Cadmium exposure is associated with increased transcript abundance of multiple heavy metal associated transporter genes in roots of hemp (Cannabis sativa L.).

Industrial hemp (Cannabis sativa L.) has demonstrated promise for phytoremediation due to an extensive root system, large biomass, and ability to survive under relatively high levels of heavy metals. However, little research has been conducted to determine the impact of heavy metal uptake in hemp grown for medicinal use. This study evaluated the potential for cadmium (Cd) uptake and its impact on growth, physiological responses, and transcript expression of metal transporter genes in a hemp variety grown for flower production. The cultivar 'Purple Tiger' was exposed to 0, 2.5, 10, and 25 mg·L-1 Cd in a greenhouse hydroponic study in two independent experiments. Plants exposed to 25 mg·L-1 Cd displayed stunted plant growth characteristics, reduced photochemical efficiency, and premature senescence suggesting Cd toxicity. At the two lower concentrations of Cd (2.5 and 10 mg·L-1 Cd), plant height, biomass, and photochemical efficiency were not affected, with chlorophyll content index (CCI) being slightly lower at 10 mg·L-1 Cd, compared to 2.5 mg·L-1 Cd. There were no consistent differences between the two experiments in total cannabidiol (CDB) and tetrahydrocannabinol (THC) concentrations in flower tissues at 2.5 and 10 mg·L-1 Cd, compared to the control treatment. Root tissue accumulated the highest amount of Cd compared to other tissues for all the Cd treatments, suggesting preferential root sequestration of this heavy metal in hemp. Transcript abundance analysis of heavy metal-associated (HMA) transporter genes suggested that all seven members of this gene family are expressed in hemp, albeit with higher expression in the roots than in the leaves. In roots, CsHMA3 was up-regulated at 45 and 68 d after treatment (DAT), and CsHMA1, CsHMA4, and CsHMA5 were upregulated only under long term Cd stress at 68 DAT, at 10 mg·L-1 Cd. Results suggest that expression of multiple HMA transporter genes in the root tissue may be upregulated in hemp exposed to 10 mg·L-1 Cd in a nutrient solution. These transporters could be involved in Cd uptake in the roots via regulating its transport and sequestration, and xylem loading for long distance transport of Cd to shoot, leaf, and flower tissues.

Arsenic removal by As-hyperaccumulator Pteris vittata from two contaminated soils: A 5-year study.

The ability of As-hyperaccumulator Pteris vittata to remove As from two contaminated soils (CCA from an As-treated wood facility and DVA from a cattle-dipping vat) over 5 years was investigated for the first time. The goal was to evaluate P. vittata's ability to continuously remove As during 10 harvests and identify how soil As was affected by P. vittata under P-sufficient (P-fertilizer) and P-limiting (phosphate rock) conditions. Sequential extraction was used to determine changes in metal distribution among different soil fractions. The high frond biomass production occurred on the 9th (62.1-63.9 and 35.6-63.5 g plant-1) and 10th harvest (58.6-60.7 and 51.9-57.1 g plant-1) for CCA and DVB soils, though frond As concentration decreased. Soil arsenic removal averaged 7-10% per harvest during the 1-6th harvests and was reduced to 0-3% during the 7-10th harvests for DVA and CCA soils. Arsenic from all fractions, excluding the residual fraction, was affected by plant uptake. The largest reduction occurred in the amorphous fraction of CCA-soil at 64-66% (61.2-61.5 to 20.8-21.8 mg kg-1) and in the crystalline fraction of DVA-soil at 50-86% (2.18-4.35 to 0.61-1.10 mg kg-1). Soil As concentrations were reduced by 37-47% from 26.7 to 129 to 15.6-16.8 and 68.9-70.1 mg kg-1 for the DVA and CCA soils, respectively. Our data indicated that P. vittata efficiently solubilized non-labile As under P-limiting conditions without impacting its As depletion.

A new method for antimony speciation in plant biomass and nutrient media using anion exchange cartridge.

A selective separation method based on anion exchange cartridge was developed to determine antimony (Sb) speciation in biological matrices by graphite furnace atomic absorption spectrophotometry (GFAAS). The selectivity of the cartridge towards antimonite [Sb(III)] and antimonate [Sb(V)] reversed in the presence of deionized (DI) water and 2mM citric acid. While Sb(V) was retained by the cartridge in DI water, Sb(III) was retained in citric acid media. At pH 6, Sb(III) and Sb(V) formed Sb(III)- and Sb(V)-citrate complexes, but the cartridge had higher affinity towards the Sb(III)-citrate complex. Separation of Sb(III) was tested at various concentrations in fresh and spent growth media and plant tissues. Our results showed that cartridge-based Sb speciation was successful in plant tissues, which was confirmed by HPLC-ICP-MS. The cartridge retained Sb(III) and showed 92-104% Sb(V) recovery from arsenic hyperaccumulator Pteris vittata roots treated with Sb(III) and Sb(V). The cartridge procedure is an effective alternative for Sb speciation, offering low cost, reproducible results, and simple Sb analysis using GFAAS.

Uptake of antimonite and antimonate by arsenic hyperaccumulator Pteris vittata: Effects of chemical analogs and transporter inhibitor.

Antimonite (SbIII) is transported into plants via aquaglyceroporin channels but it is unknown in As-hyperaccumulator Ptreis vittata (PV). We tested the effects of SbIII analogs (arsenite-AsIII, glycerol, silicic acid-Si, and, glucose), antimonate (SbV) analog (phosphate-P), and aquaglyceroporin transporter inhibitor (silver, Ag) on the uptake of SbIII or SbV by PV gametophytes. PV gametophytes were grown in 20% Hoagland solution containing 65 µM SbIII or SbV and increasing concentrations of analogs at 65-6500 µM for 2 h or 4 h under sterile condition. After exposing to 65 µM Sb for 2 h, PV accumulated 767 mg/kg Sb in SbIII treatment and 419 mg/kg in SbV treatment. SbIII uptake by PV gametophytes was not impacted by glycerol or AsIII nor aquaglyceroporin inhibitor Ag during 2 h exposure. While Si increased SbIII uptake and glucose decreased SbIII uptake by PV gametophytes, the impact disappeared during 4 h exposure. Under P-sufficient condition, P increased SbIII uptake and decreased SbV uptake during 2 h exposure, but the effect again disappeared after 4 h. After being P-starved for 2 weeks, P decreased SbIII with no effect on SbV uptake during 2 h exposure. Our results indicated that: 1) PV gametophytes could serve as an efficient model to study Sb uptake, and 2) unique SbIII uptake by PV may be related to its trait of As hyperaccumulation.

Chromate and phosphate inhibited each other's uptake and translocation in arsenic hyperaccumulator Pteris vittata L.

We investigated the effects of chromate (CrVI) and phosphate (P) on their uptake and translocation in As-hyperaccumulator Pteris vittata (PV). Plants were exposed to 1) 0.10 mM CrVI and 0, 0.25, 1.25, or 2.50 mM P or 2) 0.25 mM P and 0, 0.50, 2.5 or 5.0 mM CrVI for 24 h in hydroponics. PV accumulated 2919 mg/kg Cr in the roots at CrVI0.10, and 5100 and 3500 mg/kg P in the fronds and roots at P0.25. When co-present, CrVI and P inhibited each other's uptake in PV. Increasing P concentrations reduced Cr root concentrations by 62-82% whereas increasing CrVI concentrations reduced frond P concentrations by 52-59% but increased root P concentrations by 11-15%. Chromate reduced P transport, with more P being accumulated in PV roots. Though CrVI was supplied, 64-78% and 92-93% CrIII were in PV fronds and roots. Based on X-ray diffraction, Cr2O3 was detected in the roots confirming CrVI reduction to CrIII by PV. In short, CrVI and P inhibited each other in uptake and translocation by PV, and CrVI reduction to CrIII in PV roots served as its detoxification mechanism. The finding helps to understand the interactions of P and Cr during their uptake in PV.

Pteris vittata continuously removed arsenic from non-labile fraction in three contaminated-soils during 3.5 years of phytoextraction.

We evaluated the effectiveness of arsenic (As) hyperaccumulator Pteris vittata to continuously remove As from three contaminated-soils containing 26-126mgkg(-1) As over 7 harvests in 3.5 years. Changes in As speciation in soils, amended with P fertilizer (P-soil) or insoluble phosphate rock (PR-soil), were assessed via sequential fractionation. Arsenic in available (soluble+exchangeable), non-labile (bound to amorphous+crystalline Fe/Al oxides), and residual fractions constituted ∼12%, ∼80%, and ∼8% of soil As. Soluble As declined while exchangeable As was unchanged, likely due to replenishment from non-labile As, which accounted for ∼87% of decline in total soil As. Although plant-available As is important, the non-labile As better predicted the frond As concentration in P. vittata, with the correlation being r=0.90 and 0.64 for PR-soils and P-soils. P. vittata removed 44% of soil As from PR-soils compared to 33% from P-soils, suggesting the low-soluble P from PR was more effective than P fertilizer in enhancing As uptake by P. vittata. To facilitate acquisition of P from PR, P. vittata produced larger root biomass to solubilize non-labile As, allowing for more efficient phytoextraction.

Assessment of children's exposure to arsenic from CCA-wood staircases at apartment complexes in Florida.

Arsenic exposure from wood treated with chromated copper arsenate (CCA) remains a concern due to its presence around homes. This study evaluated children's exposure to As from CCA-treated staircases through determination of bioaccessible soil As and measurements of dislodgeable As on hand railings, steps and surfaces of household objects impacted by CCA-wood leachate. Total As concentrations in 84 soil samples from 4 apartment complexes were elevated at 1.2-66.6 mg/kg with bioaccessible As at 17-84%. Deterministic risk equations were used to estimate daily doses of As in children with estimates ranging from 0.41-54.9 µg/day from ingestion of dislodgeable As. Lifetime average daily doses from ingestion of dislodgeable As and soil ranged from 8.1×10(-6) to 3.0×10(-5) mg/kg/day, with estimated cancer risks being 1.2-4.5×10(-5). Collectively, these results highlight potential health risks in children who have near-daily exposure to As from CCA-wood and are consistent with estimates generated by USEPA's SHEDS-Wood probabilistic exposure model.

Antimony uptake, efflux and speciation in arsenic hyperaccumulator Pteris vittata.

Even though antimony (Sb) and arsenic (As) are chemical analogs, differences exist on how they are taken up and translocated in plants. We investigated 1) Sb uptake, efflux and speciation in arsenic hyperaccumulator Pteris vittata after 1 d exposure to 1.6 or 8 mg/L antimonite (SbIII) or antimonate (SbV), 2) Sb uptake by PV accessions from Florida, China, and Brazil after 7 d exposure to 8 mg/L SbIII, and 3) Sb uptake and oxidation by excised PV fronds after 1 d exposure to 8 mg/L SbIII or SbV. After 1 d exposure, P. vittata took 23-32 times more SbIII than SbV, with all Sb being accumulated in the roots with the highest at 4,192 mg/kg. When exposed to 8 mg/L SbV, 98% of Sb existed as SbV in the roots. In comparison, when exposed to 8 mg/L SbIII, 81% of the total Sb remained as SbIII and 26% of the total Sb was effluxed out into the media. The three PV accessions had a similar ability to accumulate Sb at 12,000 mg/kg in the roots, with >99% of total Sb in the roots. Excised PV fronds translocated SbV more efficiently from the petioles to pinnae than SbIII and were unable to oxidize SbIII. Overall, P. vittata displayed efficient root uptake and efflux of SbIII with limited ability to translocate and transform in the roots.

Effects of arsenate, chromate, and sulfate on arsenic and chromium uptake and translocation by arsenic hyperaccumulator Pteris vittata L.

We investigated effects of arsenate (AsV), chromate (CrVI) and sulfate on As and Cr uptake and translocation by arsenic hyperaccumulator Pteris vittata (PV), which was exposed to AsV, CrVI and sulfate at 0, 0.05, 0.25 or 1.25 mM for 2-wk in hydroponic system. PV was effective in accumulating large amounts of As (4598 and 1160 mg/kg in the fronds and roots at 0.05 mM AsV) and Cr (234 and 12,630 mg/kg in the fronds and roots at 0.05 mM CrVI). However, when co-present, AsV and CrVI acted as inhibitors, negatively impacting their accumulation in PV. Arsenic accumulation in the fronds was reduced by 92% and Cr by 26%, indicating reduced As and Cr translocation. However, addition of sulfate increased uptake and translocation of As by 26-28% and Cr by 1.63 fold. This experiment demonstrated that As and Cr inhibited each other in uptake and translocation by PV but sulfate enhanced As and Cr uptake and translocation by PV.

Sparingly-soluble phosphate rock induced significant plant growth and arsenic uptake by Pteris vittata from three contaminated soils.

We evaluated the ability of As-hyperaccumulator Pteris vittata (PV) to remove As from As-contaminated soils over five harvests in 2.5 years in raised beds (162 kg soil/bed). We tested the hypothesis that a P-limiting environment would enhance PV growth and As uptake owing its unique ability to uptake P under As-rich environment. In Dec. 2009, PV was transplanted to three As-contaminated soils (pH of 5.5-7.2) containing 25-129 mg kg(-1) As, which was amended with sparingly-soluble phosphate rock (PR-soil) or soluble P fertilizer (P-soil). During the 2.5-year, PV obtained sufficient P (1882 vs 2225 mg kg(-1)) from PR-soils, with increased root biomass (33%) and root exudation (53%) compared to P-soils. In addition, its frond biomass increased by 20% consecutively with each harvest (six month interval) from 18 to 36 g plant(-1). Its frond biomass in PR-soils (52.2 g plant(-1) year(-1) or ∼12 mt ha(-1) year(-1)) averaged 39% more than that in P-soils. To our knowledge, this represented the largest PV frond biomass reported, demonstrating the unique ability of PV in using insoluble P from PR in alkaline soils. In addition to biomass increase, PV from PR-soils had ∼1.5 times more As in fronds (2540, 780, and 920 mg kg(-1)) than those from P-soils (1740, 570, and 400 mg kg(-1)), with soils containing 129, 25, and 30 mg kg(-1) As, respectively. The low available P in PR-soils induced substantial plant growth and As uptake by PV. This translated into significantly more As removal from soil, averaging 48% reduction in PR-soils and 36% in P-soils in 2.5 years. With multiple harvests and PR amendments, our results showed As removal by PV from contaminated soils was ∼7 times faster than published studies.

Novel phytase from Pteris vittata resistant to arsenate, high temperature, and soil deactivation.

Arsenate interferes with enzymatic processes and inhibits inorganic phosphorus (Pi) uptake in many plants. This study examined the role of phytase and phosphatase in arsenate tolerance and phosphorus (P) acquisition in the arsenic hyperaccumulator Pteris vittata . Enzyme-mediated hydrolysis of phytate in P. vittata extracts was not inhibited by arsenate at 5 mM or by heating at 100 °C for 10 min. Root exudates of P. vittata exhibited the highest phytase activity (18 nmol Pi mg(-1) protein min(-1)) when available P was low, allowing its growth on media amended with phytate as the sole source of P. Phosphorus concentration in P. vittata gametophyte tissue grown on phytate was equivalent to plants grown with inorganic phosphate at 2208 mg kg(-1), and arsenic was increased from 1777 to 2630 mg kg(-1). After 2 h of mixing with three soils, P. vittata phytase retained more activity, decreasing from ∼ 26 to ∼ 25 nmol Pi mg(-1) protein min(-1), whereas those from Pteris ensiformis and wheat decreased from ∼ 18 to ∼ 1 nmol Pi mg(-1) protein min(-1). These results suggest P. vittata has a uniquely stable phytase enabling its P acquisition in P-limiting soil environments. Furthermore, the P. vittata phytase has potential use as a soil amendment, a transgenic tool, or as a feed additive supplement, reducing the need for nonrenewable, polluting P fertilizers.