Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato

Asr1, Asr2 andAsr3 are three homologous clones isolated from tomato whose expression is believed to be regulated by abscisic acid (ABA); the corresponding genes thus participate in physiological and developmental processes such as responses of leaf and root to water stress, and fruit ripening. In this report, results obtained with Near Isogenic Lines reveal thatAsr1, Asr2 andAsr3 represent three different loci. In addition, we map these genes on the restriction fragment length polymorphism (RFLP) map of the tomato genome by using an F₂ population derived from an interspecific hybrid crossL. esculentum × L. penelli. RFLP data allow us to map these genes on chromosome 4, suggesting that they belong to a gene family. The elucidation of the genomic organization of theAsr gene family may help in understanding the role of its members in the response to osmotic stress, as well as in fruit ripening, at the molecular level.     

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the β-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems.     

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the β-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems. Received: 24 March 1997 / Accepted: 26 November 1997     

Sequence Evolution and Expression Regulation of Stress-Responsive Genes in Natural Populations of Wild Tomato

The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives.     

Genetic mapping of ripening and ethylene-related loci in tomato

 The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response. Received: 26 October 1998 / Accepted: 13 November 1998     

Adaptation to drought in two wild tomato species: the evolution of the Asr gene family

Wild tomato species are a valuable system in which to study local adaptation to drought: they grow in diverse environments ranging from mesic to extremely arid conditions. Here, we investigate the evolution of members of the Asr (ABA/water stress/ripening induced) gene family, which have been reported to be involved in the water stress response. We analysed molecular variation in the Asr gene family in populations of two closely related species, Solanum chilense and Solanum peruvianum. We concluded that Asr1 has evolved under strong purifying selection. In contrast to previous reports, we did not detect evidence for positive selection at Asr2. However, Asr4 shows patterns consistent with local adaptation in an S. chilense population that lives in an extremely dry environment. We also discovered a new member of the gene family, Asr5. Our results show that the Asr genes constitute a dynamic gene family and provide an excellent example of tandemly arrayed genes that are of importance in adaptation. Taking the potential distribution of the species into account, it appears that S. peruvianum can cope with a great variety of environmental conditions without undergoing local adaptation, whereas S. chilense undergoes local adaptation more frequently.     

Fruit Development, Ripening and Quality Related Genes in the Papaya Genome

Papaya (Carica papaya L.) is the first fleshy fruit with a climacteric ripening pattern to be sequenced. As a member of the Rosids superorder in the order Brassicales, papaya apparently lacks the genome duplication that occurred twice in Arabidopsis. The predicted papaya genes that are homologous to those potentially involved in fruit growth, development, and ripening were investigated. Genes homologous to those involved in tomato fruit size and shape were found. Fewer predicted papaya expansin genes were found and no Expansin Like-B genes were predicted. Compared to Arabidopsis and tomato, fewer genes that may impact sugar accumulation in papaya, ethylene synthesis and response, respiration, chlorophyll degradation and carotenoid synthesis were predicted. Similar or fewer genes were found in papaya for the enzymes leading to volatile production than so far determined for tomato. The presence of fewer papaya genes in most fruit development and ripening categories suggests less subfunctionalization of gene action. The lack of whole genome duplication and reductions in most gene families and biosynthetic pathways make papaya a valuable and unique tool to study the evolution of fruit ripening and the complex regulatory networks active in fruit ripening.     

Molecular genetic analysis of theripening-inhibitor andnon-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes

Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato,ripening-inhibitor (rin), andnon-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning therin andnor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning therin locus is estimated to be 200–300 kb/cM, while thenor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked torin andnor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains thenot locus.     

Inheritance and genetic mapping of fruit sucrose accumulation in Lycopersicon chmielewskii

Fruit of the domestic tomato (Lycopersicon esculentum Mill.) accumulate soluble sugars primarily in the form of the hexoses, glucose and fructose. In contrast, the predominant sugar in fruit of the wild tomato relative, L. chmielewskii, is sucrose. In the present study, the inheritance and linkage relations of sucrose accumulation were examined in interspecific L. esculentum x L. chmielewskii populations. In backcrosses to either the wild or domestic tomato, segregation for sucrose accumulation permitted qualitative analysis of the trait and indicated monogenic recessive control, although deviations from Mendelian inheritance were observed in some populations. This major gene, designated sucr, was mapped in F₂, F₃, and BC₁F₂ populations using a set of 95 informative RFLP and isozyme markers covering the tomato genome. A map location near the centromere of chromosome 3 was established, with tight linkage to the genomic clone TG102. Association of sucrose accumulation with yellow fruit, encoded by an allele of the r gene, permitted alignment with the classical map, thereby confirming the map location of sucr. A linkage map of the region surrounding sucr was obtained by monitoring recombination between flanking markers in the back-crosses to tomato. A cDNA clone of tomato fruit acid invertase, TIV1, was mapped to TG102 and sucr, with no recombination between the two RFLP markers observed in over 1700 meiotic products. Despite the tight linkage, TG102 and TIV1 hybridize to distinct restriction fragments, hence do not represent the same gene. The genetic data strongly suggest that sucr is an allele of the invertase gene and thus support previous biochemical studies that demonstrated low invertase activity in sucrose-accumulating fruit. L. hisutum, another low-invertase, sucrose-accumulating species, was hybridized with L. chmielewskii and the resulting F₁ plants accumulated sucrose, indicating that genetic control of soluble sugar composition is conserved in these two species.     

Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.     

Effect of prolonged root hypoxia on the antioxidant content of tomato fruit

Tomato fruit quality depends on its metabolite content, which in turn is determined by numerous metabolic changes occurring during fruit development and ripening. The aim of this work was to investigate whether flooding affects the nutritional quality of tomato fruit, focusing on compounds essential to human health: carotenoids and ascorbate. To this end, tomato plants (Solanum lycopersicum L. cv Micro-Tom) were submitted to prolonged root hypoxia (1–2% O₂) at first flower anthesis. Fruits were harvested at five stages of the ripening process and analysed for their carotenoid and ascorbate contents. Our results showed that the ripening of fruits that developed on hypoxia treated plants was not inhibited. However, root hypoxia significantly limits carotenoid and ascorbate accumulation in pericarp during fruit ripening, the strongest effects being observed at late stages of ripening. Limitation of both carotenoids and ascorbate accumulation seems to be primarily mediated by the reduced level of expression of genes of the corresponding metabolic pathway.     

Mapping of ripening-related or -specific cDNA clones of tomato (Lycopersicon esculentum)

Summary Nineteen ripening-related or -specific clones from Lycopersicon esculentum were mapped via RFLP analysis using an F₂ population from the cross L. esculentum x L. pennellii and cDNA or genomic clones of known map location. The map produced using cDNA and genomic clones of known map location corresponded well with previously published maps of tomato. The number of loci detected for each ripening-related or-specific clone varied from one to seven. These loci were located on all 12 chromosomes of the tomato genome. There was no significant clustering of ripening-related or-specific genes. Regions of very low recombination were observed. The clone for polygalacturonase (TOM6) mapped to a single region on chromosome 10, the same chromosome as the nor and alc ripening mutants. To fine map this chromosome, two backcross populations were produced from the cross of L. esculentum x L. pimpenillifolium, in which the esculentum parents used were homozygous for either the alc or the nor. The coding region for polygalacturonase is functionally unlinked to either of these two ripening mutants.