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3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34), an enzyme catalyzing the first committed step in the mevalonic acid (MVA) pathway for the biosynthesis of isoprenoids, has been reported to be involved in the fruit size determination through the regulation of early cell division. In litchi, the cell number achieved by this early cell division determines the final fruit size, but whether HMGR plays any role in this process was unknown. In this study, we set out to address this question with gene cloning and expression analysis in fruits of different pheno- or genotypes. We found that the litchi genome includes two HMGR homologues, denoted as LcHMG1 and LcHMG2. Despite 70% sequence identity at the amino acid level, they exhibited distinct expression patterns during litchi fruit development. LcHMG1 expression was highest in the early stage of fruit development, correlated with the high level of cell division. Absolute levels of LcHMG1 expression varied among fruits of different pheno- or genotypes, with expression in large-fruited types reaching higher levels for longer duration compared to that in small-fruited types. The expression patterns for LcHMG1 strongly suggest that this gene is involved in early cell division and fruit size determination in litchi. In contrast, LcHMG2 was most highly expressed in the late stage of fruit development, in association with biosynthesis of isoprenoid compounds required for later cell enlargement. These findings provided new insights on the function of HMGR genes during fruit development.

ADDITIONAL INDEX WORDS. growth, Litchi chinensis, maturation ABSTRACT. To understand the relationship between fruit cracking and gene expression patterns, we identifi ed two expansin genes from litchi (Litchi chinensis Sonn.) fruit and then examined their expression profi les in pericarp and aril at different stages of fruit development, using the cracking-resistant cultivar Huaizhi and the cracking-susceptible cultivar Nuomici. Two full-length cDNAs of 1087 and 1010 base pairs encoding expansin, named LcExp1 and LcExp2, were isolated from expanding fruit using RT-PCR and RACE-PCR (rapid amplifi cation of cDNA ends) methods. LcExp1 mRNA could be detected from the early stage of fruit rapid growth (59 days after anthesis). The LcExp1 mRNA increased and reached to the highest level at the end of growth phase (80 days after anthesis) in pericarp of ʻHuaizhiʼ, while the mRNA could be detected at the stage of rapid fruit growth, then increased slightly and fi nally kept remained almost constant in the pericarp of ʻNuomiciʼ. Similar accumulation of LcExp2 mRNA was observed in fruit aril of ʻNuomiciʼ and ʻHuaizhiʼ, whereas LcExp2 accumulated only in pericarp of ʻHuaizhiʼ but did not appear in pericarp of ʻNuomiciʼ. The results indicate that expression of two expansin genes in litchi pericarp are closely associated with fruit growth and cracking. Litchi is a subtropical to tropical fruit of high commercial value in international trade. However, fruit cracking during growth and development is a serious problem and causes a high loss of yield and commercial value (Huang et al., 2003). In particular, ʻNuomiciʼ and ʻGuiweiʼ, which are highly appreciated cultivars, have more serious cracking than other cultivars during fruit devel- opment (Li et al., 2001, 2003a; Peng et al., 2004). Considerable research attention has been directed at the mechanism of litchi fruit cracking involved in that the expansion of the pericarp falls behind the growth of the aril (Abuhasan and Chattopadhyay, 1996; Huang and Xu, 1983; Huang et al., 1999; Li et al., 2003a; Lin, 2001; Sharma and Ray, 1987). However, the mechanisms are still not fully understood. Expansins are cell wall proteins that facilitate extension of cell walls in a pH-dependent manner and, therefore, are considered to be primary regulators of plant cell enlargement (Cosgrove, 1999a, 1999b, 2000a; Darley et al., 2001; McQueen-Mason and Cosgrove, 1994; McQueen-Mason et al., 1992; Whitney et al., 2000). Current research indicates that expansins bind to the surface of cellulose microfi brils, disrupt the hydrogen bonds formed with

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wall-related genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and beta-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening.