2019 ASHS Annual Conference

Apple (Malus x domestica) is a pome where the fruit consists of two main sections: the cortex and the pith. The true ovary of the fruit is within the pith and is not consumed. The cortex enlarges and develops into the major fleshy part of the fruit that is consumed. Cortex enlargement occurs at a rate much greater than that of the pith. Two genes that may influence the final size of the two tissues are KIP RELATED PROTEIN 4 (KRP4) and KRP5. KRPs are cyclin-dependent kinase inhibitors and negatively regulate the checkpoints in between phases of the cell cycle and thereby negatively affect the extent of cell production. Transcript abundance of these genes is low during the cell production phase within the first five weeks of fruit growth. After about five weeks, cells begin to expand and the transcript abundance of these genes begins to increase concomitant with the reduction in cell production. Further, KRP4 transcript abundance is substantially greater within the pith than in the cortex during early fruit growth, consistent with lower cell production in this tissue. Regulation of such spatio-temporal changes in transcript abundance of these genes is currently unknown. We hypothesize that promoter methylation of KRP4 and KRP5 is involved in differential spatial and temporal transcript accumulation of these genes. Hence, methylation in the promoters of KRP4 and KRP5 is being studied. Previous reports indicate specific regions within the promoters of these genes that are subject to methylation. To better understand methylation patterns, apple fruit tissue is being collected throughout fruit development, specifically from the pith and cortex tissues. Sample DNA will be extracted, treated with bisulfite, and analyzed using the Kismeth program to understand how KRP4 and KRP5 methylation patterns change over time and space. A callus system is also being developed to study methylation in vitro, as transcript accumulation patterns can change depending on growth conditions. Leaf tissue from four cultivars (‘Gala,’ ‘Honeycrisp,’ ‘Empire,’ and ‘Golden Delicious’) are being used to develop a callus culture. The most productive callus will then be placed in suspension solutions to facilitate the growth of the undifferentiated cells. Using a combination of in vitro and field grown samples, we will be able to get a more complete understanding of methylation in the promoters of KRP4 and KRP5 and its association with organ growth.