Early apple fruit development: gene identification and characterisation

Abstract

Early fruit development has an important effect on fruit yield and quality. Cell division and cell expansion make the greatest contribution to fruit size and shape. To investigate the regulation of early fruit development at the molecular level, two cDNA libraries were constructed from the fruit of apple (Malus domestica Borkh.), a globally important fruit crop. One library was from mRNA isolated from fruit two days after pollination plus GA treatment (2d cDNA library) and the other was from fruit one week after anthesis (waa) (lwk cDNA library). Differential screening of these two libraries led to the isolation of 20 cDNA clones. Sixteen cDNA clones corresponded to fruit genes that were induced or enhanced by pollination. Four cDNA clones corresponded to genes that were expressed preferentially at early stages of apple fruit development. Sequence analysis and database searches showed that most of these cDNAs corresponded to genes involved either in major developmental processes (e.g. histone H2B, which is expressed in dividing cells), or in defence/stress responses (e.g. chitinase). A pollination-enhanced cDNA clone, MilDADI, was similar to the animal defenderagainst apoptosis cell death (DADI) gene. [n apple, MdDADI transcript levelsvaried between tissues, and were enhanced by flower pollination, and duringsenescence of leaves, petals, and fruit. In situ hybridisation showed that MdDADImRNA was distributed primarily in the cells of vascular bundles. These resultscontrast with a constitutive pattern of expression reported for DADI in mammaliancells, suggesting that DAD I may be playing a different biological role in plants.Six cDNA clones were obtained from fragments generated differentially amongtissues of flower buds, I week fruit or 8 week fruit, using a modified mRNAdifferential display method. One of these clones contained a fragment similar to thehomeobox region of the Arabidopsis Bell gene. This fragment when used as a probeto screen the 2d cDNA library under high or low stringency hybridisation resulted inthe isolation of two homeobox genes, Mdhl and Mdh3 (for Malus Qomesticahomeobox). The Mdhl gene encodes a polypeptide of 810 amino acids, with ahomeodomain very similar to the Bell homeodomain. However, outside theviihomeodomain Mdhl is quite different from Bell. Additional characteristics oftranscription factors were also observed in the Mdhl protein sequence. The Mdh3gene is similar to the Phalaenopsis O39 homeobox gene involved in orchid ovuledevelopment. Both Mdhl and Mdh3 mRNAs were expressed in young leavesn flowersand young fruit. Ile rit hybridisation showed that both mRNAs accumulated in theovules of flowers.The Mdhl cDNA, in either sense or antisense orientation, was introduced intoArabidopsrs under the control of the cauliflower mosaic virus 35S promoter toinvestigate the possible function of this gene in plant development. Antisenseexpression of Mdhl led to complex changes in the morphology of transgenicArabidopsis plants and the inheritance of these changes was non-Mendelian.Overexpression of Mdhl resulted in altered silique (fruiQ size and shape, a reductionin leaf size, petiole length and flower filament length, and male sterility. Furtherexamination of the ovary/silique wall showed that there were differences in cell sizeand shape, and an irregular cell arrangement in these transgenic plants. The alterationsin ovary/silique shape are consistent with a change in cell expansion, suggesting thatthe Mdhl homeodomain protein may be a transcription factor involved in regulationof fruit development, through an effect on cell expansion.