الفهرس 
IDENTIFICATION OF DIFFERENTIALLY METHYLATED CYTOSINES OF MAIZE GENOMIC DNA UNDER DROUGHT STRESSOnly 14 pages are availabe for public view
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Abstract
The present investigation was carried out to study epigenetic changes, especially DNA
methylations induced by drought stress as well as the effect of ABA deficiency on DNA
methylation of maize. For these purposes two different maize genotypes, namely inbred W22
(which has a reasonable level of) and mutant vp10 (ABA-deficient). Seeds of maize inbred line
W22 and maize mutant vp10 were obtained from maize genetics coop stock center. All seeds
were treated with 10% sodium hypochlorite for 30 minutes, then washed with distilled water
before sowing in the soil. Each seedling was grown in a 7-litre plastic pot containing a substrate
of 90% sand and 10% clay. Maize seedlings were irrigated to field capacity until the application
of drought stress treatment.
Thirty days old seedlings were subjected to a progressive water deficit by leaving them
without irrigation for nine days (drought-stressed plants) for either maize inbred line W22
plants or maize mutant vp10 plants. Non stressed plants were watered to maintain soil water
potential close to field capacity. Relative water content (RWC) of leaves non-stressed and
drought-stressed plants was measured by using the method described by (González and
González-Vilar, 2001). Soil water potential was measured by using Tensiometer as described
by Yang et al., 1993). Leaves of non-stressed and drought-stressed plants were collected and
treated immediately with liquid nitrogen and stored at -20 ˚C until DNA extraction and
subsequent DNA analysis.
DNA extraction from leaves was carried out as described by Soltis Lab CTAB DNA
extraction protocol (2002) developed from (Doyle and Doyle, 1987) and (Cullings, 1992).
AMP was performed according to (Phutikanit et al., 2010) with modification. Genomic DNA
was digested with HpaII and MspI restriction enzymes, according to New England BioLabs
recommended protocol. The digested DNA was purified and subjected to PCR amplification.
Twenty four primers were used. To detect the presence or absence of DNA methylation, the
methylation-sensitive amplification polymorphism (AMP) technique was used. To separate
amplified PCR products, 6% polyacrylamide gel was used and stained by silver nitrate (Bassam
and Gresshoff, 2007). The images of polyacrylamide gels were analysed by image lab (Bio-
Rad) software. The resulted AMP bands had been converted right into a matrix of binary
characters, which classified into four types. Type I bands, absent from both enzymes, which
represent the case of no methylation. Type II bands, present only for HpaII (1,0), which
represent the case of hemi or full methylation of internal cytosine (CG methylation) of 5´-
CCGG (Schulz et al. 2013). The hemimethylated external cytosine containing sites (CCG
methylation), undigested by MspI (0,1), constitute type III bands, while type IV bands (1,1),
resulting from the inability of both enzymes to digest full methylation of external or both
cytosine (full methylation).
Selected 86 differentially methylated DNA bands were excised and reamplified with the
same PCR conditions. The resulting PCR products were cloned using the pGEM®-T Easy
Vector System I. White colonies (3-5) were randomly picked from the MacConkey agar plate
containing ampicillin (Jennings and Beacham, 1989), and insert length was checked by colony
PCR. Positive PCR products were sent for sequencing at Macrogen (Amsterdam). The resulting
sequences were identified by BLAST searches against Zea mays genome databases at the NCBI
(http://blast.ncbi.nlm.nih.gov) and Gramene (http://www.gramene.org/) websites. The TEs
were identified by using CENSOR (https://www.girinst.org/censor/) website. The location of
the differentially methylated regions relative to genes and transposable elements (TEs) were
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viewed in NCBI graphical displays. The obtained results can be summarized in the flowing
points:
1- There were highly significant differences in the averages in the averages of RWC of leaves of non-stressed
and drought-stressed plants of maize inbred line W22 and maize mutant vp10). However,
there were insignificant differences in the averages of relative water content between all
W22 treatments and those of vp10.
2- The percentage of the cytosine methylation at CG sequence was higher (15.94%) in the
inbred W22 than those in the mutant vp10 (6. 07%) under drought stress.
3- The percentage of external cytosine methylation at the CCG sites in maize inbred line W22
was higher (16.30%) than those in the mutant vp10 (12.5%) under drought stress.
4- The percentage of external cytosine methylation at CCG and internal at CG sites (full) in
the inbred W22 plants was higher under drought stress (49.27%) than those under non-stress
conditions (42.02%).
5- The percentage of total methylation loci in the inbred W22 was higher (81.51%) under
drought stress than those under non-stress condition (72.10% )
6- The percentage of methylation at CG and CCG under drought stress were higher in maize
inbred line W22 (17. 03 and 11.96% respectively) than those in maize mutant vp10 (7.14%
and 6.07 respectively).
7- The percentage of demethylation at CG, CCG and full under drought stress were lower
maize inbred line W22 (8.7%, 8.3%, and 1.45% respectively)) than those in maize mutant
vp10 (11.07 %, 18. 57% and 6, 07% respectively).
8- It may be concluded from previous results that maize plants may respond to drought stress
by increasing the level of DNA methylation. The gain of DNA methylation may decrease
gene expression, slowing down the metabolism of the plant, which enables to conserve
energy to overcome the deleterious effect of drought stress. The decreased level of DNA
methylation under drought stress in mutant vp10 is correlated with ABA deficiency.
Consequently, ABA may employ DNA methylation to achieve the regulation of droughtresponsive
genes.
9- Five differentially methylated segments of maize inbred line W22 are homologous to the
flowing five genes: protein ALP1-like, embryo defective 2016, zinc finger C-x8-C-x5-Cx3-
H type family protein isoform X1, 3-hydroxyisobutyrate dehydrogenase which involved
in both, valine and isoleucine degradation in Arabidopsis thaliana and 3-isopropylmalate
dehydratase large subunit 2, which involved in glucosinolate formation
10- Ten differentially methylated e sequences from maize mutant vp10 are homologous to the
flowing ten genes: zinc finger protein WIP6, acid phosphatase/vanadium-dependent
haloperoxidase-related protein, lncRNA, fumarylacetoacetate (FAA) hydrolase, cysteine
tRNA ligase chloroplastic/mitochondrial, TPLATE, NAD-dependent
epimerase/dehydratase family protein, Nijmegen breakage syndrome 1(NBS1) , protein ,
CW-type zinc and Fumarylacetoacetate (FAA) hydrolase family protein which catalyses
the final step in tyrosine degradation . The C-terminal domain of NBS1 plays a critical role
in DNA damage signalling via recruitment, hence activation of the kinase activity of ATM.
Cooperation of NBS1and DNA Methyltransferase 1 is essential to heterochromatin
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regulation during DNA repair. H3K36me2 directly binds to NBS1 for MRN complex
recruitment or indirectly to promote NHEJ repair through PHRF.
11- Most of the differentially methylated DNA of W22 and vp10 fragments were mapped to
deep intergenic regions (at least 5kb away from a known gene) on the genome.
12- A total of 40 differentially methylated fragments (DMFs) showed more than 50% sequence
similarity with transposons in both W22 and vp10 and Gypsy and Copia like retrotransposons were
observed to be the most representative in both maize inbred line W22 and maize mutant vp10.
13- The majority of sequenced demethylated DNA fragments, 17 from 27, of vp10 were homologous to
transposable elements.
14- The sequencing of five DMFs was found to be homologous to lncRNA genes in both W22
and vp10.
15- Differentially methylated fragment in both inbred W22 and mutant vp10 is found to be
homologous to peptide methionine sulfoxide reductase (MSRA4).
16- In vp10, four zinc finger genes, ZmMYB087, protein DEHYDRATION-INDUCED 19
(Di19) homolog 3, zinc finger protein WIP6, and CW-type zinc finger protein were
represented by four differentially methylated fragments. Zinc finger -CW domain enzymes
participate in the methylation of histone H3 tail and are crucial for epigenetic memory.
17- Full methylation of the DNA segment in the vp10 mutant, which is homologous to the
upstream and first exon of the TPLATE gene. Interaction of TPLATE, part of an octameric
TPLATE complex (TPC), with AP2 complex and clathrin is requisite for plant survival