Cloning and expression analysis of squalene synthase, a key enzyme involved in antifungal steroidal glycoalkaloids biosynthesis from Solanum nigrum.

Steroidal glycoalkaloids (SGAs) are a family of nitrogenous secondary metabolites produced in solanaceous plants. In our present study, γ-solamargine and its aglycone solasodine from Solanum nigrum were found to inhibit hyphae formation of Fusarium oxysporum. As phytoalexins, the formation of SGAs was significantly increased in the plants when infected with the spore of F. oxysporum. In order to understand this inducible defense mechanism, the rate-limiting enzyme squalene synthase in the biosynthesis process of SGAs was investigated well. A full-length cDNA encoding squalene synthase was isolated from S. nigrum (the squalene synthase in S. nigrum was designated as SnSS). The full-length cDNA of SnSS was 1,765 bp and contained a 1,236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Bioinformatic analysis revealed that the deduced SnSS protein had a high similarity with other plant squalene synthases. Real-time RT-PCR analysis showed that SnSS was expressed constitutively in all tested tissues, with the highest expression in stems. After treatment with the spore of F. oxysporum, the mRNA level of SnSS was significantly increased in the infected plants in accordance with the change of SGAs.

Effect of endophyte-infection on growth parameters and Cd-induced phytotoxicity of Cd-hyperaccumulator Solanum nigrum L.

The aim of this work was to evaluate effects of endophytic bacterium inoculation on plant growth and assess the possible mechanism of endophyte in heavy metal phytoremediation. Seeds of Solanum nigrum L. were inoculated with endophyte Serratia nematodiphila LRE07 and were subjected to Cd in the growing medium. Cd produced a significant inhibition on plant growth and a reduction in the content of photosynthetic pigments. The inoculation of endophytic bacterium alleviated the Cd-induced changes, resulting in more biomass production and higher photosynthetic pigments content of leaves compared with non-symbiotic ones. The beneficial effect was more obvious at relatively low Cd concentration (10 µM). Based on the alteration of nutrient uptake and activated oxygen metabolism in infected plants, the possible mechanisms of endophytic bacterium in Cd phytotoxicity reduction can be concluded as uptake enhancement of essential mineral nutrition and improvement in the antioxidative enzymes activities in infected plant.

Regulation of jasmonate metabolism and activation of systemic signaling in Solanum nigrum: COI1 and JAR4 play overlapping yet distinct roles.

• Jasmonates are ubiquitous messengers in land plants essential for the activation of defense responses. However, their signaling properties, accumulation and metabolism vary substantially among species. Solanum nigrum is a wild Solanaceous species developed as a model to study defense responses. • Solanum nigrum plants transformed to silence the expression of key genes in jasmonate production (SnLOX3), conjugation (SnJAR4) and perception (SnCOI1) were generated to analyze the function of these genes in jasmonate accumulation and metabolism (studied by a combination of LC-MS/MS and (13) C-isotope labeling methods) and in signaling [studied by the systemic elicitation of leucine aminopeptidase (LAP) activity]. • In contrast with the early single jasmonic acid (JA) burst induced by wounding in wild-type (WT) plants, elicitation with insect oral secretions induced a later, second burst that was essential for the induction of systemic LAP activity, as demonstrated by ablation experiments. This induction was dependent on SnLOX3 and SnCOI1, but not on SnJAR4. In addition, the local accumulation of JA-glucose and JA-isoleucine was dependent on SnCOI1, whereas the accumulation of hydroxylated jasmonates was dependent on both SnCOI1 and SnJAR4. • The results demonstrate that SnLOX3, SnCOI1 and SnJAR4 have overlapping yet distinct roles in jasmonate signaling, differentially controlling jasmonate metabolism and the production of a systemic signal.

Nitric oxide is associated with long-term zinc tolerance in Solanum nigrum.

Nitric oxide (NO) has been identified as a signal molecule that interplays with reactive oxygen species in response to heavy metal stresses. Roles of NO in regulating cadmium toxicity and iron deficiency have been proposed; however, the function of NO in zinc (Zn) tolerance in plants remains unclear. Here, we investigated NO accumulation and its role in plant Zn tolerance. Zn-induced NO production promoted an increase in reactive oxygen species accumulation in Solanum nigrum roots by modulating the expression and activity of antioxidative enzymes. Subsequently, programmed cell death (PCD) was observed in primary root tips. Inhibiting NO accumulation by 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (a specific NO scavenger) or N(G)-nitro-l-arginine-methyl ester (a NO synthase inhibitor) prevented the increase of superoxide radical and hydrogen peroxide as well as the subsequent cell death in the root tips, supporting the role of NO in Zn-induced PCD in the root tips. Zn-induced NO production affected the length of primary roots, the number of lateral roots, and root hair growth and thereby modulated root system architecture and activity. Investigation of metal contents in Zn-treated roots suggests that NO is required for metal (especially iron) uptake and homeostasis in plants exposed to excess Zn. Taken together, our results indicate that NO production and the subsequent PCD in root tips exposed to excess Zn are favorable for the S. nigrum seedling response to long-term Zn toxicity by modulating root system architecture and subsequent adaptation to Zn stress.

[Effect of cadmium, alone or in combination with CaCl2, on the growth, antioxidative enzyme activity and cadmium absorption of Solanum nigrum L. var pauciflorum hairy roots].

To study if Solanum nigrum hairy roots can be used for phytoremediation of Cd contamination, we investigated the effects of cadmium (Cd) alone, and in combination with different concentrations of CaCl2, on growth, activities of superoxide dismutase (SOD) and peroxidase (POD) and Cd absorption by hairy roots of S. nigrum L. var pauciflorum. The results showed that Cd concentrations of lower than 50 micromol/L enhanced the growth of hairy roots, while higher than 100 micromol/L inhibited growth and decreased the number of branched roots, also causing the root tips to become brown and shorter in length. In comparison with a control, the soluble protein content, the activities of SOD and POD in hairy roots cultures showed a trend of first increased and then gradually decreased, while the malondialdehyde (MDA) content significantly increased, when increasing the Cd concentrations. Cd concentration of 100 micromol/L or 300 micromol/L in combination with 10-30 mmol/L CaCl2 resulted in a decreased content of soluble protein and MDA in the hairy roots, but an enhanced SOD activity. The increased POD activities were observed when cultured in 100 micromol/L Cd and 10-30 mmol/L CaCl2 but decreased when cultured in 300 micromol/L Cd and 10-30 mmol/L CaCl2. Atomic Absorption Spectrometry determination showed that the Cd absorbed and adsorbed by the hairy roots increased along with the increase of Cd concentration. The exogenous addition of 10-30 mmol/L CaCl2 could reduce the toxicity of Cd. This was achieved on one hand by reducing the absorption of Cd, on the other hand by decreasing the lipid peroxidation through regulating the activities of antioxidant enzymes SOD and POD in the hairy roots.

Cadmium-induced oxidative damage and protective effects of N-acetyl-L-cysteine against cadmium toxicity in Solanum nigrum L.

The effects of cadmium (Cd) on the accumulation of hydrogen peroxide (H(2)O(2)) and antioxidant enzyme activities in roots of Solanum nigrum L. and the role of N-acetyl-l-cysteine (NAC) as a cysteine (Cys) donor against Cd toxicity were investigated. Cd at 50 and 200 microM significantly increased the contents of thiobarbituric acid-reactive substances (TBARS), the production of H(2)O(2) and superoxide anion (O(2)(-)), and the activities of catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione peroxidase (GSH-Px), glutathione reductase, and superoxide dismutase. Experiments with diphenylene iodonium as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NaN(3) as an inhibitor of peroxidase showed that the major source of Cd-induced reactive oxygen species in the roots may include plasma membrane-bound NADPH oxidase and peroxidase. In addition, the effects of NAC on plant growth, antioxidant enzyme activity, and non-protein thiol content were analyzed. Under Cd stress, the addition of 500 microM NAC decreased the contents of TBARS and production of H(2)O(2) and O(2)(-), but increased levels of Cys and reduced glutathione (GSH), phytochelatins, and activity of GSH-Px in roots. These results suggest that NAC could protect plants from oxidative stress damage, and this protection seems to be performed via increased GSH biosynthesis. Furthermore, NAC treatment also increased the contents of protein thiols in S. nigrum roots. By using size-exclusion chromatography, we found involvement of NAC in the Cd tolerance mechanism through increased biosynthesis of Cd-binding proteins.

A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling.

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS read-out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI-TOF-MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read-out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI-TOF-MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.