Calcium-Dependent Hydrogen Peroxide Mediates Hydrogen-Rich Water-Reduced Cadmium Uptake in Plant Roots1[OPEN]

Qi Wu,a,b,c,2 Liping Huang,a,2 Nana Su,b,2 Lana Shabala,c Haiyang Wang,c Xin Huang,a Ruiyu Wen,a Min Yu,a Jin Cui,b,3 and Sergey Shabalaa,c,3,4 Department of Horticulture and International Research Centre for Environmental Membrane Biology, Foshan University, Foshan 528000, China College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Tasmanian Institute of Agriculture, College of Science and Engineering, University of Tasmania, Hobart, Tasmania 7001, Australia

Cadmium (Cd) is a toxic heavy metal that is relatively mobile in the soil and has become a serious worldwide environment problem (Chaney, 2015). Cd is easily taken up by plant roots and can be loaded into the xylem for transport to above-ground tissues (Mendoza-Cózatl et al., 2011). Cd accumulation in the shoot inhibits plant growth by causing an array of morphological, physiological, biochemical, and ultrastructural changes (Romero-Puertas et al., 2004;Ali et al., 2015;Gill et al., 2015). Most plants are sensitive to even low (micromolar range) Cd concentrations. Also, even if plants do not show symptoms of toxicity and their growth is not affected, Cd accumulation in the shoot can potentially cause harm to humans through the food chain (Beccaloni et al., 2013). Thus, a better understanding of the mechanisms controlling Cd uptake and transport in plants (especially in edible leafy vegetables), and some practical solutions for minimizing Cd accumulation in above-ground plant parts, is critical to food safety.
Cytosolic free Ca 21 is a ubiquitous second messenger mediating a broad array of adaptive responses in plants (Gilroy et al., 2016). Ca 21 transport across the plasma membrane (PM) of root cells could be mediated by many ion channels with different gating properties . Cd exposure results in a decrease in Ca content in different plant species due to the competition of binding sites for proteins or transporters (Sandalio et al., 2001). Also, Cd 21 influx in plant roots could be inhibited by Ca 21 channel blockers Sun et al.,2013;He et al., 2015), suggesting a possible involvement of Ca 21 -permeable channels in Cd 21 uptake. In rice (Oryza sativa), annexin 4 (OsAAN4) and Glu receptor-like 3.4 (OsGLR3.4) channels are the most likely candidates for this role . Also, elevated Ca levels suppress Cd uptake (Lu et al., 2010) as a result of competition between Ca 21 and Cd 21 uptake for the same transporter. However, to the best of our knowledge, the role of Ca as a signaling agent in regulating Cd entry into root cells has not been explored.
Other second messengers mediating rapid systemic signaling in response to stress are reactive oxygen species (ROS; Gilroy et al., 2016;Dell'Aglio et al., 2019). The most studied ROS-producing enzymes in plants are respiratory burst oxidase homologs (RBOHs) located at the PM (Torres and Dangl, 2005;Suzuki et al., 2011). Two RBOH isoforms, RBOHD and RBOHF, play major roles in plant responses to abiotic stimuli (Kärkönen and Kuchitsu, 2015). The increase in cytosolic Ca 21 concentration activates a plethora of Ca-dependent protein kinases that phosphorylate RBOHD and RBOHF (among other substrates) to amplify ROS production (Sierla et al., 2016) via the socalled "ROS-Ca hub" . In its turn, apoplastic ROS production leads to Ca 21 influx by activating ROS-sensitive Ca 21 -influx cation channels. This self-amplification mechanism can increase the duration and amplitude of weak signals (Richards et al., 2014;. However, with excessive Ca 21 , the phosphorylation ability of calcium-dependent protein kinases decreases the production of ROS by RBOH. Among all ROSs, hydrogen dioxide (H 2 O 2 ) is often put forward as the most attractive signaling molecule because of its relatively low toxicity, long lifespan, and diffusibility (Cuypers et al., 2016). It has been reported that Cd-induced damage to rice seedlings could be reduced by pretreatment with low concentrations of H 2 O 2 or with heat shock, which is known to increase H 2 O 2 levels (Chao et al., 2009;Bai et al., 2011;Wu et al., 2015b). In the Arabidopsis (Arabidopsis thaliana) Atrboh mutant, Cd accumulation in roots was increased by 2fold (Gupta et al., 2017), suggesting a possible causal link between NADPH oxidase operation and Cd transport. The important role of NADPH oxidase in regulating ion transport activity is well accepted. For instance, RBOHC could regulate arsenic uptake in Arabidopsis plants (Gupta et al., 2013). By interacting with transition metals, RBOH-generated H 2 O 2 can form hydroxyl radicals that in turn directly activate depolarization-activated outward-rectifying K 1 (GORK) channels (Demidchik et al., 2014;Wang et al., 2017). H 2 O 2 also activates a range of cation-permeable nonselective cation channels, thus affecting intracellular K 1 and Ca 21 homeostasis and signaling (Ordoñez et al., 2014;Shabala and Pottosin, 2014;Wang et al., 2018). Taken together, it is reasonable to speculate that H 2 O 2 may play an important role in regulating Cd influx. However, the mechanistic basis of this regulation remains to be elucidated.
Hydrogen gas (H 2 ) has recently emerged as a beneficial molecule with multiple bioactive functions . It is believed that the main mechanism of H 2 action and its modulation of stress tolerance in plants might be related to the preferential scavenging of ROS, thereby reducing the oxidative damage, as reported in alfalfa (Medicago sativa), Arabidopsis, and rice (Jin et al., 2013;Xie et al., 2012Xie et al., , 2015. It is likely that H 2 does not act alone but rather interacts with other signaling molecules, such as abscisic acid, H 2 O 2 , nitrous oxide, and Ca 21 , to affect plant physiological activities. It was suggested that exogenous nitric oxide generated by nitric oxide synthase and nitrate reductase might be required for H 2 -induced adventitious root formation . Jin et al. (2016) found that under drought stress, H 2 rapidly increased H 2 O 2 and modified the apoplastic pH of leaves in alfalfa via an abscisic acid-based mechanism. Furthermore, incremental IP3dependent cytosolic Ca 21 contributes to H 2 -promoted anthocyanin biosynthesis under UV-A irradiation in radish sprouts .
Until now, alleviation of detrimental effects of Cd by H 2 was attributed to enhanced antioxidant defense mechanisms (Wu et al., 2015a;Su et al., 2019). It is also known that H 2 decreased Cd accumulation in plants, although specific mechanisms behind this phenomenon were not revealed. Recent evidence indicates that H 2 might function as an essential signaling modulator involved in regulation of cation channels or transporter operation. For example, the transcripts of two Na 1 exclusion transporters, salt overly sensitive1 (SOS1) protein and Arabidopsis H 1 -ATPase3 (AHA3), were significantly upregulated by H 2 pretreatment under salt stress (Xie et al., 2012). Similarly, H 2 treatment upregulated the transcript levels of GORK, an outwardrectifying K 1 channel in control of stomatal movements (Xie et al., 2014). For Cd 21 transport, the expression of BcIRT1 and BcZIP2 (two main transporters in Cd uptake) was found to be significantly repressed by H 2 , suggesting that H 2 may reduce Cd accumulation in plants by transcriptional regulation of these transporters. However, the question of how H 2 controls the functional activity/operation of ironregulated transporters (IRTs) remains to be answered.
Leafy vegetables accumulate more Cd than tubers and root vegetables Rizwan et al., 2017). One of the most consumed leafy vegetables in China and East Asia, pak choi (Brassica campestris ssp. chinensis), grows rapidly and can readily accumulate Cd Yu et al., 2019). Better understanding the mechanism of Cd uptake and developing methods to reduce Cd accumulation in pak choi seedlings are of great significance. In this study, we combined a range of advanced electrophysiological, biochemical, and genetic approaches to elucidate the mechanistic basis of regulation of Cd transport by H 2 gas. We show that Cd-triggered rapid H 2 production plays an essential role in stress signaling, modulating Ca 21 -dependent H 2 O 2 generation by NADPH oxidase encoded by RbohD. The latter operates upstream of IRT1 and regulates root Cd uptake at both transcriptional and functional levels.

Cd Stimulates H 2 Release and Production
Effects of Cd on endogenous H 2 production in pak choi seedlings were investigated using a hydrogen needle sensor. These measurements showed a rapid and progressive increase in endogenous H 2 concentration in leaves when roots were treated with 100 mM Cd (Fig. 1A). In comparison with the basal levels of H 2 in control samples, a rapid, significant, and sustained increase of H 2 release was detected that reached a peak value at ;300 s. Cd-triggered H 2 production was further verified by gas chromatography (GC) analysis. As shown in Figure 1B, pak choi seedlings treated with Cd had 2-fold higher H 2 concentration in their shoots following 24 h of Cd exposure, compared with controls.

HRW Alleviates Cd Stress-Induced Pak Choi Seedlings Growth Inhibition
To investigate whether H 2 had any effect on plant growth under Cd stress, phenotyping and viability staining assays were performed. Under normal growth conditions, hydrogen-rich water (HRW) treatment had no effect on the growth of pak choi seedlings ( Fig. 2A), whereas 50 mM Cd treatment for 2 d significantly inhibited the root length and fresh weight (by ;33% and 30%, respectively). In comparison with the plants challenged with Cd alone, pretreatment of Cd-stressed seedlings with 50% HRW (H 2 concentration 381 6 16 mM; Supplemental Fig. S1C) rescued Cd inhibition and increased root elongation and fresh weight by ;27% and ;19%, respectively (Fig. 2, C and D). As the O 2 concentration in the 50% HRW treatment was lower than in the control treatment, additional (hypoxic) controls were added to eliminate a possible confounding effect of hypoxia. As shown in Supplemental Figure   S2, removing oxygen from the solution by flushing it with N 2 did not alleviate the Cd-induced growth inhibition of pak choi seedlings observed in the HRW treatment, ruling out the above possibility.
At the next step, 2-d-old pak choi roots were exposed to various treatments (HRW, Cd, and diphenyleneiodonium [DPI], alone or in combination) and then double-stained with fluorescein diacetate-propidium iodide (FDA-PI; Fig. 2, B and E). Under the fluorescence microscope, viable cells fluoresced bright green, whereas dead/damaged cells fluoresced bright red. Very few dead/damaged cells were found in the root tips in treatments without Cd stress. Cd exposure for 24 h resulted in a substantial loss of cell viability in the root apex (less green signal and brighter red signal), with .37% of the root tip cells damaged. In cells pretreated with DPI (a known NADPH oxidase inhibitor) and exposed to Cd, the percentage of dead cells increased to 48%. Pretreatment with HRW alleviated Cdinduced cell damage, with only 20% of cells damaged. However, this positive effect of HRW was offset by DPI cotreatment (34% of cells damaged).

HRW Reduces Cd Influx and Accumulation in Pak Choi Roots
The microelectrode ion flux estimation (MIFE) technique was used to measure net Cd 21 flux from the pak choi roots (Fig. 3). Addition of Cd to the bath solution resulted in an instantaneous Cd 21 influx, with a peak value between 70 and 120 nmol m 22 s 21 in the elongation zone and 50 to 80 nmol m 22 s 21 in the mature zone. The influx was then gradually reduced. In both zones, net Cd 21 influx in HRW-pretreated roots was significantly (2-fold; P , 0.05) lower compared with non-pretreated controls (Fig. 3, A and B). Beneficial effects of HRW were not as strong when it was administered together with Cd, making the possibility of direct effects unlikely.
We then studied the effects of HRW incubation time on root Cd 21 uptake (1-to 48-h interval). When roots were soaked in HRW for 1 or 6 h, net Cd 21 influx fluxes were much lower (2-fold) compared with untreated roots. Longer exposures resulted in a gradual reduction in root Figure 1. Cd-stimulated H 2 release and production in pak choi seedlings. A, Real-time dynamics of H 2 release from leaves of pak choi seedlings with roots treated with 100 mM CdCl 2 . B, Endogenous H 2 production from pak choi seedlings after 24 h CdCl 2 (100 mM) treatment. Data are means 6 SE from three independent experiments. The asterisk indicates a significant difference at P , 0.05 according to Duncan's new multiple-range test. FW, Fresh weight.
Cd uptake, with small but significant net Cd 21 efflux detected for 24-and 48-h treatments (Fig. 3, C and D).
We next looked at the effect of HRW on Cd accumulation in pak choi tissues. As shown in Figure 4, A and B, Cd content increased in nontreated control roots with increasing duration of exposure to Cd; however, no significant (at P , 0.05) difference in tissue Cd content was found between 12-and 24-h Cd exposure  Cd 21 fluxes measured from the elongation and mature zones of pak choi roots . A and B, Net Cd 21 flux in 3-d-old pak choi seedlings. Seedlings were transferred into control (Con) or HRW (50% saturation) solution for 60 min, then taken out and immobilized on a slide for measurement. After 5 min of flux recordings, 50 mM CdCl 2 was added into the basic salt medium solution. 1HRW denotes the treatment when HRW was added to the plant together with Cd. C and D, For the steady Cd 21 flux, seedlings were either pretreated with HRW for 30 min or not treated, then incubated in 50 mM CdCl 2 solution for different times. Each bar represents the mean 6 SE of 8 to 10 seedlings. Asterisks indicate significant difference at P , 0.05 according to Duncan's new multiple-range test.
in roots pretreated with HRW. Consistent with the MIFE data, HRW plants showed decreased Cd accumulation in both root and shoot tissues (Fig. 4, A and B) compared with nontreated controls. Recently, yeast assays showed that the role of BcIRT1 and BcZIP2 genes was to confer Cd 21 transport activity . Here we show that under nonstress conditions, HRW pretreatment inhibited BcIRT1 expression and promoted BcZIP2 expression. After 12 h Cd exposure, the expression of BcIRT1 had decreased significantly, but BcZIP2 expression did not change dramatically. Also, Cd-induced BcIRT1 inhibition was further strengthened by the HRW pretreatment. However, the HRWinducible response was not observed in the BcZIP2 gene, which had a transcript level close to that for the ConCd treatment (Fig. 4, C and D).
NADPH Oxidase-Generated H 2 O 2 Mediates HRW-Induced Decrease in Cd 21 Uptake by Roots As Rboh-dependent H 2 O 2 generation was suggested to participate in the plant response to Cd stress (Rodríguez-Serrano et al., 2009;Cuypers et al., 2016), we used MIFE technology to examine the effect of HRW on Cd-induced H 2 O 2 efflux (Fig. 5,A and B). No H 2 O 2 flux from the elongation zone of roots was measured under Cd treatment for 30 min (Fig. 5A, white circles), while 15 min of HRW treatment resulted in a substantial increase in H 2 O 2 efflux. This increase reached its maximum value at 20 min (about 2 pmol m 22 s 22 ) and then began to decline (Fig. 5A, blue circles). About 15 min after Cd addition, H 2 O 2 efflux had considerably increased in roots pretreated with HRW compared with control roots (Fig. 5B).
As a next step, H 2 O 2 production under prolonged Cd exposure was quantified (Fig. 5C). Root incubation in HRW resulted in a significantly higher (1.7-fold; P , 0.05) H 2 O 2 fluorescent signal compared with control plants (Fig. 5D, time point 0). Exposure to Cd led to a further increase in H 2 O 2 accumulation that reached a peak value at 1 h and then gradually declined. The kinetics of Cd-induced changes in H 2 O 2 accumulation was more drastic in HRW-pretreated plants. The increase in H 2 O 2 induced by HRW treatment was completely offset by DPI cotreatment (Fig. 5D).
We then looked at the effect of Cd on the transcript levels of NADPH oxidase genes. In the family of Brassicaceae, 10 Rboh genes, RbohA to RbohJ, are known to encode NADPH oxidase Liu et al., 2019). Two prominent members, RbohD and RbohF, have been shown to play an important role in stress-induced H 2 O 2 production (Yang et al., 2018;Jakubowicz et al., 2010). Exposure to Cd resulted in a significant upregulation of BcRbohD transcripts, and this upregulation was much faster in HRW-pretreated roots than in nontreated control roots (peak accumulation after 1 and 3 h, respectively). By contrast, BcRbohD transcription was severely downregulated by HRW and DPI cotreatment (Fig. 5E). Changes in BcRbohF transcripts were not different between HRW-treated and control plants, with both peaking at ;3 h after Cd exposure; however, the expression level of BcRbohF under cotreatment of HRW and DPI was much lower at 3 h compared to the other two treatments (Fig. 5F).
Our next aim was to establish a causal link between HRW-stimulated H 2 O 2 generation and HRW-inhibited Cd uptake; this was achieved in a series of pharmacological experiments (Fig. 6). An NADPH oxidase inhibitor, DPI, was used to modulate H 2 O 2 production and reveal its role in HRW-inhibited Cd uptake. Under the control condition, Cd addition induced a fast Cd 21 influx from roots, with peak values of ;110 and 80 nmol m 22 s 21 in the elongation and mature zones, respectively. When pretreated with HRW, these peak values were reduced to ;80 and 50 nmol m 22 s 21 (Fig. 6, blue symbols). Addition of DPI eliminated the beneficial effects of HRW (Fig. 6, gray symbols). DPI pretreatment also increased peak Cd 21 uptake by non-HRW treated roots in both zones (Supplemental Fig. S3), and exogenous H 2 O 2 application reversed this process (Supplemental Fig. S3). Taken together, these results suggest that apoplastic H 2 O 2 production by NADPH oxidase mediates HRW-induced decrease of Cd 21 uptake by roots.
To confirm the involvement of NADPH oxidase as a component of the mechanism for HRW-induced decrease in Cd 21 uptake by roots, the above experiments were conducted on Arabidopsis AtrbohD and AtrbohF mutants lacking appropriate functional RBOH isoforms. No phenotypic difference was found between wild-type Col, AtrbohD, and AtrbohF plants grown under normal conditions (Supplemental Fig. S5A). The presence of Cd in the agar media caused inhibition of root elongation in the Rboh mutants, especially AtrbohD, compared to Col (Supplemental Fig.  S5, B and C). Arabidopsis plants lacking functional RbohD also showed the highest Cd 21 uptake by roots (Supplemental Fig. S5, D and E). Similar to the results observed for pak choi seedlings, HRW pretreatment reduced Cd uptake by Arabidopsis wild-type (Col-0) roots; this ameliorative effect was absent in AtrbohD and AtrbohF mutants, regardless of the root zone (Fig. 7).
As shown in Figure 4, B and C, HRW treatment downregulated BcIRT1 expression and reduced Cd accumulation in pak choi roots. To reveal a functional role of IRT1 as the downstream target of HRW, the Arabidopsis Atirt1 mutant was used in electrophysiological experiments. Consistent with previous findings, both HRW and H 2 O 2 pretreatment lowered net Cd 21 uptake by wild-type roots (in both root zones). Net Cd 21 influx was significantly lower in Atirt1 than in wild-type Col-0, peaking at ;20 and 10 nmol m 22 s 21 in the elongation and mature zones, respectively. Neither HRW nor H 2 O 2 pretreatment was able to further reduce Cd 21 influx in the Atirt1 mutant (Fig. 8). Taken together, our data are consistent with the model that HRW-induced RBOH-dependent apoplastic H 2 O 2 production operates upstream of IRT1, thus affecting Cd transport. Cytosolic Ca 21 is a ubiquitous second messenger, and changes in the cytosolic free Ca 21 concentration are reported in response to virtually every known environmental stimulus (McAinsh and Pittman, 2009;Dodd et al., 2010). In this study, HRW addition resulted in a rapid Ca 21 influx in both elongation and mature root zones, with peak values of ;50 nmol m 22 s 21 and  ;30 nmol m 22 s 21 , respectively (Fig. 9, A and B). This occurred in parallel with Ca 21 accumulation measured with a fluorescent dye, where HRW treatment for 30 min induced a stronger florescence signal in both zones (Fig. 9, A and B, insets). Application of Cd 21 , however, induced a massive leakage of Ca 21 from the root, followed by a gradual recovery; this Cd-induced Ca 21 efflux was significantly suppressed by HRW pretreatment. The changes in Ca 21 flux were consistent with the fluorescent Ca 21 signal data (Fig. 9, C and D). These results imply an involvement of Ca 21 in HRWreduced Cd uptake. H 2 O 2 addition resulted in rapid Ca 21 efflux from root elongation zones. This efflux was rather small and recovered after 10 min, with a peak of only 210 nmol m 22 s 21 (Fig. 9E). By contrast, in the mature zone, H 2 O 2 addition induced Ca 21 influx, from 8 to 25 nmol m 22 s 21 (Fig. 9F). Consistent with this, H 2 O 2 treatment for 30 min resulted in a slight decrease and increase in fluorescent Ca 21 signal in the elongation zone and mature zone, respectively (Fig. 9, E and F, insets).
Two Ca 21 channel inhibitors (Gd 31 and La 31 ) were used here to further verify the role of Ca 21 as a component of HRW signaling to Cd 21 transporters in plant roots. Compared with the control, Cd treatment conferred a 30% reduction in Ca concentration in roots, which was significantly reversed by HRW (Supplemental Fig. S6). However, the HRW-induced increase in Ca concentration was totally inhibited by Gd 31 addition (Supplemental Fig. S6), which suggests activation of Ca 21 channels by HRW under Cd exposure. As shown in Figure 10, A and B, the HRWinduced increase in BcRbohD and BcRbohF transcript levels was negated by Gd 31 and La 31 addition, as was an increase in H 2 O 2 content in roots. Also, amelioration of Cd 21 uptake in HRW-treated roots was not observed in plants treated with 0.1 mM Gd 31 (Fig. 10C), closely matching the Cd content in roots treated with Cd for 6 and 12 h (Fig. 10D). In parallel with the uptake of Cd, HRW pretreatment-induced downregulation of BcIRT1 expression under Cd stress was totally offset by Gd 31 cotreatment (Fig. 10E).

IRT1 Operates Downstream of HRW-Regulated Cd 21 Uptake by Plant Roots
Since the first report on the release of H 2 in bacteria (Stephenson and Stickland, 1931) and the discovery of hydrogenase in Clostridium pasteurianum (Nakos and Mortenson, 1971), research regarding H 2 metabolism and hydrogenase in organisms has attracted significant interest due to its multiple biological functions (Ohsawa et al., 2007;Khanna and Lindblad, 2015). In this study, using H 2 measurement, we demonstrated that Cd exposure triggered rapid and sustained H 2 production (Fig. 1, A and B). Although we did not investigate the enzymatic resource(s) for this process, this observation is consistent with previous findings that H 2 production was increased and maintained in plants treated with paraquat and salt stress (Jin et al., 2013;Xie et al., 2012). These results indicated that H 2 may play an important role in plant response to abiotic stresses.
The next question was the physiological rationale of Cd-induced H 2 production. H 2 exhibits a broad range of biological effects, of which the most common is its antioxidant function (Ohsawa et al., 2007). It is plausible, therefore, that H 2 production in pak choi roots may function uphill of the antioxidant system to inhibit Cdtriggered ROS accumulation reported elsewhere Wu et al., 2015b). In addition to that, H 2 also reduced Cd accumulation in pak choi plants (Fig. 4, A and B). There are two possible reasons for the reduction of Cd content in plants. First, lower Cd 21 influx in H 2 -treated roots (Fig. 3, A and B) could result from lower expression of the BcIRT1 gene conferring the Cd transporter (Fig. 4C) in the HRW treatment. Second, within 12 h of treatment, H 2 induced net Cd 21 efflux from roots (Fig. 3C), thus reducing Cd accumulation in plants.
In plants, many transporters of divalent transition metals have Cd 21 uptake ability (Verbruggen et al., 2009). In our previous report, BcIRT1 and BcZIP2 were shown to transport Cd 21 as well as Fe 21 in yeasts and could be regulated by HRW . Here, only BcIRT1 transcription was significantly inhibited by HRW in pak choi roots (Fig. 4C). In Arabidopsis, the Atirt1 mutant had a significantly smaller net Cd 21 influx compared with the wild type, and the ameliorative effects of HRW on root Cd 21 uptake were abolished in the Atirt1 mutant (Fig. 8). Taken together, these data suggest that IRT1 may operate downstream of HRWregulated Cd 21 uptake by plant roots.
The cytosolic free Cd 21 is the main factor behind negative effects such as membrane peroxidation, disturbance to ion homeostasis, protein cleavage, and even DNA damage in plant tissues (Bashir et al., 2015). Thus, to reduce the damage, plant cells either sequestrate Cd 21 in vacuoles or convert it into nontoxic Cd-organic acid complex via chelation (Romero-Puertas et al., 2002;Song et al., 2014;Singh et al., 2016). However, restricting Cd uptake by the root and promoting Cd efflux may be an energetically less costly option for preventing Cd toxicity. The mechanisms controlling Cd 21 transport across the root PM, however, remained elusive until now. In this work, we showed that Cd-induced H 2 production may be part of such a mechanism. It is therefore possible that H 2 should be added to the list of early response signals operating upstream of Cd transporters.

H 2 O 2 Mediates H 2 -Regulated Cd Uptake
One of the interesting observations in our work was a time lag in H 2 -induced inhibition of Cd 21 influx, with no reduction in Cd 21 uptake observed when HRW pretreatment was added together with Cd 21 (Fig. 3, A  and B). Thus, it appears that effects of H 2 on Cd uptake are indirect and most likely mediated by other components of the signaling pathway.
Consistent with previous reports (Xie et al., 2014;Jin et al., 2016), HRW treatment led to an increase in H 2 O 2 Figure 7. Transient Cd 21 flux from elongation (A) and mature (B) zones of Arabidopsis roots after different treatments. Five-day-old Col-0, Atr-bohD, and AtrbohF seedlings were pretreated with control water or HRW (50% saturation) for 60 min, then exposed to 50 mM CdCl 2 . Values are means 6 SE (n 5 8 seedlings). production, as demonstrated by both electrophysiological (Fig. 5, A and B) and fluorescence imaging data (Fig. 5, C and D). Meanwhile, DPI (an NADPH oxidase inhibitor) abolished the beneficial effects of HRW on inhibition of Cd 21 uptake by roots (Fig. 6), suggesting that NADPH oxidase H 2 O 2 operates downstream of H 2.
RbohD and RbohF are two important members of the Rboh gene family encoding NADPH oxidase (Sagi and Fluhr, 2006). Both have been shown to function in ROS signal amplification and mediation of rapid systemic signaling (Miller et al., 2009). Here we show that RbohD plays a critical role in mediating the beneficial effects of HRW on root Cd 21 uptake. Four lines of evidence support this claim. First, there was no disparity between Atrboh mutants and the wild type in H 2 release (Supplemental Fig. S4), implying that H 2 O 2 could not impact H 2 production. Second, consistent with the H 2 O 2 production data, HRW also upregulated BcRbohD transcript levels (Fig. 5E). Third, H 2 O 2 pretreatment significantly reduced both net Cd 21 influx and Cd 21 content in pak choi roots, while inhibition of NADPH activity by DPI pretreatment led to opposite results (Supplemental Fig. S3). Fourth, HRW-induced inhibition of Cd 21 influx occurred in the Arabidopsis wild type but was absent in AtrbohD mutants (Fig. 7), which had a much more sensitive phenotype (Supplemental Fig. S5). Taken together, these data strongly suggest that Rboh-dependent H 2 O 2 production is essential for HRW-suppressed Cd 21 influx, and that IRT1 operates as a downstream factor in this process, as H 2 O 2 -induced inhibition of Cd 21 influx was abolished in the Atirt1 mutant compared with the wild type (Fig. 8).

The Role of Ca 21 in H 2 -Regulated H 2 O 2 Generation
The H 2 O 2 signal could not be detected until 10 min after HRW treatment (Fig. 5, A and B). One possible explanation for this delay was that H 2 regulated H 2 O 2 production indirectly, through an intermediate signal.
The instant increase in Ca 21 influx after HRW addition (Fig. 9, A and B) prompted a hypothesis that changes in cytosolic Ca 21 levels may happen upstream of H 2 O 2 generation.
Changes in the cytosolic Ca 21 concentration are considered to be one of the earliest cellular responses to all stresses (Marcec et al., 2019), and cytosolic Ca 21 elevation is a ubiquitous denominator of the signaling network when plants are exposed to abiotic stresses, including Cd (McAinsh and Pittman, 2009;Bose et al., 2011). Compelling evidence indicates a reciprocal relationship between H 2 O 2 and Ca 21 , two crucial messengers involved in plant responses to multiple stress conditions (Tuteja and Mahajan, 2007;Mazars et al., 2010;Petrov and Van Breusegem, 2012). The mechanistic basis for this interaction lies in the so-called "ROS-Ca 21 hub" at the PM , where Ca 21activated NADPH oxidases work in concert with ROS-activated Ca 21permeable cation channels to generate and amplify stress-induced Ca 21 and ROS signals. In this study, Gd 31 and La 31 , two known blockers of Ca 21 -permeable nonselective cation channels, entirely suppressed HRW-induced changes in BcRbohD expression and H 2 O 2 production (Fig. 10, A and B). Gd 31 treatment also markedly abolished the ameliorative effects of HRW treatment on root Cd uptake and accumulation, as well as BcIRT1 expression (Fig. 10, C-E). Ca 21 directly binds to EF-hand motifs in the cytosolic N-terminal domain of the NADPH oxidase enzyme, and EF-hands in RBOHD can directly sense Ca 21 (Seybold et al., 2014).
The question of how H 2 regulates Ca 21 transport across the PM remains to be answered in future studies. One of the plausible scenarios may include H 2 -induced voltage gating of Ca 21 -permeable PM channels. Demidchik et al. (2002) proposed that voltage modulation of the coexisting nonselective cation channels and hyperpolarization-activated Ca 21 channels by the PM H 1 -ATPase would be a potent regulator for Ca 21 entry into the root cell cytoplasm. In this study, HRW treatment led to increased net H 1 efflux from plant roots (Supplemental Fig. S7), indicating the possibility of H 1 -ATPase activation by H 2. The high H 1 -pumping activity leads to hyperpolarization of the PM and thus increases Ca 21 influx through hyperpolarizationactivated Ca 21 channels.
In summary, the results of this study demonstrate the existence of a new mechanism that explains the ameliorating effect of H 2 on Cd toxicity in plants, namely H 2 control of the expression level and activity of the PMbased NADPH oxidase encoded by the RbohD gene that operates upstream of IRT1 and regulates root Cd uptake. The timing of events is summarized in the Figure 10. Analysis of gene expression, net Cd 21 , Cd content, and H 2 O 2 in roots of pak choi seedlings. A and B, BcRbohD and BcRbohF transcript levels and H 2 O 2 accumulation. Three-day-old pak choi seedlings were pretreated with HRW (50% saturation), Gd 31 (100 mM), or La 31 (50 mM), alone or in combination, for 60 min, and then incubated in 50 mM CdCl 2 solution for 60 min. Scale bars 5 0.1 cm. C, Effect of Gd 31 pretreatment on transient net Cd 21 flux in HRW-pretreated roots. Three-day-old pak choi seedlings were transferred into control (Con), HRW, or HRW 1 Gd 31 (100 mM) solution for 60 min and then exposed to 50 mM CdCl 2 . D and E, Cd content and BcIRT1 gene expression in pak choi roots. Seedlings in the solution were either not treated (Con) or supplemented with 50% HRW or HRW 1 Gd 31 (100 mM) for 48 h, followed by another 12 h treatment without or with 50 mM CdCl 2 . Values are means 6 SE (n 5 8 seedlings). Lowercase letters indicate significant difference at P , 0.05 according to Duncan's new multiple-range test. Figure 11. Tentative model explaining the ameliorating effects of H 2 on root Cd acquisition. The signaling cascade at the initial stage is the Cdinduced H 2 production. Ca 21 responds to H 2 and is elevated rapidly in the cytosol, resulting in stimulation of NADPH oxidase and subsequently inducing production of H 2 O 2 . When H 2 O 2 accumulates to high levels, it downregulates IRT1 expression, leading to inhibition of Cd 21 influx. Exogenous application of HRW effectively accelerates and amplifies this process. model in Figure 11: (1) Cd enters into the cytosol and triggers rapid H 2 production; (2) an increase in H 2 results in Ca 21 influx and leads to a rapid elevation in cytosolic free Ca 21 levels; (3) the increased cytosolic Ca 21 stimulates the activity of NADPH oxidase and subsequently induces H 2 O 2 generation; and (4) H 2 O 2 downregulates IRT1 activity, resulting in inhibition of Cd 21 influx. Exogenous application of HRW effectively leads to an increase in intracellular H 2 production, thus accelerating and amplifying the above H 2 effects.

Plant Materials, Growth Conditions, and Treatments
Pak choi (Brassica campestris ssp. chinensis 'Dongfang 2') and Arabidopsis (Arabidopsis thaliana; Col-0, rbohD, rbohF, and irt1-1) plants were used in this study. Seeds of pak choi were surface sterilized with 5% (v/v) NaClO solution, extensively rinsed with distilled water, and then soaked in deionized water at room temperature for 3 h. After that, seeds were covered with moist gauze, germinated for 36 h at 23°C, then transferred to plastic chambers containing one-quarter strength Hoagland's solution. Pak choi seedlings grown in onequarter strength Hoagland's solution (without HRW or CdCl 2 ) were used as the control. For Arabidopsis, the mutant seeds were kindly donated by Shaojian Zheng from Zhejiang University (Atirt1 mutant; Zhu et al., 2012) and Wenbiao Shen from Nanjing Agricultural University (AtrbohD and AtrbohF mutants; Xie et al., 2014). For cultivation, seeds were sown on petri dishes with one-half strength Murashige and Skoog basal salt medium supplemented with 2% (w/v) Suc and 0.8% (w/v) agar at pH 5.8. All seedlings were grown in a controlled illuminated incubator at 24 6 1°C, with a photoperiod of 16 h light/8 h dark and light intensity of 200 6 5 mmol m 22 s 21 . Uniform 4-d-old pak choi and 6-d-old Arabidopsis seedlings were selected for electrophysiological measurements or pharmacological experiments.

Determination of Cd Concentrations in Plant Tissues
Plant samples were collected, and roots were rinsed in 20 mM EDTA-Na solution for 15 min to remove Cd absorbed to the surface. After that, all samples were washed with deionized water and dried at 105°C for 24 h. Dried samples were ground to powder and digested in 2 mL HNO 3 :HClO 4 (87:13 [v/v]) solution overnight. The sample digestion was carried out in a heating block at 200°C for 8 h. After cooling, the digested solution was diluted to 15 mL with deionized water and filtered through 0.22 mm cellulose acetate membrane filters. The Cd content in the digest was determined by atomic absorption spectrophotometry (180-80, Hitachi). All assays were performed at least three times, with consistent results.

Preparation of HRW
An H 2 gas generator (SHC-500; Saikesaisi Hydrogen Energy Co.) was used to produce the purified hydrogen gas (99.99% [v/v]). H 2 was bubbled into 1.0 L of one-eighth strength Hoagland nutrition solution at a rate of 150 mL min 21 for 10 min till 100% saturation. Then, the corresponding HRW was immediately diluted to 50% (v/v) concentration. Under our experimental conditions, the H 2 concentration in the freshly prepared HRW was 830 6 10 mM; for 50% HRW this value was 381 6 16 mM (Supplemental Fig. S1, A and C). The H 2 concentration remained .100 mM for at least 12 h (Supplemental Fig. S1B). To make the HRW and N 2 solution, the one-eighth strength Hoagland nutrition solution (Con) was bubbled with H 2 and N 2 for 5 min at the rate of 150 mL min 21 .

Measurement of H 2 Release and Content
The H 2 release was measured using a needle-type hydrogen sensor (DK-8200, Unisense) following the method reported by Xie et al. (2014). The H 2specific electrode was polarized for 4 h before use. Prior to measurement, the pak choi seedlings were placed on 2% agarose gel to avoid damage to the electrode tip. For electrode analysis, the needle was directly stuck into the leaf tissues to a depth of ;200 mm using a micromanipulator. When the basal line of H 2 signal was stable, Cd treatment solution was added to immersed roots and the corresponding data were recorded. All manipulations were performed at 25 6 1°C.
For analysis of endogenous H 2 production, GC was used as described in our previous publications (Wu et al., 2015b). Approximately 1.0 g of pak choi seedlings treated with 0 or 50 mM CdCl 2 were placed in vials. A pure nitrogen gas was then bubbled into the vial to fully displace the air. Afterward, the vials were immediately capped and incubated at 25 6 1°C for 12 h to liberate H 2 from plant tissues. Nitrogen gas was used as the carrier gas, and the air pressure was 0.5 MPa.

Ion Flux Measurements
Net K 1 , Ca 21 , H 1 , and Cd 21 fluxes were measured from the elongation (;350 mm from the root tip) and mature (;1,500 mm from the root tip) root zones of 4-d-old pak choi seedlings. Prior to measurement, roots of intact seedlings were immobilized on a microscopic slide by a parafilm strip. The slide was placed in a measuring chamber containing basic salt medium (0.5 mM KCl and 0.1 mM CaCl 2 , pH 5.6) for 30 min for adaption, and tips of ion-selective microelectrodes were cofocused and positioned 40 to 50 mm above the root epidermis. During measurements, microelectrodes were moved in a 12-s square-wave cycle by a computer-controlled hydraulic micromanipulator with a travel range of 90 mm. Steady-state ion fluxes were recorded for 5 min, and then the appropriate treatment was administered followed by another 20 to 30 min of measurements. Voltage outputs of electrodes were recorded using CHART software and then converted into net flux data using the MIFEFLUX program (Shabala et al., 2006).

Measurement of Net H 2 O 2 Fluxes
An H 2 O 2 -sensititive microelectrode (tip diameter 2-3 mm; XY-DJ-502, Xuyue Science and Technology Co.) was used to monitor H 2 O 2 fluxes in the elongation zone of the roots. H 2 O 2 microelectrodes were prepared according to the method described by Zhang et al. (2017) and Twig et al. (2001). Before the measurement, the H 2 O 2 microelectrode was polarized at 10.6 V against an Ag/AgCl reference electrode. Thereafter, the microelectrodes were calibrated in the standard solutions: 0.01, 0.1, and 1 mM H 2 O 2 . Roots sampled were immobilized in the measuring solution (0.1 mM NaCl, 0.1 mM MgCl 2 , 0.1 mM CaCl 2 , and 0.5 mM KCl, with pH adjusted to 5.2 with KOH and HCl) and equilibrated for 30 min. H 2 O 2 flux was measured from the elongation root zone (;350 mm from the root tip) of 4-d-old pak choi seedlings.

Viability Assay
The viability of pak choi root cells was assessed by performing a double staining method using FDA (catalog no. F7378, Sigma-Aldrich) and PI (catalog no. P4864, Sigma-Aldrich; Koyama et al., 1995). FDA is permeable through the intact PM and shows a green color under a fluorescent microscope in viable cells after hydrolysis by the internal esterases (Rotman and Papermaster, 1966). The PI enters the dead or dying cells via large pores in the PM and shows red color upon formation of the PI-nuclear DNA conjugate. Accordingly, control and 50 mm CdCl 2 -treated roots were stained with freshly prepared FDA (5 mg mL 21 for 5 min) followed by PI (3 mg mL 21 for 10 min). Roots were then washed with distilled water several times, mounted on a slide, and observed immediately using a fluorescent microscope (Leica MZ12, Leica Microsystems) with I3-wavelength filter and UV illumination. Images were acquired by a digital camera (Leica DFC295, Leica Microsystems) and processed by LAS V3.8 software (Leica Microsystems). The camera features for all experiments were set to constant values (exposure time 1.0 s, gain 1.0, saturation 1.0, and G 1.0). Red and green fluorescence intensity was quantified using the software Image J, essentially as described by Wang et al. (2019).

Histochemical Detection of H 2 O 2 and Ca 21
The production of H 2 O 2 in root cells was detected by 29,79-dichlorofluorescein diacetate (H 2 DCFDA; catalog no. D6883, Sigma-Aldrich) staining method (Bose et al., 2014;Wang et al., 2017). The pak choi roots were collected after treatment with 50 mM CdCl 2 , washed with 10 mM Tris-HCl buffer, and immersed in 25 mM H 2 DCFDA for 30 min in the dark. The stained roots were washed thoroughly in distilled water before imaging. Fluorescent signals were visualized using a fluorescent microscope (Leica MZ12; Leica Microsystems) fitted with a high-pressure mercury lamp power (Leica HBO Hg 100 W; Leica Microsystems) and an I3-wavelength filter (Leica Microsystems). The fluorescence images were collected with excitation and emission wavelength at 488 to 525 nm for H 2 DCFDA and analyzed with Image J software.
The calcium accumulation in root cells was measured by the Fluo-3/AM (calcium fluorescent probes; catalog no. 39294, Sigma-Aldrich) based on the method of Yan et al. (2015) and Zhang et al. (2018). Briefly, the root samples were immersed in incubation solution (containing 20 mM Fluo-3/AM, 0.5 M mannitol, 4 mM MES [pH 5.7] and 20 mM KCl) for 30 min at a room temperature. The stained roots were washed thoroughly in distilled water before imaging. The green fluorescence signal was observed using a Laser-Scanning Confocal Microscope (FV1000, Olympus). At least 10 roots were imaged for each treatment.

Reverse Transcription Quantitative PCR Analysis
Total RNA was extracted from the root tissues of treated and untreated seedlings using TRIzol Reagent (catalog no. 15596018, Life Technologies) according to the manufacturer's protocol from the user guide. The first-strand complementary DNA was synthesized using the SensiFAST cDNA Synthesis Kit (catalog no. BIO-65054, Bioline). RT-qPCR reactions were performed using the SensiFAST SYBR No-ROX Kit (catalog no. BIO-98005, Bioline) and Rotor-Gene Q6000 (Qiagen). Detailed information about gene-specific primers can be found in Supplemental Table S1. The three-step cycling quantitative PCR conditions were as follows: one cycle at 95°C for 2 min followed by 40 cycles of 95°C for 5 s, 65°C for 10 s, and 72°C for 15 s. SYBR-green signals were acquired to detect amplified gene products. Data are averages of three independent biological experiments with three technical replicates for each.

Statistical Analyses
Statistical analysis was performed using SPSS Statistics 20 (IBM). Values are shown as the means 6 SE of at least three independent experiments with three replicates each. Differences among treatments were analyzed by one-way ANOVA combined with Duncan's multiple-range test at a probability of P , 0.05.

Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Changes of H 2 and O 2 concentration in H 2 -rich solution as a function of time.
Supplemental Figure S2. Effects of HRW and nitrogen gas (N 2 ) pretreatment on the alleviation of Cd stress-induced growth inhibition in pak choi (Brassica chinensis) seedlings.
Supplemental Figure S3. Net and total Cd 21 fluxes measured from the elongation and mature zones of pak choi seedling roots in response to 50 mM CdCl 2 .
Supplemental Figure S4. Analysis of H 2 release rate from leaves of Arabidopsis seedlings of three different genotypes.
Supplemental Figure S5. Phenotypes and net Cd 21 fluxes of Arabidopsis seedlings of different genotypes treated with 0 (control) or 50 mM CdCl 2 .
Supplemental Figure S6. Calcium concentration in roots of pak choi seedlings under different treatments.
Supplemental Figure S7. Effects of HRW on net proton (H 1 ) flux in the elongation and mature zones of pak choi roots.
Supplemental Table S1. Sequences of primers used for real-time reverse transcription PCR.