Journal of Experimental Botany, Vol. 51, No. 347, pp. 1037-1045,
June 2000
© 2000 Oxford University Press
Photosynthetic carbohydrate metabolism in wheat (Triticum aestivum L.) leaves: optimization of methods for determination of fructose 2,6-bisphosphate
IACR-Rothamsted, Harpenden, Herts. AL5 2JQ, UK
Received 18 November 1999; Accepted 28 February 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The accurate measurement of fructose 2,6-bisphosphate from plants such as wheat is fraught with difficulty. Extraction and assay methods for fructose 2,6-bisphosphate that give near 100% recovery of the metabolite, and a linear response with volume have therefore been developed for extracts prepared from wheat leaves of different ages. Amounts of fructose 2,6-bisphosphate in different regions of leaves generally showed a positive correlation with chlorophyll content. Measurements of sucrose and starch in third leaves harvested at different times of the diurnal cycle demonstrated that sucrose is the major form in which photosynthate is stored in the leaf, but starch can account for up to about 30% of the stored carbohydrate. Virtually all of the carbohydrate accumulated as starch and sucrose during the day was degraded at night. Amounts of fructose 2,6-bisphosphate were generally lower in extracts prepared from leaves harvested in the light than in the dark. Additionally, there was no change in either the amount of fructose 2,6-bisphosphate or the ratio of sucrose to starch in samples prepared from leaves harvested at different times of the day. These results are broadly consistent with a role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis and the partitioning of carbohydrate between sucrose and starch in wheat leaves.
Key words: Fructose 2,6-bisphosphate, photosynthesis, starch, sucrose, wheat.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The accepted roles for fructose 2,6-bisphosphate (F26BP) in both co-ordinating the rate of sucrose synthesis with the rate of photosynthesis, and in regulating the partitioning of fixed carbon between sucrose and starch are based on studies with plants that store large amounts of starch in their leaves such as spinach (Stitt et al., 1983
, 1987; Stitt, 1990a), tobacco (Kruger and Scott, 1995; Scott et al., 1995), Clarkia xantiana (Neuhaus et al., 1989) and Kalanchoë daigremontiana (Truesdale et al., 1999). Plants such as cereals and temperate grasses differ greatly from these in that they store carbon in their leaves primarily as sucrose rather than as starch. In one study of young barley leaves the amount of F26BP was inversely correlated with changes in the rate of sucrose accumulation, but had little if any relationship with the rate of starch accumulation (Sicher et al., 1984, 1986). Similarly, a comparison of the amounts of F26BP and carbohydrate partitioning in a range of other plants (Sicher et al. 1987) also suggested that the roles ascribed for F26BP in spinach and tobacco are not necessarily ubiquitous amongst higher plants.
Despite the economic importance of wheat (Triticum aestivum L.) very little is known about the role of F26BP in regulating carbohydrate synthesis and partitioning in the leaves of this plant. A prerequisite for studies to examine these questions is the possession of a reliable method for the extraction and assay of F26BP. The extraction of F26BP is complicated by the fact that it is extremely acid-labile and therefore cannot be extracted in perchloric acid. In addition, F26BP is also susceptible to hydrolysis by non-specific phosphatases. A variety of methods have been used to extract the metabolite from the leaves of a range of plants, for example, KOH with barley (Nielsen, 1992; Nielsen and Veierskov, 1990), CHCl3/MeOH with maize (Stitt and Heldt, 1985) and spinach (Stitt et al., 1982), and boiling ethanol with, amongst others, tobacco (Scott and Kruger, 1994). It appears that although a specific method may be suitable for extracting F26BP from a particular species, the extreme lability of F26BP means that the same method may give low recoveries when used with another plant (Scott and Kruger, 1994). For this reason, good evidence that the extraction method accurately recovers all of the F26BP from the tissue is absolutely essential if the results are to be accepted (Ball and ap Rees, 1989).
A number of approaches to assaying F26BP have been developed. These include (i) the release of the ATP inhibition of mammalian phosphofructokinase (PFK, EC 2.7.1.11) (Uyeda et al., 1981; Cséke et al., 1982), (ii) a competitive binding assay using mammalian PFK (Thomas and Uyeda, 1986), (iii) measurement of F6P released after acid hydrolysis of purified F26BP (Hue et al., 1982), and (iv) fluorometric determination of F6P after acid hydrolysis of NaBH4 reduced samples (Avigad, 1984). (v) However, the most widely used and the most sensitive method is a bioassay where F26BP is measured by following the activation of the enzyme pyrophosphate fructose 6-P phosphotransferase (PFP, EC 2.7.1.90) from potato tubers (Van Schaftingen et al., 1982). This enzyme has a Ka for F26BP of about 4 nM (Trevanion and Kruger, 1991), and an assay using this enzyme can measure as little as 0.1 pmol of the metabolite per sample. Since many other components of plant extracts can interact with the activation of PFP by F26BP (Van Schaftingen et al., 1982; Kombrink and Kruger, 1984; Stitt, 1989) an internal standard curve must be produced for each sample. This is generated by treating aliquots of the extract with acid to remove endogenous F26BP, neutralizing with reaction buffer, and then adding internal standards of between 0 and 2 pmol of purified F26BP (Stitt, 1990b). The rates of PFP activity measured in these reactions can then be used to calculate the amounts of F26BP in aliquots of the extract that have not been incubated with acid. Although this standard method (Stitt, 1990b) appears to work for many species, it does not give accurate results for samples prepared from maize leaves using a variety of extraction methods (Lunn, personal communication). Extracts prepared from this plant show a non-linearity of the assay with extract volume, the estimated concentration of F26BP decreasing with increasing amounts of extract. Obviously this makes accurate measurement of the amount of F26BP impossible. Unfortunately, this potential problem is not generally discussed when measurements of F26BP are published so it is not known how widespread its occurrence is. However, an inescapable consequence of this observation is that a demonstration that the amount of F26BP detected is proportional to the amount of extract assayed is required if published measurements of F26BP are to be accepted.
This paper demonstrates that existing methods for extracting and measuring F26BP from cereals do not give reliable results for wheat leaves. An extraction and assay method for F26BP from wheat leaves of a range of ages, that gives acceptable recoveries and is linear with respect to extract volume, is described. The data obtained with this method are the first reliable measurements of the metabolite in this important crop. Heterogeneity in the distribution of F26BP between different regions of wheat leaves and the potential role for F26BP in regulating sucrose synthesis and carbohydrate partitioning have been examined.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
The spring wheat (Triticum aestivum L.) cultivar Bob White was grown in a mixture of 70% compost/30% perlite in a controlled environment cabinet. The plants were grown under a 14 h day with a day-time temperature of 18 °C and a night-time temperature of 15 °C. Relative humidity was 70% during the day and 80% during the night. Light intensity was 150 µmol m-2 s-1.
Extraction of metabolites from wheat leaves
Leaves were harvested and immediately frozen and stored in liquid nitrogen. Samples were ground to fine powder in a mortar and pestle, the frozen material transferred to another mortar and rapidly homogenized with between 10 and 20 vols. of ice-cold 50 mM KOH. Two aliquots were removed, one for the measurement of chlorophyll and one for the measurement of sucrose and starch. The second aliquot was immediately incubated at 70 °C for 20 min, cooled on ice, and centrifuged for 10 min at 13 000 g. The supernatant was removed and both it and the pellet stored at -20 °C.
The remainder of the extract was heated at 80 °C for 10 min and then cooled on ice for 3 min. Activated charcoal was added to 10 mg ml-1 and the mixture incubated at 0 °C for 10 min. Insoluble material was removed by centrifuging (13 000 g for 12 min at 4 °C), and the supernatant clarified by a further brief centrifugation (13 000 g for 5 min). Samples were stored at 4 °C for up to 24 h before assaying for F26BP.
Measurement of F26BP
Before use, contaminating F26BP in the stock solution of fructose 6-P (F6P) (Sigma, Poole, UK) (Kruger et al., 1983) was removed by acid treating with 1 M perchloric acid for 10 min at 0 °C. The solution was then neutralized with KOH (5 min on ice) and insoluble potassium perchlorate removed by centrifuging for 5 min at 13 000 g. Coupling enzymes supplied as NH4(SO4)2 precipitates (Roche Diagnostics Ltd., Lewes, UK) were resuspended in buffer (100 mM TRIS/HCl [pH 8.0], 1 mM MgCl2) and desalted using NAPtm 5 columns from Amersham Pharmacia Biotech AB (Uppsala, Sweden). PFP was purified from potato tubers as described earlier (van Schaftingen et al., 1982). Purified F26BP (Sigma, Poole, UK) for the standard curve was quantified by measuring the amount of F6P released after acid hydrolysis of the stock solution (Stitt, 1990b).
F26BP was assayed using a modification of the method described by Stitt (1990b). The reaction buffer contained 100 mM TRIS/HCl [pH 8.0], 1 mM MgCl2, 100 µM NADH, 4.8 mM F6P, 0.6 IU aldolase (EC 4.1.2.13), 6 IU triose phosphate isomerase (EC 5.3.1.1), 0.4 IU glycerol 3-P dehydrogenase (EC 1.1.99.5), and 0.05 IU PFP. For the preparation of the internal standard curve, a sample of extract was mixed with 0.2 vols of 250 mM HCl and incubated at 25 °C for exactly 10 min. During this period the mixture was centrifuged briefly (2 min at 13 000 g) and aliquots pipetted into four wells of a microplate. These aliquots were then neutralized with reaction buffer and spiked with up to 2 pmol of F26BP. Samples of extract for the measurement of F26BP were pipetted directly into the microplate wells and mixed with reaction buffer before adding the 0.2 vol. of 250 mM HCl. The reactions were started by adding Na4PPi to 0.4 mM and the changes in absorbance at 340 nm followed over 15 min using a Spectromax 340 plate reader (Molecular Devices, Wokingham, UK).
Measurement of carbohydrates
Sucrose and hexoses were measured as described previously (Jones et al., 1977). Starch was measured essentially as described previously (Sonnewald et al., 1991) except that the pellet was autoclaved for 20 min before overnight digestion. Absorbance changes were measured using a Spectromax 340 plate reader. Fructans were separated by HPLC using a Carbopac CA1 column (Dionex, Camberley, UK). Elution was with a 0–1 M gradient of sodium acetate, and fructans were detected using an ED40 electrochemical detector (Dionex) by integrated amperometry employing a gold working electrode and a Ag/AgCl reference electrode. Inulin purified from chicory root (Sigma) was used as a standard.
Recovery of carbohydrates and F26BP during extraction and assay
Frozen leaf material was ground to a powder in a mortar and pestle with liquid nitrogen. The powder was divided into two and one portion extracted with 50 mM KOH as described above. The other portion was extracted with 50 mM KOH containing an amount of F26BP, sucrose, or starch comparable to that in the tissue. F26BP or carbohydrates were measured in each sample, and the recovery of the added metabolite calculated.
Measurement of chlorophyll
Aliquots of extract were immediately neutralized with an equal volume of 1 M TRIS/HCl [pH 8.0] and made to 80% (v/v) with acetone. Chlorophyll was measured as described previously. (Porra et al., 1989).
Measurement of rates of photosynthesis
The rates of photosynthesis of third leaves of wheat plants were measured using an infrared gas analyser (ADC Mark 3) (ADC, Hoddesdon, UK). Rates were measured under ambient CO2 at 18 °C with a light intensity of 150 µmol m-2 s-1.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Development of extraction and assay methods for F26BP from wheat leaves
Initial attempts to measure F26BP in wheat leaves were based on a previously published method for primary barley leaves (Nielsen, 1992
; Nielsen and Veierskov, 1990). Sections from the third, fully expanded leaves of 25-d-old plants were ground to a powder in liquid nitrogen, homogenized in 50 mM KOH, incubated with activated charcoal (10 mg ml-1) and the insoluble material removed by centrifugation. F26BP was assayed as described earlier (Stitt, 1990b) with an internal standard curve being produced by hydrolysing endogenous F26BP in aliquots of the samples with 1 vol. of 250 mM HCl, neutralizing with reaction buffer and then adding known amounts of purified F26BP. The concentrations of components of the assay were optimized to give the largest activation of PFP over the range of concentrations of F26BP used, and F6P was treated to remove contaminating F26BP. Although recoveries of added F26BP using this method were acceptable, careful analysis revealed that in some samples the amount of F26BP detected was not proportional to the amount of extract added. For example, in one experiment, extracts were prepared from samples taken from the middle region of the third leaves of wheat plants harvested at different times of the night or day. Out of 24 extracts prepared, the ratio of the amount of F26BP measured in a volume of 20 µl to that measured in 10 µl ranged from 0.96–1.92 (mean±s.d.=1.44±0.25). Additionally, replicate samples harvested at the same time of day or night varied considerably in the degree of non-linearity of the assay with extract volume (results not shown). Clearly, this extraction and assay method is unsuitable for the accurate measurement of F26BP in wheat leaves.
To investigate the possibility that a heat-sensitive component of wheat leaf extracts was responsible for the non-linearity of the assay, the method was modified to include a heat treatment (80 °C for 10 min). Although this did increase the linearity of the assay to some extent, the improvement was not enough to give reliable results (Table 1a). Next, the possibility that an acid-labile component of the extract also contributed to the non-linearity was examined by reducing the amount of 250 mM HCl added at this step, from 1 vol. to 0.2 vols. Whereas the pH of extract mixed with 1 vol. of acid was about 1, when mixed with 0.2 vols of acid the final pH was about four. A 10 min incubation under these conditions still hydrolysed all the F26BP in the extract. Again, this modification increased the linearity of the assay but it was not sufficient for the results to be reliable (Table 1b). However a combination of the two modifications, i.e. the heat treatment and a decrease in the amount of HCl used when preparing the standard curve, did give measurements of F26BP that were essentially proportional to the amount of extract used in the assay (Table 1c). As shown in Fig. 1, amounts of F26BP measured using the modified method were linear with extract volume for samples prepared from leaves of a range of ages; primary leaves of 10-d-old plants, third leaves of 4-week-old plants, and flag leaves of 10-week-old plants.
|
|
Recoveries of F26BP from wheat leaves
The efficiency of extracting F26BP from wheat using the modified method was calculated by measuring the recovery of F26BP added to the extraction buffer. Wheat leaves of different ages (primary, third and flag) were harvested 1 h before the end of the day, each sample ground in liquid nitrogen and divided into two fractions. One was extracted with 50 mM KOH alone and the other with 50 mM KOH containing an amount of F26BP comparable to that in the tissue. The recoveries for primary leaves (90±8%) and third leaves (88±6%) were considered to be good, and the recovery from flag leaves (76±19%) was judged acceptable. All values are mean±s.d. for three experiments.
Amounts of F26BP from different sections of wheat leaves
Potential heterogeneity in the distribution of F26BP within wheat leaves was examined by measuring amounts in different sections of differently aged leaves. Fully expanded wheat leaves of different ages (primary, third and flag) were divided into five regions numbered from the base (one) to the tip (five) and samples removed either 90 min before the end of the day or 90 min after the beginning of the night. Recoveries of added F26BP were similar for all regions from all leaves, and all assays were linear with extract volume (results not shown). Figure 2b, d, and f shows the amount of F26BP on a chlorophyll basis. For both flag and third leaves, there were no differences between the amounts in the different regions of the leaves, and the amounts were higher in the dark than in the light in all regions. In primary leaves, the amounts of F26BP in regions two to five were similar to each other, and did not vary much between the light and the dark. Interestingly, the base (region one) of this leaf had very high levels of F26BP, and the amounts actually decreased from light to dark. Figure 2a, c, e shows the same data expressed on a fresh weight basis. In flag leaves, the amounts were similar in all five regions, in either the light or the dark. In third and primary leaves, the amounts in different regions were similar to each other in the light, but in the dark the amounts increased from the base to the tip.
|
), or 90 min after the start of the night (
). Sections were numbered from the base (1) to the tip (5). Amounts were calculated either per fresh weight (a, c, e) or per chlorophyll (b, d, f) and are the mean ±s.d. for three replicate samples.
Diurnal changes in amounts of carbohydrates and F26BP
To examine the pathways of carbohydrate partitioning in fully expanded source leaves of wheat, the amounts of carbohydrates were measured in samples prepared from region two of third leaves taken at different times of the day or night over a 2 d period. Fructans could not be detected in any of the samples (limit of detection was about 1 µmol hexose equivalents mg-1chl) and amounts of hexoses were very low, <0.3 µmol mg-1chl (results not shown). Recoveries of glucose (84±7), fructose (90±7), sucrose (105±7) and starch (75±7) were considered as acceptable (mean±s.d., n=6). As shown in Fig. 3, carbon accumulated in the leaf during the day as both sucrose and starch. The amounts of both of these compounds decreased during the night, and there was almost complete turnover of the pools of both of them; from three experiments 93±1% of the sucrose, and 89±2% of the starch that accumulated during the day were remobilized at night. The ratio of sucrose to starch was essentially constant throughout the duration of the experiment, although there was perhaps a slight decrease towards the end of the night.
|
The role of F26BP in the regulation of carbohydrate partitioning in wheat leaves was examined by measuring the metabolite in the samples taken over the second 24 h period of the experiment described above (Fig. 4
). Twice as much F26BP was present in the dark (170 pmol mg-1chl) as in the light (80 pmol mg-1chl). The decrease in the amount of F26BP during the day occurred within the first hour after the transition from light to dark. Similarly, the increase in the amount at night occurred within the first hour after the transition from dark to light. After these initial periods the amounts did not change further and remained constant throughout the remainder of the night or day. Rates of photosynthesis measured under growth conditions were about 34 µmol mg-1chl h-1 at all times of the day.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The results presented here are the first reliable measurements of F26BP in the leaves of wheat. There are previous reports of the extraction and assay of F26BP from wheat leaves (Klechan and Buchanan, 1988
; Klechan et al., 1986; Nielsen, 1992; Reddy, 1998; Streuter et al., 1989), but none of these are accompanied by evidence of both the use of an internal standard curve and of the adequate recovery of added F26BP. The metabolite has been measured in other monocotyledons such as barley primary leaves (Sicher et al., 1986), source leaves from 4–6-week-old maize plants (Stitt and Heldt, 1985), and fourth leaves of Lolium temulentum (L.) (Pollock et al., 1989). However, none of these address the possibility of non-linearity with extract volume. The method presented here gives acceptable recoveries for the metabolite for samples prepared from a range of ages of wheat leaves, and the amounts of F26BP measured are proportional to the amounts of extract in the assays. This method may be generally applicable to other plants, such as maize, where non-linearity of the amount of F26BP with extract volume has been encountered. Excluding the bases of primary leaves, the amount of F26BP in wheat leaves grown under 150 µmol m-2 s-1 light varies from about 50 pmol mg-1chl to about 500 pmol mg-1chl. In samples harvested in the light, the amount of F26BP in wheat primary leaves is similar to the amount claimed for barley primary leaves grown under higher light (about 550 µmol m-2 s-1) with a 12/12 h light/dark cycle (Sicher et al., 1986). In darkened wheat primary leaves the amount of F26BP is about 4-fold greater than that measured in barley plants grown under continuous low light (75 µmol m-2 s-1) (Nielsen, 1992), but is a lot less than the 4 nmol mg-1chl for high light-grown barley plants claimed by Sicher et al. (Sicher et al., 1986). Whether or not this difference is a result of the different growth conditions remains to be determined. The amounts in fully expanded third leaves are similar to those measured in source leaves of maize (Stitt and Heldt, 1985) and L. temulentum (Pollock et al., 1989). In general, the amounts in wheat are at the lower end of the range of amounts measured in a selection of other species (Sicher et al., 1987; Scott and Kruger, 1994).
Generally there is a tight correlation between the amount of chlorophyll and the amount of F26BP in different regions of fully expanded primary, third, and flag leaves of wheat. This implies that there may be co-ordination of the amount of this inhibitor of sucrose synthesis with the photosynthetic capacity of a cell. The heterogeneity in the amount of F26BP in different regions that is observed when the data are expressed on a fresh weight basis reflects differences in the ratio of chlorophyll to fresh weight in the different regions of the leaves. These data contrast with expanding primary leaves of barley grown under continuous low light where the amounts of F26BP expressed on a chlorophyll basis were higher in the fully expanded tip than in the still expanding base (Nielsen, 1992). In these leaves, there was a good correlation between the amount of chlorophyll and the activity of the enzyme that synthesizes F26BP, fructose 6-P 2-kinase (F6P2K, EC 2.7.1.105) (Nielsen, 1992). The high activity of PFKII in the leaf tip is suggested to reflect a requirement for greater flexibility in regulating the amount of F26BP in tissues with high rates of photosynthesis. It is not known whether the difference between the results of Nielsen (Nielsen, 1992) and those presented in this paper is due to a difference in growth conditions, developmental stage, or is a difference between wheat and barley.
The exception to this tight correlation between amounts of chlorophyll and F26BP is in the base of young leaves. This region contains the leaf sheath, and since it contains little chlorophyll will be unable to sustain much, if any net CO2 assimilation. There was considerable F26BP in this region, especially in the light, implying a role for the metabolite in regulating carbon metabolism in non-photosynthetic areas of wheat leaves (Stitt, 1990a). Interestingly, unlike all other regions examined, darkening caused a large decrease in the amount of F26BP. The significance of a dark-induced decrease in F26BP is not clear, but may reflect a role for the metabolite in regulating non-photosynthetic carbohydrate metabolism in this area of a primary wheat leaf.
The relative roles of sucrose and starch as intermediary carbon stores in wheat source leaves were examined by measuring changes in the amounts of carbohydrates in region two of third, fully expanded leaves over a 2 d period. As expected, the majority of carbon that accumulated in the leaf during the day did so as sucrose. However, starch accumulation accounted for 20–30% of the carbohydrate stored in the leaf during the day. The pools of both sucrose and starch in the leaf were almost entirely depleted by the end of the night, demonstrating that both sucrose and starch are used as transitory stores of carbon in the wheat leaf. Significant amounts of starch (up to about 40% of the amount of sucrose) in leaves of cereals were also found in primary wheat leaves (Zulu et al., 1991), third leaves of wheat (Lawlor et al., 1987) and in primary leaves of barley (Sicher et al., 1984).
To assess the role of F26BP in the regulation of these pathways, the metabolite was measured in the samples of third, fully expanded source leaves harvested during the second day of the experiment described above. Previous studies of this kind in cereal leaves are limited to the primary leaves of young barley seedlings (Nielsen, 1992; Sicher et al., 1986). As expected for a potential inhibitor of sucrose synthesis, the amount of F26BP was lower during a period of net sucrose accumulation (during the day) than when the pool of sucrose was decreasing (at night). Supporting this, a comparison of the amount of F26BP in different regions of different ages of wheat leaves (Fig. 2
) showed that F26BP was also lower during the day than during the night in flag leaves. These results are consistent with a role for the metabolite in regulating rates of sucrose synthesis in the leaves of wheat, and are similar to results obtained with primary barley leaves (Nielsen, 1992; Sicher et al., 1986). Surprisingly, amounts of F26BP in fully expanded primary leaves did not change between the night and the day. However further experiments are required before any conclusions about the roles of F26BP in these young leaves can be drawn from this observation. Under the growth conditions used (light intensity of 150 µmol m-2 s-1, 14 h day) both the ratio of sucrose to starch and the amount of F26BP remained relatively constant throughout during the light period. These data are consistent with a role for F26BP in regulating partitioning of carbohydrate between sucrose and starch in the third leaves of wheat. However, rates of accumulation of sucrose and starch are not necessarily an indicator of rates of synthesis, and 14CO2 feeding experiments measuring the flux of carbon into these compounds are required to determine if the correlation holds under these, and other, experimental conditions. Interestingly, although the amount of F26BP in primary leaves from 8-d-old barley seedlings did not change throughout the day (Sicher et al., 1986), there were small variations in the rates of sucrose and starch accumulation in leaves from 7 d seedlings over the same period (Sicher et al., 1984).
The results presented above confirm that the method described here can accurately measure F26BP in extracts prepared from wheat leaves. However, they do not reveal the cause of the non-linearity of the original assay. One hypothesis is that wheat leaves contain an uncharacterized acid-labile inhibitor of PFP. The inhibitors of PFP identified to date act as competitive inhibitors of activation of the enzyme by F26BP (Van Shaftingen et al., 1982; Kombrink and Kruger, 1984; Stitt, 1989). Although none of these are acid-labile, the presence of an additional inhibitor that is unstable in acid and that has similar properties to those so far identified can explain the non-linearity of the assay with extract volume, as follows. When a standard curve is produced using the original assay method, a relatively strong acid treatment (pH=1) is used to remove endogenous F26BP from aliquots of the sample and an acid-labile inhibitor of PFP will therefore be degraded during this treatment. However, the amount of F26BP in the sample is measured by following the activation of PFP by aliquots of extract that have not been treated with acid. Since these will still contain the inhibitor, the activation of PFP by F26BP in these reactions will be reduced and the amount of F26BP will therefore be underestimated. As the amount of extract in the assay is increased, the inhibition of PFP activation will become more pronounced, leading to a progressive increase in the underestimation of the amount of F26BP.
This hypothesis can be reconciled with the data presented in Table 1 if it is assumed that there are in fact two acid-labile inhibitors of PFP in extracts prepared from wheat leaves. The first of these (compound A) is assumed to be less sensitive to acid hydrolysis than F26BP and, therefore, to be stable during the less acidic (pH 4) conditions of the modified assay. As a consequence, inhibition of PFP by compound A will be equal in the reactions used to generate the standard curve and in those used to estimate endogenous F26BP. However, as shown in Table 1b, this modification does not by itself give a linear response of the assay to extract volume, suggesting that the extracts contain another inhibitor (compound B) which, as described above, will be degraded under the less acidic conditions of the modified assay (pH 4). Incubating the samples at 80 °C during extraction improves the linearity of the assay (Table 1a), suggesting that compound B is heat labile and is destroyed under these modified extraction conditions. Although there is no evidence for the occurrence of these inhibitors, the possibility that they exist is significant. Current understanding of the role of PFP in plants is poor (Stitt, 1990a; Hajirezaei et al., 1994; Paul et al., 1995), and the isolation and characterization of novel inhibitors of PFP could prove significant in advancing our knowledge of the role of this enzyme in plant carbohydrate metabolism.
|
Acknowledgments |
|---|
This work was supported by a BBSRC David Phillip's Fellowship. I thank Christine Foyer and Martin Parry for their comments on the manuscript.
|
Notes |
|---|
1 Fax: +44 1582 763010. E-mail:stephen.trevanion{at}bbsrc.ac.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Avigad G.1984. Assay of D-fructose 2,6-bisphosphate in biological extracts. Analytical Letters 17, 371–384.
Ball K, ap Rees T.1989. Fructose 2,6-bisphosphate and the climacteric in bananas. European Journal of Biochemistry 177, 637–641.[Web of Science][Medline]
Cséke C, Weeden NF, Buchanan BB, Uyeda K.1982. A special regulatory fructose bisphosphate functions as a cytoplasmic regulatory metabolite in green leaves. Proceedings of the National Academy of Sciences, USA 79, 4322–4326.
Hajirezaei M, Sonnewald U, Viola R, Carlisle S, Dennis D, Stitt M.1994. Transgenic potato plants with strongly decreased expression of pyrophosphate: fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers. Planta 192, 16–30.[Web of Science]
Hue L, Blackmore PF, Shikama H, Robinson-Steiner A, Exton JH.1982. Regulation of fructose-2,6-bisphosphate content in rat hepatocytes, perfused hearts, and perfused hindlimbs. Journal of Biological Chemistry 257, 4308–4313.
Jones MGK, Outlaw WH, Lowry OH.1977. Enzymic assay of 10-7 to 10-4 moles of sucrose in plant tissues. Plant Physiology 60, 379–383.
Klechan AL, Buchanan BB.1988. Powdery mildew infection alters fructose-2,6-bisphosphate content and sucrose to starch ratio in leaves of wheat plants actively forming grain. Physiological and Molecular Plant Pathology 32, 221–227.
Klechan AL, Elliott VJ, Buchanan BB.1986. A recognition metabolite for biological stress in plants: fructose-2,6- bisphosphate. Comptes Rendus de l’Académie des Sciences, Paris 302, 51–54.
Kombrink E, Kruger NJ.1984. Inhibition by metabolic intermediates of pyrophosphate: fructose 6-phosphate phosphotransferase from germinating castor bean endosperm. Zeitschrift für Pflanzenphysiologie 114, 443–453.
Kruger NJ, Scott P.1995. Integration of cytosolic and plastidic carbon metabolism by fructose 2,6-bisphosphate. Journal of Experimental Botany 46, 1325–1333.
Kruger NJ, Kombrink E, Beevers H.1983. Fructose 2,6- bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: effect on PPi: fructose 6-phosphate phosphotransferase. Biochemical and Biophysical Research Communications 117, 37–42.[Web of Science][Medline]
Lawlor DW, Boyle FA, Young AT, Kendall AC, Keys AJ.1987. Nitrate nutrition and temperature effects on wheat: soluble components of leaves and carbon fluxes to amino acids and sucrose. Journal of Experimental Botany 38, 1091–1103.
Neuhaus HE, Kruckeberg AL, Feil R, Stitt M.1989. Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. II. Study of the mechanisms which regulate photosynthate partitioning. Planta 178, 110–122.
Nielsen TH.1992. Differences in fructose-2,6-bisphosphate metabolism between sections of developing barley leaves. Physiologia Plantarum 84, 577–583.
Nielsen TH, Veierskov B.1990. Regulation of carbon partitioning in source and sink leaf parts in sweet pepper (Capsicum annuum L.) plants. Plant Physiology 93, 637–641.
Paul M, Sonnewald U, Hajirezaei M, Dennis D, Stitt M.1995. Transgenic tobacco plants with strongly decreased expression of pyrophosphate: fructose-6-P 1-phosphotransferase do not significantly from wild type in photosynthate partitioning, plant growth or their ability to cope with limiting phosphate, limiting nitrogen and suboptimal temperatures. Planta 196, 277–283.[Web of Science]
Pollock CJ, Cairns AJ, Collis BE, Walker RP.1989. Direct effects of low temperature upon components of fructan metabolism in leaves of Lolium temulentum L. Journal of Plant Physiology 134, 203–208.
Porra RJ, Thompson WA, Kreidemann PE.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 spectroscopy. Biochimica et Biophysica Acta 975, 384–394.
Reddy AR.1998 Water stress effect on photosynthesis and fructose 2,6-bisphosphate content in triticale (Triticosecale Witt) leaves. Indian Journal of Experimental Biology 36, 403–406.
Scott P, Kruger NJ.1994. Fructose 2,6-bisphosphate levels in mature leaves of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum). Planta 193, 16–20.
Scott P, Lange AJ, Pilkis SJ, Kruger NJ.1995. Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6-bisphosphate levels. The Plant Journal 7, 461–469.[Web of Science][Medline]
Sicher RC, Baysdorfer C, Kremer DF.1987. A comparative analysis of fructose 2,6-bisphosphate levels and photosynthate partitioning in the leaves of some agronomically important crop species. Plant Physiology 83, 768–771.
Sicher RC, Kremer DF, Harris WG.1984. Diurnal carbohydrate metabolism of barley primary leaves. Plant Physiology 76, 165–169.
Sicher RC, Kremer DF, Harris WG.1986. Control of photosynthetic sucrose synthesis in barley primary leaves. Role of fructose 2,6-bisphosphate. Plant Physiology 82, 15–18.
Sonnewald U, Brauer M, von Schawaen A, Stitt M, Willmitzer L.1991. Transgenic tobacco plants expressing a yeast-derived invertase in either cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and source/sink interactions. The Plant Journal 1, 95–100.[Web of Science][Medline]
Stitt M.1989. Product inhibition of potato tuber pyrophosphate: fructose 6-phosphate phosphotransferase by phosphate and pyrophosphate. Plant Physiology 89, 628–633.
Stitt M.1990a. Fructose-2,6-bipsphosphate as a regulatory molecule in plants.Annual Review of Plant Physiology and Plant Molecular Biology 41, 153–185.[Web of Science]
Stitt M.1990b. Fructose 2,6-bisphosphate. In: Rey PM, Harborne J, eds. Methods in plant biochemistry, Vol. 3. London: Academic Press, 87–92.
Stitt M, Gerhardt R, Kürzel B, Heldt HW.1983. A role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis in spinach leaves. Plant Physiology 72, 1139–1141.
Stitt M, Heldt H.1985. Control of photosynthetic sucrose synthesis by fructose 2,6-bisphosphate. Intracellular metabolite distribution and properties of the cytosolic fructose bisphosphatase in leaves of Zea mays L. Planta 164, 179–188.
Stitt M, Huber S, Kerr P.1987. Control of photosynthetic sucrose formation. In: Hatch MD, Broadman NK, eds. The biochemistry of plants, Vol. 10. New York: Academic Press, 327–409.
Stitt M, Mieskes G, Soling H-D, Heldt, H.W.1982. On a possible role for fructose 2,6-bisphosphate in regulating photosynthetic metabolism in leaves. FEBS Letters 145, 217–222.
Streuter N, Moerschbacher BM, Fischer Y, Noli U, Reisener HJ.1989. Fructose-2,6-bisphosphate in wheat leaves infected with stem rust. Journal of Plant Physiology 134, 254–257.
Thomas H, Uyeda K.1986. A competitive binding assay for fructose 2,6-bisphosphate. Analytical Biochemistry 154, 50–56.[Web of Science][Medline]
Trevanion SJ, Kruger NJ.1991. Effect of temperature on the kinetic properties of pyrophosphate: fructose 6-phosphate phosphotransferase from potato tuber. Journal of Plant Physiology 137, 753–759.
Truesdale MR, Toldi O, Scott P.1999. The effect of elevated concentrations of fructose 2,6-bisphosphate on carbon metabolism during deacidification in the crassulacean acid metabolism plant Kalanchoë daigremontiana. Plant Physiology 121, 957–964.
Uyeda K, Furuya E, Luby LJ.1981. The effect of natural and synthetic D-fructose 2,6-bisphosphate on the regulatory kinetic properties of liver and muscle phosphofructokinases. Journal of Biological Chemistry 256, 8394–8399.
Van Schaftingen E, Lederer B, Bartrons R, Hers H-G.1982. A kinetic study of pyrophosphate: fructose 6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate. European Journal of Biochemistry 129, 191–195.[Web of Science][Medline]
Zulu JN, Farrar JF, Whitbread R.1991. Effect of phosphate supply on wheat seedlings infected with powdery mildew: carbohydrate metabolism of first leaves. New Phytologist 118, 553–558.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. N. Scofield, S. A. Ruuska, N. Aoki, D. C. Lewis, L. M. Tabe, and C. L. D. Jenkins Starch storage in the stems of wheat plants: localization and temporal changes Ann. Bot., April 1, 2009; 103(6): 859 - 868. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||