* Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires
Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina
Correspondence: E-mail: srossi{at}fbmc.fcen.uba.ar.
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Abstract |
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Key Words: tomato Lycopersicon water stress Asr genes adaptive evolution
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Introduction |
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Nowadays, drought is a major agronomic problem, resulting in reduction in yields of crops exposed to chronic or sporadic periods of drought (Boyer 1982). Therefore, this type of abiotic stress has been the focus of considerable attention, revealing physiological mechanisms of adaptation (Bohnert and Sheveleva 1998). Much research of plant responses to water deficit has been directed towards the isolation of stress-inducible genes by exposing plants to dehydration regimes (Skriver and Mundy 1990; Iusem et al. 1993). The function of only a few such genes is known (Zhu 2002). Genes related to osmotic adjustment, degradation, repair, and structural protection are up-regulated during dehydration (Ingram and Bartels 1996; Shinozaki and Yamaguchi-Shinozaki 1997).
Our interest focuses on genes responsive to limiting water availability and their evolution in lineages exposed to extreme habitats. In this context, the Asr gene family is a good working model, as it is up-regulated in leaves and roots of water-stressed plants (Maskin et al. 2001). Since Asr2 is the best-characterized member of the family, we started this study by examining this gene, originally cloned from a cultivar of commercial tomato (L. esculentum cv. Ailsa Craig) (Rossi and Iusem 1994). Asr2 encodes a putative transcription factor likely to be involved in one of the signaling pathways of ABA (Finkelstein, Gampala, and Rock 2002). The other two members, Asr1 and Asr3 share a high sequence identity with Asr2 (Maskin et al. 2001). In vitro studies showed that ASR1 has a zinc-dependant DNA-binding activity and is localized to both nuclei and cytoplasm (Kalifa and Bar-Zvi, personal communication). On the other hand, an ASR protein from Vitis vinifera (grape) was found to be associated with a hexose transporter promoter (Atanassova et al. 2003). This piece of evidence leads to the conclusion that the Asr gene family would play a role in transcriptional modulation of one or many genes related to carbohydrate mobilization and/or osmoregulation. The role of hexoses during water stress has been studied (Hare, Cress, and Van Staden 1998), but many questions still remain to be answered.
The genus Lycopersicon comprises nine species (Rick 1979) growing in western South America, from Ecuador to northern Chile. These wild tomatoes dwell in a variety of habitats, spanning a wide range of water availability (Rick 1973; Taylor 1986). All species are diploid (2n = 24) (Rick 1979). Breeding systems vary from self-incompatible, facultative self-compatible, to entirely self-compatible (Kondo et al. 2002). Tomato plants have a gametophytic type of self-incompatibility controlled by a single multiallelic "S" locus, which enables styles to recognize and reject self-pollen (Kondo et al. 2002).
In this work, we analyze the coding sequence of the Asr2 gene from several populations of tomato species living in dry, mesic, or humid habitats. L. hirsutum, L. peruvianum f. glandulosum, L. cheesmanii, L. esculentum cv. cerasiforme (likely to be the wild ancestor of cultivated tomato) and L. esculentum cv. Ailsa Craig grow in mesic or humid habitats, whereas L. peruvianum v. humifusum and L. chilense dwell in dry habitats. L. chilense is particularly interesting since it inhabits the Atacama desert, exposed to one of the driest climates in the world. Our study strongly suggests that Asr2 has been the target of positive selection during the evolution of Lycopersicon species dwelling in dry habitats.
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Materials and Methods |
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DNA Amplification and Sequencing
Genomic DNA extractions were performed following the protocol of Peralta and Spooner (2001). The following primers were used in PCR reactions to amplify the coding sequence of Asr2. Upper primer: 5'-AGAGAAGCAATACAATATGGCT-3'. Lower primer: 5'-TATTAGACAAAACATAGAGTCC-3'. Thirty-five cycles consisting of denaturation (95°C for 1 min), annealing (55°C for 1 min), and extension (72°C for 1 min) were programmed in a PTC-100 thermocycler (M. J. Research). The amplification products were run on 1% LMP agarose gels. Fragments of approximately 520 bp (as expected for the known sequence of Asr2 from L. esculentum cv. Ailsa Craig) were excised from the gel and purified using the Concert Kit (Gibco). The PCR products were sequenced using the same primers at the Biotechnology Resource Center (Cornell University) with an ABI 3700 sequencer. DNA sequences were determined on both strands. Sequences were edited using BioEdit (Hall 1999). Numbers of alleles analyzed for each species were L. peruvianum v. humifusum, n = 4; L. peruvianum f. glandulosum, n = 4; L. chilense, n = 8; L. cheesmanii, n = 6; L. hirsutum, n = 4; L. esculentum cv. Cerasiforme, n = 2; and L. esculentum cv. Ailsa Craig, n = 4. All the alleles within each species were identical.
Intron and exon boundaries were determined by comparing genomic and cDNA clones. Amino acid sequences were deduced from cDNA sequences. Deduced peptide sequences are legitimate since antibodies raised against synthetic peptides were able to recognize the ASR2 protein in Western blot experiments (N. Frankel and N. Iusem, unpublished data).
Sequence Analysis
Sequences were aligned using ClustalX (Thompson et al. 1997). Replacement versus synonymous substitution rates (Ka/Ks = ) were calculated with MEGA version 2.1 freeware (Kumar et al. 2001) using the Nei-Gojobori algorithm (Nei and Gojobori 1986). Alignment gaps were not considered for Ka/Ks calculations.
We also estimated the (Ka/Ks ) ratio between L. esculentum and L. chilense for several loci available in databases: anonymous EST clone CT268 (GenBank accession number AA824988 and nonannotated, respectively), anonymous EST clone CT251 (GenBank accession number AA824968 and nonannotated, respectively), sucr or invertase (Elliot et al. 1993 and nonannotated, respectively), dehydrin (accession numbers BF097038 and M97211, respectively), H1-like (accession numbers Z11842 and AF253416, respectively), and class I acidic endochitinase (accession numbers Z15141 and L19342, respectively). The nonannotated sequences from L. chilense were kindly provided by Thomas Städler, University of Munich, Germany.
Phylogenetic Analysis
We retrieved the complete ITS1 and ITS2 rDNA sequences from GenBank (accession numbers AJ300200, AJ300201, AJ300202, AJ300203, AJ300204, AJ300208, AJ300209, AJ300210, and AJ300215), reported by Marshall et al. (2001). In contrast to the analysis by Marshall et al. (2001), we only included fully aligned sites, eliminating trailing as well as internal gaps from the data matrix. The g1 value (1.285007) computed from 10,000 random trees was significant (P < 0.001), indicating that this data matrix has a strong phylogenetic signal (Hillis and Huelsenbeck 1992). Data were run under PAUP* version 4.0 beta (Swofford 1998) using neighbor-joining, minimum-evolution, maximum-parsimony, and maximum-likelihood algorithms. Nicotiana tabacum was used as outgroup. Maximum parsimony and minimum evolution were run using heuristic search, branch-swapping tree-bisection-reconnection, and MulTrees option in effect. Bootstrap values for ingroup nodes were above 50% in maximum-parsimonybased, neighbor-joiningbased, and minimum-evolutionbased trees. In all cases, bootstrap values were obtained with 500 pseudoreplicates. Maximum-likelihood methods gave low bootstrap values for all nodes.
Ancestral states of variable positions were inferred by means of the distance-based Bayesian approach, using the software Anc-gene (Zhang and Nei 1997). The use of a Poisson or a JTT amino acid substitution matrix did not change the inference of ancestral sequences. PAML (Yang 1997) was used to estimate the number of synonymous and nonsynonymous substitutions per branch under a free-ratio model.
Several models aimed to analyze whether certain branches of the tree have unusually high Ka/Ks () ratios were compared by means of likelihood ratio tests. In these tests, codon equilibrium frequencies were calculated from average nucleotide frequencies at each of the three codon positions and the transition/transversion ratio (
) was estimated from the data (about 1.2 for all models) (Goldman and Yang 1994). The natural logarithms of the likelihoods (LnL) associated to each one of the different models of interest (see Results for further explanation) were also calculated using PAML. The simplest model considered is one in which all branches have a background
o value (LnL = 577.08). Other models of interest are those in which the Ka/Ks ratio in the branches leading to L. chilense (
ch) and L. peruvianum f. humifusum (
ph) are equal but different from
o (
o
ch =
ph, LnL = 569.18) or different to each other but one of the
values equal to
o (
o =
ph
ch, LnL = 574.82 or
o =
ch
ph, LnL = 572.15); more complex models considering
o
ph
ch (LnL = 569.18) and the free-ratio model (LnL = 565.30) were also tested. Comparisons between models were performed by means of
2 tests with one degree of freedom.
Climatological Data
The climatological data were retrieved from the Dirección Meteorológica of Chile (http://www.meteochile.cl), the Servicio Nacional de Meteorología e Hidrología of Perú (http://www.senhami.gob.pe), and the Instituto Nacional de Meteorología e Hidrología of Ecuador (http://www.inhami.gov.ec).
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Results |
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Analysis of table 2 shows that Ka/Ks ratios higher than 1 correspond to most of the pairwise comparisons involving L. peruvianum v. humifusum. The same situation is observed in the comparison between L. chilense and L. peruvianum v. humifusum. In addition, the Ka/Ks ratios between L. chilense and L. cheesmani and both varieties of L. esculentum are slightly higher than 1. The rest of the species show Ka/Ks values lower than 1. Interestingly, L. chilense and L. peruvianum v. humifusum inhabit dry habitats, whereas the rest grow in mesic or humid environments (table 1).
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Phylogenetic Analysis
To analyze the pattern of nucleotide substitution in the Asr2 gene of tomato in a phylogenetic context, we first searched for the published phylogenies of the genus Lycopersicon. Two recent molecular phylogenetic studies on Lycopersicon were performed by Peralta and Spooner (2001), based on the sequence of a gene encoding the enzyme granule-bound starch synthase (GBSSI) and Marshall et al. (2001), based on ITS1-ITS2 rDNA. A third study by Miller and Tanksley (1990) reported dendrograms constructed using RFLP markers.
The position of L. peruvianum v. humifusum in the tree of Marshall et al. (2001) was different from those reported by Miller and Tanksley (1990) and Peralta and Spooner (2001). Given this conflicting issue, we decided to reanalyze the ITS1-ITS2 data set from Marshall et al. (2001) and to construct phylogenetic trees using alternative methods. The resulting topology using maximum-parsimony, minimum-evolution, and neighbor-joining algorithms supported the position of L. peruvianum v. humifusum as sister species of the esculentum/cheesmanii clade (fig. 2). The position of Lycopersicon peruvianum v. humifusum in all trees obtained using rDNA sequences is consistent with that obtained by Peralta and Spooner (2001) and by Miller and Tanksley (1990), indicating that L. peruvianum does not conform to a clade. The node that joins L peruvianum v. humifusum with the L. esculentum/L. cheesmanii complex (fig. 2) was supported by 94%, 74%, and 53% bootstrap values when analyzed under Neighbor-Joining, minimal-evolution and maximum-parsimony algorithms, respectively.
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Substitutions Along the Branches of the Lycopersicon Tree
We estimated the number of replacement (n) and synonymous (s) substitutions on the branches of the phylogenetic tree by different approaches. A distance-based Bayesian method (Zhang and Nei 1997) was used to infer the ancestral nucleotide sequences that allowed us to map the substitutions along the branches of the tree. Finally, we estimated the number of replacement and synonymous changes per branch using a maximum-likelihood procedure (Goldman and Yang 1994) under a "free-ratio" model (Yang 1998), which assumes different Ka/Ks () per branch. Maximum likelihood and Bayesian gave almost identical results, showing an acceleration in the rate of replacement/synonymous substitution in the terminal branches of L. chilense and L. peruvianum v. humifusum (fig. 2).
To determine if these accelerations are significant, we compared the results of likelihood ratio tests under models with different assumptions regarding branch-to-branch variation in the ratio of replacement to synonymous substitution rates (Yang 1998). Since our original aim was to explore whether lineages dwelling in arid habitats experienced accelerated rates of amino acid replacement, we a priori assumed three likely different rates:
ch (for the terminal branch leading to L. chilense),
ph (for the terminal branch leading to L. peruvianum v. humifusum), and
o (background ratio for the remaining branches of the tree). The one-ratio model (
o =
ch =
ph) was contrasted with a two-ratio model (
o
ch =
ph) to test the hypothesis of acceleration in these terminal branches. The analysis indicates that Ka/Ks in these two branches is significantly different from the background ratio (
2 = 15.8, 1df, P < 0.0001). To explore whether only one or both branches exhibited accelerated
, we performed additional tests in which only one of the ratios is compared with the background ratio (
o) while the other ratio is either allowed to vary freely or constrained to be equal to
o. The estimated
ch is significantly different from
o when
ph is constrained to be equal to
o (
o =
ph
ch versus
o =
ph =
ch,
2 = 4.52, 1df, P < 0.05) or allowed to vary freely (
o
ph
ch versus
o =
ph
ch,
2 = 11.28, 1df, P < 0.001). Analogously,
ph is significantly different from
o when
ch is either constrained to be equal to
o (
o =
ch
ph versus
o =
ph =
ch,
2 = 9.8, 1df, P < 0.01) or allowed to vary freely (
o
ph
ch versus
o =
ch
ph,
2 = 5.94, 1df, P < 0.05). This statistical evaluation of our data validates the hypothesis of an accelerated replacement substitution rate in Asr2 in L. chilense and L. peruvianum v. humifusum.
The Bayesian method permitted the mapping of each amino acid substitution onto the tree (fig. 2). We found one conservative substitution in the lower branch leading to the ancestor of the peruvianum (esculentum/cheesmanii) clade. Another conservative change is present in the esculentum/cheesmanii clade. Remarkably, the terminal branches of L. chilense and L. peruvianum v. humifusum exhibit two and three amino acid substitutions in the water stressinducible ASR2 protein, respectively, while being devoid of synonymous substitutions (according to the two methods used).
The changes in the branch leading to L. chilense are Pro63Leu and Lys18
Arg. The former is a radical substitution probably associated with alterations of protein spatial conformation. One of the three changes in the branch of L. peruvianum v. humifusum can be considered conservative: Ala52
Gly, whereas the second Val104
Ala implies a size change in the residue (fig. 2). The third one is a nonconservative change of the uncharged and small glycine by the positively charged lysine.
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Discussion |
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It is noteworthy that the branches of L. chilense and L. peruvianum v. humifusum, the only species analyzed here that grow in dry environments, display nonconservative amino acid substitutions in Asr2. None of the terminal branches leading to species living in mild conditions showed any radical amino acidic change (figs. 1B and 2). A strong piece of evidence supporting our conclusion of adaptive evolution is one radical amino acid substitution (Pro63Leu) found only in L. chilense, a species from the Atacama Desert, a habitat where rainfalls are extremely infrequent. Interestingly, the proline in position 63 is conserved in almost all Asr-like proteins known thus far, even in primitive lineages such as gymnosperms (Maskin et al. 2001). The same radical amino acid change either in position 102 or 105 of the human PrPC gene is the cause of a conformational shift associated to a prionlike disease (Prusiner 1997). Analogously, the substitution Val
Ala that occurred in the terminal branch leading to L. peruvianum v. humifusum is the cause of a similar neurodegenerative phenotype if present in the PrPC protein (Prusiner 1997). The Gly101
Lys substitution in ASR2 from L. peruvianum v. humifusum implies a change in the net charge of this small protein. Any or all of the different and nonconservative amino acid changes occurring in the L. chilense and in the L. peruvianum v. humifusum branches would generate proteins with analogous functions relevant to water-stress responses.
Van der Hoeven et al. (2002) compared a large data set of Lycopersicon ESTs with the Arabidopsis genome and concluded that genes encoding transcription factors are the fastest evolving in these two lineages, which diverged 150 MYA. This trend could be valid for plants in general. Moreover, the present work shows that Asr2, a putative transcription factor, would be clear example of such rapidly evolving genes.
It is well established that related proteins displaying a certain extent of structural diversity usually show functional differences that may have a strong impact on fitness. It is widely accepted that a protein co-opted for an emerging new function often experiences an episode of rapid sequence evolution driven by positive selection (Wallis 2001). Adaptive evolution after gene duplication has been reported in several gene families (Hughes 2002). In this context, Asr2, a member of a gene family, suggestively experienced an acceleration of the nonsynonymous rate in the two tomato lineages adapted to dry habitats. This pattern of evolution is in sharp contrast to that of other genes also known to be induced under drought in tomato, such as dehydrin and histone H1-like genes (Chen et al. 1993; Wei and O'Connell 1996). The only other available gene that displayed signs of such a type of selection was that encoding class I acidic endochitinase, a gene that is turned on by drought (Chen et al. 1994) but also involved in the defense to pathogenic fungi (Bishop, Dean, and Michael-Olds 2000).
The number of genes subjected to positive selection has been estimated to be as low as 0.5 % of the totality of genes in comprehensive DNA sequence databases (Endo, Ikeo, and Gojobori 1996). Other well-documented examples of such rapidly evolving genes are those encoding surface antigens of parasites with short generation times (Endo, Ikeo, and Gojobori 1996), ribonuclease genes in colobine monkeys (Zhang, Zhang, and Rosenberg 2002), mammal protein hormones (Wallis 2001), and proteins involved in gamete recognition (Vacquier, Swanson, and Lee 1997).
The present study also allows us to envisage possible biotechnological endeavors toward the improvement of crop yields in dry soils. A conceivable strategy to achieve that goal might well be the introduction of Asr genes from tolerant species in cultivated tomato by genetic engineering. In this regard, the task would not be straight forward because of the genetic complexity underlying the physiological response (Zhu 2002). However, it would be worth the effort, as there is evidence on the ability of certain transgenic plants overexpressing a single master transcription factor to acquire water-stress tolerance (Kasuga et al. 1999; Hsieh et al. 2002).
In summary, on grounds of the data reported in this work, we hypothesize that Asr2 genes of the wild L. chilense and L. peruvianum v. humifusum species underwent adaptive changes that might be associated to success in colonizing arid habitats. Adaptation to such stringent conditions would depend on multiple physiological and genetic factors. For instance, at the physiological level, an overt adaptive phenotype is the development of a deep root system in L. chilense able to locate water trapped in the rocky soil (Rick 1973). Other genes, as well as their patterns of evolution, are to be investigated to gain full insight into the molecular adaptation mechanisms of plants to dryness.
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Acknowledgements |
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Footnotes |
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Spencer Muse, Associate Editor
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