Studies on lead and cadmium toxicity in Dianthus carthusianorum calamine ecotype cultivated in vitro.

Information on metallophytes during reclamation of land contaminated with heavy metals is sparse. We investigated the response of D. carthusianorum calamine ecotype to Pb and Cd stress. We focused on in vitro selection of tolerant plant material for direct use in chemically degraded areas. Shoot cultures were treated with various concentrations of Pb or Cd ions. Plantlet status was estimated as micropropagation efficiency, growth tolerance index (GTI) and through physiological analysis. Moreover, determination of plant Pb, Cd and other elements was performed. The application of Pb(NO3 )2 resulted in stronger growth inhibition than application of CdCl2 . In the presence of Pb ions, a reduction was observed of both, the micropropagation coefficient to 1.1-1.8 and the GTI to 48%. In contrast, Cd ions had a positive influence on tested cultures, expressed as an increase of GTI up to 243% on medium enriched with 1.0 µm CdCl2 . Moreover, photosynthetic pigment content in shoots cultivated on media with CdCl2 was higher than in control treatment. The adaptation to Cd was associated with decreased accumulation of phenols in the order: 0.0 µm > 1.0 µm > 3.0 µm > 5.5 µm CdCl2 . It seems that high tolerance to Cd is related to K uptake, which is involved in antioxidant defence. This work presents an innovative approach to the impact of Cd ions on plant growth and suggests a potential biological role of this metal in species from metalliferous areas.

Effects of different soil remediation methods on inhibition of lead absorption and growth and quality of Dianthus superbus L.

Heavy metal pollution in soil poses a serious threat to the growth of plants used in traditional Chinese medicine. Therefore, a pot experiment was conducted to study the effects of various soil remediation methods on the performance of Herba Dianthi (Dianthus superbus L.) grown on Pb-contaminated soil. The results show that inoculation of Herba Dianthi with arbuscular mycorrhizal fungi (AMF) led to a significant reduction in Pb uptake (P< 0.05), and increased root development and root-to-shoot ratio compared to untreated control plants, along with the highest content of active components. When planting with Trifolium repens, the reduction effect of Pb absorption was insignificant. Herba Dianthi showed improved growth and active ingredients, and the lowest Pb content, with AMF inoculation. The addition of EDTA decreased the growth of Herba Dianthi, but promoted the absorption of Pb. The inhibition of tumor cells was highest in E2. In conclusion, inoculation with AMF can ensure that plant lead content meets testing standards, helping to improve the quality of medicinal herbs.

Characterization of the role of sodium nitroprusside (SNP) involved in long vase life of different carnation cultivars.

BACKGROUND: Sodium nitroprusside (SNP) has been previously shown to extend the vase life of various cut flowers; however, its positive effect on extending vase life of carnations has not been well documented. Moreover, the role of SNP in the mechanisms underlying determination of vase life of cut carnations has also not been well addressed. RESULTS: SNP increased vase life of Tico Viola carnations along with their relative fresh weight (RFW). Among the treatments, the flowers treated with 10 mg L-1 SNP had the longest vase life and maximum relative fresh weight (RFW). This was achieved through significant suppression of ethylene production via downregulation of ethylene biosynthesis and petal senescence-related genes, and through an increase in the scavenging mechanism of reactive oxygen species (ROS) by antioxidant activity during flower vase life. In addition, the positive efficacy of SNP could also be confirmed using 1-aminocyclopropane-1-carboxylic acid (ACC) and different cultivars, resulting in similar trends for both experiments. CONCLUSION: Taken together, these results suggest that SNP plays a crucial role in multiple modes of action that are associated with the longevity of cut carnation flowers.

Biosynthesis of silver nanoparticles using Artemisia annua callus for inhibiting stem-end bacteria in cut carnation flowers.

A biological method for synthesising silver nanoparticles (AgNPs) was developed using the callus extracts from Artemisia annua L. under sunlight at 25,000 lx. The AgNPs were characterised using transmission electron microscopy, atomic force microscope, X-ray diffraction and Fourier transform infrared spectroscopy. The AgNPs were mostly spherical with the size of 2.1 to 45.2 nm (average 10.9 nm). Pulse treatments of AgNPs at 125, 250 and 500 mg/l for 1 h extended vase life of cut carnation (Dianthus caryophyllus cv. Green Land) flowers. Four dominant bacteria strains Arthrobacter arilaitensis, Kocuria sp., Staphylococcus equorum and Microbacterium oxydans were isolated from the stem-ends of cut D. caryophyllus flowers. AgNP pulse inhibited significantly bacterial growth in vase solution and cut stem ends during all of the vase period. The bacteria related blockage in the stem-ends was significantly alleviated by AgNP pulse because of its higher antibacterial efficacy against the dominant bacteria. In addition, ethylene release of cut carnation flowers was inhibited in response to AgNP pulse. This is the first time that the biologically synthesised AgNPs could be applied as a promising preservative agent for cut carnation flowers.

Reversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO3 and its associated mechanism during in vitro culture.

Hyperhydricity occurs frequently in plant tissue culture and can severely affect commercial micropropagation and genetic improvement of the cultured plantlets. Hyperhydric shoots are charaterzized by high water content, but how this occurs is still a subject of investigation. Silver ion (Ag+) can reduce the extent of hyperhydricity in plants, but its effect on the reversion of hyperhydric plantlets and the underlying mechanism of reversion has not been clarified. In this study, about 67% of the hyperhydric Dianthus chinensis L. plantlets were found to revert to normal condition when the plantlets were cultured in medium supplemented with 29.4µmolL-1AgNO3. Water content and hydrogen peroxide (H2O2) content in the guard cells of these plantlets were reduced, while stomatal aperture and water loss rate were increased. AgNO3 also reduced the content of endogenous ethylene and expression of ethylene synthesis and ethylene signal transduction-associated genes. Reduced accumulation of ethylene consequently led to an increase in stomatal aperture mediated by decreased H2O2 content in the guard cells. These results adequately verified the role of AgNO3 in the reversion of hyperhydricity in D. chinensis L. and also provided clues for exploring the cause of excessive water accumulation in hyperhydric plants.

Tratamento alternativo em gatos acometidos por DITUIF / Alternative treatment in cats affected by FLUTD

This report describes two cases of male cats affected by FLUTD (Feline lower urinary tract disease). The first patient had been affected by numerous relapses after passing through various therapeutic treatments and surgical interventions, and was subjected to the alternative protocol after being recommended for euthanasia, the second patient had no history of urinary tract disorders in the past, and was not subjected to any kind of previous treatment. The two patients had a significant improvement in their clinical condition, were not affected by relapses after participation in the alternative design, and were accompanied for about a year after their treatment.(AU)

Naturally evolved enhanced Cd tolerance of Dianthus carthusianorum L. is not related to accumulation of thiol peptides and organic acids.

Two contrasting ecotypes of Dianthus carthusianorum L., metallicolous (M) and nonmetallicolous (NM), were cultivated in hydroponics at 0-50 µM Cd for 14 days to compare their Cd accumulation, sensitivity and tolerance mechanisms. While both ecotypes contained similar concentrations of Cd in the shoots and roots, the M ecotype was more Cd-tolerant (as measured by fresh weight production and root and leaf viability). Both ecotypes accumulated phytochelatins (PCs) in response to Cd with a higher amount thereof found in the NM ecotype. Concentrations of PCs remained unchanged with increasing Cd concentrations in the root tissues, but their content in the shoots increased. The addition of L-buthionine-sulfoximine (BSO) diminished glutathione (GSH) accumulation and arrested PC production, which increased the sensitivity to Cd of the NM, but not M ecotype. Organic acids (malate and citrate) as well as proline accumulation did not change significantly after Cd exposition and was at the same level in both ecotypes. The enhanced Cd tolerance of the M ecotype of D. carthusianorum cannot be explained in terms of restricted Cd uptake and differential production of PCs, organic acids or proline; some other mechanisms must be involved in its adaptation to the high Cd content in the environment.

The role of silver nano-particles and silver thiosulfate on the longevity of cut carnation (Dianthus caryophyllus) flowers.

The purpose of this study was to evaluate the efficacy of silver nano-particles (SNP) and silver thiosulfate (STS) in extending the vase life of cut carnation (Dianthus caryophyllus L. cv. 'Tempo') flowers. Pulse treatments of SNP @ 0, 5, 10 and 15 mg l(-1) and STS @ 0, 0.1, 0.2 and 0.3 mM were administered to carnation flowers for 24 hr. The longest vase life (16.1 days) was observed in flowers treated with 15 mg l(-1) of SNP + 0.2 mM STS. The least chlorophyll was destroyed in flowers treated with 15 mg I(-1) of SNP + 0.3 mM STS. Our findings showed that the 15 mg l(-1) SNP treatment inhibited bacterial growth in the preservative solution. The control flowers bloomed faster than the treated flowers. The maximum peroxidase activity and the minimum lipid peroxidation were obtained in cut flowers that were treated with 15 mg l(-1) of SNP and 0.3 mM STS. Overall, results of the study revealed that SNP and STS treatment extended the longevity of cut carnation 'Tempo' flowers by reducing oxidative stress, improving anti-oxidant system, reducing bacterial populations and delaying flowering.

Accumulation and tolerance of lead in two contrasting ecotypes of Dianthus carthusianorum.

Dianthus carthusianorum is one of the dominant plant species colonising the Zn-Pb waste deposits in Boleslaw, Southern Poland. It differs in terms of morphology and genetics from ecotypes inhabiting non-metal-polluted areas. The response of waste-heap (metallicolous, M) and reference (nonmetallicolous, NM) ecotypes of D. carthusianorum to Pb in hydroponics was investigated and compared in this study. The plants of the M ecotype were more tolerant to Pb than these of the NM ecotype in spite of accumulation of higher concentrations of Pb. In both ecotypes, about 70-78% of Pb was retained in roots. In non Pb-treated plants, a higher glutathione (GSH) level was found in the M ecotype. After the Pb exposure, the GSH level decreased and was similar in both ecotypes. Lead treatment induced synthesis of phytochelatins (PCs) only in the plant roots, with significantly higher concentrations thereof detected in the NM ecotype. Malate and citrate concentrations were higher in the M ecotype; however, they did not change significantly upon any Pb treatment in either ecotype. The results indicated that neither PCs nor organic acids were responsible for the enhanced Pb tolerance of the waste-heap plants.

Early steps of adventitious rooting: morphology, hormonal profiling and carbohydrate turnover in carnation stem cuttings.

The rooting of stem cuttings is a common vegetative propagation practice in many ornamental species. A detailed analysis of the morphological changes occurring in the basal region of cultivated carnation cuttings during the early stages of adventitious rooting was carried out and the physiological modifications induced by exogenous auxin application were studied. To this end, the endogenous concentrations of five major classes of plant hormones [auxin, cytokinin (CK), abscisic acid, salicylic acid (SA) and jasmonic acid] and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid were analyzed at the base of stem cuttings and at different stages of adventitious root formation. We found that the stimulus triggering the initiation of adventitious root formation occurred during the first hours after their excision from the donor plant, due to the breakdown of the vascular continuum that induces auxin accumulation near the wounding. Although this stimulus was independent of exogenously applied auxin, it was observed that the auxin treatment accelerated cell division in the cambium and increased the sucrolytic activities at the base of the stem, both of which contributed to the establishment of the new root primordia at the stem base. Further, several genes involved in auxin transport were upregulated in the stem base either with or without auxin application, while endogenous CK and SA concentrations were specially affected by exogenous auxin application. Taken together our results indicate significant crosstalk between auxin levels, stress hormone homeostasis and sugar availability in the base of the stem cuttings in carnation during the initial steps of adventitious rooting.

Response of carnation (Dianthus caryophyllus) cultivars to different postharvest preservatives.

Experiments were conducted to assess the effect of selected pulsing solutions on the days to flower bud shrinkage, leaf wilting and petal edge drying occurrence of carnation cultivars (Green-Go and Galy). The pulsing solutions used for this investigation were Silver Thiosulfate (STS) (0.2, 0.6, 1 mM) and also ethanol (6, 8, 10%), both received equal amount of sucrose (10%). Besides, to simulate the actual practice of the farm (0.4 mM Silver Thiosulfate (STS) plus 0.3 mM T.O.G) was used as a standard control. Senescence symptoms such as flower bud shrinkage, petal edge drying and leaf wilting were monitored. The results obtained showed that 1 mM STS plus 25 g sucrose achieved rapid petal edge drying for Green-Go cultivar. On the other hand, positive effects were also observed in days to flower bud shrinkage extended by 6 mM Silver Thiosulfate (STS) plus 25 g sucrose and being in par with 8% ethanol plus 25 g sucrose for Green-Go cultivar. Subsequently, the standard control, 0.6 mM Silver Thiosulfate (STS) plus 25 g sucrose and 8% ethanol plus 25 g sucrose attended comparable increment on the days to leaf wilting occurrences.

Transcriptional regulation of two RTE-like genes of carnation during flower senescence and upon ethylene exposure, wounding treatment and sucrose supply.

RTE1 (REVERSION-TO-ETHYLENE SENSITIVITY1) was identified as a positive regulator of ETR1 (ethylene resistant1) function in Arabidopsis; RTEs are a small gene family. Ethylene plays a crucial role in the senescence of carnation (Dianthus caryophyllus L.) flowers. Two cDNA clones encoding putative RTE-like protein (DCRTE1 and DCRTH1) were obtained from total RNA isolated from senescing carnation petals using RT-PCR and RACE techniques. The predicted proteins of DCRTE1 and DCRTH1 consist of 228 and 233 amino acids, respectively. Interestingly, the deduced DCRTE1 protein, like most other RTEs, includes two putative transmembrane domains, while the deduced DCRTH1 protein includes five putative transmembrane domains, according to the TMHMM database. Northern blots showed that the level of DCRTE1 mRNA in petals first decreased then increased remarkably after ethylene production started, and DCRTE1 expression showed an increasing trend in ovaries during natural flower senescence. The amount of DCRTH1 transcripts increased gradually in both petals and ovaries during natural senescence. Exogenous ethylene increased transcript abundance of DCRTE1 and DCRTH1 to various degrees in both petals and ovaries. STS treatment decreased the level of DCRTH1 mRNA in petals and ovaries compared with the control. DCRTE1 and DCRTH1 showed a rapid increase and then a decrease in mRNA accumulation in leaves after wounding. These results suggest that both DCRTE1 and DCRTH1 could play important roles in flower senescence-related signalling. Sucrose treatment did not remarkably affect the amount of DCRTE1 and DCRTH1 mRNAs.

Sucrose prevents up-regulation of senescence-associated genes in carnation petals.

cDNA microarrays were used to characterize senescence-associated gene expression in petals of cut carnation (Dianthus caryophyllus) flowers, sampled from anthesis to the first senescence symptoms. The population of PCR fragments spotted on these microarrays was enriched for flower-specific and senescence-specific genes, using subtractive hybridization. About 90% of the transcripts showed a large increase in quantity, approximately 25% transiently, and about 65% throughout the 7 d experiment. Treatment with silver thiosulphate (STS), which blocks the ethylene receptor and prevented the normal senescence symptoms, prevented the up-regulation of almost all of these genes. Sucrose treatment also considerably delayed visible senescence. Its effect on gene expression was very similar to that of STS, suggesting that soluble sugars act as a repressor of ethylene signal transduction. Two fragments that encoded a carnation EIN3-like (EIL) protein were isolated, some of which are key transcription factors that control ethylene response genes. One of these (Dc-EIL3) was up-regulated during senescence. Its up-regulation was delayed by STS and prevented by sucrose. Sucrose, therefore, seems to repress ethylene signalling, in part, by preventing up-regulation of Dc-EIL3. Some other transcription factors displayed an early increase in transcript abundance: a MYB-like DNA binding protein, a MYC protein, a MADS-box factor, and a zinc finger protein. Genes suggesting a role in senescence of hormones other than ethylene encoded an Aux/IAA protein, which regulate transcription of auxin-induced genes, and a cytokinin oxidase/dehydrogenase, which degrades cytokinin. Taken together, the results suggest a master switch during senescence, controlling the co-ordinated up-regulation of numerous ethylene response genes. Dc-EIL3 might be (part of) this master switch.

The role of N-lauroylethanolamine in the regulation of senescence of cut carnations (Dianthus caryophyllus).

N-acylethanolamines (NAEs) are a group of lipid mediators that play important roles in mammals, but not much is known about their precise function in plants. In this work, we analyzed the possible involvement of N-lauroylethanolamine [NAE(12:0)] in the regulation of cut-flower senescence. In cut carnation flowers of cv. Red Barbara, the pulse treatment with 5 microM NAE(12:0) slowed senescence by delaying the onset of initial wilting. Ion leakage, which is a reliable indicator of membrane integrity, was postponed in NAE(12:0)-treated flowers. The lipid peroxidation increased in carnation petals with time, in parallel to the development in activity of lipoxygenase and superoxide anion production rate, and these increases were both delayed by NAE(12:0) supplementation. The activities of four enzymes (superoxide dismutase, catalase, glutathione reductase and ascorbate peroxidase) that are implicated in antioxidant defense were also upregulated in the cut carnations that had been treated with NAE(12:0). These data indicate that NAE(12:0)-induced delays in cut-carnation senescence involve the protection of the integrity of membranes via suppressing oxidative damage and enhancing antioxidant defense. We propose that the stage from the end of blooming to the onset of wilting is a critical period for NAE(12:0) action.

The influence of EDTA application on the interactions of cadmium, zinc, and lead and their uptake of rainbow pink (Dianthus chinensis).

Soil used in this study was artificially contaminated with Cd, Zn, Pb, or applied in combinations (Cd-Zn, Cd-Pb, Zn-Pb, or Cd-Zn-Pb) to study the interactions of metals in soil contaminated with multiple metals. After planting rainbow pink (Dianthus chinensis) in these soils for 21 days, three different concentrations of ethylenedinitrilotetraacetic acid (EDTA) solutions were added to study the effect of applying EDTA on the interactions among these metals. The concentrations of Cd, Zn, and Pb in the soil solutions of different metals-treated soils increased significantly after applying 5 mmol EDTA kg(-1) soil (p<0.05). The potential of groundwater contamination will increase after applying EDTA and it is not recommended to be in situ used or have to use very carefully. The existence of Pb in the Cd-contaminated soil enhanced the uptake of Cd in rainbow pink in the treatments of control and 2 mmol EDTA kg(-1) soil. Cadmium inhibited the concentration of Zn without applying EDTA. However, whether the application of EDTA or not and the applied EDTA concentration had the greatest effect on the uptake of Pb when compared to Cd and Zn. After applying 5 mmol EDTA kg(-1) soil, Cd or Zn in the Pb-contaminated soil inhibited the uptake of Pb in rainbow pink, but there were no effect in other treatments.

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation.

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.

Repressed ethylene production in the gynoecium of long-lasting flowers of the carnation 'White Candle': role of the gynoecium in carnation flower senescence.

Ethylene production and expression of ethylene biosynthetic genes was investigated in senescing flowers of carnation (Dianthus caryophyllus L.) cultivars 'White Candle (WC)' and 'Light Pink Barbara (LPB)', with long and short vase-lives, respectively. Ethylene production from the gynoecium and petals of senescing 'WC' flowers was below the limit of detection, in agreement with the repressed ethylene production from the whole flowers. However, exogenous ethylene treatment caused the accumulation of transcripts for DC-ACS1 and DC-ACO1 genes in both the gynoecium and petals, resulting in ethylene production from the flowers. Moreover, application of ABA or IAA, which are known to exhibit their action through the induction of ethylene synthesis in the gynoecium, to 'WC' flowers from their cut stem-end induced ethylene production and wilting in the flowers. These findings suggested that, in 'WC' flowers the mechanism of ethylene biosynthesis, i.e. the induction of expression of genes for ethylene biosynthesis and the action of resulting enzymes, was not defective, but that its function was repressed during natural senescence. Transcripts of DC-ACO1, DC-ACS3, and DC-ACS1 were present in the gynoecium of senescing 'LPB' flowers. In the gynoecium of senescing 'WC' flowers, however, the DC-ACO1 transcript was present, but the DC-ACS1 transcript was absent and the DC-ACS3 transcript was detected only in a small amount; the latter two were associated with the low rate of ethylene production in the gynoecium of 'WC' flowers. These findings indicated that the repressed ethylene production in 'WC' flowers during natural senescence is caused by the repressed ethylene production in the gynoecium, giving further support for the role of the gynoecium in regulating petal senescence in carnation flowers.

[Effects of nitrogen and calcium on polyphenol oxidase, peroxidase and leaf spot in carnation].

Carnation inoculated with leaf spot was cultivated under different concentration of nitrogen and calcium in this experiment. The result showed that the nutrition state [NO3- (0.024%-0.025%), Ca2+ (0.022%-0.0342%)] was helpful to keep higher activity of POD and PPO and enhance the carnation's resistance to the germs.