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, Dcup1 and Dzrc1 Dcot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Dcup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Dzrc1 Dcot1 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 mM 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.Metal ions, including those of iron, zinc (Zn), and copper (Cu), are required for catalytic and structural properties of many proteins and are therefore essential for growth and development of all organisms. However, excessive amounts of these metals, or of nonessential metals such as cadmium (Cd) and lead, are toxic and inhibit plant growth. To maintain proper metal homeostasis, organisms are equipped with a repertoire of mechanisms to regulate the uptake and distribution of specific metal ions. Nonessential metals and excessive amounts of essential metals can be detoxified by a variety of mechanisms including secretion, compartmentalization, or chelation by metal ligands (Hall, 2002

SummaryMost 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.

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.

a b s t r a c t RNA interference has been proposed to be a promising tool for combating shrimp viruses. Antiviral double-stranded (ds)RNA has been mostly produced in Escherichia coli-expression system because of its high efficiency and inexpensive operations. However, overusing the bacteria may raise concerns regarding public health and environmental contamination, and seeking for a new dsRNA production platform would be alternative for future molecular farming. In this study, we exploited the green microalgae Chlamydomonas reinhardtii to produce dsRNA targeting the lethal shrimp yellow head virus (YHV). The expression plasmid pSL18 for C. reinhardtii was constructed to contain YHV-specific hairpin RNA expression cassette, and the successful assembly of pSL18-YHV was confirmed by PCR and enzymatic digestions. Glass bead method was employed for transformation of C. reinhardtii nuclear genome with pSL18-YHV. Microalgal expression of dsRNA-YHV, approximately 45 ng from 100-mL culture, was detected by qRT-PCR. Oral feeding experiment on postlarval shrimp revealed that the formulated feed with C. reinhardtii expressing dsRNA-YHV, at the ratio of 1 × 10 8 transformants per gram feed, improved 22% survival rate after YHV challenge. The present study suggests that C. reinhardtii can be bioengineered to produce viralspecific dsRNA for shrimp viral disease control, and the developed qRT-PCR could detect microalgal dsRNA with detection limit of subpicogram.

A tetra-manganese cluster in the photosystem II (PSII) pigment-protein complex plays a critical role in the photosynthetic oxygen evolution process. PsbY, a small membrane-spanning polypeptide, has recently been suggested to provide a ligand for manganese in PSII ([1998] Mol Gen Genet 260: 56-68). We have constructed a mutant strain of the cyanobacterium Synecho-cystis sp. PCC 6803 with an inactivated psbY gene (sml0007). Southern-blot and polymerase chain reaction analysis showed that the mutant had completely segregated. However, the psbY mutant cells grew normally under photoautotrophic conditions. Moreover, growth of the wild-type and mutant cells were similar under highlight photoinhibition conditions, as well as in media without any added manganese, calcium, or chloride, three required inorganic cofactors for the oxygen-evolving complex of PSII. Analysis of steady-state and flash-induced oxygen evolution, fluorescence induction , and decay kinetics, and thermoluminescence profiles demonstrated that the psbY mutant cells have normal photosynthetic activities. We conclude that the PsbY protein in Synechocystis 6803 is not essential for oxygenic photosynthesis and does not provide an important binding site for manganese in the oxygen-evolving complex of PSII. During oxygenic photosynthesis in plants, algae, and cyanobacteria, two reaction-center-containing integral membrane protein complexes, photosystem I (PSI) and photosystem II (PSII), are involved in the initial steps of the conversion of solar energy into usable chemical energy. Among them, PSII, a large-pigment protein, mediates electron transfer from water to plastoquinones, with simultaneous evolution of molecular oxygen. The process of water oxidation takes place in the lumen of thylakoid and is catalyzed by the oxygen-evolving complex (OEC) of PSII. Three inorganic ions, manganese, calcium, and chloride, are the known cofactors of the OEC. However, their locations in PSII and the polypeptides that coordinate these ions remain unclear (Hankamer and Barber, 1997). Isolated photoactive PSII complexes may contain as many as 22 polypeptides (Debus, 1992; Hankamer and Barber, 1997). Many of them have been suggested to constitute the OEC. The most probable candidate is the PSII reaction center protein D1 (Boerner et al.