The impact of tribenuron methyl on seeds germination
The results of the estimation of seed germination of cornflower biotypes with various types of resistance to tribenuron-methyl are shown in Table 1. Most of the seeds, namely 75 and 55 %, with the biotype of metabolic and the mutational resistance type, respectively, were germinated after the first day, whereas only 15 % of the seeds of the susceptible biotype were sprouted at the same time. In the resistant biotypes (metabolic, mutational) the values of the germination index (GI) were higher than in the susceptible biotype.
Table 1 Germination capacity of cornflower biotypes with different type of resistance to the tribenuron-metyl
Chemical composition in seeds and in tribenuron methyl treated seedlings and leaves
The differences between biotypes relating to the composition of the storage materials were clearly visible already in dry seeds. The ability of plants to survive and reproduce in response to the herbicide, and thus to develop resistance, might be related to their chemical composition. The chemical composition may affect the differences in the germination index between biotypes resistant and susceptible to tribenuron-methyl (Table 1). Chemical composition of seeds was determined on the basis of FT-Raman spectra. The identification of particular groups of compounds present in plant tissues was based on the specific marker bands (Table 2). Slight shifts comparing to theoretical data which were observed in some bands were due to the fact that, in plant tissue, the analyzed compounds exist together with others, which sometimes led to interference.
Table 2 Assignment for the most characteristic Raman bands observed for tested biotypes of dry seeds of cornflower
Analysis of the spectra obtained from dry seeds have shown the presence of marker bands, specific to several essential chemical compounds (Fig. 1). The most characteristic were those derived from mono-, di- and polysaccharides identified at 841, 872, 900, 931, 1081, 1122 and 1745 cm−1. Peaks observed at 900 and 1122 cm−1 were, to some extent, defined by lower intensity in the susceptible biotype. The presence of lipids and fatty acids could be detected at 1263, 1301, 1440 and 1745 cm−1. All the examined seeds showed a clear band at 1654 cm−1 arising from the presence of proteins. The slightly higher intensity of peaks for polyphenols (also flavonoids) was observed in biotypes with mutational and metabolic resistance (1176 and 1600 cm−1).
In addition, hierarchical analysis of similarity was performed to compare the overall chemical composition of the studied biotypes of seeds. It was demonstrated that the chemical composition of the endosperm in biotype susceptible to tribenuron-methyl was significantly different from biotypes of metabolic and mutational resistance to this compound (Fig. 2). Different effects of tribenuron-methyl on the content of particular metabolites were observed in the cotyledons of seedlings, depending on the type of resistant biotype (Fig. 3; Table 2). Tribenuron-methyl caused an increase in the amount of almost all identified metabolites, but the greatest increase was observed for sugars, lipids and carotenoids in the biotype with mutational resistance compared to the control (Fig. 3b). Tribenuron-methyl caused a decrease, relative to the control, in the content of mono-, di- and polysaccharides (1, 2, 4, 8), polyphenols (including flavonoids—10, 15) and chlorophyll (15a) in the biotype with metabolic resistance (Fig. 3a). The increase in the content of selected compounds in response to tribenuron-methyl was most evident in the mutational resistant biotype (Fig. 3b) for carotenoids (6, 9, 14) whereas in the susceptible biotype (Fig. 3c) it was observed for sugars (1, 2, 4) carotenoids (14) and chlorophyll (15a).
The cluster analysis of cotyledons of seedlings showed significant differences in chemical composition between resistant (mutational and metabolic) and susceptible biotypes, regardless of treatment ([C] or [H], Fig. 4). In addition, it was observed that each of the control objects belonging to the resistance biotypes was located on separate branches of the dendrogram. This indicates differences in chemical composition between the biotypes prior to the herbicide application. Furthermore, seedlings of the susceptible biotypes—both those treated with water [C] and with the herbicide [H]—were clustered in a separate group, so it can be concluded they are chemically similar. These results are consistent with the results obtained previously for rye-grass (Saja et al. 2014).
The structure of cornflowers’ leaves, mainly the minimal thickness of parenchyma, caused a significant increase of the noise to signal ratio, and thus difficulties in reliable statistical analysis of all FT Raman spectra. Therefore, the chemical composition of leaves of each biotype was compared only in bands originating from flavonoids and carotenoids, because these compounds may be particularly significant in response to abiotic stress.
Given that one of the plant responses to abiotic stresses is the induction of reactive oxygen species (Bray 2002), the content of flavonoids and carotenoids in leaves was analyzed. These compounds have a significant influence on the resistance of plants (Treutter 2005) constituting an essential element of the antioxidant system of cells (Pietta 2000). The analysis of the range of FT-Raman spectra comprising bands derived from flavonoids and carotenoids is shown in (Fig. 5a–c). The presence of tribenuron-methyl results in an increase in flavonoids (1608 cm−1) as well as carotenoids (carotenoid triplet—1005, 1161, 1525 cm−1) content in the leaves of the susceptible biotype relative to the control (Fig. 5c). Such an effect was not observed in biotypes of metabolic and mutational resistance types (Fig. 5a, b).
The cluster analysis of FT-Raman spectra obtained for leaves in the carotenoid-flavonoid range indicates that the control objects are clearly identified in the dendrogram based on the chemical composition resulting from their degree of resistance. Chemical stress caused by the herbicide differently alters the chemical composition of susceptible biotype as compared to resistant ones (metabolic and mutational) as shown by their clear separation in the dendrogram (Fig. 6). The described changes may indicate the launching of strong defensive responses in the leaves of more susceptible biotypes to counter the oxidative stress, which may occur as a side effect of the herbicide impact of the plant.
Efficiency of photosystem II of leaves treated with tribenuron methyl
This evaluation of the photosynthetic efficiency of PSII after herbicide application is of great importance in the diagnosis of resistance in weeds, where disruptions in photosynthesis occur as the second or an additional effect in the process of their destruction. An analysis of the kinetics of chlorophyll a fluorescence in response to the active substances of herbicides has been carried out in terms of some of the lipid biosynthesis inhibitors of the operation of acetyl-coenzyme A carboxylase (Barbagallo et al. 2003; Cowan et al. 1995), and certain inhibitors of amino acid biosynthesis–inhibitors of the operation of acetolactate synthase (Riethmuller-Haage et al. 2006). Among the analyzed parameters, the maximum photochemical efficiency of PSII—F
v/F
m was most frequently taken into consideration. In the case of stress factors, the application of the stressor, relative to the control, demonstrated the reduction of the efficiency of energy flow in PSII (Riethmuller-Haage et al. 2006; Skoczowski et al. 2011). In the presented results, however, no significant changes in the maximum of photochemical efficiency (F
v/F
m) were observed after the application of the herbicide in contrast to the plants sprayed with water.
The results of the analysis of energy flow through PSII and the chlorophyll a fluorescence kinetics in leaves sprayed with the herbicide are shown in Fig. 7 as a percentage of the control. The smallest changes in the photosynthetic efficiency of PSII after treatment with tribenuron-methyl occurred in the susceptible biotype (Fig. 7c). It may indicate that susceptible biotype is, at least partially, related to the inability to increase the efficiency of the photosynthetic apparatus under stress conditions. Significant differences in the analyzed parameters after the application of the herbicide relative to the control were observed in both resistant biotypes, with the metabolic and mutational resistance type (Fig. 7a, b). In both of these biotypes, the declines in the values of PI and Fv/F0 were accompanied by a corresponding increase in the value of the PSII antennae size indicator (ABS/RC) relative to the control. Such a chemical stress response was not observed in the susceptible biotype (Fig. 7c). However, in resistant biotypes, under conditions of impaired metabolism, the absorption of light by a single reaction center (ABS/RC) was significantly higher (by 10 % for metabolic, or even 25 % for mutational) compared to the optimal growth conditions (Fig. 7a, b). Moreover, in these biotopes a decrease in the values of the electron flow parameters (TR0/RC and ET0/RC) was accompanied by a decrease of the value of the DI0/RC parameter, which indicates that less energy is dissipated as heat and may be used for electron transport (compensation for the decline in the efficiency of electron transport, Fig. 7a, b).
Fluorescence of leaves treated with tribenuron methyl
One of the non-invasive fluorescent methods is the measurement of fluorescence of leaves, which allows the diagnosis of the physiological condition of the plant to be made based on the intensity of blue-green (F450 and F530) and red fluorescence (F690 and F735) (Lichtenthaler and Babani 2004). Previous studies (Schweiger et al. 1996, Buschmann and Lichtenthaler 1998; Hideg et al. 2002) showed that the fluorescent emission spectra of leaves may be successfully used for the detection of stress in plants. One of the possibilities presenting the differences in fluorescence intensity is using the ratios of fluorescence intensity at various wavelengths F450/F520, F450/F690, F450/F740 and F690/F740. Fluorescence ratios F450/F690 and F450/F740 are relatively easy to interpret and are particularly good indicators of stress (Schweiger et al. 1996). The ratio F450/F690 was recommended by Buschmann and Lichtenthaler (1998) as a good indicator of the level of stress already noticeable in the early stages of occurrence. The value of the F690/F740 ratio is inversely proportional to the amount of chlorophyll content (Lichtenthaler and Babani 2004).
The blue-green and red fluorescence intensity spectra are shown in Fig. 8. The shape of the blue-green fluorescence spectra in the control samples of all the biotypes was similar. At a wavelength of approximately 540 nm a peak was observed, the maximum of which, in the case of the metabolic and susceptible biotypes, was approximately 0.4 relative units (Fig. 8a, c, respectively). In the mutational resistant biotype (Fig. 8b), the intensity of green fluorescence was higher and amounted to approximately 0.5 r.u. The herbicide did not have a significant effect on the changes in blue-green fluorescence intensity, but it did in resistant biotypes with metabolic and mutational resistance types only (Fig. 8a, b, respectively).
The fluorescence emission spectra of leaves showed that the herbicide significantly increased the blue-green fluorescence intensity in the susceptible biotype (Fig. 8c). The herbicide, regardless of its mechanism of action, may lead to damage of the photosynthetic apparatus of plants by lowering their resistance to intense UV radiation. The defense mechanism may consist of the accumulation of plant phenols in the leaf tissues (Bilger et al. 2001; Schmitz-Hoerner and Weissenböck 2003). In such a case, the UV radiation is converted to blue-green fluorescence (F450 and F520) which is partially re-absorbed by the assimilation pigments. It can, therefore, be assumed that the increase in blue-green fluorescence intensity observed in the susceptible biotype under the influence of herbicide is an element of a defense strategy under which the plant protects the photosynthetic apparatus against damage by accumulating phenolic compounds in the cell walls of the epidermis.
Tribenuron-methyl decreased the intensity of red fluorescence, relative to the control, in all biotypes (Fig. 8a–c). However, while the decline was relatively small in the biotype with metabolic resistance (Fig. 8a), in the susceptible biotype the herbicide decreased the red fluorescence intensity nearly eightfold (Fig. 8c). The decrease in fluorescence intensity was accompanied by the appearance of the shoulder at 697 nm. This is particularly evident in biotypes with metabolic type of resistance (Fig. 8a). The fluorescence in the red and far red range is directly related to the content of chlorophyll in leaves; and it can be assumed that tribenuron-methyl causes disturbances in all the biotypes in the process of chlorophyll biosynthesis. This is also indicated by the emergence of the new band visible at 690 nm—F690. As the content of chlorophyll decreases, the relative intensity of red fluorescence F690 increases compared to the far red band F735. This is due to the fact that the re-absorption of red chlorophyll fluorescence decreases with its depletion. At the same time, the maximum of red fluorescence F690 shifts from longer to shorter wavelengths; the so called “blue shift” (Lichtenthaler and Babani 2004).
Tribenuron-methyl had no significant effect on the changes in fluorescence ratios relative to the control in any of the biotypes (data not shown). However, the fluorescence coefficients calculated for the control plants showed that the susceptible biotype is characterized, in comparison to other biotypes, by significantly lower values of F450/F530, F450/F690 and F450/F735 ratios (Table 3). The significance of this phenomenon, at the present stage of research, is difficult to be unequivocally interpreted. However, should further studies confirm the described dependence; a comparison of the discussed fluorescence relations may be a good indicator to assess the degree of cornflower biotypes resistance to tribenuron-methyl.
Table 3 The fluorescence ratio of leaves of cornflower biotypes with different types of resistance to tribenuron-methyl