高根沢 康一

Vacuolar sequestration via tonoplast-targeted MerC enhances Cd tolerance; expression in roots plays a primary role, while expression in mesophyll is also crucial for protecting the chloroplasts. Targeting metal transporters to the vacuole via genetic engineering offers a strategy to enhance plant tolerance to toxic metals like cadmium (Cd) by promoting vacuolar sequestration. Understanding the influence of different expression patterns of metal transporters is crucial for elucidating Cd-tolerance mechanisms. This study investigated how ubiquitous (p35S promoter) versus mesophyll-specific (pRBCS1A promoter) expression of a tonoplast-targeted bacterial metal transporter, MerC-AtVAM3 (CV), impacts Cd tolerance, nutrient homeostasis, and subcellular Cd distribution in Arabidopsis. While the short-term plate assays revealed only slight tolerance improvements, the long-term hydroponic Cd treatments (0.5 µM and 1 µM) resulted in significant enhancement in the CV-expressing lines. Notably, the p35S-CV line, which ubiquitously expresses the transgene, exhibited stronger tolerance (improved growth, mitigated chlorosis confirmed by higher SPAD values) compared to the mesophyll-specific pRBCS1A-TCV lines, particularly under 1 µM Cd. Nutritional profiling indicated that CV expression alleviated some Cd-induced nutrient imbalances. Although root Cd accumulation was similar across lines, p35S-CV shoots displayed approximately 30% lower Cd concentration compared to the wild-type (Col-0) and pRBCS1A lines under 1 µM Cd conditions. In Col-0 mesophyll cells, subcellular analysis using the Leadmium Green dye showed Cd was preferentially localized in the peripheral cytoplasm. The Leadmium Green signal also exhibited strong co-localization with chloroplasts. Conversely, CV expression effectively redirected Cd to the central vacuole, confirming efficient sequestration by the tonoplast-targeted MerC. The superior tolerance of the p35S-CV line strongly suggests that vacuolar Cd sequestration in roots plays a primary role in conferring robust Cd tolerance in Arabidopsis, while vacuolar sequestration in shoots provides supportive protection.

p62/SQSTM1 (p62) and neighbor of BRCA1 gene 1 (NBR1) are two important cargo receptors involved in selective autophagy. While p62 is known to safeguard cells against the toxic effects of the environmental toxicant methylmercury (MeHg), the specific functions of p62 and NBR1 in MeHg-exposed cells remain unclear. In this study, we aimed to elucidate the distinct roles of p62 and NBR1 in conferring protection against cytotoxicity induced by MeHg. We found that MeHg increased both the mRNA and protein levels of p62 while decreasing those of NBR1. Upon exposure to MeHg, p62-knockout (KO) cells exhibited an approximately 30 % reduction in cell viability compared to wild-type (WT) cells; however, no such reduction was observed in NBR1KO cells. Additionally, p62KO cells exhibited a 1.5-fold increase in intracellular mercury (Hg) concentration compared to the WT following MeHg exposure, whereas NBR1KO cells had Hg levels comparable to those of WT cells. Upon exposure to MeHg, Nrf2 signaling activation was significantly reduced in p62KO cells compared to that in WT cells, whereas NBR1KO cells displayed Nrf2 activation levels similar to those of WT cells. Overall, these results suggest that p62, rather than NBR1, plays a crucial role in mitigating MeHg-induced cytotoxicity by reducing intracellular Hg levels through the activation of the Nrf2 signaling pathway.

We previously reported that in Arabidopsis, the phytochelatin-mediated metal-detoxification machinery is also essential for organomercurial phenylmercury (PheHg) tolerance. PheHg treatment causes severe root growth inhibition in cad1-3, an Arabidopsis phytochelatin-deficient mutant, frequently accompanied by abnormal root tip swelling. Here, we examine morphological and physiological characteristics of PheHg-induced abnormal root tip swelling in comparison to Hg(II) stress and demonstrate that auxin homeostasis disorder in the root is associated with the PheHg-induced root tip swelling. Both Hg(II) and PheHg treatments severely inhibited root growth in cad1-3 and simultaneously induced the disappearance of starch-containing plastid amyloplasts in columella cells. However, further confocal imaging of the root tip revealed distinct effects of Hg(II) and PheHg toxicity on root cell morphology. PheHg treatment suppressed most major genes involved in auxin homeostasis, whereas these expression levels were up-regulated after 24 h of Hg(II) treatment. PheHg-triggered suppression of auxin transporters PIN1, PIN2, and PIN3 as GFP-fusion proteins was observed in the root tip, accompanied by an auxin reporter DR5rev::GFP signal reduction. Supplementation of indole-3-acetic acid (IAA) drastically canceled the PheHg-induced root swelling, however, Hg(II) toxicity was not mitigated by IAA. The presented results show that the collapse of auxin homeostasis especially in root tips is a cause for the abnormal root tip swelling under PheHg stress conditions.

Methylmercury (MeHg) is widely distributed in nature and is known to cause neurotoxic effects. This study aimed to examine the anti-MeHg activity of oleanolic acid-3-glucoside (OA3Glu), a synthetic oleanane-type saponin derivative, by evaluating its effects on motor function, pathology, and electrophysiological properties in a mouse model of MeHg poisoning. Mice were orally administered 2 or 4 mg·kg-1·d-1 MeHg with or without 100 µg·kg-1·d-1 OA3Glu 5x/week for four weeks. Motor function was evaluated using beam-walking and dynamic weight-bearing (DWB) tests. High-dose MeHg exposure significantly increased the frequency of stepping off the hind leg while crossing the beam in the beam-walking test, and increased weight on forelegs when moving freely in the DWB test. OA3Glu treatment alleviated motor abnormality caused by high-dose MeHg exposure in both motor function tests. Additionally, OA3Glu treatment reduced the number of contracted Purkinje cells frequently observed in the cerebellum of MeHg-treated groups, although cerebrum histology was similar in all experimental groups. The synaptic potential amplitude in the cerebellum decreased as MeHg exposure increased, which was restored by OA3Glu treatment. Even in the cerebrum, where the effects of MeHg were not observed, the amplitude of the field potential was suppressed with increasing MeHg exposure but was restored with OA3Glu treatment. Taken together, the study findings suggest that OA3Glu improves neurotransmission and movement disorders associated with MeHg exposure via protection of Purkinje cells in the cerebellum while ameliorating pre/post-synaptic deficits in the cerebral cortex in which no changes were observed at the tissue level, potentially providing a treatment to mitigate MeHg toxicity.