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AOBPreview originally published online on October 21, 2005
Annals of Botany 2005 96(7):1307-1314; doi:10.1093/aob/mci282
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© The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

NADP-Malate Dehydrogenase Gene Evolution in Andropogoneae (Poaceae): Gene Duplication Followed by Sub-functionalization

P. RONDEAU1, C. ROUCH1 and G. BESNARD2,*

1 Université de la Réunion, LBGM, 15 Avenue R. Cassin, 97715 St-Denis Messag Cedex 9, La Réunion, France and 2 Department of Ecology and Evolution, Biology Building, University of Lausanne, 1015 Lausanne, Switzerland

* For correspondence. E-mail gbesnar{at}hotmail.com

Received: 22 November 2004    Returned for revision: 18 May 2005    Accepted: 15 September 2005    Published electronically: 21 October 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

• Background and Aims Plastid NADP-dependent malate dehydrogenase (MDH) catalyses the conversion of oxaloacetate to malate. In C4 plants, it is involved in photosynthetic carbon assimilation. In Poaceae, one NADP-MDH gene has been identified in rice (C3; Erhartoideae) and maize (C4; Panicoideae), whereas two tandemly repeated genes have been identified in Sorghum (C4; Panicoideae). In the present study, the molecular evolution of the NADP-MDH multigene family was investigated in order to analyse how the C4 isoform has evolved over a broader range of panicoid grasses.

• Methods Polymerase chain reaction (PCR)-based cloning was used to isolate cDNAs encoding NADP-MDHs from 15 species of Panicoideae. A gene phylogeny was reconstructed based on cDNA sequences using distance and maximum parsimony methods. Episodic selection along some branches of the phylogenetic tree was tested by analysing non-synonymous and synonymous rate ratios.Transcription of NADP-MDH genes was compared in green leaves of five accessions of Saccharum, Sorghum and Vetiveria using a semi-quantitative PCR approach.

• Key Results Phylogenetic analyses of these data support the existence of two NADP-MDH gene lineages (NMDH-I and NMDH-II) in several Andropogoneae (i.e. Saccharum, Sorghum and Vetiveria). Episodic positive selection was shown along the basal branch of the NMDH-II clade. Three amino acid modifications allow the two gene lineages to be distinguished, suggesting a positive selection at these sites. In green leaves, we showed that the transcript accumulation was higher for NMDH-I than for NMDH-II.

• Conclusions It is hypothesized that the maintenance of both NADP-MDH genes in some Andropogoneae is due to a partition of the original functions across both copies. NMDH-I probably corresponds to the C4 isoform as previously suggested. Nevertheless, some C4 species (e.g. maize) only have one gene which should be selected for its high expression level in leaves. This study confirms that gene duplicates have been recruited for C4 photosynthesis but are not required in every case.

Key words: C4 photosynthesis, gene duplication, multigene family, NADP-MDH, Poaceae, positive selection, sub-functionalization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
C4 photosynthesis provides a selective advantage for plants growing in tropical areas (Sage, 2004Go). The C4 pathway serves as a pump, which provides an elevation of the CO2 concentration at the site of Rubisco, leading to limited photorespiration and high water use efficiency in C4 species (von Caemmerer and Furbank, 2003Go). This pathway has appeared several times during plant evolution from the Oligocene and Miocene, at a period when atmospheric CO2 levels were gradually declining (Edwards et al., 2001Go; Sage, 2004Go). C4 genes are of great interest since an aim of biotechnology is to introduce the C4 traits to cultivated C3 plants, possibly improving their performance under warm conditions (Matsuoka et al., 2001Go). Phylogenetic and biochemical studies of C4 genes have also provided insights into the molecular evolution of the C4 pathway (e.g. Sheen, 1999Go; Gehrig et al., 2001Go; Besnard et al., 2003Go; Monson, 2003Go; Westhoff and Gowik, 2004Go). In Poaceae, multiple appearances of C4 photosynthesis during the PACCAD (Panicoideae, Arundinoideae, Chloridoideae, Centothecoideae, Aristidoideae, Danthonioideae) clade diversification have probably occurred (Kellogg, 2000Go). Using a phylogenetic approach on the grass phosphoenolpyruvate carboxylase multigene family, it has been proposed that such multiple appearances should be associated with multiple inductions of a pre-existing system (Besnard et al., 2003Go). Study of other multigene families including a C4 isoform (NADP-malate dehydrogenase, pyruvate orthophosphate dikinase) is required to understand thoroughly how the C4 pathway evolved during grass diversification.

In plants, plastid NADP-dependent malate dehydrogenases (NADP-MDHs; EC 1.1.1.82 [EC] ) are essential enzymes involved in photosynthesis (Scheibe, 1987Go; Sheen, 1999Go). These enzymes are implicated in two main functions. First, in all plants, NADP-MDH helps control export of the reducing equivalents (malate) from the chloroplast to the cytosol (Scheibe, 1987Go; Ocheretina et al., 2000Go). Secondly, NADP-MDH is also involved in photosynthetic carbon assimilation of both C4 NADP-malic enzyme type and C4 phosphoenolpyruvate carboxykinase (PCK) enzyme type plants because it converts oxaloacetate to malate, which is then transferred from the mesophyll cells to the bundle sheath cells (von Caemmerer and Furbank, 2003Go). NADP-MDH is a redox-regulated enzyme (Miginiac-Maslow et al., 2000Go). Redox regulation appeared relatively late in evolution (probably in green algae; Ocheretina et al., 2000Go). NADP-MDH is a highly conserved enzyme in plants, and is apparently encoded by a small multigene family (Luchetta et al., 1991Go). Phylogenetic trees based on this gene family should be appropriate for increasing our understanding of the evolution of NADP-MDH isoforms and particularly to study the appearance(s) of the C4 NADP-MDH isoform during plant evolution.

In maize and sorghum, two grasses belonging to tribe Andropogoneae of subfamily Panicoideae (GPWG, 2001Go), NADP-MDH genes have been characterized (Metzler et al., 1989Go; Luchetta et al., 1991Go). Only one NADP-MDH nuclear gene was identified in maize (Metzler et al., 1989Go) and in the complete genome sequence of rice (Kikuchi et al., 2003Go). Conversely, in sorghum, the existence of two tandemly repeated genes encoding NADP-MDH was demonstrated (Luchetta et al., 1991Go). The regulation of the two genes was differential, with one being light induced (C4 gene; NMDH-I) and the other (NMDH-II) being constitutively expressed at a low level. The function of the latter was not clearly elucidated but it could be involved in the export of reducing equivalents. Based on these data, Luchetta et al. (1991)Go postulated that the grass C4 NADP-MDH gene could have been derived from a non-C4 NADP-MDH. In contrast, McGonigle and Nelson (1995)Go proposed that only changes in the regulation of a pre-existing gene were involved in the C4 NADP-MDH appearance in the genus Flaveria (Asteraceae), without gene duplication during the evolution from C3 to C4 plants. Only a few DNA sequences of NADP-MDH genes are currently available, and consequently the evolution of the NADP-MDH multigene family is still not well understood (Ocheretina et al., 2000Go).

In this study, PCR-based cloning was used to isolate cDNAs encoding NADP-MDHs from 15 species belonging to Panicoideae. From green leaves, cDNAs related to the two sorghum isoforms were isolated. A phylogenetic approach was used to provide insights into the evolution of the NADP-MDH multigene family in Panicoideae. Episodic selection along some branches of the phylogenetic tree was tested. In addition, an analysis of transcription specificities of NADP-MDH genes was conducted for three genera of Andropogoneae.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Molecular analysis
In order to isolate cDNA sequences encoding NADP-MDHs in grasses, 15 species belonging to Panicoideae were analysed (Table 1). Twelve are C4 NADP-malic enzyme type plants (representatives of Andropogoneae plus Paspalum paniculatum and Paspalidium geminatum), Urochloa maxima and Melinis repens are C4 PCK enzyme type plants, and Oplismenus compositus is a C3 plant. For each accession, green leaves were sampled at noon. Total RNA was extracted and then reverse transcription reactions were performed as previously described (Besnard et al., 2002Go).


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  		TABLE 1. Species origin, accession number and reference of NADP-MDH sequences used for analyses

 
From the available NADP-MDH sequences of sorghum [EMBL accessions X53453 [GenBank] (NMDH-I) and S55884 [GenBank] (NMDH-II); Luchetta et al., 1991Go] and maize (EMBL accession X16084 [GenBank] ; Metzler et al., 1989Go), specific primer pairs were designed to amplify cDNA segments of NADP-MDH using reverse transcription–polymerase chain reaction (RT–PCR). Primers were defined in conserved regions of available grass NADP-MDH genes. Two forward primers were defined: 5' GAGTGCTTCGGGGTCTTCTGC 3' (A) and 5' ATGGGCCTGTCAACAGYTTACTCC 5' (B). The same reverse primer was used in all reactions: 5' TACATTTCTCCTGGTAGCATTGTATC 3' (R). Long cDNA segments (of approx. 1100 bp with primers AR and 1300 bp with primers BR) were PCR generated. The PCR conditions described in Besnard et al. (2002)Go were used with an annealing temperature of 50 °C for each primer pair. RT–PCR fragments were cloned before sequencing. The pGEM®-T vector (Promega, Madison, WI, USA) and Escherichia coli strain DH5{alpha} were used according to the supplier's recommendations. For each species of Andropogoneae, distinct sequences were looked for among 15 clones using their restriction profile. The insert in each clone was amplified by PCR using SP6 and T7 primers (Promega). PCR products were restricted with HpaII, separated on a 3 % agarose gel and stained with ethidium bromide. Each clone displaying a different profile was then sequenced. Double-stranded DNA sequencing reactions were performed by the ESGS Society (Euro Sequence Gene Service, Evry, France). Sequences have been deposited in the EMBL databank.

Phylogenetic analysis
NADP-MDH cDNA sequences from Sorghum, Zea and Oryza from GenBank were added to the data (Table 1). For Saccharum and Sorghum, both cultivated forms (Saccharum officinarum and Sorghum bicolor) and close wild relatives [Saccharum spontaneum and Sorghum arundinaceum (= S. verticilliflorum)] were studied (Dillon et al., 2001Go; Hodkinson et al., 2002Go). Only coding sequences (cds) were used for analysis. In addition, the phylogenetic trees were rooted with an outgroup sequence. Since no complete NADP-MDH sequence from another monocot was available in GenBank, a cDNA sequence from Flaveria (U22533 [GenBank] ; Asteraceae) was used. A sample of 25 sequences (Table 1) displaying the major part of the cds was considered. Sequence alignments were performed using Clustal W (Thompson et al., 1994Go). Phylogenetic analyses were made using a large internal cDNA segment of 1050 bp in which indels are very infrequent (only one 3 bp deletion was detected in S. spontaneum sequences). In the sorghum sequence X53453 [GenBank] , the 1050 bp segment is located between nucleotides 220 and 1269. Phylogenetic analyses were conducted using MEGA version 2 (Kumar et al., 2004Go). Mean frequencies of each nucleotide were similar (T = 25 %, A = 29 %, G = 26 %, C = 20 %), but transitions were twice as frequent as transversions. Consequently, to consider this unequal probability of the substitution types, the Kimura 2-parameter (Kimura, 1980Go) was used to compute distances between each pair of sequences. Phylogenetic trees were then constructed using the neighbour joining algorithm (NJ; Saitou and Nei, 1987Go). Bootstrap values were computed using 1000 replicates to evaluate support for the groupings. In addition, a maximum parsimony (MP) analysis was conducted. All characters were equally weighted. Gaps were treated as missing data. A heuristic search was used to find the most-parsimonious trees. The close-neighbour-interchange algorithm was used with a search level of 3 (Kumar et al., 2004Go). Searches included 100 replications of random addition sequences. All the best trees were retained. A strict consensus tree was generated from the equally most-parsimonious trees. Bootstrap values were computed using 1000 replicates with MEGA (Kumar et al., 2004Go) to evaluate support for the branches.

Compared evolution of NADP-MDH genes in Andropogoneae
Within Andropogoneae, two NADP-MDH genes were identified (NMDH-I and NMDH-II; see below). Using all available sequences, amino acid sites providing a distinction between these two genes were investigated. A segment of 350 amino acids was used. In the Sorghum NMDH-I protein (429 amino acids; X53453 [GenBank] ), this segment is situated between amino acids 70 and 419. In addition, in order to determine possible selective divergence between the two NADP-MDH genes, the rates of non-synonymous and synonymous substitutions between pairs of sequences from S. officinarum, S. bicolor and Vetiveria zizanioides were compared independently for NMDH-I and NMDH-II. The method of Nei and Gojobori (1986)Go was applied.

Within Andropogoneae, the occurrence of episodic positive selection following gene duplication was also tested on the 1050 bp segment. Eleven sequence accessions from Saccharum, Sorghum, Vetiveria, Zea and Oryza were considered (M31965 [GenBank] , S55884 [GenBank] , X16084 [GenBank] , X53453 [GenBank] , AJ344432 [GenBank] , AJ344433 [GenBank] , AJ416567 [GenBank] , AJ512374 [GenBank] , AJ512375 [GenBank] , AJ565857 [GenBank] and AK105935 [GenBank] ). The AJ565856 [GenBank] accession was not used in this analysis since it probably encodes a non-functional enzyme (see below). Furthermore, in the genus Saccharum, only one NMDH-I sequence (AJ565857 [GenBank] ) was used because accessions AJ565857 [GenBank] and AJ833960 [GenBank] only differ by one non-synonymous substitution. The structure of the phylogenetic tree was defined based on an MP analysis as previously described. The dN/dS ratios ({omega}) were estimated for all branches of the tree by using a maximum likelihood (ML) analysis as described by Yang (1998)Go. We used the CODEML program implemented in the PAML package (Yang, 1997Go) using the ‘free ratio’ model assuming an independent dN/dS ratio for each branch of the phylogenetic tree (Yang, 1998Go). The F3X4 codon model was used; it takes into account differences in transversion vs. transition and codon usages between analysed sequences.

NADP-MDH gene transcription analysis in Andropogoneae
To quantify differential NADP-MDH RNA accumulation between different plant materials (i.e. green leaf, etiolated leaf, root and stem) in S. officinarum, we used a Northern dot-blot membrane (see Besnard et al., 2003Go). For each tissue sample of cultivar R570, 10 mg of total RNA was directly located onto an N+-Hybond nylon membrane (Amersham, Bucks, UK). An NADP-MDH segment of 357 bp (accession AJ320266 [GenBank] ) was hybridized as a probe using the procedure described in Besnard et al. (2003)Go. In Saccharum, this probe displays great similarity (at least 94 %) with both genes NMDH-I and NMDH-II. Consequently, this probe cannot be considered as specific to one NADP-MDH gene. The membrane was also independently hybridized with a ribosomal 18S probe. Ribosomal 18S gene expression was considered to be constitutive and proportional to the amount of deposited RNA. The light induction of the NADP-MDH transcription was estimated using the relative signal intensity of 18S and NADP-MDH genes on the Northern dot-blot for both green and etiolated leaves.

A semi-quantitative PCR approach was developed to compare transcript accumulation of NMDH-I and NMDH-II in green leaves of Saccharum, Sorghum and Vetiveria. One accession each for S. arundinaceum and V. zizanioides and three for S. officinarum accessions [i.e. SES14 (wild accession), Big Tana Rayé and R570 (cultivated accessions)] were characterized. Several restriction sites allowing distinction between NMDH-I and NMDH-II were identified. One primer pair was defined to amplify simultaneously an internal cds region of 253 bp of both NADP-MDH genes containing a diagnostic HpaII restriction site: For-5' GCCCTTCTTATTGGTGCTAAG 3' and Rev-5' CTAGCTGGCACTTTGCTCTAT 3'. PCR mixtures, containing 0·25 mM dNTP, 10 mM Tris–HCl (pH 8·3), 1·5 mM MgCl2 and 20 pmol of each oligonucleotide primer in a total volume of 50 µL, were added to 0·3 mg of cDNA and 1·2 U of Taq DNA polymerase (Advanced Biotechnologies, Inc., Columbia, MD, USA) and amplified in a thermocycler (PTC100-v7, MJ Research). Samples were incubated for 4 min at 94 °C, followed by 36 cycles consisting of 45 s at 94 °C, 45 s at 50 °C and 45 s at 72 °C. This PCR program was followed by steps of decreasing temperature from 94 to 50 °C on the basis of 3 min per degree, to avoid heteroduplex formation. A 10 µL aliquot of PCR product was then restricted with HpaII (Sigma-Aldrich Corp., Missouri, USA) following the supplier's recommendations. Restriction fragments were separated on a 3 % agarose gel and stained with ethidium bromide. The gels were photographed using a Kodak DC290 digital camera. Photo treatment for a relative quantification of NMDH-I or NMDH-II mRNA of each gene was carried out using the Kodak 1D Image analyser software (version 3·6). A correction was made between the ethidium bromide staining intensity and the size of the fragments [253 bp for the unrestricted fragment (NMDH-I) and 220 bp for the restricted fragment (NMDH-II)]. To test the reproducibility of the method, three independent assays were performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Data sequences and phylogenetic relationships between NADP-MDH
Nineteen 19 cDNA sequences encoding NADP-MDHs were generated in the species analysed (Table 1). One sequence from S. spontaneum (AJ565856 [GenBank] ) probably corresponds to a pseudo-gene due to the presence of a termination codon in the middle of the sequence. The phylogenetic trees based on the sequence analysis of the 1050 bp segment are presented in Fig. 1. The different methods used to reconstruct phylogenies led to similar results. Oryza, as expected with this level of sampling, is sister to Panicoideae. In a non-monophyletic tribe Paniceae, Paspalum is separated from Paspalidium, Melinis, Urochloa and Oplismenus, although with only weak bootstrap support. Paspalum is sister to Andropogoneae in the MP tree, whereas it is sister to the rest of the Panicoideae in the NJ tree. Lastly, phylogenetic reconstruction shows that the clade including all NADP-MDHs from Andropogoneae is well supported (>98 %; Fig. 1). Nevertheless, in this clade, two groups of sequences can be recognized (named NMDH-I and NMDH-II; Fig. 1B). In each of these two sub-clades, there is at least one sequence from Saccharum, Sorghum and Vetiveria. The sequence of Zea is not clearly assignable to either of these two sub-clades.



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  		FIG. 1. Phylogenetic relationships between cDNAs encoding NADP-malate dehydrogenase in grasses. The analysis is based on a 1050 bp segment (423 variable sites of which 228 are potentially parsimony informative). (A) An MP tree based on a maximum parsimony analysis (904 steps, CI = 0·64, RI = 0·57). The consensus tree is given (from the three most-parsimonious trees). Species belonging to either Andropogoneae or Paniceae are indicated. (B) An NJ tree based on Kimura 2-parameter distance and the neighbour-joining algorithm. The two gene lineages NMDH-I and NMDH-II are specified. Bootstrap values >50 % are indicated on each tree.

 
Evolution of NADP-MDH genes in Andropogoneae
The comparison of mutation rates between the same species for each NADP-MDH gene revealed that the ratio {omega} (dN/dS) is 1·3–2·0 times higher in NMDH-II than in NMDH-I (Table 2). This suggests that the evolution of NMDH-II was slightly faster than that of NMDH-I. Using an ML analysis to estimate the dN/dS ratios along phylogenetic branches, a value >1·0 was obtained at the base of the NMDH-II clade ({omega} = 1·60; Fig. 2). This probably indicates an episodic positive selection on the NMDH-II gene. Within the 350 amino acid sequence, three sites allow a distinction between NMDH-I and NMDH-II. First, at position 329 (in the sorghum sequence X53453 [GenBank] ), NMDH-I proteins have an alanine whereas all NMDH-II proteins have a valine. The other sequences from Paniceae, Zea and Oryza also have a valine. (This modification is a conservative mutation that should not induce a major change in the enzyme characteristics.) Secondly, at position 292 (in X53453 [GenBank] ), a lysine is found in proteins belonging to NMDH-I in Paniceae, Zea and Oryza, whereas a glutamine or a glutamate is found in NMDH-II proteins. Thirdly, at position 414, an asparagine is found in all proteins belonging to NMDH-I in Paniceae, Zea and Oryza, whereas an aspartate is found in NMDH-II proteins.


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  		TABLE 2. Non-synonymous (dN) and synonymous (dS) mutation rates computed by comparison of pairs of sequences from Saccharum officinarum, Sorghum bicolor and Vetiveria zizanioides

 


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  		FIG. 2. An MP tree based on a subset of 11 NADP-MDH cDNAs representative of the gene lineages NMDH-I and NMDH-II in the tribe Andropogoneae (genera Saccharum, Sorghum and Vetiveria). Only one most-parsimonious tree was obtained (289 steps, CI = 0·85, RI = 0·71). Bootstrap values >50 % are indicated. The tree topology was used to estimate dN/dS ({omega}) values along each branch based on a maximum likelihood analysis and a free ratio model. The numbers in parentheses are the maximum likelihood estimates of the numbers of synonymous and non-synonymous substitutions, respectively, along the basal branch of NMDH-II.

 
Comparative NADP-MDH gene transcription in Andropogoneae
Using a Northern dot-blot approach, higher NADP-MDH gene transcription was revealed in green leaves of sugarcane cultivar R570 (Fig 3A) than in roots, stem and etiolated leaves. This confirms that the transcription of NADP-MDH genes is light induced in Saccharum. It was estimated that the transcript accumulation was about seven times higher in green leaves than in etiolated leaves. Using the semi-quantitative PCR method, it was revealed that both NADP-MDH genes are transcribed in green leaves of Saccharum, Sorghum and Vetiveria (Fig. 3B), and it was shown that the amount of cDNA was usually higher for NMDH-I than for NMDH-II. However, similar amounts of cDNA were observed for both genes in cultivar R570 (54 and 46 %; Fig. 3B).



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  		FIG. 3. NADP-malate dehydrogenase gene transcription analyses. (A) Gene transcript accumulation according to different plant materials in sugarcane cultivar R570 revealed using Northern dot-blot analysis. A nylon membrane with RNA from four types of plant material (green leaf, etiolated leaf, stem and root) was hybridized successively with two probes: an 18S rDNA probe (30 min exposure) and an NADP-MDH probe (7 d exposure). (B) Relative frequency of NMDH-I and NMDH-II transcripts in green leaves of five accessions belonging to Saccharum, Sorghum and Vetiveria assessed using the PCR quantification method. The photograph illustrates an example of restricted DNA fragments of NMDH-I (253 bp) and NMDH-II (220 bp) separated on a 3 % agarose gel. Histograms give the relative transcript abundance of NADP-MDH genes in each plant based on three repeats (data are shown as the means±s.d.). *BTR = Big Tana Rayé.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The phylogenetic trees based on NADP-MDH sequences (Fig. 1) allow a distinction to be made between a paraphyletic Paniceae and a monophyletic Andropogoneae in sub-family Panicoideae. The separation of Paspalum from other Paniceae analysed (Paspalidium, Melinis, Urochloa and Oplismenus; Fig. 1B) was expected since paraphyly in this tribe has been demonstrated using other gene sequences (e.g. Giussani et al., 2001Go; Besnard et al., 2002Go; Duvall et al., 2003Go). In Andropogoneae, the existence of two distinct genes encoding NADP-MDH (named NMDH-I and NMDH-II) is supported by our study, and this confirms the observation in sorghum (Luchetta et al., 1991Go). These two genes are closely related, but the sequence from Zea falls in an unresolved position relative to the clades representing NMDH-I and NMDH-II (Fig. 1B). This suggests that the gene duplication event leading to the appearance of these two gene lineages is specific to some Andropogoneae (at least in species deriving from the common ancestor of Saccharum, Sorghum and Vetiveria). The duplicate gene probably exists in some of the other genera analysed (Dichanthium, Heteropogon, Hyparrhenia, Ischaemum and Pogonatherum), but this was not demonstrated in our analysis (probably due to a lower transcription level of NMDH-II than of NMDH-I).

Theoretical models predict that maintenance of duplicate genes could be associated with selective pressures via neo-functionalization, in which one copy acquires a new function, or by sub-functionalization, in which the original function is partitioned across both copies (Monson, 2003Go; Walsh, 2003Go). Gene duplication and subsequent modifications are considered to be essential for the multiple C4 pathway appearance. Many of the duplicated genes destined for C4 roles should have been shaped by selection to produce novel C3 roles, prior to being recruited for C4 photosynthesis (Monson, 2003Go). However, the C4 NADP-MDH isoform selection could be a relatively simple event compared with the selection of other C4 genes (e.g. C4 PEPC genes) since it may not always have involved gene duplication (McGonigle and Nelson, 1995Go; Monson, 2003Go). As proposed by McGonigle and Nelson (1995)Go, NADP-MDH enzymes involved in C4 photosynthesis have been selected for their high expression level in the mesophyll cells (as is probably the case in Zea in which only one NADP-MDH gene has been identified). Nevertheless, selective pressures favouring new mutations during the evolution from C3 to C4 NADP-MDH are likely to have occurred. Gene duplication should thus be an opportunity to partition the original functions of NADP-MDHs across different copies. In S. bicolor, NMDH-I was reported to have a high transcription level in green leaves and is likely to be involved in C4 photosynthesis, whereas NMDH-II, which displays a lower transcription level, could be involved in the reducing equivalent export (Luchetta et al., 1991Go). Our results (Fig. 3) confirm that the transcript accumulation in green leaves is usually stronger for NMDH-I than for NMDH-II, sustaining the hypothesis that these two genes were maintained in some Andropogoneae due to sub-functionalization. Furthermore, we expect that each isoform displays different kinetic properties since positive selection has probably occurred during the period of time following the duplication event (on the basal branch of the NMDH-II clade; Fig. 2). In this way, two amino acid sites (Lys292 and Asn414 in sorghum NMDH-I) were detected which could induce significant differences in kinetic properties between NMDH-I and NMDH-II. Different electric properties of the amino acids at these variable sites should result in conformational changes between NMDH-I and NMDH-II. To assess the importance of such sites, additional studies are needed. For example, experiments could be done using site-directed mutagenesis (Schepens et al., 2000Go) or by construction of reciprocal enzyme chimeras (Bläsing et al., 2000Go), offering new perspectives for biochemical research on the NADP-MDH enzymes (Miginiac-Maslow et al., 2000Go).


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We would like to thank Trevor Hodkinson, Gerald Edwards and Mike Fay for their constructive suggestions. We are also grateful for helpful discussions with J. P. Jacquot, N. Corradi, C. Parisod and B. Offmann.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

    Besnard G, Offmann B, Robert C, Rouch C, Cadet F. 2002. Assessment of the C4 phosphoenolpyruvate carboxylase gene diversity in grasses (Poaceae). Theoretical and Applied Genetics 105: 404–412.[CrossRef]

    Besnard G, Pinçon G, D'Hont A, Hoarau JY, Cadet F, Offmann B. 2003. Characterisation of the phosphoenolpyruvate carboxylase gene family in sugarcane (Saccharum spp.). Theoretical and Applied Genetics 107: 470–478.[CrossRef]

    Bläsing OE, Westhoff P, Svensson P. 2000. Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria: a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics. Journal of Biological Chemistry 275: 27917–27923.[Abstract/Free Full Text]

    von Caemmerer S, Furbank RT. 2003. The C4 pathway: an efficient CO2 pump. Photosynthesis Research 77: 191–207.[CrossRef][Web of Science][Medline]

    Dillon SL, Lawrence PK, Henry R. 2001. The use of ribosomal ITS to determine phylogenetic relationships within Sorghum. Plant Systematics and Evolution 230: 97–110.[CrossRef]

    Duvall MR, Saar DE, Grayburn WS, Holbrook GP. 2003. Complex transitions between C3 and C4 photosynthesis during the evolution of Paniceae: a phylogenetic case study emphasizing the position of Steinchisma hians (Poaceae), a C3–C4 intermediate. International Journal of Plant Sciences 164: 949–658.[CrossRef]

    Edwards GE, Furbank RT, Hatch MD, Osmond CB. 2001. What does it take to be C4? Lessons from the evolution of C4 photosynthesis. Plant Physiology 125: 46–49.[Free Full Text]

    Gehrig H, Heute V, Kluge M. 2001. New partial sequences of phosphoenolpyruvate carboxylase as molecular phylogenetic markers. Molecular Phylogenetics and Evolution 20: 262–274.[CrossRef][Web of Science][Medline]

    Giussani LM, Cota-Sanchez JH, Zuloaga FO, Kellogg EA. 2001. A molecular phylogeny of the grass subfamily Panicoideae (Poaceae) shows multiple origins of C4 photosynthesis. American Journal of Botany 88: 1993–2012.[Abstract/Free Full Text]

    GPWG. 2001. Phylogeny and subfamilial classification of the grasses (Poaceae). Annals of the Missouri Botanical Garden 88: 373–457.[CrossRef][Web of Science]

    Hodkinson TR, Chase MW, Lledó D, Salamin N, Renvoize SA. 2002. Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers. Journal of Plant Research 115: 381–392.[CrossRef][Web of Science][Medline]

    Kellogg EA. 2000. The grasses: a case study in macroevolution. Annual Review of Ecology and Systematics 31: 217–238.[CrossRef][Web of Science]

    Kikuchi S, Satoh K, Nagata T, Kawagashira N, Doi K, Kishimoto N, et al. 2003. Collection, mapping and annotation of over 28,000 cDNA clones from japonica rice. Science 301: 376–379.[Abstract/Free Full Text]

    Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111–120.[CrossRef][Web of Science][Medline]

    Kumar S, Tamura K, Nei M. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5: 150–163.[Abstract/Free Full Text]

    Luchetta P, Crétin C, Gadal P. 1991. Organization and expression of the two homologous genes encoding the NADP-malate dehydrogenase in Sorghum vulgare leaves. Molecular and General Genetics 228: 473–481.

    McGonigle B, Nelson T. 1995. C4 isoform of NADP-malate dehydrogenase: cDNA cloning and expression in leaves of C4, C3, and C3–C4 intermediate species of Flaveria. Plant Physiology 108: 1119–1126.[Abstract]

    Matsuoka M, Furbank RT, Fukayama H, Miyao M. 2001. Molecular engineering of C4 photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 52: 297–314.[CrossRef][Web of Science][Medline]

    Metzler MC, Rothermel BA, Nelson T. 1989. Maize NADP-malate dehydrogenase: cDNA cloning, sequence, and mRNA characterization. Plant Molecular Biology 12: 713–722.[CrossRef]

    Miginiac-Maslow M, Johansson K, Ruelland E, Issakidis-Bourguet E, Schepens I, Goyer A, et al. 2000. Light-activation of NADP-malate dehydrogenase: a highly controlled process for an optimized function. Physiologia Plantarum 110: 322–329.[CrossRef]

    Monson RK. 2003. Gene duplication, neofunctionalization, and the evolution of C4 photosynthesis. International Journal of Plant Sciences 164: S43–S54.[CrossRef][Web of Science]

    Nei M, Gojobori T. 1986. Simple methods for estimating the number of synonymous and nonsynonymous nucleotide substitutions. Molecular Biology and Evolution 3: 418–426.[Abstract]

    Ocheretina O, Haferkamp I, Tellioglu H, Scheibe R. 2000. Light-modulated NADP-malate dehydrogenases from mossfern and green algae: insights into evolution of the enzyme's regulation. Gene 258: 147–154.[CrossRef][Web of Science][Medline]

    Sage RF. 2004. The evolution of C4 photosynthesis. New Phytologist 161: 341–370.[CrossRef][Web of Science]

    Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425.[Abstract]

    Scheibe R. 1987. NADP-malate dehydrogenase in C3 plants: regulation and role of light-activated enzyme. Physiologia Plantarum 71: 393–400.[CrossRef]

    Schepens I, Ruelland E, Miginiac-Maslow M, Le Maréchal P, Decottignies P. 2000. The role of active site arginines of sorghum NADP-malate dehydrogenase in thioredoxin-dependent activation and activity. Journal of Biological Chemistry 275: 35792–35798.[Abstract/Free Full Text]

    Sheen J. 1999. C4 gene expression. Annual Review of Plant Physiology and Plant Molecular Biology 50: 187–271.[CrossRef][Web of Science]

    Thompson JD, Higgins DG, Gibson TJ. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalities and weight matrix choice. Nucleic Acids Research 22: 4673–4680.[Abstract/Free Full Text]

    Walsh B. 2003. Population-genetics models of the fates of duplicate genes. Genetica 118: 279–294.[CrossRef][Web of Science][Medline]

    Westhoff P, Gowik U. 2004. Evolution of C4 phosphoenolpyruvate carboxylase. Genes and proteins: a case study with the genus Flaveria. Annals of Botany 94: 13–23.

    Yang Z. 1997. PAML, a program package for phylogenetic analysis by maximum likelihood. Computer Applications in the Biosciences 13: 555–556.[Free Full Text]

    Yang Z. 1998. Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Molecular Biology and Evolution 15: 568–573.[Abstract]


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