin-Luther-Universität Halle–Wittenberg, Landwirtschaftliche Fakultät, 51°4′ N, 11°7′ E) in Halle (Saale), Germany. To reduce interplant competition, row spacing was 1 m and the distance between plants in the row was 0.75 m. The total number of leaves per plant ranged from 22 to 27 (median: 24). Plant height, capitulum diameter and total achene number at maturity were 126 cm, 28 cm and 1537, respectively (medians).

For 14C-labelling experiments sunflowers (cv. Rigasol) were grown in pots (potting compost: Fruhstorfer Erde; Industrie-Erdenwerk Archut GmbH & Co. KG, Lauterbach/Wallenrod, Germany) in a glasshouse at 22 ± 1/16 ± 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 at the top of the plants. Total number of leaves per plant in the greenhouse ranged from 17 to 20 (median: 19). Plant height, capitulum diameter and total achene number at maturity were 116 cm, 10 cm and 544, respectively (medians).

Transport of 13C-photoassimilates

Transport of 13C-photoassimilates from upper 15 source leaves to the capitulum was examined during anthesis and seed filling (2–26 d after first anthesis (DAF); first anthesis was defined as opening of the florets in the first two whorls). Leaves were counted from the top, with leaf 1 denoting the most apical true leaf (Fig. 1a). Standardly, a leaf chamber, 8 cm in diameter, was fixed to the centre of the leaf blade, enclosing an equal portion of the leaf blade on either side of the midvein. To study whether the position of the leaf chamber on the leaf affects the 13C-distribution in the capitulum, two leaf chambers were placed to the periphery of the leaf blade enclosing a main lateral vein each. The chamber was gas-tightly connected with a tube containing 20 mg [13C]Na2CO3 (99 atom%13C; Aldrich Chemicals Co., Milwaukee, WI, USA). 13CO2 was released by injecting 200 µl of 25% H2SO4 at 60–80°C and pumped to the leaf chamber for a 10-min exposure. After a chase period of 24 h, samples consisting of 10–20 buds or florets, or 2–5 filled achenes were taken from the capitulum. Empty achenes were not sampled. Since the upper part of the sunflower stem exhibits 3/8 phyllotaxy, the capitulum was divided into eight main sectors, numbered clockwise 1–8 (Fig. 1b, solid lines). Sector 1 aligns with leaves 1 and 9, sector 2 aligns with leaves 2 and 10 and so on. Main sectors were further divided into three subsectors, referred to, in a clockwise direction, as left, middle and right (Fig. 1b, dashed lines). In each sector, samples were taken from six concentric zones 1–6, equal in width. Zones 1 and 6 denote the most peripheral and the central zone, respectively (Fig. 1c).

image

Figure 1. Nomenclature in 13/14C-photoassimilate transport experiments. (a) Typical sunflower plant used, total number of leaves ranged from 22 to 27 per plant; c, f and s, remnants of cotyledons, first and second pair of true leaves, respectively. Leaf positions were numbered basipetally; leaf 1 is the most apical true leaf clearly different from involucral bracts. A single leaf between 1 and 15 was exposed either to 13CO2 or 14CO2. (b) For 13C-analysis the capitulum was divided into eight main sectors (1–8), corresponding to the 3/8 phyllotaxy. The orientations of leaves 1, 5, 10 and 15 are shown in relation to the sectors. Each main sector was further divided into three subsectors (left, middle and right). (c) Sectors were divided into six zones (1–6).

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Samples were frozen in liquid nitrogen, freeze dried (Lyovac GT 2; AMSCO/FINN-AQUA, Hürth, Germany), homogenized and combusted (2 mg) in an element analyser (ANCA-NT Solid/Liquid Preparation Module; Europa Scientific Ltd, Crewe, UK). Released CO2 was analysed for 13C : 12C isotope ratio using a mass spectrometer (20–20 Stable Isotope Analyser; Europa Scientific Ltd, Crewe, UK). Carbon isotope ratio, given as δ13C, is the per mil-deviation of the 13C : 12C ratio of a sample from that of the PDB (Pee Dee Belemnite) standard. Acetanilide (δ13C = −33.24‰) was used as a working standard. For each plant, δ13C of wilted petals of disk florets (sampled at the end of the chase period), ranging from −27.41‰ to −25.20‰, was used for background correction.

13C-enrichment (denoted here as Δδ13C) was calculated from:

  • Δ δ 13C [‰-points] = δ13C [‰] − δ13Cbackground[‰].

We considered differences in Δδ13C less than 2‰-points as insignificant between samples from one and the same plant. This is due to the natural variability of δ13C. In a plant not exposed to 13CO2, δ13C of peripheral florets and central achenes amounted to −27.0‰ and −25.4‰, respectively.

  • To allow quantitative comparison of distribution patterns between plants of different 13C-enrichment, relative 13C-enrichment (denoted here as Δδ13Crelative) was calculated from:

  • Δ δ 13Crelative = Δδ13C/Δδ13Cmax

where Δδ13Cmax is the highest 13C-enrichment measured in the given capitulum.

Transport of 14C-photoassimilates

14C-photoassimilate distribution in the capitulum was studied during seed filling (16–38 DAF). Plants were cut 2 d before 14CO2-application. The excised apical shoot, 50 cm in length, was kept in 1 : 20 diluted Hoagland's nutrient solution in a climate chamber (HPS 500; Heraeus-Vötsch, Balingen, Germany) under the following conditions: 06.00 h to 22.00 h/22.00 h to 06.00 h day/night, 140/0 µmol m−2 s−1 photosynthetic photon flux, 22 ± 1/16 ± 1°C temperature and 60/80% relative humidity. Analogously to 13CO2-application, 250 kBq 14CO2 was released from 10 µl aqueous solution of [14C]Na2CO3 (1.67 GBq mmol−1; Institut für Kernforschung, Dresden, Germany) by adding 20 µl of 25% H2SO4 and applied to a source leaf of defined insertion using the leaf chamber. The exposure lasted 5 min. After a chase period of 3 h the capitulum was excised, photographed and freeze dried. Every single achene was numbered, taken from the capitulum and cut in half longitudinally, between the cotyledons. To determine radioactivity, half-achenes were placed with the cut surface towards 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). Radioactivity of the half-achenes was quantified using the software Tina 2.0 (Fuji, Tokyo, Japan) in profile mode.

Sugar analysis

Both 13C- and 14C-pulse labelled plants were used to study the phloem transport sugars. After a chase period of 24 h (13C-labelling) or 1 h (14C-labelling) the following samples were analysed: leaf blade and petiole of the exposed leaf, stem tissue below and above the exposed leaf. Parenchymateous tissue (pith) was removed from petiole and stem to enrich the vascular tissue. Samples were frozen in liquid nitrogen and freeze dried. Soluble carbohydrates were extracted from 0.2 g ground samples in 80% (v/v) ethanol at room temperature for 3 h. Suspensions were filtered and, to remove lipids, partitioned against chloroform. Ionic compounds were removed through ion-exchange chromatography using AG 3-X4 and AG 50 W-X4 (Bio-Rad, Richmond, CA, USA) in Bakerbond spe columns with 20 µm pore-filters (Baker, Phillipsburg, NJ, USA). The effluent, referred to as neutral fraction, contained soluble neutral sugars and sugar alcohols, which were separated using high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC).

For HPTLC separation, 1-cm-wide bands of the neutral fraction were blotted with a micro syringe on 10 cm × 10 cm plates (Silica gel 60; Merck, Darmstadt, Germany). Two solvent systems (methods I and II) were used to develop chromatograms. Method I: Five-fold development in chloroform/glacial acetic acid/water, 30/35/5 (v/v) (Rothe et al., 1999). Method II: Two-fold development in 2-propanol/chloroform/glacial acetic acid/10% ammonia, 45/45/20/10 (v/v) followed by a five-fold development in acetonitril/water, 85/15 (v/v) (Schlesinger, 1995). Compounds were identified by co-chromatography of sugar standards (Table 1) and by re-chromatography using HPLC. The plates were stained with lead(IV)acetate-2′,7′-dichlorofluorescein reagent (Sigma-Aldrich, Steinheim, Germany; Jork et al., 1990) or orcinol reagent (Sigma, Deisenhofen, Germany) to visualize compounds in UV or daylight, respectively. Fluorochromatograms were scanned using the phosphofluoroimager Storm (Molecular Dynamics, Sunnyvale, CA, USA). To determine radioactivity, plates were placed on a storage phosphor screen for 13–20 h. Exposed screens were scanned using a phosphorimager, and relative amounts and radioactivity of carbohydrates were quantified using Tina 2.0 (see Transport of 13C-photoassimilates).

Table 1.  Chromatographic analyses of soluble carbohydrates in sunflower. HPTLC: Stationary phase, silica gel 60; mobile phase in method I, chloroform/glacial acetic acid/water, 30/35/5 (v/v), 5-fold development; mobile phase in method II, 2-propanol/chloroform/glacial acetic acid/10% ammonia, 45/45/20/10 (v/v), 2-fold development followed by acetonitril/water, 85/15 (v/v), 5-fold development. Substances were localized by staining with lead(IV)acetate-2′,7′-dichlorofluorescein-reagent. RFrc= a/b; a, migration distance of the substance; b, migration distance of fructose; n= 10. HPLC: Stationary phase, polyspher CA CH column; mobile phase, water; 0.3 mL/min, 75°C. Rt= retention time
SubstanceHPTLC Method IRFrcHPTLC Method IIRFrcHPLCRt (min)
Stachyose0.16 ± 0.0140.78 ± 0.004  8.56
Galactinol0.37 ± 0.0160.14 ± 0.00812.27
Raffinose0.37 ± 0.0160.27 ± 0.016  9.39
Trehalose0.56 ± 0.0190.51 ± 0.04810.69
Maltose0.61 ± 0.0150.61 ± 0.02010.94
Sucrose0.70 ± 0.0120.73 ± 0.02010.75
Myo-inositol0.79 ± 0.0100.37 ± 0.01816.39
Sorbitol0.86 ± 0.0030.81 ± 0.01921.58
Galactose0.88 ± 0.0080.88 ± 0.01814.85
Glucose0.93 ± 0.0070.94 ± 0.00913.44
Fructose1116.10

HPLC separation was carried out on a Polyspher CH CA column (Merck, Darmstadt, Germany) at 75°C, using water as eluent. The flow rate was 0.3 ml min−1 (for details, see Rothe et al., 1999). Retention times of standards are listed in Table 1.

13C-labelled carbohydrates were separated using preparative thin layer chromatography. Neutral fractions were blotted as 7-cm-wide bands on HPTLC plates. Five carbohydrate fractions (raffinose and oligosaccharides of identical or lower RFrc-values; trehalose; sucrose; myo-inositol; glucose and fructose) and a sugar-free silica gel sample (for δ13C background) were scraped off the HPTLC-plates. Samples were suspended in water and centrifuged. Sediments were washed twice with water and pooled supernatants were freeze-dried. Dry samples were resolved in 60–150 µl water. Aliquots were used for δ13C measurement in the mass spectrometer (see Transport of 13C-photoassimilates).

Statistics

Unless stated otherwise, data are given as means ± standard deviation and n indicates the number of plants analysed.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Results and discussion focus on three topics: photoassimilate transport from upper source leaves to the capitulum; photoassimilate distribution in the capitulum during anthesis and seed filling; and nature of the phloem transport sugars in sunflower.

Photoassimilate transport from upper source leaves to the capitulum

When 13CO2 was applied to the upper 10 leaves labelled photoassimilates moved acropetally to the capitulum. After exposing the centre of the leaf blade of leaf 1, 5 or 10 to 13CO2, 13C-label was detected in sector 1, 5 or 2, respectively (Fig. 2a–c; for sectors, see Fig. 1b). In few plants small amounts of 13C-label were also found in adjacent sectors, for instance, in sector 6 following exposure of leaf 5 (Fig. 2b). A more detailed analysis revealed that 13C-enrichment was present in four subsectors (Fig. 2d). Generally, when 13CO2 was applied to the centre of one of the upper leaves (leaf positions 1–12) at different developmental stages of the capitulum, 13C-enrichment appeared in a 2/8–3/8 sector of the capitulum (n = 64). Further, the maximum 13C-enrichment was always located inside the 1/8-sector, which had the same orientation as the insertion site of the exposed leaf. When 13CO2 was applied to leaf 15, acropetal transport to the capitulum was found in only one of six plants. In this case 13C-enrichment was detected in sector 7 (data not shown).

image

Figure 2. Transport of photoassimilates into the sunflower capitulum following 13CO2-application to leaf 1, 5 and 10 at different stages of seed filling. The leaf chamber for 13CO2-exposure was fixed to the centre of the leaf blade. After a chase period of 24 h samples were taken from the capitulum and analysed for δ13C using a mass spectrometer. For sampling and orientation of the exposed leaves, see Fig. 1. Data for the zones of maximum 13C-import are displayed for the middle subsectors (a–c) or for each subsector (d). Background (dashed lines) = 2 [‰-points]/Δδ13Cmax[‰-points]; Δδ13Cmax is the highest 13C-enrichment measured in the given capitulum.

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Positioning the leaf chamber on the midvein was the standard method. The effect of the position of the leaf chamber on the leaf was investigated using two chambers. Data in Table 2 (midvein) show a characteristic 13C-distribution pattern following 13CO2-application to the centre of the leaf 6: Most prominent 13C-accumulation was found in the middle and right subsectors of the main sector 6. Qualitatively similar results were obtained with 13CO2-application to other leaves (n = 65). Distribution patterns in the capitulum changed considerably when two leaf chambers for 13CO2-application were placed on the periphery of the leaf enclosing the main lateral veins and excluding the midvein (Table 2, side veins). 13CO2-application to the lateral areas of leaf 6 (n = 2) resulted in two separated sectors of maximum 13C-enrichment in the capitulum: in the right subsector 5 and in the left subsector 7. Minor amounts of 13C-label were measured in sector 6. Exposition of leaf 10 (n = 1) resulted in an analogous pattern.

Table 2.  Transport of photoassimilates into the sunflower capitulum following 13CO2-application to leaf 6 using one or two leaf chambers during seed filling. One leaf chamber for 13CO2-exposure was fixed to the centre of the leaf blade (midvein) or two leaf chambers were placed to the periphery of the leaf blade, enclosing one main lateral vein each (side veins). The illustrations show the approximate position and size of the leaf chambers in relation to the leaf. After a chase period of 24 h samples were taken from the capitulum and analysed for δ13C using a mass spectrometer. The capitulum was divided into main sectors, subsectors and zones (see Fig. 1). Main sector 6 aligned with leaf 6. Boxes mark highest 13C-enrichments. Similar results were obtained when leaves of other positions were exposed to 13CO2; midvein, n= 65; side veins, n= 3
Position of leaf chambersZone13C-enrichment, Δδ13C (‰-points)
Main sector
45678
Subsector
rightleftmiddlerightleftmiddleright  leftmiddlerightleft
midvein inline image1 0.030.18  0.392.4816.6225.83  3.43  1.260.53 
2 0.430.56  0.442.8221.9620.73  3.10  1.490.70 
3 0.660.38  0.922.8816.6426.25  1.82  2.070.74 
4 0.860.41  1.095.9225.6413.11  3.80  1.500.60 
5 0.740.21  0.824.536.79  5.45  4.83  1.400.55 
6  1.70  13.99   5.72   
side veins inline image10.961.095.959.862.78  3.265.76  2.93  1.821.140.72
21.061.389.3712.542.49  3.283.528.25  6.302.051.41
30.950.995.0612.594.67  3.173.797.34  5.621.200.99
41.181.364.5410.416.64  3.888.619.69  6.581.091.11
51.461.802.367.615.42  5.359.8010.7810.492.851.41
61.43  6.24   9.17  3.83

14C-photoassimilate distribution was analysed on the level of single achenes (Fig. 3). Maximum import in the capitulum was restricted to few, neighbouring, radially arranged achenes. In the capitulum shown in Fig. 3 these are three achenes with a radioactivity > 50 PSL (PSL = photostimulated luminescence, a unit defined by the manufacturer; PSL is proportional to radioactivity). The position of these achenes exactly aligned with the insertion site of the exposed leaf. On both sides of the maximum gradually decreasing amounts of radiolabel were found. Generally, radioactivity was detected in a sector comprising 2/8–3/8 of the capitulum (n = 9; 16–38 DAF), whereas maximum 14C-label appeared in different whorls.

image

Figure 3. Transport of photoassimilates into the sunflower capitulum following 14CO2-application to leaf 6 during seed filling. The leaf chamber for 14CO2-exposure was fixed to the centre of the leaf blade. The arrow indicates the orientation of the insertion site of the exposed leaf. After a chase period of 3 h every single achene was taken from the capitulum and analysed for radioactivity using a phosphorimager. The distribution pattern shown was obtained from one plant; altogether, nine plants were investigated, with similar results. Achenes without symbol, radioactivity < 1 PSL mm−1; achenes with crosses, 1 PSL mm−1 < radioactivity < 14 PSL mm−1; achenes with figures, radioactivity in PSL mm−1; PSL = quantity of photostimulated luminescence.

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Photoassimilate distribution in the capitulum during anthesis and seed filling

Studies on sink competition during the different developmental stages (Tables 3 and 4) started when anthesis of disk florets in the peripheral sixth of the capitulum was completed (Table 3, plant 1, 2 DAF). Zone 1 (for zones, see Fig. 1c) contained small, developing achenes. Florets of the adjacent whorls (zones 2 and 3) were in the pistillate and staminate stage, respectively. Zones 4–6 were filled with buds. At this stage staminate florets attracted the highest amounts of photoassimilates. The import peak, attributed to the staminate florets, was found during early stages of the anthesis (plants 1–3). In the course of further anthesis relative photoassimilate import changed, and the young achenes became the strongest sinks while the staminate florets incorporated less 13C-label (plants 4–9).

Table 3.  Transport of photoassimilates into the sunflower capitulum following 13CO2-application at different stages of anthesis
PlantDAFRelative 13C-enrichment, Δδ13Crelative
Zone 1Zone 2Zone 3Zone 4Zone 5Zone 6
  1. Leaf chamber for 13CO2-exposure was fixed to the centre of the leaf blade (leaf positions 1–15). After a chase period of 24 h samples were taken from the capitulum and analysed for δ13C using a mass spectrometer. Relative 13C-enrichments in zones 1–6 are shown for the subsector of maximum import of each plant (sectors and zones, see Fig. 1). For each plant, the zones with a relative 13C-enrichment > 0.90 are marked by boxes. Letters indicate the developmental stage of the florets in each zone: A, developing achenes; P, florets at pistillate stage, stigma lobes exerted; S, florets at staminate stage, anther tubes exerted; B, floret buds; DAF, days after first flowering.

1  20.49 A0.30 P1 S0.32 B0.20 B0.11 B
2  30.65 A0.69 A0.58 P1 S0.48 B0.44 B
3  40.52 A0.51 A0.56 A0.61 P1 S0.56 B
4  50.86 A0.81 A1 A0.27 P0.36 S0.38 B
5  61 A0.89 A0.77 P0.34 P0.24 S0.13 S
6  70.57 A1 A0.84 A0.23 P0.10 P0.04 S
7  80.38 A0.43 A0.59 A1 A0.92 P0.39 S
8  90.43 A0.62 A0.82 A1 A0.86 P0.54 P
9100.67 A0.70 A0.87 A1 A0.57 A0.26 P
Table 4.  Transport of photoassimilates into the sunflower capitulum following 13CO2-application during seed filling
PlantDAFRelative 13C-enrichment, Δδ13Crelative
Zone 1Zone 2Zone 3Zone 4Zone 5Zone 6
  1. The end of anthesis was 11 DAF. All zones contained developing achenes. DAF, days after first flowering. For further details, see Table 3.

1160.53     0.980.7910.620.41
2160.200.210.220.220.491
3160.771     10.820.580.44
4160.510.750.660.89     10.84
5220.770.8110.980.570.23
6220.470.490.580.83     10.71
7220.340.480.69     10.750.48
8220.72     0.910.96     10.640.21

During seed filling, monitored between 12 DAF (anthesis of the whole capitulum completed) and 26 DAF (late stage of seed filling, back of the capitulum yellow), relative photoassimilate import into different zones of the capitulum showed high variability (Table 4). Comparing the 13C-distribution in the capitulum of plants that were grown under the same conditions and analysed at the same developmental stage, maximum 13C-import appeared in different zones.

Records of cumulative 13C-import activity, calculated on the basis of the relative 13C-enrichment, showed the following characteristics (Fig. 4): (i) Import activities in zones 1–3 and 5 were nearly identical, and constant between 12 and 23 DAF. Notably, the hierarchy, zone 1 < zone 2 < zone 3, did not change during the period monitored. (ii) Relative import into zone 4 was similar to that of zones 1–3 up to 18 DAF. Thereafter, import into zone 4 increased, while relative import into zones 5 and 6 decreased. (iii) Zone 6 always exhibited the lowest 13C-import. It was considerably lower than import into zones 1–5.

image

Figure 4. Transport of photoassimilates into the sunflower capitulum following 13CO2-application to a leaf during seed filling. Leaf chamber for 13CO2-exposure was fixed to the centre of the leaf blade (leaf positions 1–12). After a chase period of 24 h samples were taken from the capitulum and analysed for δ13C using a mass spectrometer. Relative 13C-enrichments were calculated for zones 1–6 of the sector of highest 13C-enrichment (see Fig. 1). Curves show the cumulative relative 13C-enrichment in each zone. If more than one plant per day were analysed, data were averaged across plants; n = 45. DAF, days after first flowering.

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Phloem transport sugars

Chromatographic separation of the neutral extracts obtained from leaf blade, petiole and stem of sunflower revealed that glucose (32–40%), fructose (20–28%) and sucrose (12–22%) were the most abundant sugars (Table 5; Fig. 5, thin lines). Lower amounts of myo-inositol (6–17%), trehalose (0.3–7%) and raffinose (0.9–3%) were found in most extracts. However, trehalose was absent in the petiole.

Table 5.  Chromatographic analyses of soluble carbohydrates from sunflower. 14CO2 was applied to the leaf 5 during seed filling. Leaf blade, petiole and stem were extracted after a chase period of 1 h. Neutral fractions of ethanol extracts were separated using HPTLC (method I); plates were dyed with lead(IV)acetat-2′,7′-dichlorofluorescein-reagent. Fluorescent traces were recorded using a phosphofluoroimager. Radioactivity was detected using a phosphorimager. Optical density and radioactivity were corrected for background; n= 3
TissueRelative content (%)
RaffinoseTrehaloseSucroseMyo-inositolGlucoseFructoseRest
Exposed leaf blade1.1 ± 1.00.3 ± 0.211.7 ± 2.116.5 ± 3.233.4 ± 2.224.9 ± 2.312.1 ± 5.3
Petiole of the exposed leaf0.9 ± 0.4021.5 ± 1.7  9.3 ± 1.139.8 ± 1.020.3 ± 2.6  8.2 ± 4.6
Stem above exposed leaf3.0 ± 1.16.9 ± 2.819.8 ± 1.7  6.6 ± 2.831.6 ± 0.726.7 ± 1.9  5.3 ± 2.4
Stem below exposed leaf2.6 ± 1.26.5 ± 3.419.1 ± 1.6  6.2 ± 2.632.2 ± 2.028.4 ± 2.0  5.0 ± 2.3
TissueRelative radioactivity (%)
RaffinoseTrehaloseSucroseMyo-inositolGlucoseFructoseRest
Exposed leaf blade0.8 ± 0.5080.5 ± 8.2  0.9 ± 0.78.4 ± 3.56.9 ± 3.1  2.5 ± 1.2
Petiole of the exposed leaf0093.9 ± 2.9  03.0 ± 1.72.8 ± 1.5  0.4 ± 0.3
Stem above exposed leaf1.6 ± 1.6094.9 ± 3.0  02.3 ± 1.21.6 ± 0.4  0.1 ± 0.2
Stem below exposed leaf0.6 ± 0.4097.5 ± 2.4  01.7 ± 0.71.2 ± 0.4  0.1 ± 0.1
image

Figure 5. Chromatographic analyses of soluble carbohydrates from sunflower. 14CO2 was applied to the leaf 5. Leaf blade (a), petiole (b) and stem (c) were extracted after a chase period of 1 h. Neutral fractions of ethanol extracts were separated using HPTLC (method I); plates were dyed with lead(IV)acetat-2′,7′-dichlorofluorescein-reagent. Fluorescent traces were recorded using a phosphofluoroimager (thin line). Radioactivity was detected using a phosphorimager (bold line). Optical density and radioactivity were corrected for background; n = 3. Raf, raffinose; Tre, trehalose; Suc, sucrose; Ino, myo-inositol; Glc, glucose; Frc, fructose.

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Phloem transport sugars were analysed on the basis of 14C- and 13C-pulse labelled carbohydrates. 14C-labelling provides a lower detection limit to analyse minor amounts of sugars but it can be applied to greenhouse plants in laboratory conditions only. 13C-labelling can be carried out in the field but the technique provides less sensitive analyses.

Following 14CO2-application all analysed carbohydrates (except trehalose) from the leaf blade contained 14C-label 1 h after exposure (Table 5; Fig. 5, bold lines). In petiole and stem sucrose contained the highest 14C-proportion (94–98%). Less radioactivity was found in glucose (2–3%), fructose (1–3%) and raffinose (0.6–2%). The latter sugar was not 14C-labelled in the petiole. Labelled myo-inositol was absent outside of the leaf blade.

Following 13CO2-application glucose and fructose were the most prominent labelled sugars in the leaf blade after 24 h (δ13C from 3.4‰ to 237‰; Table 6). Sucrose, myo-inositol and raffinose exhibited minor 13C-label in leaf blade extracts. In the petiole sucrose was the only sugar enriched above background level.

Table 6.  Chromatographic analyses of soluble carbohydrates from sunflower. 13CO2 was applied to the leaf 1, 5, 6 or 10 during seed filling. Leaf blade and petiole were extracted after a chase period of 24 h. Neutral fractions of ethanol extracts were separated using HPTLC (method I). Substance bands were scraped off the HPTLC-plates, eluted in water and analysed for δ13C using a mass spectrometer; background =−17.7%; n.d., not determined
TissuePlantCarbon isotope ratio, δ13C (‰)
Neutral fractionRaffinoseTrehaloseSucroseMyo- InositolGlucose + Fructose
Exposed leaf blade1287  13.9n.d.  32.6  33.6237
2  11.5 −12.9n.d. −12.7 −16.9    8.9
3     −0.3   −7.7 −14.6 −14.1 −15.2    3.4
Petiole of the exposed leaf4 −19.3 −19.0 −20.1    5.9 −20.5 −21.8
5 −18.1 −16.8 −18.7    5.1n.d. −20.8
6 −18.0 −18.1 −19.7   −9.0 −16.9 −19.2
7 −17.6 −17.9 −20.5   −1.1 −17.9 −19.8
8 −15.1n.d.n.d.   −7.9 −19.2 −15.6

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Assimilate import is accompanied by accumulation of soluble carbohydrates and starch in younger achenes and by an increased oil synthesis in older ones (Luthra et al., 1991; Goffner et al., 1988). During seed filling, dry mass increase is either linear (Connor & Hall, 1997) or it reaches a maximum after 3–4 wk (Goffner et al., 1988; Luthra et al., 1991).

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Moritz Knoche for critical discussions. Further, we are grateful to Suzanne Roß and Edith Fuß for skilled technical assistance. The research was supported by the Deutsche Forschungsgemeinschaft.

References

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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