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Running Title: Plant ASPPs and starch biosynthesis

Subject area: Proteins, enzymes and metabolism

Corresponding authors: Javier Pozueta-Romero, Francisco José Muñoz

Instituto de Agrobiotecnología, Universidad Pública de Navarra, Gobierno de Navarra

and Consejo Superior de Investigaciones Científicas, Ctra. de Mutilva s/n, 31192

Mutilva Baja, Navarra, Spain

E-mail: javier.pozueta@unavarra.es

Tel: (34) 948168030, FAX: (34) 948232191

Number of figures: 4

Number of tables: 4

Supplemental data: 1 Table, 6 figures and legends to figures

Plant and Cell Physiology 2006 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.

Plant and Cell Physiology Advance Access published June 13, 2006

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Title: Cloning, expression and characterization of a Nudix hydrolase that catalyzes the

hydrolytic breakdown of ADP-glucose linked to starch biosynthesis in Arabidopsis

thaliana

Authors: Francisco José Muñoz, Edurne Baroja-Fernández, María Teresa Morán-

Zorzano, Nora Alonso-Casajús, Javier Pozueta-Romero

Address: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo

Superior de Investigaciones Científicas/Gobierno de Navarra, Ctra. Mutilva s/n, 31192

Mutilva Baja, Navarra, Spain.

Abbreviations: ASPP, adenosine diphosphate sugar pyrophosphatase; AtASPP,

Arabidopsis thaliana ASPP; FW, fresh weight; StASPP, Solanum tuberosum ASPP, U,

unit of enzyme activity, WT, wild type.

Footnotes: The nucleotide sequence of AtASPP and StASPP encoding cDNAs have

been submitted to EMBL database under accession number AJ748742 and AM180509,

respectively. F.J.M and E.B.F. have equally contributed to this work. Corresponding

authors: Javier Pozueta-Romero, E-mail, javier.pozueta@unavarra.es; Fax, (34)

948232191; Francisco José Muñoz, E-mail, francisco.munoz@unavarra.es; Fax, (34)

948232191

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ABSTRACT �Nudix� hydrolases are widely distributed nucleotide pyrophosphatases that possess a

conserved GX5EX7REUXEEXGU motif where U is usually Ile, Leu or Val. Among

them, Escherichia coli ADP-sugar pyrophosphatase (ASPP) has been shown to catalyze

the hydrolytic breakdown of ADP-glucose linked to bacterial glycogen biosynthesis

(Moreno-Bruna et al. 2001). Comparisons of the 31 different Nudix encoding

sequences of the Arabidopsis genome with those coding for known bacterial and

mammalian ASPPs identified one sequence possessing important divergences in the

Nudix motif that, once expressed in E. coli, produced a protein with ASPP activity.

This protein, designated as AtASPP, shares strong homology with hypothetical rice and

potato proteins, indicating that ASPPs are widely distributed in both mono- and di-

cotyledonous plants. As a first step to test the possible involvement of plant ASPPs in

regulating the intracellular levels of ADP-glucose linked to starch biosynthesis we

produced and characterized AtASPP-overexpressing Arabidopsis plants. Source leaves

from these plants exhibited a large reduction of the levels of both ADP-glucose and

starch indicating that plant ASPPs catalyze the hydrolytic breakdown of a sizable pool

of ADP-glucose linked to starch biosynthesis. No pleiotropic changes in maximum

catalytic activities of enzymes closely linked to starch metabolism could be detected in

AtASPP-overexpressing leaves. The overall informations provide a first evidence for

the occurrence of plant Nudix hydrolases that have access to an intracellular pool of

ADP-glucose linked to starch biosynthesis.

Keywords: Arabidopsis thaliana, Carbohydrate metabolism, Nudix hydrolase,

Solanum tuberosum, Starch

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INTRODUCTION

The Nudix hydrolases constitute a family of metal-requiring phosphoanhydrases that

catalyze the hydrolytic breakdown of Nucleoside diphosphates linked to some other

moiety such as a phosphate, sugar, or nucleoside (Bessman et al. 1996). They are

characterized by the following conserved array of 23 amino acids,

GX5EX7REUXEEXGU, where U represents a bulky, hydrophobic amino acid (usually

Ile, Leu, or Val). Nudix hydrolases are widely distributed among organisms ranging

from viruses to mammals and have been suggested to act as �housecleaning� enzymes

that prevent accumulation of reactive nucleoside diphosphate derivatives, cell

signalling molecules or metabolic intermediates by diverting them to metabolic

pathways in response to biochemical and physiological needs (Bessman et al. 1996,

Jambunhathan and Mahalingam 2005). Compared to mammalian cells and bacteria,

little is known about the functions of Nudix hydrolases in plants.

During the course of our investigations on bacterial glycogen metabolism we

identified a bacterial Nudix hydrolase, designated as adenosine diphosphate sugar

pyrophosphatase (ASPP), that cleaves ADP-sugars such as ADP-ribose, ADP-mannose

and the precursor molecule for glycogen biosynthesis, the ADP-glucose (Moreno-

Bruna et al. 2001). Changes on ASPP activity were accompanied by changes in

bacterial glycogen accumulation, strongly indicating that ASPP controls intracellular

levels of ADP-glucose linked to the glycogen biosynthetic process in Escherichia coli.

Rodríguez-López et al. (2000) reported the occurrence in plants of a widely

distributed ADP-glucose hydrolytic activity whose pattern showed inverse correlation

with respect to starch accumulation. This activity is catalysed by protein entities

displaying different properties and subcellular localizations (Rodríguez-López et al.

2000, Baroja-Fernández et al. 2000). As a first step to test whether a part of this activity

is catalysed by a Nudix hydrolase, we have identified, cloned and expressed in E. coli

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sequences of plant genomes that code for proteins sharing homology with ASPPs

occurring in both prokaryotic and mammalian cells. In addition, we have produced and

characterized ASPP-overexpressing transgenic plants. We discuss the possible

involvement of plant ASPPs in controlling the intracellular levels of ADP-glucose

linked to starch biosynthesis and in connecting starch metabolism with other metabolic

pathways in response to biochemical needs.

RESULTS AND DISCUSSION

Identification of putative ASPP encoding Nudix genes from Arabidopsis

Searches in the InterPro database (http://www.ebi.ac.uk/interpro) revealed the existence

of at least 31 different sequences that code for Nudix hydrolases in the Arabidopsis

genome. As shown in Supplemental Table 1, most of these sequences code for

proteins of still unknown functions. To identify which sequence(s) code for proteins

with ASPP activity, we compared them with ASPP encoding Nudix genes from

bacterial and mammalian species (Dunn et al. 1999, Gasmi et al. 1999, Yang et al.

2000, Moreno-Bruna et al. 2001). This analysis allowed us to identify four sequences

(At2g42070, At4g11980, At1g68760 and At5g20070) sharing significant homology

with the aforementioned ASPP encoding genes (Supplemental Fig. 1).

At4g11980 codes for an ADP-glucose hydrolase

Complete At4g11980, At1g68760 and At5g20070 cDNAs available in the RIKEN

Arabidopsis collection were expressed in E. coli as described in �Materials and

Methods�. Purification and SDS-PAGE of the recombinant proteins resulted in single

bands that were not detectable in extracts from control bacterial cells (not shown). The

sizes of the recombinant proteins purified from bacterial cells transformed with pET-

At4g11980, pET-At1g68760 and pET-At5g20070 were ca. 38, 20 and 52 kDa,

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respectively, which roughly correspond to the molecular mass predicted from their

amino acid sequences.

Recombinant proteins encoded by At4g11980, At1g68760 and At5g20070 were

tested for their hydrolytic activity against ADP-glucose in the presence of 5 mM Mg2+.

These analyses revealed that only the At4g11980 product, also designated as

AtNUDT14 (Ogawa et al. 2005), was capable of hydrolyzing ADP-glucose.

Substrate specificity of the recombinant protein encoded by At4g11980

Substrate specificity of the purified At4g11980 encoding recombinant protein was

tested using a wide range of compounds at a concentration of 2 mM. The protein,

designated as AtASPP, recognized ADP-sugars such as ADP-glucose, ADP-mannose

and ADP-ribose, and poorly hydrolysed other nucleotide-sugars such as UDP-glucose,

CDP-glucose, GDP-glucose and UDP-galactose (Table 1). AtASPP does not recognize

PPi, synthetic phosphodiester-bond-containing compounds such as bis-p-nitrophenyl

phosphate, diadenosine polyphosphates, CoA or phosphomonoester-bond-containing

compounds such as p-nitrophenyl phosphate, sugar-phosphates and nucleotide mono-,

di- and triphosphates.

Properties of AtASPP

The catalytic properties of AtASPP were studied on ADP-glucose, ADP-ribose and

ADP-mannose. ADP-ribose and ADP-mannose concentration curves followed a typical

Michaelis-Menten pattern (not shown) whereas, similar to other enzymes displaying

atypical kinetic behavior (Yu et al. 1988), ADP-glucose kinetics displayed a non-

saturable pattern (Supplemental Fig. 2). Km and Vmax values for ADP-ribose were 42.8

µM and 0.71 U/mg protein, respectively, whereas Km and Vmax values for ADP-

mannose were 130 µM and 0.32 U/mg protein, respectively.

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When subjected to gel filtration chromatography, AtASPP appeared as a

symmetrical peak in a region expected for a ca. 70-75 kDa protein. Taking into account

that AtASPP migrates as a ca. 38 kDa protein in SDS-PAGE, it is quite possible that

the native enzyme occurs as a homodimer. In this respect, we must emphasize that a

common feature of other ASPPs is the formation of homodimers (Gasmi et al. 1999,

Yang et al. 2000, Moreno-Bruna et al. 2001) that are required for substrate recognition

and catalytic activity (Gabelli et al. 2001).

ASPPs are widely distributed in mono- and di-cotyledonous plants

Computer searches of data banks showed that AtASPP shares high sequence similarity

with Q9SNS9 and POADP80, two �hypothetical� proteins from rice and potato,

respectively (Supplemental Fig. 3). A POADP80 cDNA was isolated and expressed in

E. coli to produce a recombinant protein designated as StASPP. Analyses of glycogen

content in these cells revealed that StASPP expression leads to a glycogen-less

phenotype (Fig. 1) thus providing a first indication that StASPP catalyses the hydrolytic

breakdown of ADP-glucose. In agreement with this presumption, substrate specificity

analyses revealed that StASPP cleaves ADP-glucose and displays both substrate

recognition pattern and a kinetic behavior with respect to ADP-glucose similar to

AtASPP (Table 1, Supplemental Fig. 2). The overall information thus indicates that

Nudix hydrolases with ASPP activity are widely distributed in both mono- and di-

cotyledonous species.

Structural divergences of the Nudix signature sequence of plant ASPPs

Nudix hydrolases comprise a large family of proteins that are defined by the

GX5EX7REUXEEXGU motif, where U is usually Ile, Leu or Val. Although this highly

conserved Nudix motif has been shown to be essential for the metal binding and

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pyrophosphatase activity (Gabelli et al. 2001), there are numerous examples of non-

consensus motifs among the Nudix hydrolases, including extra amino acids and missing

Glu or Gly residues (O´Handley et al. 1998, Yagi et al. 2003). As presented in Fig. 2,

AtASPP, StASPP and rice Q9SNS9 contain important modifications in the Nudix

motif, including an extra amino acid, and the substitution of the conserved Glu at

position 7 by Lys or Gln. These differences are not responsible for the specific

recognition of ADP-sugars, since they are not present in bacterial and mammalian

ASPPs, and strengthen the view that the specificity for individual substrates lies outside

the Nudix motif (Gabelli et al. 2001).

AtASPP-overexpression leads to a large reduction of both ADP-glucose and

transitory starch levels in leaves

Arabidopsis plants were transformed with 35S-AtASPP-NOS via Agrobacterium-

mediated gene transfer. We then compared ADP-glucose hydrolytic activities in leaves

of AtASPP-overexpressing plants with those of wild type (WT) plants. As illustrated in

Fig. 3, ADP-glucose hydrolytic activities in each of the AtASPP-overexpressing lines

were several fold higher than those occurring in the control plants. At no stage during

development could we detect any phenotypic difference between the 35S-AtASPP-

NOS and the control plants (not shown). No significant differences were observed in

protein and chlorophyll contents, dry weight, plant height, flowering time, and leaf

number or size between 35S-AtASPP-NOS and control plants (not shown).

Leaves from control and AtASPP-overexpressing plants were then characterized

for their ADP-glucose and starch contents after 7 h of illumination. As it is our

experience that biochemical analyses are subject to considerable variation, we analysed

10 plants per line to obtain reliable data. As illustrated in Fig. 4A, leaves from 35S-

AtASPP-NOS plants showed a ca. 50% reduction of ADP-glucose content. Most

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significantly, AtASPP-overexpression leads to ca. 60-70% reduction of the starch

content (Fig. 4B). The overall data thus indicate that AtASPP has access to an

intracellular pool of ADP-glucose linked to starch biosynthesis. The fact that

irrespective of the ADP-glucose hydrolytic activity ADP-glucose content is nearly the

same in every line tested is consistent with a previoius report pointing to the occurrence

of different subcellular localizations of ADP-glucose (Baroja-Fernández et al. 2004).

Measurement of key enzymes of sucrose and starch metabolism

Starch-deficient Arabidopsis mutants exhibit large changes in activities of enzymes

involved in sucrose and starch metabolism (Caspar et al. 1985, Lin et al. 1988), likely

reflecting a regulated response to the absence of metabolic flux towards starch. To

identify possible pleiotropic effects of AtASPP-overexpression we measured the

maximum catalytic activities of enzymes closely connected to starch and sucrose

metabolism in leaves from both WT and 35S-AtASPP-NOS plants. As shown in Table

2, these analyses revealed no significant changes in ADP-glucose pyrophosphorylase,

alkaline pyrophosphatase, UDP-glucose pyrophosphorylase, sucrose synthase,

phosphoglucomutase, hexokinase, acid invertase and total amylolytic activity. By

contrast, some but not all AtASPP-overexpressing lines displayed a significant

reduction of total starch synthase.

Levels of soluble sugars and sugar phosphates

One of the distinguishing characteristics of some starch deficient Arabidopsis mutants

is that, because they are unable to store net photosynthate in starch, they accumulate

relatively large quantities of sucrose and hexose in leaves (Caspar et al. 1985, Jones et

al. 1986, Lin et al. 1988, Neuhaus and Stitt 1990). To know whether AtASPP-

overexpression leads to increasing levels of soluble sugars, we measured the

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intracellular content of sucrose, glucose and fructose. In addition, we measured the

levels of sugar-phosphates. As shown in Table 3, leaves from both WT and 35S-

AtASPP-NOS plants accumulated nearly identical amounts of total soluble sugars.

Significantly however, AtASPP-overexpression leads to both a significant decrease of

sucrose and increase of fructose, whereas glucose remained unaltered. Measurements of

glucose-1-phosphate and glucose-6-phosphate revealed a trend-wise increase in the

levels of glucose-1-phosphate and a decrease in the levels of glucose-6-phosphate.

Additional remarks on possible functions of plant ASPPs

This is the first report describing the cloning, expression in plants and characterization

of genes coding for plant ADP-glucose cleaving enzymes. Results presented in this

work showing that enhancement of plant ASPP activity leads to a concomitant

reduction of ADP-glucose and starch levels (Fig. 4) provide evidence that plant ASPPs

have access to an intracellular pool of ADP-glucose that is linked to starch

biosynthesis. This is not exclusive of Arabidopsis since plant ASPP-overexpressing

potato leaves accumulate low levels of both ADP-glucose and starch when compared to

WT leaves (Supplemental Fig. 4). This and the fact that maximum ASPP catalytic

activities are similar to those of enzymes responsible for ADP-glucose synthesis and

utilization (Table 2) strongly suggest that, essentially similar to the suggested role of

bacterial ASPP (Moreno-Bruna et al. 2001), plant ASPPs may play a role in both

regulating intracellular levels of ADP-glucose and in connecting the starch biosynthetic

process with other metabolic pathways in response to biochemical and physiological

needs.

Whether plant ASPPs exert a strong control on the starch biosynthetic process

will require studies on regulation of both gene expression and enzyme activity,

metabolic flux analyses in both ASPP-overexpressing and -deficient mutants as well as

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subcellular localization studies of plant ASPPs. In this last respect, using the TargetP

prediction program (http://www.cbs.tu.dk/services/TargetP) Ogawa et al. (2005) have

classified the At4g1980 product (AtASPP) as a mitochondrial protein. In clear contrast

however, ChloroP (http://www.cbs.dtu.dk) predicts that both StASPP and AtASPP have

a plastidial localization, whereas both TargetP and Psort (http://psort.nibb.ac.jp) predict

that StASPP has a cytosolic localization.

Predictions of protein localization based on in silico analyses are questionable

and need experimental verification (Soltys and Gupta 1999, Koroleva et al. 2005,

Villarejo et al. 2005). As a first step to investigate whether AtASPP is a mitochondrial

protein we have performed subcellular fractionation studies. Employing the

centrifugation method of Leaver et al. (1983), mitochondrial preparations were

obtained from leaves of 35S-AtASPP-NOS plants. As shown in Table 4, comparisons

of enzyme activities in fractions obtained at the end of the preparation with those in the

initial lysate as well as in the centrifugation step guaranteed no loss of activity during

the preparation for any of the enzymes analysed. Judging by the activities of fumarase,

a mitochondrial matrix space marker (Nishimura et al. 1982, Pádua et al. 1996), ca. 70

% of the mitochondria originally present in the homogenates of 35S-AtASPP-NOS

were recovered in the final mitochondrial preparations. By contrast, the activities of

ASPP and the contaminating cytosolic marker sucrose phosphate synthase in the

mitochondrial preparations were found to be respectively ca. 15% and 8 % of those

occurring in the initial homogenates. The data thus strongly indicate that in contrast to

the predictions of Ogawa et al. (2005), AtASPP is not a mitochondrial protein.

Plant ASPPs recognize both ADP-glucose and ADP-ribose (Table 1). To know

whether intracellular ADP-ribose levels can interfere with the ADP-glucose cleaving

reaction catalysed by ASPP in the cell we analysed the ADP-ribose content in different

plant organs. Despite the fact this nucleotide-sugar can be artifactually formed from

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both NAD and NADH during the process of nucleotide extraction (Jacobson et al.

1997) we failed to detect ADP-ribose (Supplemental Fig. 5), indicating that the

intracellular levels of this nucleotide-sugar must be very low in the plant cell. Taking

into account that ADP-glucose is one of the most abundant nucleotide-sugars in starch

storing organs (Feingold and Avigad 1980, Baroja-Fernández et al. 2003), it is highly

conceivable that ADP-ribose does not significantly prevent the ADP-glucose cleaving

reaction catalysed by plant ASPPs in the cell.

2. MATERIALS AND METHODS

Plants, bacterial strains and culture medium

The work was carried out using WT Arabidopsis (ecotype Columbia) plants and plants

transformed with 35S-AtASPP-NOS (see below). Plants were grown in pots at ambient

CO2 (350 p.p.m.) in growth chambers under a 16 h light (300 µmol photons sec�1 m-2,

20 ºC) / 8 h dark (22 ºC) regime. For biochemical analyses, fully expanded source

leaves were harvested after 7 h of illumination, immediately quenched in liquid

nitrogen, and stored at �80 oC for up to 2 months before use.

All plasmid constructs were electroporated and propagated in E. coli XL1Blue.

E. coli BL21(DE3) cells transformed with either pET-AtASPP or pET-StASPP (see

below) were grown in LB medium at 37oC. Agrobacterium tumefaciens cells, strain

EHA105, transformed with pBIN35S-AtASPP-NOS were grown in LB medium at

28oC. In every case, the bacterial cells were grown with rapid gyratory shaking.

Gene cloning and expression in E. coli cells

Complete cDNAs corresponding to three Nudix encoding Arabidopsis genes

(At1g68760, At4g11980 and At5g20070) were obtained from the RIKEN Arabidopsis

cDNA collection (Seki et al. 1998, 2002) and amplified by PCR using specific primers.

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The amplified products were cloned in the pGEM-T Easy vector (Promega, Madison,

WI, USA). The NheI-XhoI fragments were then extracted and cloned into the

corresponding restriction sites of the pET-28c(+) expression vector (Novagen, Madison,

WI, USA). The resulting plasmids were designated as pET-At1g68760, pET-

At4g11980, pET-At5g20070.

A StASPP encoding cDNA was obtained from 1 µg of total RNA which was

isolated from potato leaves using a ULTRASPEC RNA Isolation System (BIOTECX

Company, Houston, TX, USA). After DNase I treatment, poli-A+ transcripts were

reversed-transcribed using the Expand Reverse Transcriptase system (Roche

Diagnostics, Mannheim, Germany) and (dT)18 as a primer. The first- cDNA strand was

amplified by PCR using the primers 5�-

CAAGTGCGGCTAGCATGAGACTAACAGTGTCGCGTTG-3� (forward) and 5�-

CAACTGCTCGAGTCAAGGCAACAGTCCATCTCTTTTAG-3� (reverse). The

amplified product was cloned in the pET-28c(+) expression vector to produce pET-

StASPP.

BL21(DE3) cells transformed with either pET-At1g68760, pET-At4g11980,

pET-At5g20070 or pET-StASPP were grown at 37°C in 100 ml of LB medium

supplemented with 50 µg/ml kanamycin to an attenuance at 600 nm of 0.6, and then 1

mM isopropil-D-thiogalactopyranoside was added to the culture medium. After 5

hours, 100 ml of cultured cells were centrifuged at 6,000 g for 10 min. The pelleted

bacteria were resuspended in 6 ml of His-bind binding buffer (Novagen, Madison, WI,

USA), sonicated and centrifuged at 10,000 g for 10 min. The supernatant thus obtained

was subjected to His-bind chromatography (Novagen, Madison, WI, USA). The eluted

hexahistidine-tagged recombinant proteins were then rapidely desalted by ultrafiltration

on Centricon YM-10 (Amicon, Bedford, MA, USA).

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Production of AtASPP-overexpressing transgenic plants of Arabidopsis

For the production of 35S-AtASPP-NOS plants, the NcoI�XhoI fragment of pET-

At4g11980 was digested successively with XhoI, T4 DNA polymerase and NcoI. The

fragment thus released was ligated into NcoI-SmaI restriction sites of p35S-NOS

(Baroja-Fernández et al. 2004) to produce p35S-AtASPP-NOS (Supplemental Fig. 6).

This construct was digested with HindIII-EcoRI and the fragment thus released was

cloned into the pBIN20 plant expression vector (Hennegan and Danna 1998) previously

digested successively with the enzymes HindIII-EcoRI to produce pBIN35S-AtASPP-

NOS. Transfer of this construct into Agrobacterium tumefaciens was carried out by

electroporation. Subsequent transformation of Arabidopsis plants was conducted as

described by Clough and Bent (1998). Transgenic plants were selected on kanamycin-

containing medium.

Enzyme Assays

All enzymatic reactions were carried out at 37 oC. Harvested leaves were immediately

freeze-clamped and ground to a fine powder in liquid nitrogen with a pestle and mortar.

To assay enzyme activity, 1 g of the frozen powder was resuspended at 4 oC in 5 ml of

100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol. The suspension was

desalted and assayed for enzymatic activities. We checked that this procedure did not

result in loss of enzymatic activity as evidenced by comparing activity in extracts

prepared from the frozen powder and extracts prepared by homogenizing fresh tissue in

extraction medium. Measurements of nucleotide hydrolytic activities were performed

essentially as described elsewhere (Moreno-Bruna et al. 2001, Baroja-Fernández et al.

2004). ADP-glucose pyrophosphorylase, acid invertase, phosphoglucomutase,

hexokinase, UDP-glucose pyrophosphorylase, alkaline pyrophosphatase, total starch

synthase, total amylolytic activity, and sucrose-phosphate-synthase activities were

15

assayed as described by Baroja-Fernández et al. (2004). Fumarase was measured

essentially as described by Pádua et al. (1996). We define 1 U of enzyme activity as the

amount of enzyme that catalyzes the production of 1 µmol of product per min. Kinetic

parameters were evaluated by Lineweaver-Burk plots.

Determination of soluble sugars

Fully expanded leaves were harvested and immediately ground to a fine powder in

liquid nitrogen with a pestle and mortar. 0.5 g of the frozen powdered tissue was

resuspended in 0.4 ml of 1.4 M HClO4, left at 4 oC for 2 h and centrifuged at 10,000 g

for 5 min. The supernatant was neutralized with K2CO3 and centrifuged at 10,000 g.

ADP-glucose content in the supernatant was determined as described by Muñoz et al.

(2005) by using either one of the following methods:

Assay A: by HPLC on a system obtained from P.E. Waters and Associates fitted with a

Partisil-10-SAX column.

Assay B: by HPLC with pulsed amperometric detection on a DX-500 system (Dionex)

fitted to a CarboPac PA10 column.

To further confirm that measurements of ADP-glucose were correct, ADP-

glucose eluted from either the Partisil-10-SAX or CarboPac PA10 columns were

enzymatically hydrolysed with purified E. coli ASPP and assayed for conversion into

AMP and G1P.

Glucose, sucrose, fructose, glucose-1-phosphate and glucose-6-phosphate were

determined by HPLC with pulsed amperometric detection on a DX-500 system as

described by Baroja-Fernández et al. (2003).

16

Isolation of mitochondria

Mitochondria were prepared essentially as described by Leaver et al. (1983). The final

pellet was resuspended in 50 mM Tris, pH 7.5 / 5 mM MgCl2 / 1 mM EDTA/ 2 mM

DTT / 1% (vol/vol) Triton X-100.

Analytical procedures

Bacterial growth was determined spectrophotometrically by attenuance at 600 nm.

Protein content was determined by the Bradford method using Bio-Rad prepared

reagent (Bio-Rad Laboratories, Hercules, CA, USA). Starch in desalted plant extracts

obtained by precipitation with 70% ethanol was measured by using an

amyloglucosydase�based test kit (Sigma-Aldrich Chemical Co, St. Louis, MO).

Chlorophyll was quantified according to the method of Wintermans and De Mots.

(1965). The native molecular mass of AtASPP was determined by gel filtration on a

Superdex 200 column (Pharmacia LKB) using a Bio-Rad kit of protein standards.

ACKNOWLEDGEMENTS: This research was supported by the grant BIO2004-

01922 from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo

de Desarrollo Regional (Spain) and by the government of Navarra. M.T.M-Z.

acknowledges the Spanish Ministry of Culture and Education for a pre-doctoral

fellowship. We thank Beatriz Zugasti for expert technical support.

17

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LEGENDS FOR FIGURES

Figure 1: ADP-glucose hydrolytic activities and glycogen levels of BL21(DE3) cells

cells transformed with either pET or pET-StASPP. Results are given as mean ± SEM.

Figure 2: Structural divergences of the Nudix signature sequence of plant ASPPs.

Partial sequence alignment of the Nudix signature of ASPP proteins. The conserved

Nudix box is in bold type.

Figure 3: ADP-glucose hydrolytic activity in source leaves of WT and 35S-AtASPP-

NOS plants. Results are given as mean ± SEM of 10 independent plants per line.

Figure 4: Plant ASPPs have access to an intracellular pool of ADP-glucose linked to

starch biosynthesis in Arabidopsis leaves. ADP-glucose and starch levels in source

leaves from 6-week-old WT and AtASPP-overexpressing Arabidopsis plants. Leaf

samples were taken and quenched in liquid nitrogen at 7 h after the beginning of the

light period. Results are given as mean ± SEM of 10 independent plants per line.

24

Table 1: Substrate specificity of AtASPP. The reaction mixture contained 50 mM Hepes,

pH 7/5 mM MgCl2, 2 mM of the indicated compound and recombinant plant ASPP. After

30 min of incubation at 37oC, the reaction was stopped by boiling in a dry bath for 2 min.

The resulting products were then measured as described by Moreno-Bruna et al. (2001)

and Rodríguez-López et al. (2000). Ap3A, adenosine(5´)triphospho(5´)adenosine. Ap4A,

adenosine(5´)tetraphospho(5´)adenosine.

Substrate Substrate specificity (% activity relative to ADP-glucose)

AtASPP StASPP

ADP-glucose 100 100

ADP-ribose 256 152

ADP-mannose 29 not determined

CDP-glucose <1 <1

GDP-glucose <1 <1

UDP-glucose 1.7 7.1

UDP-galactose <1 <1

NAD+ <1 <1

NADP+ <1 <1

AMP <1 <1

NDP <1 <1

NTP <1 <1

Ap3A <1 <1

Ap4A <1 <1

CoA <1 <1

Bis-p-nitrophenyl phosphate <1 <1

p-nitrophenyl phosphate <1 <1

25

Table 2: Enzyme activities (given in mU/g FW) in WT and 35S-AtASPP-NOS Arabidopsis leaves. The activities were determined in samples from source leaves of plants grown in chambers at ambient CO2 conditions, 20 oC and at an irradiance of 300 µmol photons sec-1 m-2. Leaf samples were taken and quenched in liquid nitrogen 7 h after the beginning of the light period. The results are the mean ± SE of extracts from 10 independent plants per line.

WT 35S-AtASPP-NOS

3 5 7 8

ADP-glucose pyrophosphorylase 150 ± 21.1 139 ± 9.1 157 ± 4.8 150 ± 7.2 138 ± 6.9

UDP-glucose

pyrophosphorylase 124 ± 12.4 131 ± 7.8 164 ± 17.7 137 ± 9.4 116 ± 7.8

Acid invertase

634 ± 43 644 ± 21 563 ± 38 589 ± 45 559 ± 98

Phospho-glucomutase 455 ± 45 450 ± 10 532 ± 30 350 ± 55 448 ± 36

Hexokinase

34.2 ± 5.6 35.5 ± 3.9 35.6 ± 3.6 29.2 ± 3.6 28.8 ± 6.6

Sucrose phosphate synthase 178 ± 37 180 ± 38 192 ± 61 180 ± 10 192 ± 39

Alkaline

pyrophosphatase 3,870 ± 444 3,420 ± 540 3,760 ± 660 3,440 ± 220 3,380 ± 615

Amylolytic activity

42.0 ± 3.5 42.1 ± 4.9 41.8 ± 3.8 41.6 ± 5.8 38.2 ± 5.2

Total starch synthase 98.8 ± 7.9 14.4 ± 2.6 43.3 ± 7.9 21.8 ± 3.9 84.6 ± 7.9

26

Table 3: Metabolite levels (given in nmol/g FW) in control (WT) and 35S-AtASPP-NOS source leaves. Source leaf samples were taken from plants grown in chambers at ambient CO2 conditions, 20 oC and at an irradiance of 300 µmol photons sec-1 m-2. Leaves were taken and quenched in liquid nitrogen 7 h after the beginning of the light period. The results are the mean ± SE of extracts from 10 independent plants per line. Values that are significantly different from the control plants are marked in bold type.

Control 35S-AtASPP-NOS

WT 3 5 7 8

Glucose 234 ± 31 183 ± 21 231 ± 23 296 ± 25 295 ± 26

Fructose

2,843 ± 243 4,812 ± 367 3,501 ± 217 3,727 ± 310 3,717 ± 258

Sucrose 1,175 ± 27 602 ± 28 855 ± 36 450 ± 38 876 ± 25

Σ soluble sugars

4,252 ± 431 5,597 ± 798 4,633 ± 453 3,473 ± 399 3,888 ± 435

Glucose-6-phosphate

299 ± 15 253 ± 10 251 ± 21 217 ± 8 207 ± 13

Glucose-1-phosphate 63.9 ± 5.1 87.3 ± 2.1 98.5 ± 7.5 93.4 ± 9.1 107.6 ± 5.5

27

Table 4: Subcellular localization of AtASPP in leaves of 35S-AtASPP-NOS potato plants. Mitochondria were prepared as described by Leaver et al. (1983). Data are given as mean ± SEM of three independent experiments Centrifugation

Lysate Supernatant Mitochondrial preparation

Enzyme Activity Activity Activity

(mU/ g FW) (mU/ g FW) % of lysate (mU/ g FW) % of lysate Recovery (%)

ASPP 342 ± 29 274 ± 25 80.2 ± 5.3 50.0 ± 3.2 14.6 ± 1.5 94.9 ± 7.3

Fumarase 3558 ± 462 1988 ± 439 45.9 ±10.8 2386 ± 423 67.1 ± 4.6 113 ± 14.7

Sucrose phosphate synthase 742 ± 23.3 624 ± 10.5 84.2 ± 2.6 58.6 ± 2.9 7.9 ± 0.2 92.1 ± 6.5