Domestication of many plants has correlated with dramatic increases in fruit size. In tomato, one quantitative trait locus (QTL), fw2.2, was responsible for a large step in this process. When transformed into large-fruited cultivars, a cosmid derived from the fw2.2 region of a small-fruited wild species reduced fruit size by the predicted amount and had the gene action expected for fw2.2. The cause of the QTL effect is a single gene, ORFX, that is expressed early in floral development, controls carpel cell number, and has a sequence suggesting structural similarity to the human oncogene c-H-ras p21. Alterations in fruit size, imparted by fw2.2 alleles, are most likely due to changes in regulation rather than in the sequence and structure of the encoded protein.

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2. Using an F(2) nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) x Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 +/- 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be </= 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

fw2.2 is a quantitative trait locus responsible for approximately 30% of the difference in fruit size between large, domesticated tomatoes (Lycopersicon esculentum Mill.) and their small-fruited wild relatives. The gene underlying this quantitative trait locus was cloned recently and shown to be associated with altered cell division in ovaries (Frary et al., 2000). However, it was not known whether the change in fruit size is associated with other changes in plant morphology or overall fruit yield-changes that could potentially cause the fruit weight phenotype. To shed light on this issue, a detailed comparison was made between nearly isogenic lines differing for alleles at this locus to search for pleiotropic effects associated with fw2.2. Field observations show that although the small-fruited nearly isogenic line produced smaller ovaries and fruit as expected, this was compensated by a larger number of fruit-due mainly to a significantly greater number of inflorescences-but with no net change in total fruit mass yield. This strongly suggests that fw2.2 may have a pleiotropic effect on how the plant distributes photosynthate among fruit. In a flower removal experiment to control for differences in inflorescence size and number, fruit size remained significantly different between the nearly isogenic lines. These observations indicate that the primary effect of fw2.2 is in controlling ovary and fruit size, and that other associated phenotypic effects are secondary.

Sequence variation was sampled in cultivated and related wild forms of tomato at fw2.2--a fruit weight QTL key to the evolution of domesticated tomatoes. Variation at fw2.2 was contrasted with variation at four other loci not involved in fruit weight determination. Several conclusions could be reached: (1) Fruit weight variation attributable to fw2.2 is not caused by variation in the FW2.2 protein sequence; more likely, it is due to transcriptional variation associated with one or more of eight nucleotide changes unique to the promoter of large-fruit alleles; (2) fw2.2 and loci not involved in fruit weight have not evolved at distinguishably different rates in cultivated and wild tomatoes, despite the fact that fw2.2 was likely a target of selection during domestication; (3) molecular-clock-based estimates suggest that the large-fruit allele of fw2.2, now fixed in most cultivated tomatoes, arose in tomato germplasm long before domestication; (4) extant accessions of L. esculentum var. cerasiforme, the subspecies thought to be the most likely wild ancestor of domesticated tomatoes, appear to be an admixture of wild and cultivated tomatoes rather than a transitional step from wild to domesticated tomatoes; and (5) despite the fact that cerasiforme accessions are polymorphic for large- and small-fruit alleles at fw2.2, no significant association was detected between fruit size and fw2.2 genotypes in the subspecies--as tested by association genetic studies in the relatively small sample studied--suggesting the role of other fruit weight QTL in fruit weight variation in cerasiforme.

fw2.2 is a major quantitative trait locus that accounts for as much as 30% of the difference in fruit size between wild and cultivated tomatoes. Evidence thus far indicates that fw2.2 alleles modulate fruit size through changes in gene regulation rather than in the FW2.2 protein itself. To investigate the nature of these regulatory changes and the manner in which they may affect fruit size, a pair of nearly isogenic lines has been subjected to detailed developmental, transcriptional, mitotic, and in situ hybridization studies. The results indicate that the large- and small-fruited alleles of fw2.2 differ in peak transcript levels by approximately 1 week. Moreover, this difference in timing of expression is associated with concomitant changes in mitotic activity in the early stage of fruit development. The changes in timing of gene expression (heterochronic allelic variation), combined with overall differences in total transcript levels, are sufficient to account for a large portion phenotypic differences in fruit weight associated with the two alleles.

It has been proposed that fw2.2 encodes a negative fruit-growth regulator that underlies natural fruit-size variation in tomato (Lycopersicon spp.) via heterochronic allelic variation of fw2.2 expression, rather than by variation in the structural protein itself. To further test the negative regulator and the transcriptional control hypotheses, a gene dosage series was constructed, which produced a wider range of fw2.2 transcript accumulation than can be found in natural tomato populations. Fruit developmental analyses revealed that fw2.2 transcript levels were highly correlated (negatively) with fruit mass, supporting the negative regulator and transcriptional regulation hypotheses. Further, the effect of fw2.2 on fruit mass was mediated by repressing three- and two-dimensional cell division in placental and pericarp tissues, respectively. Finally, fw2.2 had little effect on fertility and seed size/number, indicating that fruit size effects of fw2.2 are due largely to expression in the maternal tissues of developing fruit and not mediated through fertility or seed-setting-related processes.

fw2.2 is one of the few QTLs thus far isolated from plants and the first one known to control fruit size. While it has been established that FW2.2 is a regulator (either directly or indirectly) of cell division, FW2.2 does not share sequence homology to any protein of known function (Frary et al. Science 289:85-88, 2000; Cong et al. Proc Natl Acad Sci USA 99:13606-13611, 2002; Liu et al. Plant Physiol 132:292-299, 2003). Thus, the mechanism by which FW2.2 mediates cell division in developing fruit is currently unknown. In an effort to remedy this situation, a combination of yeast two-hybrid screens, in vitro binding assays and cell bombardment studies were performed. The results provide strong evidence that FW2.2 physically interacts at or near the plasma membrane with the regulatory (beta) subunit of a CKII kinase. CKII kinases are well-studied in both yeast and animals where they form part of cell cycle related signaling pathway. Thus while FW2.2 is a plant-specific protein and regulates cell division in a specialized plant organ (fruit), it appears to participate in a cell-cycle control signal transduction pathway that predates the divergence of single- and multi-cellular organisms. These results thus provide a glimpse into how ancient and conserved regulatory processes can be co-opted in the evolution of novel organs such as fruit.