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Therefore we propose that uracil salvage is of major importance for plant development. Keywords: ..... analysed the publicly available CATMA transcriptome data.
The Plant Journal (2009) 60, 280–291

doi: 10.1111/j.1365-313X.2009.03963.x

Uracil salvage is necessary for early Arabidopsis development Samuel E. Mainguet1, Bertrand Gakie`re2, Amel Majira3, Sandra Pelletier1, Franc¸oise Bringel4, Florence Gue´rard5, Michel Caboche1, Richard Berthome´1,* and Jean Pierre Renou1,* 1 URGV, UMR INRA 1165 - CNRS 8114 - UEVE, 2, Rue Gaston Cre´mieux, CP5708, 91057 Evry cedex, France, 2 Institut de Biotechnologie des Plantes, Bat 630, Universite´ Paris Sud XI, 91405 Orsay cedex, France, 3 Laboratoire de Biologie Cellulaire, INRA, Route de Saint-Cyr, 78026 Versailles cedex, France, 4 UMR7156 Universite´ Louis Pasteur/CNRS, Ge´ne´tique Mole´culaire, Ge´nomique, Microbiologie, De´partement Microorganismes, Ge´nomes, Environnement, 28 Rue Goethe, 67083 Strasbourg, France, and 5 Plateforme Me´tabolisme-Me´tabolome, IFR87, Bat 630, Universite´ Paris Sud XI, 91405 Orsay cedex, France Received 22 April 2009; revised 2 June 2009; accepted 8 June 2009; published online 23 July 2009. *For correspondence (fax +33160874549; e-mail [email protected] or fax +3316087454; e-mail [email protected]).

SUMMARY Uridine nucleotides can be formed by energy-consuming de novo synthesis or by the energy-saving recycling of nucleobases resulting from nucleotide catabolism. Uracil phosphoribosyltransferases (UPRTs; EC 2.4.2.9) are involved in the salvage of pyrimidines by catalyzing the formation of uridine monophosphate (UMP) from uracil and phosphoribosylpyrophosphate. To date, UPRTs are described as non-essential, energy-saving enzymes. In the present work, the six genes annotated as UPRTs in the Arabidopsis genome are examined through phylogenetic and functional complementation approaches and the available T-DNA insertion mutants are characterized. We show that a single nuclear gene encoding a protein targeted to plastids, UPP, is responsible for almost all UPRT activity in Arabidopsis. The inability to salvage uracil caused a lightdependent dramatic pale-green to albino phenotype, dwarfism and the inability to produce viable progeny in loss-of-function mutants. Plastid biogenesis and starch accumulation were affected in all analysed tissues, with the exception of stomata. Therefore we propose that uracil salvage is of major importance for plant development. Keywords: Uracil phosphoribosyltransferase, pyrimidine salvage, Arabidopsis, plastid, early development.

INTRODUCTION Nucleotides are immediate precursors of nucleic acids and some coenzymes, as well as essential co-substrates of numerous endergonic reactions such as phosphorylation. They are also necessary for the activation of sugars to permit the synthesis of polysaccharides, glycolipids and glycoproteins (for review see Loffler et al., 2005). Nucleotides are formed by a (deoxy)ribose moiety associated with a purine or a pyrimidine nucleobase and one or more phosphate groups. The nucleotides can be produced either by: (i) de novo synthesis, which starts with small molecules and requires multiple energy-consuming steps; or (ii) the energy-saving ‘salvage pathway’, which utilizes pre-formed nucleobases or nucleosides coming from the catabolism of nucleotides. In the pyrimidine biosynthesis pathway, uridine monophosphate (UMP) is the precursor of each pyrimidine (deoxy-)nucleotide (Figure 1). It is known that a number of ubiquitous metabolic pathways, including RNA and DNA 280

metabolism, cell division and energy transfer reactions, are connected through their utilization of nucleotides. In this context, we propose that UMP biosynthesis needs to be taken into consideration in order to fully understand many aspects of plant physiology. De novo synthesis of pyrimidines is strongly conserved among species and kingdoms, although the number of enzymes involved in the different metabolic steps varies. For example, in mammals the single tri-functional CAD enzyme is responsible for the first three steps of de novo synthesis (Coleman et al., 1977), whereas these reactions are performed by three distinct enzymes in bacteria and plants (Stasolla et al., 2003). Further disparities are observed when considering salvage activities. Mammals lack uracil phosphoribosyltransferase (UPRT) activity, and thereby are unable to perform one-step salvage of uracil with phosphoribosylpyrophosphate (PRPP) (Cleary et al., 2005). However, they are able to recycle uridine via uridine kinases (UKs) ª 2009 INRA Journal compilation ª 2009 Blackwell Publishing Ltd

Arabidopsis development requires UPP 281 Figure 1. Metabolism of uracil and related compounds in plants. Abbreviations: UMP, uridine monophosphate; RNA, ribonucleic acid; UDP, uridine diphosphate; UTP, uridine triphosphate; UMPS, UMP synthase; UMK, UMP kinase; NDK, nucleotide diphosphate kinase; RNApol, RNA polymerase; UK, uridine kinase; URH, uridine hydrolase; UPRT, uracil phosphoribosyltransferase; PRPP, phosphoribosyl-pyrophosphate; PPi, pyrophosphate; Pi, inorganic phosphate; ATP, adenosine triphosphate; ADP, adenosine diphosphate.

De novo synthesis Orotate

Catabolism

PRPP O

O

CO2 NH3

β-alanine

N H

PPi

UMPS

NH OH

NH O

Ribose

Uracil

O

N

O

CO2 Pi

H2 O

URH

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Uridine

UMP ATP

O HO P O O–

ADP

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UMK

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NDK PPi

N

OH OH

UK

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NH O

UTP

Other pyrimidine nucleotides and deoxynucleotides UDP-Glucose

RNApol

Salvage RNA

(Simmonds, 1995; Connolly and Duley, 1999). On the other hand, bacteria, lower eukaryotes and plants are able to recycle pyrimidines at the base and nucleoside level (Natalini et al., 1979; Kern et al., 1990; Jensen and Mygind, 1996; Schumacher et al., 2002; Stasolla et al., 2003). Importantly, these differences in salvage abilities have been exploited to devise therapies that are selectively lethal for a number of human parasitic pathogens. Such an example involves the use of the fluorinated uracil analogue 5-fluorouracil (5FU), which becomes toxic when metabolized, used in the eradication of the protozoan Toxoplasma gondii in vivo (Donald and Roos, 1995; Fung and Kirschenbaum, 1996). In addition, fluorinated pyrimidines have become the major cytotoxic agents in the treatment of cancer (de Bono and Twelves, 2001), since cancer cells metabolize uracil more avidly than normal tissue (Heidelberger et al., 1957; Rich et al., 2004). In plants, pyrimidine salvage activities are far from thoroughly described. Pyrimidine recycling activities have been measured in several plant species, including Pisum sativum (Bressan et al., 1978), Solanum tuberosum (Katahira and Ashihara, 2002), Pinus radiata (Stasolla et al., 2007) and Picea glauca (Stasolla et al., 2006). In S. tuberosum tubers, partial inhibition of de novo pyrimidine synthesis leads to the stimulation of the salvage pathway, surprisingly improving the synthesis of starch and cell wall components (Geigenberger et al., 2005), suggesting that this energy-saving route can have a dramatic influence on biosynthetic performance. In Arabidopsis, such an influence has also been demonstrated by the study of purine recycling pathway apt mutants impaired in adenine phosphoribosyltransferase (APRT), that exhibited growth retardation and male sterility (Moffatt and Somerville, 1988). Most recently, the tight regulation of pyrimidine nucleoside breakdown via uridine ribohydrolase (URH; Figure1) has been shown to be important in the early phase of Arabidopsis development (Jung et al., 2009).

To our knowledge, no mutation affecting the pyrimidine salvage pathway has yet been reported to alter Arabidopsis development. Only one gene putatively involved in the salvage of pyrimidines has been cloned and the corresponding insertion mutant analysed (Islam et al., 2007). This gene (named here UKL1, At5g40870) was first annotated to encode a bi-domain UK/UPRT protein, and its corresponding knock-out (KO) mutant does not display any visible growth alteration, although it is resistant to 5FU and 5-fluorouridine. These results lead to the prevalent idea that UKL1 is the unique pyrimidine salvage gene. However, there was no examination of the four other members in the gene family (At1g55810, At3g27190, At3g27440, At4g26510), or the gene encoding a single domain UPRT (At3g53900). Here we report the global analysis of UPRT annotated genes in Arabidopsis. In addition to phylogenetic analyses, UPRT activity has been investigated in vivo for each candidate, and the available T-DNA insertion mutants have been characterized. Our results demonstrate that the monodomain UPP gene encodes a functional UPRT enzyme, while the tested dual-domain genes lack this activity. The characterization of two insertion mutants in the UPP gene revealed that loss of UPRT activity strongly affects early Arabidopsis development. Furthermore, we show that UPP is targeted to plastids, and propose that plant metabolism is highly reliant on uracil salvage. RESULTS Evolution of putative UPRT genes of Arabidopsis In Arabidopsis, six homologous genes are predicted to encode proteins with UPRT domains (http://www.arabidopsis. org/, Figure 2). Five of these genes (At5g40870, At3g27190, At1g55810, At4g26510 and At3g27440) show a high level of identity, and are annotated as also containing a N-terminal UK domain. These genes are herein referred to as UKL1 (UK-like 1), UKL2, UKL3, UKL4 and UKL5, respectively.

ª 2009 INRA Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 280–291

282 Samuel E. Mainguet et al. In this AGI article accession

Protein domains TP

UPRT

c

UPP

Other name

At3g53900

TP

UK

UPRT

c

UKL1

At5g40870 AtUK/UPRT1

TP

UK

UPRT

c

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At3g27190

UK

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UKL3

At1g55810

UK

UPRT

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UKL4

At4g26510

UK

UPRT

c

UKL5

At3g27440

UPRT

Figure 2. Structure and names of uracil phosphoribosyltransferase (UPRT) annotated genes of Arabidopsis.

Uracil phosphoribosyltransferase domain according to TAIR and PFAM

UPRT Uracil phosphoribosyltransferase domain according to TAIR but not PFAM UK

Uridine kinase domain according to TAIR and PFAM

TP

Plastid transit peptide according to targetP

The UPRT gene family arose from recent duplication events, revealed by near identical exon patterns, both in size and localization (Figure S1a in Supporting Information). The sixth gene analysed (At3g53900, herein named UPP) is annotated as encoding a putative mono-domain UPRT protein. An intron insertion site at the +1 position within a codon of the annotated UPRT domain is conserved between UPP, the five UKLs and their homologs in rice and grapevine, confirming a common ancestor (Figure S1b). We constructed a phylogenetic tree to compare the UPRT domains of genes, putative or functionally validated, of plants and other phyla (Figure 3). Plant mono-domain UPRTs appear to be the closest to prokaryotic UPRT, whereas the UKL family members, together with other plant homologs, form a distinct and distant group. These results suggest that during plant evolution mono-domain UPRTs have retained the prokaryotic UPRT structure, whereas the UPRT domain of UKLs diverged significantly from its prokaryotic ancestor. Indeed, the Pfam UPRT domain (PF00156) is not significantly detected in the UKL family (Figure 2). Additionally, to our knowledge, no UPRT activity has been measured for proteins included in the plant UPRT group, and it is uncertain if the derived UPRT domain present in the UKL proteins is able to catalyze the formation of UMP from uracil. The mono-domain gene UPP encodes a protein with UPRT activity in vivo We performed functional complementation assays in Escherichia coli to identify Arabidopsis genes encoding a functional UPRT. The E. coli mutant strain BM604 (Andersen et al., 1992) lacks the enzyme (pyrF30) responsible for the last step of the de novo pyrimidine synthesis pathway, as well as uridine phosphorylase (udp) and UPRT (upp) activities. This mutant can only grow on complete medium or, due to its UK activity, on minimal medium supplemented with uridine. We transformed E. coli BM604 both with the fulllength UPP coding sequence (CDS) and with the CDS of the four UKLs that are expressed (UKL1, -2, -3, -4). Possibly due

to its very low expression (http://www.genevestigator.com/), we were unable to isolate a full-length UKL5 cDNA. The results clearly show that only the plasmid carrying the UPP CDS enabled the mutant to grow with uracil as the sole pyrimidine source (Figure 4a). The same result was obtained with the control plasmid pUC18_pLpupp expressing the Lactobacillus plantarum UPRT gene (Arsene-Ploetze et al., 2006). Importantly, as the expressed UKLs were unable to complement the upp mutation this suggests that they lack UPRT activity. UPP is the major functional UPRT in Arabidopsis In order to investigate the importance of the UPP gene, relative to the contribution of the UKL gene family, we analysed T-DNA insertion mutants for the UPP, UKL1, UKL2 and UKL3 genes (Figure 4b). Two allelic mutations of UPP were analysed (upp-1 in the Col-0 background and upp-2 in the WS background). Insertions were validated by PCR genotyping and mutants were backcrossed for further analyses. In each line, transcripts of the mutated gene were not detected by RT-PCR in homozygous plants (Figure 4c). A measurement of the enzymatic activity of UPRT indicates that both upp-1()/)) and upp-2()/)) mutant lines have a 99 and 93% lower UPRT activity, respectively, compared with that of the wild type. No significant decrease in UPRT activity was measured in the mutants disrupted at UKL loci (Figure 4d). These results show that the disruption of the single UPP gene is sufficient to abolish UPRT function in Arabidopsis. In accordance with the UPRT activity test, tolerance to the uracil analogue 5FU was only observed for upp(+/)) heterozygous plants (the drastic phenotype of homozygous upp()/)) does not allow the observation of a 5FU resistance phenotype). Indeed, no increase in 5FU tolerance was seen in the homozygous ukl1, ukl2 and ukl3 mutants compared with the wild type (Figure S2I). Interestingly, a graduation in the tolerance to 5FU was observed among the upp(+/)) heterozygous plants. To test whether this graduation is due to differential expression of

ª 2009 INRA Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 280–291

Arabidopsis development requires UPP 283 81 28 23

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the UPP gene among heterozygous upp-1(+/)) plants, individuals showing tolerance to 200 lM 5FU were grouped into three phenotypic classes (A, B and C; Figure S2III) and RTquantitative (q)PCR was performed. The results showed that expression levels of the UPP gene are inversely correlated to 5FU tolerance (Figure S2II). Taken together, these results show that Arabidopsis uracil recycling function relies on the unique UPP gene but not on each individual UKL gene. UPP is necessary for early plant development In the homozygous state, the three UKL KO mutants displayed no clear phenotypic alteration when compared with wild-type plants (data not shown). This result suggests that each mutated UKL is dispensable for the proper development of Arabidopsis; however, this may betray a functional redundancy between members of this gene family. In contrast, both upp()/)) mutants displayed dramatic growth

Plant monodomain

67 100 100 91 99 69

Procaryotic mono-domain

86 42

Arabidopsis UKL4 Arabidopsis UKL3 Oryza NP 001046530 Oryza NP 001067657 Vitis CAO14676 Arabidopsis UKL5 Oryza EAZ09672 Oryza NP 001062322 Vitis CAO68279 Arabidopsis UKL1 Arabidopsis UKL2 Chlamydomonas XP 001691 Homo Q96BW1 Macaca Q95KB0 Leishmania XP 001468477 Trypanosoma XP 811495 Dictyostelium XP 647128 Toxoplasma Q26998 (Donald et al., 1995) Saccharomyces FUR1 (Kern et al., 1990) Neurospora XP 955968 Sclerotinia XP 00159810 Anopheles XP 558608 Brugia EDP32190 Xenopus NP 001088880 Danio XP 686775 Tetraodon CAG11268 Gallus XP 001234874 Opossum XP 001375670 Rattus NP 001102682 Mus NP 081041 Bos XP 870700 Macaca XP 001083686 Homo BAD96279 Paramecium XP 001458774 Sulfolobus NP 341784 (Jensen et al., 2005) Aspergillus XP 00121150 Botritys XP 001551957 Chloroflexus ZP 00766720 Agrobacterium NP 353171 Bradyrhizobium YP 00124 Bacillus (Jensen et al., 1997) Lactobacillus NP 785838 (Arsene-Ploetze et al., 2006) Xanthomonas NP 642836 Yersinia NP 406337 Escherichia NP 289051 (Andersen et al., 1992) Nostoc ZP 00108189 Nostoc NP 486103 Crocosphaera ZP 00516647 Chlamidomonas XP 001699 Oryza NP 001055725 Arabidopsis UPP (This article) Vitis CAO15711 Vitis CAN72274 Porphyra YP 537035 Chloroflexus A0H4P6 Crocosphaera Q4CB49

Plant bi-domain

Figure 3. Phylogenetic tree based on the alignment of the uracil phosphoribosyltransferase (UPRT) annotated domain in various species. Terminal branch labels indicate the genus and the accession number of the protein. Dots indicate UPRT annotated genes of Arabidopsis; cyanobacterial UPRTs are highlighted. Branch labels indicate the consensus support (%).

retardation, a pale-green to albino phenotype and flimsy roots with less branching (Figure 5a). To confirm that the phenotypic alterations were due to upp, we performed an allelism test by crossing reterozygous plants carrying the upp-1 allele with reterozygous plants carrying the upp-2 allele (Figure 6c). The results revealed that retarded palegreen plantlets were always reterozygous for both alleles, thus KO for the gene. These results clearly demonstrate that the UPP mutation is responsible for the dwarf pale-green phenotype in both mutant alleles studied. The transmission efficiency of upp alleles in selfed progenies of reterozygous plants is shown in Table 1. The proportion of homozygous plants obtained was slightly lower than expected for a single Mendelian trait, at 10–13% of viable seeds. Although not significant (chi-squared test, Table 1) this difference could be due to germination defects of the homozygous plants in our culture conditions.

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284 Samuel E. Mainguet et al.

(a) Disposition on plates

M9 + URI

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Figure 4. UPP is the major functional uracil phosphoribosyltransferase (UPRT) in Arabidopsis. (a) Functional complementation. Escherichia coli BM604 was transformed with the Lactobacillus plantarum upp gene (Lp upp) cloned into the pUC18 vector. UPP, UKL1, UKL2, UKL3 and UKL4 coding sequences (CDS) were cloned into the pCR2.1 vector. pUC18 empty vector and pCR2.1 ligated with control DNA (pCR2.1-) were used as negative controls. M9, M9 medium, supplemented with uridine (+ URI) or uracil (+ URA). (b) Schematic representation of the UPRT annotated genes studied and T-DNA insertion locations in each mutant. (c) Reverse transcriptase-PCR on each mutant and their respective wild-type to detect Actin2, UPP, UKL1, UKL2 and UKL3 transcripts. (d) The UPRT activity in planta of each studied mutant. Solid bars, Col-0 background; open bars, WS background. *Significantly different from wild type (P < 0.05).

The addition of sucrose to the growth medium is normally dispensable for the germination of wild-type seeds; however, it was necessary for upp-1()/)) and upp-2()/)) mutants (Figure 6b). When germinated on a growth medium without sucrose, homozygous upp mutants displayed albino cotyledons and failed to develop beyond this stage. Supplying sucrose to the medium up to 2% (w/v) allowed the mutants to develop pale-green cotyledons and small pale-green leaves. These results suggest that mutant embryos may lack the storage of, or are unable to mobilize, the reduced carbon necessary for the establishment of an operational photosynthetic apparatus. To address this question, we analysed the publicly available CATMA transcriptome data for the germination process (http://urgv.evry.inra.fr/CATdb,

project RA03-04). The results support the second hypothesis, because a time-course experiment between 6 and 50 h after seed imbibition indeed reports an inverse correlation between the transcript levels of UPP and photosynthesisrelated genes (Figure S3). Such opposing expression profiles may reveal the need to produce pyrimidines at a lower energetic cost during germination. UPP loss of function causes chloroplastic disorders The pale-green phenotype and the heterotrophy of homozygous upp mutants grown in vitro suggested that these mutants present some kind of chloroplastic disorder. We found no differences in the photomorphogenic response of upp mutants and their wild-type siblings grown for 15 days

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Arabidopsis development requires UPP 285

(a)

(c)

upp-1 (–/–)

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upp-2 (–/–)

Col0

WS

upp-1

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(i) % chloroplasts

1%

Sucrose

Hemispheric suface (µm2) 50 40 30 20 10

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52

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72

Hemispheric suface (µm2) Figure 5. Phenotype of upp()/)) mutants. (a–c) Macroscopic phenotype. (a) Phenotype of upp-1 and upp-2 homozygous mutants compared with their wild-type counterpart. Plants were photographed after 2 weeks of growth in vitro. (b) Progeny of reterozygous upp-1 and upp-2 were grown in vitro on medium supplemented with various sucrose concentrations. Plants were photographed after 2 weeks. Arrows indicate homozygous mutants. Scale bars = 5 mm. (c) Phenotype of double reterozygous upp-1(+/)) upp-2(+/)) plants grown in vitro for 20 days. Arrows indicate double reterozygous. (d–i) Mutation at the UPP locus affects chloroplast arrangement and size in cotyledons. (d–g) Confocal micrographs, showing fluorescing chlorophyll of 2-week-old plantlet cotyledons: (d) Col-0; (e) upp-1()/)); (f) WS; (g) upp-2()/)). Scale bars = 50 lm. (h, i) Graphs representing the distribution of hemispherical surfaces of chloroplast populations (n ‡ 72) in Col-0 (h) or WS (i) cotyledons (thin-dotted line), leaves (wide-dotted line) and upp()/)) cotyledons (solid line).

in the dark on 1% sucrose (Figure S4). Therefore, this first result reveals that UPP is not required during etiolation, a process that does not rely on functional chloroplasts. We then performed microscopic analysis of upp-1()/)) and upp-2()/)) mutants to determine whether chloroplast development or morphology was affected. Confocal microscope analyses of 2-week-old cotyledons revealed an increased chaotic distribution of red fluorescing organelles in both mutants compared with their wild-type counterpart (Figure 5d–g). Also, as observed with chloroplast hemispherical surface measurements, the size of the chloroplasts was reduced in the mutants (Figure 5h,i). We concluded that UPP loss-of-function mutations disturbed the biogenesis or structure of chloroplasts. Chloroplast ultrastructure of the first leaves was compared in the upp-1()/)) mutant and wild type by transmis-

sion electron microscopy (TEM). To avoid artefacts linked to the development lag between mutants and wild-type plants, we compared 3-week-old upp-1()/)) second leaves with 3-week-old wild-type second and fourth leaves. The TEM observations showed that, as in the wild type, the mutant displayed chloroplasts with differentiated thylakoids. However, thylakoids displayed increased irregularities and fewer grana stacks in the mutant (Figure 6b–e). In the wild type, one or more starch grains were visible in most observed chloroplasts of the second and fourth leaves (Figure 6a,b,f). In contrast, no starch grains were seen in the mutant leaf samples (Figure 6c), except in stomata guard cells where the chloroplasts appeared no different from those of the wild type (Figure 6f,g). The lack of starch grains in upp-1()/)) mutants was also supported by TEM observation of 3-weekold plantlet root tips (Figure 6h,i). While wild-type root tips

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286 Samuel E. Mainguet et al.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Table 1 Results of upp-1 and upp-2 transmission in the self progeny of backcrossed reterozygous upp-1 and upp-2 (+/)) plants He mutant parent

NG

He progeniesa

Ho progeniesa

WT progeniesa

v2 for 3/1b

upp-1.6 upp-1.9 upp-1.12 upp-2.3

2 4 7 5

57 55 53 53

10 11 12 13

27 28 24 29

0.35 0.67 0 1.24

Ho, homozygous; He, heterozygous; WT, wild type; NG, not germinated. a Plants were scored by PCR genotyping as described in Experimental Procedures. b Chi-squared obtained for 3/1 segregation (not significant with an alpha risk of P < 0.001, one degree of freedom).

displayed numerous amyloplasts, only proplastids with no, or no more than two, starch granules were observed in upp-1()/)). Therefore, loss of UPP function is associated

Figure 6. Transmission electron microscopy observations. (a) Second leaves of Col-0. (b) Fourth leaves of Col-0. (c) Second leaves of upp-1()/)). (d, e) Higher magnifications of (b) and (c). (f) Guard cells from second leaves of Col-0. (g) Guard cells from second leaves of upp-1()/)). (h) Root tips of Col-0. (i) Root tips of upp-1()/)). Bars = 1 lm.

with disturbed plastid starch accumulation in initial leaves and root tips. This disturbed accumulation may be linked to the observed aberrant plastid biogenesis. The UPP locus encodes a plastidial UPRT enzyme According to putative transcription start sites, UPP is predicted to encode two transcripts that differ in the length of their 5¢ end and in the length of the encoded protein: 296 amino acids (aa) versus 231 aa (Figure 7a). In order to validate the existence of different transcripts, we carried out RT-PCR on total RNA from 3-week-old Arabidopsis seedlings with primer pairs designed to specifically amplify each of the predicted transcripts. The PCR products corresponding to the spliced long transcript were detected, but only a very weak signal corresponding to the short transcript was observed. While these results suggest that the long transcript is the major one, the presence of the short transcript is inconclusive. The weak signals observed with S1, S2 and S3

ª 2009 INRA Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 280–291

Arabidopsis development requires UPP 287 Figure 7. The UPP transit peptide is sufficient to address GFP to chloroplasts. (a) Top: schematic representation of the two predicted UPP transcripts. Dark grey, untranslated regions (UTRs); light grey, coding sequences (CDS); box, predicted transit peptide. Bottom: PCRs performed on cDNA (RT-PCR), genomic DNA or water, with forward primers identified above each line. Rev, the same reverse primer was used in every reaction. (b–d) Confocal micrographs of tobacco epidermal cells transiently expressing UPPtp–GFP chimeric protein: (b) red chlorophyll autofluorescence; (c) green GFP fluorescence; (d) Overlay of (b) and (c). Scale bars = 20 lm.

(a)

L3

L2

L1

S3

200 bp --

DISCUSSION The UPRTs are key enzymes in the salvage of the ubiquitous pyrimidine nucleotides. Using functional complementation and enzymatic activity assays performed on different Arabidopsis mutants, we clearly demonstrate that the loss of function of the single UPP gene is sufficient to abolish almost

fw

PCR on genomic DNA PCR on water

Primers --

primers could be due to the occurrence of some residual unspliced long transcripts or tissue-specific expression of the short transcript that is difficult to detect in total RNA obtained from a whole plantlet. The longer UPP transcript encodes a protein of 296 aa with a predicted N-terminal 61-aa plastid transit peptide according to TargelP V1.1 (Emanuelsson et al., 2000) (Figure 7a). To investigate the subcellular localization of the UPP protein, we constructed a translational fusion between the first 125 aa of the UPP protein and the green fluorescent protein (GFP) under the control of the 35S promoter (UPPtp–GFP). Transient expression experiments were performed in Nicotiana benthamiana leaves. Figure 7(b–d) shows examples of the typically observed patterns in epidermal cells. Both GFP and red autofluorescence of the chlorophyll exhibited very similar distribution patterns in cells and could be superimposed in an overlay (Figure 7b–d). Both signals co-localized in optical sections indicating that UPPtp–GFP protein is targeted to plastids. Thus, the 125-aa N-terminal sequence of UPP is sufficient to address GFP to chloroplasts in planta. In addition, the Plant Proteome Database (http://ppdb.tc. cornell.edu/) reports the identification of several UPPderived peptides in samples coming specifically from chloroplast stroma. Taken together, these results support the plastidial targeting of the UPP protein.

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(c)

(d)

all UPRT activity in planta (Figure 4). In addition, we show that lack of uracil salvage leads to dramatic developmental alterations in Arabidopsis (Figures 5 and 6). We also show that disruption of three members of the UKL family has no significant influence on either Arabidopsis UPRT activity or on plant development (Figures 4 and S2). While we might still hypothesize that the five members of the UKL family could be partially redundant, these results show that the disruption of the UPP gene alone is sufficient to suppress almost all UPRT activity in planta. In contrast, however, Islam et al. (2007) claim that UKL1 (AtUK/UPRT1) is an important UPRT in Arabidopsis, based on resistance of the ukl1 Arabidopsis mutant to 5FU. This reported 5FU resistance has been interpreted as a loss of UPRT activity. However, this result is definitely inconsistent with our UPRT activity measurements that have shown the ukl1 mutant not to be impaired at all for the UPRT activity (Figure 4). Therefore an increased tolerance of ukl1 plants to 5FU is unexpected. Here, we tested several culture conditions and 5FU concentrations, but we were never able to reproduce Islam et al. (2007) result (Figure S2). Instead, we observed 5FU tolerance correlating with UPP transcript level on upp heterozygous mutants (Figure S2). Islam et al. (2007) only used a negative functional complementation assay with E. coli 5FU-insensitive strains mutated for the upp gene, but still having a functional de novo pyrimidine synthesis. When transformed with a plasmid carrying the UKL1 coding sequence, the bacteria were no longer able to grow in the presence of 5FU. However, the results shown do not definitely prove the UPRT activity of UKL1. Indeed, many well-known factors can lead to the inability of bacteria to multiply (for example, recombinant protein toxicity; Fernandez et al., 2007).

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288 Samuel E. Mainguet et al. Therefore, we chose a positive functional complementation assay using E. coli BM604 (Andersen et al., 1992), which can only grow in the presence of uracil as the sole pyrimidine source following transformation with a functional UPRT. None of UKL1, UKL2, UKL3, or UKL4 succeeded in complementing the BM604 upp mutation, although UPP did. Altogether, our results shed light on the essential role of UPP protein in Arabidopsis uracil salvage, which seems to be the major, if not the only, functional UPRT in Arabidopsis. From an evolutionary point of view, protein sequence similarities revealed that the UPRT domains of UPP and uncharacterized UPP-like genes in Vitis vinifera, Oryza sativa and Chlamydomonas reinhardtii appear to be the closest relatives to the cyanobacterial homologue found in Nostoc punctiforme or Crocosphaera watsonii (Figure 3). Together with the plastidial localization of UPP demonstrated in this study (Figure 7), it can be speculated that plant UPRTs have an endosymbiotic origin, as do many other nuclear genes in plants, arising from gene transfer from plastid to nucleus. Interestingly, we found an intron insertion site at the +1 position within a codon of the annotated UPRT domain to be conserved between UPP, the five UKLs and their orthologs in plants (Figure S1b). Considering that in plants 55–56% of intron insertion sites are at position 0 of the codons (i.e. between two codons), and only 23–24% are at position +1, this insertion constitutes strong evidence for a common gene ancestor (Long et al., 1995; Tomita et al., 1996). Nonetheless, our analysis show that the UPRT domains of UPP and UKLs diverged significantly after the duplication event (Figure 3). Therefore, based on functional complementation and analysis of UPRT activity (Figure 4), it is tempting to speculate that UKLs have only retained UK activity, with the UPRT moiety simply acting as a regulatory domain sensing UMP and/or uracil and/or PRPP. Indeed, UPRT gene duplication followed with the adaptation of one UPRT paralogue to a regulatory function has previously been described in Bacillus subtilis. Two genes encoding UPRTs, upp and pyrR, are present in the B. subtilis genome. The disruption of upp leads to a 99% reduction of UPRT activity, whereas upp pyrR double mutants lack any detectable UPRT activity (Martinussen et al., 1995). Interestingly, it has also been demonstrated that PyrR displays typical UPRT activity under non-physiological conditions, although under physiological conditions it only senses PRPP and UMP, and acts as an attenuation protein regulating the pyrimidine biosynthesis operon (Tomchick et al., 1998). Unexpectedly, as the de novo synthesis pathway is thought to be able to provide pyrimidine nucleotides in the absence of salvage activity, our results show that early plant development is highly reliant on this salvage pathway. We characterized two allelic mutations in the UPP gene that have lost almost all UPRT activity (Figure 4) and displayed the same dramatic phenotype of heterotrophy, growth retardation, pale-green to albino cotyledons and leaves

and inability to produce viable progeny (Figure 5). Mutants impaired in photosynthesis and/or chloroplast development display a similar developmental phenotype (de Longevialle et al., 2008; Myouga et al., 2008). Microscopic observations revealed alterations in plastid biogenesis, growth and starch content in all tissues except stomata guard cells, where chloroplasts of mutant plants were indistinguishable from those of the wild type (Figures 5 and 6). These dramatic developmental alterations observed in the Arabidopsis upp mutants could arise from: (i) abnormal embryogenesis and seed formation and/or (ii) an impaired germination process. Since no macroscopic abnormalities have been observed during seed formation in the siliques of reterozygous mutants, embryogenesis does not appear to be strongly affected by UPP loss of function. This is in accordance with the pyrimidine synthesis activities measured for Picea glauca, in which 80% of pyrimidine synthesis occurs via a de novo synthesis pathway during embryo maturation (Ashihara et al., 2001). Ashihara and colleagues also demonstrated that the salvage pathway is operative during embryogenesis, with uridine being much more efficiently salvaged (through UK activity) than uracil (through UPRT activity). Therefore, germination may be heavily reliant on uracil salvage. As reviewed (Stasolla et al., 2003), pyrimidine metabolism during germination has two distinct phases: ‘salvage synthesis’ at the inception of germination and ‘de novo synthesis’ at later stages. Indeed, transcriptomic data indicate that UPP transcript levels decrease at the onset of photosynthesis (see the expression data presented in Figure S3). In Arabidopsis, inhibition of de novo synthesis at the ATCase step leads to a five-fold increase in UPP transcript (Chen and Slocum, 2008). Similarly, inhibition of de novo synthesis at the UMPS step resulted in the stimulation of the uracil salvage pathway in potato (Solanum tuberosum; Geigenberger et al., 2005). Taken together, these results suggest that the uracil salvage pathway is essential when de novo synthesis is limiting, which may be the case during initiation of germination. One obvious unique feature of plants is the central role of sucrose in carbohydrate metabolism. As UDP is required for both optimal sucrose synthesis and breakdown, the deregulation of pyrimidine metabolism can have far-reaching consequences for sugar metabolism. Indeed, in potato tubers, when pyrimidine salvage was stimulated, a larger UDP–glucose pool increased the flux toward the cell wall and starch biosynthesis was improved (Geigenberger et al., 2005). The starch grain deficiency observed in plastids of roots and leaves in upp()/)) mutants (Figure 6), together with their need for an external sucrose supply to survive (Figure 4b), corroborate the hypothesis that uridine nucleotides are limiting factors in sucrose and starch synthesis. In upp mutants, the only plastids with apparently normal starch grains are located in stomata guard cells

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Arabidopsis development requires UPP 289 (Figure 6f,g). The high mitochondrial density that has also been observed is in accordance with the well-known specialization of guard cells towards respiration (Zhu et al., 2009). Furthermore, the lack of uracil salvage in upp mutants is likely to cause an energy deficit, because of the higher energetic cost of de novo pyrimidine synthesis. In that situation, the high mitochondrial activity of guard cells could provide enough energy to build their apparently normal chloroplasts. Nonetheless, de novo synthesis may require functional chloroplasts for optimal rates, based on the plastidial location of the initial step enzymes and its high energy demand (Zrenner et al., 2006). In this scenario, this study suggests that the salvage of uracil could be critical for the establishment of de novo pyrimidine synthesis during early development, presumably by improving energetic balance and plastidial biogenesis. This could explain the fact that upp()/)) mutants do not recover at later stages, even when grown in a sucrose-supplemented medium. This work provides new insight into the tight energy control that is behind the very first stages of Arabidopsis development. Plants may need to recycle pyrimidines to preserve the fragile energetic balance that is necessary to an efficient autotrophic growth. EXPERIMENTAL PROCEDURES Plant material and growth conditions Arabidopsis T-DNA insertion mutants were identified using the online database FlagDB++ (http://urgv.evry.inra.fr/projects/FLAGdb++/) (Samson et al., 2004). Seeds of Arabidopsis thaliana (L.) heynh, ecotype Col-0, carrying mutations in the genes At3g53900 (SALK_086006: upp-1), At5g40870 (SALK_108486: ukl1; atuk/uprt1), At3g27190 (SALK_058257: ukl2), At1g55810 (SAIL_156D06: ukl3) and seeds of A. thaliana (L.) heynh, ecotype Ws, carrying the upp-2 mutation (FLAG_191A02) were obtained from the Salk Institute collection (Alonso et al., 2003) and the Versailles collection (Bechtold et al., 1993) of T-DNA insertion mutants, respectively. Seeds grown in in vitro assays were surface sterilized and sown on Arabidopsis medium (Estelle and Somerville, 1987) with or without kanamycin (100 mg L)1) and germinated in a growth chamber (16-h light/8-h dark cycle, 21C, 50% hygrometry) after cold treatment for 48 h at 4C. The 1% (w/v) sucrose concentration was modified when mentioned. Mature plants were grown in the greenhouse at 20–25C, under a 16-h light/8-h dark cycle. To study photomorphogenic effects, seeds were sown on plates covered by three sheets of aluminium foil. For the 5FU toxicity experiments, 5FU was dissolved in DMSO to obtain 1000· stock solutions. Just before filling the plates, 0.1% (v/v) of the stock solution was added to the medium.

Molecular analyses The DNA for PCR genotyping was extracted according to (Edwards et al., 1991) and as described in (Bouchez et al., 1996) for Southern blots. Total RNA was extracted from aerial parts of 4-week-old plants using the RNeasy Plant mini kit (Qiagen, http://www.qiagen. com/) without the DNase step. The quality of the RNA was verified by gel electrophoresis or on an Agilent 2100 Bioanalyser (Agilent Technologies, http://www.home.agilent.com/). After PCR genotyping when needed, RNA was subjected to a DNase treatment of 2 U per microgram of RNA (Invitrogen, http://www.invitrogen.

com/) in 10 ll. The reaction was stopped by the addition of EDTA. The RNA was quantified using Ribogreen (Invitrogen) and a FluoStar Galaxy 96-well plate fluorescence reader (BMG Labtech, http://www.bmglabtech.com/), according to the manufacturer’s recommendations.

PCR genotyping. The T-DNA insertions were confirmed using the primer pairs 11/23, 24/14, 21/16, 22/18 and 22/20 for upp-1, upp-2, Sail_156D06, SALK_058257 and SALK_108486 lines, respectively and the primer pairs 11/12, 13/14, 15/16, 17/18 and 19/20 for the wildtype alleles of upp-1, upp-2, Sail_156D06, SALK_058257 and SALK_108486 lines, respectively (Table S1). The PCR amplifications were performed on 100 ng of genomic DNA, in 25 ll and 1 U of Taq DNA polymerase, under the following conditions: 5 min 95C, 35 cycles (30 sec 95C, 30 sec at specific Tm, 1 min kbp)1 at 72C) followed by 10 min at 72C. Insertion-containing amplification products were sequenced. RT-PCR experiments. First-strand cDNA was synthesized from 500 ng of total RNA in 19 ll and 200 U of SuperScript II (Invitrogen) for 1 h at 42C. The reaction was stopped with RNaseH (Invitrogen). SuperScript II was replaced by water in negative controls. The PCR reactions were run as described above using 1 ll of first-strand cDNA and specific primer sets 26/27 (At3g53900), 36/37 (At5g40870), 38/39 (At3g27190), 40/41 (At1g55810) and 9/10 (Actin2). The absence of DNA contamination was tested in negative controls with Actin2specific primers 9/10. Real-time RT–PCR was carried out using the ABI PRISM 7900HT (Applied Biosystems, http://www3.appliedbiosystems.com/). A specific primer set (5/6) was designed to amplify a 132-bp fragment of the UPP cDNA. A standard curve was generated from duplicate series of five DNA template dilutions to test PCR efficiencies. The PCR was conducted in duplicate in the presence of 1 ng cDNA, 3 ll of primer mix 1 lM each, 5 ll Mesa GREEN qPCR Mastermix (Eurogentec, http://www.eurogentec.com/) in 10 ll. The PCR conditions were as described above with 10 min at 95C, then 40 cycles (10 sec 95C, 10 sec 60C). The results were standardized by comparing the data obtained for three housekeeping genes: At2g36060, At1g16460 and At3g18780 (primer pairs 3/4, 7/8 and 9/ 10) (Table S1). The quantification of gene expression was performed using the comparative cycle threshold number (Ct) method. Transient expression experiments The sequence coding for the putative targeting peptide was amplified from genomic DNA using the primer pair 1/2 (Table S1). Using the Gateway cloning technology (Invitrogen), the 804-bp amplification product was shuttled to the pDONR207 entry vector by a BP recombination reaction, sequenced and subsequently transferred to the pGWB5 binary vector (Nakagawa et al. 2007) by a LR recombination reaction. The resulting binary vector was electroporated into Agrobacterium tumefaciens C58C1 strain (Koncz et al., 1984) harbouring the pCH32 helper (Bendahmane et al., 2000). The UPPtp– GFP fusion protein was transiently expressed in N. benthamiana leaves via agroinfiltration as described by (Bendahmane et al., 2000). Leaves were harvested 72 h after infiltration and small pieces placed on a glass slide. Fluorescent proteins were visualized in adaxial leaf cells using a spectral Leica SP2 AOBS confocal microscope (Leica Microsystems, http://www.leica-microsystems.com/) equipped with an argon laser and a HeNe laser. Different signals were detected using the 488 and 543 nm laser lines. The images were coded green (GFP) and red (chlorophyll autofluorescence), giving a yellow co-localization when signals overlap in merged images. Microscopic observations were carried out using a Leica

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290 Samuel E. Mainguet et al. HC-PL-APO633/1.20-Water-Corr/0.17-Lbd.BL lens. Each image represents a projection of individual optical section taken as a Z series. Sequential scans were performed using between line mode.

Functional complementation assay The CDS from the four studied genes were respectively PCR-amplified with primer sets 26/27, 28/29, 30/31, 32/33 and 34/35 (Table S1) using 1 ll of cDNA produced as described above with total RNA extract from wild type Arabidopsis Col-0 plants and the Accuprime Taq polymerase (Invitrogen) under the following conditions: 5 min 95C, then 35 cycles (30 sec 95C, 30 sec specific Tm, 1 min kbp)1 68C), then 5 min 68C. Amplification products were cloned into the pCR2.1 cloning vector using the TOPO TA cloning kit (Invitrogen). The ligation mixture was electroporated into E. coli-deficient upp udp pyrF strain BM604 (Andersen et al., 1992). Transformants obtained on solid Luria–Bertani medium plates containing ampicillin (100 mg L)1) were selected by PCR. Five clones for each gene were sequenced. The UPP function was tested in the E. coli strain BM604 on minimal medium M9 supplied with either uracil or uridine at 25 lg ml)1 as described by (Arsene-Ploetze et al., 2006). Clones containing pUC18 empty vector, pCR2.1 ligated with supplier’s control DNA, as well as pLpupp vector, harbouring Lactobacillus plantarum upp gene known to complement E. coli-deficient strain BM604 (Arsene-Ploetze et al., 2006), were used as controls.

Transmission electron microscopy Samples were infiltrated for 1 h with fixation buffer [cacodylate buffer 0.1 M pH = 7.4; 3% glutaraldehyde; 1% paraformaldehyde; 1% (w/v) tannic acid; 2% (w/v) sucrose; 2 mM CaCl2], under vacuum. After overnight fixation at 4C with gentle shaking, samples were post-fixed in cacodylate 0.1 M pH = 7.4 with OsO4 2% for 90 min at 4C with gentle shaking. Samples were dehydrated in a graded series of ethanol, embedded in Spurr resin and sectioned in 90-nm slices. Observations were made on a Philips EM208 TEM (Philips, http://www.fei.com).

UPRT activity measurements Protein isolation Protein extracts were prepared from aerial parts of 2-week-old plants. Following grinding in liquid nitrogen, plant tissue samples were homogenized in cold buffer A containing 100 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)–HCl (pH 7.5), 20 mM MgCl2, 1 mM DTT and 1 mM PMSF. After 15-min centrifugation (18 000 g), the supernatant was collected and desalted through a NAP-5 column (GE Healthcare, http://www. gehealthcare.com/) equilibrated in buffer A. Protein concentrations were estimated by the method of Bradford (Bradford, 1976) using the Bio-Rad protein assay reagent with bovine c-globulin as standard (http://www.bio-rad.com/). The method developed to measure UPRT activity was based on the formation of UMP. The UPRT activity was measured in a volume of 100 ll containing 100 mM TRIS–HCl (pH 7.5), 20 mM MgCl2, 2 mM uracil and 2 mM 5-phospho-D-ribose-1-diphosphate (Sigma-Aldrich, http://www.sigmaaldrich.com/). Assays were initiated by adding 60 lg of protein extract. After incubation at 30C for 15–45 min, the reaction was stopped at 100C for 2 min. The precipitated protein was removed by centrifugation at 18 000 g for 15 min. The supernatant was subjected to reverse-phase ultraperformance chromatography (Acquity UPLC, Waters, http://www.waters.com/) using a UPLC HSS T3 1.8 lm (100 · 2.1 mm internal diameter) column (Waters). The UMP was eluted using a linear gradient of 5% methanol in 15 mM triethylamine pH 7.9 (0.6 ml per min) and detected at 260 nm using a diode array detector (Waters 2696 PDA detector). Peak areas were calculated using EMPOWER software (Waters).

To ensure that steady-state conditions applied, at least four time points were used for the determination of product. Product formation was linear for at least 45 min and velocities were proportional to the concentration of protein in the assay.

Phylogenetic analysis All sequences were retrieved from the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), except the amino acid sequence of the Bacillus caldolyticus UPRT which was retrieved from Jensen et al. (1997). Sequence alignment was done using Muscle (Edgar, 2004) included in Geneious software (Biomatters Ltd, http://www.biomatters.com/), with default parameters. The phylogenetic tree was built using MEGA version 4 (Tamura et al., 2007) on the extracted alignment of the 249-aa C-terminal consensus region shared by all the sequences, using the neighbourjoining method and a resampling with the bootstrap method (1000 replicates, amino acid Poisson correction).

ACKNOWLEDGEMENTS Samuel Mainguet is funded by the University of Paris-Sud 11 (Doctoral school ‘Sciences du Ve´ge´tal’ ED145, Orsay, France). The authors thank Olivier Granjean (LCC, INRA Versailles, France) and Danielle Jaillard (CCME Orsay, France) for technical assistance and advice; Se´bastien Aubourg (URGV, Evry, France) for helpful discussions and Alison Winger and Jennifer Yansouni (URGV, Evry, France) for correcting the manuscript.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Conservation of intron insertion sites between uracil phosphoribosyltransferase (UPRT) annotated genes in plants. Figure S2. 5-Fluorouracil (5FU) toxicity assay on T-DNA mutants impaired in uracil phosphoribosyltransferase (UPRT) annotated genes. Figure S3. Opposite transcript profiles for UPP and photosynthesisrelated genes during germination, from publicly available CATMA transcriptome data (project RA03-04). Figure S4. UPP loss of function does not alter Arabidopsis growth in the dark. Table S1. Primers used in this study. Table S2. Annealing temperatures of PCR reactions and amplification product sizes for primer combinations used in this work. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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ª 2009 INRA Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 280–291