The enclosed and exposed part of the peduncle of wheat (Triticum aestivum) – spatial separation of fructan storage

Authors

  • Thomas Gebbing

    Corresponding author
    1. Grassland Science, Technische Universität München, 85350 Freising, Germany; Present address: Institute for Plant Production, University of Bonn, 53115 Bonn, Germany
      Author for correspondence: Thomas Gebbing Tel: +49 228736849 Fax: +49 228732870 Email: t.gebbing@uni-bonn.de
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Author for correspondence: Thomas Gebbing Tel: +49 228736849 Fax: +49 228732870 Email: t.gebbing@uni-bonn.de

Summary

  • • Although fructan accumulation is reported in photosynthetically active organs, the long-term storage of fructan mainly occurs in more heterotrophic tissues. Significant amounts of fructan are stored in the internodes during grain filling of wheat (Triticum aestivum). The uppermost internode (peduncle) of wheat consists of a lower unexposed (i.e. enclosed by the flag leaf sheath and thus heterotrophic part, Pl) and an upper exposed autotrophic part (Pu).
  • • Diurnal and long-term changes of fructan and sucrose (the precursor of fructan synthesis) contents were studied in Pl and Pu of potted wheat plants.
  • • At mid grain-filling the sucrose concentration in Pu increased almost threefold during the light period and decreased in the following night. Diurnal changes in sucrose concentration were much less expressed in Pl. Fructan concentration was significantly higher in Pl than in Pu and did not change during the light period.
  • • In another experiment, field grown wheat plants were sampled at regular intervals between 5 d before anthesis and grain maturity. At the time of maximum fructan content, 88% of the fructans in the total peduncle were stored in the heterotrophic Pl. Within Pl, fructan accumulation started in the older segments. The reason for the sharp separation of fructan storage between Pl and Pu remains unclear.

Introduction

Fructan and sucrose are the main water-soluble carbohydrates (WSC) in the vegetative plant parts of temperate grasses (Pollock, 1986; Pollock & Cairns, 1991). In vegetative parts of wheat the WSC contents reach considerable amounts (Spiertz & Ellen, 1978) and these carbohydrates are supposed to buffer grain yield as a long-term storage reserve (Schnyder, 1993; Gebbing et al., 1999).

In general, long-term storage of fructans occurs in heterotrophic sink tissues, which are dependent on the import of carbohydrates, e.g. in the tubers of Jerusalem artichoke (Kaeser, 1983), the root of chicory (Van den Ende & Van Laere, 1996), or in the internodes of wheat (Bonnett & Incoll, 1993). Significant fructan accumulation can also be found in growing tissues of grasses, e.g. leaf growth zones (Schnyder & Nelson, 1989) and wheat grains (Schnyder et al., 1988), which are also tissues of relatively low photosynthetic activity. From the occurrence of fructan metabolism in these tissues one may conclude that the role of fructan is not restricted to long-term carbohydrate storage only, as indicated by several authors (e.g. Bieleski, 1993; Pilon-Smits et al., 1995; Demel et al., 1998; Vijn & Smeekens, 1999; Vergauwen et al., 2000), though the role of fructan in increasing stress tolerance of, for example transgenic plants is still discussed controversially (Cairns, 2003). In a recent review fructan synthesis is assumed to be a mechanism to sustain sucrose gradients and thus control carbon metabolism in leaves (Pollock et al., 2003).

The internodes are the major storage sites where fructans are deposited until approximately mid grain filling (Bonnett & Incoll, 1993). The lower internodes of wheat (and other cereals) are mainly enclosed by the surrounding leaf sheaths and thus photosynthetic activity of these tissues is low or negligible (Wardlaw, 1965). But the uppermost internode, i.e. peduncle below the ear, is only partly enclosed by the flag leaf sheath and is thus partly exposed to a high irradiance (only shaded by the ear). This different light environment causes a more heterotrophic metabolism in the lower enclosed part (Wardlaw, 1965) and a more ‘leaf-like’ autotrophic carbohydrate metabolism in the upper part, respectively, and the upper part of the peduncle may contribute to the significant photosynthesis of the stem (including leaf sheaths) shown by Evans & Rawson (1970). Fructan contents in the total peduncle have been reported to be lower than in the penultimate internode below the peduncle (Willenbrink et al., 1998), but in this study the peduncle was taken as a whole, and whether the different light environment affects the distribution of fructan within the peduncle was not shown.

In photosynthetically active tissues, diurnal changes in sucrose content of leaves during the light and dark period (Jenner & Rathjen, 1972) were observed. Thus, the sucrose pool is often attributed to a reserve pool, buffering short-term fluctuations in assimilate supply (Schnyder, 1993). One might expect a similar diurnal change of the sucrose content in the (photosynthetically active) exposed peduncle part, but this had not yet been demonstrated.

In the present study the effect of the different light environment of the lower and upper peduncle part on carbohydrate storage and diurnal changes of carbohydrate contents were investigated.

Materials and Methods

Experiment A

Approx. 230 main tillers of field grown plants of wheat (cv. Sappo) (Poppelsdorf experimental field, Bonn, Germany) were selected for homogeneity in size and developmental stage (stage 51 on the Zadoks scale; first spikelet of inflorescence just visible; Zadoks et al., 1974) and tagged with a piece of wire. In the morning of the next day (5 d before anthesis) three replicates were sampled containing 4 peduncles each. Tillers were cut close to ground level. During later sampling at anthesis (stage 65; Zadoks et al., 1974), 3, 10, 16, 23, 30 and 36 days after anthesis (DAA)) 8 peduncles per replicate were collected. Sampling was conducted before 09:00 h at all sampling occasions. Tillers were cooled in boxes and immediately transported to the laboratory. Peduncles were cut above the node of the flag leaf sheath and the length of each peduncle was determined. To separate the growing tissues of the lower peduncle part from the more mature tissues, the unexposed part of the peduncle was dissected into five 2 cm segments (Pl1–Pl5) and one segment of variable length (depending on the length of the flag leaf sheath, Pl6), while the exposed part (Pu) was used as a whole according to the scheme in Fig. 1. Dissection started within approx. 15 min after tillers were cut. Peduncle segments were weighed, frozen in liquid nitrogen and stored at −27°C until freeze drying.

Figure 1.

Dissection of the peduncle of field grown wheat (Triticum aestivum) plants (Experiment A). The segments Pl1–Pl5 were each 2 cm in length.

Growth conditions were favourable for wheat during the grain filling period. The daily mean air temperature ranged between 15 and 28°C. Monthly mean temperature was 20°C in June and 21.4°C in July and thus slightly above the 30-year long-term average. The above average rainfall ensured sufficient water supply during the experiment.

Experiment B

Experiment B was detailed previously (Experiment 2 in Gebbing & Schnyder, 2001). Briefly, spring wheat plants were established singly in pots. At dusk of day 13 after anthesis (Anthesis: stage 65 on the Zadoks scale (Zadoks et al., 1974)) plants were transferred to a growth chamber (E15; Conviron, Winnipeg, Canada). Plants were kept in the dark for 8 h followed by a 16-h light period with a photosynthetic photon flux density (PPFD) of 700 µmol m−2 s−1 at ear height. Temperature and humidity were controlled at 20/14°C and 70/80% (light/dark), respectively. During the light and the second dark period plants (n = 3, four plants per replicate) were harvested and the main tillers of sampled plants were dissected. The internode below the ear (peduncle) was separated into the upper exposed (Pu) and the lower part (Pl). The f. wt was determined, than samples were frozen with liquid nitrogen and stored at −27°C until freeze drying.

Distribution of growth

In experiment A at −6 and −5 DAA the length of growth zone was determined using the pin hole technique (Schnyder et al., 1987). The base of the flag leaf sheath (and thus the growth zone of the peduncle) was punctured with a fine needle. The first hole was made right above the flag leaf node and consecutive holes were made at approx. 3 mm intervals in the acropetal direction. After 8 h punctured tillers were sampled and the distance of the holes in the flag leaf sheath and peduncle was measured using a magnifying glass with a micrometer scale. Displacement of holes in the peduncle was compared to the holes in the (nongrowing) flag leaf sheath to determine the growth distribution and length of the growth zone.

WSC content

The WSC contents were determined using procedures described previously (Thome & Kühbauch, 1985; Gebbing & Schnyder, 2001). Dried plant material was ground in a ball mill. For extraction of WSC, ground plant material was weighed in 100 ml screw capped plastic bottles and 40 ml of hot deionised water was added. Bottles were sealed, vigorously shaken, and put for 10 min in a water bath at 85°C. Afterwards, bottles were placed on a horizontal shaker for 30 min at room temperature. The aqueous extract was drained off through filter paper into a round bottom flask and dried in a vacuum rotary evaporator at 40°C. The dry residue was dissolved in 2 ml deionised water and after centrifugation, an aliquot of the supernatant was applied to a chromatographic column (Ionpack S802; Macherey-Nagel, Düren, Germany) for separation of fructan and sucrose. Carbohydrates were eluted with bi-distilled, degassed water and quantified by measuring the reducing power of hydrolysed carbohydrates with a potassium ferricyanide solution (Suzuki, 1971). Aqueous solutions (range 180–3000 ppm) of commercially available carbohydrates (inulin, sucrose, Merck, Germany) analysed between the samples were used as reference.

Results

Growth of the peduncle (Experiment A)

At −5 DAA peduncle length was 12.5 cm and totally enclosed by the flag leaf sheath (Fig. 2). Between −5 and 3 DAA the peduncle grew almost linearly at a mean rate of 28 mm d−1. The pinhole technique applied to the peduncle base showed that the length of the growth zone was 32 mm (data not shown). Thus, the growth zone approximately reproduced itself within 1 d during this period. Linear growth ceased after 3 DAA and the peduncle reached a final length of c. 40 cm (mean of all mature peduncles, n = 24). Variation of the length of the flag leaf sheath between replicates was small and ranged between 17.9 and 18.4 cm. The mean length of the flag leaf sheath was 18.2 cm. Thus, slightly more than half of the total peduncle length was exposed above the flag leaf sheath.

Figure 2.

Elongation of the peduncle during grain filling of wheat (Triticum aestivum). The shaded area indicates the mean length of the flag leaf sheath which encloses the lower peduncle part. SE were smaller than symbols.

WSC content

Short-term changes (Experiment B) At the beginning of the light period sucrose concentration was lower in Pu (9.5 mg g−1 f. wt) than in Pl (13.4 mg g−1; Fig. 3a). During the light period the sucrose concentration in Pu increased approx. threefold at a mean rate of 1.1 mg g−1 f. wt h−1 (r2 = 0.93). This accumulation of sucrose was followed by a decrease in subsequent dark (r2 = 0.89). Diurnal changes of the sucrose concentration were less in Pl. Sucrose concentration remained unchanged during the first 11 h of the light period. After 16 h of light, the sucrose concentration had increased by 7 mg g−1 f. wt. Thus, almost all of this increase occurred during the latter part of the light period.

Figure 3.

Diurnal change of the sucrose (a) and fructan (b) concentration in Pu (open circles) and Pl (closed circles) in potted wheat (Triticum aestivum) plants (Experiment B). Dark periods are shown by the black lines at the bottom. Vertical bars represent 2SE. The slope of the regression line was 1.1 mg g−1 f. wt h−1 (P < 0.01) during the light and −1.3 mg g−1 f. wt h−1 (P < 0.02) during the dark period, respectively.

In both peduncle parts, no diurnal change of the fructan concentration was found (Fig. 3b). On average, fructan concentration was 50.2 mg g−1 f. wt in Pl and thus about four times higher than in Pu (12.2 mg g−1 f. wt).

Long-term changes (Experiment A) The sucrose content in the peduncle increased 3.5-fold shortly after anthesis (Fig. 4a). When elongation had stopped (c. 10 DAA) the sucrose content was on average 12.8 mg per peduncle and remained unchanged during the mid grain filling period. During mid grain filling approx. two thirds of total sucrose were located in Pl. After 30 DAA the sucrose content decreased in both peduncle parts.

Figure 4.

Sucrose (a) and fructan (b) content in the peduncle during grain filling of field grown wheat (Triticum aestivum) (Experiment A). Carbohydrate content in Pl is indicated by the shaded area. Bars represent 2SE.

The fructan content in the peduncle was low at anthesis (1.3 mg per tiller) but increased rapidly to 24 mg per tiller at 16 DAA (Fig. 4b). Almost all of this fructan (88%) was stored in Pl. The following decrease in fructan content started sooner and was more rapid than the decrease of the sucrose content. At 30 DAA already 78% of the fructan in the total peduncle were mobilized.

Spatial distribution of sucrose and fructan in the peduncle (Experiment A)

At anthesis, sucrose concentration was < 3 mg g−1 f. wt in the lower segments of Pl (Pll−5, Fig. 5). A slight increase of the sucrose concentration in the more mature tissues of Pl6 occurred and in Pu the sucrose concentration was much higher (8.9 mg g−1 f. wt). At 10 DAA sucrose concentration had increased considerably in all segments. Highest concentrations were observed in the more mature peduncle segments. At 16 DAA, sucrose concentration was uniformly distributed in all Pl segments and the average concentration was 14.6 mg g−1 f. wt. During the later stages of grain filling, when the sucrose concentration began to decrease in Pl, the sucrose concentration in Pu reached its maximum at 15.5 mg g−1 f. wt. At maturity, sucrose concentrations had decreased in all peduncle segments and were on average 2.7 mg g−1 f. wt in Pl and 5.5 mg g−1 f. wt in Pu, respectively.

Figure 5.

Sucrose concentrations in the single peduncle segments during grain filling of wheat (Triticum aestivum) (experiment A). Bars indicate 2SE. DAA, days after anthesis. For definition of segments see annotations given in Fig. 1 from lowermost (Pl1) to uppermost (Pu) segment.

At anthesis the mean concentration of fructan was 1.5 mg g−1 f. wt in all peduncle segments (Fig. 6). At 10 DAA the concentration of fructan started to increase in the older segments of Pl. In the two youngest segments (Pl1 and Pl2) the fructan concentration remained still low. At mid grain filling when fructan content reached its maximum the fructan concentration was similar in all Pl segments and on average 36 mg g−1 f. wt, whereas in Pu it was only 4.8 mg g−1 f. wt. The decrease of the fructan concentration during late grain filling was similar in all Pl segments and thus at maturity fructan concentrations were uniformly distributed among the whole peduncle.

Figure 6.

Fructan concentrations in the single peduncle segments during grain filling of wheat (Triticum aestivum) (experiment A). Bars indicate 2SE. DAA, days after anthesis. For definition of segments see annotations given in Fig. 1 from lowermost (Pl1) to uppermost (Pu) segment.

Discussion

Growth of the peduncle

Elongation of the peduncle is similar to leaf elongation in grasses in that it is restricted to its tissues at the base of the internode, which is enclosed by the flag leaf sheath. At anthesis, the peduncle grew linearly in length at a rate of 28 mm d−1 (c. 1.2 mm h−1). Leaf elongation rate of wheat seedlings was reported to be higher and reached values up to 3 mm h−1 (e.g. Christ, 1978; Hu et al., 2000). In those studies, leaf elongation rate was measured at 20°C, which is within the optimum temperature range for growth of most temperate grasses (20–25°C; Cooper & Tainton, 1968). During peduncle elongation, mean daily air temperature ranged between 17 and 25°C. Lower temperatures during night might have caused a reduction of the mean peduncle elongation rate. Whether the peduncle elongation rate is always lower than leaf elongation rate is unclear. A high leaf elongation rate is crucial for the rapid development of light intercepting area in seedlings. Peduncle elongation is probably not primarily associated with the development of photosynthetically active area, but with exposure of the ear above the leaves. An increased distance between the upper leaves and the ear could lessen the risk of pathogen infection of the ear by ‘leaf borne’ diseases. This could be achieved by a high elongation rate, an extended duration of elongation, or a combination of both.

Elongation of the peduncle is followed by cell wall deposition (i.e. structural growth) to strengthen the newly built tissue (Lev-Yadun et al., 1999), which increases DM of the stem after anthesis (Gebbing et al., 1998). The lower sucrose concentration in the basal Pl segments until 10 DAA and the delayed accumulation of fructan in Pl1 and Pl2 might be attributable to this utilization of carbohydrates in structural growth. At mid grain-filling, Pu and Pl were approximately equal in mass (data not shown). Thus, the differences in WSC storage in these parts were almost exclusively attributable to differences in carbohydrate concentration.

Fructan and sucrose contents in the peduncle during grain filling

In the present field experiment the well described pattern of fructan accumulation in vegetative plant parts after anthesis and the mobilization of these reserves during later stages of grain filling was found (e.g. Blacklow et al., 1984; Kühbauch & Thome, 1989, for a review see Schnyder, 1993). The highest fructan content was observed at 16 DAA which is well in the range of 10–20 DAA given for temperate climate and favourable growth conditions (Schnyder, 1993). Higher carbohydrate (fructan) contents than in the peduncle were usually observed in the lower inserted internodes (Bonnett & Incoll, 1993; Willenbrink et al., 1998). But in most studies the peduncle was taken as a whole. In the present study the observed fructan concentrations in Pl were similar to fructan concentrations reported for the penultimate internode (Willenbrink et al., 1998).

At 14 DAA no diurnal changes in fructan concentration could be found, whereas the sucrose concentration changed significantly. This corroborates the assumed role of fructan in the wheat stem as a long-term reserve (Schnyder, 1993), which is not subject to turnover (Winzeler et al., 1990). Mobilization of fructans during late grain filling is associated with an increased activity of fructan exohydrolase (Simpson & Bonnett, 1993; Willenbrink et al., 1998). It has often been concluded from sink-source manipulation that this mobilization of reserves might buffer insufficient photosynthate production during late grain filling (Kühbauch & Thome, 1989).

Fructans do not accumulate in Pu

As expected from photosynthesising organs like leaves (Kalt-Torres et al., 1987) or glumes (Gebbing & Schnyder, 2001) the sucrose content in Pu showed significant diurnal variation. The photosynthetic activity of the peduncle is likely to contribute to stem photosynthesis observed by Evans & Rawson (1970). The increase of sucrose concentration during the light might be attributable to temporary sucrose storage in photosynthesising tissues, whereas the following decrease during the dark period is likely to be associated with export of sucrose or with respiratory metabolism.

The rate of sucrose accumulation during the light period of 1.1 mg g−1 f. wt h−1, was substantial. For comparison: in excised wheat leaves subjected to continuous light, sucrose concentration increased by c. 1.5 mg g−1 f. wt h−1 (at 300 µmol m−2 s−1 PPFD and 20°C (Penson & Cairns, 1994)), but in these leaves export of photosynthate was blocked completely by excision.

In both experiments, fructan concentrations were significantly higher in Pl than in Pu. Results indicate an abrupt discontinuity in fructan concentration between Pl6 and Pu during the fructan storage phase (16–23 DAA). Unfortunately, this discontinuity was not studied in spatial detail, but graphical interpolation (i.e. connecting the data points of Pl6 and Pu) excludes a linear decrease of the fructan concentration along these two compartments. Furthermore, changes of fructan concentrations in Pu were small between 10 and 23 DAA, whereas in Pl, the fructan concentration increased by a factor of c. 3. This also indicates a nonlinear decrease of fructan concentration between Pu and Pl, otherwise the fructan concentration in Pu would also have increased .

Much knowledge about fructan metabolism in grasses has been derived from experiments with excised leaf blades kept in a sucrose solution or exposed to continuous light (e.g. Penson & Cairns, 1994). Usually, fructan concentrations in leaf blades of grasses are low (Housley et al., 1989). But such treatments induce a rapid accumulation of WSC (mainly sucrose) followed by a change in gene expression (Winters et al., 1994) associated with fructan synthesis and subsequent accumulation of fructan (Wagner et al., 1983, 1986, Cairns & Pollock, 1988; Penson & Cairns, 1994). Thus, in these experiments fructan accumulated in tissues of high photosynthetic activity. Fructan accumulation is supposed to be a mechanism to sustain sucrose gradients between different cell types (Pollock et al., 2003). An increase of the sucrose concentration is assumed to be the signal for switching from export of carbohydrates to pathways associated with storage of fructan (Winters et al., 1994; Müller et al., 2000). Thus, fructan accumulation is induced by a certain sucrose concentration, which is in line with the definition of the fructan pool in leaves as an extension of the sucrose pool (Wiemken et al., 1995). The triggering sucrose concentration is considered to be c. 14–20 mg g−1 f. wt (Cairns & Pollock, 1988; Penson & Cairns, 1994).

In the present experiment, linear regression (Fig. 3a) showed that high (triggering) sucrose concentrations were reached after 10 h of light and stayed above this level for the next 10 h. In a study with excised leaves of Festuca, the application of inhibitors indicated that de novo synthesis of fructan synthesising enzymes started within 8 h after onset of sucrose accumulation and later (> 8 h) applications of inhibitors did not prevent accumulation of fructan (Winters et al., 1994). The peduncle contains the conducting tissues which transport all the photosynthate exported from the leaves to the growing grains, and part of the sucrose in the peduncle may be associated with the phloem (Willenbrink et al., 1998). The sucrose concentration in Pl increased during the latter part of the light period, which could be attributed to an increased flux of sucrose through the transport pathways, because photosynthesis of Pl is supposed to be negligible (Wardlaw, 1965). This may also hold for part of the sucrose accumulation observed in Pu. When this increase of sucrose in Pl was subtracted from the sucrose in Pu, thus correcting the sucrose concentration in Pu for this ‘transport sucrose’, the residual concentration of sucrose in Pu was still higher than 15 mg g−1 f. wt for more than 9 h (data not shown).

At 10 DAA the sucrose concentrations were similar in Pu and in Pl6. These concentrations were observed at 09:00 h and it is likely that the sucrose concentration in Pu had increased further during the day as in the growth chamber experiment. Thus, in both experiments the sucrose concentration seemed to be sufficient to induce fructan accumulation in both peduncle parts. But it is not known whether a higher sucrose concentration in Pu than in Pl is required to trigger fructan synthesis.

At least in part, the observed increase in sucrose concentration occurred in tissues which do not store fructan. For example the transport tissues are probably less sensitive to sucrose concentration in terms of triggering fructan synthesis, because besides one exception in Agave deserti (Wang & Nobel, 1998) fructans are virtually absent from the phloem. We are not aware of any study which determines the fraction of transport tissues in Pu and Pl. But the outer diameter of the peduncle decreases in the upper region (data not shown) and, assuming a constant conductivity for assimilates along the peduncle (i.e. a constant size of the transport pathways), this fraction of transport tissues might be higher in Pu than in Pl. Thus, there may be more of this nonfructan storing tissues in the upper part of the peduncle. Recent developments in single cell sampling and carbohydrate analysis showed different patterns of carbohydrate accumulation in different cell types of barley leaves (Koroleva et al., 1998). To our knowledge, such experiments with different cells of the peduncle have not yet been conducted.

The present study showed a strong spatial separation of the long-term storage of fructan within the peduncle. It remains unclear why fructan did not accumulate in the exposed photosynthetically active peduncle part. Fructan storage can rapidly be induced in photosynthetically active tissues, when the export of carbohydrates is restricted. But in most fructan storing species, under more ‘natural’ growth conditions, long-term fructan storage occurs mainly in tissues of low or negligible photosynthetic activity. The present findings corroborate the circumstantial evidence for a negative relationship between ‘photosynthesis’ and (long-term) fructan storage. Although still rather speculative, the assumption that metabolic steps associated with photosynthesis may hinder the long-term storage of fructan deserves further investigation.

Acknowledgements

I gratefully acknowledge support for this study by the DFG. Thanks are due to Dr A. Cairns (IGER, Aberystwyth, UK) for helpful discussions and to Dr H. Schnyder (Technische Universität München, Germany) for continued support.

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