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Methylation Imprinting R Paramutation B Paramutation Pl-Blotched

Assay Data

The following is a description of our assays. Data are available at the tabs in the sub-menu above.

Effects on epimutation at pl. The purple1 (pl) gene encodes transcription factors that activate synthesis of flavonoid pigments and regulates anthocyanin production in vegetative tissues, such as leaf sheaths [Dooner et al., 1991 Annual Rev. Genetics 25: 193-199] The epigenetically silenced allele, Pl-Blotched leads to variegated—rather than uniform—pigmentation, and this phenotype coincides with decreased mRNA levels, alterations in DNA methylation, and more tightly packed chromatin structure [Cocciolone and Cone, 1993 Genetics 135: 575-588; Hoekenga et al, 2000 Genetics 155: 1889-1902; Das and Messing, 1994 Genetics 136: 1121-1141; Lund et al, 1995 Plant J. 7: 797-807]. Thus, this gene serves as a reporter for activities important for epigenetic regulation. To ask if any of the chromatin-gene IR lines alter Pl-Blotched expression, Basta-resistant T1 plants are crossed to stocks carrying this epiallele. Backcross progeny are grown in the field and scored for Basta resistance. Genotyping is conducted to identify homozygous Pl-Blotched plants and leaf sheaths are sampled for anthocyanin extractions.

Effects on paramutation. Paramutation, an interaction between specific alleles that leads to a heritable change in gene expression, is another epigenetic phenomenon associated with chromatin changes [Stam et al, 2002 Genes Dev 16: 1906-1918]. As the phenomena are distinct at different genes (reviewed in [Chandler et al, 2000 Plant Mol. Biol. 43: 121-145]), we assay affects of the IR lines on paramutation at b and r, both encoding transcription factors activating the anthocyanin pigment pathway [Dooner et al., 1991 Annual Rev. Genetics 25: 193-199; Radicella et al, 1991 Plant Mol. Biol. 17: 127-130; Perrot and Cone, 1989 Nucleic Acids Research 17: 8003].

Assays of paramutation at B: Paramutation at B occurs when B-I (B-Intense; dark purple pigment) is converted to B' (light purple pigment). The high penetrance of the B-I system makes it a particularly reliable screen for lines with defects in paramutation. To test establishment (the ability of the B' allele to change B-I into B'), T1 IR plants (Basta-resistant, b/b, green) are crossed with B-I/B-I plants (purple). The b allele in the IR lines is recessive and does not participate in paramutation. In the next generation, Basta-resistant purple (B-I/b, IR/-) plants are crossed with B'/B' plants. If gene silencing by the IR has no effect on establishment of paramutation, all of the progeny will be B'. If gene silencing by the IR affects establishment of paramutation, the Basta-resistant plants will have a purple B-I phenotype. To test maintenance (the ability of the B allele to heritably retain its low expression state), T1 IR plants (Basta-resistant, b/b, green) are crossed to B'/B' plants (light purple). If gene silencing by the IR has no effect on maintenance of paramutation, all progeny should be B'. If gene silencing by the IR affects maintenance, the Basta-resistant plants will have increased pigment, i.e. a B-I phenotype.

Assays of paramutation and imprinting at R. Paramutation at r is quite distinct from that at b [Chandler et al, 2000 Plant Mol. Biol. 43: 121-145], and one of the r alleles used in our research is also subject to another epigenetic phenomenon, imprinting [Kermicle, 1978 Maize Breeding and Genetics, pp. 357-371 Wiley, NY publisher; Alleman and Doctor, 2000 Plant Mol. Biol. 43: 147-161]. Paramutation and imprinting can be tested by examining kernel pigment levels using the same crosses. When R-stippled (R-st) is crossed with R-r:standard (R-r), R-r is changed into R-r', which has reduced pigment. R-r is also imprinted; conferring dark purple pigment when maternally transmitted and mottled pigment when paternally transmitted. The IR lines are homozygous for recessive r, which does not participate in paramutation. To test for effects on paramutation, T1 IR plants are crossed with W22:R-r/R-r, the resulting Basta-resistant R-r/r plants are crossed with W22:R-st/R-st producing progeny kernels that are R-st/R-r (purple) and R-st/r (stippled). By observing the genetic ratios of stippled, nearly colorless, and colored kernels, we can determine the effect of knocking down a given chromatin gene, e.g., whether it affects establishment or maintenance of paramutation or both. Effects on imprinting are also being tested in reciprocal crosses of W22 R-r/R-r with the IR lines. Increased expression of male-transmitted R-r or reduced activation of female-transmitted R-r is visualized as a change in the frequency of purple versus mottled kernels.

DNA methylation: DNA methylation in T2 IR transgenic plants is being compared to nontransgenic sibs, with DNA isolated from the same tissue sample used to assay reduced expression. DNA is digested with isoschizomer pairs HpaII/MspI (CG and CCG methylation) and EcoRII/BstNI (CA/TG methylation). DNA gel blots are probed with highly repetitive (ribosomal, centromere), moderately repetitive (retrotransposon, transposon) and low copy genic sequences with known methylation patterns. Any samples that show altered methylation will be analyzed with HPLC to quantify the total methylation change [Papa et al, 2001 Plant Cell 13: 1919-1928].

This material is based upon work supported by the National Science Foundation under Grant No. NSF-DBI-0421619. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. © 2006–2009 The University of Arizona. All contents copyrighted. All rights reserved.