Transcription of putative tonoplast transporters in response to glyphosate and paraquat stress in Conyza bonariensis and Conyza canadensis and selection of reference genes for qRT-PCR

Abstract

Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines. Transcription of ABC transporters also increased after paraquat treatment in all three lines of C. bonariensis. However, in C. canadensis, paraquat treatment increased transcription of only one ABC transporter gene in the susceptible line. The increase in transcription of ABC transporters after herbicide treatment is likely a stress response based on similar response observed across all Conyza lines regardless of resistance or sensitivity to glyphosate or paraquat, thus these genes do not appear to be directly involved in the mechanism of resistance in Conyza spp.

Gene expression in response to glyphosate treatment in fleabane (Conyza bonariensis) – glyphosate death response and candidate resistance genes

“…In our C. bonariensis lines, however, expression was slightly higher in the susceptible lines than in the resistant lines, and we categorise these genes as part of the glyphosate death response. Our interpretation is in line with that of the authors of a recent study who observed a similar pattern of expression in both C. canadensis and C. bonariensis in susceptible and resistant lines, and in response to both glyphosate and paraquat stress . Even when working from mechanistic principles, the identification of NTSR genes can be hampered by stress‐related genes that are activated by the herbicide.…”

“…This revealed that EPSPS expression (of all three genes) was reduced following glyphosate treatment, but not as dramatically as the effect on EPSPS1 alone. A slight (often not significant) decrease in EPSPS expression following glyphosate treatment has been detected in other studies . The reduced expression of EPSPS1 , but not EPSPS2 and EPSPS3 indicates that transcription of the different genomic copies is controlled differently, transcription of EPSPS1 may be affected by some aspect of a stress‐response pathway, for example.…”

“…However, the expression data for these two copies are susceptible to read mismapping across sections of them. Previous qPCR expression studies on C. bonariensis and C. canadensis have tended to deploy primers that amplify all copies of EPSPS . We therefore calculated the sum of RPKM values for all EPSPS copies (Fig.…”

“…Resistant populations of C. canadensis from Crete showed no expression differences for the EPSPS gene, whereas populations from mainland Greece showed 2–4‐fold overexpression of the EPSPS gene following glyphosate treatment. Conversely, EPSPS expression was found to be stable in resistant and susceptible American populations of both C. canadensis and C. bonariensis . Basal EPSPS expression was, however, found to be higher in some resistant populations from Spain .…”

“…vacuolar or apoplast sequestration). Resistant lines of C. bonariensis from California show reduced translocation of the herbicide out of the leaf . There are also reports that Spanish populations of C. canadensis have reduced glyphosate mobility within the plant and can also metabolise glyphosate …”