Functional characterization of metallothionein-like genes from Physcomitrella patens: expression profiling, yeast heterologous expression, and disruption of PpMT1.2a gene.

MAIN CONCLUSION: Physcomitrella patens contains four metallothionein-like genes. Three were shown to confer metal tolerance in yeast. Transcript profiling suggests their roles in senescence and reproductive development or cadmium and oxidative stress. Metallothioneins (MTs) have been suggested to play various roles including metal detoxification, nutrient remobilization, ROS scavenging, stress tolerance, and plant development. However, little is known about the forms and functions of MTs in bryophytes. The moss Physcomitrella patens genome was found to contain four MT-like genes. Amino acid sequence composition showed that the P. patens MTs (PpMTs) were clustered with Type 1 plant MTs, and could be further classified into two sub-types, herein referred to as sub-type 1: PpMT1.1a and PpMT1.1b and sub-type 2: PpMT1.2a and PpMT1.2b. Transcript abundance of PpMT1.1b and PpMT1.2b was upregulated in the gametophore compared to protonema, and all, except PpMT1.2a, were highly induced in senescing gametophytes. PpMT1.1a and PpMT1.1b transcripts were upregulated in protonema treated with cadmium and hydrogen peroxide. Unlike many higher plant MTs, the PpMT transcript abundance was not strongly induced in response to copper and zinc. These results suggest that PpMTs may play a role in protecting P. patens from cadmium and oxidative stress and may be involved in tissues senescence and reproductive development. The PpMTs, except PpMT1.2b, were also able to confer metal tolerance and accumulation when heterologously expressed in the ∆cup1 yeast. A P. patens mutant lacking PpMT1.2a through targeted gene disruption was generated. However, it did not show any alteration in growth phenotypes under senescence-induced conditions or hypersensitivity to cadmium, copper, zinc, H2O2, and NaCl stresses. Further characterization of additional P. patens mutants lacking single or multiple PpMTs may provide insight into the physiological roles of bryophytic MTs.

Performance of packed bed column using Chara aculeolata biomass for removal of Pb and Cd ions from wastewater.

Biosorption of Pb and Cd from aqueous solution by biomass of Chara aculeolata was studied in a continuous packed bed column. C. aculeolata in the fixed bed column is capable of decreasing Pb and Cd concentrations from 10 mg/L to a value below the detection limit of 0.02 mg/L. Selective uptake of Pb and Cd in a binary solution resulted in Pb having much higher relative affinity than Cd. The experiments were conducted to study the effects of column design parameters, bed depth, and flow rate on the metal biosorption. Pb uptake capacity of C. aculeolata increased with increased bed depth and decreased flow rate, while Cd uptake capacity increased with increased bed depth but remained constant at any flow rate. The Thomas model was found in a suitable fitness with the experiment data for Pb and Cd (R2 > 0.90). The efficiency of biosorbent regeneration achieved by 0.1 M HCl was very high, that was, 98% for Pb and 100% for Cd in the third reused cycle. It can be concluded that C. aculeolata is a good biosorbent for treating wastewater having low concentrations of Pb and Cd contamination.

Metallothionein deficiency impacts copper accumulation and redistribution in leaves and seeds of Arabidopsis.

Most angiosperm genomes contain several genes encoding metallothionein (MT) proteins that can bind metals including copper (Cu) and zinc (Zn). Metallothionein genes are highly expressed under various conditions but there is limited information about their function. We have studied Arabidopsis mutants that are deficient in multiple MTs to learn about the functions of MTs in plants. T-DNA insertions were identified in four of the five Arabidopsis MT genes expressed in vegetative tissues. These were crossed to produce plants deficient in four MTs (mt1a/mt2a/mt2b/mt3). The concentration of Cu was lower in seeds but higher in old leaves of the quad-MT mutant compared to wild-type plants. Experiments with stable isotopes showed that Cu in seeds came from two sources: directly from roots and via remobilization from other organs. Mobilization of Cu out of senescing leaves was disrupted in MT-deficient plants. Tolerance to Cu, Zn and paraquat was unaffected by MT deficiency but these plants were slightly more sensitive to cadmium (Cd). The quad-MT mutant showed no change in resistance to a number of microbial pathogens, or in the progression of leaf senescence. Although these MTs are not required to complete the plant's life cycle, MTs are important for Cu homeostasis and distribution in Arabidopsis.

Phytoremediation potential of charophytes: bioaccumulation and toxicity studies of cadmium, lead and zinc.

The ability for usage of common freshwater charophytes, Chara aculeolata and Nitella opaca in removal of cadmium (Cd), lead (Pb) and zinc (Zn) from wastewater was examined. C. aculeolata and N. opaca were exposed to various concentrations of Cd (0.25 and 0.5 mg/L), Pb (5 and 10 mg/L) and Zn (5 and 10 mg/L) solutions under hydroponic conditions for 6 days. C. aculeolata was more tolerant of Cd and Pb than N. opaca. The relative growth rate of N. opaca was drastically reduced at high concentrations of Cd and Pb although both were tolerant of Zn. Both macroalgae showed a reduction in chloroplast, chlorophyll and carotenoid content after Cd and Pb exposure, while Zn exposure had little effects. The bioaccumulation of both Cd and Pb was higher in N. opaca (1544.3 microg/g at 0.5 mg/L Cd, 21657.0 microg/g at 10 mg/L Pb) whereas higher Zn accumulation was observed in C. aculeolata (6703.5 microg/g at 10 mg/L Zn). In addition, high bioconcentration factor values (> 1000) for Cd and Pb were observed in both species. C. aculeolata showed higher percentage of Cd and Pb removal (> 95%) than N. opaca and seemed to be a better choice for Cd and Pb removal from wastewater due to its tolerance to these metals.

Examination of the Metallothionein Gene Family in Greater Duckweed Spirodela polyrhiza.

Duckweeds are aquatic plants that proliferate rapidly in a wide range of freshwaters, and they are regarded as a potential source of sustainable biomass for various applications and the cost-effective bioremediation of heavy metal pollutants. To understand the cellular and molecular basis that underlies the high metal tolerance and accumulation capacity of duckweeds, we examined the forms and transcript profiles of the metallothionein (MT) gene family in the model duckweed Spirodela polyrhiza, whose genome has been completely sequenced. Four S. polyrhiza MT-like genes were identified and annotated as SpMT2a, SpMT2b, SpMT3, and SpMT4. All except SpMT2b showed high sequence homology including the conserved cysteine residues with the previously described MTs from flowering plants. The S. polyrhiza genome appears to lack the root-specific Type 1 MT. The transcripts of SpMT2a, SpMT2b, and SpMT3 could be detected in the vegetative whole-plant tissues. The transcript abundance of SpMT2a was upregulated several-fold in response to cadmium stress, and the heterologous expression of SpMT2a conferred copper and cadmium tolerance to the metal-sensitive ∆cup1 strain of Saccharomyces cerevisiae. Based on these results, we proposed that SpMT2a may play an important role in the metal detoxification mechanism of duckweed.

Generation of microalga Chlamydomonas reinhardtii expressing shrimp antiviral dsRNA without supplementation of antibiotics.

RNA interference (RNAi) is an effective way of combating shrimp viruses by using sequence-specific double-stranded (dsRNA) designed to knock down key viral genes. The aim of this study was to use microalgae expressing antiviral dsRNA as a sustainable feed supplement for shrimp offering viral protection. In this proof of concept, we engineered the chloroplast genome of the green microalga Chlamydomonas reinhardtii for the expression of a dsRNA cassette targeting a shrimp yellow head viral gene. We used a previously described chloroplast transformation approach that allows for the generation of stable, marker-free C. reinhardtii transformants without the supplementation of antibiotics. The generated dsRNA-expressing microalgal strain was then used in a shrimp feeding trial to evaluate the efficiency of the algal RNAi-based vaccine against the virus. Shrimps treated with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection (dpi), whereas 84.1% mortality was observed in control groups exposed to the YHV virus. RT-PCR using viral specific primers revealed a lower infection rate in dsRNA-expressing algae treated shrimp (55.6 ± 11.1%) compared to control groups (88.9 ± 11.1% and 100.0 ± 0.0%, respectively). Our results are promising for using microalgae as a novel, sustainable alternative as a nutritious, anti-viral protective feedstock in shrimp aquaculture.

The biological responses and metal phytoaccumulation of duckweed Spirodela polyrhiza to manganese and chromium.

The phytoaccumulation ability of duckweed Spirodela polyrhiza on manganese (Mn) and chromium (Cr) was assessed by exposing the plant to various concentrations of single or dual metals (5-70 mg L-1 Mn, 2-12 mg L-1 Cr(VI)) under laboratory conditions. The results showed that S. polyrhiza can tolerate Mn at high concentrations of up to 70 mg L-1, and its growth rate was barely affected by Mn. The effects of Cr on S. polyrhiza growth were dose-dependent, and the growth was completely inhibited in the presence of 12 mg L-1 Cr. Analysis of metal content in the plant biomass revealed a high accumulation of Mn (up to 15.75 mg per g of duckweed dry weight). The Cr bioaccumulation (from below detection limit to 2.85 mg Cr (11.84 mg Cr2O72-) per g of duckweed dry weight) increased with cultivation time and metal concentration in the medium. Further study with the concurrence of Mn and Cr showed increased toxicity to plant growth and photosynthesis. The metal accumulations in the dual metal treatments were also significantly decreased as compared to the single metal treatments. Nevertheless, the phytoaccumulation of these two metals in S. polyrhiza in the dual metal treatments were still comparable to or higher than in previous reports. Thus, it was concluded that duckweed S. polyrhiza has the potential to be used as a phytoremediator in aquatic environments for Mn and Cr removal.

Increasing the Triacylglycerol Content in Dunaliella tertiolecta through Isolation of Starch-Deficient Mutants.

The production cost of biodiesel from microalgae is still not competitive, compared with that of petroleum fuels. The genetic improvement of microalgal strains to increase triacylglycerol (TAG) accumulation is one way to reduce production costs. One of the most promising approaches is the isolation of starch-deficient mutants, which have been reported to successfully increase TAG yields. To date, such a stable mutant is not available in an oleaginous marine microalga, despite several advantages of using marine species for biodiesel production. Algae in the genus Dunaliella are known to tolerate high salt concentration and other environmental stresses. In addition, the cultivation processes for large-scale outdoor commercialization have been well established for this genus. In this study, Dunaliella tertiolecta was used to screen for starch-deficient mutants, using an iodine vapor-staining method. Four out of 20,016 UV-mutagenized strains showed a substantial reduction of starch content. A significantly higher TAG content, up to 3-fold of the wild-type level, was observed in three of the mutants upon induction by nitrogen depletion. The carotenoid production and growth characteristics of these mutants, under both normal and oxidative stress conditions, were not compromised, suggesting that these processes are not necessarily affected by starch deficiency. The results from this work open up new possibilities for exploring Dunaliella for biodiesel production.

The Effect of Light Stress and Other Culture Conditions on Photoinhibition and Growth of Dunaliella tertiolecta.

This work aimed to examine the effects of high light stress as well as other culture conditions including HCO3 − concentration, temperature, salinity, and pre-acclimation on photoinhibition and growth of halotolerant alga Dunaliella tertiolecta. Significant photoinhibition of D. tertiolecta was observed during a short period of exposure (6 hours) to high intensity of lights (1000, 1500, and 2000 µmol photons m-2 s-1); however, after 2 days of continuous light exposure, the alga adapted to high light stress and reached similar growth rates as low light exposure. The increase in HCO3 − concentration in the culture medium did not reduce photoinhibition, but the growth rate and chlorophyll contents increased with increasing HCO3 − concentrations. Temperature had significant effects on photoinhibition. Combined high temperature and high light intensity led to more serious photoinhibition and reduced cell growth rates, so did combined low salinity and high light intensity. Pre-acclimation by 50, 200, or 500 µmol photons m-2 s-1 each for 1, 3, or 6 hours (a total of nine treatments) did not significantly influence photoinhibition or cell growth of D. tertiolecta, probably because the acclimation periods were not long enough.

Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance.

Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.