Detection of DNA hypomethylation mediated floral induction in longan and spinach using the HAT-RAPD technique

American Journal of Applied Sciences, Feb, 2009 by S. Anuntalabhochai, R. Chundet, N. Buapong, R.W. Cutler

INTRODUCTION

Many plants in temperate zones require a low temperature stress (vernalization) to induce flowering and fruit production. In some plants such as Arabidopsis and Cichorium intybus the vernalization response has been linked to changes in DNA methylation (1), (2), while the seedlings of other plants like Oryzia sativa are regularly treated with 5-azacytidine which results in dwarfism and a high tillering number due to hypomethylation of the cytosine nucleotide (3), (4). Alternatively, treatment of petunia seedlings with 5-azacytidine results in inhibition of shoot bud induction which is also mediated, at least partly, by the alteration of cytosine DNA methylation patterns (5). The parallels between the inheritance of the vernalization signal and DNA methylation patterns indicates that one common biochemical signaling system for vernalization is DNA methylation dependent. To investigate the mechanism of vernalization in other tropical plants, particularly Longan (Dimocarpus longan Lour.) and spinach (Spinacea oleracea L.) the demethylation agent 5-azacytidine and KCl[O.sub.3] application, in addition to natural temperature stress, was used to test for the presence of altered DNA methylation patterns.

The process of DNA methylation is a common modification of cytosine nucleotides seen in many eukaryotic organisms and in plant DNA, up to 30% of all the cytosine residues that exist as CpG or CpNpG sequence can have detectable levels of methylation (6). This modification directly affects DNA structure and hence can interrupt the interaction between a DNA template and RNA polymerase increasing the possibility of aborted transcription (3). Although DNA methylation seems to be a widespread mechanism present in eukaryotic organisms, the level and effect of DNA methylation can vary depending on species and developmental status. In plants, the alteration of methylation patterns is of particular interest since it can lead to morphological changes which can affect flowering patterns and overall plant development.

Due to the low temperature requirement for flower induction of many tropical fruits, the availability of these fruits is limited to narrow seasons which exactly match the required environmental conditions. One remarkable advancement in tropic fruit production is the recent determination that potassium chlorate can induce out of season flowering in Longan (7). This has lead to the widespread and year-round availability of Longan both domestically and as a growing product for export (8). By understanding the mechanism of action inducing this out of season fruit production, insight into other tropical fruits may lead to further advances making other fruits such as Lychee or Longong similarly available. Recently, molecular markers linked to the temperature independence for fruit production phenotype in both Lychee (9) and Longan (10) have been reported. In spinach, potassium chlorate and 5-azacytidine-treated spinach both develop an early flowering phenotype (11), indicating that potassium chlorate might also play a similar role to 5-azacytidine (12).

In this study, we investigated DNA methylation patterns in longan and spinach which were induced by 5-azacytidine, potassium chlorate (KCl[O.sub.3]) and low temperatures, using the HAT-RAPD methodology (High Annealing Temperature-Random Amplified Polymorphic DNA (13). This method is based on the random amplified polymorphic DNA (RAPD) technique combined with the application of isochizomers (HpaII and MspI) that differ in their sensitivity to methylation of their recognition sequences. In particular, both enzymes recognize the nucleotide sequence 5'-CCGG-3', where HpaII is inactive if one or both cytosines are methylated otherwise HpaII cleaves the recognition site, whereas MspI is inactive if the outer cytosine is methylated but cleaves if the inner cytosine is methylated (14). If a site interior to an RAPD amplified region is cleaved by a restriction enzyme then the DNA cannot be amplified and the corresponding band is absent. By characterizing altered amplification products due to the demethylation effects of 5-azacytidine upon the genome, the presence or absence of methylation linked to phenotypic alterations in longan and spinach can be studied using the RAPD technique.

MATERIALS AND METHODS

Plant materials: For each of the four species under study, samples were obtained and processed at the University of Chiang Mai in Chiang Mai Thailand. The 10 year old longan (Dimocarpus longan Lour. Var. Daw) trees were kindly provided by a private local orchard in the Hangdong district of the Chiang Mai province. The spinach (Spinacea oleracea L.) F1 hybrid var HI-UP 6901 seeds were obtained commercially (Know-you seeds Co., Ltd). Samples of Thai jasmine Rice (Oryza sativa indica cv. KDML 105) were provided by the Sun Pah Tong Rice Research Center in the Chiang Mai province. The petunia (Petunia hybrida var Border Gem) seeds were obtained from Arthur Yates and Co Ltd., Australia.