VOZ1 and VOZ2 transcription factors regulate arsenic tolerance and distribution in rice and Arabidopsis.

Rice is the major source of arsenic (As) intake in humans, as this staple crop readily accumulates As in the grain. Identifying the genes and molecular mechanisms underlying As accumulation and tolerance is a crucial step toward developing rice with reduced As levels. We identified 25 rice genes that improve As tolerance in yeast cells by expressing a complementary DNA (cDNA) library generated from As-treated rice roots. Among them, a zinc finger-type transcription factor VASCULAR PLANT ONE- ZINC FINGER 1 (OsVOZ1) (OsVOZ1) conferred the most pronounced As tolerance. OsVOZ1 inhibits As accumulation in yeast via activation of As efflux transporter Acr3p by post-transcriptional modification in yeast. The Arabidopsis voz1 voz2 double-knockout mutant exhibited As hypersensitivity, altered As concentrations in various tissues, and reduced As transport activity via the phloem. Arabidopsis and rice VOZs were highly expressed in phloem cells in various tissues, which are critical for As distribution in plant tissues. The double-knockdown and single-knockout plants of OsVOZ1 and OsVOZ2 reduced As accumulation in their seeds. These findings suggest that rice and Arabidopsis VOZs regulate the translocation of As into tissues by regulating the phloem loading of this element.

Toward an experimental system for the examination of protein mannosylation in Actinobacteria.

The Actinobacterial species Cellulomonas fimi ATCC484 has long been known to secrete mannose-containing proteins, but a closer examination of glycoproteins associated with the cell has never been reported. Using ConA lectin chromatography and mass spectrometry, we have surveyed the cell-associated glycoproteome from C. fimi and collected detailed information on the glycosylation sites of 19 cell-associated glycoproteins. In addition, we have expressed a previously known C. fimi secreted cellulase, Celf_3184 (formerly CenA), a putative peptide prolyl-isomerase, Celf_2022, and a penicillin-binding protein, Celf_0189, in the mannosylation capable host, Corynebacterium glutamicum. We found that the glycosylation machinery in C. glutamicum was able to use the recombinant C. fimi proteins as substrates and that the glycosylation matched closely that found in the native proteins when expressed in C. fimi. We are pursuing this observation as a prelude to dissecting the biosynthetic machinery and biological consequences of this protein mannosylation.

Role of ubiquitination in arsenic tolerance in plants.

Arsenic (As) is harmful to all living organisms, including humans and plants. To limit As uptake and avoid its toxicity, plants employ systems that regulate the uptake of As from the soil and its translocation from roots to grains. Ubiquitination, a highly conserved post-translational modification (PTM) in all eukaryotes, plays crucial roles in modulating As detoxification mechanisms in budding yeast (Saccharomyces cerevisiae), but little is known about its roles in As tolerance and transport in plants. In this opinion article we review recent findings and suggest that ubiquitination plays a crucial role in regulating As transport in plants. We also propose ideas for future research to explore the importance of ubiquitination for enhancing As tolerance in crops.

Comparative Analysis of Arsenic Transport and Tolerance Mechanisms: Evolution from Prokaryote to Higher Plants.

Arsenic (As) is a toxic metalloid for all living organisms and can cause serious harm to humans. Arsenic is also toxic to plants. To alleviate As toxicity, all living organisms (from prokaryotes to higher plants) have evolved comprehensive mechanisms to reduce cytosolic As concentration through the set of As transporters localized at the plasma and tonoplast membranes, which operate either in arsenite As(III) extrusion out of cells (via ArsB, ACR3, and aquaporins) or by sequestering arsenic into vacuoles (by ABC transporters). In addition, a special arsenate resistance mechanism found in some bacterial systems has evolved in an As hyperaccumulating fern Pteris vittata, which involves transforming arsenate As(V) to an As(V) phosphoglycerate derivative by a glyceraldehyde 3-phosphate dehydrogenase and transporting this complex by an efflux transporter. In the present review, we summarize the evolution of these arsenic resistance mechanisms from prokaryotes to eukaryotes and discuss future approaches that could be utilized to better understand and improve As resistance mechanisms in plants.

Correction: Characterization of Brassica rapa metallothionein and phytochelatin synthase genes potentially involved in heavy metal detoxification.

[This corrects the article DOI: 10.1371/journal.pone.0252899.].

Characterization of Brassica rapa metallothionein and phytochelatin synthase genes potentially involved in heavy metal detoxification.

Brassica rapa is an important leafy vegetable that can potentially accumulate high concentrations of cadmium (Cd), posing a risk to human health. The aim of the present study was to identify cadmium detoxifying molecular mechanisms in B. rapa using a functional cloning strategy. A cDNA library constructed from roots of B. rapa plants treated with Cd was transformed into the Cd sensitive yeast mutant strain DTY167 that lacks the yeast cadmium factor (YCF1), and resistant yeast clones were selected on Cd containing media. Two hundred genes potentially conferring cadmium resistance were rescued from the surviving yeast clones and sequenced. Sequencing analysis revealed that genes encoding for metallothionein (MT)1, MT2a, MT2b and MT3, and phytochelatin synthase (PCS)1 and PCS2 accounted for 35.5%, 28.5%, 4%, 11.3%, 18.7% and 2%, respectively of the genes identified. MTs and PCSs expressing DTY167 cells showed resistance to Cd as well as to Zn. PCS1 expressing yeast cells were also more resistant to Pb compared to those expressing MTs or PCS2. RT-PCR results showed that Cd treatment strongly induced the expression levels of MTs in the root and shoot. Furthermore, the different MTs and PCSs exhibited tissue specific expression. The results indicate that MTs and PCS genes potentially play a central role in detoxifying Cd and other toxic metals in B. rapa.

Ideal Cereals With Lower Arsenic and Cadmium by Accurately Enhancing Vacuolar Sequestration Capacity.

Cereals are a staple food for many people around the world; however, they are also a major dietary source of toxic metal(loid)s. Many agricultural regions throughout the world are contaminated with toxic metal(loid)s, which can accumulate to high levels in the grains of cereals cultivated in these regions, posing serious health risks to consumers. Arsenic (As) and cadmium (Cd) are efficiently accumulated in cereals through metal transport pathways. Therefore, there is an urgent need to develop crops that contain greatly reduced levels of toxic metal(loid)s. Vacuolar sequestration of toxic metal(loid)s is a primary strategy for reducing toxic metal(loid)s in grains. However, until recently, detailed strategies and mechanisms for reducing toxic metal(loid)s in grain were limited by the lack of experimental data. New strategies to reduce As and Cd in grain by enhancing vacuolar sequestration in specific tissues are critical to develop crops that lower the daily intake of As and Cd, potentially improving human health. This review provides insights and strategies for developing crops with strongly reduced amounts of toxic metal(loid)s without jeopardizing agronomic traits.

Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii.

In many eukaryotes, endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) via the transmembrane endoribonuclease IRE1 to maintain ER homeostasis. The ER stress response in microalgae has not been studied in detail. Here, we identified Chlamydomonas reinhardtii IRE1 (CrIRE1) and characterized two independent knock-down alleles of this gene. CrIRE1 is similar to IRE1s identified in budding yeast, plants, and humans, in terms of conserved domains, but differs in having the tandem zinc-finger domain at the C terminus. CrIRE1 was highly induced under ER stress conditions, and the expression of a chimeric protein consisting of the luminal N-terminal region of CrIRE1 fused to the cytosolic C-terminal region of yeast Ire1p rescued the yeast ∆ire1 mutant. Both allelic ire1 knock-down mutants ire1-1 and ire1-2 were much more sensitive than their parental strain CC-4533 to the ER stress inducers tunicamycin, dithiothreitol and brefeldin A. Treatment with a low concentration of tunicamycin resulted in growth arrest and cytolysis in ire1 mutants, but not in CC-4533 cells. Furthermore, in the mutants, ER stress marker gene expression was reduced, and reactive oxygen species (ROS) marker gene expression was increased. The survival of ire1 mutants treated with tunicamycin improved in the presence of the ROS scavenger glutathione, suggesting that ire1 mutants failed to maintain ROS levels under ER stress. Together, these results indicate that CrIRE1 functions as an important component of the ER stress response in Chlamydomonas, and suggest that the ER stress sensor IRE1 is highly conserved during the evolutionary history.

Engineering rice with lower grain arsenic.

Arsenic (As) is a poisonous element that causes severe skin lesions and cancer in humans. Rice (Oryza sativa L.) is a major dietary source of As in humans who consume this cereal as a staple food. We hypothesized that increasing As vacuolar sequestration would inhibit its translocation into the grain and reduce the amount of As entering the food chain. We developed transgenic rice plants expressing two different vacuolar As sequestration genes, ScYCF1 and OsABCC1, under the control of the RCc3 promoter in the root cortical and internode phloem cells, along with a bacterial γ-glutamylcysteine synthetase driven by the maize UBI promoter. The transgenic rice plants exhibited reduced root-to-shoot and internode-to-grain As translocation, resulting in a 70% reduction in As accumulation in the brown rice without jeopardizing agronomic traits. This technology could be used to reduce As intake, particularly in populations of South East Asia suffering from As toxicity and thereby improve human health.

Mesocotyl Elongation is Essential for Seedling Emergence Under Deep-Seeding Condition in Rice.

BACKGROUND: Direct-seeding cultivation by deep-seeding of seeds (drill seeding) is becoming popular due to the scarcity of land and labor. However, poor emergence and inadequate seedling establishment can lead to yield loss in direct-seeding cultivation by deep-sowing. In rice, mesocotyl and coleoptile are primarily responsible for seedling emergence from deeper levels of soil. RESULTS: Quantitative trait loci (QTLs) for mesocotyl and coleoptile length at 5-cm seeding depth were detected using 98 backcross inbred lines from a cross between Kasalath and Nipponbare. Three QTLs qMel-1, qMel-3, and qMel-6 for mesocotyl length were identified on chromosomes 1, 3, and 6, respectively, in two independent replicates. At two QTLs, qMel-1 and qMel-3, the Kasalath alleles increased mesocotyl length, whereas Nipponbare allele increased at qMel-6. The Nipponbare alleles at two QTLs (qCol-3 and qCol-5) increased the coleoptile length. Further, seeds of 54 chromosome segment substitution lines (CSSLs) from the cross between Kasalath and Nipponbare sown at 5 cm soil depth showed a significant positive correlation between seedling emergence and mesocotyl elongation (r > 0.6, P < 0.0001), but not with coleoptile elongation (r = 0.05, P = 0.7). Seedling emergence of Nipponbare, Kasalath, and the 3 of the 54 CSSLs rapidly decreased with increasing sowing depth. Seedling emergence at seeding depths of 7 and 10 cm was faster in Kasalath and CSSL-5 that harbored the Kasalath alleles across the qMel-1 and qMel-3 regions than in the other two CSSLs that contained a single QTL and Nipponbare alleles. CSSL-5 showed the longest mesocotyl among the 3 CSSLs, but no difference in coleoptile length was observed among the 3 CSSLs at seeding depths of 7 and 10 cm. CONCLUSION: Variation of mesocotyl elongation was found to be associated with seedling emergence at the seeding depth of 5 cm. To our knowledge, this is the first study performed using CSSLs to detect QTLs for mesocotyl or coleoptile elongation and to determine the effect of mesocotyl elongation on seedling emergence in rice. Our findings provides a foundation for developing rice cultivars that show higher seedling emergence after direct seeding by introgressing QTLs for mesocotyl elongation in rice breeding.

Analysis of 1-Aminocyclopropane-1-Carboxylic Acid Uptake Using a Protoplast System.

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene. The movement of ACC across the plasma membrane (PM) has been implicated in various physiological contexts during environmental adaptation and differentiation in higher plants. A PM-localized transporter in Arabidopsis thaliana, LYSINE HISTIDINE TRANSPORTER1 (LHT1) participates in the uptake of ACC, implicating a class of amino-acid transporters in the transport of ACC. Here, we describe the method for assaying uptake of ACC into the plant cells using an Arabidopsis mesophyll protoplast system.

Identification of amino acid residues important for the arsenic resistance function of Arabidopsis ABCC1.

The Arabidopsis ATP-Binding Cassette (ABC) transporter ABCC1 sequesters arsenic (As)-phytochelatin conjugates into the vacuole, thereby conferring As resistance. Here, we report the results of a screen for phosphorylation-dependent regulation sites of AtABCC1. Variants of AtABCC1 harboring mutations that replaced amino acid residues Tyr682 , Tyr709 , Tyr822 , Ser846 , Ser1278 , or Thr1408 with alanine confer reduced resistance and decrease the intracellular As content relative to wild-type AtABCC1 when heterologously expressed in the SM7 yeast strain. This suggests that these mutations compromise the vacuolar sequestration of As by AtABCC1. Furthermore, through a phosphomimic mutant study, we found that phosphorylation of Ser846 is required for the As resistance function of AtABCC1. Our analysis provides a first clue as to the phosphorylation-mediated regulation of AtABCC1 activity.

Root avoidance of toxic metals requires the GeBP-LIKE 4 transcription factor in Arabidopsis thaliana.

Plants reorganize their root architecture to avoid growth into unfavorable regions of the rhizosphere. In a screen based on chimeric repressor gene-silencing technology, we identified the Arabidopsis thaliana GeBP-LIKE 4 (GPL4) transcription factor as an inhibitor of root growth that is induced rapidly in root tips in response to cadmium (Cd). We tested the hypothesis that GPL4 functions in the root avoidance of Cd by analyzing root proliferation in split medium, in which only half of the medium contained toxic concentrations of Cd. The wild-type (WT) plants exhibited root avoidance by inhibiting root growth in the Cd side but increasing root biomass in the control side. By contrast, GPL4-suppression lines exhibited nearly comparable root growth in the Cd and control sides and accumulated more Cd in the shoots than did the WT. GPL4 suppression also altered the root avoidance of toxic concentrations of other essential metals, modulated the expression of many genes related to oxidative stress, and consistently decreased reactive oxygen species concentrations. We suggest that GPL4 inhibits the growth of roots exposed to toxic metals by modulating reactive oxygen species concentrations, thereby allowing roots to colonize noncontaminated regions of the rhizosphere.

Tilianin Inhibits MUC5AC Expression Mediated Via Down-Regulation of EGFR-MEK-ERK-Sp1 Signaling Pathway in NCI-H292 Human Airway Cells.

In the human airway, mucus exists to protect the respiratory system as a primary barrier of the innate immune system. However, hyperexpressed mucus limits airflow, resulting in a decrease of lung function. Among more than 20 mucin family members, MUC5AC and MUC5B are major glycoproteins in human airway mucus. The epidermal growth factor receptor (EGFR) signaling pathway is one of the mechanisms of these mucins expression and specificity protein-1 (Sp1) transcription factor is the downstream signal of this pathway, playing pivotal roles in mucin expression. Even though there are some drugs for treating mucus hypersecretion, no drug has proven effects on humans. We found that the flavonoid tilianin regulated MUC5AC expression and also inhibited Sp1 phosphorylation. In this study, we investigated how tilianin would modulate EGFR signaling and regulate mucin production. In conclusion, tilianin inhibited MUC5AC expression mediated via modulating the EGFRMEK-ERK-Sp1 signaling pathway in NCI-H292 human airway epithelial cells. This study may provide the basis for the novel treatment of mucus hypersecretion.

Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

Plant ABC Transporters Enable Many Unique Aspects of a Terrestrial Plant's Lifestyle.

Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us to propose that diverse ABC transporters enabled many unique and essential aspects of a terrestrial plant's lifestyle, by transporting various compounds across specific membranes of the plant.

Arsenic Uptake and Translocation in Plants.

Arsenic (As) is a highly toxic metalloid that is classified as a non-threshold class-1 carcinogen. Millions of people worldwide suffer from As toxicity due to the intake of As-contaminated drinking water and food. Reducing the As concentration in drinking water and food is thus of critical importance. Phytoremediation of soil contaminated with As and the reduction of As contamination in food depend on a detailed understanding of As uptake and transport in plants. As transporters play essential roles in As uptake, translocation and accumulation in plant cells. In this review, we summarize the current understanding of As transport in plants, with an emphasis on As uptake, mechanisms of As resistance and the long-distance translocation of As, especially the accumulation of As in grains through phloem-mediated transport.

Rice PCR1 influences grain weight and Zn accumulation in grains.

Proteins containing a placenta-specific 8 domain (PLAC8) function as major organ size regulators in Solanum lycopersicum and Zea may, and putative metal ion transporters in Arabidopsis thaliana, Oryza sativa and Brassica juncea. However, it is unknown how PLAC8 domain-containing proteins fulfill such diverse roles. Here, we found that plant cadmium resistance 1 (PCR1) influences both zinc (Zn) accumulation and grain weight in rice. OsPCR1 knockout and knockdown lines produced lighter grains than the wild type, while OsPCR1 overexpression lines produced heavier grains. Furthermore, the grains of OsPCR1 knockdown lines exhibited substantially higher Zn and lower cadmium (Cd) concentrations than the control, as did yeast heterologously expressing OsPCR1. Through sequence analysis, we showed that the amino acid sequence of japonica-type PCR1 was distinct from that of indica-type and wild rice accessions. This difference was correlated with distinct Zn-related phenotypes. Japonica-type PCR1 had a shorter N-terminus than did PCR1 in the other rice types, and yeast heterologously expressing japonica-type PCR1 was more sensitive to Zn than was yeast expressing indica-type PCR1. Furthermore, japonica-type grains accumulated less Zn than did indica-type grains. Our study suggests that rice PCR1 maintains metal ion homeostasis and grain weight and might have been selected for during domestication.

The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii.

Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels. Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources. However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive. Here, we report that the fungicide fenpropimorph rapidly causes high levels of neutral lipids to accumulate in Chlamydomonas reinhardtii cells. When treated with fenpropimorph (10 µg mL(-1)) for 1 h, Chlamydomonas cells accumulated at least fourfold the amount of triacylglycerols (TAGs) present in the untreated control cells. Furthermore, the quantity of TAGs present after 1 h of fenpropimorph treatment was over twofold higher than that formed after 9 days of nitrogen starvation in medium with no acetate supplement. Biochemical analysis of lipids revealed that the accumulated TAGs were derived mainly from chloroplast polar membrane lipids. Such a conversion of chloroplast polar lipids to TAGs is desirable for biodiesel production, because polar lipids are usually removed during the biodiesel production process. Thus, our data exemplified that a cost and time effective method of producing TAGs is possible using fenpropimorph or similar drugs.

Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana.

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana.