Sequential removal of cation/H+ exchangers reveals their additive role in elemental distribution, calcium depletion and anoxia tolerance.

Multiple Arabidopsis H+ /Cation exchangers (CAXs) participate in high-capacity transport into the vacuole. Previous studies have analysed single and double mutants that marginally reduced transport; however, assessing phenotypes caused by transport loss has proven enigmatic. Here, we generated quadruple mutants (cax1-4: qKO) that exhibited growth inhibition, an 85% reduction in tonoplast-localised H+ /Ca transport, and enhanced tolerance to anoxic conditions compared to CAX1 mutants. Leveraging inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence (SXRF), we demonstrate CAX transporters work together to regulate leaf elemental content: ICP-MS analysis showed that the elemental concentrations in leaves strongly correlated with the number of CAX mutations; SXRF imaging showed changes in element partitioning not present in single CAX mutants and qKO had a 40% reduction in calcium (Ca) abundance. Reduced endogenous Ca may promote anoxia tolerance; wild-type plants grown in Ca-limited conditions were anoxia tolerant. Sequential reduction of CAXs increased mRNA expression and protein abundance changes associated with reactive oxygen species and stress signalling pathways. Multiple CAXs participate in postanoxia recovery as their concerted removal heightened changes in postanoxia Ca signalling. This work showcases the integrated and diverse function of H+ /Cation transporters and demonstrates the ability to improve anoxia tolerance through diminishing endogenous Ca levels.

From dawn 'til dusk: daytime progression regulates primary and secondary metabolism in Cannabis glandular trichomes.

Cannabis sativa L. glandular trichomes (GTs) synthesise large amounts of secondary metabolites, predominantly cannabinoids and terpenoids. The associated demand for carbon and energy makes GTs strong sink tissues with indications that their secondary metabolism is coupled to the availability of photoassimilates. Many metabolites show diurnal patterns of flux, but it is unknown whether cannabinoids and terpenoids are regulated by time of day. We quantified cannabinoids, terpenoids and the GT proteome over a 12-hour light period in flowers of Hindu Kush, a high-tetrahydrocannabinol (THC) cultivar. Major cannabinoids changed significantly over the course of day, resulting in an increase in total measured cannabinoids. Major terpenoids also changed, with sesquiterpenes generally decreasing with day progression. While monoterpenes generally did not decrease, the second most abundant, α-pinene, increased. The GT proteome changed the most within the first six hours of the day and analysis of differentially abundant proteins indicated upregulation of primary metabolism. Surprisingly, key cannabinoid biosynthetic enzymes decreased with daytime progression despite increases in cannabinoid content, which indicate that daytime increases of photoassimilates are the main driver of cannabinoid regulation. This first reporting of variability of cannabinoid and terpenoid biosynthesis over the course of the day has implications for Cannabis research and production.

Brassicaceae Mustards: Phytochemical Constituents, Pharmacological Effects, and Mechanisms of Action against Human Disease.

The Brassicaceae genus consists of many economically important mustards of value for food and medicinal purposes, namely Asian mustard (Brassica juncea), ball mustard (Neslia paniculata), black mustard (B. nigra), garlic mustard (Alliaria petiolata), hedge mustard (Sisymbrium officinale), Asian hedge mustard (S. orientale), oilseed rape (B. napus), rapeseed (B. rapa), treacle mustard (Erysimum repandum), smooth mustard (S. erysimoides), white ball mustard (Calepina irregularis), white mustard (Sinapis alba), and Canola. Some of these are commercially cultivated as oilseeds to meet the global demand for a healthy plant-derived oil, high in polyunsaturated fats, i.e., B. napus and B. juncea. Other species are foraged from the wild where they grow on roadsides and as a weed of arable land, i.e., E. repandum and S. erysimoides, and harvested for medicinal uses. These plants contain a diverse range of bioactive natural products including sulfur-containing glucosinolates and other potentially valuable compounds, namely omega-3-fatty acids, terpenoids, phenylpropanoids, flavonoids, tannins, S-methyl cysteine sulfoxide, and trace-elements. Various parts of these plants and many of the molecules that are produced throughout the plant have been used in traditional medicines and more recently in the mainstream pharmaceutical and food industries. This study relates the uses of mustards in traditional medicines with their bioactive molecules and possible mechanisms of action and provides an overview of the current knowledge of Brassicaceae oilseeds and mustards, their phytochemicals, and their biological activities.

Comparative transcriptomic analysis of wheat cultivars differing in their resistance to Fusarium head blight infection during grain-filling stages reveals unique defense mechanisms at play.

Fusarium head blight (FHB) is a devastating fungal disease that poses a significant threat to wheat production, causing substantial yield losses. Understanding the molecular mechanisms of wheat resistance to FHB is crucial for developing effective disease management strategies. This study aimed to investigate the mechanisms of FHB resistance and the patterns of toxin accumulation in three wheat cultivars, Annong8455, Annong1589, and Sumai3, with different levels of resistance, ranging from low to high respectively, under natural field conditions. Samples were taken at three different grain-filling stages (5, 10, and 15 DPA) for gene expression analysis and phenotypic observation. Results found that toxin concentration was inversely correlated with varietal resistance but not correlated with disease phenotypes, indicating that toxin analysis is a more accurate measure of disease status in wheat ears and grains. Transcriptomic data showed that Sumai3 exhibited a stronger immune response during all stages of grain filling by upregulating genes involved in the active destruction of pathogens and removal of toxins. In contrast, Annong1589 showed a passive prevention of the spread of toxins into cells by the upregulation of genes involved in tyramine biosynthesis at the early stage (5 DPA), which may be involved in cell wall strengthening. Our study demonstrates the complexity of FHB resistance in wheat, with cultivars exhibiting unique and overlapping defense mechanisms, and highlights the importance of considering the temporal and spatial dynamics of gene expression in breeding programs for developing more resistant wheat cultivars.

Salt stress alters membrane lipid content and lipid biosynthesis pathways in the plasma membrane and tonoplast.

Plant cell membranes are the sites of sensing and initiation of rapid responses to changing environmental factors including salinity stress. Understanding the mechanisms involved in membrane remodeling is important for studying salt tolerance in plants. This task remains challenging in complex tissue due to suboptimal subcellular membrane isolation techniques. Here, we capitalized on the use of a surface charge-based separation method, free flow electrophoresis, to isolate the tonoplast (TP) and plasma membrane (PM) from leaf tissue of the halophyte ice plant (Mesembryanthemum crystallinum L.). Results demonstrated a membrane-specific lipidomic remodeling in this plant under salt conditions, including an increased proportion of bilayer forming lipid phosphatidylcholine in the TP and an increase in nonbilayer forming and negatively charged lipids (phosphatidylethanolamine and phosphatidylserine) in the PM. Quantitative proteomics showed salt-induced changes in proteins involved in fatty acid synthesis and desaturation, glycerolipid, and sterol synthesis, as well as proteins involved in lipid signaling, binding, and trafficking. These results reveal an essential plant mechanism for membrane homeostasis wherein lipidome remodeling in response to salt stress contributes to maintaining the physiological function of individual subcellular compartments.

The vacuolar H+/Ca transporter CAX1 participates in submergence and anoxia stress responses.

A plant's oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice (Oryza sativa) cultivation, and submergence tolerance of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters to this process is poorly understood. Four functional cation/proton exchangers (CAX1-4) in Arabidopsis (Arabidopsis thaliana) help regulate Ca homeostasis around the vacuole. Our results demonstrate that cax1 mutants are more tolerant to both anoxic conditions and submergence. Using phenotypic measurements, RNA-sequencing, and proteomic approaches, we identified cax1-mediated anoxia changes that phenocopy changes present in anoxia-tolerant crops: altered metabolic processes, diminished reactive oxygen species production post anoxia, and altered hormone signaling. Comparing wild-type and cax1 expressing genetically encoded Ca indicators demonstrated altered cytosolic Ca signals in cax1 during reoxygenation. Anoxia-induced Ca signals around the plant vacuole are involved in the control of numerous signaling events related to adaptation to low oxygen stress. This work suggests that cax1 anoxia response pathway could be engineered to circumvent the adverse effects of flooding that impair production agriculture.

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum.

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes' protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

Identification, characterization and epitope mapping of proteins encoded by putative allergenic napin genes from Brassica rapa.

BACKGROUND: Brassica rapeseed crops contain high concentrations of oil in the seed. The remaining meal, following oil extraction, has a high protein content, but is of low value due to the presence of high amounts of napin seed storage proteins. These 2S albumin-like proteins are difficult to digest and have been identified as major allergens in humans. OBJECTIVE: To comprehensively characterize the napin gene (NG) family in Brassica rapa and to gain an understanding of the structural basis of allergenicity of the expressed proteins. METHODS: To identify candidate napin genes in B rapa, 2S albumin-like napin genes of Arabidopsis thaliana were used as query sequences to search for similarity against the B rapa var. pekinensis Chiifu-401 v2 and the var. trilocularis R-o-18 v1.5 genomes. Multiple sequence alignment (MSA) and epitope modelling was carried out to determine structural and evolutionary relationships of NGs and their potential allergenicity. RESULTS: Four candidate napin genes in R-o-18 and ten in Chiifu-401 were identified with high sequence similarity to A thaliana napin genes. Multiple sequence alignment revealed strong conservation among the candidate genes. An epitope survey indicated high conservation of allergenic epitope motifs with known 2S albumin-like allergens. CONCLUSION: Napin is thought to be responsible for a  high prevalence of food allergies. Characterization of the napin gene family in B rapa will give important insight into the protein structure, and epitope modelling will help to advance studies into allergenicity including the development of precise diagnostic screenings and therapies for this potential food allergy as well as the possible manipulation of napin levels in the seed by gene editing technology.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

Impact of imidacloprid on the nutritional quality of adult black tiger shrimp (Penaeus monodon).

Neonicotinoid insecticides, including imidacloprid, are increasingly being used to control insect pests in agricultural and urban areas, and are often detected in aquatic environments. The effects of neonicotinoids on non-target insects have been investigated with respect to behavioural, biochemical, physiological and population-level responses, but information of their effects on crustaceans is limited. This study investigated the adverse effects of both acute and chronic exposure to sublethal concentrations of imidacloprid on the nutritional quality of adult Black Tiger Shrimp (Penaeus monodon). Shrimp were continually exposed to imidacloprid in water (5 µg L-1 and 30 µg L-1), or through their food (12.5 µg g-1 and 75 µg g-1), for the entire exposure period. Imidacloprid concentrations in water and residues in tissues were quantified using liquid chromatography-mass spectrometry after solid-phase extraction and QuEChER extraction respectively. Within 4 days, shrimp accumulated imidacloprid at up to 0.350 µg imidacloprid per g body weight from water and food exposure. Chronic exposure resulted in a significant decrease in body weight and total lipid content. Fatty acid composition in exposed shrimp was modified relative to controls. Overall, these results demonstrate that neonicotinoid exposure could lead to nutritional deficiency, which has implications for the productivity and food quality of shrimp.

Bridging the food security gap: an information-led approach to connect dietary nutrition, food composition and crop production.

BACKGROUND: Food security is recognized as a major global challenge, yet human food-chain systems are inherently not geared towards nutrition, with decisions on crop and cultivar choice not informed by dietary composition. Currently, food compositional tables and databases (FCT/FCDB) are the primary information sources for decisions relating to dietary intake. However, these only present single mean values representing major components. Establishment of a systematic controlled vocabulary to fill this gap requires representation of a more complex set of semantic relationships between terms used to describe nutritional composition and dietary function. RESULTS: We carried out a survey of 11 FCT/FCDB and 177 peer-reviewed papers describing variation in nutritional composition and dietary function for food crops to identify a comprehensive set of terms to construct a controlled vocabulary. We used this information to generate a Crop Dietary Nutrition Data Framework (CDN-DF), which incorporates controlled vocabularies systematically organized into major classes representing nutritional components and dietary functions. We demonstrate the value of the CDN-DF for comparison of equivalent components between crop species or cultivars, for identifying data gaps and potential for formal meta-analysis. The CDN-DF also enabled us to explore relationships between nutritional components and the functional attributes of food. CONCLUSION: We have generated a structured crop dietary nutrition data framework, which is generally applicable to the collation and comparison of data relevant to crop researchers, breeders, and other stakeholders, and will facilitate dialogue with nutritionists. It is currently guiding the establishment of a more robust formal ontology. © 2019 Society of Chemical Industry.

Making Epidermal Bladder Cells Bigger: Developmental- and Salinity-Induced Endopolyploidy in a Model Halophyte.

Endopolyploidy occurs when DNA replication takes place without subsequent mitotic nuclear division, resulting in cell-specific ploidy levels within tissues. In plants, endopolyploidy plays an important role in sustaining growth and development, but only a few studies have demonstrated a role in abiotic stress response. In this study, we investigated the function of ploidy level and nuclear and cell size in leaf expansion throughout development and tracked cell type-specific ploidy in the halophyte Mesembryanthemum crystallinum In addition to developmental endopolyploidy, we examined the effects of salinity stress on ploidy level. We focused specifically on epidermal bladder cells (EBC), which are modified balloon-like trichomes, due to their large size and role in salt accumulation. Our results demonstrate that ploidy increases as the leaves expand in a similar manner for each leaf type, and ploidy levels up to 512C were recorded for nuclei in EBC of leaves of adult plants. Salt treatment led to a significant increase in ploidy levels in the EBC, and these cells showed spatially related differences in their ploidy and nuclear and cell size depending on the positions on the leaf and stem surface. Transcriptome analysis highlighted salinity-induced changes in genes involved in DNA replication, cell cycle, endoreduplication, and trichome development in EBC. The increase in cell size and ploidy observed in M. crystallinum under salinity stress may contribute to salt tolerance by increasing the storage capacity for sodium sequestration brought about by higher metabolic activity driving rapid cell enlargement in the leaf tissue and EBC.

Remobilization and fate of sulphur in mustard.

BACKGROUND AND AIMS: Sulphur (S) is an essential macronutrient involved in numerous metabolic pathways required for plant growth. Crops of the plant family Brassicaceae require more S compared with other crops for optimum growth and yield, with most S ultimately sequestered in the mature seeds as the storage proteins cruciferin and napin, along with the unique S-rich secondary metabolite glucosinolate (GSL). It is well established that S assimilation primarily takes place in the shoots rather than roots, and that sulphate is the major form in which S is transported and stored in plants. We carried out a developmental S audit to establish the net fluxes of S in two lines of Brassica juncea mustard where seed GSL content differed but resulted in no yield penalty. METHODS: We quantified S pools (sulphate, GSL and total S) in different organs at multiple growth stages until maturity, which also allowed us to test the hypothesis that leaf S, accumulated as a primary S sink, becomes remobilized as a secondary source to meet the requirements of GSL as the dominant seed S sink. KEY RESULTS: Maximum plant sulphate accumulation had occurred by floral initiation in both lines, at which time most of the sulphate was found in the leaves, confirming its role as the primary S sink. Up to 52 % of total sulphate accumulated by the low-GSL plants was lost through senesced leaves. In contrast, S from senescing leaves of the high-GSL line was remobilized to other tissues, with GSL accumulating in the seed from commencement of silique filling until maturity. CONCLUSION: We have established that leaf S compounds that accumulated as primary S sinks at early developmental stages in condiment type B. juncea become remobilized as a secondary S source to meet the demand for GSL as the dominant seed S sink at maturity.

Membrane Lipid Remodeling in Response to Salinity.

Salinity is one of the most decisive environmental factors threatening the productivity of crop plants. Understanding the mechanisms of plant salt tolerance is critical to be able to maintain or improve crop yield under these adverse environmental conditions. Plant membranes act as biological barriers, protecting the contents of cells and organelles from biotic and abiotic stress, including salt stress. Alterations in membrane lipids in response to salinity have been observed in a number of plant species including both halophytes and glycophytes. Changes in membrane lipids can directly affect the properties of membrane proteins and activity of signaling molecules, adjusting the fluidity and permeability of membranes, and activating signal transduction pathways. In this review, we compile evidence on the salt stress responses of the major membrane lipids from different plant tissues, varieties, and species. The role of membrane lipids as signaling molecules in response to salinity is also discussed. Advances in mass spectrometry (MS)-based techniques have largely expanded our knowledge of salt-induced changes in lipids, however only a handful studies have investigated the underlying mechanisms of membrane lipidome regulation. This review provides a comprehensive overview of the recent works that have been carried out on lipid remodeling of plant membranes under salt treatment. Challenges and future perspectives in understanding the mechanisms of salt-induced changes to lipid metabolisms are proposed.

Membrane Proteomic Insights into the Physiology and Taxonomy of an Oleaginous Green Microalga.

Ettlia oleoabundans is a nonsequenced oleaginous green microalga. Despite the significant biotechnological interest in producing value-added compounds from the acyl lipids of this microalga, a basic understanding of the physiology and biochemistry of oleaginous microalgae is lacking, especially under nitrogen deprivation conditions known to trigger lipid accumulation. Using an RNA sequencing-based proteomics approach together with manual annotation, we are able to provide, to our knowledge, the first membrane proteome of an oleaginous microalga. This approach allowed the identification of novel proteins in E. oleoabundans, including two photoprotection-related proteins, Photosystem II Subunit S and Maintenance of Photosystem II under High Light1, which were considered exclusive to higher photosynthetic organisms, as well as Retinitis Pigmentosa Type 2-Clathrin Light Chain, a membrane protein with a novel domain architecture. Free-flow zonal electrophoresis of microalgal membranes coupled to liquid chromatography-tandem mass spectrometry proved to be a useful technique for determining the intracellular location of proteins of interest. Carbon-flow compartmentalization in E. oleoabundans was modeled using this information. Molecular phylogenetic analyses of protein markers and 18S ribosomal DNA support the reclassification of E. oleoabundans within the trebouxiophycean microalgae, rather than with the Chlorophyceae class, in which it is currently classified, indicating that it may not be closely related to the model green alga Chlamydomonas reinhardtii A detailed survey of biological processes taking place in the membranes of nitrogen-deprived E. oleoabundans, including lipid metabolism, provides insights into the basic biology of this nonmodel organism.

Single cell-type analysis of cellular lipid remodelling in response to salinity in the epidermal bladder cells of the model halophyte Mesembryanthemum crystallinum.

Salt stress causes dramatic changes in the organization and dynamic properties of membranes, however, little is known about the underlying mechanisms involved. Modified trichomes, known as epidermal bladder cells (EBC), on the leaves and stems of the halophyte Mesembryanthemum crystallinum can be successfully exploited as a single-cell-type system to investigate salt-induced changes to cellular lipid composition. In this study, alterations in key molecular species from different lipid classes highlighted an increase in phospholipid species, particularly those from phosphatidylcholine and phosphatidic acid (PA), where the latter is central to the synthesis of membrane lipids. Triacylglycerol (TG) species decreased during salinity, while there was little change in plastidic galactolipids. EBC transcriptomic and proteomic data mining revealed changes in genes and proteins involved in lipid metabolism and the upregulation of transcripts for PIPKIB, PI5PII, PIPKIII, and phospholipase D delta suggested the induction of signalling processes mediated by phosphoinositides and PA. TEM and flow cytometry showed the dynamic nature of lipid droplets in these cells under salt stress. Altogether, this work indicates that the metabolism of TG might play an important role in EBC response to salinity as either an energy reserve for sodium accumulation and/or driving membrane biosynthesis for EBC expansion.

Brassicaceae Mustards: Traditional and Agronomic Uses in Australia and New Zealand.

Commonly cultivated Brassicaceae mustards, namely garlic mustard (Alliaria petiolata), white mustard (Brassica alba), Ethiopian mustard (B. carinata), Asian mustard (B. juncea), oilseed rape (B. napus), black mustard (B. nigra), rapeseed (B. rapa), white ball mustard (Calepina irregularis), ball mustard (Neslia paniculata), treacle mustard (Erysimum repandum), hedge mustard (Sisymbrium officinale), Asian hedge mustard (S. orientale), smooth mustard (S. erysimoides) and canola are the major economically important oilseed crops in many countries. Mustards were naturalized to Australia and New Zealand and Australia is currently the second largest exporter of Brassicaceae oilseeds to meet the global demand for a healthy plant-derived oil, high in polyunsaturated fats. Apart from providing edible oil, various parts of these plants and many of their phytochemicals have been used traditionally for both agronomic as well as medicinal purposes, with evidence of their use by early Australian and New Zealand settlers and also the indigenous population. This review provides an overview of the current knowledge of traditional and agronomic uses of Brassicaceae oilseeds and mustards with a focus on their importance in Australia and New Zealand.

An alternative mode of CD43 signal transduction activates pro-survival pathways of T lymphocytes.

CD43 is one of the most abundant co-stimulatory molecules on a T-cell surface; it transduces activation signals through its cytoplasmic domain, contributing to modulation of the outcome of T-cell responses. The aim of this study was to uncover new signalling pathways regulated by this sialomucin. Analysis of changes in protein abundance allowed us to identify pyruvate kinase isozyme M2 (PKM2), an enzyme of the glycolytic pathway, as an element potentially participating in the signalling cascade resulting from the engagement of CD43 and the T-cell receptor (TCR). We found that the glycolytic activity of this enzyme was not significantly increased in response to TCR+CD43 co-stimulation, but that PKM2 was tyrosine phosphorylated, suggesting that it was performing moonlight functions. We report that phosphorylation of both Y105 of PKM2 and of Y705 of signal transducer and activator of transcription 3 was induced in response to TCR+CD43 co-stimulation, resulting in activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway. ERK5 and the cAMP response element binding protein (CREB) were activated, and c-Myc and nuclear factor-κB (p65) nuclear localization, as well as Bad phosphorylation, were augmented. Consistent with this, expression of human CD43 in a murine T-cell hybridoma favoured cell survival. Altogether, our data highlight novel signalling pathways for the CD43 molecule in T lymphocytes, and underscore a role for CD43 in promoting cell survival through non-glycolytic functions of metabolic enzymes.

Cadmium and zinc activate adaptive mechanisms in Nicotiana tabacum similar to those observed in metal tolerant plants.

MAIN CONCLUSION: Tobacco germinated and grew in the presence of high concentrations of cadmium and zinc without toxic symptoms. Evidence suggests that these ions are sequestered into the vacuole by heavy metal/H + exchanger mechanisms. Heavy metal hyperaccumulation and hypertolerance are traits shared by a small set of plants which show specialized physiological and molecular adaptations allowing them to accumulate and sequester toxic metal ions. Nicotiana tabacum was used to test its potential as a metal-accumulator in a glass house experiment. Seed germination was not affected in the presence of increasing concentrations of zinc and cadmium. Juvenile and adult plants could concentrate CdCl2 and ZnSO4 to levels exceeding those in the hydroponic growth medium and maintained or increased their leaf dry weight when treated with 0.5- or 1-mM CdCl2 or 1-mM ZnSO4 for 5 days. Accumulation of heavy metals did not affect the chlorophyll and carotenoid levels, while variable effects were observed in cell sap osmolarity. Heavy metal-dependent H+ transport across the vacuole membrane was monitored using quinacrine fluorescence quenching. Cadmium- or zinc-dependent fluorescence recovery revealed that increasing concentrations of heavy metals stimulated the activities of the tonoplast Cd2+ or Zn2+/H+ exchangers. Immunodetection of the V-ATPase subunits showed that the increased proton transport by zinc was not due to changes in protein amount. MTP1 and MTP4 immunodetection and semiquantitative RT-PCR of NtMTP1, NtNRAMP1, and NtZIP1 helped to identify the genes that are likely involved in sequestration of cadmium and zinc in the leaf and root tissue. Finally, we demonstrated that cadmium and zinc treatments induced an accumulation of zinc in leaf tissues. This study shows that N. tabacum possesses a hyperaccumulation response, and thus could be used for phytoremediation purposes.

Genome and transcriptome sequencing characterises the gene space of Macadamia integrifolia (Proteaceae).

BACKGROUND: The large Gondwanan plant family Proteaceae is an early-diverging eudicot lineage renowned for its morphological, taxonomic and ecological diversity. Macadamia is the most economically important Proteaceae crop and represents an ancient rainforest-restricted lineage. The family is a focus for studies of adaptive radiation due to remarkable species diversification in Mediterranean-climate biodiversity hotspots, and numerous evolutionary transitions between biomes. Despite a long history of research, comparative analyses in the Proteaceae and macadamia breeding programs are restricted by a paucity of genetic information. To address this, we sequenced the genome and transcriptome of the widely grown Macadamia integrifolia cultivar 741. RESULTS: Over 95 gigabases of DNA and RNA-seq sequence data were de novo assembled and annotated. The draft assembly has a total length of 518 Mb and spans approximately 79% of the estimated genome size. Following annotation, 35,337 protein-coding genes were predicted of which over 90% were expressed in at least one of the leaf, shoot or flower tissues examined. Gene family comparisons with five other eudicot species revealed 13,689 clusters containing macadamia genes and 1005 macadamia-specific clusters, and provides evidence for linage-specific expansion of gene families involved in pathogen recognition, plant defense and monoterpene synthesis. Cyanogenesis is an important defense strategy in the Proteaceae, and a detailed analysis of macadamia gene homologues potentially involved in cyanogenic glycoside biosynthesis revealed several highly expressed candidate genes. CONCLUSIONS: The gene space of macadamia provides a foundation for comparative genomics, gene discovery and the acceleration of molecular-assisted breeding. This study presents the first available genomic resources for the large basal eudicot family Proteaceae, access to most macadamia genes and opportunities to uncover the genetic basis of traits of importance for adaptation and crop improvement.