Glyphosate (N-phosphonomethyl glycine) is the most widely used herbicide in the world due to its high efficiency, broad-spectrum capacity and systemic mode of action. It binds to the active site of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), a key enzyme of the shikimate pathway, antagonizing its natural substrate phosphoenol pyruvate. Thus, it inhibits the synthesis of crucial amino acids and other compounds causing subsequent plant death [1,2,3].
The wide use of glyphosate-resistant (GR) crops has led to an enormous increase in the application of glyphosate, as it constitutes a low-cost and highly effective weed control technology. However, its repeated and intensive use has exerted a high selection pressure on weed populations and has led to the evolution of glyphosate resistance weeds. To date, 25 weed species have evolved resistance to glyphosate worldwide , jeopardizing the efficiency of weed management programs in modern agriculture . Thus, understanding the mechanism of glyphosate resistance in weeds is a prerequisite to guarantee its continued use .
Horseweed (Conyza canadensis L.), which belongs to the Asteraceae family, was the first broadleaf weed to evolve glyphosate resistance . Especially in Mediterranean regions including Greece, Conyza spp. is the most difficult to tackle weed in perennial crops. Prolonged and exclusive use of glyphosate, combined with the lack of integrated weed management approaches, have mainly contributed to the evolution of tolerant and/or resistant biotypes in many orchard regions containing among others olives, grapes and citrus [8,9,10].
According to Sammons and Gaines  the glyphosate resistance mechanisms reported so far include: (1) mutations at the target site of the key enzyme EPSPS [12,13]; (2) gene amplification of EPSPS ; (3) limited absorption and/or translocation of the herbicide [15,16]; (4) changes in the sequestration of glyphosate to vacuoles ; (5) faster metabolism of the herbicide in resistant plants ; (6) rapid mature leaf necrosis resulting in reduced translocation in ragweed (Ambrosia trifida) ; and (7) the recently proposed synchronization of the overexpression of EPSPS and ABC-transporter genes . As indicated in several reports, resistant weeds could combine several glyphosate resistance mechanisms within populations and within individuals .
High light intensity and elevated temperatures ameliorate glyphosate performance by enhancing the rapid absorption by the plant, as well as its accumulation and translocation . Regarding the influence of environmental conditions to glyphosate efficacy, as a general rule it can be pointed out that glyphosate is more effective under higher temperatures and ambient light conditions due to elevated levels of plant metabolism as a result of an increased vegetative growth [21,22,23]. On the contrary, decreased glyphosate absorption and translocation is manifested in sub-optimal environmental conditions, resulting in a lower glyphosate efficacy on treated plants. Earlier studies have addressed how environmental conditions might affect levels of glyphosate resistance in various other weeds. Researchers have shown that resistance to both glyphosate and paraquat (thought to be dependent on vacuolar sequestration) is diminished at low temperatures [24,25]. Moreover, the analysis of an Arabidopsis GR mutant that was dysfunctional in perceiving light, further supported previous observations that light quality and intensity differentiates herbicide efficacy .
Results from our previous study in C. canadensis, clearly revealed that the glyphosate resistance mechanism involves a synchronized induction of EPSPS and ABC-transporter genes , supporting the concept that glyphosate resistance mechanisms can be quite complex . Former studies showed that subjecting GR-plants to low temperatures could make those plants sensitive to glyphosate due to its higher vacuolar sequestration . However, there has been no report so far regarding the effects of environmental conditions on the expression levels of the aforementioned key genes. Therefore, our main objective was to elucidate how two environmental conditions (temperature, light) affect the induction of key genes such as EPSPS and ABC-transporters in glyphosate susceptible (GS) and GR plants aiming to further understand the mechanism of glyphosate resistance in weed species such as C. canadensis.
Glyphosate, being a non-selective systemic herbicide, requires full and active growth of treated plants in order to show its highest efficacy. The effects of environmental factors on glyphosate performance on targeted plants have been documented in earlier studies mainly regarding changes in its uptake and translocation [22,23,27]. The inhibition of the shikimate pathway, located in chloroplasts, has long been validated as glyphosate’s mode of action .
Measuring shikimate levels has long been proposed as a discrimination test between the GR and GS plants; either as an in vivo test [8,29] or as a leaf disk test . However, clear cut differences in shikimate accumulation between GR and GS plants were not always identified, possibly due to the growing conditions, the amount of glyphosate applied and the plant growth rates [20,30,31,32]. Optimum temperature and light is considered to have a positive effect on the shikimate pathway, thus shikimate accumulation was more evident in plants maintained under light conditions after glyphosate treatment [28,33]. In our study, under dark conditions, shikimate accumulation was lower than in light conditions, presumably due to less flux in the shikimate pathway either directly and/or less efficient photosynthesis (Figure 1). Increased temperature (35 °C) and dark conditions resulted in significant shikimate accumulation on either GS- and GR-plants (Figure 1b), emphasizing the predominant role of temperature compared to light; this result is in agreement with previous reports [24,33].
As previously mentioned, the usefulness of the shikimate test has been frequently undermined by false-positive and false-negative results  stressing the need for standardization. In our study, it was shown that higher glyphosate load (4×, 8×) could minimize differences in shikimate accumulation between GR and GS population at either low (8 °C) and normal (24 °C) temperature conditions (Figure 1a,c). On the contrary, it was clearly shown that the most discrete differences (between GR and GS population) in shikimate accumulation were measured at 35 °C and light conditions regardless of the glyphosate dose applied (Figure 1b). At those conditions, it is expected to have the maximum flux in the shikimate pathway and, therefore, the confounding factor of glyphosate load is eliminated and the maximum differences were recorded between GR and GS population. For this reason, the above conditions are proposed as the standard ones for conducting shikimate analysis as a discriminating biochemical test to detect glyphosate resistant in C. canadensis plants.
Gene expression analysis was performed on GR and GS biotypes for EPSPS, M10 and M11 genes. Under light conditions at normal (24 °C) and high (35 °C) temperatures, the following points could be made:
(a) The EPSPS gene was significantly induced at normal glyphosate doses in the GR biotype, whereas at 35 °C no significant differences was observed between the two biotypes, suggesting that the synchronization theory of EPSPS and ABC-transporter gene expression as a glyphosate resistance mechanism is applicable only at normal temperatures;
(b) At an early stage (one DAT), across most glyphosate rates, the GR plants had a higher expression rate for both M10 and M11 genes compared to the GS plants (Figure 2e,f and Figure 3b,c). This result further supports previous reports about the possible role of the ABC-transporter genes in glyphosate resistance [30,34];
(c) GR plants had constantly higher overexpression of the above key genes only at an early stage (one DAT), regardless of the temperature. Therefore, the early time of initiation of overexpression is critical for the resistant mechanism itself, since this early overexpression (immediately after glyphosate application) of the genes secures glyphosate inactivation due to vacuolar sequestration. This result is in agreement with our previous findings . Moreover, the highest M10 and M11 gene expression of the (1×) GS plants at four DAT (Figure 2d,f) indicates that it might be too late (at such a late stage) for the ABC-transporters overexpression to offer resistance protection. Also, this finding is in agreement with our previous findings .
Regarding gene expression at low temperature (8 °C), if GR horseweed plants are made sensitive then they should behave (biochemically and molecularly) like GS plants. In accordance to this hypothesis, two important facts were pointed out:
(a) Documented GR plants could be indeed “sensitized” (become S-GR plants) when subjected to cold and light conditions;
(b) The process of such “sensitization” is clearly correlated to the mechanism of glyphosate resistance in C. canadensis.
In our study, the sensitized S-GR plants were correlated with the inversion of M10 and M11 gene expression, but not that of EPSPS (Figure 4a,c,e). This finding is in agreement with previous reports about the fate of glyphosate in such plants: when horseweed GR biotypes were in cold conditions (similar to our own ones), less glyphosate was sequestered to vacuoles suggesting a role of putative
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