Effect of silicon addition on soybean (Glycine max) and cucumber (Cucumis sativus) plants grown under iron deficiency.

Silicon is considered an essential element in several crops enhancing growth and alleviating different biotic and abiotic stresses. In this work, the role of Si in the alleviation of iron deficiency symptoms and in the Fe distribution in iron deficient plants has been studied. Thus, soybean and cucumber plants grown in hydroponic culture under iron limiting conditions were treated with different Si doses (0.0, 0.5 and 1.0 mM). The use of a strong chelating agent such as HBED avoided Fe co-precipitation in the nutrient solution and allowed for the first time the analysis of Si effect in iron nutrition without the interference of the iron rhizospheric precipitation. SPAD index, plant growth parameters and mineral content in plant organs were determined. For soybean, the addition of 0.5 mM of Si to the nutrient solution without iron, initially or continuously during the experiment, prevented the chlorophyll degradation, slowed down the growth decrease due to the iron deficiency and maintained the Fe content in leaves. In cucumber, Si addition delayed the decrease of stem dry weight, stem length, node number and iron content in stems and roots independently of the dose, but no-effect was observed in chlorosis symptoms alleviation in leaves. The observed response to Si addition in iron deficiency was plant-specific, probably related with the different Fe efficiency strategies developed by these two species.

Assessment of biomarkers of cadmium stress in lettuce.

Laboratory and field studies have provided encouraging insights into the capacity of plants to act as biomonitors of environmental quality through the use of biomarkers. However, a better understanding of the overall process of Cd-induced senescence, describing the cascade of Cd effects in plants is needed for a selection of relevant biomarkers of Cd stress. In order to approach this, 5-week old Lactuca sativa L. were exposed for 14 days to 100muM Cd(NO(3))(2) and harvested at days 0, 1, 3, 7 and 14. The parameters measured included classical endpoints (shoot and root growth) and biochemical endpoints related to photosynthesis, nutrients content, and oxidative stress. Cadmium-exposed plants displayed nutrient imbalances in leaves and roots. Photosynthetic efficiency was significantly decreased and lipid peroxidation was enhanced. Antioxidant enzymes were significantly altered during exposure-catalase was inhibited by the end of exposure and peroxidase was induced at day 1 in young leaves. These alterations culminated in a decrease in shoot growth after 14-days exposure to Cd. Biochemical alterations could be used in integrative approaches with classical endpoints in ecotoxicological tests for Cd and after further testing in real scenarios conditions, they could form the basis of a plant biomarkers battery for monitoring and predicting early effects of exposure to Cd.

Expression of ethylene biosynthetic and receptor genes in rose floral tissues during ethylene-enhanced flower opening.

Ethylene production, as well as the expression of ethylene biosynthetic (Rh-ACS1-4 and Rh-ACO1) and receptor (Rh-ETR1-5) genes, was determined in five different floral tissues (sepals, petals, stamens, gynoecia, and receptacles) of cut rose (Rosa hybrida cv. Samantha upon treatment with ethylene or the ethylene inhibitor 1-methylcyclopropene (1-MCP). Ethylene-enhanced ethylene production occurred only in gynoecia, petals, and receptacles, with gynoecia showing the greatest enhancement in the early stage of ethylene treatment. However, 1-MCP did not suppress ethylene production in these three tissues. In sepals, ethylene production was highly decreased by ethylene treatment, and increased dramatically by 1-MCP. Ethylene production in stamens remained unchanged after ethylene or 1-MCP treatment. Induction of certain ethylene biosynthetic genes by ethylene in different floral tissues was positively correlated with the ethylene production, and this induction was also not suppressed by 1-MCP. The expression of Rh-ACS2 and Rh-ACS3 was quickly induced by ethylene in gynoecia, but neither Rh-ACS1 nor Rh-ACS4 was induced by ethylene in any of the five tissues. In addition, Rh-ACO1 was induced by ethylene in all floral tissues except sepals. The induced expression of ethylene receptor genes by ethylene was much faster in gynoecia than in petals, and the expression of Rh-ETR3 was strongly suppressed by 1-MCP in all floral tissues. These results indicate that ethylene biosynthesis in gynoecia is regulated developmentally, rather than autocatalytically. The response of rose flowers to ethylene occurs initially in gynoecia, and ethylene may regulate flower opening mainly through the Rh-ETR3 gene in gynoecia.

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.

Chitinase induced by jasmonic acid, methyl jasmonate, ethylene and protein phosphatase inhibitors in rice.

Chitinase is a pathogenesis-related protein that hydrolyzes chitin, a major component of fungal cell walls. Two-week-old rice seedling leaf, leaf sheath and root tissues responded to an exogenous treatment by jasmonic acid (JA) with induction of the chitinases as determined by immunoblot analysis using an anti-endochitinase antibody. Induced accumulation of these chitinases was observed within 24 to 48 h in the leaf sheaths, leaves and roots. Besides, ethylene generator ethephon and abiotic stressor copper could also induce chitinases accumulation among various plant hormones and stress agents examined. Cycloheximide effectively blocked their accumulation by JA, suggesting that de novo protein synthesis is required. Partial blockage of the induced accumulation of chitinases by NADPH oxidase inhibitor and free radical scavengers suggested involvement of reactive oxygen species. Moreover, induced accumulation of these chitinases also by methyl jasmonate and certain protein phosphatase inhibitors indicated their potential importance and wider role in rice seedlings.

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.

Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the synthesis of glycine betaine.

Previously, we showed that transformation with the codA gene for choline oxidase allows plants to synthesize glycine betaine (GB) and enhances their ability to tolerate various kinds of stress during germination and vegetative growth. In this study, we examined the tolerance of transformed plants to salt stress at the reproductive stage, which is the stage at which plants are most sensitive to environmental stress. Salt-shock treatment of wild-type plants for 3 days resulted in the abortion of flower buds and decreased the number of seeds per silique. These deleterious effects were clearly visible 6 days after the termination of salt-shock treatment. Microscopic examination of floral structures revealed that salt stress inhibited the development of anthers, pistils, and petals. In particular, the production of pollen grains and ovules was dramatically inhibited. These effects of salt stress were significantly reduced by transformation with the codA gene, and our observations suggested that the enhanced tolerance of the transgenic plants was a result of the accumulation of GB in the reproductive organs. Indeed, levels of GB in flowers, siliques, and inflorescence apices were about five times higher than in leaves.

Characterization of cadmium uptake by the water lily Nymphaea aurora.

This study characterizes cadmium (Cd) uptake by the waterlily Nymphaea aurora, (Nymphaeaceae) in two systems: a model hydroponic Cd solution and heavily polluted sludge from two sites in Israel. The uptake of Cd from hydroponic solution resulted in Cd storage in petioles and laminae of Nymphaea, as well as in the roots. The pH of the solution affected Cd solubility and availability, with pH 5.5 yielding maximum Cd content in the plant (140 mg Cd per g DW). Cd uptake was reduced by the addition of EDTA to the hydroponic growth medium, although EDTA enhanced heavy metal uptake by terrestrial plants. Nymphaea efficiently reduced the concentration of Cd in heavy metal polluted urban and industrial sludge and the amount of Cd uptake was enhanced by the addition of KCl to the sludge and by adjustment of the pH to 5.5. The inherent growth patterns of Nymphaea plants allowed Cd uptake by the shoot and root, and resulted in maximum contact between the various plant parts and the growth media. Thus, Nymphaea has potential as an optimal, highly effective phytoremediation tool for the removal of Cd from polluted waste sources.

Phytotoxicity of the tetramic acid metabolite trichosetin.

Trichosetin, a tetramic acid-containing metabolite produced in the dual culture of Trichoderma harzianum and Catharanthus roseus (L.) G. Don callus, was subjected to phytotoxicity assays. In seedling growth assays, trichosetin inhibited root and shoot growth of all five plant species tested by damaging the cell membrane, as evidenced by the dose-dependent increase in electrolyte leakage and lipid peroxidation. Vital staining of trichosetin-treated Nicotiana tabacum BY-2 cells, with rhodamine 123, showed a weaker green fluorescence compared to controls indicating damaging effects on mitochondria. FDA-PI staining, to determine cell viability, indicated that cells of the trichosetin-treated roots were mostly dead.

Abscisic acid and cytokinins in the root exudates and leaves and their relationship to senescence and remobilization of carbon reserves in rice subjected to water stress during grain filling.

The possible regulation of senescence-initiated remobilization of carbon reserves in rice (Oryza sativa L.) by abscisic acid (ABA) and cytokinins was studied using two rice cultivars with high lodging resistance and slow remobilization. The plants were grown in pots and either well-watered (WW, soil water potential = 0 MPa) or water-stressed (WS, soil water potential = -0.05 MPa) from 9 days after anthesis until they reached maturity. Leaf water potentials of both cultivars markedly decreased at midday as a result of water stress but completely recovered by early morning. Chlorophyll (Chl) and photosynthetic rate (Pr) of the flag leaves declined faster in WS plants than in WW plants, indicating that the water deficit enhanced senescence. Water stress accelerated starch remobilization in the stems, promoted the re-allocation of pre-fixed (14)C from the stems to grains, shortened the grain-filling period and increased the grain-filling rate. Sucrose phosphate synthase (SPS, EC 2.4.1.14) activity was enhanced by water stress and positively correlated with sucrose accumulation in both the stem and leaves. Water stress substantially increased ABA but reduced zeatin (Z) + zeatin riboside (ZR) concentrations in the root exudates and leaves. ABA significantly and negatively, while Z+ZR positively, correlated with Pr and Chl of the flag leaves. ABA, not Z+ZR, was positively and significantly correlated with SPS activity and remobilization of pre-stored carbon. Spraying ABA reduced Chl in the flag leaves, and enhanced SPS activity and remobilization of carbon reserves. Spraying kinetin had the opposite effect. The results suggest that both ABA and cytokinins are involved in controlling plant senescence, and an enhanced carbon remobilization is attributed to an elevated ABA level in rice plants subjected to water stress.

Effect of ethylene on flower abscission: a survey.

The effect of ethylene on flower abscission was investigated in monocotyledons and eudicotyledons, in about 300 species from 50 families. In all species studied except Cymbidium, flower abscission was highly sensitive to ethylene. Flower fall was not consistent among the species in any family studied. It also showed no relationship with petal senescence or abscission, nor with petal colour changes or flower closure. Results suggest that flower abscission is generally mediated by endogenous ethylene, but that some exceptional ethylene-insensitive abscission occurs in the Orchidaceae.

Accumulation and detoxification of lead ions in legumes.

This study focuses on lead accumulation in roots, stems and leaves of three plant species of the Fabacea family: Vicia faba, Pisum sativum and Phaseolus vulgaris grown hydroponically in a medium supplemented with 1 mM concentration of lead. The largest amount of lead, up to 75 mg Pb/g dry weight, was accumulated in roots of P. vulgaris. The highest rate of Pb ions uptake from the medium took place during the first 10 h of incubation with lead and after 96 h of incubation lead content in the medium decreased by half. Thus, it was suggested that P. vulgaris could be used in rhizofiltration--the use of plant roots to absorb pollutants from water contaminated with lead. At the same time we studied the influence of lead on acid soluble thiol, glutathione, homoglutathione contents and the synthesis of phyto- and homophytochelatins in roots of V. faba, P. sativum and P. vulgaris grown hydroponically. Activation of the detoxicative-phytochelatin system was observed in the cytosol of root cells of the tested plants. This system was composed of phytochelatins (PCs) in roots of V. faba, homophytochelatins (hPCs) in P. vulgaris roots and both PCs and hPCs in P. sativum roots. The total content of PCs and hPCs in roots of P. sativum was very high and reached around 4800 (expressed in nmol SH x g(-1)FW) and induction of their synthesis occurred after only 2 h of treatment with 1 mM Pb.

Evidence for high activity of xylem parenchyma and ray cells in the interface of host stem and Agrobacterium tumefaciens-induced tumours of Ricinus communis.

Rapidly developing tumours at hypocotyls of Ricinus communis, induced by Agrobacterium tumefaciens strain C58, were characterized by strong differentiation of vascular bundles and their functional connection to the host bundles. The stem/tumour interface showed increased xylem, with numerous vessels accompanied by multiseriate unlignified rays. To know how nutrients efficiently accumulate in the tumour sink tissue, cell electropotentials (E(m)) in cross-sections were mapped. The measured cells were identified by injected Lucifer Yellow. Xylem and phloem parenchyma cells and stem/tumour-located rays hyperpolarized to E(m) values of about -170 mV, which suggest high plasma membrane proton pump activities. Rapidly dividing cells of cambia or small tumour parenchyma cells had low E(m). The tumour aerenchyma and the stem cortex cells displayed values close to the energy-independent diffusion potential. The lowest values were recorded in stem pith cells. Cell K(+) concentrations largely matched the respective E(m). The pattern of individual cell electropotentials was supplemented by whole organ voltage measurements. The voltage differences between the tumour surface and the xylem perfusion solution in stems attached to the tumours, the trans-tumour electropotentials (TTP), confirm the findings of respiration-dependent and phytohormone-stimulated high plasma membrane proton pump activity in intact tumours, mainly in the xylem and phloem parenchyma and ray cells. TTPs were inhibited by addition of NaN(3), CN(-) plus SHAM or N(2) gas in the xylem perfusion solution and by external N(2) flushing. The data provide functional evidence for the structural basis of priority over the host shoot in nutrient flow from the stem to the tumour.

Ethylene response to pollen tube growth in Nicotiana tabacum flowers.

In flowers of Nicotiana tabacum L., pollination induces a transient increase in ethylene production by the pistil. The characteristic dynamics of the increase in ethylene correspond to the main steps of the pollen-tube journey into the pistil: penetration into the stigma, growth through the style, entry into the ovary and fertilization. Ethylene is synthesized de novo in the pistil, and its production is reduced in the dark. Ethylene production was monitored in tobacco flowers after pollination with incongruous pollen from three different Nicotiana species, N. rustica, N. repanda and N. trigonophylla, and with pollen from Petunia hybrida. Pollen from all of these different sources can germinate on the stigma surface but each pollen type shows a different behavior and efficiency in penetrating the pistil tissues. Thus, these different crosses provided a model with which to study the response of the pistil to pollination and fertilization. Ethylene evolution upon pollination in tobacco differed in each cross, suggesting that ethylene is correlated with the response to pollen tube growth in the tobacco flower.

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.

The abscisic acid-related SNARE homolog NtSyr1 contributes to secretion and growth: evidence from competition with its cytosolic domain.

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion, and secretion. Previously, we isolated the syntaxin-related protein NtSyr1 (NtSyp121) from tobacco in a screen for abscisic acid-related signaling elements, demonstrating its role in determining the abscisic acid sensitivity of K(+) and Cl(-) channels in stomatal guard cells. NtSyr1 is localized to the plasma membrane and is expressed normally throughout the plant, especially in root tissues, suggesting that it might contribute to cellular homeostasis as well as to signaling. To explore its functions in vivo further, we examined stably transformed lines of tobacco that expressed various constructs of NtSyr1, including the full-length protein and a truncated fragment, Sp2, corresponding to the cytosolic domain shown previously to be active in suppressing ion channel response to abscisic acid. Constitutively overexpressing NtSyr1 yielded uniformly high levels of protein (>10 times the wild-type levels) and was associated with a significant enhancement of root growth in seedlings but not with any obvious phenotype in mature, well-watered plants. Similar transformations with constructs encoding the Sp2 fragment of NtSyr1 showed altered leaf morphology but gave only low levels of Sp2 fragment, suggesting a strong selective pressure against plants expressing this protein. High expression of the Sp2 fragment was achieved in stable transformants under the control of a dexamethasone-inducible promoter. Sp2 expression was correlated positively with altered cellular and tissue morphology in leaves and roots and with a cessation of growth in seedlings. Overexpression of the full-length NtSyr1 protein rescued the wild-type phenotype, even in plants expressing high levels of the Sp2 fragment, supporting the idea that the Sp2 fragment interfered specifically with NtSyr1 function by competing with NtSyr1 for its binding partners. To explore NtSyr1 function in secretion, we used a green fluorescent protein (GFP)-based section assay. When a secreted GFP marker was coexpressed with Sp2 in tobacco leaves, GFP fluorescence was retained in cytosolic reticulate and punctate structures. In contrast, in plants coexpressing secreted GFP and NtSyr1 or secreted GFP alone, no GFP fluorescence accumulated within the cells. A new yellow fluorescent protein-based secretion marker was used to show that the punctate structures labeled in the presence of Sp2 colocalized with a Golgi marker. These structures were not labeled in the presence of a dominant Rab1 mutant that inhibited transport from the endoplasmic reticulum to the Golgi. We propose that NtSyr1 functions as an element in SNARE-mediated vesicle trafficking to the plasma membrane and is required for cellular growth and homeostasis.

Actomyosin promotes cell plate alignment and late lateral expansion in Tradescantia stamen hair cells.

Cytokinesis in higher-plant cells involves the formation of a cell plate in the interzone between the separating chromatids. The process is directed by the phragmoplast, an array of microtubules, actin filaments, and membranous elements. To determine if the role of actin in cytokinesis is dependent on myosin, we treated stamen hair cells of Tradescantia virginiana L. with 2,3-butanedione monoxime (BDM), an inhibitor of myosin ATPase and ML-7, a specific inhibitor of myosin light-chain kinase. Treatment with BDM resulted in a tilted cytokinetic apparatus during early initiation and a wavy cell plate with curved phragmoplasts during late lateral expansion. Treatment with ML-7 also resulted in inefficient late lateral expansion of the cell plate, with effects ranging from slower expansion to complete inhibition. Taken together, these results implicate myosin in the control of cell plate expansion and alignment.