Planta (1992)188:238-244

P l a n t a 9 Springer-Verlag1992

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol Till Jelitto 1, Uwe Sonnewald 2, Lothar Willmitzer 2, Mohammad Hajirezeai 1, and Mark Stitt 3. 1 Lehrstuhl ffir Pflanzenphysiologie,Universitfit Bayreuth,W-8580 Bayreuth, Federal Republic of Germany 1 Institut ffir genbiotogischeForschung Berlin GmbH, Ihnestrasse 63, W-1000 Berlin 33, Federal Republic of Germany 3 Botanisches Institut, Im Neuenheimer Feld 360, W-6900 Heidelberg, Federal Republic of Germany Received 17 February; accepted 10 April 1992

Abstract. Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role. Key words: Inorganic pyrophosphate - Nicotiana - Sinksource - S o l a n u m - Sucrose metabolism Transgenic plants * To whom correspondence should be addressed; FAX 49 (6221) 565859 Abbreviations: Fru2,6bisP=fructose-2,6-bisphosphate; Fru6P= fructose 6-phosphate; FW = fresh weight; GlclP= glucose-Iphosphate; Glc6P= glucose-6-phosphate; PEP = phosphoenolpyruvate; 3PGA= glycerate-3-phosphate; PFK = phosphofructokinase; PFP=pyrophosphate: fructose-6-phosphate phosphotransferase; Pi=inorganic phosphate; PPi=inorganic pyrophosphate; UDPGlc= UDP-glucose

Introduction Pyrophosphate (PPi) is produced in a wide range of biosynthetic reactions and is usually hydrolysed by pyrophosphatase, making these reactions effectively irreversible in vivo (Stryer 1990). However, recent research indicates that PPi may have other roles in higher plants. Alkaline pyrophospatase is restricted to the plastid (Gross and ap Rees 1986; Weiner et al. 1987), and the cytosol contains a substantial pool of PPi (Weiner et al. 1987). Three reactions in the cytosol could utilise PPi as an energy source, namely (i) UDP-glucose (UDPGlc) pyrophosphorylase when it is operating in combination with sucrose synthase to degrade sucrose; (ii) phosphorylation of fructose-6-phosphate (Fru6P) by fructose-6-phosphate: phosphotransferase (PFP; see review by Stitt 1990); and (iii) a PPi-dependent proton pump on the tonoplast membrane which could be involved in tonoplast energisation and ion uptake into the vacuole (Rea and Sanders 1987). These three PPi-utilising enzymes are widely distributed, and are often present at high activities (ap Rees 1984; Rea and Sanders 1987; Stitt 1990). They are also strongly regulated. For example, PFP is regulated by the signal metabolite fructose-2,6-bisphosphate (Fru2,6bisP) in plants (Stitt 1990), instead of phosphofructokinase as is the case in almost all other eukaryotes. Sucrose synthase is strongly regulated by post-transcriptional mechanisms (Taliercio and Chourey 1989) and at the level of gene expression by developmental and environmental signals (Salanoubat and Belliard 1989; Maas et al. 1990; Doehlert and Chourey 1991). The tonoplast PPi-proton pump is regulated by Ca 2§ (D. Sanders, Biology Department, University of York, UK, personal communication). It is therefore tempting to propose that PPi metabolism is a central and distinctive feature of plants. The precise role of PPi in plants, however, is still unclear. Two major problems have made it difficult to carry out definitive and unambigous experiments. Firstly, each of the PPi-dependent reactions is, in principle,

T. Jelitto et al. : Genetic manipulation of PPi r e d u n d a n t b e c a u s e it c a n be r e p l a c e d by a n A T P - d e p e n d e n t e n z y m e , viz. sucrose m o b i l i s a t i o n via i n v e r t a s e a n d h e x o k i n a s e s , p h o s p h o r y l a t i o n o f F r u 6 P b y the classical glycolytic e n z y m e p h o s p h o f r u c t o k i n a s e , a n d energisat i o n o f the t o n o p l a s t b y a n A T P - d e p e n d e n t p r o t o n p u m p . Secondly, the P P i - d e p e n d e n t reactions a p p e a r to o p e r a t e quite close to their t h e r m o d y n a m i c e q u i l i b r i u m ( W e i n e r et al. 1987), m a k i n g it difficult to decide w e t h e r a p a r t i c u l a r r e a c t i o n is c o n s u m i n g o r g e n e r a t i n g PPi. S o n n e w a l d (1992) h a s used r e v e r s e d genetics to investigate the significance o f PPi for p l a n t growth. P o t a t o a n d t o b a c c o p l a n t s were t r a n s f o r m e d with a l k a l i n e p y r o p h o s p h a t a s e f r o m Escherichia coli, t a r g e t e d to the c y t o sol to decrease the PPi p o o l . T h e t r a n s f o r m a n t s s h o w e d a n i n c r e a s e d s u c r o s e : s t a r c h r a t i o , a c c u m u l a t i o n o f carb o h y d r a t e in the leaf, a n d p e t u r b e d " s i n k " g r o w t h revealed b y s t u n t i n g in t o b a c c o , a n d i n c r e a s e d s i d e - s h o o t d e v e l o p m e n t a n d large n u m b e r s o f small l o w - s t a r c h t u b e r s in p o t a t o . T h e f o l l o w i n g e x p e r i m e n t s investigate the b i o c h e m i c a l basis for these s t r i k i n g p h e n o t y p i c changes. W e h a v e i n v e s t i g a t e d w h e t h e r PPi is i n d e e d d e c r e a s e d a n d then a s k e d h o w this affects the r e a c t i o n s i n v o l v e d in the synthesis o f sucrose in the source leaves a n d the use o f sucrose in the sink organs.

Materials and methods Tobacco (Nicotinia tabacum L., cv. Sansun NN) and potato (Solanum tuberosum cv. Desir6e; both from Vereinigte Saatzuchten, Ebstorf, FRG) were transformed with PPi from Escherichia eoli, under the control of the constitutive 35S CaMV promotor and with the octopine synthase poly-A site, using a binary vector system from Agrobacterium (Sonnewald 1992). Regenerated plants were tested for expression of pyrophosphate by Northern blots and activity gels of heated extracts (to inactivate the endogenous plant enzyme; as in Sonnewald 1992). In the reported experiments, plants from at least four independent tobacco transformations and four independent potato transformations were investigated to check that the results were not an artefact caused by integration of the transferred DNA (T-DNA) into a critical genome sequence. Tobacco and potato plants were grown in a glasshouse in Berlin under 14 h supplementary lighting as in Sonnewald (1992) for PPi and metabolite determinations. Leaf discs (diameter 10 mm) were removed from leaves (see Table legends for details) and frozen in liquid N2. Tobacco and potato were grown in a growth chamber in Bayreuth (12 h/12 h light/darkness, 10/15 ° C, 4.0/3.0 Pa" kPa water deficit), using high-pressure mercury-.vapour lamps (HQJD 200 W; Osram, Mfinchen, FRG), and adjusting the irradiance to 350 p,mol photons" m -2- - t with green netting. The plants were grown in pots I0 cm diameter, 10 cm deep cm 20 cm diameter, 20 cm deep potato in a soil/peat mixture containing stow-release fertiliser pellets. (Osmocote 14-14-14; Meyer, Rellingen, FRG.) Leaf discs were used for determination of sucrose-phosphatesynthase activity and for 14C incorporation. Potato tubers were harvested for metabotite measurements by boring a cylinder longitudinally through the tuber and immediately slicing discs (1-2 nm thick) into liquid N2. Tobacco plantlets were also grown in agar containing Hoagland nutrient solution (10 mM NH4NO3, 3 mM KH2PO4, 1 mM MgSO4, 1 mM CaSO4, 50mM KC1, 25mM H3BO3, 2 gM MnSO4, 2gM ZnSO4, 0.5 gM CuSO4, 0.5 gM MoO> 20 ~tM Fe-EDTA) in sterile plastic boxes (Phytatray TMII; Sigma, Deisenhofen, FRG) in a growth chamber with a day/night regime of 14/10 h (250 gmol photons • m -2 " s 1), 25/20 ° C. Small holes in the upper side of the box allowed gas exchange. Samples were taken between 20-30 d for pyrophosphatase activity, growth analysis and carbohydrate content.

239 To measure pyrophosphatase activity, 100-200 mg tobacco leaf or potato tuber was homogenised in 1 mt 100 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)-KOH (pH7.5), 4raM MgClz, 1 mM EDTA, 1 mM ethylene glycol-bis(13aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 5 mM mercaptoethanol, and centrifuged. The supernatant was diluted 1:5 (v/v, wild type) or 1:20 (transformant), and 10 gl assayed in 200 gl 50 mM Tris-HC1 (pH 8.0), 10 mM MgC12 and 1.3 mM NaPPi for 10 min at 25 ° C before heating at 95 ° C for 5 rain, and assaying for release of inorganic phosphate (PPi) as in Murphy and Riley (1962). The assay was linear with time and amount of extract. To measure metabolites, a 200- to 300-rag sample of frozen material was first homogenized to a fine powder in liquid N 2 in a mortar standing on powdered dry ice (solid C02). A 15-ml aliquot of 16% trichloroacetic acid (TCA) in diethylether (v/v), precooled to the temperature of dry ice, was then added and the sample homogenised again. After leaving for 20 rain on dry ice, the homogenate was added to 0.8 ml of 16% TCA in water (v/v) containing 5 mM NaF, warmed to 4 ° C and then left for 3 h. The mixture was subsequently extracted four times with diethylether and neutralised with KOH/triethanolamine as in Weiner et al. (1987). All mortars and materials were prewashed for 12 h in 2 N HCt, and pseudoextracts were prepared in parallel to check that the reagents and apparatus were not contaminated with PPi. Before assaying metabolites in potato or tobacco leaf extracts, 400 tal of extract was added to 400 gl of cation exchanger (Serva, Heidelberg, FRG; Dowex AG 50 x 8, 100-200 Mech, preequilibrated with 2 N HC1, brought to pH 5 with water, and then dried for 12h at room temperature), mixed for 20 s, and centrifuged to remove compounds in the extract which interfered with the metabolite assays. Inorganic pyrophosphate was assayed photometrically as in Weiner et al. (1987); UDPGlc, glucose-l-phosphate (GlclP), glucose-6-phosphate (Glc6P), Fru6P, glycerate-3-phosphate (3-PGA), phosphoenolpyruvate (PEP), ATP and ADP were determined as in Stitt et al. (1989). Sucrose, glucose, fructose and starch were measured in the soluble and residual fractions of an ethanol extract as in Sonnewald et al. (1991) or in a TCA extract as in Stitt et al. (1989). Sucrosephosphate synthase was extracted and assayed as in Quick et al. (1988) using 4 mM Fru6P, 10 mM Gtc6P and zero Pi in the assay. The labelling of sucrose, starch and other cell components from 14CO2 was investigated by illuminating three leaf discs (diameter 10 mm) in a leaf-disc O2-electrode (Hansatech, Kings Lynn, Norfolk, UK) at 150 gmot photons -m 2. s-1 and 20°C for 25 min. The 14C02 was supplied from 300 gl of 1 M bicarbonate buffer (10 s Bq 14C • nmol -~) placed on a mat in the base of the electrode. Distribution of label was analysed as in Quick et al. (1988). The reliability of the extraction and assay of metabolites was checked by adding a small representative amount (two- to threefold the endogenous content) of each metabolite to the plant material in the killing mixture of TCA and diethylether. The recovery of metabolites from tobacco leaves was (as a percentage of the amount added): PPi, 110%, Glc6P, 141%; Fru6P, 115%; GlclP, 101%; UDPGlclc, 120%; and, in potato leaves, PPi, 76%; Glc6P, 83%; Fru6P, 81% ; GlclP, 88 % ; UDPGIc, 82%. Recovery of metabolites from potato tubers by our methods has already been documented (Hajirezaei and Stitt 1991).

Results

How high is pyrophosphatase activity in the transformants. Overall pyrophosphatase activity i n c r e a s e d f r o m 11.7 ! 0.6 ~tmol Pi released • g fresh weight (FW)-I .min-~ in w i l d - t y p e t o b a c c o leaves to 22.3-35.7 g m o l Pi - g F W - 1 . m i n - 1 in v a r i o u s t r a n s f o r mants. M o s t o f the p y r o p h o s p h a t a s e in the wild t y p e will be in the p l a s t i d ( W e i n e r et al. 1987). T h e p y r o p h o s p h a tase activity i n t r o d u c e d into the c y t o s o l in the t r a n s f o r -

240

T. Jelitto et al. : Genetic manipulation of PPi

mants is therefore comparable to that found normally in the chloroplasts. In potato tubers, pyrophosphatase activity was lower in the wild type (154131 nmol P i ' g F W - i . h-1, n = 6), and increased proportionately more in the transformants, lying in the range 270 to 1170 nmol P i . g FW -1- min-< For comparison, the activities of PFP and sucrose synthase in developing potato tubers were about 600 and 1-2000 nmol • g FW- rain- i, respectively (data not shown). Decreased PPi content in the leaves and tubers of transformants. Measurement of PPi in plant extracts is technically difficult because the endogenous pool is very small, whereas pyrophosphatase activity is extremely high and is relatively acid-stable (We±net et al. 1987). To allow penetration and killing to occur at low temperature, all extracts were made by adding 16% (v/v) TCA in diethylether at - 60 ° C. We demonstrated the reliability of our extraction and assay by adding representative amounts of PPi to the plant material in the killing mixture and showing that the added PPi was recovered through the extraction, storage and assay (see Materials and methods). Another potential source of error arises because many commercial reagents and laboratory articles contain PPi as a contaminant. All laboratory articles were routinely prewashed in 2 N HC1 for 24 h, and pseudoextracts were prepared in parallel with the plant extracts to check that the reagents and equipment did not contain PPi. The contamination in the pseudoextracts was equivalent to less than 1 nmol PPi'g FW-1 in the data presented. Table 1 summarizes the changes in the levels of PPi in the leaves of five different tobacco transformants and three different potato transformants. The PPi content was decreased on average threefold in tobacco leaves, and twofold in potato leaves in the transformants. Leaves on the tobacco transformant were smaller than on the wild type (data not shown). We checked that the decreased PPi contents were still found, irrespective of

Table 1. Pyrophosphate content of wild-type and transformed tobacco and potato plants. Tobacco and potato leaves were harvested in the light and had reached over 60 % of the final size. Potato tubers were harvested from 50-d-old plants. Results are given as mean ± SE (n) PPi content (nmol • g F W - 0 Wild type

Transformant

Tobacco

leaf

9.44- 1.3 (5)

2.9±0.9 (6)

Potato

Leaf Tuber

9.7 ± 2.5 (3) 0.8+0.2 (6)

5.0 ± 0.6 (9) 0.44-0.1 (4)

whether leaves of equal age or equal size were compared (data not shown). The PPi content of potato tubers from four different transformants decreased on average twofold (see below for individual data). Although most of the PPi is located in the cytosol, small amounts may be present in other organelles. During extraction, there is also a possibility that small amounts of PPi are released by acid hydrolysis of pyrophosphate-containing metabolites like phosphoribosyl pyrophosphate (Dancer and ap Rees 1989). The decrease in the overall PPi content will therefore be a minimum estimate of the change in the cytosolic pool. Perturbation of sucrose synthesis in leaves. Decreased PPi could favour sucrose synthesis because it will displace the reaction catalysed by UDPGlc pyrophosphorylase in favour of UDPGlc formation. We therefore investigated metabolite levels, and sucrose and starch formation in leaves. In potato leaves, there was a twofold decrease in the levels of the major hexose-phosphates (Glc6P and Fru6P), a smaller decrease of GlclP, and UDPGlc rose slightly (Table 2). As a result, the UDPGlc/hexosephosphate ratio increased. In immature tobacco leaves, the decrease of PPi was accompanied by a twofold increase of U D P G l c , while the major hexose-phosphates

Table 2. Metabolite contents of potato leaves. Fully expanded leaves were harvested in the light and quenched in liquid N2. The results are given as m e a n ± SE ( n = 5 for the wild type, 25 for the transformants) Metabolite (nmol - g F W - 1 )

Wild type Transformant

Carbohydrate (gmol • g F W - 1 )

UDPGlc

GlclP

Glc6P

Fru6P

Sucrose

Glucose

Fructose

Starch

72±3,3 80±3.1

2313.0 234-2.1

260±44 105± 5.0

81 4-9.2 36.4±2.2

8.84- 1,0 24.7±3.5

0.84-0.1 3.0±0.5

1.3±0,2 5.9±1.4

200±23 118±28

Table 3. Metabolite contents of tobacco leaves. Leaves were harvested into liquid N2 in the light. Young leaves were about 30% of final size. The results are given as mean ± SE (n = 4) Carbohydrate (gmol • g F W - 1 )

Metabolite (nmol • g F W - 1)

Leaf age

U D P G lc

GlclP

G|c6P

Fru6P

Sucrose

Glucose < 1 18.44- 8.9

Young

Wild type Transformant

82+21 178+25

25+8 36=1:3

144:t: 12 1004- 6

4 8 + 12 354- 4

6.9:t:3.0 14.54-3.8

Fully expanded

Wild type Transformant

39± 4 140±16

12±3 364-4

9 9 ± 10 99±10

33± 4 40± 3

3.14-0.5 24.3±5.0

Fructose 1.6±0.3 8.7:t:4.1

4.7± 2.1 7.3±2.2 107.7±23 55.04-6.4

T. Jelitto et al. : Genetic m a n i p u l a t i o n o f PPi

241

chlorophyll (Sonnewald 1992). Nevertheless, the rate of photosynthesis was already lower in the transformant (data not shown). The transformant synthesised starch at a rate 40 % lower than that of the wild type, but also synthesised sucrose 20% more slowly. A similar decrease in sucrose synthesis was seen in saturating (800 gmol. m -2 9s -1) irradiance (data not shown).

(Glc6P and Fru6P) decreased slightly (Table 3). Glucose-l-phosphate behaved in an intermediate manner, rising in level but not as much as UDPGlc. Similar results were obtained with fully expanded leaves, except that the increase of UDPGlc was larger (3.6-fold) and hexosephosphates remained constant rather than declining slightly. In potato, there was a twofold decrease of starch and a threefold increase of the sucrose content of transformant leaves, compared with the wild type (Table 2). This indicates that photosynthetic carbon partitioning has changed in favour of sucrose. In tobacco the ratio of sucrose/starch in the leaf changed in favour of sucrose (Table 3), but there was a very large, general accumulation of carbohydrate which included in increase in starch as well as sucrose and hexose, especially in leaves of older plants (Sonnewald 1992, see also below). We carried out 14COz labelling experiments to distinguish short-term fluxes from the accumulated storage pools (Table 4). With potato leaves, there was a small increase in the rate of sucrose synthesis, and a decrease of starch synthesis. On average, the ratio of sucrose synthesis/starch synthesis increased by 30% in the transformants. In the experiments with tobacco, leaves were used which had only reached 50-60% of their final size, to avoid additional complications due to the loss of

Effect on metabolism in sink tissues. Potato transformants produced more tubers, but they were much smaller than the wild type (Sonnewald 1992). Compared with those of the wild type, tubers from the transformant contained 30% more sucrose (62.5 4- 9.9 compared with 46.8 + 7.4 gmol 9g FW -1) and 30% less starch (4704-86 compared with 6304- 198 gmol -g FW -1) (n= 8 and 12, respectively). It follows that the decreased tuber size is the consequence of a decreased use of the sucrose. Glycolytic metabolites were measured in tubers from four different transformants (Table 5). The transformants contained up to three fold more UDPGIc, and two- to threefold less hexose-phosphates than wild-type tubers. There was an eight- to tenfold increase in the ratio of UDPGlc to hexose-phosphate. The contents of other glycolytic metabolites, including 3PGA and PEP also decreased two- to threefold. There was no large change in the ratio of hexose-phosphates/C-3 metabolites. The reg-

Table 4. Labelling o f sucrose a n d starch after s u p p l y i n g 14CO2 for 30 min. L e a f discs f r o m e x p a n d e d leaves were i l l u m i n a t e d in limiting irradiance (150 lxmol, m - 2 . s - 1 ) at 20 ~ in a leaf-disc O2-electrode. T h e results are given as mean_+ SE (n = 4 for t o b a c c o a n d p o t a t o ) C 0 2 - f i x a t i o n (lamol C 9 m - 2

9 S -1)

into:

Ratio of

Organic acids

Amino acids

Sucrose

Starch

Sucrose/ Starch

Tobacco

W i l d type Transformant

1.1 -+ 0.20 1.3_+0.10

1.0 _ 0.10 1.1 __0.10

2.2 _ 0.40 1.8+0.30

3.1 + 0.50 2.1 _ _ _ 0 . 5 0

0.70 + 0.13 0.90_+0.21

Potato

Wild type Transformant

0.4 + 0.02 0.6 -+ 0.10

0.5 _ 0.02 0.9 + 0.10

4.6 __+0.80 5.3 _ 0.60

3.5 -+ 0.40 3.3 _ 0.50

1.40 -+ 0.10 1.70 -+ 0.20

Table 5. M e t a b o l i t e levels in p o t a t o tubers. T h e t u b e r s were h a r v e s t e d f r o m f o u r wild-type p l a n t s or f r o m f o u r i n d e p e n d e n t t r a n s f o r m a n t s with differing levels o f expression o f p y r o p h o s p h a t a s e . T h e results are given as m e a n _+ SE (n = 16 for wild t y p e ; n = 4 for e a c h t r a n s f o r m a n t ) W i l d type

Transformants 3

Pyrophosphatase ( m m o l 9 g F W - 1 . m i n - 1) Metabolites ( n m o l 9 g F W -1) PPi UDPGlc GlclP Glc6P Fru6P 3-PGA PEP Pyruvate ATP ADP Fru2,6bisP (pmol - g FW-1)

0.15-4-0.03

1.0 30.5 6.2 92.9 24.6 26.3 8.5 6.6 29.9 13.1 68.4

_+0.08 -+1.50 +0.80 ___3.40 +1.30 +2.10 ___1.10 +0.80 -+1.40 -I-0.50 -+4.00

1 0.27__+ 0.03

0.62___ 0.08 47.4 + 4.10 5.7 _ 1.30 78.1 -I- 0.80 25.9 __+ 5.50 25.5 _+ 5.50 10.6 -+ 1.30 5.2 _+ 1.00 32.1 _+ 2.80 12.3 ___ 0.50 111.0 -+30.0

0.69+

4

2

0.07

0.84+__ 0.07

1.17__ 0.26

0.37-t- 0.06 92.5 _+ 5.50 5.0 ___ 0.50 40.7 _+ 1.60 12.3 _ 0.70 10.3 -+ 1.80 2.4 -+ 0.30 3.3 ___ 0.30 33.1 _ 3.50 9.7 _+ 1.00 440.0 _+48.0

0.37-t- 0.02 101.0 _ 3.70 6.0 _+ 0.80 45.7 -+ 3.00 14.3 _+ 1.80 10.3 -I- 1.00 3.0 -I- 3.00 4.0 + 1.20 42.3 _+ 3.9 12.5 _+ 0.60 526.0 + 7 4 . 0

0.41-+ 0.10 86.50-+ 5.40 5.6 4- 0.60 34.3 ___ 3.60 10.5 _+ 0.40 8.3 ___ 1.00 1.7 _ 0.30 2.7 -+ 0.40 32.4 _+ 1.50 9.7 ___ 0.30 345.0 ___42.0

242

T. Jelitto et al. : Genetic manipulation of PPi

Table 6. Growth inhibition and accumulation of carbohydrate in stems and roots of tobacco. Plants were on agar-containing nutrient medium in sterile containers for 30 d before harvest FW (g) Leaves Stem Root

Wild type Transformant Wild type Transformant Wild type Transformant

1246.0 108.0 313.0 7.7 170.0 10.0

Carbohydrate (gmol - g FW- 1) Sucrose Glucose 1.2+0.1 15.7• 1.2 0.9 • 0.1 4.5+0.9 2.5 • 0.3 4.6+0.9

ulator metabolite, Fru2,6bisP, increased six- to sevenfold. One of the transformants (T3) contained lower pyrophosphatase activity and had a smaller reduction of PPi. The changes in all the metabolites were also smaller in this transformant, emphasising that there is a proportionality between the decrease of PPi and the effect on metabolism. The transformed potato tubers contained slightly lower ATP and higher A D P levels. Rapid hydrolysis of PPi would be expected to lead to dissipation of energy and the decreased A T P / A D P ratio therefore is rather unexpected. The higher ATP/ADP ratio does not, however, necessarily imply that the phosphorylation potential is higher in the transformed tubers. The levels of all the phosphorylated metabolites decreased and it is probable that Pi increases. The changes in the levels of carbohydrates in sink tissues of tobacco are summarised in Table 6. In this experiment, seedlings were grown in agar-containing nutrient medium, to allow ready sampling of the roots. Two features o f this experiment are noteworthy. Firstly, there was a very strong inhibition of root growth and stem growth. Sonnewald (1992) also reported that when tobacco plants were grown in soil, there was an especially strong inhibition o f stem extension. Secondly, the growth inhibition was accompanied by an accumulation of carbohydrates in the root and stem. In this case, starch and hexoses accumulated as well as sucrose. Discussion

The results presented by Sonnewald (1992) and in the present article show that expression of pyrophosphatase from E. coli in the cytosol o f higher plants leads to a twoto threefold decrease o f the PPi content, with dramatic consequences for metabolism and growth, thus demonstrating that PPi plays an important role in plant metabolism. As we will now discuss, the detailed changes in the levels o f carbohydrates and metabolites in these plants also provide information about the sites at which PPi is required.

Source-leaf metabolism. The reaction catalysed by U D P G l c pyrophosphorylase in the cytosol is reversible (Weiner et al. 1987; Roberts 1990). Removal of PPi should displace this reaction in the direction of U D P G l c

1.2• 38.2+3.3 1.2 + 0.2 5.4• 1.1 2.7 • 0.6 4.7+ 1.2

Fructose 0.6• 22.3• 0.7 • 2.6• 2.1 • 2.9•

1.1 0.1 0.5 1.2

Starch 15.3• 1.1 35.0• 10.0 2.7 + 0.7 5.0• 0.9 1.3 + 0.4 4.4+ 1.3

formation, and might therefore stimulate sucrose synthesis. In agreement, the transformants had an increased UDPGlc/hexose-phosphate ratio in their leaves. The contents of sucrose and starch as well as the partitioning of newly fixed 14C revealed a small shift towards sucrose synthesis, at the expense o f starch synthesis. These results are the mirror image of the changes observed after feeding fluoride or imidodiphosphate to spinach leaves to inhibit PPi hydrolysis and increase PPi levels, when sucrose synthesis was inhibited (Quick et al. 1988 ; Neuhaus and Stitt 1991). The increased level of U D P G l c will be accompanied by a decrease of free uridine nucleotides; indeed, since the contents of U T P and U D P are much lower than that of U D P G I c (see e.g. Quick et al. 1988; Roberts 1990), the relative decrease of the free-nucleotide pools is likely to be larger than the increase of UDPGIc. Thus, even though removal of PPi favours U D P G l c formation, a decline of U T P could nevertheless eventually restrict the activity of U D P G l c pyrophosphorylase. This would provide an explanation for the small rise in the amount of G l c l P in the transformants, relative to the other hexose-phosphates. The need to correctly "poise" the uridine-nucleotide pool may constrain the extent to which sucrose synthesis can be stimulated by removing PPi. In agreement, the change of flux in the pyrophosphatase transformants was rather modest, and was much smaller than the change in the UDPGlc/hexose-phosphate ratio (two- to threefold). In evaluating the changes in leaf metabolism we also need to note that decreased growth in the rest of the transformed plant (see below) may lead to indirect effects which superimpose on the direct effects of low PPi. Decreased growth will be accompanied by decreased export of sucrose from the leaf, leading to accumulation of carbohydrate and feedback inhibition. These indirect effects were particularly marked in tobacco leaves, where free hexoses increased, sucrose-phosphate-synthase activity decreased (data not shown), starch accumulated, and the leaves gradually bleached. These changes show many similarities to those observed when export out of the leaf was inhibited by expressing invertase in the cell wall to block phloem loading (von Schaewen et al. 1990; Stitt et al. 1991; Sonnewald et al. 1991).

Metabolism in sink tissues. Transformants from both potato and tobacco showed peturbed sink growth. Clear-

T. Jelitto et al. : Genetic manipulation of PPi ly the sinks were not able to utilise the available carbohydrate for growth. Removal of PPi could, in principle, affect growth in several ways including inhibition of sucrose synthesis via sucrose synthase, inhibition of glycolysis via PFP, or decreased tonoplast energisation and ion uptake into the vacuole (see Introduction). The relative importance o f these processes can now be evaluated. Growth o f individual tubers and the accumulation of starch was inhibited in transformed potato plants even though the sucrose concentration in the tubers increased. This demonstrates that sucrose mobilisation had been inhibited. The observation that U D P G I c increased dramatically and that all other glycolytic intermediates were depleted provides strong evidence that mobilisation via sucrose synthase and U D P G I c pyrophosphorylase had been inhibited by low PPi. Inhibition of glycolysis at the reaction catalysed by PFP would be expected to lead to an increased hexosephosphate : 3PGA ratio in the transformant (3PGA is the main representative of the pool o f C-3 metabolites in glycolysis). This was not observed. Apparently, the tubers are able to compensate for any effect o f low PPi on net glycolytic flux. Two factors might be involved. Firstly, the decrease of PEP (and probable increase of Pi) will activate P F K (Dennis and Greyson 1987). Secondly, there was a large increase of Fru2,6bisP, which is a potent activator o f PFP. The increased Fru2,6bisP in potato tubers is probably the consequence of a decrease of the 3PGA/Pi ratio, which will activate the enzyme responsible for its synthesis, Fru6P,2-kinase (Stitt 1990). We will report elsewhere that potato plants transformed with "antisense" to PFP can compensate for a 20- to 50-fold decrease of PFP by increasing the Fru2,6bisP content in their tubers. Evidently, changes of Fru2,6bisP provide a very effective way o f modulating PFP in vivo. We conclude that the PPi-requirement of PFP does not represent a major site where PPi is required during growth. In tobacco, root growth and stem extension was strongly inhibited, even though free hexoses and starch increased. In this case the mobilisation o f sucrose was not limiting. It is noteworthy that leaf number was only slightly decreased in the transformants (data not shown), indicating that cell extension was affected more than meristem activity. It is tempting to speculate that the tonoplast PPi-dependent proton pump had been inhibited, and that this protein plays a key role in tonoplast energisation and the generation o f turgor. Further experiments are needed, however, to provide direct evidence for this proposal.

Comparison of potato and tobacco. Transformation of potato and tobacco with the same enzyme leads to visually different phenotypes. In tobacco, plant growth is strongly inhibited, carbohydrate accumulates in the leaf, and the leaves yellow. In potato, individual tuber and shoot growth is decreased, but the accumulation of carbohydrate is less marked. Instead, more sinks are initiated, by producing.sideshoots and a larger number o f tubers per plant (Sonnewald 1992). Similar differences were found when tobacco (von Schaewen et al. 1990) and

243 potato (data not shown) were transformed with invertase. The different responses may result from the contrasting sink-source relationships in these two species: tobacco has one major stem producing large leaves and has relatively little sink tissue, whereas potato has a large number of storage sinks.

In conclusion, these plants provide direct evidence for the importance o f PPi plant metabolism and growth. They also provide a novel experimental system in which the role o f PPi can be investigated. Our results already show that sucrose mobilisation via sucrose synthase represents one key site where PPi is required, but that this is certainly not the only point at which PPi plays a crucial role in growth. In the future, it will be possible to study the impact of low PPi on metabolism and physiology in these plants in a range of different developmental stages and in different environmental conditions, to elucidate the contributions of PPi- and ATP-dependent reactions. It will also be important to generate a new generation of plants in which the pyrophosphatase is expressed in a tissue- or time-dependent manner, to allow us to distinguish between direct effects of PPi on metabolism and indirect effects caused by changes in the sink-source status of the whole plant. This research was supported by the Deutsche Forschungsgemeinschaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister fiir Forschung und Technologie (U.S., L.W.).

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