Journal of Experimental Botany, Vol. 53, No. 377, pp. 2023-2030,
October 1, 2002
© 2002 Oxford University Press
Use of genomics tools to isolate key ripening genes and analyse fruit maturation in tomato
Received 22 April 2002; Accepted 10 June 2002
USDA-ARS Plant, Soil and Nutrition Laboratory and Boyce Thompson Institute for Plant Research, Tower Road, Cornell Campus, Ithaca, NY 14853, USA
1 To whom correspondence should be addressed. Fax: +1 607 255 1132. E-mail: jjg33{at}cornell.edu
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Abstract |
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 Top Abstract IntroductionTomato as a model...Ethylene and non-ethylene...Tomato ripening-inhibitor and...Tools for gene expression...Expression profiling of tomato...References |
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Development, maturation and ripening of fruits has received considerable experimental attention, primarily due to the uniqueness of such processes to plant species and the importance of fruit as a significant aspect of human dietary intake and nutrition. Molecular and genetic analysis of fruit development, and especially ripening of fleshy fruits, has resulted in significant gains in knowledge over recent years, especially with respect to understanding ethylene biosynthesis and response, cell wall metabolism and, to a lesser extent, environmental cues which impact ripening. Tomato has proved to be an excellent model system for the analysis of fruit ripening and development, in part due to the availability of well characterized ripening mutants. Especially interesting are the non-allelic ripening-inhibitor (rin) and non-ripening (nor) mutations which result in non-ripening fruit. Fruit from both mutants are deficient in climacteric respiration and the associated burst in ethylene biosynthesis. Exogenous ethylene does not restore ripening yet does induce expression of ethylene-regulated ripening genes, suggesting both mutations block necessary aspects of ripening outside the realm of ethylene’s influence. Both mutations therefore represent genes upstream of ethylene control and additional non-ethylene mediated aspects of ripening. Both genes have recently been isolated through positional cloning strategies and it was shown that ripening is regulated, in part, by a MADS-box transcription factor at the rin locus. Recent development of tools for tomato genomics summarized here have further expanded the potential of the tomato system for the elucidation of genetic regulatory components impacting fruit development, ripening and nutritional quality.
Key words: Key words: ESTs, map-based cloning, microarrays, mutant complementation.
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Introduction |
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TopAbstractIntroductionTomato as a model...Ethylene and non-ethylene...Tomato ripening-inhibitor and...Tools for gene expression...Expression profiling of tomato...References |
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Ripe fruit demonstrate a wide range of diversity in form, pigmentation, texture, aroma, flavour, and nutrient composition. Fruit of many species undergo modification of cell wall ultrastructure and texture, conversion of starch to sugars, increased susceptibility to post-harvest pathogens, alterations in pigment biosynthesis/accumulation, and heightened levels of flavour and aromatic volatiles during the maturation and ripening processes (for reviews see Rhodes, 1980; Seymour et al., 1993).
From a practical viewpoint, a number of ripening characteristics result in negative quality attributes including decreased shelf-life and high input harvest, shipping and storage practices. Particularly important, in this respect are the changes in firmness and the overall decrease in resistance to microbial infection brought about by the ripening process and associated tissue deterioration. Significant advances in understanding the molecular regulation of individual ripening parameters, especially cell wall metabolism and ethylene biosynthesis and response have occurred in the last 15 years (reviewed in Giovannoni, 2001). The resulting knowledge has contributed to a more complete view of molecular ripening control and has, additionally, yielded molecular tools for addressing problems in fruit production and quality.
Two major classifications of ripening fruit, climacteric and non-climacteric, have been used to distinguish fruit on the basis of respiration and ethylene biosynthesis rates. Climacteric fruit (e.g. tomato, avocado, apple, banana) are distinguished from non-climacteric fruits (e.g. strawberry, grape, citrus) by their increased respiration and ethylene biosynthesis rates during ripening (Lelievre et al., 1997). While non-climacteric fruits do not require ethylene for ripening of their fruits, ethylene has been shown to be necessary for the co-ordination and completion of ripening in climacteric fruit. This has been demonstrated in a number of ways including the analysis of inhibitors of ethylene biosynthesis and perception (Tucker and Brady, 1987; Yen et al., 1995), transgenic plants altered in ethylene biosynthesis (Klee et al., 1991; Oeller et al., 1991; Picton et al., 1993), and through the analysis of the tomato Never-ripe (Nr) ethylene receptor mutant (Lanahan et al., 1994; Wilkinson et al., 1995).
Fruit are an important component of the human diet. Ripening has an impact on fibre content and composition, lipid metabolism, and the levels of vitamins and various antioxidants (Ronen et al., 1999). The ability to understand and manipulate, through breeding or biotechnology, key control points in the global control of ripening or regulatory points of specific ripening process such as carotenoid, flavonoid, vitamin, and flavour volatiles, will allow the manipulation of nutrition and quality characteristics associated with ripening. Possibly the most convincing argument for the promotion of plant genetic engineering will be the development of modified plants or plant-derived products with direct consumer appeal such as increased quality and nutrition.
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Tomato as a model system for fruit ripening |
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TopAbstractIntroductionTomato as a model...Ethylene and non-ethylene...Tomato ripening-inhibitor and...Tools for gene expression...Expression profiling of tomato...References |
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Tomato has long served as the most studied model for fruit ripening, in part because of its importance as a food crop species. This practical importance combined with diploid inheritance, ease of seed and clonal propagation, efficient sexual hybridization, a relatively short generation period, and year-round growth potential (in greenhouses) has fostered tomato as the primary model for ripening research. From the standpoint of genetic and molecular investigations tomato has the additional advantages of a relatively small genome (0.9 pg/haploid genome; Arumuganathan and Earle, 1991) on which nearly 2000 molecular markers have been mapped (Tanksley et al., 1992; Solanaceae Genome Network http://www.sgn.cornell.edu/). High molecular weight insert genomic libraries are available in both YAC (Martin et al., 1992; Bonnema et al., 1996) and BAC (Budiman et al., 2000) vector systems to facilitate positional cloning. A recently added tool to the repertoire of tomato and other plant science researchers is the National Science Foundation sponsored development of a tomato EST database. 23 cDNA libraries from various tissues have been created, followed by single-pass 5' sequencing of over 150 000 clones result ing in approximately 28 000 non-redundant sequences (http://www.tigr.org/tdb/lgi/). The database includes approximately 40 000 sequences derived from fruit at various stages of development with an emphasis on ripening. In addition, years of breeding and focus on tomato as an agricultural crop have resulted in a valuable germplasm resource representing genes influencing multiple aspects of fruit development and ripening. A summary of ripening mutants of tomato is listed in Table 1.
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Ethylene and non-ethylene ripening control |
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TopAbstractIntroductionTomato as a model...Ethylene and non-ethylene...Tomato ripening-inhibitor and...Tools for gene expression...Expression profiling of tomato...References |
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Considerable research has been based on the tomato system specifically for the analysis of ethylene synthesis and signalling during ripening. The role of ethylene in facilitating climacteric ripening has been shown through the analysis of ethylene-inducible gene expression in tomato fruit (Lincoln et al., 1987; Maunders et al., 1987; Zegzouti et al., 1999). Reduced ethylene evolution resulted in ripening inhibition in fruit of ACC synthase and ACC oxidase antisense lines (Oeller et al., 1991; Hamilton et al., 1990) and the mutation of the Nr ethylene receptor results in non-ripening, ethylene-insensitive fruit (Wilkinson et al., 1995). Furthermore, the introduction of a dominant mutant allele of the NR ethylene receptor resulted in tomato, Arabidopsis and petunia plants inhibited in virtually every measurable ethylene response including fruit ripening (Wilkinson et al., 1997).
Careful analysis of transgenic and mutant tomato lines inhibited in ethylene biosynthesis or perception suggests that climacteric ripening represents a combination of ethylene regulation and developmental control. The term ‘developmental control’ is used here to signify aspects of ripening regulation operating independently from ethylene. For example, the gene encoding the rate-limiting activity in ethylene biosynthesis, ACC synthase, is itself initially induced during ripening by a signalling system other than ethylene (Theologis et al., 1993; Barry et al., 2000). Gene expression analysis indicates that ‘developmental’or ‘non-ethylene mediated’ regulation has an impact on a number of ripening-related genes in tomato (Giovannoni, 2001).
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Tomato ripening-inhibitor and non-ripening mutants |
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TopAbstractIntroductionTomato as a model...Ethylene and non-ethylene...Tomato ripening-inhibitor and...Tools for gene expression...Expression profiling of tomato...References |
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The strongest evidence for non-ethylene-mediated ripening control comes from the analysis of gene expression in fruit of the rin (ripening-inhibitor) and nor (non-ripening) tomato mutants that fail (a) to produce autocatalytic ethylene, (b) to ripen, and (c) to ripen in response to exogenous ethylene, yet display signs of ethylene sensitivity and signalling, including the induction of some ethylene-regulated genes (Tigchelaar et al., 1978; Yen et al., 1995). These results have been interpreted to indicate that higher order regulatory constraints are placed on climacteric fruit maturation in addition to general ethylene biosynthesis and signalling. Such regulatory mechanisms could include fruit-specific regulation of certain subsets of ethylene-regulated genes or regulatory mechanisms that operate separately and in addition to ethylene. Genes corresponding to both the rin and nor mutations have been recently cloned, and while unrelated at the level of DNA or peptide sequence, both have features suggestive of roles in regulation of gene transcription (Vrebalov et al., 2002; J Giovannoni et al., unpublished results). Both genes were isolated via positional cloning strategies. The details of NOR gene isolation are preliminary and will not be discussed further.
The rin locus was mapped to high resolution in an F2 population resulting from an initial cross between L. esculentum (rin/rin) and the wild relative of tomato L. cheesmannii (Rin/Rin). L. cheesmannii was selected over the more divergent (and thus more likely to be polymorphic at marker loci) L. pennellii used in the development of the tomato genetic map, as F2 progeny derived from L. esculentum by L. pennellii crosses have a high incidence of sterility resulting in few ripe fruit (Tanksley et al., 1992). Tightly linked RFLP markers were used both to isolate and to map a high molecular weight tomato genomic clone harbouring the targeted rin locus. This clone was subsequently labelled in total and used as a hybridization probe to identify cDNA sequences derived from this cloned segment of the tomato genome. Specifically, a breaker fruit cDNA library was screened and several classes of independent sequences were isolated. Two cDNAs resulting from this screen yielded different size RNA gel-blot hybridization signals when RNA from normal and nearly isogenic rin fruit were compared (Fig. 1). This result was the first clue that sequences derived from the mutant locus had indeed been isolated (Vrebalov et al., 2002).
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RT-PCR and subsequent DNA sequencing of the two cDNAs which yielded alternate mRNA transcript sizes in rin fruit indicated that these sequences were fused into a chimeric gene in the rin mutant as a result of a genome deletion. Both genes are members of the MADS-box family of transcriptional regulators (Fig. 2). A combination of mutant complementation and antisense gene expression in rin/rin and Rin/Rin genotypes, respectively, indicated that one gene (LeMADS-RIN) regulates ripening while the other (LeMADS-MC) is responsible for the large sepal (macrocalyx) phenotype associated with the rin mutation (Vrebalov et al., 2002).
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Phylogenetic analysis combined with detailed phenotypic characterization indicates that LeMADS-MC is likely to be the tomato orthologue of the AP1 and SQUA genes of Arabidopsis and Anthirhinum, respectively (Fig. 2). LeMADS-RIN is most similar to the SEP1 and AGL3 genes of Arabidopsis (Fig. 2), but the expression patterns of these latter genes, and the phenotype attributed to SEP1 (there is no reported phenotype for an AGL3 mutation), are inconsistent with LeMADS-RIN expression or function. To assess whether LeMADS-RIN is likely to represent a conserved function among diverse fruit species a strawberry cDNA library was screened with the tomato cDNA and a gene (FvMADS-23) was recovered which demonstrates ripening-related gene expression (Fig. 2; Vrebalov et al., 2002). This result is especially interesting in that strawberry is a non-climacteric fruit with very different fruit morphology and development compared with tomato.
The cloning of these ripening regulatory genes should now foster analysis of steps in the ripening regulatory hierarchy preceding ethylene. These discoveries should also permit an assessment of whether or not such genes represent regulatory mechanisms common to both climacteric and non-climacteric fruit species. In addition, as many of the ripening-related genes which have undergone promoter analysis are impacted by the rin and nor mutations, the recent cloning of these putative transcription factors will provide opportunities to test for specific interactions of the RIN and NOR proteins with functionally characterized regulatory sequences. The tools for gene expression profiling described below will also facilitate characterization of the unique and overlapping regulatory effects of the RIN and NOR genes.
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Tools for gene expression profiling |
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TopAbstractIntroductionTomato as a model...Ethylene and non-ethylene...Tomato ripening-inhibitor and...Tools for gene expression...Expression profiling of tomato...References |
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The increasing availability of efficient, high-throughput methodologies for cloning and sequencing have driven the development of novel discovery platforms able to exploit the increasing amounts of available genome data (Rounsley and Briggs, 1999, and references therein). Until recently, significant gene sequence and functional data for a given biological system was virtually non-existent or the result of painstakingly piecing together studies conducted over many years using classical approaches. New methodologies now allow for the expansion of the traditional platforms of using forward and reverse genetics to those that facilitate the examination of the behaviour of hundreds or thousands of genes simultaneously. In tomato, a collaborative NSF-funded effort has resulted in the construction and sequencing of cDNA libraries from a multitude of tissues and conditions, and the creation of a tomato EST database (Fig. 3; Quackenbush et al., 2000; Van der Hoeven et al., 2002). This information provides the foundation for parallel gene studies for the detection and quantitation of gene expression levels. Parallel studies can provide both static (e.g. examination of gene expression in a single tissue) and dynamic (comparative) information. There are multiple methodologies to achieve parallel analysis, ranging from traditional RNA gel blots and RT-PCR to those providing a more global view including differential display (Liang and Pardee, 1992), serial analysis of gene expression (SAGE, Velculescu et al., 1995), and microarrays (Schena et al., 1995),
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Microarrays allow for the analysis of expression patterns of thousands of genes within the confines of a single experiment (Fig. 4). Arrays are descendants of DNA gel-blot (Southern)-based assays that capitalize on interactions between complementary strands of DNA (Southern, 1975). The inclusion of a solid glass substrate, precision robotics, and fluorescence-based detection methods provide expression arrays with increased accuracy, speed, and scale over their filter- and radioactivity-based relatives.
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Microarrays can be constructed using either PCR-amplified cDNAs or oligonucleotides. Arrays based on amplified expressed sequence tags (ESTs) are the most popular candidates for micro-spotting. ESTs are usually generated by single-pass sequencing 300–900 bases from the 5' end of cDNA clones. EST sequence and homology information provide a distinct and obvious advantage in expression studies compared with the use of anonymous clones, as immediate functional implications can often be made based on sequence homologies.
Unique DNAs are printed onto chemically coated glass microscope slides to create microarrays. Glass provides an excellent platform with low inherent fluorescence resulting in negligible intrinsic background levels. Glass also presents a non-porous surface important for preventing diffusion of deposited samples and thus allowing utilization of minimum hybridization volumes. Glass slides also allow for miniaturization and the easy storage of arrays (Schena, 1999; Duggan and Bittner, 1999; Zammatteo et al., 2000).
Probes for transcript analysis are constructed by incorporating fluorescent molecules into cDNAs created from a single round of reverse transcription (Schena et al., 1995; DeRisis et al., 1996). Our group utilizes protocols and procedures provided by Genisphere Inc. Probes from two tissues to be compared (e.g. wild type and mutant or +/– a given treatment) are labelled with different fluorescent dyes and applied simultaneously to the array. Visualizing hybridized arrays requires excitation of bound fluorochromes by a laser source, collection of the emitted fluorescence through a series of filters which block reflected and scattered excitation energy, and conversion of the focused energy to an electrical signal by a photomultiplier (Montagu and Weiner, 1999; Brignac et al., 1999).
The output from scanning a hybridized array is typically a simple TIFF or bitmap image. Perhaps the most difficult and challenging aspect of microarray experiments is data analysis, as a single experiment appropriately replicated can typically produce thousands of data points. There are currently multiple software programs available and many more under development. Many available programs are variations of similar themes and have features to facilitate the following data manipulations, locate spots, normalize signal, quantify intensities, subtract out background, and generate a report which can be downloaded in a simple format for subsequent analysis in general or customized software packages (Ermolaeva et al., 1998; Chen et al., 1997; Bard, 1999; Baldwin et al., 1999; Bowtell, 1999).
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Expression profiling of tomato fruit ripening |
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TopAbstractIntroductionTomato as a model...Ethylene and non-ethylene...Tomato ripening-inhibitor and...Tools for gene expression...Expression profiling of tomato...References |
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Important new resources that are available for tomato include substantial sequence information, the EST database, and microarray technology (http://www.sgn.cornell. edu/). These tools are now allowing tomato and Solanaceae researchers to expand the platforms available for answering general biological questions. A cDNA microarray has recently been constructed with the purpose of answering questions about fruit development and ripening. A time-course of ten intervals has been established, spanning fruit development from 7 d post anthesis to 15 d past breaker. The time-points were selected to represent biologically significant stages in the fruit developmental process (e.g. cell division, cell expansion, onset and continuation of ripening). Initially, the focus was on using Ailsa Craig (Ac) to establish a baseline of wild-type gene expression. However, an investigation into multiple ripening-related mutants has been started in order to expand information on their specific functions and effects. Mutations targeted for comparative analysis to the normal expression profile include those known to be altered in their perception to light (hp-1), ethylene (Nr) and other aspects of ripening (rin, nor) (Table 1; Giovannoni, 2001, and references therein).
Probes for array experiments were constructed for each stage and used in step-wise dual hybridizations (e.g. 1 d versus 10 d, 10 d versus 20 d, etc). The hybridizations were performed in multiple replications and included ‘dye-swap’ experiments in an attempt to compensate for any variability in signal intensity due solely to the characteristics of the individual fluorochrome. The resulting data has allowed a comparative analysis of genome-wide transcript accumulation during fruit ripening and development for a subset of genes to begin. Although there are defined patterns of differential expression for each stage in development, there is also a dramatic increase in the number of differentially expressed genes that corresponds with the onset of ripening. This set includes ESTs with putative homology to genes known to be involved in ethylene synthesis, carotenoid accumulation and cell wall modifications, in addition to others known to be ripening regulated (Table 2). As analysis of the initial developmental profile is completed, it may become possible to make predictions about ESTs with little or no known homology, based upon their expression patterns and how they relate to genes that have been extensively characterized. By developing expression profiles and co-ordinating them with other tools such as analysis of the tomato proteome, further elucidation of the underlying genetic and molecular events contributing to fruit development and ripening phenomenon will be possible.
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Acknowledgements |
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We are exceptionally grateful to the following who funded this research, USDA-NRI (92-37300-7653; 95-37300-1575), NSF (IBN-9604115; DBI-9872617), Zeneca Agrochemicals (Syngenta), Texas Agricultural Experiment Station, and USDA-ARS.
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 M. Causse, P. Duffe, M. C. Gomez, M. Buret, R. Damidaux, D. Zamir, A. Gur, C. Chevalier, M. Lemaire-Chamley, and C. Rothan A genetic map of candidate genes and QTLs involved in tomato fruit size and composition J. Exp. Bot., August 1, 2004; 55(403): 1671 - 1685. [Abstract] [Full Text] [PDF]
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A. Itai, K. Ishihara, and J. D. Bewley Characterization of expression, and cloning, of {beta}-D-xylosidase and {alpha}-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit J. Exp. Bot., December 1, 2003; 54(393): 2615 - 2622. [Abstract] [Full Text] [PDF]
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L. Trainotti, D. Zanin, and G. Casadoro A cell wall-oriented genomic approach reveals a new and unexpected complexity of the softening in peaches J. Exp. Bot., August 1, 2003; 54(389): 1821 - 1832. [Abstract] [Full Text] [PDF]
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P. J. White Recent advances in fruit development and ripening: an overview J. Exp. Bot., October 1, 2002; 53(377): 1995 - 2000. [Abstract] [Full Text] [PDF]
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G. B. Seymour, K. Manning, E. M. Eriksson, A. H. Popovich, and G. J. King Genetic identification and genomic organization of factors affecting fruit texture J. Exp. Bot., October 1, 2002; 53(377): 2065 - 2071. [Abstract] [Full Text] [PDF]
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