Molecular Mechanism Of Root Development In Edt1 Mutant & Mechanism Of Paraquat Tolerance In Pqt24-1

We previously reported the edtl (enhanced drought tolerance 1) mutant showing improved drought tolerance and well-developed root system with a deeper primary root and more lateral roots. The mutant phenotypes were caused by an activated expression of HDG11, a HD-Zip family transcription factor. The edtl mutant apparently has its own root development pattern but its underlying mechanism is unknown. Through root transcriptome comparison between the mutant and the wild type (Col-0), we found that the genes coding for cell wall-associated proteins, oxidative stress response, oxygen and radical detoxification, osmosensing and response, and anion transport were significantly up-regulated and enriched. Those genes might work together to improve root development. Most of the cell wall-associated proteins are cell wall-loosening-related and belong to expansins, xyloglucan endotransglucosylase/hydrolases (XTHs), extensins, pectin lyases, pectin methylesterase inhibitors (PMEIs) and endo-(1,4)-/β-D-glucanase gene families. Nearly half of those genes contain HD cis elements in their promoters. The predominant cis element is AAATTAAA, which can be bound by HDG11. HDG11 bound to different HD cis elements with different affinities by yeast-one-hybrid assay, with the highest to AAATTAAA. ChIP assay showed that HDG11 bound in vivo to the segments containing HD-binding cis-element in EXPA5 and EXPB3 promoter. Over-expression of AtEXPA5, one of the most significantly up-regulated in edtl, produced longer primary roots resulted from more pronounced cell elongation. These results demonstrated that HDG11 transcriptionally regulated cell wall-associated protein genes. The cell wall-associated proteins are likely reguired to loosen cell wall and increase the elasticity of cell wall during root development. An enhanced such capacity in edt1 would help to improve root architecture that contribute to the drought-tolerance of the mutant.Paraquat is one of the most widely used herbicides in the world and is often used in experiments as an oxidant. However, its transporter has not been isolated in plants. Here we report a paraquat tolerant mutant pqt24-1 isolated from an activation tagging library, which contains 5,5000 lines, for its tolerance to 2 μM paraquat in the seedling stage. Molecular analysis revealed that the T-DNA was inserted in the thirteenth exon of AT1G66950 gene that encodes AtPDR11, a member of the ATP-binding cassette transporter superfamily. As a result, AtPDRll was knocked out in the mutant. Loss-of-function mutations of AtPDRll led to a reduced paraquat accumulation in plant cells. In addition, AtPDR11 protein was specifically localized in the plasmalemma, suggesting AtPDR11 as a potential transporter of paraquat. This was supported by kinetic analysis of paraquat import. Further studies showed that the transcript level of AtPDRll could be strongly induced by paraquat and other abiotic stresses including H2O2, indicating a possible up-regulation of AtPDR11 expression by oxidative stress signaling. Thus, our data suggests that paraquat is a substrate of AtPDR11 and the enhanced paraquat tolerance of pqt24-1 is due to the reduced uptake of paraquat into plant cells.