RESUMO
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.
Assuntos
Mesembryanthemum/citologia , Mesembryanthemum/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Poliploidia , Plantas Tolerantes a Sal/citologia , Tamanho Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Germinação , Mesembryanthemum/fisiologia , Células Vegetais , Folhas de Planta/citologia , Raízes de Plantas/genética , Salinidade , Estresse Salino/genética , Estresse Salino/fisiologia , Plantas Tolerantes a Sal/crescimento & desenvolvimento , Plantas Tolerantes a Sal/fisiologiaRESUMO
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.
Assuntos
Metabolismo dos Lipídeos , Mesembryanthemum/metabolismo , Epiderme Vegetal/citologia , Plantas Tolerantes a Sal/metabolismo , Lipídeos de Membrana/metabolismo , Mesembryanthemum/citologia , Ácidos Fosfatídicos/metabolismo , Fosfatidilcolinas/metabolismo , Epiderme Vegetal/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Caules de Planta/citologia , Caules de Planta/metabolismo , Estresse Salino , Plantas Tolerantes a Sal/citologia , Sódio/metabolismo , Triglicerídeos/metabolismoRESUMO
Mesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation. We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants. The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineage-specific genes of unknown function, in response to salt treatment. The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell type-specific resolution in understanding stress adaptive mechanisms.
Assuntos
Mesembryanthemum/citologia , Mesembryanthemum/genética , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Salinidade , Transcriptoma/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Redes Reguladoras de Genes/efeitos dos fármacos , Mesembryanthemum/efeitos dos fármacos , Anotação de Sequência Molecular , Epiderme Vegetal/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2 (â¢-)) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3'-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2 (â¢-) was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2 (â¢-) is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth.
Assuntos
Mesembryanthemum/enzimologia , NADPH Oxidases/metabolismo , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rizoma/enzimologia , Células Cultivadas , Mesembryanthemum/citologia , Mesembryanthemum/crescimento & desenvolvimento , Estresse Oxidativo , Transporte Proteico , Rizoma/crescimento & desenvolvimento , Rizoma/ultraestruturaRESUMO
In plant Mesembryanthemum crystallinum, which has the inducible crassulacean acid metabolism (CAM), isoforms of plastidic phosphate translocators (pPTs) are categorized into three subfamilies: the triose phosphate/phosphate translocator (McTPT1), the phosphoenolpyruvate/phosphate translocator (McPPT1), and the glucose 6-phosphate/phosphate translocator (McGPT1 and McGPT2). In order to elucidate the physiological roles of these pPTs in M. crystallinum, we determined the substrate specificity of each pPT isoform. The substrate specificities of McTPT1, McPPT1, and McGPT1 showed overall similarities to those of orthologs that have been characterized. In contrast, for glucose 6-phosphate, McGPT2 showed higher selectivity than McGPT1 and other GPT orthologs. Because the expression of McGTP2 is specific to CAM while that of McGTP1 is constitutively expressed in both the C3- and the CAM-state in M. crystallinum, we propose that McGPT2 functions as a CAM system-specific GPT in this plant.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mesembryanthemum/citologia , Mesembryanthemum/metabolismo , Fosfatos/metabolismo , Fotossíntese , Plastídeos/metabolismo , Cinética , Filogenia , Especificidade por SubstratoRESUMO
Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+)-transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism.
Assuntos
Mesembryanthemum/citologia , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Plantas Tolerantes a Sal/citologia , Mesembryanthemum/química , Mesembryanthemum/metabolismo , Proteômica , Plantas Tolerantes a Sal/metabolismo , Espectrometria de Massas em TandemRESUMO
The effect of free cadaverine (Cad) on its conjugates formation was analyzed in roots of the common ice plants (Mesembryanthemum crystallinum L.). It was found for the first time that Cad could induce oxidative burst in the roots of adult plants, as was evident from the sharp decrease in the content of Cad soluble or insoluble conjugates. This unusual effect was associated with the increased oxidative degradation of exogenous Cad (1mM, 1.5h) and intense H(2)O(2) production in the root cells of adult plants. Root treatment of both juvenile and adult plants with H(2)O(2) (1mM, 1.5h) reduced the content of soluble Cad conjugates and increased the content of their components, free Cad and phenols. We also found that one of the possible reasons of the negative effect of exogenous diamine on the formation of conjugated forms in adult roots was alkalization of the root apoplast at Cad addition to nutrient medium and the unusual O(2)(-) synthase function as a pH-dependent guaiacol peroxidase in the presence of a high content of H(2)O(2). This was confirmed by the data on the accumulation of O(2)(-) and enhanced superoxide dismutase activity in adult roots under treatment with Cad. It is possible that the accumulation of O(2)(-) together with H(2)O(2) was also responsible for oxidative burst, which induced a decrease in the content of Cad conjugates in adult roots of the common ice plants.
Assuntos
Cadaverina/metabolismo , Cadaverina/farmacologia , Mesembryanthemum/citologia , Mesembryanthemum/efeitos dos fármacos , Explosão Respiratória/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Mesembryanthemum/enzimologia , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Peroxidase/metabolismo , Fenóis/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Cloreto de Sódio/farmacologia , Solubilidade/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
The aerial surfaces of the common or crystalline ice plant Mesembryanthemum crystallinum L., a halophytic, facultative crassulacean acid metabolism species, are covered with specialized trichome cells called epidermal bladder cells (EBCs). EBCs are thought to serve as a peripheral salinity and/or water storage organ to improve survival under high salinity or water deficit stress conditions. However, the exact contribution of EBCs to salt tolerance in the ice plant remains poorly understood. An M. crystallinum mutant lacking EBCs was isolated from plant collections mutagenized by fast neutron irradiation. Light and electron microscopy revealed that mutant plants lacked EBCs on all surfaces of leaves and stems. Dry weight gain of aerial parts of the mutant was almost half that of wild-type plants after 3 weeks of growth at 400 mM NaCl. The EBC mutant also showed reduced leaf succulence and leaf and stem water contents compared with wild-type plants. Aerial tissues of wild-type plants had approximately 1.5-fold higher Na(+) and Cl(-) content than the mutant grown under 400 mM NaCl for 2 weeks. Na(+) and Cl(-) partitioning into EBCs of wild-type plants resulted in lower concentrations of these ions in photosynthetically active leaf tissues than in leaves of the EBC-less mutant, particularly under conditions of high salt stress. Potassium, nitrate, and phosphate ion content decreased with incorporation of NaCl into tissues in both the wild type and the mutant, but the ratios of Na(+)/K(+) and Cl(-)/NO(3)(-)content were maintained only in the leaf and stem tissues of wild-type plants. The EBC mutant showed significant impairment in plant productivity under salt stress as evaluated by seed pod and seed number and average seed weight. These results clearly show that EBCs contribute to succulence by serving as a water storage reservoir and to salt tolerance by maintaining ion sequestration and homeostasis within photosynthetically active tissues of M. crystallinum.
Assuntos
Mesembryanthemum/metabolismo , Cloreto de Sódio/metabolismo , Homeostase , Mesembryanthemum/citologia , Mesembryanthemum/genética , Mutação , Nitratos/metabolismo , Fenótipo , Fosfatos/metabolismo , Fotossíntese , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Caules de Planta/citologia , Caules de Planta/genética , Caules de Planta/metabolismo , Potássio/metabolismo , Reprodução , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismoRESUMO
We report on the sub-cellular localisation and function of m-Rab(mc), a N-myristoylated plant-specific Rab-GTPase previously characterised at the molecular level and also by structural analysis in Mesembryanthemum crystallinum. By confocal laser scanning microscopy, we identified m-Rab(mc) predominantly on the prevacuolar compartment of the lytic vacuole but also on the Golgi apparatus in various plant cell types. Two complementary approaches were used immunocytochemistry and cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP)-fusion proteins. Co-localisation studies of m-Rab(mc) with a salinity stress modulated integral calcium-ATPase suggest involvement of m-Rab(mc) in a plant-specific transport pathway to the prevacuolar compartment of the lytic vacuole. This hypothesis was strengthened by the inhibition of the transport of aleurain fused to green fluorescent protein (GFP), a marker of the lytic vacuole, in the presence of the dominant negative mutant m-Rab(mc)(N147I) in Arabidopsis thaliana protoplasts. The inhibitory effect of m-Rab(mc)(N147I) was specific for the transport pathway to the lytic vacuole, since the transport of chitinase-YFP, a marker for the neutral vacuole, was not hindered by the mutant.
Assuntos
Complexo de Golgi/metabolismo , Mesembryanthemum/citologia , Mesembryanthemum/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Fusão Gênica Artificial , ATPases Transportadoras de Cálcio/metabolismo , Quitinases/metabolismo , Genes de Plantas , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Mesembryanthemum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico/fisiologia , Protoplastos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Uptake and transport of inorganic nitrogen and allocation of amino acids are essential for plant growth and development. To study the effects of salinity on the regulation of transporters for nitrogenous compounds, we characterized the putative nitrate transporter McNRT1 and the amino acid transporters McAAT1 and McAAT2 from Mesembryanthemum crystallinum. By transcript analyses, McAAT1 was found in leaves, McAAT2 in roots, and McNRT1 in both tissues. By in situ PCR McNRT1 was localized, for example, to epidermal and vascular cells whereas McAAT2 was abundant in most cell types in mature roots and McAAT1 in the mesophyll and cells neighbouring xylem vessels in leaves. In response to salt stress, expression of McAAT2 and McNRT1 was stimulated in the root vasculature. In addition, McNRT1 and McAAT1 signals increased in the leaf phloem. Growth of yeast mutants deficient in histidine uptake was restored by McAAT2 whereas both McAAT1 and McAAT2 complemented a yeast mutant carrying a defect in proline uptake. The differential and cell-specific transcriptional activation of genes encoding nitrogen and amino acid transporters under salt stress suggest complex coordinated regulation of these transporter families to maintain uptake and distribution of nitrogenous compounds and amino acids under conditions of high salinity in plants.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Proteínas de Transporte de Ânions/genética , Mesembryanthemum/genética , Cloreto de Sódio/farmacologia , Aminoácidos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Teste de Complementação Genética , Mesembryanthemum/citologia , Mesembryanthemum/efeitos dos fármacos , Mesembryanthemum/metabolismo , Mutação , Transportadores de Nitrato , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Prolina/metabolismo , RNA de Plantas/efeitos dos fármacos , RNA de Plantas/genética , RNA de Plantas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
Mesembryanthemum crystallinum, a facultative halophyte and C(3)-Crassulacean acid metabolism (CAM) intermediate plant, has become a favoured plant for studying stress response mechanisms during C(3)-CAM shifts. One hour of exposure to excess light (EL) caused inhibition of photosynthetic electron transport in M. crystallinum leaves as indicated by chlorophyll a fluorescence measurements. This was accompanied by an increase in NADP-malic enzyme (ME), one of the key cytosolic enzymes involved in CAM, and by a general increase in superoxide dismutase (SOD) activity. In contrast, NAD-ME activity (the mitochondrial form of ME) was not affected by EL. Exposure to EL and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) treatment of a whole plant in low light induced hydrogen peroxide (H(2)O(2)) and C(3) to CAM transition. In contrast, treatment with 3-3,4-dichlorophenyl-1,1-dimethyl urea (DCMU) has blocked high light-induced H(2)O(2) accumulation and C(3)-CAM transition. Moreover, the abundance of transcripts encoding different SODs, ascorbate peroxidase and SOD activity was differently regulated by DCMU and DBMIB. Results of applying EL or high light, H(2)O(2) and photosynthetic electron transport inhibitors suggest that the redox events in the vicinity of PSII and/or PSI and photo-produced H(2)O(2) play a major role in the regulation of C(3)-CAM transition and photooxidative stress responses in M. crystallinum.