Indian Journal of Experimental Biology Vol. 37, June 1999, pp. 523-529

Review Article

Higher plant sucrose-phosphate synthase: Structure, function and regulation

Debasis Pattanayak

Division of Crop Physiology and Biochemistry, Central Potato Research Institute, Shimla 171 00 I, India. Fax: ++91-0177-224460

Sucrose-phosphate synthase (SPS) in higher plants catalyzes the penultimate and pivotal step of sucrose synthesis. SPS has a broad pH optimum for its activity and 'catalyzes a order bi bi reaction. SPS shows hyperbolic substrate saturation kinetics for both the substrates, UDP-glucose and fructose-6-phosphate. The enzyme is allosterically activated by glucose-6-phosphate and inhibited by Pi. These two effectors change the enzyme's affinity for both the substrates. SPS is believed to be a homodimer of 120-\38 kd subunits with no interchain disulfide bridges. It has a conserved N-terminal portion with variable C-terminal part. Cloning of SPS gene has been accomplished from some plant spe'.:ies. SPS shows high sequence homology among the plant species. A glycine-rich region in the N-terminal portion is a distinctive feature of SPS from monocot plant species but is absent in dicots. Rice SPS gene has been mapped on chromosome 1. Genome organization study reveals that rice SPS gene consists of 12 exons and 11 introns. Light modulates SPS activity by covalent modification (coarse control) and allosteric regulation (fine control). In dark SPS is phosphorylated at a specific serine residue (Ser-158 in case of spinach SPS) by a specific protein kinase and becomes less active. ' Light activation of SPS activity involves dephosphorylation by a type 2A protein phosphatase. Covalent modification alters the kinetic properties of the enzyme. The diurnal and circadian rhythm of SPS activity is because of light regulation of de novo expression of SPS-phosphatase. The tissue metabolites, glucose-6-phosphate and Pi, not only alter the SPS activity but also affect the enzymes involved in covalent modulation of SPS. Glucose-6-phosphate inhibits SPS protein kinase and Pi inhibits SPS-phosphatase. The fluctuation of leaf Pi concentrations during light-dark transitions has been proposed to play a tylajor role in the signal amplification of SPS regulation. The prospects of generation of transgenic plants overexpressing SPS have also been discussed.

Sucrose, being non-reducing in nature, is the major form of photoassimilate for export from the leaves and also a major effector of gene expression 1,2 . The rate of sucrose synthesis in leaves determines the availability of carbon for export from leaves3 and also affects the rate of photosynthesis. Sucrose-phosphate synthase (SPS) catalyzes the penultimate and rate­limiting step of sucrose synthesis. Studies with transgenic plants have shown that SPS activity is the major determinant of partitioning of photosynthate between sucrose and starch. Transgenic tomato plants expressing high activity of maize SPS reached saturation of net photosynthetic rate at higher CO2

concentration and had lower concentration of starch and higher concentration of sucrose in leaves4

-7

.

Consequently, · manipulation of this growth limiting enzyme has been considered to be a means to increase plant yield potentialS. Therefore, a thorough understanding of structure and function of SPS, and elucidation of the mechanisms of regulation of the enzyme activity in vivo is of primary importance. Since the first discovery of SPS from wheat germ

9

considerable efforts have been made to gain an insight into the details of biochemistry of this enzyme. During the last few years, research on SPS

has got momentum because of cloning, characterization and heterologous expression of the SPS gene either in Escherichia coli 10 or in transgenic plants ll

. The purpose of the present review is to highlight the recent developments related to structure, function and regulation of SPS in higher plants.

Purification and characterization of SPS-In higher plants SPS (EC 204 .1. 104) is present mainly in the mesophyll cell cytoplasm 12 and catalyz~s the formation of sucrose phosphate:

UDP-glucose + Fructose-6-phosphate <=> Sucrose-6-phosphate + UDP + H+. Although, this is a freely reversible reaction, the rapid removal of phosphate group from sucrose-phosphate by sucrose-phosphate phosphatase (SPP) renders the reaction essentia lly irreversible. The calculated mass action ratio for the reaction in vivo also confirmed that the reaction is fa r from equilibrium 13. In fact it has been observed that SPS and SPP actually form a complex in ViVOI4 .

Recent evidence suggests that SPP either activates or stabilizes SPS activity without altering its kinetic properties 14, 15.

Because of the low abundance and relative instability of the enzyme, progress on the purification and characterization of SPS from different plant

524 INDIAN J EXP BIOL, JUNE 1999

sources has been slow. Till date, SPS has been purified and characterized from spinach leaves lO

•16,

b I 17 .. d f · 18· soya ean eaves , germmatmg see s 0 pea , nce leaves9, corn leaves2o

, potato tubers21 and leaves of a tree species, Prosopis juliflora 22 by a combination of anion exchange, affinity and immunoaffinity chromatography. Although, spinach, corn, soyabean and P. juliflora contain a single isoform of SPS, plant species differ with respect to the presence of different isoforms of SPS. Cloning and sequencing o( SPS cDNA from many plant species have also confirmed this observation. Two isoforms of SPS, designated as SPS I and SPS II, have been purified from rice leaves. These two isofurms differ in s.ubstrate specificity and allosteric regulation 19.

SPS has a broad pH optimum (PH 6.5 -7.5) for its activityl2. Substrate saturation profiles for both the substrates, UDP-glucose and fructose-6-phosphate, are hyperbolic l8

, 21.24. The reaction mechanism for the enzyme involves a sequential addition of substrates (i.e. order bi bi reaction) without the formation of a glucosyl enzyme intermediates22,25. Kinetic data for the forward reaction and both products and dead-end inhibition indicate that the enzyme is allosterically activated by glucose-6-phosphate and inhibited by Pi. These effectors have a large effect on the affinities for both UDP-glucose and fructose-6-phosphate with Pi acting antagonistically to glucose-6-phos-

h t 21,22,26 P a e . However, in case of soyabean leaf SPS, neither

glucose-6-phosphate nor Pi has much effect on the enzyme activityl7. SPS is found to be inhibited by anions and activated by divalent cations, Mn2+ or Mg2+. Although, no inhibition of the enzyme activity is detected wit-h either sucrose or sucrose-phosphate, one of the products, UDP, is inhibitory to SPS activity. This inhibition is competitive with UDP­glucose (Ki 0.7-3 .6 mM) 18,22,25,17 .

Immunochemical studies and photoaffinity labelling of the active site of the enzyme with substrate analogue revealed that the enzyme is a homodimer of 120-138 kd subunits I6

.27

. N-terminal part of subunit of SPS, in general , is highly conserved. A stretch of I I amino acid residues, Asp 197 to Glu 206 (corresponding to the amino acid sequence of spinach SPS), within the conserved N­terminal portion has been observed to resemble the glycine-rich motif of phosphate binding domains. It has been proposed that these residues might be involved in binding of fructose-6-phophate to SPS28.

The identity of the catalytic subunit and active site of SPS has been confirmed by photoaffinity labelling with substrate analogue 5-N3-UDP-Glucose27

. Photo­affinity labelling of a recombinant spinach SPS frag­ment using U3_32p] 5-N3-UDP-Glucose determined the portion of the primary sequence in the vicinity of the uridine moiety of the substrate molecule. It has been observed that the 5 position of the uridine ring is near to the residues GIn 227 to Glu 239 which is proximal to the residues Asp 197 to Glu 206, involved in binding of fructose-6-ph·osphat~28. This uridine binding region of SPS has been found to be highly conserved among the plant species sequenced so far26

.

However, the domains containing the effector sites (allosteric site) involved in glucose-6-phosphate and Pi binding have yet to be identified. Although, SPS molecule does not have any inter-subunit disulfide bridges, it has 10 conserved cysteine residues. One or more of these Cys residues have been found to be present at the effector sites which are essential for

11 . l' 29 a ostenc regu atlOn. Cloning and characterization of SPS gene-Inspite

of the slow progress of purification and characteri­zation of SPS enzyme, cloning and characterization of the SPS gene (either partial or full length cDNA or genomic clone) have gained momentum during the last four or five years either by us ing conserved SPS sequence as primer for selective amplification of total cDNA pool and screening of the cDNA library with the help of this partial SPS sequence30

,31 or by antibody screening of cDNA library4. Cloning of the SPS gene has been achieved from the plant species:

. 4 . hI 0 32 . 33 34 b 35 36 maize ; splnac ' ; nce ' ; sugar eet ; sugarcane ; . 31 V" fi b 37 A . 'd ' d I" 38 d CitruS, lew a a; cllnl w e IClOsa an

Craterostigma plantagineum3o. In all these plant

species (except citrus, sugarcane and C. planta­gineum) only one type of expressed SPS gene has been reported. Plant species differ with regard to the presence of gene copy number of SPS . Although, SPS is present as either low copy or single copy gene in maize, spinach, sugarbeet and V faba , in sugarcane it . d d b I ' I 410 3) 35·17 C . IS enco e y mu tip e genes ' . -, ' . ompansons among the published SPS DNA sequences reveal a high degree of homology which indicates that the enzyme is conserved evolutionarilly. Deduced amino acid sequences show that this enzyme has a well conserved region comprising its first 700 amino acids and a variable C-terminal part. However, SPS from monocots differs from to that of dicots: a glycine rich region in the N-terminal portion is one of the

PA IT ANA Y AK: HIGHER PLANT SUCROSE-PHOSPHATE SYNTHASE 525

distinctive features of the former SPS polypeptide and . b . hi' 4 33 34 IS a sent In t e atter species' . .

SPS expression in the leaves is in part regulated at the mRNA level by transcriptional activation or mRNA stability specially during leaf development, i.e., transition from sink to source status32. However, diurnal variation of SPS activity in leaves is not regulated by gene expression but by post-translational (covalent) modification of the existing protein molecule26. Glucose has been found to activate SPS gene expression' in sugarbeet leaves. Incubation of detached leaves of sugarbeet in glucose-containing media leads to an accumulation of the SPS transcript, while sucrose feeding reduces the steady-state level of the mRNA35 . Organ specific expression of SPS tran~cript has been reported from plant species: SPS transcripts were detected mainly in leaves of spinach, maize and rice 4,32-34 and tap roots of sugarbeee5,

Cloning of the genomic DNA of SPS and its characterization have, so far, been accomplished in rice33.34 . With the help of RFLP mapping rice SPS gene has been mapped on chromosome I (ref. 33). Rice SPS gene consists of 12 exons and II introns. Both nucleotide and deduced amino acid sequc;nces of rice leaf SPS have high homology with that of monocot SPS but relatively low similarity with that of dicot SPS. An 'additional sequence of 48 bp encoding 15 glycine residues has been found in exon I. This sequence ,is composed of GC-rich repeats typical of monocot SPS gene, as already mentioned. No consensus promoter sequences are found to be present at the proximal upstream of the putative initiation codon. A GC-rich sequence was found approximately 750 bp upstream from the putative initiation codon which harbors a G-box like sequence, CACGTG, suggested to be involved in the light response39 and a GAT A motif in the light response element. Several

,promoter like sequences, TAT A and CAA T, were also found around this sequence. Expression of rice SPS gene was found to be low because of the weakness of the promoter33

.34 .

Light regulation of SPS--Considering the important role SPS plays in carbon metabolism, the regulation of SPS activity has appropriately received considerable attention. The enzyme activity is regulated by a complex interplay of different factors and also show~ considerable interspecific variations. SPS activity is found to vary diurnally in many plant species in response to light-dark transitions40-43. Ligbt activates SPS activity and light modulation of the

enzyme activity involves covalent modification of the enzyme molecule by phosphorylation-dephosphoryla­tion26.44 and allosteric regulation by glucose-6-phosphate (activator) and Pi (inhibitort5.46. These two mechanisms are often referred to as 'coarse' and 'fine' control, respectively.

The mechanism underlying covalent modification of SPS is protein phosphorylation and, dephos­phorylation. In the dark, SPS is phosphorylated/ inactivated by a specific protein kinase. Upon illumination, phospho-SPS is dephosphorylated/ activated by type 2A protein phosphatase (SPS-PP) which is sensitive to the specific inhibitor okadaic acid47.50 (Fig. I). Covalent modulation of SPS in spinach leaves alters the enzyme's affinity for both the substrates and allosteric effectors but has no effect on maximal catalytic activity. Kinetically two distinct forms of SPS, corresponding to the phospho and dephospho-SPS have been demonstrated to be present in spinach leaves. Dephospho-SPS has higher affinity for both substrates and activator, glucose-6-phosphate but lower affinity for inhibitor, Pi and phospho-SPS has lower affinity for both substrates and activator but higher affinity for inhibitor. Covalent modification of SPS has been found to induce a two fold change in affinities for substrates but about four fold change in affinities for allosteric effectors26.45 . Thus the effect of covalent modification of SPS is primarily reflected at the allosteric site where glucose-6-phosphate and Pi have antagonistic effects.

SPS is phosphorylated in vivo when leaves are fed with [32p] Pi and in vitro by using [y 32p] ATP and protein kinase with the partially purified SPS51. Two dimensional peptide maps of tryptic digested 32p_SPS_ phosphopeptide revealed that the enzyme is phosphorylated at multiple seryl residues both in vivo and in vitr051

•52

• The major phosphorylation site which determines the activation state of SPS in situ is found to be Ser-158 (in the sequence of spinach SPS). Phosphorylation of Ser-158 alone inactivates SPS in vitro. Phosphorylation of the other seryl residues are not associated with the modulation of the enzyme activity and appear to be phosphorylated consti­tutively (Fig. I). Phosphorylation of Ser-158 has different turnover rates than the nonregulatory phosphorylation sites. It is rapidly phosphorylated in vitro than the other nonregulatory phosphorylation sites. Using partially purified maize SPS, the apparent Km for" MgA TP for SPS kinase was estimated to be

526 !NDIAN] EXP BIOL, JUNE 1999

Dark

~er-p

~

15«( ' .. Ser-~58 · :'-162

PI

1 C>

SPS-PP

Light

Pi

hloropla.t

. . .' Calvin '.

" .. cycle .. '

.. Triose-P +--~ ..... - Trlose-P

Pi -.-""""""""

"41

~

F-6-P +

UDP-Glu Pi

~ Sucrose

Less active Active ~ Sucrose-P

+ UDP

<::::) .. G-6-P

SPSK

ADP ATP

Fig. I-A model for the light regulation of higher plant sucrose-phosphate synthase (SPS). SPS is shown with expanded regulatory phophorylation site (Ser-158 and conserved amino acids surrounding it) corresponding to the sequence of spinach leaf SPS. F-6-P, fructose-6-phosphate; G-6-P, glucose-6-phosphate; UDP-Glu, UDP-glucose; SPSK, SPS kinase; SPS-PP, SPS protein phosphatase: SPP, sucrose-phosphate phosphatase.

approximately 10 mM. Phosphorylation of maize leaf SPS appears to induce a conformational change in the protein molecule through either electrostatic or hydrophobic interactions. High ionic strength in the assay mixture . reversed this conformational change and activated the phospho-SPS48.

SPS protein sequences: analyzed so far, revealed that SPS from di fferent plant sources contains a homologous seryl res idue corresponding to Ser-158 in spinach: Ser-150 in potato, Ser-145 in sugarbeet and Ser-162 in maize and rice4,26,33-35 . Several amino acid residues have been found to be present surrounding the regulatory phosphorylation site, Ser-158. Three basic residues at three, six and eight amino acids upstream and a hydrophobic residue at five amino acids upstream and four amino acids downstream, respectively, relative to the regulatory seryl residue have been found to play an important role for the

. . f SPS . k' 26 33 34 5253 T recogl1ltJOn 0 protell1 lI1ase ' . . , -. wo

forms of SPS protein kinase with apparent molecular masses of 45 and 150 kD have been purified from spinach leaves with the help of a synthetic peptide based on the phosphorylation site sequence of spinach SPS 53 . These two kinases differ in the requirement of divalent cation, .Ca2. , for the catalytic activity. The smaller kinase is strictly Ca2

+ dependent, whereas, the larger kinase is Ca2

+ independent53 . In potato, indirect evidence suggests the presence of two forms of SPS protein kinase24 . However, only a single form of SPS protein kinase (strictly Ca2

+ dependent) is present in maize leaves 26. SPS protein kinases have requirement for peptide substrates with basic residues at three and six. amino acid residues upstream and a hydrophobic residue five amino acid residues downstream, respectively, from the phosphorylation site26

,53 . Glucose-6-phosphate inhibits the activity of SPS protein kinase54 (Fig. I).

Although, regulation of SPS activity by phos-

PA IT ANA Y AK: HIGHER PLANT .sUCROSE-PHOSPHATE SYNTHASE 527

phorylation at the regulatory seryl residue plays a major role, recent evidences suggest that phosphorylation at other sites may be required to maintain the catalytically active conformation of the enzyme molecule55 .56. Inactivation of activated SPS was correlated with loss of 32p from the enzyme and both the inactivation and loss of 32p were inhibited by KF, a nonspecific inhibitor of protein phosphatase55 . The A TP mediated activation of SPS from bundle sheath cells of C4 plants was found to be due to an increase in the affinity for the substrate, UDP­glucose56. Phosphorylation of Ser-424 of spinach leaf SPS by a Ca2

+ dependent protein kinase (different from that .of inactivating protein kinase which phosphorylates Ser-158) was found to activate phosphoserine (Ser-158) SPS during osmotic stress . Although, Ser-424 has same type of conserved amino acid residues around it as Ser-158, this residue is not recognized by the inactivating protein kinase that phosphorylates Ser-15857 . So, the multiple phos­phorylation sites of SPS may be responsible for differential regulation of the enzyme molecule at different situations.

It has already been mentioned that light activation of SPS involves dephosphorylation of phospho-SPS by a type 2A protein phosphatase (SPS-PP) which is inhibited by p/O.54.58-60 (Fig. 1). The coarse control of light activation of SPS involves an apparent light activation of SPS-PP. Total extractable SPS kinase activity did not vary diurnally whereas pronounced light activation of SPS-PP activity was observed. SPS dephosphorylation/activation in light was prevented and reversed independently by inhibitors of trans­cription (cordycepin), cytoplasmic protein synthesis (cyloheximide) and type I and 2A phosphatase (okadaic acid). These results suggested that SPS-PP that dephosphorylates/activates SPS in light is itself activated by light by a process that inv.olves protein synthesisI6.54.59-61. Recent evidences suggest that diurnal and circadian rhythms of SPS are the result of protein phosphorylation state and not because of deactivation of SPS. Using inhibitors directed against protein kinases, phosphatases and transcription and translation it has been clearly shown that de novo expression of SPS-PP is under the control of circadian rhythm which is in turn responsible for the circadian fluctuation of SPS activity62.

The allosteric regulation (fine cOf1trol) of SPS by tissue metabolites, glucose-6-phosphate and Pi, provides a way of linking the activity of SPS to the

rising rate of photosynthesis. Both the activation of SPS and the level of its substrates and effectors have been found to vary with photosynthesis in vivo and . d ' h f h . 63 64 mteract to etermme t e rate 0 sucrose synt eSls . . Sucrose synthesis in the cytosol depends on the export of triose phosphate, generated during carbon assimilation of photosynthesis in chloroplast, to cytosol. Export of triose phosphate to cytoso l is coupled to an influx of Pi, generated in the cytosol, to chloroplast (Fig. 1 ). Triose phosphates in the cytosol are converted to hexose phosphate pool to be utilized for sucrose synthesis during day time 12

. It is likely that increase in the leaf concentration of phosphate esters, such as glucose-6-phosphate, would corres­pond to decrease in the Pi concentration to maintain a constant phosphate pool in. the cytosol65. The cyto­plasmic Pi cORcentration in leaves in the dark has been estimated as 20-30 rnM6 and measurements of phosphorylated intermediates provided indirect evidences that Pi concentration could decline by as much as 15 ruM during photosynthesis63.64 . It has already been fnentioned that glucose-6-phosphate increases the affinity of SPS for both the substrates and thereby increases the activation state of the enzyme but Pi decreases the activation state by lowering the enzyme's affinity for the substrates. Moreover, SPS kinase is inhibited by glucose-6-phosphate and SPS-PP is inhibited by p?6.45,46 (Fig. I). So both these tissue metabolites are allosteric effectors of SPS itself and also the effectors of the interconverting enzymes. Therefore, a small increase in glucose-6-phosphate coupled with a small decrease in Pi will have a much larger effect on the activity of ~PS in situ. It has been proposed that changes in the availability of Pi play a major role in the signal amplification of SPS activation24.42.43. In fact, it has been observed that leaf Pi concentration follows an exactly opposite pattern to that of diurnal variation of potato leaf SPS24.

Quantitative differences have been found to exist with respect to light modulation of SPS. Plant species have been classified into three groups based on the occurrence and characteristics of light activation of SPS. In group I species (e.g. mainly monocots such as maize) SPS is allosterically regulated and covalent modification of the enzyme affects its V max and sensitivity to effectors23.43. SPS from group II species (e.g. spinach, potato) is also allosterically regulated but covalent modification changes the enzyme's affinity for both substrates (Km) and effectors but not

528 INDIAN J EXP BIOL, JUNE 1999

Ymax24,43. In group III species (e,g, soyabean) SPS is

weakly regulated by effectors and the enzyme is not modulated by covalent modification and does not h I· h . , 1743

S ow Ig t activatIOn ' . Cloning and characterization of SPS gene from

different plant species and heterologous expression of SPS gene in transgenic plants have opened a new horizon of plant biology that would enable us to get new information about metabolism and com part­mentation of sugars in leaves, and to improve crops, SPS overexpression holds promise for achieving the goal of if!creased plant yield potential through altered

photoassimilate partitioning8. In the fi ·e ld of trans­

genic biology, maize SPS has been overexpressed in tomato and Arabidopsis, and spinach SPS has been overexpressed in tobacc067

. However, among the three transgenic plant species only tomato showed positive correlation between overexpression of SPS and increased photosynthetic rate, biomass accumulation and fruit development68. The cause of no effect of overexpression of SPS in tobacco and Arabidopsis, both phenotypically and metabolically, is down regulation of SPS protein by phosphorylation. So, there is a need to generate transgenic plants with genetically modified SPS protein molecule specially by changing the phosphorylation site and inhibitor binding site, With the present pace of research, achievement of this goal seems imminent.

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