In the aim to study the contribution of phenolic compounds in the inhibition of seeds germination of wheat ( Triticum aestivum L. ) under water deficit, we have forwarded quantitatively and qualitatively the change of the phenolic compounds levels in the seeds of two varieties (Achtar and Tigre) submitted to water stress induced by PEG-6000 during the germination stage. The results revealed significant differences between the stress intensity and duration and the polyphenol content. Some germination traits were significantly correlated with water stress and polyphenols in the both varieties studied. The polyphenol content in Tigre was much higher than those noted in Achtar. Analysis of phenolic extracts by HPLC showed the dominance of hydroxycinnamic derivatives (97.2% and 94.5% in Tigre and Achtar, respectively). Regarding phenolic acid, the ferulic acid was the most dominant, and seemed to be related mainly to the inhibition of germination. Quantitatively, the Achtar variety was characterized by the presence of acid sinapic which was absent in Tigre variety. Furthermore, the water deficit appeared to stimulate the induction of salicylic acid accumulation in both varieties.
Because of the global climate change and the increasing scarcity of water resources, wheat production is strongly influenced [
Water stress is one of the most important limitations of the development and the growth of plants because of the role of water in plant metabolism during germination. Indeed, the water deficit imposes itself as extrinsic factor that negatively influences the germination of seeds due to the essential and vital role of water in the induction of recovery of metabolic activity during germination process. In fact, this stage represents the critical and crucial step of plant growth. Many studies showed that the rate and speed of germination are reduced when the water potential of the medium decreases [
The phenolic compounds in seeds were often implicated in the regulation of germination. They are known by their inhibitory effect. Given that the outer layers of mature seeds are rich in phenolic compounds, the content and nature of these compounds appear to be indexes for explain their involvement in the inhibition of seed germination. However, germination is generally improved when the levels of phenolic acids in seeds decreased during the natural evolution and following treatments applied to seeds [
The biological material for the present study consisted of seeds of Triticum aestivum L. So, the major aim of the study has been to investigate whether phenolic compounds are involved in the inhibition of wheat seed germination under water stress simulated with PEG-6000.
Selected seeds of two Moroccan varieties of wheat (Triticuma estivum L.) Tigre and Achtar, kindly provided by the National Seed Marketing Company (SONACOS-Société Nationale de Commercialisation de Semences), were studied. The seeds were germinated in glass petri dishes (90 mm diameter), sterilized beforehand and not hermetically closed, containing the special medium. Each Petri dish contained 50 seeds (one replication). Our experiments were performed in control and PEG treatment conditions. We induced stressful conditions by different concentrations of PEG-6000: 20, 40, 60, 80 and 100 g/l PEG. Petri dishes were placed in incubator maintained at 25˚C ± 1˚C and a photoperiod of 16:8 h (light: dark). The experiment consisted of a randomized block design with six replications.
The number of germinated seeds was determined once a day until the percentage of germination was stable. For each treatment, we characterized the germination of two wheat varieties by four parameters: the maximum percentage of germination (MPG), the time to starting germination (TSG), the time to 50% germination corresponding to 50% of total germinated seeds in the Petri dish (T50% G) and the time to maximum germination (TMG).
The finely ground powder was ground on ice in 80% methanol (MeOH) and then sonicated for 10 minutes in ultrasonic bath (Elma, Germany) containing 700 ml ultrapure water. The homogenate was centrifuged at 12,000 x g for 10 min at 4˚C. The concentration of polyphenols in the supernatants was determined with Folin-Ciocalteu reagent following the colorimetric method by adopting a differential assay in the presence/absence of 1% (w/v) polyvinylpolypyrrolidine (PVPP) as described in [
HPLC analysis of phenolic acids of wheat seeds extracts was performed with a Waters 600 system equipped with a photodiode array detector (Waters 2996) and a Waters 600 gradient pump. The column used as stationary phase was an Alltima C18 reversed-phase column (250 mm × 20 mm, 5 μm) with a C18 Guard-pak pre-column (Waters Assoc.). The mobile phase was composed of mixture of water-acetic acid (98: 2, v/v) (solvent A) and acetonitrile (solvent B). Phenolic acid separation was achieved using a 60-min linear solvent gradient at a flow rate of 1.0 mL/min. The solvent gradient was as follows: 0% - 15% B (10 min), 15% - 20% (10 min), 20% - 30% (10 min), 30% - 40% (5 min), 40% - 55% (5 min), 55% - 80% (5 min), 80% - 100% (2 min), 100% (8 min) and 0% (5 min). After filtration through a syringe filter (25 mm GHP 0.45 µm, Acrodisc GHP), the injection volume was 20 µL. The different phenolic compounds were detected by absorption at the range of 240 - 400 nm. Then, they were determined by comparing the retention times of known peaks of the following pure standards: galic, p-hydroxybenzoic, trans-cinnamic, caffeic, ferulic, sinapic and p-coumaric acids as well as vanillin and salicylic acid (Sigma Aldrich, USA). Concentrations of phenolic compounds were expressed as μg ferulic acid equivalent per gram of wheat, using the height and the area under peaks in quantitative calculations.
Each data pointed the mean of three separate replicates, and mean values and standard deviations were calculated. Results were examined by the analysis of variance (ANOVA) in order to examine the effect of ecotype, time, watering regime and their interactions in each of the physiological and biochemical study variables. Means were compared using the Tukey’s Post hoc test after checking the normality and the homoscedasticity of data. Pearson correlation analysis was done for some variables for each ecotype after checking the assumptions of parametric tests.
The results of the variation of parameters related to the germination time showed significant differences depending on the variety (P < 0.05) (
According the time taken to maximum germination for each variety (3 and 6 days for Achtar and Tigre, respectively), we followed the kinetic change of total polyphenols (
The quantitative and qualitative analysis of phenolic extracts of two wheat varieties studied was performed by HPLC. We chose phenolic extracts corresponding to the first, third and sixth day of the period of germination
PEG-6000 g/L | Variety | Germination parameters | ||
---|---|---|---|---|
TSG (h) | T50%G (h) | TMG (h) | ||
0 | Achtar | 30 | 36 | 72 |
Tigre | 50 | 100 | 144 | |
20 | Achtar | 30 | 36 | 72 |
Tigre | 52 | 108 | 144 | |
40 | Achtar | 30 | 36 | 72 |
Tigre | 53 | 108 | 144 | |
60 | Achtar | 32 | 36 | 72 |
Tigre | 57 | 120 | 144 | |
80 | Achtar | 34 | 40 | 72 |
Tigre | 58 | 128 | 144 | |
100 | Achtar | 34 | 40 | 72 |
Tigre | 58 | 128 | 144 |
for the Tigre variety, while for the Achtar variety, we studied the phenolic extracts relating the first and third day. Thus, we have considered only the treatments of water stress corresponding to concentrations: 60 and 100 g/L of PEG-6000 by simulating moderate and severe water treatment, respectively. The HPLC-UV qualitative approach revealed two series of phenolic acids, including hydroxybenzoic and hydroxycinnamic derivatives (Ta- ble 2 and
Germinated seeds | Seeds control | |||||||
---|---|---|---|---|---|---|---|---|
Days | 1 | 3 | ||||||
PEG (g/L) | 0 | 60 | 100 | 0 | 60 | 100 | ||
Benzoic acid derivatives | Gallic acid | 6.8 | 2.9 | 3.8 | 13.7 | 8.3 | 10.7 | 2.4 |
OH-benzoic acid | 4.1 | 2.1 | 2.9 | 5.3 | 3.9 | 4.8 | 1.4 | |
Vanillin | 2.4 | 1.0 | 0.6 | 1.3 | 0.3 | 0.7 | 0.6 | |
Cinnamic acid derivatives | Caffeic acid | 4.7 | 4.9 | 4.4 | nd | nd | nd | 1.8 |
Sinapic acid | 3.4 | 3.6 | 3.9 | 1.2 | 0.5 | 0.6 | 1.1 | |
p-coumaric acid | 1.2 | 1.0 | 1.3 | 0.9 | 0.9 | 1.1 | 1.5 | |
Salycilic acid | nd | nd | nd | 16.89 | 6.54 | 11.97 | nd | |
Cinnamic acid | 61.1 | 28.9 | 45.9 | 35.4 | 28.8 | 30.2 | 45.8 | |
Ferulic acid | 241.9 | 203.5 | 208.4 | 123.9 | 112.8 | 117.7 | 242.8 | |
PT (HPLC) | 325.7 | 248.2 | 271.2 | 198.5 | 162.1 | 177.7 | 297.3 | |
PT (FC) | 341.7 | 253.3 | 313.3 | 218.3 | 210.0 | 240.0 | 366.7 |
phenolic composition identified in Tigre and Achtar varieties, respectively (
Germinated seeds | Seeds control | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Days | 1 | 3 | 6 | ||||||||
PEG (g/L) | 0 | 60 | 100 | 0 | 60 | 100 | 0 | 60 | 100 | ||
Benzoic acid derivatives | Gallic acid | 3.9 | 1.5 | 2.1 | 9.0 | 6.2 | 8.9 | 5.3 | 6.7 | 4.5 | 3.3 |
OH-benzoic acid | 4.3 | 1.5 | 4.6 | 4.4 | 2.6 | 2.9 | 2.5 | 2.1 | 2.7 | 7.2 | |
Vanillin | 1.8 | 4.3 | 1.8 | 0.8 | 0.7 | 0.7 | nd | nd | nd | 2.7 | |
Cinnamic acid derivatives | Caffeic acid | 5.2 | 6.2 | 6.2 | 5.5 | 3.1 | 3.9 | nd | nd | nd | 2.9 |
Sinapic acid | nd | nd | nd | nd | nd | nd | nd | nd | nd | nd | |
p-coumaric acid | 1.5 | 1.8 | 2.5 | 1.1 | 1.2 | 1.8 | nd | nd | nd | 1.5 | |
Salycilic acid | nd | nd | nd | nd | nd | nd | 4.58 | 3.72 | 5.96 | nd | |
Cinnamic acid | 119.5 | 102.7 | 120.9 | 46.2 | 59.2 | 75.2 | 41.9 | 64.6 | 82.9 | 131.5 | |
Ferulic acid | 285.7 | 279.7 | 262.0 | 229.3 | 257.5 | 250.5 | 218.4 | 245.5 | 243.5 | 275.2 | |
PT (HPLC) | 421.9 | 397.7 | 400.0 | 296.3 | 330.5 | 343.8 | 272.7 | 322.6 | 339.6 | 424.2 | |
PT (FC) | 486.7 | 486.7 | 493.3 | 435.0 | 458.3 | 481.7 | 281.7 | 303.3 | 405.0 | 495.0 |
According to the chromatograms obtained from the both varieties, we have highlighted a rich phenolic composition, mainly with phenolic acids. In fact, the phenolic acids identified in Tigre and Achtar varieties were: gallic acid, hydroxybenzoic acid, vanillin, caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid and salicylic acid. However, the cinnamic acid was detected with high content after the acid ferulic, the most dominant in the phenolic composition of seeds (
Exposure of seeds of both wheat varieties, especially Tigre variety, to water stress induced by PEG-6000 was manifested by a significant decrease of the final percentage germination. Indeed, water stress seriously limits the rate of germination [
Our results also showed changes of total polyphenols contents during the germination period, under water stress conditions. Thus, varietal differences, in terms of the variation of polyphenol contents, were recorded. Water stress as abiotic factor seriously influences the content of phenolic compounds in plant material [
The majority of phenolic compounds that we have detected in the both varieties were phenolic acids and precisely hydroxycinnamic acid derivatives whose fraction of hydroxybenzoic acid derivatives was minor. Thus, the phenolic composition identified in the present work corroborates with other studies on wheat, with some differences (presence or absence of certain phenolic acid), according the variety effect [
It is well known that salicylic acid plays an essential role in the induction of resistance genes as a long distance chemical signal responsible for systemic acquired resistance [
The quantitative and qualitative analysis of phenolic compounds in seeds of two varieties of wheat, under water stress conditions during germination, allowed to characterize the main phenolic acids, dominated by ferulic acid, in order to explain the involvement of these compounds in the germination inhibition. Varietal differences were noted for the contents of phenolic compounds and the germination parameters related to time, depending on the intensity and duration of stress. The induced water stress significantly affected MPG, TSG, T50% G and TMG. In parallel, the polyphenol contents also varied significantly during the germination stage. The both varieties studied Achtar and Tiger were qualitatively distinguished by sinapic acid. However, a late induction of salicylic acid has been recorded in both varieties. Future researches on the role of phenolic compounds during wheat germination seem to be necessary to reduce their contents in order to enhance and accelerate germination.
The authors would like to thank entire team of “Laboratoire de Biotechnologie Valorisation et Protection des Agroressources”, Faculté des Sciences et Techniques Guéliz, Université Cadi Ayyad, Marrakech, Maroc.
Abdelghani Chakhchar,Salama Aissam,Cherkaoui El Modafar, (2016) Quantitative and Qualitative Study of Phenolic Compounds Involved in Germination Inhibition of Wheat under Water Deficit. Technology and Investment,07,86-95. doi: 10.4236/ti.2016.73011