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Keywords:

  • Ricinus communis L ;
  • ADP-glucose pyrophosphorylase;
  • elevated CO2;
  • starch accumulation;
  • sucrose signalling

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Ricinus communis plants were grown under normal (350 ppm) and elevated (700 ppm) CO2 atmosphere and the growth and carbohydrate status of leaf 2 (first leaf above the pair of primary leaves) was studied. Elevated carbon dioxide stimulated the growth of leaves 1·7-fold. The glucose and fructose concentrations exhibited the same diurnal rhythm under both growth conditions. The sucrose concentrations stayed relatively constant and at 700 ppm were one-third higher than at 350 ppm. The starch content increased steadily during the day and disappeared overnight at 350 ppm CO2, but remained partially in plants at 700 ppm CO2. Consequently at 700 ppm CO2, the leaves accumulated starch continuously over their life time. The rate of starch synthesis was correlated to the activity of ADP-glucose pyrophosphorylase, which was related to the sucrose concentration in the leaf. It is concluded that sucrose controls the expression of ADP-glucose pyrophosphorylase, leading to a shift of carbohydrate partitioning into starch when more sucrose is produced than consumed or exported, a situation which is especially pertinent at elevated CO2. These results show that the previously experimentally observed transscriptional regulation of starch synthesis by sucrose occurs in vivo in the daily life of a leaf.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

It is a common observation that plants grown at elevated carbon dioxide concentrations develop a larger plant biomass and have higher starch levels in the leaves ( Wong 1990; Lawlor & Mitchell 1991; Ziska et al. 1991 ) than plants grown at the natural, ambient CO2 concentration of 350 ppm. The higher proportion of ‘non-structural carbohydrate’ ( Wong 1990) increases continuously with the life time of the leaves and leads to a higher ‘residency time’ of freshly assimilated carbon in the mesophyll ( Vessey et al. 1990 ). It is therefore concluded that the stimulation of primary carbon assimilation at elevated CO2 cannot be fully exploited by the plants because of the lack of a similar increase in carbon export or in carbon consumption. The question is how the shift of assimilate partitioning under elevated CO2 into starch is achieved.

The triose-phosphate/phosphate exchange carrier may decrease assimilate exit from the chloroplast if there is a shortage of cytosolic phosphate. Production and accumulation of sucrose, however, do not change the cytosolic phosphate pool and therefore this carrier may not be the target for control of the shift of assimilate partitioning from sucrose into starch. Literature reports point to a control of starch synthesis via transcriptional control of ADP-glucose pyrophosphorylase by cytosolic sucrose. The transcript level of ADP-glucose pyrophosphorylase was increased 24 h after excision of a leaf or by feeding of sucrose to an excised leaf ( Müller-Röber et al. 1990 ). Also the promoter region of ADP-glucose pyrophosphorylase responded positively to addition of sucrose ( Dujardin et al. 1997 ). Metabolic studies on potato tuber slices fed with sucrose indicated a sucrose-specific stimulation of ADP-glucose pyrophosphorylase and starch synthesis ( Geiger et al. 1998 ). All these reports dealt with systems under a strong experimental impact (leaf excision or feeding of high concentrations of exogenous sucrose to tubers). The experiments with castor bean reported in the following should show whether the mechanism of sucrose-regulated ADP-glucose pyrophosphorylase also works in intact, non-manipulated (except the ambient increase of carbon dioxide) source leaves. The studies were performed in the framework of research on carbohydrate synthesis and allocation in plants under elevated CO2.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Growth

Seeds of Ricinus communis L. var. sanguineus were soaked in water overnight, surface-sterilized in quinoline and germinated on vermiculite at 27 °C. After 9–10 days, when the cotyledons had unfolded and became green, the plantlets were transferred onto quartz sand culture in growth chambers (BBC-York, York, UK). The light regime was a 13·5 h light/9·5 h dark cycle with 700–750 μE m−2 s−1 intensity (lamps EYE Iwasaki-type, MT400 DL BH−1) and a dusk period of 30 min in between consisting of a stepwise increase or decrease of light in five equal steps. The temperature cycle was 26 °C/20 °C; the humidity was regulated at a dew point of 19 °C in light and 16 °C in darkness. The chambers were equipped for maintenance of a stable CO2 concentration of 350 ppm or 700 ppm. The plants were potted in 4 litre pots and were fertilized with a modified Hoagland’s nutrient solution (4 mol m−3 CaSO4, 2 mol m−3 MgSO4, 0·09 mol m−3 Fe-EDTA, 3 mol m−3 K2HPO4, 6 mol m−3 NH4NO3, 0·1% micronutrients, pH 6·0). The fertilization regime was adapted to the biomass production of the plants, therefore 500 cm3 day−1 pot−1 were used in the first 5–6 days, 600 cm3 in the following 4 days, 800 cm3 for the next 4 days and 1500 cm3 for the residual growth period. Since under 700 ppm CO2 the plants were approximately 3 days ahead in development compared to 350 ppm plants, the increase of fertilizer supply was adjusted a few days earlier in that case.

Day 0 of a leaf was defined as the day when the lamina tips first extended out of the husk and became visible.

Collection of plant material

Leaf material was only taken from leaves 2, i.e. the first leaf above the primary leaf pair. The plants had been transferred to the defined CO2 atmosphere at least 11 days prior the first sampling, the leaf age at harvest was 7–8, 9–11, 12–14 and 15–17 days. Material was taken at the start of the light (dusk) period (0 h), and 5, 9·5 and 14 h after onset of light. For each sample, five discs of 6·5 mm diameter (33·2 mm2) were punched from the middle sector of the lamina of each leaf, excluding the mid-rib, frozen at –20 °C and lyophilized. For determination of enzyme activities, the samples were immediately frozen in liquid nitrogen.

Dry weight and leaf area

The dry weight of leaves or leaf samples was determined after lyophilization of the freshly harvested material.

The leaf area was determined by an area meter (Licor, Lincoln, NR, USA).

Extraction and carbohydrate determination

The freeze-dried samples were finely ground to a powder and extracted in 0·5 cm3 80% ethanol in 1 mol m−3 HEPES, pH 7·5, for 45 min at 70 °C. The extract was centrifuged at 3000 g for 10 min. The supernatant was analysed for soluble sugars. Glucose and fructose were determined enzymatically with hexokinase, glucose phosphate dehydrogenase and phosphoglucose isomerase ( Bergmeyer 1984). Sucrose was first hydrolysed by acid invertase; the resulting hexoses were determined as described above.

Starch was measured from the pellet after extraction. The pellet was washed with 70% ethanol and then twice with distilled water, then suspended in 0·8 cm3 water and autoclaved for 2 h. The suspension (0·2 cm3) was incubated at 37 °C in 0·5 cm3 100 mol m−3 Na-acetate pH 4·8, 5 mm3 amyloglucosidase (0·7 μ), 5 mm3α-amylase (1 μ) and 0·29 cm3 water for 2 h. The liberated glucose was determined enzymatically as described above.

Photosynthesis measurements

The photosynthetic capacity of leaf discs was measured in a leaf disc oxygen electrode (Hansatech, King’s Lynn, UK) at 26 °C and saturating CO2 (2%) as described by Walker (1988).

Chlorophyll was determined according Porra et al. (1989) in N, N-dimethylformamide.

Determination of ADP-glucose pyrophosphorylase

The frozen leaf material was powdered in liquid N2 with 1 cm3 extraction buffer ( Neuhaus & Stitt 1990), placed on ice, suspended and stored on ice for further 20 min, then centrifuged at 3000 g for 10 min and the supernatant recovered. The enzyme assay was conducted according Müller-Röber et al. (1992) . It is based on NADPH formation by oxidation of glucose-6-phosphate to 6-phosphogluconate (via glucose-1-phosphate) and a second oxidation to ribulose-5-phosphate.

Protein was determined according Bradford.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Leaf growth and development

The morphological and physiological parameters of leaf 2, which is the first leaf above the pair of primary leaves, were investigated in this study. Leaf area increased until day 11 when full expansion was reached. There was a small difference in expansion rate between plants grown at 350 ppm CO2 or 700 ppm CO2 ( Fig. 1a). Chlorophyll content per leaf area increased until day 10 ( Fig. 1b). There was no difference between the two CO2 treatments on dry weight basis, but the 700 ppm leaves had a slightly higher chlorophyll content on leaf area basis. Photosynthetic capacity was more or less constant from day 12 onwards ( Fig. 1c). It was only slightly higher for 700 ppm plants compared to 350 ppm plants when both were tested at saturating CO2 concentration. The actual photosynthetic rate at 700 ppm CO2 was, however, 40% higher than the photosynthetic rate at 350 ppm CO2 (data not shown).

image

Figure 1. . Lamina area of leaf (a), chlorophyll content (b) and capacity for net photosynthesis (c) of leaf 2 at different leaf age (bottom axis) from plants grown under 350 or 700 ppm CO2. The photosynthetic capacity was measured as oxygen evolution at saturating CO2 (each point is the mean of three replicates).

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The growth pattern of the Ricinus leaf is a gradient from tip to base of the leaf, the tip area of the lobes being approximately 4 days ahead in development relative to the basal lamina part ( Heckenberger et al. 1998 ; K. Hartig, Bayreuth, unpublished data). Since the leaf samples for the following carbohydrate analysis were taken from the lobes and not from the base of the lamina, the samples obtained from leaves of 9–11 days can be regarded as representing a leaf area just after sink–source transition, whereas the samples from 12–14 days and 15–17 days represent source areas with several days of functional assimilate export.

The dry weight of leaves continued to increase beyond the time of maximal leaf expansion and the increase was 1·7 times faster at 700 ppm than at 350 ppm ( Fig. 2).

image

Figure 2. . Dry weight of leaf 2, measured at the start of the light period (0 h) during growth under 350 or 700 ppm CO2 (r2 = 0·86).

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Diurnal carbohydrate levels in leaves

Glucose and fructose concentrations exhibited a clear rhythm during the daytime, with the highest level at 5 h after onset of light ( Fig. 3). The concentrations at the end of the light period were often (but not always) lower than those at the end of the dark period. The daily rhythm appeared more pronounced in the 350 ppm plants than in 700 ppm plants, mostly because the values at the start and at the end of the light period were consistently lower, whereas the peak values were similar in both atmospheres. The leaves of 350 ppm plants had the highest hexose levels at age 12–14 days with some decline later, whereas the samples from 700 ppm plants were more similar throughout the leaf age.

image

Figure 3. . Glucose, fructose and sucrose concentration (in hexose units) and starch content (in hexose units) in leaf 2 samples during the light period. Leaf discs were harvested from leaves of different age and at different times during the light period from plants grown at 350 or 700 ppm CO2 (mean ± SD, n = 5).

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The sucrose concentrations in the leaf samples were several-fold higher than the hexose concentrations and exhibited only a small (relative) diurnal rhythm ( Fig. 3). With increasing leaf age, a small increase of sucrose concentration was noticed. Consistently the sucrose level was (on average one-third) higher in the 700 ppm plants than in the 350 ppm plants.

The starch level increased steadily during the light period and disappeared overnight in the case of 350 ppm CO2, whereas in 700 ppm CO2, a considerable starch fraction remained until the next day ( Fig. 3). Obviously not all of the starch could be mobilized during the night, and consequently the starch level measured at the end of the night period increased continuously over the leaf age ( Fig. 4). Thus in 700 ppm plants, the hexose content in starch exceeded the hexose content of sucrose, while in plants grown at 350 ppm the hexose levels present in starch and sucrose were about equal.

image

Figure 4. . Increase of starch content (measured at the start of the light period, 0 h) in leaf 2 samples at different leaf age from plants grown at 350 or 700 ppm CO2 (each point is the mean of two replicates).

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The net rate of starch synthesis during the light phase was age-dependent in the sense that it increased with leaf age ( Fig. 5). It appeared that the net synthesis rate was higher at 700 ppm CO2 than at 350 ppm CO2 at all studied leaf age levels.

image

Figure 5. . Rate of starch synthesis at different leaf age of plants grown at 350 ppm (r2 = 0·81) and at 700 ppm (r2 = 0·47).

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Starch synthesis and ADP-glucose pyrophosphorylase activity

ADP-glucose pyrophosphorylase is considered to regulate starch synthesis ( Preiss 1988). The ADP-glucose pyrophosphorylase activity measured in vitro was usually less in 350 ppm-grown plants than in 700 ppm-grown plants, although great variation existed. There was some correlation between the rate of net synthesis of starch in the light period and the ADP-glucose pyrophosphorylase activity measured in the same leaf sample ( Fig. 6). A strong correlation, however, existed between the sucrose concentration in the leaf and the ADP-glucose pyrophosphorylase activity ( Fig. 7) and between the sucrose concentration in the leaves and the net rate of starch synthesis ( Fig. 8). These correlations were valid for both CO2 conditions, 350 and 700 ppm, although the correlation coefficient was weaker at 350 ppm due to the data scatter (not shown).

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Figure 6. . Increase of starch content of leaves during the 13·5 h full light period versus the in vitro activity of ADP-glucose pyrophosphorylase in the same leaf samples. The data were obtained from plants grown under 350 or 700 ppm CO2 (r2 = 0·45); each point is the mean of two replicates.

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image

Figure 7. . Relationship between sucrose content and ADP-glucose pyrophosphorylase activity in leaf samples from plants grown under 350 or 700 ppm CO2 (adjusted r2 = 0·76); each point is the mean of two replicates.

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image

Figure 8. . Correlation between sucrose concentration and starch content in leaves from plants grown under 350 or 700 ppm CO2 (each point is the mean of two replicates).

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The different carbohydrate fractions in the leaves showed distinct differences in their diurnal behaviour. The starch pool increased steadily during the day and disappeared entirely (under 350 ppm) or partially (under 700 ppm) during the night. In contrast the sucrose level was kept relatively constant over the day (and night) phase. The hexose levels fluctuated considerably during the day and this fluctuation was not inversely correlated to a fluctuation of the sucrose levels, although the 1:1 ratio of glucose and fructose suggested sucrose as precursor of the hexoses. It appeared that the leaf cells were set for a relatively stable, intracellular sucrose concentration, probably because sucrose is a main osmotic substance for the maintenance of cell turgor.

The correlation between ADP-glucose pyrophosphorylase activity and sucrose levels indicated that the transcriptional control of assimilate partitioning by sucrose, previously measured under strong experimental impact, does indeed take place in the daily life of a source leaf. This regulation works under 350 ppm CO2 as well as under 700 ppm; in the latter it became more obvious, however, leading to high and persisting starch levels in the leaves. By this mechanism, the mesophyll cells may avoid a rise in sucrose concentration to harmful levels in a situation when (at 700 ppm CO2) the carbon assimilation rate is stimulated more than the sucrose export rate. Since with age higher starch levels and higher sucrose levels accumulate in the leaves, leading to higher starch synthesis rates in older leaves, a developmental correlation between leaf age and the rate of starch synthesis was also observed.

The regulation of assimilate partitioning between sucrose and starch is direct and local by the sucrose concentration in the mesophyll. Whether the back-up of sucrose in the leaves is the consequence of limited phloem loading capacity in the source leaves (which may also be influenced by sucrose via a transcriptional control of a sucrose transporter ( Chiou & Bush 1998)) or the result of sugar recirculation due to limited assimilate consumption by the sink tissues, is unknown. Future experiments will need to show whether substantial sugar recirculation via the xylem takes place under conditions of assimilate accumulation in the source leaves.

ACKNOWLEDGEMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

This work was supported by the Deutsche Forschungsgemeinschaft.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  • Bergmeyer H.U. (1984) In Methods of Enzymatic Analysis, Volume VI 3rd edn, p 97. Verlag Chemie, Weinheim.
  • Chiou T.J. & Bush D.R. (1998) Sucrose is a signal molecule in assimilate partitioning. Proceedings of the National Academy of Sciences of the USA 95, 4784 4788.
  • Dujardin P., Harvengt L., Kirsch F., Le V.Q., Nguyen-Quoc B., Yelle S. (1997) Sink cell specific activity of a potato ADP-glucose pyrophosphorylase B subunit promoter in transgenic potato and tomato plants. Planta 203, 133 139.
  • Geiger M., Stitt M., Geigenberger P. (1998) Metabolism in slices from growing potato tubers responds differently to addition of sucrose and glucose. Planta 206, 234 244.
  • Heckenberger U., Roggatz U., Schurr U. (1998) Effect of drought stress on the cytological status in Ricinus communis. Journal of Experimental Botany 49, 181 189.
  • Lawlor D.W. & Mitchell R.A.C. (1991) The effects of increasing carbon dioxide on crop photosynthesis and productivity. A review of field studies. Plant, Cell and Environment 14, 807 818.
  • Müller-Röber B.T., Koßmann J., Curtis H.L., Willmitzer L., Sonnewald U. (1990) One of two different ADP-glucose pyrophosphorylase genes from potato responds to elevated levels of sucrose. Molecular and General Genetics 224, 136 146.
  • Müller-Röber B., Sonnewald U., Willmitzer L. (1992) Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO Journal 11, 1229 1239.
  • Neuhaus E. & Stitt M. (1990) Control analysis of photosynthate partitioning. Planta 182, 445 454.
  • Porra R.J., Thompson W.A., Kriedemann P.E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectrometry. Biochimica Biophysica Acta 975, 384 394.
  • Preiss J. (1988) Biosynthesis of starch and its regulation. In The Biochemistry of Plants, Volume 14 (ed. J. Preiss), pp. 181–254. Academic Press, San Diego.
  • Vessey J.K., Henry L.T., Raper C.D. (1990) Nitrogen nutrition and temporal effects of enhanced carbon dioxide on soybean growth. Crop Science 30, 287 294.
  • Walker D. (1988) The Use of the Oxygen Electrode and Fluorescence Probes in Simple Measurements of Photosynthesis, 2nd edn. Oxygraphics, Sheffield.
  • Wong S.C. (1990) Elevated atmospheric partial pressure of carbon dioxide and plant growth. II. Non-structural carbohydrate content in cotton plants and its effect on growth parameters. Photosynthesis Research 23, 171 180.
  • Ziska L.H., Hogan K.B., Smith A.P., Drake B.G. (1991) Growth and photosynthetic response of nine tropical species with long term exposure to elevated carbon dioxide. Oecologia 86, 383 389.