Glyphosate was suggested to cause inhibition of photosynthetic CO2 assimilation as well as a decrease in intermediates of the photosynthetic carbon reduction cycle. Shikimic acid, one of the main products in the EPSPS pathway, is a precursor of pigments, defense compounds, lignin and other important molecules in plants. Interestingly, glyphosate injury was also found to be correlated with chlorophyll content. This research was conducted to examine the joint effects of increased temperature and elevated CO2 level on the sensitivity of weeds to glyphosate. To accomplish this objective, we chose two weed species, C. canadensis and C. album, that differ in leaf surface characteristics, flowering phenology and plant architecture. The specific research objectives were to examine the influence of increased temperatures, elevated CO2 levels, and the combination of both factors on the sensitivity of C. canadensis and C. album to glyphosate and to investigate the mechanistic basis of plant response to glyphosate treatment under these environmental conditions. For both species and all populations, plant survival was highest under the combined high temperature and elevated CO2 treatment. Two out of four C. canadensis populations had a considerably higher percentage of plants surviving treatment with glyphosate under the combination of low temperature and ambient CO2 level than all others . Thus, differences in plant survival between the LT/ACO2 and the HT/ECO2 were not statistically significant for these two populations. However, vertical grow racks for the remaining six populations of both C. album and C. canadensis, the survival percentage differed significantly between the LT/ACO2 and HT/ECO2 treatments .
Large differences in plant survival between current and projected environmental conditions were recorded for populations CA1, CA3 and CCS in which no plants survived glyphosate treatment under LT/ACO2 but 61.1%, 69.0% and 64.0% of the plants tested, respectively, survived under HT/ECO2 conditions . In addition, a higher percentage of glyphosate-treated plants survived under high temperature than under elevated CO2 level . Loss of apical dominance and outgrowth of multiple lateral shoots were observed in glyphosate-treated plants grown under high temperature alone and the combination of both high temperature and elevated CO2 level . This phenotype was consistently observed for C. album , but only approximately 10% of the C. canadensis plants exhibited a loss of apical dominance under HT/ACO2 and HT/ECO2. Despite using the same photoperiod for all treatments, variation in flowering phenology among C. album plants under different temperatures was detected. At the end of the experiment, 21 days after glyphosate treatment, both treated and untreated CA1 plants grown under HT/ACO2 or HT/ECO2 conditions had flower buds or flowers while plants grown under LT/ACO2 and LT/ECO2 did not have visible reproductive structures .Over the four days following herbicide treatment, leaves of glyphosate-treated plants grown under HT/ECO2 exhibited more rapid reduction in chlorophyll content than leaves of plants grown under LT/ACO2 . Te differences in SPAD measurements between the two environmental treatments were statistically significant for both C. album and C. canadensis . Interestingly, differences between species were also observed as glyphosate-treated C. album plants grown under HT/ECO2 exhibited faster reduction in chlorophyll than C. canadensis plants treated and grown under the same conditions . Five days after glyphosate application, leaves of treated plants grown under HT/ECO2 exhibited severe chlorosis and turgor loss thus preventing further measurements. Phosphor images of 14C-glyphosate translocation from the treated leaf to the rest of the plant revealed differences in the distribution of glyphosate within plants grown under different environmental conditions . For both species, differences in glyphosate translocation were mainly observed at 12, 24 and 48 hours after treatment . Higher 14C-glyphosate signal intensity was detected in the shoot and roots of C. album plants grown under HT/ECO2 than plants grown under LT/ACO2 conditions at both 12 and 24 HAT .
A similar pattern of glyphosate distribution was observed in C. canadensis although the differences in glyphosate distribution among plants grown under the different environmental conditions were not as visually distinguishable as in C. album plants . For both species, apparent differences in glyphosate translocation were also observed at 48 HAT. Based on the phosphor imaging results described above, which indicate that the largest differences in 14C-glyphosate translocation between plants grown under different environmental conditions, occur at 12, 24 and 48 HAT, we investigated the absorption and quantified the distribution of 14C-glyphosate in different plant parts of C. album and C. canadensis under different environmental conditions at these time points. Glyphosate absorption differed markedly between the two species . C. album plants grown under HT/ECO2 conditions absorbed 14C-glyphosate in a significantly greater amount than plants grown under LT/ ACO2 within 12 and 24 HAT . However, at 48 HAT, no statistically significant difference in glyphosate absorption was observed between plants grown under the different environmental conditions. Although less 14C-glyphosate was absorbed by C. canadensis plants grown under HT/ECO2, differences in absorption between plants grown under different environmental conditions were not statistically significant . Overall, C. album absorbed substantially more 14C-glyphosate than C. canadensis. Quantification of 14C-glyphosate translocation into different plant parts revealed that significantly more glyphosate was retained in the treated leaf of C. album plants grown under LT/ACO2 than HT/ECO2 conditions at both 24 and 48 HAT . In foliage leaves , low amounts of 14C-glyphosate were found in plants grown under both LT/ACO2 and HT/ECO2 with no statistically significant differences between the environmental conditions . Higher amounts of 14C-glyphosate were found in plant stems under HT/ECO2 than LT/ACO2 although a statistically significant difference between treatments was only observed at 24 HAT . More glyphosate was found in both the shoot apical meristems and the roots of plants grown under HT/ECO2 compared with plants grown under LT/ACO2. For both shoot apical meristems and roots, differences between environmental conditions were statistically significant at 24 and 48 HAT . In C. canadensis, significantly more 14C-glyphosate was translocated out of the treated leaf of plants grown under HT/ECO2 at all harvest time points . No significant differences were observed in the amount of 14C-glyphosate found in the rosette leaves of plants grown under different environmental conditions . However, more 14C-glyphosate was observed in both shoot meristems and the roots of plants grown under HT/ECO2 compared with plants grown under LT/ACO2 at all harvest time points . Significant differences in the quantity of 14C-glyphosate between environmental conditions were observed at both 12 and 24 HAT for shoot meristems , whereas in the roots, significant differences were observed for all harvest time points .Taken together, the results of our study clearly indicate that the control of two major weeds in California agriculture by glyphosate could be reduced under the projected changes in climatic conditions. Compared to current conditions, both C. canadensis and C. album plants were less sensitive to glyphosate under the higher temperatures, elevated CO2 levels and the combination of both environmental conditions, which are predicted for the future. To the best of our knowledge, 4×4 plastic tray this research provides the first experimental evidence of the joint effects of both high temperatures and elevated CO2 levels on weed sensitivity to glyphosate. Reduced glyphosate sensitivity under high temperature and CO2 conditions was observed for all four populations of each species. Although the populations used for this study were primarily chosen from herbicide-free habitats, two C. canadensis populations exhibited a higher percentage of plants surviving glyphosate treatment at low temperature combined with ambient CO2 level than all others.
The wind-mediated seed dispersal, combined with the evolution and spread of glyphosate resistant C. canadensis populations across the Central valley of California, may account for the higher percentage of plants surviving glyphosate under current conditions. The rapid reduction in chlorophyll content , loss of apical dominance, and early initiation of reproductive structures observed in glyphosate-treated plants grown under high temperature combined with elevated CO2 level provide insights into the mechanistic basis of the reduced plant sensitivity to glyphosate under climate change scenarios. It is generally claimed that glyphosate controls weedy plants by binding to, and inhibiting the EPSPS enzyme, which is essential for the biosynthesis of branched-chain amino acids. Interestingly, several recent studies, in addition to this study, have revealed changes in phenological and physiological plant traits caused by glyphosate. Outgrowth of lateral shoots, delayed flower development and reduced stomatal conductance, have been observed in response to glyphosate treatment. Additionally, as a phloem-mobile herbicide, glyphosate exhibits a classic source-to-sink translocation pattern. The influence of glyphosate on photosynthesis-related processes, such as carbon fixation, starch accumulation and general carbohydrate formation, can eventually lead to self-induced limitation of glyphosate translocation. Our fndings suggest that most of the glyphosate that was not retained in the treated leaves was translocated into shoot apical meristems and young leaves , which caused rapid leaf decay and thus reduced glyphosate translocation to other plant organs. Glyphosate absorption differed between the two species. In C. album, significantly higher 14C-glyphosate absorption was observed in plants grown under HT/ECO2 compared to LT/ACO2 conditions. In C. canadensis, 14C-glyphosate absorption was marginally higher, but not significantly, under LT/ACO2 conditions. However, despite the differences in glyphosate absorption between species, the translocation and distribution pattern of 14C-glyphosate within plants, once absorbed, was similar. In glyphosate-treated plants grown under HT/ECO2, glyphosate was translocated more quickly out of the treated leaf to other plant tissues than in plants grown under LT/ACO2 conditions. Moreover, in plants grown under HT/ECO2, glyphosate translocation from the treated leaf into strong sinks was rapid for both C. album and C. canadensis . The rapid movement of glyphosate into shoot apical meristems and roots may reduce the mobility of the herbicide to other parts of the plant thereby reducing the overall sensitivity of plants to glyphosate under higher temperature and CO2 conditions. It has been hypothesized for many glyphosate-resistant weeds that less glyphosate is translocated from the treated leaf to other plant parts compared to glyphosate-sensitive plants. Interestingly, our results suggest a mechanistic basis for reduced plant sensitivity to glyphosate that differs from the altered glyphosate translocation mechanism hypothesized for many glyphosate-resistant weeds. For both C. album and C. canadensis, reduced translocation of glyphosate from the treated leaf was proposed as the mechanism for glyphosate tolerance. Our results suggest that the mechanism leading to reduced glyphosate sensitivity under high temperatures and elevated CO2 levels may differ from that conferring evolved glyphosate resistance in weeds. The pattern of glyphosate translocation observed in C. canadensis and C. album in this study can also explain the loss of apical dominance and the initiation of lateral shoots in glyphosate-treated plants grown under HT/ECO2. It is well-known that auxin moves basipetally from the apical shoot in order to suppress lateral bud growth. Glyphosate translocation into the shoot apical meristem may cause severe damage to this tissue and, as a result, constrain auxin production. Low quantities of auxin and glyphosate at the whole plant level may enable the outgrowth of lateral shoots which, in turn, could lead to increased plant survival after glyphosate treatment and the phenotype observed in this study. In conclusion, we have shown that glyphosate-treated plants grown under increased temperature and elevated CO2 level exhibit reduced glyphosate sensitivity. Thus, the continued over reliance on glyphosate for weed control under changing climatic conditions may result in more weed control failures. In addition, from a practical point of view, the loss of apical dominance and early initiation of reproductive structures, as observed in glyphosate-treated plants grown under high temperature in this study, could further exacerbate weed problems by resulting in an unexpected increase in seed production per plant and rapid replenishment of the soil seed bank. Our translocation studies have revealed variation in glyphosate distribution pattern between plants grown under different environmental treatments. Thissue-specific glyphosate sequestration may be the leading cause for sub-lethal glyphosate quantities at the whole plant level reducing the overall efficacy of the herbicide. Further research is required to determine the exact mechanism leading to the reduced plant sensitivity to glyphosate under altered environmental conditions.Farmers in the region of the central Valley of California usually treat C. album and C. canadensis with glyphosate after seeds germinate and seedlings emerge in February or March during which the daily current temperatures averaged 18°C and current maximum temperatures averaged 27°C.Based on Intergovernmental Panel on Climate Change predictions, future projected extreme temperatures are estimated to be 3–5 °C higher than current maximum temperatures. Thus, the two temperature treatments chosen for this study were 18/12 °C as the current average and 32/26 °C as the projected maximum.