Conservation of dual-targeted proteins in Arabidopsis ... - Springer Link

Mol Genet Genomics (2009) 281:525–538 DOI 10.1007/s00438-009-0429-7

ORIGINAL PAPER

Conservation of dual-targeted proteins in Arabidopsis and rice points to a similar pattern of gene-family evolution Carolina V. Morgante · Ricardo A. O. Rodrigues · Phellippe A. S. Marbach · Camila M. Borgonovi · Daniel S. Moura · Marcio C. Silva-Filho

Received: 15 December 2007 / Accepted: 25 January 2009 / Published online: 13 February 2009 © Springer-Verlag 2009

Abstract Gene duplication followed by acquisition of speciWc targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter. Keywords Gene duplication · Dual targeting · Genome evolution

C. V. Morgante and R. A. O. Rodrigues contributed equally to this work. Communicated by S. Hohmann. Electronic supplementary material The online version of this article (doi:10.1007/s00438-009-0429-7) contains supplementary material, which is available to authorized users. C. V. Morgante · R. A. O. Rodrigues · P. A. S. Marbach · C. M. Borgonovi · D. S. Moura · M. C. Silva-Filho (&) Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Av. Pádua Dias, 11, C.P. 83, Piracicaba, SP 13400-970, Brazil e-mail: [email protected]

Introduction Evolution of eukaryotic cells was driven through the segregation of distinct metabolic pathways by the development of a complex system of sub-compartments surrounded by membranes, the nature of each compartment being predominantly determined by the set of proteins that it contained. Although the multi-compartmentalization of eukaryotic cells enabled distinct roles for the organelles, functional overlaps have been observed in many processes in plants, indicating that some enzymes are required in more than one compartment. The acquisition of both mitochondria and chloroplasts was determinant in the evolution of plant cells, supplying them with energy and essential metabolic and biosynthetic pathways that are common to both organelles (Dyal et al. 2004). Most proteins that are required in more than one compartment are encoded by distinct genes and equipped with speciWc targeting information within the protein sequence (Schatz and Dobberstein 1996). It is likely that gene duplication events coincided with the establishment of eYcient and eVective protein translocation systems capable of targeting duplicated gene products to the diVerent organelles in which they were required. Preservation of duplicate genes appears to be related and is common to genes that are expressed in many tissues or encode multidomain proteins (Davies and Petrov 2004). Exceptions to this distinct gene strategy have been observed in increasing numbers of studies that show single gene products targeted simultaneously to more than one compartment by a wide and sophisticated range of mechanisms (Silva-Filho 2003; Mackenzie 2005; Millar et al. 2006). Important insights into genome evolution have emerged from multiple genome sequence analyses. IdentiWcation of evolutionary forces that shaped genomes and impinged

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principles upon gene organization is one of the major challenges of comparative genomics. Gene duplications are a primary force in evolution and more than a third of higher eukaryotic genomes consist of duplicate genes and gene families (Lynch and Conery 2000) Thus, large-scale gene or even entire-genome duplications have played major roles in the evolutionary history of eukaryotes (Vandepoele et al. 2003). Plants have about a 20-fold variation of DNA content and how this reXects on gene content is yet to be fully understood. It has also been shown that either developmental constraints or alternative splicing may reduce the level of persistent gene duplications (Castillo-Davis and Hartl 2002; Yang and Li, 2004; Su et al. 2006). We raised the hypothesis that conservation of dual targeting of proteins in plant species may have had important implications for macro evolutionary changes. To address this issue, we conducted a comparative analysis of the number of dual-targeted (DT) proteins and their corresponding gene family sizes in Arabidopsis thaliana and Oryza sativa. Taken together, our data point out to a similar pattern of genefamily evolution in Arabidopsis and rice.

Materials and methods Protein sequence analyses For genes encoding Arabidopsis isoforms, amino acid sequences of previously described DT proteins were used as primary input inquiries in BLAST (Altschul et al. 1997) searches at “The Arabidopsis Information Resource database” (TAIR, http://www.arabidopsis.org). The Arabidopsis isoforms recovered were used to search the rice genome annotated database at “The Institute for Genomic Research” (TIGR, http://tigrblast.tigr.org). Hits with an e value above 1.0e-65 and protein sequences lacking a complete functional domain were ignored in our analysis. Exceptions were made for the aminoacyl-tRNA synthetase, ascorbate peroxidase and peptide deformylase families (Duchêne et al. 2005; Chew et al. 2003; Teixeira et al. 2006; Giglione et al. 2000; Dinkins et al. 2003). TAIR Microarray Expression Search (http://www.arabidopsis. org), and TIGR, and a TBLASTN were performed against EST databases from NCBI (Altschul et al. 1997) in order to verify if the recovered isoforms were expressed. Predictions of targeting sequences were carried out with Predotar (Small et al. 2004), TargetP (Emanuelsson et al. 2000), and iPSORT (Bannai et al. 2002). The entire amino acid sequences were aligned using CLUSTALW (Thompson et al. 1994). Phylogenetic trees were built using the SplitsTree program (Huson and Bryant 2006) and the neighbour joining method (Saitou and Nei 1987). The number of bootstrap replicates used was 100.

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Mol Genet Genomics (2009) 281:525–538

Cloning of rice dual-targeting sequences N-terminal targeting sequences of methionine aminopeptidase (Os02g52420), monodehydroascorbate reductase (Os08g05570), glutamyl-tRNA synthetase (Os10g22380), and tyrosyl-tRNA synthetase (Os01g31610) from O. sativa were ampliWed by RT-PCR. The reaction was carried out on RNA from leaves of three-month old rice plants. The cDNA was synthesised with ImProm-II™ Reverse Transcription System (Promega), using oligo(dT). The following forward and reverse primers were used: AAG GTACCACACACACACGCAGTCAGGGAGT and TAG GATCCCCAGGTCTTTGACGAGCATTGACATATGG for methionine aminopeptidase; ATGGTACCATGACCTC AGAGGTGGCCGTAGCTCTC and TTGGATCCGTCA GTGCTGGTCGCTCATATGG for monodehydroascorbate reductase; AAGGTACCCAATCTCCGGAGATGATGGCG and TAGGATCCGCGAAGAGGTAGTTGAAGAGC for glutamyl-tRNA synthetase, and CAGGTACCCGCACA GCAAATTTCCTCCTAATCCGTTTC and ATGGATCC CCGATGAGCGATTTGATAGCGTTGGAG for tyrosyltRNA synthetase. Forward primers had a KpnI site at the 5⬘ end and reverse primers had a BamHI site. The PCR products included the Wrst 331, 341, 233 and 455 bp of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase and tyrosyl-tRNA synthetase cDNA sequences, respectively. Because of the high content of GC after the Wrst ATG, and the diYculty in designing primers at these regions, 12 and 41 bp of the 5⬘ untranslated region were included for glutamyl-tRNA synthetase and tyrosyl-tRNA synthetase, respectively. PCR products were digested with BamHI and KpnI restriction enzymes and ligated in frame with the GFP coding sequence of pUCAP-GFP vector, pUCAP containing the expression cassette of pCK-GFP3 (Menand et al. 1998), previously digested with the same enzymes. The expression cassettes, including double 35S promoter, targeting sequence, GFP sequence and terminator, were transferred to pBinPLUS binary vector (van Engelen et al. 1995) by digestion with AscI and Pac I restriction enzymes, followed by ligation. The constructions were used to transform Agrobacterium tumefaciens GV3101 cells. Transient expression in Nicotiana tabacum protoplasts and confocal microscopy analysis Agrobacterium-mediated transient transformation of N. tabacum cv. SR1 leaves was carried out: a single colony of Agrobacterium was inoculated into 5 ml of LB medium containing antibiotics, 10 mM MES (pH 5.6) and 50 M acetosyringone. After incubation at 28°C for 16 h, 1.5 ml culture was centrifuged at 13,000 rpm for 1 min, the pellet was resuspended in 1 ml 10-mM MgCl2 and the OD600 of


527

Table 1 A. thaliana dual-targeted proteins encoded by single genes and its homologous in O. sativa

Alanyl-tRNA synthetase

Asparaginyl-tRNA synthetase

Aspartyl-tRNA synthetase

Cysteinyl-tRNA synthetase

Glycyl-tRNA synthetase

Glutamyl-tRNA synthetase

Histidyl-tRNA synthetase

Lysyl-tRNA synthetase

Methionyl-tRNA synthetase

Loci

Predotar

Target P

iPSORT

Experimental localization

References

Mireau et al. (1996)

At1g50200

M

NC

M

M/C/Cyt

At5g22800

E

C

M

M/C

Os10g10244

M

M

M

–

Os06g13660

C

C

M

–

Os07g15440

E

E

M

–

At4g17300

C

C

M

M/C

At1g70980

E

E

E

–

At5g56680

E

E

E

–

Os07g30200

M

NC

M

–

Os01g27520

E

E

E

–

Os12g22600

E

C

E

–

At4g33760

M

NC

M

M/C

At4g31180

E

C

E

–

At4g26870

E

E

E

–

Os01g06020

E

E

E

–

Os02g46130

E

NC

E

–

Os02g04700

E

E

E

–

At2g31170

C

C

M

M/C

At3g56300

E

E

E

–

At5g38830

E

E

E

–

Os09g38420

ER

M

M

–

Os10g32570

E

E

E

–

Os03g04960

E

E

E

–

At1g29880

E

M

M

Cyt/M

At3g48110

ER

C

C

M/C

Os08g42560

E

E

E

–

Os04g32650

M

M

M

–

At5g64050

E

M

M

M/C

At5g26710

E

C

S

–

Os10g22380

C

C

E

–

Os01g16520

E

E

E

–

Os02g02860

E

NC

M

M/C

At3g46100

M

M

S

M/C

At3g02760

E

E

E

–

Os02g51830

M

NC

M

–

Os05g05840

C

E

S

–

At3g13490

C

M

M

M/C

At3g11710

E

E

E

–

Os02g41470

E

NC

M

–

Os03g38980

E

E

E

–

At3g55400

C

C

C

M/C

At4g13780

E

E

E

–

Os03g11120

M

M

M

–

Os10g26050

E

E

M

–

Os06g31210

E

E

C

–

Duchêne et al. (2005)

(Duchêne et al. 2005)







123

528


Table 1 continued

Phenylalanyl-tRNA synthetase

Prolyl-tRNA synthetase

Seryl-tRNA synthetase

Threonyl- tRNA synthetase

Tryptophanyl-tRNA synthetase

Tyrosyl-tRNA synthetase

Loci

Predotar

Target P

iPSORT


References

At3g58140

E

C

C

M/C


At4g39280

E

E

E

–

At1g72550

E

E

E

–

Os12g34860

M

NC

M

–

Os10g26130

E

E

E

–

Os05g48510

E

E

E

–

At5g52520

E

NC

M

M/C

At3g62120

E

E

E

–

Os07g07060

E

M

M

–

Os12g25710

E

E

E

–

At1g11870

E

M

S

M/C

At5g27470

E

E

E

–

Os03g10190

E

E

E

–

Os11g39670

E

NC

M

–

Os01g37837

E

E

E

–

At2g04842

C

C

C

M/C

At5g26830

M

M

M

–

At1g17960

E

E

E

–

Os08g19850

M

M

M

–

Os02g33500

NC

NC

M

–

At2g25840

E

C

C

M/C

At3g04600

E

E

E

–

Os01g54020

E

NC

M

–

Os12g35570

E

NC

E

–

Os07g17770

E

M

E





At3g02660

C

C

M

M/C

At2g33840

E

C

E

–

At1g28350

E

E

E

–

Os01g31610

M

NC

M

M/C

Os08g05490

E

C

E

–

Os08g23110

E

C

E

–

Os08g09260

E

E

E

–

tRNA-dependant amidotransferase subunit A

At3g25660

M

C

C

M/C

Os04g55050

M

M

M

–

tRNA-dependant amidotransferase subunit B

At1g48520

C

M

M

M/C

Pujol et al. (2008)

Os11g34210

M

M

M

tRNA-dependant amidotransferase subunit C

At4g32915

M

C

M

M/C

Pujol et al. (2008)

Os03g44820

M

C

C von Braun et al. (2007)

tRNA nucleotidyltransferases AtPreP metalloprotease

AtSufE

123

At1g22660

M

C

ER

M/C/Cyt

Os12g07350

ER

M

M

–

At3g19170

M

C

M

M/C

At1g49630

M

C

M

M/C

Os02g52390

M

M

M

–

At4g26500

C

C

C

M/C

Os09g09790

C

C

M

–


Pujol et al. (2008)

Bhushan et al. (2005)

Xu and Moller (2006)


529

Table 1 continued Loci

ATXR methyltransferase

Phage-type RNA polymerase

-type DNA polymerase

DNA gyrase DNA ligase I

Predotar

Target P

iPSORT


References

Raynaud et al. (2006)

At5g09790

C

C

M

C/Nucleus

At5g24330

E

M

M

Nucleus

Os01g73460

NC

C

M

–

Os02g03030

E

M

C

–

At5g15700

E

C

C

M/C

At2g24120

C

C

C

C

At1g68990

M

M

E

M

Os09g07120

C

C

C

M

Os06g44230

C

C

C

C

At3g20540

E

NC

M

M/C

At1g50840

C

C

C

M/C

Os08g07840

C

C

C

–

Os08g07850

E

NC

C

–

Os04g54500

C

C

C

–

At3g10690

C

C

C

M/C

Os03g59750

M

M

M

–

At1g08130

M

C

M

M/Nucleus

At1g49250

E

E

E

–

At1g66730

E

C

E

–

Os10g34750

E

E

E

–

Os01g49180

C

C

M

–

AP2 domain-containing transcription factor TINY

At2g44940

C

C

M

C/Nucleus

OSB Proteins

At5g44785

E

M

M

M/C

At1g47720

E

C

M

M

At4g20010

E

E

C

C

At1g31010

E

C

M

–

Os01g72049

M

M

M

–

Os03g43420

C

C

C

–

Os03g41530

E

M

M

–

At5g35630

C

C

M

M/C

At5g37600

E

E

E

Cyt

At1g66200

E

E

E

Cyt

At3g17820

E

E

E

Cyt

At5g16570

E

E

E

Cyt

At1g48470

E

E

E

Cyt

Os04g56400

C

C

C

–

Os02g50240

E

E

E

–

Os03g12290

E

E

E

–

Os03g50490

E

E

E

–

At3g25740

E

C

C

M/C

At4g37040

E

M

M

M/C

At2g45240

E

NC

E

Cyt

At1g13270

C

C

C

C

Os02g52420

C

M

M

M/C

Os04g52100

E

C

C

–

Os07g25410

E

E

E

–

Glutamine synthetase

Methionine aminopeptidase

Os10g41130

Hedtke et al. (1999, 2000)

Kusumi et al. (2004) Elo et al. (2003) and Christensen et al. (2005)

Wall et al. (2004) Sunderland et al. (2006)

Schwacke et al. (2007)

– Zaegel et al. (2006)

Taira et al. (2004)

Giglione et al. (2000)

123

530


Table 1 continued

Peptide deformylase

Pseudouridine synthase Cryptochrome Preprotein and Amino Acid Transporter

Formate dehydrogenase

Ascorbate peroxidase

Monodehydroascorbate reductase

Glutathione reductase

123

Loci

Predotar

Target P

iPSORT


Os10g36470

E

E

M

–

Os07g32590

C

E

M

–

At1g15390

E

C

E

M/C

References

At5g14660

E

C

C

M/C

Giglione et al. (2000) and Dinkins et al. (2003)

Os01g45070

M

M

M

M/C

Moon et al. (2008)

Os01g44980

M

NC

M

–

Os01g37510

ER

C

E

C

At2g30320

E

M

C

M/C

Os03g21980

M

M

M

–

At5g24850

E

E

E

M/C

Os06g45100

E

E

M

–

At5g24650

E

E

E

M/C

At3g49560

E

E

E

C

Os04g33220

E

M

M

–

At5g14780

E

M

M

M/C

Os06g29180

M

M

M

–

Os06g29220

M

M

M

At4g08390

C

C

M

M/C

At1g77490

C

C

C

–

At4g35000

E

E

E

–

At4g35970

E

E

E

–

At3g09640

E

E

E

–

At1g07890

E

E

E

–

At4g32320

E

C

M

–

At4g09010

C

C

C

–

Os04g35520

C

C

M

–

Os02g34810

C

C

C

–

Os12g07820

M

M

M

M

Os12g07830

M

M

M

–

Os04g14680

E

E

E

P

Os08g43560

E

NC

E

–

Os03g17690

E

E

E

–

Os07g49400

E

E

E

–

Os08g41090

C

C

M

–

At1g63940

C

C

C

M/C

At3g52880

E

E

E

–

At3g27820

ER

E

S

–

At5g03630

E

E

E

–

At3g09940

E

E

E

–

Os08g05570

C

NC

C

M/C

Os09g39380

E

E

E

–

Os08g44340

E

E

E

–

Os02g47800

E

M

S

–

Os02g47790

E

E

S

–

At3g54660

C

C

C

M/C

At3g24170

E

E

E

–

Peeters and Small (2001) Kleine et al. (2003) Murcha et al. (2007)

Herman et al. (2002)

Chew et al. (2003)

Teixeira et al. (2006)

Obara et al. (2002)

Chew et al. (2003)


531

Table 1 continued

Mercaptopyruvate sulfurtransferase

Phosphatidylglycerophosphate synthase I

NAD(P)H dehydrogenase A

NAD(P)H dehydrogenase B

NAD(P)H dehydrogenase C Glutathione S-transferase F8

Holocarboxylase synthetase 1

Ribosomal protein S16

Aldolase

Loci

Predotar

Target P

iPSORT


Os03g06740

C

C

C

–

Os10g28000

E

E

M

–

Os02g56850

E

E

E

–

At1g79230

NC

C

M

M/C

At1g16460

E

E

M

Cyt

Os12g41500

E

E

E

–

Os02g07040

E

NC

E

–

At2g39290

E

C

M

M/C

At3g55030

E

E

E

–

Os03g17520

C

C

M

–

At1g07180

M

M

C

M/P

At2g29990

M

M

C

M/P

Os01g61410

M

M

M

–

Os07g37730

M

M

M

–

At4g28220

M

M

E

M/P

At4g21490

ER

ER

E

–

At4g05020

E

E

E

–

At2g20800

E

M

M

–

Os06g47000

M

M

M

–

Os05g26660

M

M

M

–

Os08g04630

ER

M

M

–

At5g08740

C

C

C

M/C

Os06g11140

C

C

M

–

At2g47730

E

C

C

C/Cyt

At4g02520

E

E

C

–

Os01g27390

E

E

E

–

At2g25710

E

E

M

C/Cyt

At1g37150

E

E

E

–

Os02g07040

M

M

E

–

At4g34620

M

M

M

C

At5g56940

NC

M

M

M/C

Os09g32274

M

M

M

M/C

Os08g40610

M

M

M

M/C

At3g52930

E

E

E

M/Cyt

At2g36460

E

E

M

–

At5g03690

M

M

M

–

At4g26530

E

E

M

–

At4g26520

E

E

E

–

At2g21330

C

C

M

–

At4g38970

C

C

M

–

At2g01140

C

C

C

–

Os01g67860

E

E

E

–

Os05g33380

E

E

E

–

Os10g08022

E

E

E

–

Os08g02700

E

E

E

–

Os06g40640

E

E

M

–

References

Nakamura et al. (2000)

Babiychuk et al. (2003)

Carrie et al. (2008)

Carrie et al. (2008)

Carrie et al. (2008) Thatcher et al. (2007)

Puyaubert et al. (2008)

Ueda et al. (2008) Ueda et al. (2008) Giegé et al. (2003)

123

532


Table 1 continued

Enolase

Thiazole biosynthetic enzyme Vacuoleless 1 Vacuolar protein sorting 11 Vacuolar protein sorting 33

Loci

Predotar

Target P

iPSORT


Os01g02880

C

M

M

–

Os11g07020

C

E

M

–

At2g36530

E

E

E

M/Cyt

At1g74030

E

C

C

–

At2g29560

E

E

E

–

Os03g14450

E

E

E

–

Os10g08550

E

C

C

–

Os06g04510

E

E

E

–

Os09g20820

C

M

M

–

Os03g15950

E

E

E

–

At5g54770

C

C

C

M/C

Os07g34570

C

C

M

–

At2g38020

E

E

E

T/PVC

Os01g47650

E

E

E

–

At2g05170

E

E

E

T/PVC

Os04g31390

E

E

E

–

At3g54860

E

E

E

T/PVC

Os04g14650

E

E

E

–

References

Giegé et al. (2003)

Chabregas et al. (2003) Rojo et al. (2003) Rojo et al. (2003) Rojo et al. (2003)

C chloroplast, Cyt cytosol, E elsewhere (programs are not able to assign a subcellular location with values equal or above 0.5), ER endoplasmatic reticulum, M mitochondria, NC no consensus (programs are assigning two subcellular locations with values equal or above 0.5), P Peroxissome, PVC prevacuolar compartment, S secretory pathway, T tonoplast

the mixture was adjusted to 0.2–0.3. Acetosyringone was added to a Wnal concentration of 100 M. Abaxial surfaces of leaves were inWltrated by using a syringe without needle. Mesophyll protoplasts were prepared from the inWltrated tobacco leaves as described by Carneiro et al. (1993). Samples were analyzed at 4 and 5 days after inWltration with an Olympus FV1000 confocal laser scanning microcospe. Excitation Wlters and emission were as follows: GFP, 488 nm excitation, 510–550 nm emission; MitoTracker Red FM, 543 nm excitation, 590–640 nm emission; chlorophyll autoXuorescence, 635 nm excitation, 670–700 nm emission. Images were captured using Olympus Fluoview FV10-ASW software. To speciWcally dye mitochondria, MitoTracker was added to the protoplasts suspension, at 300 nM Wnal concentration, for 40 min prior to observation.

Results In order to understand the evolution of genes encoding DT proteins and how it aVected gene duplication, we performed a search of the complete annotated Arabidopsis and rice genomes for orthologues and paralogues of experimentally proved DT proteins. The identiWcation of the rice closest orthologue of an Arabidopsis thaliana DT protein would indicate a candidate for a protein with a similar function

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and probably with a similar localization. At present, there are 58 Arabidopsis proteins that have been shown, experimentally, to translocate to more than one subcellular compartment, mostly to mitochondria and chloroplasts. These 58 DT proteins can be arranged into 52 gene families (Table 1). All genes found in Arabidopsis have homologues in the rice genome. Combining sequence homology analysis, presence of functional domains and expression data allowed us to establish orthology between rice and Arabidopsis sequences. Twenty-seven out of the 52 families displayed the same number of paralogous proteins in both Arabidopsis and rice, with a similar pattern of gene expansion. The remaining 25 families could be divided into two groups: one with 12 families presenting a higher number of paralogous proteins in Arabidopsis compared to rice and the other with 13 families with a higher number of paralogous proteins in rice than in Arabidopsis. However, the number of paralogues found in rice and Arabidopsis did not highly diVer despite the fact that the rice genome is estimated to be fourfold larger than the Arabidopsis genome and that rice has over 40% more unique expressed sequences than Arabidopsis (Plant Gene Indices at http:// www.tigr.org). Prediction of the subcellular location of members of DT protein families using available softwares, as well as the experimentally conWrmed localization of the paralogous


533

Fig. 1 Phylogenetic trees of Arabidopsis thaliana and Oryza sativa (a) monodehydroascorbate reductase (b) methionine aminopeptidase (c) glutamyltRNA synthetase, and (d) tyrosyl-tRNA synthetase. The trees are based on amino acid sequence alignments and generated by neighbour joining. Numbers on the branches indicate the bootstrap values

proteins are indicated in Table 1. Comparison of the output of these programs shows that they often disagree on the Wnal location of the protein. Therefore, combining of bioinformatics targeting algorithms, comparative genomic analysis and experimental approaches is required to validate of protein location (Heazlewood et al. 2005).

To ascertain the accuracy of paralogous determination (see “Materials and methods” for details), we reconstructed the phylogeny for all families listed in Supplementary material. Phylogenetic distribution of randomly selected monodehydroascorbate reductase, methionine aminopeptidase, glutamyl-tRNA synthetase and tyrosyl-tRNA synthetase

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534


Fig. 1 continued

gene families (Table 1) revealed that Arabidopsis DT proteins form unique clusters with the rice isoforms (Fig. 1). Arabidopsis monodehydroascorbate reductase, encoded by At1g63940, translocate to both mitochondria and chloroplasts (Obara et al. 2002). It belongs to a gene family that encodes Wve paralogous proteins in both Arabidopsis and rice (Fig. 1a). Phylogenetic reconstruction showed that rice Os08g05570 clustered with Arabidopsis At1g63940. This suggests that the rice orthologue also translocates to mitochondria and chloroplasts. Arabidopsis methionine aminopeptidases are also dual-targeted to both mitochondria and chloroplast (Giglione et al. 2000). The rice methionine aminopeptidase Os02g52420 gene product branches speciWcally with an orthologous Arabidopsis protein (Fig. 1b), encoded by At4g37040 as observed with the monodehydroascorbate reductase family. The same occurs to Arabidopsis tyrosyl-tRNA synthetase At3g02660, dualtargeted to mitochondria and chloroplasts (Duchêne et al. 2005), and its homologue Os01g31610 in rice (Fig. 1c), and to Arabidopsis glutamyl-tRNA synthetase At5g64050, also imported to these two organelles (Duchêne et al. 2005), and its homologue Os02g02860 in rice (Fig. 1d). The intracellular localization of the rice orthologues suggested by the phylogenetic trees (Fig. 1) was conWrmed by using recombinant fusion proteins in which the N-terminal coding sequences of the rice cDNAs were fused to GFP

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reporter genes. The subcellular localization of the chimeric proteins was determined by transient transformation of N. tabacum protoplasts. This in vivo approach avoids the problems associated with in vitro targeted systems, particularly when considering proteins simultaneously translocated to mitochondria and chloroplast (Silva-Filho et al. 1997; Cleary et al. 2002). As predicted by the phylogenetic reconstruction, the rice orthologues were also transported to both mitochondria and chloroplast (Fig. 2).

Discussion Despite the asymmetric evolution of gene families by increased gene duplications of otherwise paralogous genes, our data demonstrated conservation of a dual targeting strategy between orthologous proteins (methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase) of Arabidopsis and rice. The methionine aminopeptidase gene family was shown to be composed by four paralogous genes in Arabidopsis whereas the rice gene family was organized into Wve paralogues, indicating that it contains more gene duplication than its Arabidopsis counterpart (Fig. 1b). This is supported by studies that showed a more extensive diploidization in rice than Arabidopsis and also that gene


535

Fig. 2 Dual localization of rice glutamyl-tRNA synthetase-GFP (GluRS Os02g02860), tyrosyl-tRNA synthetase-GFP (TyrRS Os01g31610), methionine aminopeptidase-GFP (MetAP Os02g52429), and monodehydroascorbate reductase-GFP (MDHAR Os08g05570) fusion proteins. Chimerci proteins were introduced by Agrobacterium tumefaciens–mediated transfection in tobacco protoplasts. Fluorescence images were taken using a confocal laser scanning microscope.

GFP corresponds to the Xuorescence detected in the green channel; Chlorophyll, detected in far-red channel, corresponds to the chloroplast autoXuorescence signal; Mitotracker signal was detected in the red channel; Mitotracker + GFP corresponds to the merging of red and green channels; Chlorophyll + GFP corresponds to the merging of farred and green channels; yellow represents the co-localization of green and red signals. All scale bars represent 5 m

loss is not a random event (Bowers et al. 2003; Paterson et al. 2004). Analyzing the DT data recorded in the literature, examples supporting conservation of the DT of homologous proteins become evident among dicots and also between monocots and dicots. The Arabidopsis -type DNA polymerases (At3g20540, At1g50840) (Elo et al. 2003; Christensen et al. 2005) and its two homologues in N. tabacum (Ono et al. 2007) are dual-targeted to mitochondria and chloroplasts. The same occurs to DNA gyrase A, which is dual-targeted to these two organelles in A. thaliana (Wall et al. 2004) and N. benthamiana (Cho et al. 2004). The protein glutathione reductase also presents DT to mitochondria and chloroplasts in Arabidopsis (Chew et al. 2003) and pea (Creissen et al. 1995). The phage-type RNA polymerase (At5g15700) (Hedtke et al. 2000) and its homologs in N. tabacum (Hedtke et al. 2002) and N. sylvestris (Kobayashi et al. 2001) are dual-targeted to mitochondria and chloroplasts. Interestingly, DT of this polymerase was also reported in the moss Physcomitrella patens (Richter et al. 2002). DT to mitochondria and chloroplasts has been

reported for homologous proteins in monocots and dicots: ribosomal protein S16 of Arabidopsis (At5g56940) and rice (Os09g32274) (Ueda et al. 2008); peptide deformylase 1B in Arabidopsis (At5g14660) (Dinkins et al. 2003) and rice (Os01g45070) (Moon et al. 2008), and seryl-tRNA synthetase in Arabidopsis (Duchêne et al. 2005) and maize (Rokov-Plavec et al. 2008). The fact that Arabidopsis and rice genomes, despite the fourfold diVerence in size, represent small genomes among plants prompted us to search for homologues of DT proteins in maize, with a larger 2,365 Mb genome and a representative amount of sequences in public databases. The maize genome evolved by complete genome duplication due to allotetraploidy and by segmental duplications, and expanded rapidly by retrotransposition events (Jorgensen 2004). A complete survey of the maize genome was not possible given that some sequence information was still missing, but for some gene families analysed we could identify homologues for all the Arabidopsis and rice paralogues. In spite of its larger genome size, the number of maize isoforms was nearly the same as observed for

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536

Arabidopsis and rice (data not shown). It is thus likely that the prevalence of dual targeted proteins is not increased in expanded genomes. The understanding of the evolutionary forces that shape gene family sizes is far from complete. Based on the comparison of currently known gene families containing DT members, on the experimentally conWrmed DT conservation of aminopeptidase, monodehydroascorbate, glutamyl-tRNA synthetase, tyrosyl-tRNA synthetase, peptide deformylase, and ribosomal protein S16 in rice and Arabidopsis, and on the double role of DT proteins in a cell, we propose that DT proteins may be considered one of the evolutionary forces shaping gene family size. Although the relatively small number of 50 gene families currently known to have DT members limits, the data presented here indicate an evolutionary tendency for gene families containing dual-targeting members. Detailed analysis of each known family of DT genes and certainly the characterization of novel and still unknown DT proteins will further expound what is here proposed. Acknowledgments This research was supported by FAPESP and CNPq, Brazil. C.V.M. was supported by a fellowship from CNPq. R.A.O.R., P.A.S.M. and C.M.B. were supported by fellowships from FAPESP. M.C·S.F. is a research fellow of CNPq. The authors thank Carlos Menck and Christine Stock for helpful suggestions and critical read of the manuscript.

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