CSIRO, Division of Plant Industry

Cold treatment does not appear to enhance the activity of a methylation sensitive Ac element

--Richard Brettell and Elizabeth Dennis

Previous work with wx-m9 Ds-cy, an inactive derivative of Ac in wx-m9, has shown that recovery of activity is associated with demethylation of cytosine residues in the 5' region of the transposase gene (Schwartz and Dennis, Mol. Gen. Genet. 205: 476-482, 1986). Recently we have shown that the frequency of reactivation is high among plants regenerated from tissue cultures initiated from embryos with wx-m9 Ds-cy (Dennis and Brettell, Phil. Trans. R. Soc. Lond. B 326: 217-229, 1990) and that the altered patterns of methylation are heritable (unpublished). As part of the study we examined the effects of other environmental influences on the activity of Ac in wx-m9 Ds-cy. Preliminary results of an experiment in which germinating seedlings were subjected to various cold treatments suggest that the methylation status and activity of the Ac element are not influenced by steady low temperature regimes.

Two lines of maize designated B and F, homozygous for bz2-m, were chosen from cobs which showed a low level of Ac activity (2-5% of kernels showing variegation, characteristic of an active Ac causing excision of Ds from bz2). The parent plants were later shown to be segregating for wx-m9 Ds-cy. Only kernels without spots were selected for planting, and were subjected to the following treatments: I, sown in a warm glasshouse (18 - 30 C) and after one week transferred to 3 C under low light for three weeks, then returned to the glasshouse; II, sown in a warm glasshouse and after one week transferred to 8 C under low light for three weeks, then returned to the glasshouse; III, sown at 8 C and maintained at 8 C in low light for four weeks before transfer to the glasshouse; IV, sown and maintained in the glasshouse.

The seedlings from treatment I succumbed to fungal rot and did not survive. For the other treatments DNA was extracted from young leaf tissue when the plants had reached a height of 300mm. The DNA was cut with the methylation-sensitive restriction endonuclease HpaII and subjected to Southern analysis using the internal HindIII fragment of Ac as a probe. The size of the hybridising band gives a measure of the level of cytosine methylation within the transposable element (Schwartz and Dennis, 1986). No differences were seen in the hybridisation patterns between the samples taken for the different treatments, although a slightly smaller band was seen in the samples from line B compared to those from line F.

The plants were grown to maturity, self-pollinated and the resulting cobs scored for Ac activity. The data are given in Table 1 and show that there is no increase in the level of Ac activity for the cold temperature treatments. Our conclusion is that moderate cold treatment at an early stage of development does not enhance the activity of the inactive Ac element in wx-m9 Ds-cy.

Table 1. Percentage of kernels showing Ac activity in cobs harvested from wx-m9 Ds-cy plants exposed to different temperature regimes.

    Numbers of plants in each class defined by percentage of kernels showing Ac activity
    0% 0.1-1% 1-5% >5%
Line Treatment        
B II 3 0 2 0
  III 8 1 2 1
  IV (control) 7 2 1 2
F II 1 1 0 0
  III 10 2 1 0
  IV (control) 8 2 0 2

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