Analysis of physiological traits under different soil water availability
The results showed that the two Einkorn genotypes had significant differences (p < 05.0) for stomatal conductance and photochemical efficiency of photosystem II (Table 2) after 14 days of water deficiency. RWC, stomatal conductance and Fv/Fm were significantly different among different soil water availability treatments (Table 2). The results of mean comparisons of two Einkorn genotypes after 14 days of water deficiency showed the highest values of stomatal conductance and Fv/Fm was shown in S10 genotype (Table 2). In addition, the results of LSD test of four soil water availability treatments after 14 days of water deficiency showed that the lowest RWC, stomatal conductance Fv/Fm were related to the severe stress (Table 2).
Table 2
Analysis of variance and mean comparison of measured traits in two Einkorn wheat genotypes under four soil water availability treatments after application of soil water availability treatments
Treatment | Relative water content (%) | Stomatal conductance (mmolH2O m− 2 s− 1) | Photochemical efciency of photosystem II |
Genotypes | ns | ** | ** |
Soil water availability treatments | ** | ** | ** |
Genotypes × Soil water availability treatments | ns | ns | ns |
Irrigation at (% FC) | |
100 (control) | 73.10a | 26.25a | 0.746a |
75 | 68.80b | 22.18b | 0.738a |
50 | 63.60c | 20.66c | 0.715b |
25 | 59.30d | 19.81c | 0.658c |
Genotypes | |
S10 | 66.8a | 23.22a | 0.738a |
H9 | 65.5a | 21.23b | 0.690b |
ns, *, and ** indicate non-significant, significant at P < 0.05 and P < 0.01, respectively. Values with different letters in superscript in a column indicate significant difference at P ≤ 0.05. |
Responses Of Physiological Traits After Re-watering
Analysis of variance of measured physiological traits in two Einkorn genotypes after 14 days of re-watering showed that H9 and S10 genotypes significantly were different for stomatal conductance and Fv/Fm. Significant difference observed between soil water availability treatments for all measured traits (Table 3). The results LSD test for two Einkorn wheat genotypes after re-watering showed that S10 genotype was significantly superior to H9 in terms of all traits (Table 3). Also, the results of LSD test for measured traits in the four soil water availability treatments after re-watering showed that RWC in severe stress treatment was significantly lower than other treatments. The highest and lowest stomatal conduction rates were obtained in control and severe stress treatments, respectively (Table 7).
Table 3
Analysis of variance and mean comparison of measured traits in two Einkorn wheat genotypes under four soil water availability treatments after re-watering
Treatment | Relative water content (%) | Stomatal conductance (mmolH2O m− 2 s− 1) | Photochemical efciency of photosystem II |
Genotypes | ns | ** | ** |
Soil water availability treatments | ** | ** | ** |
Genotypes × soil water availability treatments | ns | ns | ns |
Irrigation at (% FC) | |
100 (control) | 74.5a | 24.28a | 0.768a |
75 | 72.1ab | 21.63b | 0.745b |
50 | 69.8b | 19.65c | 0.726c |
25 | 62.4c | 18.06d | 0.671d |
Genotypes | |
S10 | 70.6a | 20.97a | 0.740a |
H9 | 68.8a | 20.84b | 0.710b |
ns, *, and ** indicate non-significant, significant at P < 0.05 and P < 0.01, respectively. Values with different letters in superscript in a column indicate significant difference at P ≤ 0.05. |
Results Of Molecular Analyses
In molecular studies, four soil water availability treatments in two stages included two weeks of water deficiency and two weeks after re-watering in two Einkorn wheat genotypes (S10 and H9) in three replications were examined individually using five RAPD primers and CRED-RA assay.
CRED-RA assay’s results in two Einkorn wheat genotypes after two weeks water deficiency and two weeks re-watering
Opc-12 Primer
Amplification of cleaved products with HpaII and MspI enzymes in S10 genotype show different patterns of methylation (including methylation, methylation in external cytosine and methylation in internal cytosine) in different replications of four soil water availability treatments (Fig. 1). This difference in different replications can be attributed to the variability in the response of individuals to stress and the specificity of the methylation pattern in different individuals. Cleavage with methylation-sensitive restriction enzymes in H9 genotype leading to fragments amplification that show different methylation patterns (including methylation, change of methylation pattern from complete methylation to methylation in external cytosine and methylation in internal cytosine and methylation of genome) in different replications of moderate and severe stress treatments and in response to severe moisture stress.
The results of cleavage with HpaII and MspI enzymes in S10 genotype after re-watering showed the return and emergence of deleted fragments in irrigation treatments at moderate and severe stress treatments, except for one fragment of about 250 bp in the first replication of moderate and severe stress treatments is still cleaved during digestion with HpaII enzyme and shows methylation in external cytosine. Methylation changes are reversible as the stress disappears. Also, the results of cleavage with HpaII and MspI enzymes in H9 genotype indicated that the methylation changes observed after 14 days of water deficiency, were reversible.
Opa-019 Primer
Cleavage results with restriction enzymes (MspI and HpaII) in S10 genotype after after 14 days of water deficiency showed that the only difference between the treatments was 900 bp fragment, which was observed in one of the replications in control treatment. Therefore, it was not considered as a change in methylation pattern in response to water deficiency (Fig. 2). Amplification of cleaved products with HpaII and MspI in H9 genotype after 14 days of water deficiency, showed removal of bands in the sizes of 230 and 250 bp in severe stress treatment during digestion with HpaII enzyme. Also, 550 bp fragment in moderate and severe stress treatments was cleaved by HpaII enzyme and converted from hypermethylation to methylation in foreign cytosine (Bolukbasi and Aras, 2016). These results indicate the response of H9 genotype to severe stress.
PCR-amplified DNA after endonuclease digestion after re-watering in S10 genotype showed that the removed fragment, when treated with MspI enzyme, which was observed in some replications after 14 days of water deficiency, appeared after re-watering. It seems that this fragment is related to a specific growth stage in the plant and after reaching this stage of cytosine external methylation growth, it has become completely methylated. In addition, the results of cleavage with restriction methylation-sensitive enzymes after re-watering in H9 genotype indicate the reappearance of deleted fragments of 230, 250 and 550 bp after stress relief and reversibility of genome methylation changes.
Opc-08 Primer
The results of digestion with HpaII enzyme in S10 genotype after 14 days of water deficiency indicated the presence of a fragment of about 2000 bp in moderate stress treatment and change of methylation pattern from complete methylation to methylation of external cytosine (Bolukbasi and Aras 2016). This fragment was removed by MspI and HpaII enzymes in severe stress treatment, which indicates its methylation in response to severe water deficiency. A fragment of 1700 bp in severs stress treatment was removed by HpaII enzyme and showed the change of methylation pattern to methylation of foreign cytosine in irrigation treatments in moderate and sever stress treatments and in response to severe water deficiency.
The results of CRED-RA assay for H9 genotype in different treatments of moisture stress, showed the difference a 1400 bp completely de-methylated in severe stress treatment (irrigation at 25% of field capacity) but in two replications of irrigation treatment in moderate stress treatment and one replication of mild stress treatment when cut with MspI enzyme showed methylation in external cytosine (Fig. 3).
The results of cleavage with HpaII and MspI enzymes after re-watering in S10 genotype show a stable genomic pattern and no polymorphism was observed among the amplification fragments. On the other hand, digestion with HpaII and MspI enzymes after re-watering in H9 genotype indicates the emergence of a 1400 bp fragment in severe stress treatment, but the remarkable point is the methylation pattern in internal cytosine in most repetitions in the mild and moderate stress treatments, so that it seems that the methylation pattern of this fragment can be related to other unknown factors.
Ops-07 Primer
CRED-RA assay in S10 genotype after two weeks water deficiency showed a fragment of 900 bp that was completely removed in the control treatment, and in mild stress treatment by the MspI was removed and shows a pattern of methylation in internal cytosine, whereas this fragment was not digested in moderate and severe stress treatments in three replications when treated with any of the enzymes and showed a complete methylation pattern. A fragment of 1500 bp was also removed by both MspI and HpaII in severe stress treatment (Fig. 4). Cleavage with methylation-sensitive enzymes in H9 genotype after two weeks water deficiency show that a 750 bp fragment was removed both MspI and HpaII enzymes and de-methylated by in severe stress treatment. It is from changing the methylation pattern to de-methylation.
PCR-amplified DNA after methylation-sensitive cleavage after re-watering in S10 genotype showed that 250 and 1500 bp fragments removed in severe stress treatment. While a 900 bp fragment in control and mild stress treatments is removed by both enzymes and in moderate and severe stress treatments when digestion with MspI enzyme is removed and shows the methylation pattern in internal cytosine. The genomic region appears to be completely methylated in response to severe moisture stress, and the methylation pattern changes due to milder moisture stress. Probably after re-watering, the effects of stress on moisture treatment are still observed in severe stress treatment. The methylation investigation results in H9 genotype show that the 750 bp fragment removed in severe stress treatment after two weeks water deficiency, re-emerged and the methylation pattern changed from methylation to hypermethylation.
Opb-07 Primer
The results of CRED-RA assay after two weeks water deficiency show that a fragment with a size of about 1000 bp in moderate stress treatment was digested by HpaII enzyme, which indicates a change in methylation pattern from hypermethylation status to methylation in external cytosine. This fragment was removed and completely methylated in the severe stress treatment by MspI and HpaII. The change of methylation pattern from complete methylation to de-methylation seems to have taken place in response to intense moisture stress.
The results of digestion with methylation-sensitive enzymes in the stress application stage in H9 genotype showed that a fragment of 760 bp in severe stress treatment was removed by both MspI and HpaII and completely methylated indicates the change of methylation pattern in severe stress from hypermethylation to de-methylation (Fig. 5).
The results of CRED-RA technique in S10 genotype after re-watering show that all removed fragments reappeared in response to moisture stress, no digestion and elimination took place and the stability of the genome pattern at this stage is 100%. Also, the results of CRED-RA technique after re-watering in H9 genotype showed the absence of 760 bp fragment in severe stress treatment during cutting with MspI and HpaII, which indicates the maintenance of methylation pattern in severe stress after relieving stress and recording changes in plant memory.