Summary: The genetic regulation underlying the effect of arsenic (As(III)) on the model symbiosis Medicago–Ensifer was investigated using a combination of physiological (split‐roots), microscopy and genetic (microarrays, qRT‐PCR and composite plants) tools. Nodulation was very sensitive to As(III) (median inhibitory dose (ID50) = 20 μM). The effect on root elongation and on nodulation was local (nonsystemic). A battery of stress (salt, drought, heat shock, metals, etc.)‐related genes were induced. Glutathione played a pivotal role in tolerance/detoxification, together with secondary metabolites ((iso)flavonoids and phenylpropanoids). However, antioxidant enzymes were not activated. Concerning the symbiotic interaction, molecular evidence suggesting that rhizobia alleviate As stress is for the first time provided. Chalcone synthase (which is involved in the first step of the legume–rhizobia cross‐talk) was strongly enhanced, suggesting that the plants are biased to establish symbiotic interactions under As(III) stress. In contrast, 13 subsequent nodulation genes (involved in nodulation factors (Nod factors) perception, infection, thread initiation and progression, and nodule morphogenesis) were repressed. Overexpression of the ethylene responsive factor ERN in composite plants reduced root stress and partially restored nodulation, whereas overexpression of the early nodulin ENOD12 enhanced nodulation both in the presence and, particularly, in the absence of As, without affecting root elongation. Several transcription factors were identified, which could be additional targets for genetic engineering aiming to improve nodulation and/or alleviate root stress induced by this toxic.

The aim of this work was to develop a biotechnological tool to hyperaccumulate high copper (Cu) concentrations from wastewaters. Transgenic tobacco hairy roots were obtained by expressing, either the wild-type version of the gene copC from Pseudomonas fluorescens in the cytoplasm of plant cells (CuHR), or a modified version targeted to the vacuole (CuHR-V). Control hairy roots transformed with the empty vector (HR) were also generated. The roots were incubated in the presence of solutions containing Cu (from 1 to 50 mM). At 5 mM external copper, transgenic hairy roots accumulated twice the amount of copper accumulated by control hairy roots. However, at 50 mM Cu, accumulation in both transgenic and control roots reached similar values. Maximum Cu accumulation achieved by transgenic hairy roots was 45,000 µg g⁻¹ at 50 mM external Cu. Despite the high Cu accumulation, transgenic hairy roots, particularly CuHR-V, showed less toxicity symptoms, in correlation with lower activity of several antioxidant enzymes and lower malondialdehyde (MDA) levels. Moreover, CuHR-V roots displayed low values of the oxidative stress index (OSI) – a global parameter proposed for oxidative stress – indicating that targeting CopC to the vacuole could alleviate the oxidative stress caused by Cu. Our results suggest that expressing copC in transgenic hairy roots is a suitable strategy to obtain Cu-hyperaccumulator hairy roots with less toxicity stress symptoms. Abbreviations: APX: ascorbate peroxidase; ATSDR: Agency for Toxic Substances and Disease Registry (U.S.); BCF: bioconcentration factor; CuHR: copper-hairy roots; EDTA: ethylenediamine tetracetic acid; EPA: Environmental Protection Agency (U.S.); GSH: glutathione; HM: heavy metals; HR: control hairy roots; ICP-OES: Inductively Coupled Plasma/Optical Emission Spectrometry; MDA: malondialdehyde; NBT: nitroblue tetrazolium; OD: optical density; OSI: oxidative stress index; PCR: polymerase chain reaction; PVP: polyvynilpirrolidone; PX: peroxidase; ROS: reactive oxygen species; SOD: superoxide dismutase GRAPHICAL ABSTRACT