Reduced Cytosolic Fructose Bisphosphatase - Plant Physiology

Received for publication February 20, 1992 Accepted April 9, 1992

Plant Physiol. (1992) 100, 210-215 0032-0889/92/100/0210/06/$01 .00/0

Carbon Partitioning in a Flaveria linearis Mutant with Reduced Cytosolic Fructose Bisphosphatase' Thomas D. Sharkey*, Leonid V. Savitch, Peter 1. Vanderveer, and Barry J. Micallef Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT Oxygen sensitivity and partitioning of carbon was measured in a mutant line of Flaveria linearis that lacks most of the cytosolic fructose-1,6-bisphosphatase found in wild-type lines. Photosynthesis of leaves of the mutant line was nearly insensitive to 02, as found before. The mutant plants partitioned 2.5 times less carbon into sucrose than the wild type in a pulse chase experiment, with the extra carbon going mainly to starch but also to amino acids. From 10 to 50 min postlabeling, radioactivity chased out of the amino acid fraction to starch in both lines. In the middle of the light period, starch grains were larger in the mutant than in the wild type and covered 30% of the chloroplast area as seen with an electron microscope. Starch grains were found in both mesophyll and bundle sheath chloroplasts in both lines in these C3-C4 intermediate plants. At the end of the dark period, the starch levels were considerably reduced from what they were in the middle of the light period in both lines. The concentration of sucrose was higher in the mutant line despite the lack of cytosolic fructose-1,6bisphosphatase. The amino acid fraction accounted for about 30% of all label following a 10-min chase period. In the mutant line, most of the label was in the glycine + serine fraction, with 10% in the alanine fraction. In wild-type leaves, 35% of the label in amino acids was in alanine. These results indicate that this mutant survives the reduced cytosolic fructose-1,6-bisphosphatase activity by partitioning more carbon to starch and less to sucrose during the day and remobilizing the excess starch at night. However, these results raise two other questions about this mutant. First, why is the sucrose concentration high in a plant that partitions less carbon to sucrose, and second, why is alanine heavily labeled in the wild-type plants but not in the mutant plants?

All of the common transported sugars made during photosynthesis require FBPase for synthesis. Sucrose is the primary sugar transported from most leaves, and the raffinose sugars are made from sucrose. Sucrose synthesis ordinarily depends upon FBPase activity in the cytosol (29). Some plants make and transport sugar alcohols and these too depend upon cytosolic FBPase for synthesis (8). F. linearis partitions a large fraction of its photosynthate to sucrose relative to other Flaveria species (18). It is unknown how the mutant line of Flaveria with reduced FBPase activity makes sugars for transport out of the leaf. F. linearis has photosynthetic characteristics intermediate between C3 and C4 (11). Such C3-C4 intermediates in the genera Alternanthera, Moricandia, Mollugo, Neurachne, and Partheneum generally have Kranz-like leaf anatomy (6, 11, 13, 15, 32). It is believed that these plants shuttle photorespiratory glycine from the mesophyll cells to the bundle sheath-type mitochondria, where glycine decarboxylase releases CO2 (9). Hylton et al. (14) have shown that glycine decarboxylase is found in bundle sheath mitochondria but not in mesophyll cell mitochondria. Moore et al. (19) have shown that CO2 builds up inside the leaves of C3-C4 intermediates, and Bauwe et al. (3) have shown that these plants reassimilate CO2 better than C3 plants, although not as well as C4 plants. In addition to the glycine shuttle, many of the Flaveria intermediate species appear to operate a C4 cycle to a greater or lesser degree (15, 18). F. linearis exhibits labeling of C4 acids when fed radioactive CO2 (18), especially when the CO2 concentration is near the compensation point (7) but the label chases out very slowly (7, 18), indicating little or no true C4 function in this species. Nevertheless, there is a division of labor between mitochondria in the bundle sheath cells and mesophyll cells (17). This raises the possibility of compartmentalization of carbohydrate metabolism, just as sucrose is made primarily in the mesophyll cells in maize (29). We have investigated the partitioning of photosynthates after a 5-min pulse of 14CO2 or 13CO2 followed by a 10-min (or longer) chase with 12CO2 in this F. linearis mutant and compared the result with a wild-type line. We used transmission EM to determine where within the leaf starch is made in the mutant and wild-type lines. We measured total starch and sucrose levels in the middle of the light period and near the end of the dark period. Because we found the amino acid fraction to be heavily labeled, we investigated which amino acids were labeled.

A mutant or variant line of Flaveria linearis with reverse oxygen sensitivity was discovered by Brown et al. (5). Sharkey et al. (26) showed that the activity of cytosolic FBPase2 was substantially reduced in this line of Flaveria and could not find any compensatory enzymic activity in pyrophosphatedependent phosphofructokinase. Subsequently, Bouton et al. (4) have demonstrated that there may be two genes involved in the reverse oxygen sensitivity of this line of Flaveria. The loss or reversal of oxygen sensitivity is believed to be caused by sucrose or starch synthesis limiting photosynthesis (24), and the finding of reduced cytosolic FBPase in this line of Flaveria is consistent with this belief. 1 Research supported by Department of Energy grant FG0287ER13785. 'Abbreviations: FBPase, fructose-1,6-bisphosphatase; SPS, sucrose phosphate synthase.

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PARTITIONING IN A FRUCTOSE-1 ,6-BISPHOSPHATASE MUTANT

MATERIALS AND METHODS

Plant Material and Experimental Conditions Flaveria linearis lines were obtained from R.H. Brown, University of Georgia, Athens. The plant identified as 84-9, the mutant that lacks most of its cytosolic FBPase activity, was grown alongside plants of line 85-1, a control line. During this study, we measured cytosolic FBPase levels as described earlier (4) and found them to be normal in our wild-type line (146 ± 35 nmol g-' fresh weight min-f) and below our limits of detection (6.5 nmol g-' fresh weight min-') in the mutant line. Plants were propagated by cuttings during the course of the experiment. Plants were grown in a Conviron E-15 growth chamber in 1-L pots containing a 3:3:3:2 (v/v/v/v) mixture of soil:perlite:peat:rice hulls. The photoperiod was 16 h with lights coming on over 2 h to a peak of 440 smol photons m-2 s-1. Temperature was 26/ 180C day/night, and the RH was 60%. All experiments were begun between 10 AM and 12 noon unless stated otherwise and were carried out between January and September 1991.

Gas-Exchange Measurements and Labeling Procedure Four to six leaves (5 mm wide) were laid across a circular chamber made of aluminum with Saran wrap windows to admit light. Air was mixed from N2, 02, and 5% CO2 in air. After humidification, the air was dried to 100C dew point and then passed over the leaf. The temperature of the cuvette was controlled by water circulating within the aluminum and the leaf temperature was monitored with a copper-constantan thermocouple appressed to the abaxial side of the leaf. Leaf temperature was kept at 260C. Photosynthesis was measured as depletion of CO2 as the air stream passed over the leaves. A Li-Cor 6251 IR gas analyzer was used to measure the partial pressure of CO2 before and after the cuvette and humidity of air was measured with a General Eastern Dew10 hygrometer. Further details of this gas-exchange system are reported in Loreto and Sharkey (16). Calculations of photosynthetic parameters were done using the equations of von Caemmerer and Farquhar (31). Leaves were left to equilibrate in 1000 ,umol photons m2 s-i and O2 partial pressure of 200 mbar for at least 30 min. In some experiments, a low 02 partial pressure of 20 mbar was used. Labeling was carried out by switching the CO2 supply from a tank of 5% '2CO2 in air to a tank of 5% "3CO2 in N2 (99% '3CO2) or to a tank of 5% '4CO2 in air (specific activity 0.024 Ci/mol). The "3CO2 was purchased from Sigma and distilled through a glass vacuum line into a lecture bottle, which was subsequently filled with N2 to dilute the CO2 to 5% to match the CO2 tank used in the gas-exchange system. Because 13CO2 has a different absorption spectrum in the IR, we could not use the IRGA when "3CO2 was in the system. The concentration of '3CO2 was measured by trapping all of the CO2 in 5 mL of the mixture and then measuring the pressure in a closed gas-trapping train upon warming the C02. The "'CO2 was generated from NaH"'CO3 and pulled by vacuum into a 2-L metal cylinder. Then the 5% '2CO2 tank was used to pressurize the 2-L cylinder until the desired specific activity was obtained. Specific activity was deter-

211

mined by measuring the CO2 in a calibrated vacuum line and counting the radioactivity contained in an equivalent volume. All feedings were done under steady-state conditions and the CO2 partial pressure was maintained at 350 ,ubar before, during, and after the labeling. The rate of photosynthesis was measured just before starting the feeding and again after the feeding during the chase period. If the rate before and after differed by more than 10%, the leaves were discarded as not being at steady state. In all experiments, leaves were fed for 5 min and then the CO2 supply was switched back to '2CO2 for 10, 30, or 50 min. At the end of the chase, leaves were frozen by clamping with liquid N2-cooled copper heads. Leaves were stored at -800C until analyzed. Extraction and Amino Acid Analysis Leaves were extracted by plunging them into boiling ethanol:water:formic acid (33:7:2) (25). After extraction, the leaves were ground and the extract was filtered. The starch in the filter cake was digested as described by Rufty and Huber (22). The filtered ethanol solution containing plant extract was dried at 550C under an air stream. The soluble fraction was obtained by adding water to the residue. After filtration, a faintly yellow solution remained. The solution was passed through a Dowex 50 (H+ form) and the eluent from this column was put through Dowex 1-Cl. The soluble, neutral fraction was eluted by washing both columns with water, anions (mostly phosphorylated compounds) were eluted with 2 N hydrochloric acid from Dowex 1-Cl, and cations (amino acids) were eluted with 6 N NH40H from Dowex 50. All fractions were concentrated to dryness under a stream of dry air and resuspended in 1 mL of H20. Then 200 gL of the sample was put in 5-mL Bio-Safe 2 (Research Products International Corp.) scintillation cocktail and counted with a Beckman LS 7800. Amino acids were separated by HPLC for determination of labeling. We used a Beckman Ultrasphere column ODS dp 5 gm, 4.6 mm diameter by 25 cm long. Radioactivity was detected with a Flo-one/Beta (Radiomatic Instruments and Chemical Co., Inc.). Amino acids were derivatized with phenylisothiocyanate. Derivatives were prepared by mixing 100 gL of sample with 500 ,L of methanol and 100 MAL of derivatization cocktail. The derivatization cocktail contained 700 uL of methanol, 100 ,uL of triethylamine, and 100 gL of phenylisothiocyanate. The mixture was kept at room temperature for 30 min, then 200 gL of heptane was added. The solution was vortexed a few times and the heptane was removed after 5 min. The solution was dried on a vacuum centrifuge and redissolved in 200 ML of 50% methanol. Mobile phases were: A, 0.05 M sodium acetate (pH 6.8), B, 100% acetonitrile. The flow rate was 1 mL/min. Amino acids were eluted with a 30-min gradient from 5 to 25% B, and the column was cleaned between runs with a 10-min gradient to 40% B, followed by a 10-min equilibration at 5% B. Amino acids were detected by UV absorption at 254 nm. Flo-Scint 5 (Radiomatic) was used for counting. All radioactivity was found in two peaks, but a lack of resolution of the radioactivity counter prevented us from determining which of several possible amino acids were radioactive.

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To determine which amino acids were labeled, we fed '3CO2 and used GC/MS (GC Carlo Erba-Kratos MS 25) because we found greater resolution with this system. During feeding, the CO2 partial pressure was always 350 ,bar, regardless of which isotope was used. After feeding "3CO2 for 5 min, we switched back to 12CO2 for a 10-min chase, then extracted the amino acids and derivatized for GC/MS. We compared the mass spectra of each amino acid with the spectrum of a standard to find which amino acids were labeled with 13C as judged by an increase in mass of some of the mass fragments. Derivatization and GC analyses of Nheptafluorobutyryl isobutyl derivatives were performed as described by Rhodes et al. (21). We used a 30-m fused silica capillary column, SPB-5 (0.32 mm i.d.). Column temperature was initially 1000C for 4 min, rising to 2200C at 6°C/min. The injector temperature was 2350C. Amino acid concentrations were determined by injecting heptafluorobutyl isobutyl derivatives on a Shimadzu gas chromatograph and detecting the amino acids with a photoionization detector. The detector temperature was 2450C. Peak areas were determined by a Spectra Physics SP 4400 peak integrator and peak areas were compared with standards. The reported data for amino acid analysis is the mean ± SE of five to six samples.

Biochemical Determinations Metabolite pool sizes and SPS and FBPase activity was determined using leaves harvested at 3:00 PM and at the end of night. Metabolite pool sizes were determined enzymically as described previously (22, 25). For SPS assay, leaf extracts were prepared as described by Huber et al. (12) with minor modifications. Microscopy Leaf tissue for microscopy was taken from the middle of the lamina at approximately the same location used for biochemical and gas-exchange analysis. Samples were taken at 3 PM to see where starch was located and at 6 AM (the lights in the growth chamber come on at 6:30 AM) to observe leaf sections at the end of the night. Leaf sections were fixed and embedded as described by Sharkey et al. (27). The reported data are the means ± SE of 45 to 60 chloroplasts.

Table I. Photosynthetic Rate in Normal and Low 02 in Two Lines of F. linearis in Normal and Low 02 Partial Pressure Values are mean + SE, n = 5. 02= 20 mbar 02 = 200 mbar Mutant Wild type UmoI m-2 s-1 Photosynthesis 15.8 ± 0.8 18.3 ± 1.1 23.8 ± 1.0 19.8 ± 1.2

Wild type

Mutant

exported in 10 min but most of the label is out of the photosynthetic carbon reduction cycle (25). After 50 min of chase, almost half of the radioactivity left the wild-type leaves, whereas essentially none of the radioactivity left the mutant leaves. In wild-type leaves, most of the label was in water-soluble carbohydrates with a substantial amount also found in the cationic fraction throughout the chase. The decline in total label between 10 and 50 min was accounted for by declines in these two fractions. In the mutant, most label was found in the cationic fraction following a 10-min chase, with substantial labeling in the starch fraction. Over 50 min, label washed out of the cationic fraction and into the starch fraction, so that following 50 min of chase, about half of all label was in starch. A similar shift of label from the cationic fraction to the starch fraction was also seen in the wild type, although this was not as dramatic as in the mutant

(Fig. 1).

The ratio of label in starch relative to that in the watersoluble fraction was 1.29 ± 0.07 (average ± SE) for the mutant but just 0.245 ± 0.002 in the wild type after 10 min; the amount of label in sucrose was 2.5 times greater in the wild type. After 50 min, the ratio was 1.93 ± 0.01 in the mutant and 0.66 ± 0.01 in the wild type. The increase in the ratio was caused by the shift in the label from the cationic fraction to starch in both lines, and in wild type the additional effect Mutant

Wild type

225 200 1 75 S

RESULTS Partitioning of Carbon Following a 5-min Pulse of 14C02 In normal 02, the mutant plant had, if anything, a higher rate of photosynthetic carbon assimilation than did the wildtype plant on a leaf area basis (Table I). However, in low 02 photosynthesis was stimulated in the wild type but not in the mutant. This confirms that these plants were behaving as described by Brown et al. (5). It has already been shown that the reduced, extractable FBPase activity is correlated with a large pool of FBP (26). Following 5 min of labeling and 10 min of chase, the total amount of radioactivity was the same in the mutant and wild-type leaves (Fig. 1). We chose 10 min for the first time point because earlier work showed that little radioactivity is

cr

1 50

U,

1 25

0a ._

0

1 00

75 0

50

25

4

0 20

40

:;T 0

A 20

40

60

Time of chase, min

Figure 1. Total radioactivity and radioactivity in four fractions of F. linearis leaves from wild-type or mutant plants following a 10-, 30-, or 50-min chase. Error bars are ± 1 SE of the mean, n = 5.

PARTITIONING IN A FRUCTOSE-1,6-BISPHOSPHATASE MUTANT

of the drop in water-soluble carbohydrates, presumably reflecting transport out of the leaf. Sugars and Starch at the Middle of the Light Period and the End of the Dark Period We next examined the amount of soluble sugars and starch in the middle of the light period (3:00 PM) and at the end of the dark period. The amount of glucose and fructose was lower in the wild type than in the mutant, and for both lines glucose and fructose were lower in the dark than in the light (Table II). There was much more sucrose and starch in the mutant than in the wild-type leaves (Table II). We determined the location of the starch within the leaf by EM. We found large starch grains in mesophyll cell chloroplasts of the mutant and smaller but still prominent starch grains in chloroplasts from bundle sheath areas (data not shown). At the end of the dark period, there were very few starch grains. The distribution of starch was similar in leaves of a wild-type plant, although the starch grains were usually smaller (data not shown). This distribution was quantified by measuring the area of chloroplasts taken up by starch. Nearly one-third of the chloroplast volume was starch in mutant mesophyll cells, but only 18% was starch in the wild type (Table III). Less but still substantial amounts of starch were found in the bundle sheath chloroplasts. The proportion of starch in the mesophyll chloroplasts relative to that in the bundle sheath chloroplasts was similar in the two lines (Table III). We measured the activity of SPS in leaves during the middle of the light period and at the end of the dark period. When measured in the presence of saturating levels of substrates and without Pi present, the activity of SPS was between 21 and 24 Mmol sucrose g-1 fresh weight h-1 and did not vary between the two lines or between day and night. When measured in the presence of limiting amounts of substrate and in the presence of Pi, the activity was marginally higher in the mutant plants. The ratio between these two assays during the day was 78% for mutant and 63% for the wild type. Similar numbers were found when measurements were made at the end of the night period (data not shown).

Labeling of Amino Acids We investigated the amino acid (cationic) fraction because it accounted for such a large proportion of the label. The

213

Table l1l. Starch as a Proportion of Chloroplast Area Data from 45 to 60 chloroplasts per condition, SE of all of the light data were less than 5%. Dark

Light

Mesophyll Bundle sheath Mesophyll Bundle sheath

Wildtype Mutant

18 30

11 18

2 0

2 0.3

most abundant amino acids were glutamate, serine, and aspartate + asparagine (Table IV). These three amino acids accounted for 80 to 85% of the total. When leaves were harvested while in low 02, the level of alanine more than doubled relative to the amounts found in normal 02 partial pressure, and there was also a substantial increase in the amount of glutamine and y-amino butyric acid in both lines. The amount of glycine in leaves harvested in low 02 was less than in leaves harvested in normal air. The other amino acids showed much less or no difference between normal and low-

02 treatments. We found three amino acids with noticeable labeling: alanine, glycine, and serine (Fig. 2). Alanine was labeled as evidenced by the increased strength of the 241 and 242 ions. The labeling of alanine occurred whether the "CO2 was fed while the leaf was in normal or low oxygen. Glycine was labeled in normal oxygen but not in low oxygen, and serine showed some label in low oxygen but more in normal oxygen

(Fig. 2).

We used HPLC and 14CO2 to determine the amount of labeling in alanine, serine, and glycine because it was not possible to measure the amount of labeling with the mass spectrometer. Elution of the amino acids gave two peaks of radioactivity, one peak came around the time of elution of alanine, histidine, and threonine, and a second peak came at the time of elution of glycine, serine, glutamine, and asparagine. From the work with the GC/MS, we knew that histidine, threonine, glutamine, and asparagine were not labeled under the conditions we used and so they are not considered here. In wild-type leaves, one-third of the label of the cationic fraction was in alanine, but in the mutant, only one-tenth was in alanine following a 10-min chase (Table V).

DISCUSSION Table II. Total Amounts of Soluble Sugars and Starch in Mutant and Wild-Type Flaveria Data are mean ± SE, n = 5. Starch is reported as glucose equivalents. Samples labeled "Light" were taken at 3 PM; "Dark" samples were taken at 6 AM, before the lights came on. Light Sugar

Wild type Mutant iAmol g-' fresh weight 2.62 ± 0.34 0.37 ± 0.10 0.28 ± 0.09 0.91 ± 0.06 0.16 ± 0.03 0.09 ± 0.02 3.8±0.5 3.0±0.2 8.7±1.1 55.0 ± 3.5 19.1 ± 3.6 21.7 ± 3.6 Mutant

Glucose Fructose Sucrose

Starch

Dark Wild type

0.03 ± 0.01 0.02 0.5 ±0.1 5.6 ± 0.3

The mutant line of F. linearis studied here partitioned 2.5 times less carbon into water-soluble carbohydrates than did the wild-type line. Thus, the reduced activity of cytosolic FBPase in the mutant does appear to reduce the flux of carbon through the sucrose biosynthetic pathway. The extra carbon is partitioned primarily into starch with a small amount also partitioned into serine and glycine. This is consistent with the view that starch serves as an overflow for carbon that cannot be made into sucrose as fast as it can be fixed (25, 28, 29), but that when starch synthesis is also at its maximum, switching to low oxygen will no longer stimulate, and can even inhibit, photosynthesis (10, 24). In these experiments, there was no significant difference in photosynthesis

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Table IV. Amounts of Selected Amino Acids in Two Lines of F. linearis in Normal and Low 02 Partial Pressure

Other amino acids did not vary substantially between treatments. 02 = 200 mbar Wild type

02 = 20 mbar Wild type

Mutant

520 ± 76 432 ± 10 1449 ± 133 3057 ± 41 432± 18 78 ± 33 304 ± 5 107 ± 5 2025 ± 136

566 ± 7 237 ± 21 1557 ± 93 3649 ± 120 207± 19 100 ± 6 264 ± 52 12 ± 2

Mutant

nmol g-'

Alanine

y-Amino butyric acid Aspartate and asparagine Glutamate Glutamine Glycine Phenylalanine Pipecolicacid Serine

256 ± 3 190 ± 10 1473 ± 154 3308 ± 343 141 ±28 172 ± 5 291 ± 42 181 ± 9 2119 ± 390

rate between normal and low 02 in the mutant plants (Table I). The wild-type line of F. linearis partitioned about 50% of photosynthate into water-soluble carbohydrates at the 10min chase time (Fig. 1). Monson et al. (18) have reported that F. linearis partitions more carbon into sucrose than most other C3-C4 intermediate Flaveria species. Partitioning to water-soluble carbohydrates was reduced in the mutant line but the amount of sucrose in the leaf was substantially higher. This surprising result indicates that phloem loading or transport may be affected in the mutant, causing higher levels of sucrose in the leaf. There was evidence from the 14CO2 feeding (Fig. 1) that export of water-

100

-240

240

240

240

240

0)0

B

100

226

226

226

226

226

0

U)

100 nd239

200 mbar

20 mbar

150 ± 7 96 ± 9 2028 ± 155 4380 ± 362 119±25 131 ± 16 468 ± 97 50 ± 13 2375 ± 99

soluble carbohydrates was reduced in the mutant. We have no information on localization of the sucrose within the leaf or cell. It may be that the mutant stores more sucrose in the vacuole than does the wild type. We were also surprised to find that the activity of SPS was not different between the mutant and the wild type. In many studies, SPS activity has been found to be related to the rate of photosynthesis, and presumably the rate at which carbon is converted to sucrose (2, 20, 30). The mutant lost twice as much starch during the dark period as did the wild type (Table II). The level of starch in the leaves decreased in both lines by the end of the dark period (Table II). The difference in starch levels between the middle of the light period and the end of the dark period was 2.5 times greater for the mutant. If we assume that the difference in starch level seen in the middle of the day was still present at the end of the day, then the chloroplasts of mutant plants may supply sugars to the rest of the plant at a greater rate or for a longer period during the night than do the wild-type chloroplasts. Because this is a C3-C4 intermediate, we checked whether mesophyll cell chloroplasts or bundle sheath chloroplasts accumulated more starch. We found that starch was more prevalent in both tissue types in the mutant line; no cellspecific differences in starch accumulation were found between the mutant and wild type. We were surprised to find so much labeling of alanine in wild-type plants; however, substantial labeling of alanine was reported for Flaveria ramosissima, another C3-C4 intermediate (23). One explanation could be that alanine is made from pyruvate, which can be synthesized by Rubisco (1). The FBPase-lacking mutant partitioned much less carbon into

Table V. Proportion of Label in Cation Fraction Found in Alanine or Glycine Plus Serine following a 10-min Chase

M/Z

Figure 2. Selected portion of mass spectra of alanine, glycine, and serine. The labeling with 13C is indicated by a shift of some of the ions to higher mass numbers. In no case was the labeling complete enough to change the mass of the most prominent ion.

2111 ± 106

Alanine

Glycine + serine

Wild Type

Mutant

36 64

11 89

PARTITIONING IN A FRUCTOSE-1 ,6-BISPHOSPHATASE MUTANT

alanine and had a lower level of alanine at 200 mbar 02 than did the wild type. At lower 02 partial pressure, the concentration of alanine increased in both lines to nearly equivalent levels. The elimination of photorespiration can lead to loss of 02 sensitivity as it does in C4 plants. However, in this study we found large pools of serine and glutamate in both lines, which is consistent with the diffusion of photorespiratory intermediates to and from the bundle sheath chloroplasts and a nitrogen transport system. Also, in the tracer experiments much of the label in amino acids was in serine and glycine, and in low 02 the level of glycine decreased in both lines. These results confirm that the loss of 02 sensitivity in the mutant is not caused by the elimination of photorespiration (24). In summary, these results show that partitioning of photosynthate to water-soluble carbohydrates, presumably sucrose, is reduced in plants lacking cytosolic FBPase. This is the first report that SPS activity is normal in this mutant and that sucrose occurs at high concentration despite the reduced activity of cytosolic FBPase and rate of labeling of sucrose. These results raise two other questions about this mutant. First, why is the sucrose concentration high in a plant that partitions less carbon to sucrose, and second, why is alanine heavily labeled in the wild-type plants but not in the mutant plants? LITERATURE CITED

1. Andrews TJ, Kane HJ (1991) Pyruvate is a by-product of catalysis by ribulosebisphosphate carboxylase/oxygenase. J Biol Chem 266: 9447-9452 2. Battistelli A, Adcock MD, Leegood RC (1991) The relationship between the activation state of sucrose-phosphate synthase and the rate of CO2 assimilation in spinach leaves. Planta 183: 620-622 3. Bauwe H, Keerberg 0, Bassuner R, Parnik T, Bassuner B (1987) Reassimilation of carbon dioxide by Flaveria (Asteraceae) species representing different types of photosynthesis. Planta 172: 214-218 4. Bouton JH, Brown RH, Byrd GT, Sharkey TD (1990) Inheritance of the reversal of 02 response of photosynthesis in a Flaveria linearis mutant. Plant Physiol 92: 186-190 5. Brown RH, Boulton JH, Evans PT (1986) Oxygen stimulation of apparent photosynthesis in Flaveria linearis. Plant Physiol 81: 212-215 6. Brown RH, Hattersley PW (1989) Leaf anatomy of C3-C4 species as related to evolution of C4 photosynthesis. Plant Physiol 91: 1543-1550 7. Chastain CJ, Chollet R (1989) Interspecific variation in assimilation of "4CO2 into C4 acids by leaves of C3, C4 and C3-C4 intermediate Flaveria species near the CO2 compensation concentration. Planta 179: 81-88 8. Davis JM, Fellman JK, Loescher WH (1988) Biosynthesis of sucrose and mannitol as a function of leaf age in celery (Apium graveolens L.). Plant Physiol 86: 129-133 9. Edwards GE, Ku MSB (1987) Biochemistry of C3-C4 intermediates. In MD Hatch, NK Boardman, eds, The Biochemistry of Plants, Vol 10, Photosynthesis. Academic Press, San Diego, pp 275-325 10. Harley PC, Sharkey TD (1991) An improved model of C3 photosynthesis at high C02: reversed 02 sensitivity explained by lack of glycerate reentry into the chloroplast. Photosynth Res 27: 169-178 11. Holaday AS, Lee KW, Chollet R (1984) C3-C4 intermediate species in the genus Flaveria: leaf anatomy, ultrastructure, and

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the effect of 02 on the CO2 compensation concentration. Planta 160: 25-32 12. Huber JL, Hite DRC, Outlaw WH Jr, Huber SC (1991) Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation. Plant Physiol 95: 291-297 13. Huber WE, Brown RH, Bouton JH, Stemnberg LO'R (1989) CO2 exchange, cytogenetics, and leaf anatomy of hybrids between photosynthetically distinct Flaveria species. Plant Physiol 89: 839-844 14. Hylton CM, Rawsthorne S, Smith AM, Jones DA, Woolhouse HW (1988) Glycine decarboxylase is confined to the bundlesheath cells of leaves of C3-C4 intermediate species. Planta 175: 452-459 15. Ku MSB, Monson RK, Littlejohn RO, Nakamoto H, Fisher DB, Edwards GE (1983) Photosynthetic characteristics of C3-C4 intermediate Flaveria species. Plant Physiol 71: 944-948 16. Loreto F, Sharkey TD (1990) A gas-exchange study of photosynthesis and isoprene emission in Quercus rubra L. Planta 182: 523-531 17. Monson RK, Edwards GE, Ku MSB (1984) C3-C4 intermediate photosynthesis in plants. BioScience 34: 563-574 18. Monson RK, Moore BD, Ku MSB, Edwards GE (1986) Cofunction of C3- and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species. Planta 168: 493-502 19. Moore Bd, Ku MSB, Edwards GE (1987) C4 photosynthesis and light-dependent accumulation of inorganic carbon in leaves of C3-C4 and C4 Flaveria species. Aust J Plant Physiol 14: 657-668 20. Quick WP, Siegl G, Neuhaus E, Feil R, Stitt M (1989) Short term water stress leads to a stimulation of sucrose synthesis by activating sucrose phosphate synthase. Planta 177: 535-546 21. Rhodes D, Hogan AL, Deal L, Jameson GC, Haworth P (1987) Amino acid metabolism in Lemna minor L. II. Responses to chlorsulfuron. Plant Physiol 84: 775-780 22. Rufty TW Jr, Huber SC (1983) Changes in starch formation and activities of sucrose phosphate synthase and cytoplasmic fructose-1,6-bisphosphatase in response to source-sink alterations. Plant Physiol 72: 474-480 23. Rumpho ME, Ku MSB, Cheng S-H, Edwards GE (1984) Photosynthetic characteristics of C3-C4 intermediate Flaveria species. Plant Physiol 75: 993-996 24. Sharkey TD (1990) Feedback limitation of photosynthesis and the physiological role of ribulose bisphosphate carboxylase carbamylation. Bot Mag Tokyo Special Issue 2: 87-105 25. Sharkey TD, Berry JA, Raschke K (1985) Starch and sucrose synthesis in Phaseolus vulgaris as affected by light, CO2, and abscisic acid. Plant Physiol 77: 617-620 26. Sharkey TD, Kobza J, Seemann JR, Brown RH (1988) Reduced cytosolic fructose-1,6-bisphosphatase activity leads to loss of 02 sensitivity in a Flaveria linearis mutant. Plant Physiol 86: 667-671 27. Sharkey TD, Vassey TL, Vanderveer PJ, Vierstra RD (1991) Carbon metabolism enzymes and photosynthesis in transgenic tobacco (Nicotiana tabacum L.) having excess phytochrome. Planta 185: 287-296 28. Stitt M (1990) Fructose-2,6-bisphosphate as a regulatory molecule in plants. Annu Rev Plant Physiol Plant Mol Biol 41: 153-185 29. Stitt M, Huber S, Kerr P (1987) Control of photosynthetic sucrose formation. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants. Academic Press, New York, pp 327-409 30. Vassey TL, Quick WP, Sharkey TD, Stift M (1991) Water stress, carbon dioxide, and light effects on sucrose-phosphate synthase activity in Phaseolus vulgaris. Physiol Plant 81: 37-44 31. von Caemmerer S, Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376-387 32. Winter K, Usuda H, Tsuzuki M, Schmitt M, Edwards GE, Thomas RJ, Evert RF (1982) Influence of nitrate and ammonia on photosynthetic characteristics and leaf anatomy of Moricandia arvensis. Plant Physiol 70: 616-625