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Keywords:

  • Dahlia;
  • fructans;
  • inulin;
  • photoperiod;
  • carbohydrate partitioning

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
  • • The effect is reported here of photoperiod on fructan accumulation in the tuberous roots of Dahlia sp. cv. Sunny Rose seedlings.

  • • Growth parameters were measured of shoots and roots on glasshouse-grown dahlia seedlings subjected to either short day (SD) or long day (LD; 4 h night photoperiod interruption) light regimes. The carbohydrate concentrations of tuberous roots was analysed by high performance anion exchange chromatography.

  • • Total plant dry weight was unaffected by photoperiod. The LD treatment inhibited tuberous root development but increased shoot dry weight. Tuberous root tissue of SD seedlings showed a 156% increase in total fructan (inulin) concentration compared with LD tuberous root tissue, which had higher reducing sugar concentrations than SD tuberous roots. A wide range of oligomers increased during the SD treatment.

  • • Sucrose appears to be the regulating factor in fructan metabolism in dahlia. Photoperiod is a valuable tool for studying fuctan metabolism in vivo, as it provides a nondestructive means of regulating sucrose partitioning.


Introduction

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

Fructan accumulation in vascular plants is enhanced by conditions that increase photosynthesis (i.e. long photoperiods) while decreasing the demand for carbon (i.e. low temperatures, low nitrogen availability, and drought) (Hendry, 1987). The relationship between fructans and numerous environmental and cultural factors has been investigated. The effects of temperature on fructan metabolism, accumulation, and partitioning have been widely reported (Chatterton et al., 1989b; Pollock et al., 1989; Solhaug, 1991; Bancal & Triboï, 1993; Prud’Homme et al., 1993). Other reports include the effects of drought stress (Pilon-Smits et al., 1995), hypoxia (Albrecht et al., 1993, 1997), carbon dioxide (Smart et al., 1994), and nitrate concentration (Améziane et al., 1995, 1997).

Few studies have investigated the effects of photoperiod on fructan accumulation. Solhaug (1991) attributed reports of LD promotion of fructan accumulation (Hendry, 1987) to increased irradiance levels. Young Bluegrass (Poa pratensis) plants accumulated more total fructan under short photoperiods (8 h). This was due primarily to the allocation of assimilates for storage in leaf sheaths and stems. Plants grown under long photoperiods showed increased growth resulting from greater partitioning of assimilates to the shoot. As the photosynthetic capacity of LD plants increased, due primarily to greater leaf area, the rate of fructan accumulation in LD plants increased compared with SD plants and differences in fructan accumulation between treatments disappeared (Solhaug, 1991). Short photoperiods (8 h) induced fructan acumulation in leaf blades, sheaths, stems, and roots of Phippsia algida compared with LD (8 h + 16 h low irradiance extension). Fructan accounted for 15–20% of root d. wt compared to 2–3% in LD plants (Solhaug & Aares, 1994). Isejima et al. (1991) observed that short photoperiods (8–10 h) inducing flowering in Viguiera discolor caused greater accumulation of fructans (degree of polymerisation (DP) > 10) in tuberous roots compared with plants grown under noninductive photoperiods (> 10 h). Photoperiod altered fructan composition without affecting total fructose.

Fructans of the inulin series accumulate in the tuberous roots of dahlia. Zimmerman & Hitchcock (1929) determined that SD promoted tuberization in Dahlia variabilis (Willd.) Desf. with the critical day-length estimated to be between 11 and 12 h (Moser & Hess, 1969). Dahlia seedlings should provide a reproducible and rapidly growing system to study the effects of photoperiod on fructan accumulation because they produce tuberous roots at a relatively young age. The objective of this study was to investigate the effects of photoperiod on fructan accumulation in the tuberous roots of dahlia seedlings.

Materials and Methods

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

Plant material

Dahlia sp. cv. Sunny Rose seeds (Ball Seed Co., West Chicago IL., USA) were sown on 7 February (two seeds per cell) in plug trays containing 80 2.5 cm cells filled with Fafard Superfine Germinating Mix (Fafard, Inc., Anderson, SC, USA). The trays were misted, covered with plastic film and placed in a growth chamber with fluorescent lights at 18°C. Germination occurred in approx. 4–5 d, after which the plastic film was removed. One wk after sowing, plants were thinned to one seedling per cell and moved to a glasshouse for photoperiod treatments. Glasshouse temperatures were approx. 27°C day/17°C night. During the second week of growth, trays were subirrigated and fertilized by immersion in a 50-mg N l−l solution of 20–10–20 fertilizer (Grace-Sierra Horticultural Products Co., Milpitas, CA, USA). Fertilizer concentrations were increased to 150 mg N l−1 at each watering following the second week of production as seedlings became established enough for overhead watering. Weekly applications of 33 ppm ancymidol spray were made beginning on 28 February (a standard practice for seed propagated dahlia production).

Photoperiod treatments

All plants received 9 h of natural photosynthetically active radiation (PAR), provided by covering all plants with a blackcloth curtain from 18:00 hours to 09:00 hours. Seedlings under LD treatments were given a night photoperiod interruption provided by two incandescent bulbs illuminated from 22:00 hours to 02:00 hours (Moser & Hess, 1969). There were five replications (trays) for each photoperiod treatment. Each tray was randomly divided into three equal sections to be harvested at biweekly intervals for 6 wk following the start of photoperiod treatment.

Harvesting procedures

Five seedlings (the experimental unit) were harvested for each photoperiod-time combination, constituting a replication. All harvests began at approx. 13:00 hours. Shoot height and number of true leaf pairs were recorded. Shoots were cut at the soil surface and fresh weights and leaf area were recorded. Shoot tissues were then frozen in liquid nitrogen, and freeze-dried. Root tissue was harvested at approx. 15:00 hours. Growing media was rinsed from root tissues with tap water and the tissues rinsed again with distilled water and patted dry with paper towel. Tuberous and fibrous roots were separated and fresh weights were recorded for each. Tissues were then frozen and freeze-dried as described earlier. Before carbohydrate extraction, d. wt of shoots and roots (fibrous and tuberous) were recorded.

Carbohydrate extraction

Freeze-dried tuberous root tissues were ground to a fine powder and samples (25 mg) were placed in 15 ml centrifuge tubes. Carbohydrates were extracted in 8 ml of HPLC grade water (pH adjusted 8.0 with CaCO3) in a 70°C water bath for 1 h, then at room temperature for 3 h. Samples were then centrifuged at 5000 g at 20°C for 20 min. The supernatant was deionized by passing through columns containing 1 ml of Dowex-50 H+ and Amberlite-1-acetate ion exchange resins and brought to a final volume of 10 ml. This preparation is referred to as the ‘final extract.’

Carbohydrate analysis

Carbohydrate separation was accomplished using high performance anion exchange chromatography (HPAEC) using a CarboPac PA-1 column on a Dionex DX-300 chromatography system (Dionex, Sunnyvale, CA, USA), utilizing a pulsed amperometric detector (PAD). A flow rate of 1 ml min−1 was used at an operating pressure of c. 1500 psi. Applied PAD potentials were +0.05 V (E1); +0.60 V (E2); and −0.80 V (E3). Typically, the final extract was diluted 10 times before analysis. Fructans were separated using a sodium acetate gradient with a running time of 32 min (Fig. 1). Glucose, fructose and sucrose concentrations were quantified externally from peak areas. Total carbohydrate was determined colourimetrically by the phenol-sulphuric acid procedure using fructose as the standard (Dubois et al., 1956). Once total carbohydrate concentrations were determined for each sample, glucose, fructose and sucrose concentrations (determined from HPAEC-PAD) were subtracted to give total fructan. Quantification of 1-kestose and higher DP fructan was not possible due to the unavailability of standards. Therefore, data on individual fructan polymers were expressed as peak area per gram d. wt of tissue for comparison of fructans of identical DP and are referred to as a peak area concentration. Since 1-kestose standards were not available, 1-kestose was synthesized so its retention time could be determined and compared with tuberous root extracts. In vitro synthesis of 1-kestose was achieved through the action of purified yeast invertase on 0.5 M sucrose as described by Chatterton et al. (1989a).

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Figure 1. Sodium acetate gradient utilized for high performance anion exchange chromatography utilizing a pulsed amperometric detector (HPAEC-PAD) separation and detection of soluble carbohydrates in extracts of dahlia tuberous roots, eluent I (0.2 M NaOAc in 0.2 M NaOH) (closed triangles); eluent II (0.2 M NaOH) (open triangles).

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Results

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

There was no visible macroscopic evidence of tuberous root development after 2 wk of photoperiod treatment (week 2). By week 4 there was noticeable swelling at the stem bases of both LD and SD seedlings. Adventitious roots originated from the swollen base of the stems, independent of fibrous roots, and greater in diameter. These tuberous roots were noticeably larger on SD seedlings compared with LD seedlings. Differences in d. wt were observed after the week 4 harvest, with LD causing slightly higher shoot d. wt and SD resulting in greater root d. wt. These differences coincided with visible signs of tuberization. By week 6, SD seedlings had 50% more total root d. wt but approx. 40% less shoot d. wt compared to LD seedlings (Fig. 2). Short day seedlings developed large rounded tuberous roots compared with slender elongated structures under LD. No differences in total plant d. wt were observed throughout the treatment period, indicating that photoperiod directly affects assimilate partitioning (Fig. 2).

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Figure 2. Variation in the dry weights of shoots, roots, and shoots plus roots (total plant d. wt) of Dahlia. Shoots long day (LD) (closed squares): short day (SD) (open squares); roots LD (closed circles), SD (open circles); total plant d. wt LD (closed triangles), SD (open triangles). Bars indicate ± SE, n = 5.

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Fig. 3a shows an HPAEC-PAD chromatogram of a Dahlia tuberous root extract from week 6 of a seedling grown under SD. Fig. 3b is an expansion of the same chromatogram showing DP 9 to approx. DP 30. Chromatograms were similar to the homologous inulin series found in Jerusalem artichoke (Helianthus tuberosus) (Chatterton et al., 1993). No differences in tuberous root glucose, fructose, and sucrose concentration were observed between LD and SD seedlings at week 4, but week 6 LD tuberous roots had 175% more glucose and 56% more fructose compared with SD tuberous roots (Fig. 4). SD tuberous roots had a 20% greater sucrose concentration over LD tuberous roots. Differences in fructan concentration were observed between LD and SD tuberous roots at week 4 and 6 coinciding with increases in tuberous root d. wt (Fig. 5). Short day tuberous roots showed a 68% increase in fructan concentration over LD tuberous roots at week 4, increasing to 156% by week 6.

image

Figure 3. High performance anion exchange chromatography utilizing a pulsed amperometric detector (HPAEC-PAD) chromatogram of soluble carbohydrates in an extract of Dahlia tuberous roots (a), and an expansion showing fructans DP 9 and higher (b). Approximately 1 week following germuriation, plants were grown under short photoperiods (8 hr) for 6 weeks. Glu, glucose; Fru, fructose; Suc, sucrose; 1-Kes, 1-kestose; DP 4, degree of polymerization, 4; DP 5, degree of polymerization, 5; DP 6, degree of polymerization, 6; DP 7, degree of polymerization, 7.

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image

Figure 4. Variation in reducing sugar and sucrose concentrations of tuberous roots of Dahlia grown under different photoperiod regimes. Glucose long day (LD) (closed squares), short day (SD) (open squares); fructose; LD (closed circles), SD (open circles); sucrose LD (closed triangles), SD (open triangles). Bars indicate SE (n = 5).

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image

Figure 5. Variation in total fructan concentrations and dry weights of tuberous roots of Dahlia grown under different photoperiod regimes. Fructan long day (LD) (closed squares), short day (SD) (open squares); tuberous root dry weight LD (closed circles), tuberous roots SD (open circles). Bars indicate SE (n = 5).

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Peak area concentrations (peak area g−1 d. wt) for fructans of DP 3 to DP 30 were greater in SD tuberous roots at week 4 and 6. Fig. 6a, b, c shows comparisons of peak areas for selected fructans, namely 1-kestose, 1,1-nystose, DP 5, DP 10, DP 15, DP 20, DP 25, and DP 30 in LD and SD tuberous roots. Comparisons are only valid between fructans of identical DP. Fig. 7 shows the percent increase of peak area of SD fructans compared with LD fructans (DP 3–DP 25). Percent increase of peak area increased with DP. The data show that when comparing inductive and noninductive photoperiods, the largest differences occur in the synthesis of longer chain fructans (DP > 20) and the smallest differences in the synthesis of shorter chain fructans (DP < 7).

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Figure 6. Variation in peak area concentrations of tuberous root fructans of Dahlia grown under different photoperiod regimes. (a) 1-kestose long day (LD) (closed square), short day (SD) (open square); 1,1 nystose (degrees of polymerization (DP 4)) LD (closed circle), SD (open circle). (b) DP 5 LD (closed square), SD (open square); DP 10 LD (closed circle), SD (open circle); DP 15 LD (closed triangle), SD (open triangle). (c) DP 20 LD (closed square), SD (open square); DP 25 LD (closed circle), SD (open circle); DP 30 LD (closed triangle), SD (open triangle). Bars indicate ± standard error (n = 5).

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image

Figure 7. Percent increase of Dahlia tuberous root fructans (DP3 to DP25) at week 6 due to inductive photoperiods (short day).

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Discussion

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

Results indicate that short photoperiods promote fructan accumulation in tuberous roots of dahlia seedlings by affecting assimilate partitioning. This is clearly seen with the identical total d. wt of SD and LD seedlings, but substantially more d. wt partitioned to tuberous root tissue in SD plants (Figs 2, 5). This SD accumulation is similar to that observed in young Bluegrass plants and P. algida (Solhaug, 1991; Solhaug & Aares, 1994). While fructan accumulation under LD was not as large compared with SD, a significant accumulation was observed. We speculate that LD accumulation may be the result of increased photosynthetic capacity due to increased leaf area in LD seedlings as suggested by Solhaug (1991). Although both LD and SD seedlings accumulated fructan in their tuberous roots, accumulation was always greater under short photoperiods. Total fructose (free and combined) was altered by photoperiod. These results differ from the findings of Isejima et al. (1991) who observed no effect of photoperiod on total fructose (including fructan) in adventitious roots of V. discolor, but did observe changes in fructan composition (DP). Age of the plants must be considered however, as their plants were over 1-yr-old compared with ours being only a few weeks old. It would be interesting to induce tuberization with SD, then move plants to LD and observe changes in fructan concentration and composition. When P. algida plants grown under SD were moved to LD, a large decrease in fructan concentration was observed in leaf blades, sheaths, stems, and roots (Solhaug & Aares, 1994). Our results are in agreement with Solhaug (1991) and Solhaug & Aares (1994) in that short photoperiods promote greater fructan synthesis compared with long photoperiods when plants received identical amounts of PAR.

The carbohydrate status of actively tuberizing roots is characterized by the influx of sucrose, its incorporation into fructan, and the liberation of glucose (Edelman & Jefford, 1968). Although total fructan concentration was less in LD seedlings (Fig. 5), glucose concentrations were much greater compared with SD seedlings (Fig. 4). We propose the following to explain the fate of glucose in SD tuberous roots:

Cell wall synthesis: free glucose is incorporated into structural components required for increased tuberous root growth in SD plants. Vieira & Figueiredo-Riberiro (1993) examined fructan accumulation in the tuberous roots of Gomphrena macrocephala at various phenological stages. During early dormancy, they observed an increase in structural component (residue left after soluble carbohydrate and starch analysis) corresponding with highest concentrations of fructose containing polysaccharides. With this observation, they did not observe an increase in monosaccharides.

Respiratory substrate: perhaps glucose is utilized more rapidly in SD tuberous root tissue to meet increased respiration demands associated with increased tuberous root growth.

Conversion back to sucrose: Edelman & Jefford (1968) observed that tubers of Jerusalem artichoke could freely convert free hexose to sucrose at all stages of development. When [14C]-hexose was injected into growing tubers, the labelled carbon was present in glucose 6-phosphate, fructose 6-phosphate, and UDP-glucose (all precursors to sucrose). We speculate that the conversion of free hexose to sucrose in the vacuole may aid in the suppression of fructan exohydrolase (FEH) (Edelman & Jefford, 1968; Simpson et al., 1991; Marx et al., 1997).

We assume that there is increased sucrose transport to the roots of SD seedlings. This is supported by the large increase in tuberous root d. wt of SD seedlings compared with LD seedlings (Fig. 5); however, at week 6, SD seedlings showed only a 20% increase in sucrose concentration over LD seedlings compared with a 156% increase in fructan concentration (Figs 4, 5). The data suggest a rapid turnover of sucrose to fructan. The percent increase in fructan peak area in SD roots compared with LD roots increased with molecular weight. The resulting graph (Fig. 7) is characterized by a sharp increase from DP 3 to DP 7 (20% to 132%), a gradual increase from DP 7 to DP 18 (132% to 172%), then another sharp increase from DP 18 to DP 25 (172% to 256%). We may only speculate why a positive correlation between the percent increase in peak area and fructan DP exists. Perhaps higher DP fructans have a greater affinity for free fructose during fructosyl transfer via fructosyl transferase. Van den Ende et al. (1996) determined that longer chain fructans (DP > 10) were relatively more efficient acceptors in the 1-FFT reaction compared to oligofructans. Dickerson & Edelman (1966) observed that 50 h after supplying 14CO2 to leaves of growing Jerusalem artichoke, 40% of the 14C was present in the inulin fraction containing fructans DP 25 and higher.

Sucrose appears to be the regulating factor in fructan metabolism. Winters et al. (1994) observed that inducing sucrose accumulation and subsequent fructan synthesis in leaves of Lolium temulentum resulted in the synthesis of specific size-classes of proteins and transcripts. The synthesis of fructans and these specific proteins was inhibited when leaves were fed with transcription inhibitors (cordycepin and cycloheximide). The pathway by which photoperiod regulates the translocation of sucrose and affects fructan metabolism warrants further study. Our findings suggest that photoperiod is a valuable tool for studying fructan metabolism in vivo, as it provides a nondestructive (i.e. compared to leaf excision) means of regulating sucrose partitioning.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
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  • Améziane RE, Deléens Noctor G, Morot-Gaudry JF & Limami MA. 1997. Stage of development is an important determinant in the effect of nitrate on photoassimilate (13C) partitioning in chicory (Cichorium intybus). Journal of Experimental Botany 48: 2533.
  • Améziane R, Limami MA, Noctor G & Morot-Gaudry JF. 1995. Effect of nitrate concentration during growth on carbon partitioning and sink strength in chicory. Journal of Experimental Botany 46: 14231428.
  • Bancal P & Triboï E. 1993. Temperature effect on fructan oligomer contents and fructan-related enzyme activities in stems of wheat (Triticum aestivum L.) during grain filling. New Phytologist 123: 247253.
  • Chatterton NJ, Harrison PA, Thornley WR & Bennett JH. 1989a. Purification and quantification of kestoses (fructosylsucroses) by gel permeation and anion exchange chromatography. Plant Physiology and Biochemistry 27: 289295.
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  • Solhaug KA & Aares E. 1994. Remobilization of fructans in Phippsia algida during rapid inflorescence development. Physiologia Plantarum 91: 219225.
  • Van den Ende W, Van Wonterghem D, Verhaert P, Dewil E & Van Laere A. 1996. Purification and characterization of fructan: fructan fructosyl transferase from chicory (Chichorium intybus L.) roots. Planta 199: 493502.
  • Vieira CCJ & Figueiredo-Riberiro RCL. 1993. Fructose-containing carbohydrates in the tuberous root of Gomphrena macrocephala St.-Hil. (Amaranthaceae) at different phenological phases. Plant, Cell & Environment 16: 919928.
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