ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 714-719



УДК 581.1


© 2015 S. F. Li*, G. J. Zhang**, J. H. Yuan*, C. L. Deng*, L. D. Lu*, W. J. Gao*

*College of Life Sciences, Henan Normal University, Xinxiang, China **Research Center for Immunology, Department of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China Received December 28, 2014

Spinach (Spinacia oleracea L.) is a diploid dioecious plant with a pair of heteromorphic sex chromosomes X and Y Plant DNA methylation, a known process for genome epigenetic modification, regulates gene expression in plants. To explore the effects of DNA methylation on spinach growth and sexual development, spinach seeds were treated with the demethylating reagent 5-azaC. The resulting phenotypes were then investigated, including germination percentage, root length, plant height, flowering time, and sexual phenotype. Results showed that 5-azaC at a low concentration (30 цМ) only slightly influenced spinach development but promoted seed germination. The germination percentage, root length, and plant height negatively correlated with 5-azaC at 100—1000 цМ. The flowering time significantly reduced at all four treatments with 5-azaC. In addition, 5-azaC influenced the sexual phenotype of spinach and remarkably increased the percentage of monoecious individuals. These results may suggest that vegetative and reproductive growth are both epigenet-ically regulated by DNA methylation.

Keywords: Spinacia oleracea — 5-azaC — DNA methylation — flowering time — plant growth — sexual phenotype

DOI: 10.7868/S0015330315050115


DNA methylation, particularly cytosine methylation (5mC) in eukaryotes has received considerable attention in recent years as an important epigenetic regulation mechanism. The DNA modification of cy-tosine at position 5 primarily occurs at symmetrical CG dinucleotides in the animal genome; by contrast, this modification is also observed in the plant genome at non-CG sites, such as the symmetrical CNG and asymmetrical CNN trinucleotides (where N is A, C, or T) [1, 2]. Plant nuclear DNA usually possesses high levels of 5-methylcytosine; the total methylation levels of CG, CNG, and CNN in the Arabidopsis thaliana genome are 24%, 6.7%, and 1.7%, respectively [3]. DNA methylation, particularly cytosine methylation in plants, has been associated with several fundamental cellular activities, such as gene expression, genomic integrity maintenance, plant development, immune

1 This text was submitted by the authors in English.

Abbreviations'. 5-azaC — 5-azacytidine; 5mC — cytosine methylation; MS-ISSR — methylation-sensitive-inter-simple sequence repeats.

Corresponding author. Wu-Jun Gao. College of Life Sciences, Henan Normal University, No. 46, East Jianshe Road, Xinxiang 453007, P.R. China; fax. 86-0373-3329102; e-mail. gaowujun1@163.com

response, chromatin remodeling, and genomic imprinting [4—7].

Hypomethylating agents are widely used to study the consequences of genome-wide demethylation. 5-azacytidine (5-azaC) is a cytosine analog in which ring carbon 5 is replaced by nitrogen [8, 9]. Assessment of the effects of 5-azaC treatment on the morphological, molecular, and cytological properties of plant species is important to investigate the functions and mechanisms of DNA methylation. For example, 5-azaC treatment can improve the phenotypic plasticity of tuber-bearing Solanum species and the inter-relation between DNA methylation and microRNA accumulation in a wide range of species [10].

DNA methylation participates in sex determination and differentiation. Most of these studies investigated the sex chromosomes of human and other mammals [11—14]. Only a few studies have analyzed the relationship between DNA methylation and plant sex determination or sex chromosome evolution. Reproducible differences in the DNA methylation patterns between two X chromosomes in female somatic cells were investigated in the model dioecious species Melandrium album [15]. Treatment of M. album seeds with 5-azaC induces a sex change to androhermaph-roditism (andromonoecy) in approximately 21% of male plants, whereas no apparent phenotypic effects

have been observed in female plants [16]. Thus, female sex suppression in M. album males is dependent on the methylation of specific DNA sequences, which can be heritably modified by hypomethylating drugs. DNA methylation and heterochromatinization play essential roles in the early evolution of sex chromosomes [17].

Spinach is a diploid dioecious plant that has been cultivated as a highly nutritious vegetable in many regions worldwide. A 1 : 1 ratio of males and females has been found in many spinach varieties; however, monoecious individuals with both male and female flowers also exist at low frequencies [18]. Spinach has 2n = 12 chromosomes, and the sex of this species is controlled by the X and Y chromosomes [18]. The genotype XX is pistilate, XmX and XmXm are potentially monoecious, and XY and XmY are staminate [19]. Spinach is a model dioecious plant species for genetic and physiological studies on sex determination and expression [18—20]. We have recently identified various altered methylation patterns between untreated and 5-azaC-treated spinach plants via MS-ISSR (methylation-sensitive-inter-simple sequence repeats) technique [21]. However, there was no other report on the study of DNA methylation in spinach.

In the present study, spinach seeds were treated with the DNA demethylating agent 5-azaC to explore the effects on spinach growth and sex development. The resulting phenotypes were investigated, including germination percentage, root length, plant height, flowering time, and sexual phenotype.


Plant material and 5-azaC treatment. Spinach (Spinacia oleracea L.) seeds from full-sibling progenies were sterilized with 75% ethyl alcohol and immersed in sterilized water for 12 h. The soaked seeds were transferred to filter papers in Petri dishes. Different concentrations of 5-azaC in sterile distilled water (0, 30, 100, 500, and 1000 |M; "Sigma", USA) were added to the Petri dishes. Five treatments were prepared, and each treatment was performed in triplicate. The treatment lasted for 14 days in all plant groups. Fresh 5-azaC solutions were prepared and applied daily. During the treatment period, the Petri dishes containing the spinach seeds were gently shaken in dim light conditions at room temperature. The plantlets were then transferred into pots and grown in a greenhouse in Henan Normal University under a 14 h day/10 h night cycle.

Effects of 5-azaC on root induction. The roots of the spinach seedlings without treatment (control) and treated with 5-azaC at four concentrations were observed every other day before transplantation. The germination percentage (number of germinated seeds at the given time point divided by the total number of seeds and multiplied by 100) was measured on the 3rd

100 90 ^ 80 §70 60 I 50 I40

13 30 $ 20 10

0 30 100 500 1000

Concentration of 5-azaC, p.M

Fig. 1. Seed germination of spinach treated by 5-azaC in different concentrations.

1 — 3 days; 2 — 7 days. The one-way ANOVA was used to analyze the significance of the results. Asterisks represent significant differences between the 5-azaC-treated and the control plants: *p < 0.05; **p < 0.01. Values are expressed as the means based on three independent experiments. Error bars indicate SD.

and 7th day. The root lengths of each treatment group were measured on the 3rd, 5th, and 7th day by randomly selecting 20 germinated seeds.

Effects of 5-azaC on spinach development and sexual phenotype. For each treatment, we randomly selected 20 plants and recorded the following data: (1) plant height after the germinated seeds were transferred to pots at 7, 20, and 50 days; and (2) flowering time, which is defined as the number of days between the start of the greenhouse experiment and the first appearance of a flower. To investigate sexual phenotype, the flower structure of individual plants was checked during the flower development stage to distinguish the male, female, and monoecious plants. Data for at least 150 individuals from each treatment were randomly recorded.

Statistical analyses. Data were analyzed by one-way ANOVA with the SPSS statistical program (v. 18.0) to determine the significant differences (p < 0.05 or p < 0.01) between the 5-azaC-treated samples and the control ones. Separate analyses were performed for each sample.


Effects of 5-azaC on the spinach seed germination

The germination percentage of spinach seeds on the 3rd day was greater than 70% even with 5-azaC treatment at different concentrations; the germination percentage on the 7th day was greater than 80% for all treatments (fig. 1). Compared with the control group, the group treated with 30 ^M 5-azaC had significantly higher germination percentage on the 3rd and 7th day. However, the seed germination percentage gradually


9 8


° 6 - 5

M 5




30 100 500 Concentration of 5-azaC,


80 70

J? 60

e, 50



40 30 20 10



55.7** —»-


30 100 500 1000 Concentration of 5-azaC, p.M

Fig. 2. Root length of spinach seedlings treated by 5-azaC in different concentrations.

1 — 3 days; 2 — 5 days; 3 — 7 days. Asterisks represent significant differences between the 5-azaC-treated and the control plants. *p < 0.05; **p < 0.01.

decreased with increasing 5-azaC concentration (fig. 1). Compared with the control group, the group treated with the highest concentration of 5-azaC (1000 ^M) demonstrated 15.1 and 13.5% lower seed germination percentage on the 3rd and 7th day. Therefore, low concentrations of 5-azaC can improve the germination of spinach seeds, whereas high concentrations of this demethylating agent suppressed seed germination.

Root development analysis of spinach after 5-azaC tr

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