The fate of duplicated genes in a polyploid plant genome.

Polyploidy is generally not tolerated in animals, but is widespread in plant genomes and may result in extensive genetic redundancy. The fate of duplicated genes is poorly understood, both functionally and evolutionarily. Soybean (Glycine max L.) has undergone two separate polyploidy events (13 and 59 million years ago) that have resulted in 75% of its genes being present in multiple copies. It therefore constitutes a good model to study the impact of whole-genome duplication on gene expression. Using RNA-seq, we tested the functional fate of a set of approximately 18 000 duplicated genes. Across seven tissues tested, approximately 50% of paralogs were differentially expressed and thus had undergone expression sub-functionalization. Based on gene ontology and expression data, our analysis also revealed that only a small proportion of the duplicated genes have been neo-functionalized or non-functionalized. In addition, duplicated genes were often found in collinear blocks, and several blocks of duplicated genes were co-regulated, suggesting some type of epigenetic or positional regulation. We also found that transcription factors and ribosomal protein genes were differentially expressed in many tissues, suggesting that the main consequence of polyploidy in soybean may be at the regulatory level.

Growth response of Pinus densiflora seedlings inoculated with three indigenous ectomycorrhizal fungi in combination

Pinus densiflora seedlings were inoculated with three indigenous ectomycorrhizal fungi (Cenococcum geophilum, Rhizopogon roseolus and Russula densifolia) in single-, two-, and three-species treatments. After 8 months, the colonization rates of each ectomycorrhizal species, seedling growth and the nutrition were assessed in each treatment. P. densiflora seedlings inoculated with different ECM species composition showed an increase in height and basal diameter and improved seedling root and shoot nutrition concentrations compared to control treatment. Generally, combined inoculation had a more positive influence on the seedlings than the single inoculation. The three-species inoculation presented the highest growth and basal diameter and concentration of most nutrients except potassium. In conclusion, the results provided strong evidence for benefits of combined inoculation with the indigenous ectomycorrhizal fungi on P. densiflora seedlings under controlled conditions.

Preferential blockade of dioxin-induced activation of the aryl hydrocarbon receptor by Antrodia camphorata.

Halogenated and polycyclic aromatic hydrocarbons are widely distributed pollutants in environments. These toxic substances activate the aryl hydrocarbon receptor (AhR) and thereby cause a broad spectrum of pathological changes. Development of AhR inhibitors will be useful for prevention of diseases caused by AhR activation. Using the dioxin responsive element (DRE)-based sensing via secreted alkaline phosphatase (DRESSA), we examined effects of Antrodia camphorata, a mycerial extract, on the activation of AhR by halogenated and polycyclic aromatic hydrocarbons. We found that Antrodia camphorata markedly suppressed activation of AhR triggered by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, activation of AhR by polycyclic aromatic hydrocarbons (benzo[a]pyrene and 3-methylcholanthrene) was inhibited only modestly by this mycelium. Similarly, Antrodia camphorata only mildly attenuated activation of AhR by cigarette smoke that contains polycyclic aromatic hydrocarbons. Consistent with these results, Northern blot analysis revealed that DRE-driven exogenous and endogenous gene expression triggered by TCDD was abolished by Antrodia camphorata, whereas it did not substantially affect DRE-induced transcription triggered by benzo[a]pyrene, 3-methylcholanthrene or cigarette smoke. We also found that the inhibitory effect of Antrodia camphorata on TCDD-induced AhR activation was ascribed to neither down-regulation of AhR, down-regulation of the AhR nuclear translocator, nor up-regulation of the AhR repressor. These results suggest that Antrodia camphorata preferentially inhibits AhR activation and DRE-dependent gene expression triggered by dioxin.

Induction of callus from a metal hypertolerant fern, Athyrium yokoscense, and evaluation of its cadmium tolerance and accumulation capacity.

The callus of a metal hypertolerant fern, Athyrium yokoscense, was induced from the spores generated on a small sectioned frond in vitro. The callus grew vigorously with the periodical medium change, especially in a liquid culture. When the callus and regenerated tissues were exposed to Cd, every tissue tolerated at least 1 mM Cd for >1 month. These tissues accumulated high levels of Cd (maximum 3.3 mg g(-1) dry weight in roots) in accordance with the Cd concentration of the medium, and the Cd concentrations of all parts, except roots, were at a similar level. The data suggest that the Cd tolerance of this fern is basically independent of the plant parts and the developmental stages, although the accumulation ability is higher in roots than in the other plant parts.

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments.

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses. Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK), phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms. In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid). Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions. In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.

Iron transport and signaling in plants.

Cellular and whole organism iron homeostasis must be balanced to supply enough iron for metabolism and to avoid excessive, toxic levels. To perform iron uptake from the environment, iron distribution to various organs and tissues, and iron intracellular compartmentalization, various membranes must be crossed by this metal. The uptake and transport of iron under physiological conditions require particular processes such as chelation or reduction because ferric iron has a very low solubility. The molecular actors involved in iron acquisition from the soil have recently been characterized. A few candidates belonging to various gene families are hypothesized to play major roles in iron distribution throughout the plant. All these transport activities are tightly regulated at transcriptional and posttranslational levels, according to the iron status of the plant. These coordinated regulations result from an integration of local and long-distance transduction pathways.

Communication between animal cells and the plant foods they ingest: phyto-zooidal dependencies and signaling (Review).

The beneficial effect of plant foods on human health is unmistakable. Time and time again, studies have found foods of plant origin to reduce the risk of most major chronic illnesses suffered by the human population. Possible mechanisms for the preventative effects of these foods are discussed. Each of the plant groups reviewed was found to reduce the risk of one or more of the following: cardiovascular disease, cancer (lung, breast, colon, rectal, prostate, epithelial, stomach, esophageal, oral, pharynx, larynx, urinary tract, endometrium, pancreas, thyroid, liver, ovary, gallbladder, bladder, and kidney), diabetes, hypertension, bone degeneration, diverticulitis, constipation, gallstones, age-related blindness. Almost no evidence was found to suggest a negative effect on health due to consumption of these plant foods. Based on this material and a review of conserved animal signaling molecules we surmise that animals require these chemicals to enhance specific mammalian cellular processes, demonstrating phyto-zooidal signaling. Further, this diet dependency coupling between plants and animals probably evolved because of the abundance of a particular plant material in a local environment, which is now broken because of technological advances. In conclusion, the overwhelming majority of evidence shows that people may significantly decrease their risks of the aforementioned diseases by increasing their intake of these foods since they represent a natural method to enhance animal processes and signaling.

Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch.

The phototropins are flavoprotein kinases that control phototropic bending, light-induced chloroplast movement, and stomatal opening in plants. Two flavin mononucleotide binding light, oxygen, or voltage (LOV) domains are the sites for initial photochemistry in these blue light photoreceptors. We have determined the steady state, photoexcited crystal structure of a flavin-bound LOV domain. The structure reveals a unique photochemical switch in the flavin binding pocket in which the absorption of light drives the formation of a reversible covalent bond between a highly conserved Cys residue and the flavin cofactor. This provides a molecular picture of a cysteinyl-flavin covalent adduct, the presumed signaling species that leads to phototropin kinase activation and subsequent signal transduction. We identify closely related LOV domains in two eubacterial proteins that suggests the light-induced conformational change evident in this structure is an ancient biomolecular response to light, arising before the appearance of plants.

Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem.

The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H(+)-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-beta-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is beta-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.

Role of DIVARICATA in the control of dorsoventral asymmetry in Antirrhinum flowers.

Dorsoventral asymmetry of the Antirrhinum corolla depends on expression of the CYC and DICH genes in dorsal petals. One role of these genes is to inhibit DIVARICATA (DIV), a determinant of ventral identity. Therefore, in cyc;dich double mutants ventral identity spreads all around the flower. We show that DIV encodes a protein belonging to the MYB family of transcription factors. Early on in corolla development, DIV affects specifically the growth of ventral and lateral petals but is transcribed in all petals. Analysis of a closely related gene suggests that the lack of effect on dorsal petals is not due to redundancy. More likely, therefore, DIV is regulated posttranscriptionally through a mechanism that depends on CYC and DICH. Later on, DIV affects growth and cell types and is transcribed mostly in a single layer of cells of ventral and lateral petals. This late pattern may itself depend on DIV activity because it fails to be established in a transcribed but inactive div mutant and, conversely, spreads all around the flower in cyc;dich double mutants.

Engineering for drought avoidance: expression of maize NADP-malic enzyme in tobacco results in altered stomatal function.

Water is a principal limitation to agricultural production during drought and in arid regions of the world. Mechanisms that plants use to cope with drought can be grouped into two different strategies: drought tolerance and drought avoidance. Previous efforts toward engineering plants for improved performance during drought have focused on drought tolerance, the ability to adjust to dry conditions. This report addresses the engineering of a drought-avoidance phenotype, which allows for the conservation of water during plant growth. The majority of water lost from plants occurs through stomata. When stomata are open, potassium, chloride and/or malate are present at high concentrations in guard cells. The accumulation of large numbers of ions during stomatal opening increases the turgor pressure of the guard cells, which results in increased pore size. Expression of a single gene from maize, NADP-malic enzyme (ME), which converts malate and NADP to pyruvate, NADPH, and CO(2), resulted in altered stomatal behaviour and water relations in tobacco. The ME-transformed plants had decreased stomatal conductance and gained more fresh mass per unit water consumed than did the wild type, but they were similar to the wild type in their growth and rate of development. Providing chloride via the transpiration stream partially reversed the effects of ME expression on stomatal aperture size, which is consistent with the interpretation that expression of ME altered malate metabolism in guard cells. These results suggest a role for malic enzyme in the mechanism of stomatal closure, as well as a potential mechanism for genetically altering plant water use.

Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus.

Restriction of long-distance movement of tobacco etch virus (TEV) in Arabidopsis ecotype Col-0 plants requires the function of at least three genes: RTM1 (restricted TEV movement 1), RTM2, and RTM3. The mechanism of TEV movement restriction remains poorly understood, although it does not involve a hypersensitive response or systemic acquired resistance. A functional characterization of RTM1 and RTM2 was done. The RTM1 protein was found to be soluble with the potential to form self-interacting complexes. The regulatory regions of both the RTM1 and RTM2 genes were analyzed using reporter constructs. The regulatory sequences from both genes directed expression of beta-glucuronidase exclusively in phloem-associated cells. Translational fusion proteins containing the green fluorescent protein and RTM1 or RTM2 localized to sieve elements when expressed from their native regulatory sequences. Thus, components of the RTM system may function within phloem, and sieve elements in particular, to restrict TEV long-distance movement.

Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis.

The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis.

From milliseconds to millions of years: guard cells and environmental responses.

During the past year, significant advances have been made in our understanding of stomatal development and its response to climate change, and in our knowledge of how guard cell Ca(2+) oscillations encode environmental signals. Recent studies on (de)phosphorylation mechanisms have provided new information on how guard cells respond to abscisic acid and blue light.

Direct ligand-receptor complex interaction controls Brassica self-incompatibility.

Many higher plants have evolved self-incompatibility mechanisms to prevent self-fertilization. In Brassica self-incompatibility, recognition between pollen and the stigma is controlled by the S locus, which contains three highly polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also called S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma, and SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SP11 is localized in the pollen coat. It is thought that, during self-pollination, SP11 is secreted from the pollen coat and interacts with its cognate SRK in the papilla cell of the stigma to elicit the self-incompatibility response. SLG is a secreted stigma protein that is highly homologous to the SRK extracellular domain. Although it is not required for S-haplotype specificity of the stigma, SLG enhances the self-incompatibility response; however, how this is accomplished remains controversial. Here we show that a single form of SP11 of the S8 haplotype (S8-SP11) stabilized with four intramolecular disulphide bonds specifically binds the stigma membrane of the S8 haplotype to induce autophosphorylation of SRK8, and that SRK8 and SLG8 together form a high-affinity receptor complex for S8-SP11 on the stigma membrane.

The cytoskeleton facilitates a three-dimensional symplasmic continuum in the long-lived ray and axial parenchyma cells of angiosperm trees.

The microtubule (MT), microfilament (MF) and myosin components of the cytoskeleton were studied in the long-lived ray and axial parenchyma cells of the secondary xylem (wood) and secondary phloem of two angiosperm trees, Aesculus hippocastanum L. (horse-chestnut) and Populus tremula L. x P. tremuloides Michx. (hybrid aspen), using indirect immunofluorescence localisation and transmission electron microscopy. MTs and MFs were bundled and oriented axially (parallel to the cell's long axis) within all parenchyma cell types after they had fully differentiated. Additionally, actin and myosin were immunolocalised at the thin-walled membranes of the pits, which linked cells in neighbouring files of both ray and axial parenchyma, and at the pits between axial and ray parenchyma cells themselves. Anti-callose antibody immunolocated the plasmodesmata at the pit membranes, and in the same pattern as that of anti-myosin. Ray cells are important symplasmic pathways between the xylem and the phloem throughout the life of trees. We hypothesise that the MT and MF components of the cytoskeleton in the ray and axial parenchyma cells are involved in the transport of materials within those cells, and, in association with the acto-myosin of plasmodesmata at pit fields, are also important in intercellular transport. Thus, the symplasmic coupling between ray cells, between axial parenchyma cells, and between axial parenchyma and ray cells represents an extensive three-dimensional communication pathway permeating the tree from the phloem through the cambium into the wood. We suggest that this cytoskeletal pathway has an important role in delivery of photosynthate, and mobilised reserves, to the actively dividing cambium, and in the movement of materials to sites of reserve deposition, principally within the wood. This pathway could also have an important role in co-ordinating developmental processes throughout the tree.

Hydrogel control of xylem hydraulic resistance in plants.

Increasing concentrations of ions flowing through the xylem of plants produce rapid, substantial, and reversible decreases in hydraulic resistance. Changes in hydraulic resistance in response to solution ion concentration, pH, and nonpolar solvents are consistent with this process being mediated by hydrogels. The effect is localized to intervessel bordered pits, suggesting that microchannels in the pit membranes are altered by the swelling and deswelling of pectins, which are known hydrogels. The existence of an ion-mediated response breaks the long-held paradigm of the xylem as a system of inert pipes and suggests a mechanism by which plants may regulate their internal flow regime.

Regulation of ethylene biosynthesis in response to pollination in tomato flowers.

Pollination of many flowers leads to an increase in ethylene synthesis and flower senescence. We have investigated the regulation of pollination-induced ethylene synthesis in tomato (Lycopersicon esculentum) using flowers of the dialytic (dl) mutant, in which pollination can be manipulated experimentally, with the aim of developing a model system to study tomato flower senescence. Ethylene synthesis increased rapidly in dl pistils following pollination, leading to accelerated petal senescence, and was delayed in ethylene-insensitive Never-ripe (Nr) pistils. However, Nr pistils eventually produced more ethylene than dl pistils, suggesting the presence of negative feedback regulation of ethylene synthesis following pollination. LEACS1A expression correlated well with increased ethylene production in pollinated dl pistils, and expression in Nr revealed that regulation is via an ethylene-independent mechanism. In contrast, the induction of the 1-aminocyclopropane-1-carboxylic acid oxidases, LEACO1 and LEACO3, following pollination is ethylene dependent. In addition, the expression profiles of ACS and ACO genes were determined during petal senescence and a hypothesis proposed that translocated 1-aminocyclopropane-1-carboxylic acid from the pistil may be important for regulating the initial burst of ethylene production during petal senescence. These results are discussed and differences between tomato and the ornamental species previously studied are highlighted.

Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda.

Nicotiana repanda Wildenow ex Lehmann acylates nornicotine in its trichomes to produce N-acyl-nornicotine (NacNN) alkaloids which are dramatically more toxic than nicotine is to the nicotine-adapted herbivore, Manduca sexta. These NacNNs, like nicotine, were induced by methyl jasmonate (MeJA) and wounding, but the 2-fold increase in NacNN pools was much faster (within 6 h) than the MeJA-induced increase in nornicotine pools (24 h to 4 d), its parent substrate. When 15NO(-)3 pulse-chase experiments with intact and induced plants were used to follow the incorporation of 15N into alkaloids in different plant parts over the plant's lifetime, it was found that the root nicotine pool was most rapidly labeled, followed by the shoot nornicotine and NacNN pools. After 3 d, 3.12% of 15N acquired was in nicotine (0.93%), nornicotine (0.32%) and NacNNs (1.73%) while only 0.14% was in anabasine. Once NacNNs are externalized to the leaf surface, they are not readily re-distributed within the plant and are lost with senescing leaves. The wound- and MeJA-induced N-acylation of nornicotine is independent of induced changes in nornicotine pools and the rapidity of the response suggests its importance in defense against herbivores.