Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 6, 2002
Publication Date: February 1, 2003
Citation: LAW, R.D., SUTTLE, J.C. TRANSIENT DECREASES IN METHYLATION AT 5'-CCGG-3' SEQUENCES IN POTATO (SOLANUM TUBEROSUM L.) MERISTEM DNA DURING PROGRESSION OF TUBERS THROUGH DORMANCY PRECEDE THE RESUMPTION OF SPROUT GROWTH. PLANT MOLECULAR BIOLOGY. 2003. V. 51. P. 437-447. Interpretive Summary: For an indeterminate period of time following harvest, potatoes will not sprout and are physiologically dormant. Dormancy is gradually lost during postharvest storage and the resultant sprouting is detrimental to the nutritional and processing qualities of potatoes. Because of this, sprouting results in severe financial loss to producers. Currently sprouting is controlled through the use of synthetic sprout inhibitors. The research being conducted in this project is directed towards 1) identifying key cellular and physiological processes that naturally regulate tuber dormancy and, ultimately, 2) modifying these processes genetically thereby eliminating the need for artificial sprout suppression. At the cellular level, our research is directed toward identifying the molecular bases for low rates of RNA synthesis that accompany and possibly regulate tuber dormancy. In this paper, the effects of tuber dormancy on DNA methylation have been determined. DNA methylation is a reversible process that has been linked to the inhibition of RNA synthesis in other organisms. Our research is the first to demonstrate that significant changes in DNA methylation occur as tuber dormancy ends and RNA synthesis increases. Further studies are in progress to determine other changes in DNA structure and function during tuber dormancy. When complete, these studies will provide new knowledge concerning the cellular bases of dormancy-imposed growth arrest and may provide insight into new mechanisms to regulate this economically important process in potatoes.
Technical Abstract: The 5-methylcytosine (5mC) content in DNA of tuber meristems isolated from field-grown potatoes (Solanum tuberosum L.) Was determined during an 8- month postharvest storage period at 3 degree C for three growing/harvest seasons. Genome-wide 5mC levels were not significantly different in dormant versus non-dormant meristem DNA. Similarly, no significant changes sin 5mC content within CG sequences were seen after incorporation of tritiated methyl groups into DNA by SssI methylase. Following cleavage with restriction enzymes whose activities are inhibited by 5mC within their CG and /or CNG-containing recognition sequences (MspI, HpaII, EcoRII and BssHII), a nucleotide extension assay showed no change in 5mC content within CG islands or CA(T)G trinucleotide sequences. However, a consistent but transient 50 to 70% decrease in cytosine methylation at CCGG sequences was detected 110 to 140 days postharvest. This observation was corroborated by amplified fragment length polymorphism analysis after EcoR and HpaII digestion. Cytosine methylation status was also followed in vitro-generated potato microtubers throughout dormancy. While no changes in genome-wide 5mC content were noted, the SssI methylase assay indicated that microtubers underwent a transient 46% drip in methylation at CG dinucleotides 98 days after the start of storage. This decrease was concomitant with 30 to 60% declines in mCCG and mCGG and levels. As microtubers exited dormancy, a three-fold increase in transcriptional activity was observed using (3H) uridine incorporation into RNA. Together, these data demonstrate that CCG and CGG methylation in meristems and microtubers declines during storage of potatoes, and may be important in the activation of transcription of genes during tuber dormancy break.