INTRODUCTION

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco),¹ in many but not all plant species, is modulated by the transition state analogue, 2-carboxy-D-arabinitol 1-phosphate (CA1P) in response to decreased light intensity (1, 2, 3). CA1P, synthesized in chloroplasts during darkness or low irradiance, is a potent tight binding inhibitor of carbamylated Rubisco. CA1P is a branched chain, phosphorylated sugar acid (1, 4) also known as hamamelonic acid 2¹-phosphate (5), whose parent sugar is commonly known as hamamelose. At dawn, CA1P is released from the catalytic site of Rubisco by Rubisco activase (6) and rendered noninhibitory by a specific CA1P phosphatase, which converts it to 2-carboxy-D-arabinitol (CA) and inorganic phosphate (7, 8, 9). CA occurs in many plants and is not confined to the chloroplast (10). CA, administered to leaves through the petiole, is converted to CA1P in a subsequent period of darkness (11). However, since CA is present in species that make little CA1P, such as wheat (Triticum aestivum L.) (10, 12), it may serve other purposes, apart from its role as a precursor of CA1P.

The ready conversion of CA into CA1P in the leaf tissue of some plants, taken together with the identification of CA1P phosphatase, led to the conclusion that CA1P and CA participate in a metabolic substrate cycle in vivo, remote from mainstream metabolism (11). However, Andralojc et al. (13) demonstrated that up to 8% of recently assimilated carbon could be incorporated into CA1P and CA. This indicates a considerable flux of new carbon from Calvin cycle intermediates into CA and CA1P. Of the Calvin cycle intermediates, the most obvious potential precursor is fructose 1,6-bisphosphate (FBP) which undergoes intramolecular rearrangement in vivo and in vitro to form hamamelose 2¹,5-bisphosphate (HBP) (14). Consistent with this suggestion is the increased incorporation of newly assimilated radiolabel into CA1P when assimilation takes place at low irradiance (13); conditions under which FBP concentrations would be relatively high (15). In leaves of an alpine primrose (Primula clusiana Tausch), HBP is metabolized to hamamelose 2¹-phosphate, hamamelose 5-phosphate and hamamelose (14, 16). Hamamelose (2-hydroxymethyl-D-ribose) is very widely distributed among plant species (17).

In this study, radiolabeled hamamelose was prepared in order to explore its metabolism in leaves. It was specifically metabolized to CA in the light and to CA and CA1P in the dark. These results therefore provide a link, through hamamelose, between CA1P (or CA) and reports of the synthesis of HBP from FBP.

MATERIALS AND METHODS

Hamamelitannin (2 molecules of gallic acid esterified to hamamelose) (18) was isolated from the bark of witch hazel (Hamamelis virginiana) according to the method of Gilck et al. (19). The tannin was hydrolyzed using tannase (ICN Biomedicals Ltd., UK), and the hydrolysate was treated with Dowex 50-H⁺, followed by neutralization with Dowex-1 carbonate. Such treatment removed the gallic acid and further decolorized the sample. Approximately 800 mg of the liberated sugar (dissolved in 40 ml of H₂O) were resolved chromatographically using a (60 g/60 g) charcoal/Celite column (46 cm × 2.5-cm diameter), eluted with a gradient of 0-10% (v/v) ethanol in water at 0.7 ml·min¹, in 24 h, at room temperature. Approximately 300 mg of pure hamamelose were recovered from the effluent collected between 11 and 14 h. The ¹H and ¹³C NMR spectra obtained for this purified product (in D₂O) were diagnostic of hamamelose (20).

We adapted the methods of McFadden et al. (21) and Serianni et al. (22). 0.2 ml of 80 mM K¹⁴CN (58 mCi·mmol¹; Amersham, UK), 100 mM triethanolamine (TEA)-acetate, pH 7.2, was combined with an equal volume of 80 mM D-ribulose 5-phosphate,² 100 mM TEA-acetate, pH 7.5. After 60 min at 25 °C, the mixture was acidified (pH 1.5) with Dowex 50-H⁺. Glacial acetic acid was added to a concentration of 0.3 M and the mixture transferred to a glass tube with a Rotaflo stopcock. 2.9 mg of hydrogenated Pd/BaSO₄ catalyst (Sigma, UK) in H₂O were added, and the tube was flushed repeatedly with hydrogen gas, finally being left under 1 atmosphere of hydrogen for 6 h at 25 °C with constant agitation, recharging with hydrogen after the 1st h. The catalyst was sedimented, and the supernatant was adjusted to pH 4.0 with 1 M TEA. The sample was frozen in liquid nitrogen, then stored at 70 °C, until HPLC separation of the resulting diastereoisomers (see below).

French bean (Phaseolus vulgaris cv. Tendergreen) and wheat (Triticum aestivum cv. Alexandria) were grown in a glasshouse with supplementary lighting to ensure a photoperiod of 16 h, with a minimum photon flux density (PFD) of approximately 200 µmol·m²·s¹. Leaves were harvested between 14 and 21 days after sowing.

10 discs were cut from seedling leaves of P. vulgaris using a cork borer of 13-mm diameter. Stacks of 10 discs accumulated in the cork borer were transferred to the bottom of a 10-ml plastic syringe barrel. Just prior to infiltration, the plunger of the syringe was pushed into place to exert gentle pressure on the stack of discs. Syringes containing the disc samples were supported vertically in a large vacuum desiccator with their nozzles submerged in 0.5 ml of [¹⁴C]hamamelose (4 × 10⁶ dpm; 58 mCi·mmol¹) or [¹⁴C]CA (2.2 × 10⁶ dpm; 55.3 mCi·mmol¹) held in a small glass cup. (In some experiments, inhibitors were infiltrated along with hamamelose; see Table II.) A vacuum was applied until the substrate solution began to boil (2.3 kPa, 20 °C). The desiccator was held at this vacuum for 20 s, and then air was readmitted. The solution filled the space below the plunger and entered all the leaf discs, giving complete infiltration. The discs were quickly removed and spread out in plastic Petri dishes, with their upper surfaces downward, without removing adhering substrate solution (which served to attach the discs to base of the dish). To prevent desiccation, the lids of the Petri dishes were lined with a wet filter paper. The dishes were inverted under metal halide lamps, providing a PFD of 200 µmol·m²·s¹, followed by a period of darkness (as indicated) prior to freezing in liquid nitrogen, to await analysis. The samples of 10 discs weighed approximately 0.25 g, fresh weight (fw). In experiments using wheat seedlings, 1-cm leaf strips (totalling 0.25 g, fw, per sample) were treated in the same way.

Table II. Inhibition of CA and CA1P synthesis from hamamelose by DTT and sodium fluoride 0.25 g, fw, samples of leaf discs of P. vulgaris were vacuum infiltrated with [14C]hamamelose and the indicated concentrations of DTT or sodium fluoride (NaF). They were then illuminated for 2 h (PFD = 200 µmol·m2·s1), followed by 6 h of darkness. Proportion of radiolabel in CA and CA1P (the only radiolabeled products detected) was then determined by HPLC analyses of the leaf extracts. Total (100%) radioactivity recovered was 1.2-1.5 × 106 dpm g1, fw. Treatment [14C]Hamamelose [14C]CA [14C]CA1P % Control 31.2 64.3 4.5 DTT  10 mM 80.5 18.2 1.3  50 mM 98.1 1.9 0 NaF  20 mM 93.5 6.3 0.1  100 mM 98.9 1.1 0

Infiltrated, frozen leaf material was ground to a fine powder in liquid nitrogen and mixed rapidly with 1 ml of ice-cold 3.5% (v/v) trifluoroacetic acid, 0.15% (w/v) 8-hydroxyquinoline. Extract clarification, and removal of hydrophobic and cationic compounds using C₁₈ and Dowex 50-H⁺ columns, respectively, was as described previously (13). The treated extracts were evaporated to dryness in vacuo over NaOH pellets and anhydrous CaCl₂, then immediately rehydrated and stored at 25 °C, awaiting HPLC. Extracts to be analyzed for hamamelose were neutralized with Dowex 1 carbonate directly after the Dowex 50 treatment, before freezing.

Leaf material infiltrated with [¹⁴C]CA was subsequently analyzed for [¹⁴C]CA1P. In this case, CA1P was extracted as a complex with Rubisco, after which the protein was separated from low molecular weight components by gel filtration (1). Protein-bound metabolites were then released by thermal denaturation and resolved by anion exchange HPLC (13).

We used a Bio-LC chromatography system (Dionex, UK) with a pulsed amperometric detector (PAD; Dionex, UK) or a radioisotope detector (RID; Berthold LB507A fitted with solid scintillant flow cell YG 150; EG & G Berthold, UK). The absolute radioactivity (disintegrations/min) of resolved solutes was determined by subjecting the fractionated eluate to liquid scintillation counting, in Ultima Gold scintillation mixture (Canberra Packard, UK). Anion exchange HPLC utilized a CarboPac PA1 column (4 mm × 250 mm; Dionex, UK) with a flow rate of 1 ml·min¹, equilibrated, and developed as follows. (i) Isocratic elution using 0.04 M NaOH. Sample (in H₂O) was applied through a 0.05-ml injection loop, and the column was regenerated with 0.20 M NaOH (10 min), then 0.04 M NaOH (10 min) (see Fig. 1). (ii) Gradient elution, 0.05 M sodium acetate, pH 7.0 (0-5 min), 0.05-1.00 M sodium acetate, pH 7.0 (5-15 min), 1.00 M sodium acetate, pH 7.0 (15-20 min). The sample (in H₂O) was carefully neutralized with NaOH and applied through a 1.0-ml injection loop. The column was regenerated for 10 min with 0.05 M sodium acetate, pH 7.0 (see Fig. 3). (iii) Isocratic elution using 0.05 M sodium acetate, pH 6.0. Samples (in H₂O) were applied through a 1.0-ml injection loop. After sample elution, no further regeneration needed (see Fig. 4). (iv) Gradient elution with 0.15 M NaOH (0-2 min), 0.15-0.30 M NaOH (2-12 min), 0.30 M NaOH (12 min-end). Samples (in 0.01 M NaOH, pH 10) were applied through a 0.1-ml injection loop. The column was regenerated for 20 min with 0.15 M NaOH (see Fig. 5). (v) Gradient elution with 0.10-1.0 M sodium acetate, pH 7.0 (0-30 min), a sample was applied through a 1-ml injection loop. The column was regenerated for 10 min with 0.10 M sodium acetate. (These conditions were used in the separation of products during [¹⁴C]hamamelose synthesis.)

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Ion-moderated partitioning (IMP) HPLC utilized a Bio-Rad Aminex HPX-87H column, equilibrated and developed with 5 mM H₂SO₄, at a flow rate of 0.4 ml·min¹. Samples (in H₂O, unless stated otherwise) were applied through a 0.05-ml injection loop (Figs. 2 and 6).

In spite of differences in sample size and detector, HPLC elution profiles obtained using the same column chemistry were displayed with common axes, by expressing the detector signal as a percentage of the highest signal in the same chromatogram.

A known weight (between 0.90 and 1.50 g, fw) of leaf material from P. vulgaris was extracted as described above in 2 ml of 3.5% (v/v) trifluoroacetic acid, containing 10.5 nmol of radiolabeled hamamelose (2.8 mCi mmol¹). Hamamelose was purified from these extracts and the endogenous hamamelose deduced from the dilution of radiolabel, as evidenced by the altered PAD/radioactivity ratio, using a similar approach to that described for the quantitation of CA1P (12).

The peak of radioactivity with an identical retention time to CA, derived from leaf material infiltrated with radiolabeled hamamelose (Fig. 3, track B), was collected and treated with an excess of Dowex 50-H⁺. Hydrochloric acid was then added, to a final concentration of 10 mM (pH  3), and the sample was evaporated in vacuo over NaOH pellets and anhydrous CaCl₂. Just before subsequent HPLC analyses, the samples were rehydrated in water (Fig. 4) or 5 mM H₂SO₄ (Fig. 6).

The peak of radioactivity with an identical retention time to CA1P, derived from leaf material infiltrated with radiolabeled hamamelose (Fig. 3, track B), was treated with Dowex 50-H⁺, followed by evaporation in vacuo, over NaOH pellets and anhydrous CaCl₂. The sample was dissolved in 0.3 ml of 10 mM TEA. Two units of alkaline phosphatase (bovine intestine, Sigma, UK) were added, and the mixture was kept at 25 °C for 2 h, after which it was mixed with 0.02 ml of Dowex 50-H⁺. The resulting solution was analyzed by HPLC (Fig. 5).

Acid-stable neutral plus anionic metabolites, derived from hamamelose-infiltrated leaf material, were resolved by anion exchange HPLC (Fig. 3) and collected in a series of 0.5-ml fractions as they emerged from the column. Aliquots (35 µl) were incubated for 5 min in 0.5 ml of 100 mM Bicine, pH 8.2 (NaOH), 20 mM MgCl₂, 10 mM NaHCO₃, and 10 µg of Rubisco (previously carbamylated by exposure to the same concentrations of Bicine, MgCl₂, and NaHCO₃, for 40 min at 37 °C). Rubisco activity was then determined, following addition of 0.5 ml of 100 mM Bicine, pH 8.2 (NaOH), 20 mM MgCl₂, 10 mM NaH¹⁴CO₃ (0.5 mCi mmol¹), and 0.66 mM ribulose 1,5-bisphosphate. The assay was stopped with 0.1 ml of 10 M formic acid after 5 min. The samples were oven-dried, and the acid-stable ¹⁴C was determined by liquid scintillation counting.

Witch hazel bark was a generous gift from Berk (Materials for Industry) Ltd., Basingstoke, Berkshire, UK. [2¹-¹⁴C]-Radiolabeled and nonradiolabeled CA, CA1P, and 2-carboxy-D-arabinitol 1,5-bisphosphate (CABP) were synthesized as described previously (9) from ribulose 1,5-bisphosphate and either potassium [¹⁴C]cyanide (Amersham, UK) or nonradiolabeled potassium cyanide. NaH¹⁴CO₃ was obtained from Amersham, UK. Dowex resins (AG 50W-X8 and AG 1-X8) were supplied by Bio-Rad, UK. All other chemicals were of analytical grade and used without further purification.

RESULTS

Authentic hamamelose, from hamamelitannin, was resolved from common sugars by two distinct HPLC procedures. Thus, using anion exchange HPLC with 40 mM NaOH as eluent, hamamelose (retention time 16.0 min; Fig. 1, track C, peak 5) was fully resolved from galactose, glucose, fructose, and sucrose. It was also resolved from glucose, sucrose, and ribose by means of IMP HPLC, using 5 mM H₂SO₄ as eluent (retention time 15.5 min; Fig. 2, track A). These two HPLC procedures exploit different properties of hamamelose, and so their combined use provides a powerful tool in the separation of this sugar from complex mixtures (see below).

Reduction of the products of the reaction of K¹⁴CN and D-ribulose 5-phosphate (see ``Materials and Methods'') yielded two components that were resolved by anion exchange HPLC, with retention times similar to sugar monophosphates and each accounting for 20-25% of the detected radiolabel. Radiolabel was also incorporated into an unresolved mixture of the 5-phosphates of 2-carboxyarabinitol and 2-carboxyribitol (30-40% of radiolabel) and an unidentified neutral component (10-15% of label). The sugar constituent of only one of the sugar phosphate peaks had identical chromatographic properties to hamamelose, as evidenced by HPLC (Figs. 1, track B, and 2, track D) and also by TLC (not shown) and so was identified as 2-C-(hydroxymethyl)-D-ribose 5-phosphate (hamamelose 5-phosphate). The sugar liberated by phosphatase treatment of this compound was therefore [1-¹⁴C]hamamelose (using nomenclature adopted by Beck et al. (5)) and was used in the following experiments.

Leaf discs were vacuum-infiltrated with solutions of the [¹⁴C]hamamelose, illuminated (PFD 200 µmol·m²·s¹) for 2 h, then placed in total darkness for 6 h. Analysis of the acid extracts by anion-exchange HPLC at neutral pH revealed three radiolabeled peaks (Fig. 3, track B). The first radiolabeled component to emerge from the column was neutral, having the same retention time as hamamelose (approximately 2.8 min; peak 1). This was followed by a weakly anionic component (peak 2), then a strongly anionic component (peak 3), and these had retention times identical to CA (6.2 min) and CA1P (15.7 min), respectively. Each of these peaks was subjected to further analysis to confirm their identities.

Material from the neutral peak (Fig. 3, track B) was shown exclusively to be hamamelose, by means of IMP (Fig. 2, track C). This demonstrates that the hamamelose administered had not been metabolized to any other neutral component in the course of the experiment.

When the component with the same retention time as CA (Fig. 3, track B, peak 2) was dehydrated in acid and then rehydrated immediately before anion exchange HPLC (at pH 6.0) it had a shorter retention time, consistent with a neutral compound. This is illustrated in Fig. 4, which shows the elution profiles of [¹⁴C]CA standards before (track A) and after (track B) such treatment, as well as of the treated product of [¹⁴C]hamamelose metabolism (track C). This behavior is consistent with the formation of a lactone (which is uncharged) and would be expected if the compound were CA.

Anion exchange HPLC at pH > 12 (i.e. at or above the pK_a of sugar hydroxl groups) (23), using sodium hydroxide as eluent, gives baseline resolution of CA from other weakly anionic components (Fig. 5, track A), including gluconic acid (GA), which can also form a lactone. Under these conditions, the weakly anionic compound derived from [¹⁴C]hamamelose (Fig. 5, track C) runs as a single peak with the same retention time as the [¹⁴C]CA standard (Fig. 5, track B).

Finally, both authentic CA and the weakly anionic product of hamamelose metabolism were dehydrated/rehydrated in dilute mineral acid (to ensure complete lactonization) prior to IMP. Again, the compound in question yielded a single peak (Fig. 6, track C) with the same elution profile as the CA (lactone) control (Fig. 6, track B). Furthermore, 96% of the applied radiolabel was recovered in each of the peaks (of tracks B and C). We concluded that this compound was exclusively CA.

The component with the same retention time as CA1P (Fig. 3, track B, peak 3) was collected as it emerged from the column, in a series of fractions, collected at 0.5-min intervals. Analysis of these fractions showed that radioactivity and ability to inhibit Rubisco coincided in three separate experiments (Fig. 7, A, B, and C), suggesting that radiolabeled CA1P had been formed. The amounts of CA1P present (60-80 nmol·g¹, fw) were estimated by reference to a standard CA1P-inhibition curve, constructed contemporaneously, and are similar to published values (12, 13).

Phosphatase treatment of this compound, followed by anion exchange HPLC, yielded a single peak of radioactivity (Fig. 5, track E) with a retention time identical to that of an authentic sample of CA1P treated in the same way (track D), and of authentic CA (track B). Additionally, 97% of the radiolabel associated with the strongly anionic component was accounted for beneath the peak of track E (Fig. 5), following phosphatase treatment. This confirms the identity of the strongly anionic peak as exclusively CA1P.

The conversion of infiltrated hamamelose exclusively into CA and CA1P by leaves of P. vulgaris strongly suggests that hamamelose is a precursor of these compounds in vivo, for which it must be present in the leaves of P. vulgaris. A combination of anion-exchange and IMP HPLC of the acid-stable, neutral compounds extracted from leaf material demonstrated the presence of hamamelose and allowed an estimate of its amount. An acid extract of leaves was treated with Dowex 50-H⁺ and then neutralized using excess Dowex 1 carbonate, prior to HPLC. The supernatant was first analyzed by anion exchange HPLC at high pH, yielding several components with short retention times, followed by three large peaks corresponding to glucose, fructose and sucrose. Finally, a single, small peak emerged with a retention time identical to that of hamamelose (Fig. 1, track A). This peak was collected and treated immediately with excess Dowex 50-H⁺ to remove Na⁺ ions and acidify. This was further resolved by IMP HPLC, revealing 2 major peaks (Fig. 2, track B). The first to emerge had a retention time similar to sucrose (track A), while the second, larger, peak had a retention time identical to that of hamamelose and was symmetrical, indicating base line resolution from other components. We concluded that this (latter) peak was hamamelose.

A radioisotope dilution assay was conducted to measure the hamamelose content of leaves. This involved addition of ¹⁴C-labeled hamamelose of known specific radioactivity to the leaf material at the (initial) acid extraction, followed by isolation of the hamamelose in the extract by the two sequential HPLC fractionations. The specific radioactivity of the purified hamamelose was measured and used to determine the endogenous hamamelose. Table I shows that the hamamelose content of P. vulgaris was between 35 and 72 nmol·g¹, fw. Consistent with its proposed role as a precursor of CA1P (which accumulates in the dark) the hamamelose content of dark adapted leaves was 50% lower than that of light adapted leaves (treatment 3). Transfer from normal to low irradiance (treatment 2) did not significantly alter the hamamelose content (Table I).

Table I. Hamamelose content of P. vulgaris leaves kept in normal light, low light, or darkness Quantitation by means of radioisotope dilution assay. Values shown are averages ± standard deviation, of three separate determinations. Pretreatment Hamamelose content nmol·g1fw 1. 2 h:310 µmol·m2·s1 68.2  ± 8.2 2. As (1), then 0.5 h:60 µmol·m2·s1 71.2  ± 5.1 3. As (2), then 4 h in dark 35.4  ± 2.3

We have shown that hamamelose is specifically converted into both CA and CA1P in leaf discs of P. vulgaris. Similarly, [¹⁴C]CA administered to leaf discs by vacuum infiltration is also converted into [¹⁴C]CA1P, in a subsequent period of darkness (Fig. 8). Since no other radiolabeled compounds were detected following infiltration of [¹⁴C]CA (not shown), we can conclude that CA is converted directly into CA1P in infiltrated leaf discs.

One approach to determining whether the conversion of hamamelose into CA and CA1P is sequential, and to establish a likely order of synthesis, is to find out if either CA or CA1P can be derived from hamamelose without the accompanying synthesis of either CA1P or CA, respectively. Moore and Seemann (10) reported that wheat contains substantial levels of CA, but has very little CA1P (12). We infiltrated wheat leaves with [¹⁴C]hamamelose and found that it was converted exclusively into CA (Fig. 3, track C and inset). The identity of [¹⁴C]CA, formed from hamamelose in wheat, was established as described above. This shows that hamamelose can be converted directly into CA in the absence of CA1P synthesis. In addition, DTT and fluoride have been shown to inhibit the conversion of CA into CA1P (11). We assessed the effect of these reagents on the conversion of hamamelose into CA and CA1P in leaf discs from P. vulgaris (Table II). Both DTT and fluoride reduce the conversion of hamamelose into CA and CA1P at both concentrations. However, at the higher concentrations of each inhibitor (50 mM DTT and 100 mM NaF), no radiolabel could be detected in CA1P, although significant amounts (1.5-3.0 × 10⁴ dpm g¹, fw) still appeared in CA. In other words, in P. vulgaris as in wheat, conversion of hamamelose into CA does not require the synthesis of CA1P.

Leaf discs were infiltrated with [¹⁴C]hamamelose, then incubated at room temperature in the light (PFD 200 µmol· m²·s¹) for up to 4 h, after which they were frozen in liquid nitrogen immediately and subsequently analyzed for radiolabeled products. Radiolabeled CA was apparent in illuminated leaf discs after only 30 min and was produced faster as time increased, but no [¹⁴C]CA1P was detected (Fig. 9, open symbols).

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After 2 h of illumination, a proportion of the leaf discs were transferred to complete darkness for the indicated times, after which the amounts of ¹⁴C-labeled products were determined (Fig. 9, solid symbols). Synthesis of [¹⁴C]CA continued at the rate which had been established in the preceding period of light, for at least 2 h, after which the rate declined. A small but significant amount of [¹⁴C]CA1P was present after the first hour of darkness (Fig. 9). Although the appearance of [¹⁴C]CA1P was considerably slower than the initial rate of [¹⁴C]CA synthesis in the dark, it was sustained for at least 6 h. As in preceding experiments, ¹⁴C was detected exclusively in CA or CA and CA1P, depending on the conditions.

DISCUSSION

Hamamelose was not metabolized to any other neutral component, following leaf infiltration (Fig. 2, track C). Therefore, the radiolabeled CA and CA1P must have been derived directly from hamamelose, rather than (indirectly) from some other neutral hamamelose derivative. The present work shows that CA and CA1P are the only significantly radiolabeled compounds to be derived from [¹⁴C] hamamelose. Thus, hamamelose is a specific precursor of CA and CA1P, and in P. vulgaris is almost exclusively metabolized into these two compounds.

A small amount of radiolabel occasionally became incorporated into a component with a retention time consistent with a bisphosphate (Fig. 3, track B). However, the presence of ¹⁴C in this component was not always apparent, and the incorporation of radiolabel into CA or CA1P from hamamelose did not depend on its appearance.

Since the only compounds to consistently become radiolabeled, following the administration of hamamelose, were CA and CA1P (Fig. 3 track B), either CA and CA1P are both derived directly from hamamelose or only one is derived directly from hamamelose, the other being derived from the product of the initial conversion.

Hamamelose is converted exclusively into CA in the leaves of T. aestivum (Fig. 3, track C). The same applies using P. vulgaris in the presence of high concentrations of DTT or sodium fluoride (Table II). It therefore appears that hamamelose is converted directly into CA. The exclusive conversion of hamamelose into CA in the light (Fig. 9) supports this notion, but is not sufficient proof for a direct conversion of hamamelose into CA, since any CA1P made as an intermediate could be hydrolyzed to CA by the light-activated CA1P phosphatase (7, 9) and so escape detection. CA is converted exclusively into CA1P in darkened leaf discs, following vacuum infiltration of [¹⁴C]CA (Fig. 8). This is in agreement with observations of Moore and Seemann (11), who investigated the metabolism of CA, following its uptake through the petiole. Since the interconversions, hamamelose  CA, and CA  CA1P have been demonstrated, but no conversion of hamamelose into CA1P has been observed in which CA was not also formed, the reaction sequence, hamamelose  CA  CA1P is likely. Absolute confirmation must await purification of the associated enzymes.

The occurrence of hamamelose in P. vulgaris would be expected, in the light of a survey by Sellmair et al. (17), which concluded that hamamelose was distributed widely, if not universally, throughout the plant kingdom. This survey depended on detecting radiolabeled hamamelose in plants following photosynthesis in ¹⁴CO₂.

We describe a method using HPLC for detecting and measuring hamamelose in leaf tissue directly. The smaller hamamelose content of darkened leaves (Table I) is consistent with its role in the (dark-dependent) synthesis of CA1P. Since hamamelose conversion into CA can take place at similar rates in the light and dark (Fig. 9), the lower hamamelose content of dark-adapted leaves (Table I) probably reflects a fall in the rate at which hamamelose is synthesized, rather than an accelerated conversion into CA/CA1P. Since hamamelose is converted almost exclusively into CA and CA1P (Fig. 3, track B), the difference between the hamamelose content of light and dark adapted leaves (of 36 nmol·g¹, fw) represents a minimum estimate for the amount of carbon converted into CA/CA1P at night. Our previous observation, that carbon assimilated at low irradiance is preferentially incorporated into CA1P (13), is not paralleled by an increase in the total pool size of hamamelose in leaves transferred from normal to low irradiance (Table I). However, it is possible that precursors of hamamelose accumulate, following such a transition (see below).

[¹⁴C]Hamamelose is converted into [¹⁴C]CA in the light and in the dark at rates much greater than that of its dark-dependent conversion into [¹⁴C]CA1P (Fig. 9). This explains why the absolute amount of [¹⁴C]CA formed was greater than that of [¹⁴C]CA1P (Fig. 3, inset). Only after a prolonged period in the dark does the rate of [¹⁴C]CA synthesis fall to that of [¹⁴C]CA1P (Fig. 9). This is presumably due to substrate ([¹⁴C]hamamelose) depletion having a greater effect on [¹⁴C]CA synthesis than on [¹⁴C]CA1P synthesis (consistent with the notion that CA is derived from hamamelose, but that CA1P is derived from CA).

The conversion of hamamelose into CA in wheat (Fig. 3, track C) is in agreement with the precursor/product relationship proposed for hamamelose and CA, and would be expected, judging by the considerable amounts of CA in wheat (10), the widespread distribution of hamamelose (17), and the structural similarity of the two molecules. The fact that no ¹⁴C was detected in CA1P in wheat is possibly due to the relatively low levels of CA1P present in wheat (10-fold lower than in P. vulgaris (12)) and/or a larger contribution of preexisting CA to its synthesis.

We propose the following sequence of reactions for the biosynthesis of CA and CA1P:

(Eq. 1)

This proposal is based on (a) the conversion of FBP into HBP by illuminated chloroplast material (Gilck and Beck (14)), (b) the present demonstration that hamamelose (H) can be metabolized to CA and CA1P, and (c) the phosphorylation of CA to CA1P in isolated leaves (More and Seemann (11)).

This reaction sequence is also supported by our previous observation of a lag in the appearance of radiolabeled CA1P (compared to total CA1P), following brief exposure to ¹⁴CO₂, in a pulse-chase experiment (13). Such a delay would correspond to the time taken for recently assimilated carbon to pass through the sequence of intermediates, following its initial incorporation into intermediates of the Calvin cycle. The flux of carbon from CO₂ through this pathway can be considerable: more than 8% of carbon assimilated at low irradiance became incorporated into CA + CA1P, in a subsequent period of darkness (13).

FBP is converted into HBP by chloroplast components (14), and CA1P has been shown to occur exclusively within the chloroplast (24). Therefore, the intracellular site of the FBP  HBP conversion and of the phosphorylation of CA, is thought to be the chloroplast. Neither hamamelose nor any hamamelose monophosphate have been detected in chloroplasts (16), and so it has been suggested that the site(s) of HBP dephosphorylation is/are outside the chloroplast (16). The site of the conversion, hamamelose  CA, remains to be established, although the proposed occurrence of hamamelose outside the chloroplast and the detection of CA in cytosol and vacuole (10) suggest that this reaction is either cytosolic or vacuolar. None of the enzymes of CA1P biosynthesis have been purified or characterized, except perhaps the CA1P phosphatase, which may be found to have an additional role in the dephosphorylation of HBP. However, this enzyme is located exclusively in the chloroplast (24).

The reaction sequence shown implies that factors influencing the amounts of FBP in the chloroplast may affect the flux of photosynthate into CA and/or CA1P. Sassenrath-Cole and Pearcy (15) have shown that FBPase responds rapidly to changes in light intensity, and that this is reflected in the amounts of fructose and sedoheptulose bisphosphates. In particular, increased amounts of these two metabolites were shown to accompany a fall in light intensity (15). These observations are consistent with the greater incorporation of recently assimilated carbon into CA and CA1P at low PFD than at high PFD, reported previously (13). Hence, changes in the ambient concentration of FBP may well affect the amount of recently assimilated carbon incorporated into CA and CA1P.

If HBP is synthesized in the chloroplast, then its structural similarity to the transition state intermediate of the carboxylation reaction of Rubisco would suggest that it, too, would inhibit Rubisco. Indeed, it may prove to be the daytime inhibitor reported previously (25). This may be another reason why Rubisco activase is necessary, ensuring that Rubisco does not become irreversibly inhibited during the day.

It is puzzling that, in the light, there is a net flow of carbon away from CA1P (dephosphorylation to CA) while de novo precursors of CA1P are simultaneously being synthesized. CA1P, dephosphorylated to CA in the light, should provide sufficient carbon skeletons (along with the relatively large pool of vacuolar CA) (10) to meet the demand for rephosphorylation to CA1P, in an ensuing period of darkness. Perhaps these branch chain sugars serve some other purpose as well, for which a regular supply from recently assimilated carbon is needed.