lnfluence of elevated fructose-2,6-bisphosphate levels on ... · plant physiol. (1 995) 108: 1569-1...

Plant Physiol. (1 995) 108: 1569-1 577

lnfluence of Elevated Fructose-2,6-Bisphosphate Levels on Starch Mobilization in Trans enic Tobacco Leaves

in the Dark f Peter Scott and Nicholas J. Kruger*

Department of Plant Sciences, University of Oxford, Oxford 0x1 3RB, United Kingdom

In contrast to the well-defined role of Fru-2,6-bisP during photosynthesis, our understanding of the influente of Fru- 2,6-bisP on nonphotosynthetic carbon metabolism is limited. In darkened leaves, Fru-2,6-bisP has been proposed to contribute to the regulation of both respiration and starch mobilization. However, the evidente that Fru-2,b-bisP plays a role in leaf respiration is at best questionable. With the exception of a single report on a direct correlation between the rate of dark respiration and Fru-2,6-bisP levels in primary leaves Of barley (Baysdorfer et 1987)r evidence that Fru-2,6-bisP is involved in respiration is con- fined to nonPhotosYnthetic Plant tissues and cell-susPen- sion cultures. In ripening banana there are suggestions that an increase in Fru-2,6-bisP correlates with an increase in the rate of respiration (Beaudry et al., 1989). However, other workers have cast serious doubts on the observation

ap R ~ ~ ~ , 1988). ln addition, the activity of p ~ p , which is suggested to catalyze the increase in the rate of respiration in banana (Beaudry et al., 1989), decreases during the phase of ripening in which respiration is greatest (Beaudry et al., 1987). Work with heterotrophic cell-suspension cultures of Chenopodium rubrum demonstrates that, under certain cir- cumstances, there is a correlation between increasing Fru- 2,6-bisP levels and the rate of respiration (Hatzfeld and Stitt, 1991). However, the same study shows that changes in the rate of respiration can occur independently of changes in the Fru-2,6-bisP level. Such data argue that there is no obligatory link between the rate of glycolysis and the leve1 of Fru-2,6-bisP.

Similarly, evidence that Fru-2,6-bisP can affect starch metabolism in darkened leaves is limited and exclusively correlative. In primary leaves of barley and pea and mature leaves Of maize, Fru-2,6-bisP levels increase dramatically at the beginning of the dark period (Sicher et al., 1986, 1987). Starch degradation in leaves of these species did not begin until the levels of Fru-2h-bisP had decreased appreciably from the initial peak leve1 in the dark. However, in potato and soya leaves Fru-2,6-bisP levels increased at the beginning of the dark Period at the Same time as starch degradation was initiated (Sicher et al., 1986; Scott and Kruger, 1994). Thus, there is no consistent relationship between Fru-2,6-bisP levels and starch degradation. In species in

Abbreviations: FBPase, Fru-1,6-bisphosphatase; Fru-2,6-P2ase, Fru-2,6-bisphosphatase; 6-PF-2-K, 6-phosphofructo-2-kinase; PFP, PPi:Fru-6-P 1-phosphotransferase; SPGA, 3-phosphoglyceric acid.

l h e aim of this work was to study the effect of elevated fructose- 2,6-bisphosphate (Fru-2,6-bisP) levels on carbohydrate metabolism in leaves in the dark. In transgenic tobacco (Nicotiana fabacum 1.) lines containing mammalian 6-phosphofructo-2-kinase activity there is an inverse relationship between the leve1 of Fru-2,6-bisP in leaves and the rate of starch breakdown in the dark. Estimates of the flux response coefficient for the rate of net starch degradation with respect to changes in Fru-2,C-bisP level are -0.57 for whole leaves and -0.69 to -0.89 for excised leaf discs. We suggest that this decrease in the net rate of starch breakdown is caused, at least in part, by stimulation of unidirectional starch synthes;s. M ~ ~ ~ ~ ~ ~ - ments of the levels of metabolic intermediates and the metabolism of [U-'4Clglucose indicate that the stimulation of starch synthesis in the dark is a result of high Fru-2,6-bisP levels, increasing the 3-phos- phog1ycerate:inorganic phosphate ratio in leaves. We argue that the observed response to changes in the level of Fru-2,6-bisP are ef-

1-phosphotransferase. However, the extent to which changes in Fru-2,L-bisP inftuence starch metabolism in wild-type plants is not known.

fected through activation of pyrophosphate:fructose-6-phosphate that Fru-2,6-bisP levels change during ripening (Ball and

Fru-2,6-bisP is an important metabolic effector of two enzymes involved in the interconversion of Fru-6-P and Fru-1,6-bisP in plants. First, it is a potent inhibitor of the cytosolic FBPase (EC 3.1.3.11), which catalyzes the following irreversible reaction: Fru-1,6-bisP -+ Fru-6-P + Pi. Sec- ond, Fru-2,6-bisP stimulates the cytosolic enzyme PFP (EC 2.7.1.90), which catalyzes the following readily reversible reaction: Fru-6-P + PPi % Fru-1,fj-bisP + Pi. Current views of the role of Fru-2,6-bisP in photosynthetic carbon metabolism have centered almost exclusively on its ability to modulate cytosolic FBPase activity in vivo. It is through inhibition of FBPase that Fru-2,6-bisP is thought to contribute to the regulation of the partitioning of photosyntheti- cally fixed carbon between Suc and starch. Work on spinach, Clarkia, and transgenic tobacco (Nicotiana tabacum L.) has confirmed that Fru-2,6-bisP m&es an important contribution to the control of this partitioning during the early stages of the light period (Neuhaus et al., 1989, 1990; Scott et al., 1995).

This research was supported by the Agriculture and Food Research Council (grant No. PG43/531) and the Royal Society of London, UK.

* Corresponding author; e-mail krugerQtrans.plants.ox.ac.uk; fax 44-1865-275074.

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1570 Scott and Kruger Plant Physiol. Vol. 108, 1995

which there is a correlation between high Fru-2,6-bisP levels and low rates of starch degradation it is difficult to assess whether changes in Fru-2,6-bisP regulate starch degradation or whether Fru-2,6-bisP levels simply reflect changes in the levels of other metabolites.

Thus, from these data it is not possible to establish whether Fru-2,6-bisP has a role in leaves in the dark. To address this problem we have studied carbohydrate metabolism in darkened leaves of transgenic plants containing a modified mammalian gene for 6-PF-2-K (EC 2.7.1.105) (Scott et al., 1995). In these plants we have observed up to a 4-fold increase in the extractable 6-PF-2-K activity in leaves. Transformation with the mammalian gene resulted in transgenic plant lines with Fru-2,6-bisP levels ranging from 115 to 230% of those observed in leaves of wild-type tobacco. Here we show that in transgenic tobacco leaves there is an inverse correlation between Fru-2,6-bisP levels in leaves and the rate of starch mobilization in the dark. We argue that this reduction in the net rate of starch breakdown is due, at least in part, to an increase in the rate of unidirectional starch synthesis in the transgenic plants. We suggest that this effect is mediated through the modulation of PFP activity in the dark. The possibility that Fru-2,6-bisP levels could influence starch mobilization in wild-type leaves is considered.

MATERIALS AND METHODS

Plant Material and Crowth Conditions

Tobacco (Nicotiana tabacum L. cv Samsun) was obtained from the Department of Plant Sciences (Cambridge, UK). The transgenic tobacco lines used were generated as described by Scott et al. (1995). Plants were grown under illumination of 200 pmol PAR m-' s-' at 25°C. Photope- riod was varied as specified in the text and figure and table legends. Seeds obtained from primary transformants were germinated in the presence of 200 pg mL-l kanamycin for 1 week. The seedlings were then grown in a greenhouse for 6 weeks in a 12-h photoperiod using 200 pmol PAR m-' s-' illumination at 25°C. From the age of 4 weeks plants were fed fortnightly with 10 mL of a preparation of 3 g L-' of Miracle-Grow (ICI Chemicals, Farnham, Surrey, UK). Plants were transferred to the desired growth conditions in a growth cabinet 2 weeks before metabolic measurements were made. Tobacco plants used in experiments were between 6 and 10 weeks old, except for those used in Tables I and 111, which were 18 weeks old. A11 measurements were performed on healthy, fully expanded leaf tissue.

The transgenic plant lines used in this work were A(31), A(46), B(44), B(114), C(102), and D(86). A large range of plant lines was used to demonstrate that our observations were consistent over a wide range of transformants. How- ever, our study has concentrated on the plant lines A(31) and D(86), in which photosynthetic metabolism was studied in earlier work (Scott et al., 1995). The principal metabolic characteristics of the transgenic plants used in this study were described by Scott et al. (1995).

Extraction and Measurement of Metabolites

Leaf material was freeze-clamped between two alumi- num blocks bolted onto the end of a pair of blacksmith's tongs, which had been precooled in liquid nitrogen (ap Rees et al., 1977). Each sample was homogenized in liquid nitrogen in a mortar and pestle. For a11 metabolites except Fru-2,6-bisP, the homogenate was immediately suspended in 2 mL of 1.4 M perchloric acid and left on ice for 2 h. The extract was then neutralized with 5 M KCO,, and the insoluble debris was removed by centrifugation at 10,OOOg. The pellet was then resuspended in 0.5 mL of water and centrifuged at l0,OOOg. This was repeated a second time, and the supernatants were pooled. Glc-6-P and Fru-6-P were measured in the extract as described by Michal (1984a). Triose phosphates and 3PGA were measured as described by Michal (1984b).

Starch was extracted and measured as described by Scott and Kruger (1994). Fru-2,6-bisP levels in tobacco leaves were determined as described by Scott and Kruger (1994).

To ensure the reliability of the extraction and measurement of the metabolic intermediates we performed recovery experiments. For these, an amount of the authentic metabolic intermediate similar to that present in tobacco leaves was added to the freeze-clamped sample prior to extraction. Estimates of the recovery (mean 5 SE, where n = 3) of the metabolites added to the tobacco tissue were: 100.0 2 3.1% for Glc-6-P, 100.6 2 3.1% for Fru-6-P, 94.0 .+- 13.3% for triose phosphate, 111.4 _f 9.8% for 3PGA, and 106.4 2 9.7% for Fru-2,6-bisP.

Extraction and Measurement of Enzyme Activity

Enzymes were extracted as detailed by Hajirezaei and Stitt (1991). The activities of FBPase, PFP, and 6-PF-1-K were measured as described by Scott et al. (1995). ADP- Glc pyrophosphorylase was measured as described by Hajirezaei et al. (1994) using 50 mM Hepes-KOH (pH 8.2), 5 mia MgCl,, 2.4 mM NAD, 5 mM glycerate-3-P, 2 mM ADP-Glc, 1 unit of Glc-6-P dehydrogenase (from Leuconos- toc), 1 unit of phosphoglucomutase in a volume of 250 pL. Starch phosphorylase was measured using 50 mM Hepes- KOH (pH 7.0), 5 mM MgCI,, 2.0 mM NAD, 1.2 mM Na,HPO,, 4 FM Glc-1,6-bisP, 4 mg of amylopectin, 1.4 units of Glc-6-P dehydrogenase (from Leuconostoc), and 4 units of phosphoglucomutase in a volume of 250 FL. Activities of a11 of these enzymes were measured spectrophotometri- cally with an EL340 microplate Bio Kinetics Reader (Bio- Tek Instruments, Winooski, VT) at 30°C.

Metabolism of 14C-Starch

Starch in tobacco leaf discs (10 mm diameter) was radiolabeled by illuminating the discs in a leaf-disc oxygen electrode (Hansatech, Kings Lynn, Norwich, UK) at 200 pmol PAR m-' s-' at 20°C for 20 min. The CO, was supplied from 200 pL of 1 M NaH'*CO, (specific activity, 7.4 GBq mol-'1, pH 9.0, placed on a felt mat at the base of the inner chamber of the electrode. Leaf discs were then harvested and extracted or floated in the dark on 20 mM NaCl (adjusted to pH 7.4 with 1 M NaOH) for 1 h (Quick et

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Fru-2.6-bisP lnhibits Starch Mobilization 1571

al., 1989). After this period leaf discs were extracted and the distribution of radioactivity was analyzed as detailed by Quick et al. (1989).

Metabolism of ['4ClGlc

Leaf discs (10 mm) were cut from leaves submerged in 20 mM NaCl (pH 7.4) to prevent xylemic embolism. The discs were then floated in the presence of 0.7 /LM [U-'"CjGlc (11.0 TBq mol-') in 20 mM NaCl (pH 7.4) for 1 h in the dark. After this period the fate of the radioactivity metabolized by the leaf discs was determined as described by Quick et al. (1989). Labeling of starch was determined by treating ethanol-insoluble material with a-amylase and amyloglu- cosidase as described by Scott and Kruger (1994). Only 14C that was solubilized and then recovered as [14C]Glc was attributed to starch. Glc was separated from other sugars by TLC in a solvent of formic acid:ethyl methyl ketone: tertiary butano1:water (30:60:80:30) as described by Chaplin (1986).

Metabolic Control Analysis

Metabolic control was analyzed quantitatively within the theoretical framework proposed by Kacser and Porteous (1987). The effects of Fru-2,6-bisP levels on starch degradation during the dark period is expressed as the flux response coefficient (X):

R = (SJ/n/(Sx/x)

where SJ/J is the fractional change in carbon flux from starch, and S X / X is a fractional change in the level of Fru-2,6-bisP.

RESULTS

The levels of starch at the end of the night period in wild-type and transgenic tobacco plants after 2 weeks of growth under a 10-h light period are shown in Figure 1.

WT A(31) B(44) A(46) C(102) B(114)

Figure 1. Starch levels at the end of the dark period in transgenic tobacco leaves. The amount of starch at the end of the night in leaves of 6- to 10-week-old wild-type (WT) and transgenic tobacco lines was determined for plants grown under a 10-h light period. The levels of Fru-2,6-bisP (O) in the leaves at the same time are also shown. Values are means 2 SE of measurements from three different leaves. FW, Fresh weight.

These data show that there was little accumulation of starch in leaves of wild-type tobacco and transgenic lines A(31) and B(44). In contrast, appreciable levels of starch were present immediately before the beginning of the light period in the other transgenic lines. There was an approx- imate correlation between the level of Fru-2,6-bisP in the dark and the total amount of accumulated starch. The only exception to this generalization was in plant line B(44) (although in subsequent experiments conducted with a shorter light period, starch accumulated in this line as well; see below).

To discover the reason for the appearance of large amounts of starch at the end of the 14-h dark period in the leaves of transgenic lines A(46), C(102), and B(1141, we conducted the following studies. Initially, we tested whether the accumulation of starch was a result of the transgenic plants synthesizing more starch than wild-type plants during the day. Measurements of starch production in leaves of the wild-type and four transgenic lines during a 12-h light period are shown in Figure 2. We observed no significant difference in the amount of starch synthesized by the transgenic and wild-type plants.

Since accumulation of starch in leaves of transgenic plants could not be accounted for by a change in the amount of starch made by the different tobacco lines during the photoperiod, we investigated whether the rate of starch degradation in the leaves at night differed between the transgenic and wild-type plants. First, we determined the pattern of starch breakdown throughout the dark period. The levels of starch in leaves of wild-type and eight transgenic lines were determined during the first 8 h of a 12-h dark period. For each plant line starch levels decreased progressively throughout the dark period (data not shown). Thus, in subsequent experiments measurements of starch in tobacco leaves at the beginning and end of the dark period were used to estimate the rate of starch breakdown. To determine the rate of breakdown of starch in leaves from different tobacco lines, we placed plants under a 12-h photoperiod. To eliminate variation in the basal levels of starch in the different plant lines, a11 plants were placed in the dark for 72 h prior to the start of the experiments. This treatment completely depleted leaf starch in a11 of the plant lines in this study. The starch levels in leaves during the first 3 d are shown for three plant lines in Figure 3. Under a 12-h light period, starch accumulated in leaves of a11 of the plants investigated. These results contrast with our observations in tobacco plants grown under an 8-h light period in Figure 1. Thus, it appears that the shorter, 12-h dark period used in Figure 3 was not sufficient to enable the plants to mobilize a11 of the starch synthesized during the daytime. Under such conditions the accumulation of starch at the start of d 2 and d 3 was significantly greater (P c0.05) in the transgenic tobacco lines B(44) and C(102) than in the wild type (Fig. 3).

The plant lines studied in Figure 3 and two other transgenic tobacco lines were used to investigate the effect of altered Fru-2,6-bisP on the rate of starch breakdown during the 12-h dark period. We measured the amount of starch in leaves at the beginning and the end of the light period on



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Scott and Kruger Plant Physiol. Vol. 108, 1995

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Figure 2. Starch synthesis in wild-type (WT) and transgenic tobacco leaves. Plants were left in the dark for 72 h to remove starch from leaves and then were grown under a 12-h light period. The amount of starch synthesized in leaves of different lines of transgenic and wild-type tobacco was determined over 1, 2, 3, and 6 d from the start of the experiment. Starch synthesis was determined from three leaf disc samples taken at the beginning and end of the light period. The values for total starch synthesized (O) are means % SE from the four light periods. The mean level of Fru-2,6-bisP (O) from three different leaves at the end of the light period are also shown. FW, Fresh weight.

the lst, 2nd, 3rd, and 6th d after destarching the plants. A comparison of the mean rate of starch degradation during the dark and the mean rate of net daily starch accumulation in the leaves is shown in Figure 4, respectively. These data show that there was an inverse relationship between the rate of starch degradation in leaves and the level of Fru- 2,6-bisP. The decrease in the net rate of starch degradation in the transgenic lines was sufficient to account for the additional accumulation of starch in the leaves of the transgenic plants compared to wild type (Fig. 4b), taking into

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account the minor fluctuations in total starch synthesized in the tobacco lines (Fig. 2).

To establish whether the reduction in rate of starch breakdown reported in Figure 4 was due to changes in the levels of Fru-2,6-bisP in the leaves of the transgenic plants or was a secondary effect caused by modification of metabolism in tissues other than leaves, we measured the rate of starch breakdown in isolated leaf discs of transgenic plants. To ensure that the leaf discs from different lines were comparable, we grew plants under an 8-h daylength to prevent differential accumulation of starch in the transgenic plants (data not shown). As an additional precaution, the plants were kept in the dark for 24 h prior to the start of the experiment. No starch was detected in the samples from the leaves of any of the plants at the outset of the experiment. Starch was labeled to a known specific activity by incubating the leaf discs in saturating levels of 14C0, at 200 kmol PAR m-'s-' at 20°C for 20 min. The redistribu- tion of radioactivity between ethanol-soluble and -insoluble components during an ensuing period of darkness was used to estimate the net rate of starch mobilization (Table I). Preliminary studies confirmed that the release of radioactivity from starch was linear for at least the 1st h of darkness (data not shown). The results presented in Table I again revealed an inverse relationship between the rate of starch degradation in the leaf discs and the level of Fru- 2,6-bisP. These data suggest that elevated Fru-2,6-bisP levels in the transgenic plants inhibited starch mobilization in the leaves.

To investigate the mechanism by which the rate of starch degradation in leaves of the transgenic plants was reduced by elevated Fru-2,6-bisP levels, we determined the effect of changes in Fru-2,6-bisP levels on metabolic intermediates in leaves 2 h into the night (Table 11). The transgenic plants contained significantly lower levels of Glc-6-P and higher levels of C3-phosphorylated intermediates than wild-type plants. One consequence of an increase in C3-phosphory-

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Figure 3. Starch levels in transgenic and wild-type (WT) tobacco leaves at the start and end of three consecutive light periods. Plants were incubated in the dark for 72 h to remove starch from leaves and then were grown under a 12-h light period. The amount of starch present in transgenic and wild-type tobacco leaves was determined at the beginning and end of the first, second, and third light periods. The values are means -C SE of the starch level in three leaf discs from different leaves. FW, Fresh weight. www.plantphysiol.orgon February 13, 2019 - Published by Downloaded from

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Fru-2,6-bisP lnhibits Starch Mobilization 1573

-1 Fructose 2,6-bisphosphate (pmol. g FW)

30 40 50 60 70 80

I 1 0 30 40 50 60 70 80

Fructose 2,6-bisphosphate (pmol. g -' FW)

Figure 4. Rate of starch degradation and accumulation in leaves of transgenic and wild-type tobacco relative to Fru-2,6-bisP levels. Plants were incubated in the dark for 72 h to remove starch from leaves and then were grown under a 12-h light period. The rates of starch degradation (A) and starch accumulation (B) were determined for four independent lines of transgenic and wild-type tobacco over 1, 2, 3 , and 6 d following the start of the experiment. The levels of starch were determined from three leaf discs taken at the beginning and end of the light period on each day. The values for total starch degraded and accumulated are means f SE from measurements made on separate days. The rates of starch degradation and accumulation in the leaves are plotted against the level of Fru-2,6-bisP in leaves at the end of the dark period. FW, Fresh weight.

lated intermediates in leaves of transgenic plants compared to wild-type plants could be to stimulate starch synthesis (see "Discussion"). To test this possibility we supplied 0.7 /-LM [U-'4ClGlc in the dark to leaf discs that were breaking down starch. The fate of ['4C]Glc metabolized by leaf discs is shown in Table 111. The extent of uptake and metabolism of [14C]Glc by leaf discs was similar in the wild type and line A(31), whereas line D(86) absorbed and metabolized significantly more ["CIGlc. The fate of the metabolized ['4C]Glc differed between the lines. The percentage of ["CIGlc metabolized to starch was proportional to the level of Fru-2,6-bisP in the leaves. In addition, there were significant increases in the percentages of the ['4C]Glc metabolized to amino acids in both transgenic lines and to CO, in line D(86). Consequently, the percentage of [14C]Glc metabolized to organic acids was significantly lower in the transgenic leaves than wild type.

To quantify the effects of changes in Fru-2,6-bisP on different parameters in leaves we have calculated the proportional changes in fluxes and metabolite levels in response to elevations in Fru-2,6-bisP levels (Table IV). These data suggest that the effects of increases in Fru-2,6-bisP on net starch degradation in leaf discs and whole leaves are similar. Furthermore, the effects of elevated Fru-2,6-bisP on net starch degradation are quantitatively similar to the converse effects of elevated Fru-2,6-bisP levels on net starch synthesis during the dark period. These data imply that the effects of Fru-2,6-bisP levels on net starch mobilization in leaves of transgenic plants (Figs. 1 and 3; Table I) may result from the indirect stimulation of starch synthesis by elevated Fru-2,6-bisP levels.

To establish whether changes in the levels of Fru-2,6-bisP were the major factor that contributed to alterations in starch metabolism in the transgenic plants (Fig. 3), the activities of FBPase, PFP, 6-PF-l-K, ADP-Glc pyrophosphorylase, and starch phosphorylase in leaves were measured (Table V). The only significant change in enzyme activity observed was an increase in the activity of FBPase in the transgenic line D(86). The activities of other enzymes fluc- tuated, but no trends were evident.

DISCUSSION

The data we have obtained from transgenic plants containing elevated Fru-2,6-bisP levels compared to wild-type plants clearly demonstrate that this metabolite can influente starch metabolism in darkened leaves. The estimated response coefficient for net starch mobilization in whole leaves with respect to changes in Fru-2,6-bisP levels was -0.57, and in isolated leaf discs it was between -0.69 and -0.89 (Table IV). These values are as great, if not greater, than the coefficients estimated for SUC synthesis with respect to changes in Fru-2,6-bisP levels in the light (-0.39 to -0.48) (Scott et al., 1995; P. Scott and N.J. Kruger, unpublished results). We stress that in the present work comparable results were obtained in whole plants and excised leaf discs, suggesting that the decrease in the rate of net starch mobilization in transgenic plants is a result of increases in the levels of Fru-2,6-bisP in the leaves and is not a result of modification of metabolism in other parts of the plant. We attribute the differential accumulation of large quantities of starch throughout the growth of the plants to a decrease in the rate of net degradation of starch at night rather than an increase in starch synthesis during the photoperiod. The latter did not occur under the growth conditions used in our experiments. Although we have previously shown that Fru-2,h-bisP levels have a major influence on photosynthetic carbon partitioning at the start of the day (Scott et ai., 1995), more recent data reveal that the contribution of Fru-2,6-bisP to the regulation of carbon partitioning de- clines throughout the photoperiod. As a consequence, we have been unable to detect any significant difference between the total amount of starch synthesized during the day by different lines.

We cannot exclude the possibility that the observed changes in starch degradation in the transgenic plants result from pleiotropic effects on the activities of enzymes




Table 1. Breakdown of ‘‘C-labeled starch in darkened leaf discs Tobacco plants grown in an 8-h light period were left in the dark for 24 h. Starch was radiolabeled

by supplying leaf discs with 14C0, for 20 min at the start of the light period. Then the leaf discs were floated on 20 mM NaCI, pH 7.4, at 20°C in the dark for 1 h. The amount of I4C-starch in the leaf discs at the start and end of the dark treatment was determined. The leve1 of Fru-2,6-bisP in the leaves 2 h into the dark period is also shown. Values shown are means t SE from three replicate measurements. Signficant differences between wild-type and transgenic lines were assessed using Student’s t test. Fischer’s values were ”P < 0.05, ‘P < 0.01, ‘P < 0.005.

Plant Line Parameter

Wild type N 3 1 ) D(86) Total 14C metabolized 1636 % 127 1663 t 157 2033 ? 128‘

Percentage I4C metabo- 43.9 t 3.0 50.6 t 1.8a 55.8 2 2.4b (Bq)

lized present in starch

during 1 h in dark

(nmol C m-’s-’)

fresh wt)

Percent 14C-starch lost 21.4 5 1.8 13.2 f 2.4b 5.5 ? 1.5‘

Rate of starch breakdown 57.7 5 4.8 41.7 2 7.5b 23.4 2 6.3‘

Fru-2,6-bisP (pmol g 35.0 t 2.7 45.8 2 4.7b 65.1 ? 11.3‘

other than those directly involved with Fru-2,6-bisP metabolism. However, we have observed consistent results in a wide range of transgenic tobacco lines. In addition, in leaves of transgenic tobacco we failed to detect any significant changes in the activities of FBPase, PFP, and 6-PF-1-K in a majority of the transgenic plants (Table V). Measure- ments of the activities of ADP-Glc pyrophosphorylase and starch phosphorylase in the transgenic lines showed that the activities of these enzymes were identical in a11 of the plant lines. Therefore, the influence of elevated Fru-2,6- bisP levels on the rate of starch mobilization in tobacco leaves is unlikely to be a direct result of altered maximum catalytic activities of these enzymes, which are thought to be important in starch metabolism (Stitt, 1990b).

Current understanding of the pathways and regulation of starch breakdown in leaves is limited. However, our measurements show that Fru-2,6-bisP levels can pro- foundly affect starch metabolism in the dark. Our data support two possible mechanisms by which changes in Fru-Z,6-bisP levels could influence the rate of starch mobilization in the dark. First, lower levels of Glc-6-P in leaves of transgenic plants may reduce the rate of Suc synthesis relative to that in wild type by decreasing the stimulation of Suc phosphate synthase (Doehlert and Huber, 1983).

Second, changes in the levels of metabolic intermediates may influence starch synthesis. There is an increase in the amount of 3PGA and total phosphorylated intermediates in leaves of transgenic plants with respect to wild type (Table 11). Since an increase in phosphorylated intermediates is likely to be accompanied by a decrease in Pi levels (Stitt, 1990a), these changes will result in an increase in the ratio of 3PGA:Pi in transgenic tobacco leaves in the dark. An increase in this ratio, if transmitted to the chloroplast, would activate ADP-Glc pyrophosphorylase and stimulate starch synthesis (Copeland and Preiss, 1981), resulting in a decrease in the net rate of starch degradation by stimulat- ing starch turnover. Support for the latter view is provided by our measurements of [I4C1G1c metabolism in tobacco leaves in the dark (Table 111).

These results demonstrate, first, that starch synthesis can occur in tobacco leaves during a period of net starch degradation and, second, that the rate of unidirectional starch synthesis is increased by elevated Fru-2,6-bisP levels. This increase in the extent of starch turnover in the transgenic plants could account for some, if not all, of the decrease in the net degradation of starch in leaves. We stress that in these experiments considerable care was taken to establish that the radioactivity was incorporated into starch. Only

Table I I . Metabolite levels in tobacco leaves 2 h into the dark period Three replicate sets of measurements of metabolites taken from separate leaves of tobacco plants

grown under an 8-h photoperiod. The results are means ? SE. Significam differences between wild-type and transgenic lines were assessed using Student’s t test. Fischer’s values were: aP < 0.01, ‘P < 0.005.

Plant Line Metabolite

Wild type , 4 3 1 ) D(86) nmol g-’ fresh wt

G I C-6-P 57.1 t 5.3 24.2 t 11 .7b 39.0 t 4.3b Fru-6-P 8.8 ? 3.5 6.5 t 3.5 7.7 ? 0.8 Triose phosphate 5.0 k 2.1 18.9 t 6.8b 22.6 ? O.gb 3PCA 146.4 2 12.8 202.7 2 25.8b 261.3 ? 16.8b Total phosphorylated intermediates 216.6 t 14.6 252.2 2 20.6a 330.6 t 11.6b



Fru-2,6-bisP lnhibits Starch Mobilization 1575

Table 111. Metabolism of [‘4C]GIc by leaf discs in the dark Tobacco plants were grown under an 8-h light period. Three replicate leaf discs were cut from

different leaves 2 h into the dark period. These were incubated at 20°C for 1 h in 0.7 /.LM [U-’4ClGlc, 20 mM NaCI, pH 7.4. After this period the extent of metabolism the Clc was determined. The values are means 5 SE of three replicate leaf discs from different leaves. Significant differences between wild-type and transgenic lines were assessed using Student’s ttest. Fischer‘s values were: aP < 0.05, bP < 0.01, ‘P < 0.005.

Parameter Wild tvDe

Plant Line

A(31) D(86)

14C taken up (Bq) 14C metabolized (Bq) Percentage 14C metabolized present in

Ethanol soluble SUC

Fru Amino acids Organic acids CO,

Ethanol insoluble Starch

283.4 ? 31.6 104.4 5 11.6

17.1 k 1.5 12.8 5 1.5 4.0 5 0.7

17.8 t- 2.4 3.8 ? 0.3

43.9 2 3.0 20.5 2 0.6

301.1 ? 33.6 96.2 t- 10.4

17.9 2 5.6 5.6 2 2.8” 9.1 t- 4.6“

11.8 k 3.4” 5.0 k 1.0

50.6 k 1.8” 27.0 2 1.2“

338.0 t- 24.9‘ 123.4 2 8.5”

20.8 k l.la 4.9 5 1.3b 9.2 k 0.7b 4.5 -C 2.7b 5.2 2 0.1”

55.8 -C 2.4b 31.2 2 0.3‘

that proportion of label released as [14C]G1~ following en- zymic digestion of the ethanol-insoluble fraction of the leaf material was considered to be starch. Labeling of starch from [14C]Glc in the dark has been observed previously in leaves of pea and Arabidopsis (Stitt and ap Rees, 1978; Trethewey and ap Rees, 1994a), suggesting that starch turnover during periods of net starch mobilization may be common. However, in previous studies the proportion of the label incorporated into starch was much lower than that which we observed in tobacco. These variations may reflect differences between plant species or between the experimental protocols used for feeding radioisotope to the tissues.

Our data suggest that starch is mobilized in darkened tobacco leaves via a pathway that is influenced by the level of C3-phosphorylated intermediates. The mechanism by which the level of 3PGA is influenced by Fru-2,6-bisP will depend on the form in which carbon from starch is ex- ported to the cytosol for Suc synthesis. Two possibilities have been suggested. First, starch could be metabolized to triose phosphates and then translocated into the cytosol and converted into Suc by the pathway used during photosynthesis (Stitt, 1990b). Alternatively, starch could be metabolized to Glc and then translocated into the cytosol, phosphorylated, and converted to Suc (Trethewey and ap Rees, 1994a). If the former pathway is the dominant route

Table IV. Fractional changes in fluxes and response coefficients of different metabolic parameters to changes in Fru-2,6-bisP levels

The values for fluxes for starch degradation (Starch,,,) were determined in intact leaves during the whole night (Fig. 5) and in isolated leaf discs during a I - h period (Table I). The fractional changes in Fru-2,6-bisP level (SX/X) and flux from starch (S//J) were calculated for both A(31) and D(86) relative to wild-type using the formula 6X/X = (XTransgenic - xwJ/ X,, and SI// = UTransgenic - /,,)//,.,, respectively, where wt indicates wild type. Response coefficients were estimated from the same data by expressing fractional changes in the flux to specific photosynthetic parameters between the wild type (WT) and the transgenic line A(31) and between wild type and D(86), as a proportion of the fractional change in Fru-2,6-bisP. A negative sign indicates an inhibition of flux through a pathway in response to an increase in the level of Fru-2,6-bisP. A similar treatment has been used to calculate the changes in fluxes for net starch synthesis (Starch,,,) with respect to changes in Fru-2,6-bisP from data presented in Table 111. -, Not determined.

Fractional Change Response Coefficient

Tissue

Leaves (Fig. 4) - - - -0.57 -

WT-A(31) +0.31 -0.28 - -0.89 - WT-D(86) +0.86 -0.59 - -0.69 -

WT-A(3 1 ) +0.31 - +0.31 - +1 .o2 WT-D(86) +0.86 - +0.52 - +0.61

Leaf discs (Tables I and II)

Leaf discs (Table 111)




Table V. Enzyme activities in leaves of transgenic tobacco containing elevated Fru-2,B-bisP levels

Tobacco plants were grown under a 12-h light period. Values are means t SE of three replicate leaf discs from different leaves harvested at the end of the dark period. Significam differences between wild-type and transgenic lines were assessed using Student's t test. The Fischer's value was: 'P < 0.05. -, Not determined.

Enzyme

FBPase PFP

ADP-Clc pyrophosphorylase

Starch phosphorylase

6-PF-1 -K

Plant Line

Wild type A(31) W44) D(86)

nmol niin- g- fresh wr 38.1 ? 8.3 50.3 t 7.3 30.6 ? 1.7 57.9 ? 8.9" 37.2 ? 3.3 35.0 t 10.0 31.1 ? 0.5 35.4 t 2.7 52.6 t 6.4 41.3 It 7.0 45.6 t 6.0 55.8 2 10.6

307.6 t 55 324.1 ? 54 - 385.9 C 37

21.1 t 4.0 17.8 t 0.6 17.2 t 3.9 32.0 t 7.5

for starch mobilization, the influence of Fru-2,6-bisP on flux of carbohydrate can be explained by a direct inhibition of the cytosolic FBPase. This will restrict the conversion of triose phosphates to hexose phosphates in the cytosol in a manner analogous to that in leaves in the light (Scott et al., 1995). An increase in cellular C3-phosphorylated intermediates would prevent export of carbon from the chloroplast, resulting in an increase in stromal 3PGA levels, which would decrease the net rate of starch degradation through a stimulation of starch synthesis (see earlier discussion).

However, there is a growing body of evidence that ar- gues against the pathway for starch mobilization being via a triose phosphate intermediate. First, in vitro measurements of the cytosolic FBPase from spinach leaves suggest that Fru-2,6-bisP is largely a competitive inhibitor with respect to the substrate Fru-1,6-bisP (Stitt, 1990a). In spinach leaves, Fru-2,6-bisP levels are high and Fru-1,6-bisP levels are low in the dark (Stitt et al., 1983), ensuring that cytosolic FBPase is largely inactive at night. Second, chloroplasts from both spinach and pea contain a Glc/maltose transporter (Schafer et al., 1977; Herold et al., 1981). This transporter may account for the export of more than one- half of the carbohydrate mobilized in isolated chloroplast in the dark (Stitt, 1990b). Third, the Arabidopsis mutant TC265, lacking the ability to transport Glc from the chloroplast in the dark, is unable to mobilize the majority of its starch reserves in the dark (Trethewey and ap Rees, 199413). Fourth, analysis of metabolism of ['4Clglycerol by the same Arabidopsis mutant suggests that in darkened leaves there is little net conversion of C3 to C6 intermediates in darkened leaves (Trethewey and ap Rees, 1994a). Finally, the view that Glc can be transported across the chloroplast envelope in tobacco is supported by our demonstration that an appreciable proportion of the ['4ClGlc supplied to leaf discs is metabolized to starch (Table 111). It is unlikely that Glc entered the chloroplast via a triose phosphate, since the chloroplastic FBPase, which would be required to convert the triose phosphate to a hexose phosphate for starch synthesis, is largely inactive in the dark (Laing et al., 1981). This evidence would argue that a substantial proportion of the products of starch breakdown in the dark is

transported by the Glc transporter rather than the phosphate translocator.

Given the appreciable evidence that Glc could be the major form in which starch is mobilized from chloroplasts in the dark, inhibition of cytosolic FBPase by Fru-2,6-bisP is unlikely to account for the effects of this signal metabolite on 3PGA levels. Therefore, we suggest that the increase in 3PGA levels in darkened leaves is a result of activation of PFP by Fru-2,6-bisP, leading to an adjustment in the relative levels of hexose and triose phosphates. Two recent studies support this view. First, reduction of PFP activity in potato tubers through antisense inhibition is accompanied by a decrease in the ratio of 3PGA:Fru-6-P (Hajirezaei et al., 1994). Second, this result is complemented by studies from our laboratory in which potato tubers containing elevated Fru-2,6-bisP levels exhibit an increase in the ratio of 3PGA: Fru-6-P (P. Scott, M. Bettey, L.N. Donath, and N.J. Kruger, unpublished results). Since there is no detectable FBPase in extracts from potato tubers (Entwistle and ap Rees, 1990), these results strongly suggest that increasing Fru-2,6-bisP levels in the tubers has stimulated PFP activity operating in the glycolytic direction and resulted in a perturbation of the 3PGA:Fru-6-P ratio. The limited data for tobacco leaves are consistent with this proposal. Not only is there an increase in the proportion of [l4C1G1c respired in transgenic compared with wild-type plants (Table 111) but the rate of respiration in tobacco leaf discs at the beginning of the dark period increases in response to increases in Fru-2,6-bisP (P. Scott and N.J. Kruger, unpublished results). In combina- tion, these data suggest that PFP operates in a net glycolytic direction in tobacco leaves in the dark and is responsive to changes in the level of Fru-2,6-bisP.

We have argued from data obtained with transgenic plants that Fru-2,6-bisP levels can influence starch mobilization in darkened leaves. To complement this study we have evidence that tobacco plants that contain reduced levels of Fru-2,6-bisP, through transformation with a mammalian Fru-2,6-P2ase (EC 3.1.3.46), show higher rates of starch degradation than wild-type plants (P. Scott and N.J. Kruger, unpublished results). These data suggest that changes, either up or down, in the level of Fru-2,6-bisP in transgenic leaves in the dark affect the net rate of starch



Fru-2.6-bisP lnhibits Starch Mobilization 1577

degradation. However, in wild-type plants the level of Fru-2,6-bisP is regulated through allosteric control of 6-PF- 2-K/Fru-2,6-P2ase by levels of 3PGA, Pi, and Fru-6-P (Stitt, 1990a). If Fru-2,6-bisP levels play any role in influencing starch mobilization in wild-type leaves, its effect will be mediated in response to changes in the relative levels of the aforementioned metabolic intermediates. The work presented in this paper demonstrates that starch turnover in darkened leaves may be modified by manipulating the level of Fru-2,6-bisP. However, the extent to which Fru-2,6- bisP influences starch metabolism in wild-type leaves re- mains unclear.

ACKNOWLEDCMENTS

We thank Prof. S.J. Pilkis and Dr. A.J. Lange (University of Minnesota, Minneapolis) for generously providing the mammalian cDNA used to produce the transgenic plants studied in this work and V. Tekin for his technical assistance.

Received February 6, 1995; accepted May 2, 1995. Copyright Clearance Center: 0032-0889/95/l08/1569/09.

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