Elsevier

Plant Science

Volume 175, Issue 3, September 2008, Pages 217-225
Plant Science

A comparative analysis of developmental profiles for DNA methylation in 5-azacytidine-induced early-flowering flax lines and their control

https://doi.org/10.1016/j.plantsci.2008.03.023Get rights and content

Abstract

Earlier experiments demonstrated that DNA from young plants of 5-azacytidine-induced flax (Linum usitatissimum) lines that flower earlier-than-normal is hypomethylated relative to DNA from their control lines and detected differences in methylation level between plants sampled at different ages, which suggested that the methylation level in flax changes during development. To investigate this possibility, and its potential impact on the difference in methylation level between early-flowering and control lines, developmental profiles were established for the cytosine methylation levels in DNA from post-germination seedlings and from the shoot tips of main stems and the cotyledons sampled throughout vegetative phase. The methylation profiles for two early-flowering lines and their control lines were compared. The methylation profiles were then compared to profiles for DNA content, tissue weight and chlorophyll content (green tissues); these additional parameters provided information on tissue status in terms of cell division, tissue expansion and/or photosynthetic maturity. With one exception, methylation levels were either static or increased with plant age and/or tissue maturity; the highest methylation levels were seen in senescent cotyledons. Although DNA from immature plants or tissues of the early-flowering lines was usually hypomethylated, the hypomethylation was not always apparent in tissues from older plants.

Introduction

The phenotypic plasticity of plants is thought to involve epigenetic regulation, and DNA methylation and chromatin organization may be responsible for reinforcing changes in patterns of gene expression that occur during plant development [1], [2], [3]. Many processes, including gene silencing [4], [5], transposon regulation [6], [7], [8], gametic imprinting [9] and nucleolar dominance [10], involve changes in gene expression that are moderated by changes in cytosine methylation. In some cases, methylated cytosine residues in promoter and enhancer regions may directly prevent the binding of transcription factors but, in most cases, the presence of methylated cytosine is thought to attract methyl-cytosine-binding proteins which recruit histone deacetylases and chromatin remodelling proteins that, in turn, compact the chromatin and restrict access of the transcription machinery [11], [12]. Thus, heterochromatin is usually characterized by hypermethylation and gene inactivation and euchromatin by hypomethylation and activation [3]. Furthermore, changes in chromatin may simultaneously regulate the expression of more than one gene [13] and RNA may mediate the formation of silent chromatin by directing the modification of DNA and histone methylation [14], [15].

In some plant species, phenotypic and/or developmental changes have been induced by the DNA demethylating agent 5-azacytidine (azaC), and in a smaller subset of these species the transmission of these changes into subsequent generations, together with altered methylation status, has been observed. For example, heritable azaC-induced changes have been reported in Oryza sativa [16], triticale [17], [18], Brassica oleracea [19], Nicotiana tabaccum [20], Melandrium album [21], and Linum usitatissimum (flax) [22].

In flax, azaC treatments applied to germinating seeds produced a number of early-flowering lines. Four of the azaC-induced, early-flowering flax lines from two different genetic backgrounds (inbred cultivars) have been maintained, by inbreeding, for 8–10 generations. Lines of the progenitor cultivars from untreated plants that were grown in the treatment generation and with each subsequent generation serve as their “control lines”. The early-flowering lines flower 8–13 days earlier than their control lines. The early-flowering phenotype of each azaC-induced line is regulated by at least two independent loci [22] and crosses between early-flowering lines have demonstrated that some, but not all, of the loci are the same in more than one line (Fieldes, unpublished). Although the genes involved have not yet been elucidated, the early-flowering phenotype has been characterised [23] and hypomethylation of total DNA from the early-flowering lines, compared to that from controls, has been demonstrated [24].

The first generation progeny of the azaC-treated flax plants were hypomethylated, and flowering age and methylation level cosegregate in the F2 and subsequent generations of crosses between early-flowering and control lines [24]. During these earlier studies, which used DNA from the cotyledon and epicotyl region of 4-day-old plants and from the shoot tips of the main stems of 14-day-old plants, differences in methylation levels were observed between the tissues/ages and were sometimes seen between experiments that used the same tissue/age [24]. This suggested the level of methylation in flax changes during plant growth and development and was consistent with other reports of temporal and spatial differences in methylation levels in other plant species [25], [26], [27], [28], [29], [30], [31], [32]. It also indicated a need for ontogenetic profiles of methylation level in the flax lines.

The heteroblasty of stem and leaf characteristics delineates three periods of vegetative development in the main stems of control and early-flowering lines [23]. The three periods are likely to correspond to the juvenile, transition and adult phases of the vegetative phase, respectively. In addition, the first 6–8 leaves at the base of the stem appear to make up a separate category [23], and are referred to, here, as the “early juvenile phase” with the remainder of the juvenile leaves constituting the “late juvenile phase”. During the vegetative phase, the major difference between early-flowering and control lines lies in the duration of adult phase, which is extremely truncated in the early-flowering lines [23]. Under greenhouse conditions, the shifts from juvenile-to-transition and transition-to-adult occur at approximately 28.5 and 35 days, respectively, in both types of line.

In establishing the developmental profiles for DNA methylation, the primary objective was to determine the changes in methylation level that occur in flax and whether they relate to changes in tissue maturity or differentiation, and to compare the profiles for early-flowering and control lines. Based on the association between cytosine methylation and heterochromatin formation, it was predicted that the methylation levels would be low in immature tissues and increase with age/tissue maturity, reflecting declining levels of cell division and/or increasing cellular or functional differentiation. In addition, it was predicted that the early-flowering lines would be hypomethylated in all tissues and at all ages except, perhaps, at the onset of flowering. This possible exception related to the idea that the azaC-induced hypomethylation in the early-flowering lines is permanently associated with loci that control the early-flowering phenotype, whereas, in the control lines the same loci have to be demethylated as part of the normal developmental program that leads to flowering [24].

Two plant regions were emphasized; the cotyledons, which provide a well-defined, single source of tissue for looking at growth, differentiation and senescence, and the epicotyls and shoot tips, which provide a source of consistently young tissue but may also reflect the changes in leaf form that occur during vegetative growth. Profiles for tissue weight and DNA content were used to assess the contribution of cell division to increases in fresh weight and changes in chlorophyll content were used to assess the developmental status of some green tissues, in particular, the cotyledons.

Section snippets

Plant lines, growing conditions, planting and sampling

Three inbred lines of flax were used: RE1, RE2 and RC. The azaC-induced early-flowering lines, RE1 and RE2, were derived from the inbred oilseed cultivar Royal (R) and came from early-flowering progeny of plants that had been grown from germinating seeds that were treated with 5-azacytidine [22], [33]. RE2 was previously called RE2’ [24]; the control line, RC, came from an untreated R plant in the azaC-treatment generation.

The seeds for the seedling experiments sampled 16–76 h from sowing were

Developmental profiles for the cotyledons: early post-germination

In the flax lines used the radicle normally emerges as early as 16 h after the start of imbibition, and early post-germination, from 16 h to day 3, is characterised by radicle elongation followed by elongation of both hypocotyl and radicle. By day 3, the epigeic cotyledons begin to enlarge and “green” and the seed coats are shed indicating depletion of the endosperm reserves. The early post-germination of RC and RE1 was examined. The seeds used were surface sterilized and their germination and

Discussion

The DNA methylation levels in flax ranged from about 10% to 20%, they changed systematically within each different type of tissue, and the changes with plant age or tissue location were almost invariably associated with increasing tissue age and/or maturity. The methylation profiles suggested that either shoot tips or cotyledons, from plants that are 14–24 days old, would be most suitable for comparing the methylation levels in RC, RE1 and RE2, and probably also other flax lines. That is,

Acknowledgements

The research was supported by a Natural Sciences and Engineering Research Council of Canada Undergraduate Student Research Award (J.C.L.B.) and a Discovery Grant (M.A.F.), and by infrastructure funded by the Canadian Foundation for Innovation, the Ontario Innovation Trust, Wilfrid Laurier University, and Varian Canada. The technical assistance provided by M.R. Martin and A.V.D. Johnson is acknowledged with thanks.

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