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Solanaceae plants produce two major classes of valuable sterol-derived natural products-steroidal glycoalkaloids and steroidal saponins-from a common cholesterol precursor. Attempts to heterologously produce these molecules have consistently failed, although the genes responsible for each biosynthetic step have been identified. Here we identify a cellulose synthase-like protein, an unexpected biosynthetic component that interacts with the early pathway enzymes, enabling steroidal scaffolds production in plants. Moreover, knockout of this gene in black nightshade, Solanum nigrum , resulted in plants lacking both steroidal alkaloids and saponins. Unexpectedly, these knockout plants also revealed that steroidal saponins deter serious agricultural insect pests. This discovery provides the missing link to engineer these high-value steroidal molecules and also pinpoints the ecological role for steroidal saponins.

Cadmium (Cd) is a nonessential element in plants and has adverse effects on the growth and development of plants. However, the molecular mechanisms of Cd phytotoxicity, tolerance and accumulation in hyperaccumulators Solanum nigrum L. has not been well understood. Here, physiology, transcriptome, and metabolome analyses were conducted to investigate the influence on the S. nigrum under 0, 25, 50, 75 and 100 µM Cd concentrations for 7 days. Pot experiments demonstrated that compared with the control, Cd treatment significantly inhibited the biomass, promoted the Cd accumulation and translocation, and disturbed the balance of mineral nutrient metabolism in S. nigrum, particularly at 100 µM Cd level. Moreover, the photosynthetic pigments contents were severely decreased, while the content of total protein, proline, malondialdehyde (MDA), H 2 O 2 , and antioxidant enzyme activities generally increased first and then slightly declined with increasing Cd concentrations, in both leaves and roots. Furthermore, combined with the previous transcriptomic data, numerous crucial coding-genes related to mineral nutrients and Cd ion transport, and the antioxidant enzymes biosynthesis were identified, and their expression pattern was regulated under different Cd stress. Simultaneously, metabolomic analyses revealed that Cd treatment significantly changed the expression level of many metabolites related to amino acid, lipid, carbohydrate, and nucleotide metabolism. Metabolic pathway analysis also showed that S. nigrum roots activated some differentially expressed metabolites (DEMs) involved in energy metabolism, which may enhance the energy supply for detoxification. Importantly, central common metabolism pathways of DEGs and DEMs, including the "TCA cycle", "glutathione metabolic pathway" and "glyoxylate and dicarboxylate metabolism" were screened using conjoint transcriptomics and metabolomics analysis. Our results provide some novel evidences on the physiological and molecular mechanisms of Cd tolerance in hyperaccumulator S. nigrum plants., (© 2024. The Author(s).)

Virus infections cause devastative economic losses for various plant species, and early diagnosis and prevention are the most effective strategies to avoid the losses. Exploring virus genomic evolution and constructing virus infectious cDNA clones is essential to achieve a deeper understanding of the interaction between host plant and virus. Therefore, this work aims to guide people to better prevent, control, and utilize the youcai mosaic virus (YoMV). Here, the YoMV was found to infect the Solanum nigrum under natural conditions. Then, an infectious cDNA clone of YoMV was successfully constructed using triple-shuttling vector-based yeast recombination. Furthermore, we established phylogenetic trees based on the complete genomic sequences, the replicase gene, movement protein gene, and coat protein gene using the corresponding deposited sequences in NCBI. Simultaneously, the evolutionary relationship of the YoMV discovered on S. nigrum to others was determined and analyzed. Moreover, the constructed cDNA infectious clone of YoMV from S. nigrum could systematically infect the Nicotiana benthamiana and S. nigrum by agrobacterium -mediated infiltration. Our investigation supplied a reverse genetic tool for YoMV study, which will also contribute to in-depth study and profound understanding of the interaction between YoMV and host plant.

Solanum nigrum, which belongs to the Solanaceae family, is an essential plant for food and medicine. It has many important secondary compounds, including glycoproteins, glycoalkaloids, polyphenolics, and anthocyanin-rich purple berries, as well as many ideal characteristics such as self-fertilization, a short life cycle and a small genome size that make it a potential model plant for the study of secondary metabolism and fruit development. In this study, we report a highly efficient and convenient tissue culture, transformation and genome editing method for S. nigrum using leaf segments after 8 weeks of tissue culture, with a required period from transformation initiation to harvest of about 3.5 months. Our results also show multi-shoot regeneration per leaf segment and a 100% shoot regeneration efficiency in a shoot regeneration medium. Moreover, over 82% of kanamycin-resistant plants exhibited strong green fluorescence marker protein expression, with genetic integration confirmed by PCR results and green fluorescence protein expression in their T 1 progeny. Furthermore, we successfully applied this transformation method to achieve an average of 83% genome editing efficiency of SnMYB1, a gene involved in regulating the anthocyanin biosynthetic pathway of S. nigrum in response to missing nutrients. Taken together, the combination of highly efficient tissue culture, transformation and genome editing systems can provide a powerful platform for supporting fundamental research on the molecular mechanisms of secondary metabolism, fruit development, and production of important compounds by biotechnology., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Vacuole compartmentalization plays an important role in the storage of heavy metals in hyperaccumulators. Is the vacuolar compartmentation a simple shielding process or a dynamic process that continuously consumes cell sap resources? How does glutathione affect the process of vacuolar compartmentalization? These unknown questions are very important to understand the mechanism of vacuole compartmentalization and can provide a guide for the design of hyperaccumulator plants by genetic engineering. Therefore, this study explored the enzyme activities, total cadmium, Cd 2+ , glutathione, oxidized glutathione, and reactive oxygen species contents in protoplasts and vacuoles of leaf cells in Solanum nigrum L. through subcellular separation. The results showed that vacuolar compartmentalization was a dynamic process that actively induced the related substances produced by cell sap to enter the vacuole for detoxification. When regulating the decreased glutathione content with buthionine sulfoximine, the total cadmium and combined cadmium in protoplasm decreased significantly, but the vacuole still maintained a high proportion of cadmium content and stable ROS content, which indicated that various external resources were preferentially used to maintain cadmium storage and homeostasis in vacuole rather than outside vacuole. These findings could guide the use of genetic engineering to design hyperaccumulator plants., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2022 Zhishuai Li et al.)

Cadmium (Cd) triggers molecular alterations in plants, perturbs metabolites and damages plant growth. Therefore, understanding the molecular mechanism underlying the Cd tolerance in plants is necessary for assessing the persistent environmental impact of Cd. In this study, Solanum nigrum was selected as the test plant to investigate changes in biomass, Cd translocation, cell ultrastructure, metabolites and genes under hydroponic conditions. The results showed that the plant biomass was significantly decreased under Cd stress, and the plant has a stronger Cd transport capability. Transmission electron microscopy revealed that increased Cd concentration gradually damaged the plant organs (roots, stems and leaves) cell ultrastructure, as evidenced by swollen chloroplasts and deformed cell walls. Additionally, metabolomics analyses revealed that Cd stress mainly affected seven metabolism pathways, including 19 differentially expressed metabolites (DEMs). Moreover, 3908 common differentially expressed genes (DEGs, 1049 upregulated and 2859 downregulated) were identified via RNA-seq among five Cd treatments. Meanwhile, conjoint analysis found several DEGs and DEMs, including laccase, peroxidase, D-fructose, and cellobiose etc., are associated with cell wall biosynthesis, implying the cell wall biosynthesis pathway plays a critical role in Cd detoxification. Our comprehensive investigation using multiple approaches provides a molecular-scale perspective on plant response to Cd stress., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Black nightshade (Solanum nigrum) belongs to the Solanaceae family and is used as a medicinal herb with health benefits. It has been reported that the black nightshade plant contains various phytochemicals that are associated with antitumor activities. Here we employed a genetic approach to study the effects of overexpression of Arabidopsis thaliana production of anthocyanin pigment 1 (AtPAP1) in black nightshade. Ectopic expression of AtPAP1 resulted in enhanced accumulation of anthocyanin pigments in vegetative and reproductive tissues of the transgenic plants. Analysis of anthocyanin revealed that delphinidin 3-O-rutinoside-5-O-glucoside, delphinidin 3,5-O-diglucoside, delphinidin 3-O-rutinoside, petunidin 3-O-rutinoside ( cis - p -coumaroyl)-5-O-glucoside, petunidin 3-(feruloyl)-rutinoside-5-glucoside, and malvidin 3-(feruloyl)-rutinoside-5-glucoside are highly induced in the leaves of AtPAP1 overexpression lines. Furthermore, ectopic expression of AtPAP1 evoked expression of early and late biosynthetic genes of the general phenylpropanoid and flavonoid pathways that include phenylalanine ammonia-lyase ( PAL ), cinnamate-4-hydroxylase ( C4H ), 4-coumarate CoA ligase ( 4CL ), chalcone isomerase ( CHI ), and quinate hydroxycinnamoyl transferase ( HCT ), which suggests these genes might be transcriptional targets of AtPAP1 in black nightshade. Concomitantly, the total content of anthocyanin in the transgenic black nightshade plants was higher compared to the control plants, which supports phenotypic changes in color. Our data demonstrate that a major anthocyanin biosynthetic regulator, AtPAP1, can induce accumulation of anthocyanins in the heterologous system of black nightshade through the conserved flavonoid biosynthesis pathway in plants., Competing Interests: The authors declare no competing financial interest.

Plant immunity has mainly been studied under controlled conditions, limiting our knowledge regarding the regulation of immunity under natural conditions where plants grow in association with multiple microorganisms. Plant pathology theory, based on laboratory data, predicts complex biochemical plant-pathogen interactions leading to coevolution of pathogen infectivity vs. plant recognition of microbes in multiple layers over time. However, plant immunity is currently not evaluated in relation to ecological time-scales and field conditions. Here we report status of immunity in plants without visible disease symptoms in wild populations of nightshades, Solanum dulcamara and Solanum nigrum, and in agricultural fields of potato, Solanum tuberosum. We analysed presence of pathogenesis-related proteins in over 500 asymptomatic leaf samples collected in the field in June, July and August over three years. Pathogenesis-related proteins were present in only one-third of the collected samples, suggesting low activity of the immune system. We could also detect an increase in pathogenesis-related proteins later in the growing season, particularly in S. tuberosum. Our findings, based on pathogenesis-related protein markers, indicate major gaps in our knowledge regarding the status and regulation of plant immunity under field conditions., Competing Interests: The authors have declared that no competing interests exist.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Solanum nigrum fruits have been conventionally used in beverages due to their nutritional substances such as minerals, vitamins, amino acids, proteins, sugars, polyphenols, and anthocyanins. The characterization of components and regulatory mechanism of anthocyanins in S. nigrum fruits have rarely been reported. In this study, we determined that the peel and flesh of S. nigrum fruits shared similar HPLC profiles but different contents and total antioxidant activities for anthocyanins. After an efficient purification method, mainly including extraction with pH 1.0 distilled water and then desorption with pH 1.0 95% ethanol after a DM-130 resin adsorption step to obtain more pure anthocyanin extracts, the purity of anthocyanins extracted from S. nigrum fruits reached 56.1%. Moreover, eight anthocyanins from S. nigrum fruit were identified with HPLC-MS/MS for the first time. A typical R2R3-MYB transcription factor gene, SnMYB , was also cloned for the first time by rapid amplification of cDNA ends (RACE)-PCR from S. nigrum. Moreover, the contents of anthocyanins were shown to correlate well ( r = 0.93) with the expression levels of SnMYB gene during the fruit's developmental stages. Most significantly, SnMYB gene successfully produced high anthocyanin content (1.03 mg/g) when SnMYB gene was transiently expressed in tobacco leaves. Taken together, S. nigrum fruits are a promising resource for anthocyanin extraction, and SnMYB gene is an activator that positively regulates anthocyanin biosynthesis in S. nigrum., Competing Interests: The authors declare no conflict of interest.

In this study, complete chloroplast genome of Solanum nigrum, a wild relative of potato and tomato being important for its medicinal features, was sequenced. The genome size is 154 671 bp in length, with 40% GC content. A pair of inverted repeats, IRa and IRSb, were separated by a large single-copy region and a small single-copy region of 82 315 bp and 33 411 bp, respectively. The genome harbored 73 protein-coding genes, 36 tRNA genes, and three rRNA genes. The evolutionary relationships in our phylogenetic analysis revealed that S. nigrum is closer to S. tuberosum when compared with those of known relatives' species belonging to Solanum genus.

Key Message: SnYSL3 encodes a plasma-localized transporter delivering various metal-nicotianamine complexes. The expression of SnYSL3 is up-regulated by excess Cd, suggesting an important role for SnYSL3 in response to Cd stress. The Yellow Stripe-Like (YSL) transporters have been proposed to participate in metal uptake and long-range transport in model plants. In this study, we isolated and characterized a novel member of the YSL gene family, SnYSL3, from the cadmium hyperaccumulator Solanum nigrum. SnYSL3 was constitutively expressed and encodes a plasma membrane-localized protein. In situ RNA hybridization localized the SnYSL3 transcripts predominantly in vascular tissues and epidermal cells of the roots and stems, while in leaves, the mRNA levels were high in the vasculature. The SnYSL3 expression level was up-regulated by excess Cd, excess Fe and Cu deficiency. Heterologous expression of SnYSL3 in yeast revealed that SnYSL3 transports nicotianamine complexes containing Fe(II), Cu, Zn and Cd. SnYSL3 overexpression in Arabidopsis thaliana decreased Fe and Mn concentrations in the roots and increased the root-to-shoot translocation ratios of Fe and Mn. Under Cd exposure, the transgenic plants showed increased translocation ratios of Fe and Cd, but no difference was observed in Mn translocation from roots to shoots between the transgenic and wild-type lines. Although the accurate function of SnYSL3 remains to be confirmed, these results suggest that SnYSL3 is a transporter delivering a broad range of metal-nicotianamine complexes and is potentially important for the response to heavy metal stress, especially due to Cd and Fe.

Different ecotypes of crop hybridization can produce heterosis effects and have wide applications in plant breeding. In this study, seedlings of cadmium (Cd) hyperaccumulator Solanum nigrum were collected from two different climate-ecology regions of the western Sichuan Basin, China, to carry out reciprocal hybridizing and to study the Cd accumulation characteristics of F1 hybrids of S. nigrum. In the two pot experiments (high and low soil Cd concentration), the biomass and Cd extraction of reciprocal hybridizing F1 hybrids were higher than those of the parents, but the Cd content in different organs was lower than those of the parents. These results indicate that the biomass and Cd extraction of F1 hybrids show over-parent heterosis, and the Cd content shows hybrid weakness. In the field experiment, the variety of the biomass, Cd content, and Cd extraction of reciprocal hybridizing F1 hybrids were the same as the pot experiments, and the Cd extraction by shoots of reciprocal hybridizing F1 hybrids increased by 17.20 and 23.08 %, relative to the two higher parents. Therefore, the reciprocal hybridizing S. nigrum of different climate-ecology regions could be efficiently used to improve the phytoremediation ability of S. nigrum to Cd-contaminated soil.

We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd(2+) in this study. The transformed yeast strains showed much higher resistance ability to Cd(2+) compared with the control. Strikingly, their Cd(2+) accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd(2+) under a wide range of pH levels, from 3 to 7, without disturbing the Cu(2+) and Hg(2+). Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd(2+)-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants.

Lunasin, a 44 amino acid soybean bioactive peptide, exhibits anticancer and anti-inflammatory properties. All soybean varieties that have been examined contain lunasin. It has also been reported in a few other plant species including amaranth, black nightshade, wheat, barley, rye, and triticale. Interestingly, detailed searches of transcriptome and DNA sequence databases of cereals failed to identify lunasin-coding sequences, raising questions about the authenticity of lunasin in cereals. To clarify the presence or absence of lunasin in cereals and other plant species, an immunological investigation was conducted utilizing polyclonal antibodies raised against the first 20 amino acid N-terminal peptide (SKWQHQQDSCRKQLQGVNLT) and a 15 amino acid C-terminal peptide (CEKHIMEKIQGRGDD) of lunasin. Protein blot analyses revealed the presence of proteins from several plants that reacted against the lunasin N-terminal peptide antibodies. However, the same proteins failed to react against the lunasin C-terminal peptide antibodies. These results demonstrate that peptides identical to soybean lunasin are absent in seeds of diverse plants examined in this study.

A new osmotin-like protein gene from Solanum nigrum L. var indica (SindOLP) was cloned and overexpressed in Escherichia coli. The full-length intron-less gene is 744 bp, encoding a mature protein of 247 amino acids with a molecular mass of 26 kDa. The protein has an N-terminal cleavable signal sequence of 21 amino acids. There is the Thaumatin family signature pattern, with one each of amidation, N-myristoylation, casein kinase II phosphorylation, tyrosine kinase phosphorylation, and protein kinase C phosphorylation sites. Hydropathy plot showed that it has six transmembrane helices. It has antifungal activity and can permeabilize fungal hyphae and spores. SindOLP is most active at pH 8, 25 °C and its antifungal activity is retained after 75 °C for 30 min. SindOLP inhibits fungal spore germination. The protein however lacks glucanase activity. The potential for SindOLP in developing fungus-resistant, transgenic crops is discussed.

Background: Drought is by far the most important environmental factor contributing to yield losses in crops, including soybeans [Glycine max (L.) Merr.]. To address this problem, a gene that encodes an osmotin-like protein isolated from Solanum nigrum var. americanum (SnOLP) driven by the UBQ3 promoter from Arabidopsis thaliana was transferred into the soybean genome by particle bombardment., Results: Two independently transformed soybean lines expressing SnOLP were produced. Segregation analyses indicated single-locus insertions for both lines. qPCR analysis suggested a single insertion of SnOLP in the genomes of both transgenic lines, but one copy of the hpt gene was inserted in the first line and two in the second line. Transgenic plants exhibited no remarkable phenotypic alterations in the seven analyzed generations. When subjected to water deficit, transgenic plants performed better than the control ones. Leaf physiological measurements revealed that transgenic soybean plants maintained higher leaf water potential at predawn, higher net CO2 assimilation rate, higher stomatal conductance and higher transpiration rate than non-transgenic plants. Grain production and 100-grain weight were affected by water supply. Decrease in grain productivity and 100-grain weight were observed for both transgenic and non-transgenic plants under water deficit; however, it was more pronounced for non-transgenic plants. Moreover, transgenic lines showed significantly higher 100-grain weight than non-transgenic plants under water shortage., Conclusions: This is the first report showing that expression of SnOLP in transgenic soybeans improved physiological responses and yield components of plants when subjected to water deficit, highlighting the potential of this gene for biotechnological applications.

Peanut (Arachis hypogaea L.) is the sixth most important oil seed crop in the world. Yield loss due to Cercospora leaf spot (early and late leaf spots) is a serious problem in cultivating this crop. Non-availability of resistant genes within crossable germplasms of peanut necessitates the use of a genetic engineering strategy to develop genetic resistance against various biotic stresses. The pathogenesis-related (PR) proteins are a group of plant proteins that are toxic to invading fungal pathogens, but are present in trace amounts in plants. The PR proteins, PR-5 and defensins, are potent antifungal proteins. A double gene construct with SniOLP (Solanum nigrum osmotin-like protein) and Rs-AFP2 (Raphanus sativus antifungal protein-2) genes under separate constitutive 35S promoters was used to transform peanut plants. Transgenic peanut plants expressing the SniOLP and Rs-AFP2 genes showed enhanced disease resistance to late leaf spot based on a reduction in number and size of lesions on leaves and delay in the onset of Phaeoisariopsis personata leaf spot disease. PCR, RT-PCR, and Southern hybridization analyses confirmed stable integration and expression of these genes in peanut transgenics. The results demonstrate the potential of SniOLP and Rs-AFP2 genes in developing late leaf spot disease resistance in transgenic peanut.

• Solanum nigrum is a cadmium (Cd) accumulator, whereas Solanum torvum is a low Cd-accumulating plant. The molecular mechanisms that are responsible for differential cadmium (Cd) accumulation in the two Solanum species are poorly understood. • Here, grafting experiments confirmed that increased Cd loading into the root xylem was responsible for the differential Cd accumulation in the two Solanum species. An iron (Fe) supply assay indicated that low Fe accumulation in S. torvum leaves is related to its Cd sensitivity. • Transcriptome analyses revealed higher expression of the genes that encode several metal transporters as well as antioxidant-related genes, and several organic and amino acid biosynthesis/metabolism-related genes in Cd-treated S. nigrum. Our data also indicated that the different responsive mechanisms of the transporter genes to Fe deficiency might be responsible for differential uptake and redistribution of metals in the two Solanum species • These results form a basis upon which to further explore the molecular mechanisms of Cd accumulation and tolerance, and provide an insight into novel strategies that can be used for phytoremediation and food safety., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

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.

Some heavy metals (HM) are highly reactive and consequently can be toxic to living cells when present at high levels. Consequently, strategies for reducing HM toxicity in the environmental must be undertaken. This work focused on evaluating the Nickel (Ni) accumulation potential of the hyperaccumulator Solanum nigrum L., and the participation of metallothioneins (MT) in the plant Ni homeostasis. Metallothioneins (MT) are gene-encoded metal chelators that participate in the transport, sequestration and storage of metals. After different periods of exposure to different Ni concentrations, plant biometric and biochemical parameters were accessed to determine the effects caused by this pollutant. Semi-quantitative RT-PCR reactions were performed to investigate the specific accumulation of MT-related transcripts throughout the plant and in response to Ni exposure. The data obtained revealed that Ni induced toxicity symptoms and accumulated mostly in roots, where it caused membrane damage in the shock-treated plants, with a parallel increase of free proline content, suggesting that proline participates in protecting root cells from oxidative stress. The MT-specific mRNA accumulation analysis showed that MT2a- and MT2d-encoding genes are constitutively active, that Ni stimulated their transcript accumulation, and also that Ni induced the de novo accumulation of MT2c- and MT3-related transcripts in shoots, exerting no influence on MT1 mRNA accumulation. These results strongly suggest the involvement of MT2a, MT2c, MT2d and MT3 in S. nigrum Ni homeostasis and detoxification, this way contributing to the clarification of the roles the various types of MTs play in metal homeostasis and detoxification in plants., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C(19)-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C(19)-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA(1) content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA(1) content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth.

Jasmonate signalling plays a central role in activating the plethora of responses that are elicited by herbivory. Solanum nigrum plants silenced in the expression of genes involved in jasmonic acid biosynthesis (irlox3), conjugation (irjar4) and perception (ircoi1) were used to study the function of these genes in the field and in the regulation of transcriptional and metabolic responses. In the field, damage from Noctuidea larvae was four- to fivefold higher on irlox3 and ircoi1 than on wild-type (WT) plants, whereas damage to irjar4 plants was similar to WT levels. Damage rates reflected plant survival rates; fewer irlox3 (78%) and ircoi1 (22%) plants survived compared with irjar4 and WT plants of which all plants survived. Gene expression profiling in leaves 3 h after simulated herbivory revealed differential regulation of ∼700 genes in irlox3 and ircoi1 plants but of only six genes in irjar4 compared with WT plants. Surprisingly, transcriptional responses were not reflected in metabolomic responses; 48 h after simulated herbivory, irjar4 plants showed a 50% overlap in their metabolic profile with ircoi1 plants. Together, these results reveal that SnJAR4 does not play a direct role in herbivore defence, but suggests that SnJAR4 is involved in responses other than those to herbivory., (© 2011 Blackwell Publishing Ltd.)

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development.

• 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., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Solanaceaeous taxa produce diverse peptide serine proteinase inhibitors (SPIs), known antidigestive defenses that might also control endogenous plant proteases. If and how a plant coordinates and combines its different SPIs for the defense against herbivores and if these SPIs simultaneously serve developmental functions is unknown. We examine Solanum nigrum's SPI profile, comprising four different active inhibitors, of which the most abundant proved to be novel, to understand their functional specialization in an ecological context. Transcript and activity characterization revealed tissue-specific and insect-elicited accumulation patterns. Stable and transient gene silencing of all four SPIs revealed different specificities for target proteinases: the novel SPI2c displayed high specificity for trypsin and chymotrypsin, while two other SPI2 homologs were highly active against subtilisin. In field and lab experiments, we found all four SPIs to display herbivore- and gene-specific defensive properties, with dissimilar effects on closely related species. However, we did not observe any clear developmental phenotype in SPI-silenced plants, suggesting that SPIs do not play a major role in regulating endogenous proteases under the conditions studied. In summary, specific single SPIs or their combinations defend S. nigrum against generalist herbivores, while the defense against herbivores specialized on SPI-rich diets requires other unknown defense mechanisms.

By using genetic transformation of Agrobacterium rhizogenes and liquid culture, induction and culture conditions for Solanum nigrum L. var. pauciflorum Liou hairy roots and its solasodine production and consumption changes of N resource and calcium in the medium during liquid culture were investigated. The results showed that hairy roots could be initiated from the cut edges of leaf explants 5 days after inoculation with the strain of A. rhizogenes ATCC15834. The percentage of rooted leaf explants 25 days after infection was more than 90%. Hairy roots could grow rapidly on solid or liquid growth regulator-free MS medium. The PCR amplification of rol genes and virC gene showed that rol genes of Ri plasmid of A. rhizogenes were integrated in the genome of transformed hairy roots of S. nigrum L var pauciflorum. The hairy roots could produce medicinal secondary metabolites solasodine and the amount of solasodine in the hairy root cultures reached a level of 582.05 microg/g dry weight and was 1.31 times as much as those in the untransformed roots. The hairy roots grew very slowly in 0-5 days in the liquid medium, then, very fast from 5 to 15 days. During liquid culture, NO3(-) and NH4(+) in the medium were gradually absorbed and utilized by hairy roots. NH4(+) -N of the medium was used up at day 15 of the culture, while NO3(-) in the medium was not used up, the content of which was 44.7% of the initial amount. Ca2+ of the medium was gradually absorbed and utilized during liquid culture and it was not used up at day 25, the content of which was still 43.5% of the initial amount. The results presented here had provided the possibilities on how to prepare optimum medium for large scale cultivation and production of solasodine from S. nigrum L. var. pauciflorum hairy roots.

We extend Ryan's seminal work on the 18-amino acid polypeptide systemin in tomato's (Solanum lycopersicum) systemic wound response to the closely related solanaceous species Solanum nigrum. We compared wild-type plants to plants transformed with an inverted repeat prosystemin construct (IRSys) to silence the expression of the endogenous S. nigrum prosystemin gene. In wild-type plants elicited with wounding + oral secretions from Manduca sexta larvae, trypsin-proteinase inhibitors (TPIs) accumulated even though prosystemin transcripts were down-regulated. Neither reducing the endogenous systemin levels by RNAi nor complementing the plants with systemin by exogenously supplying the polypeptide through excised stems significantly increased TPI activity, indicating that systemin and TPIs are not correlated in S. nigrum. The performance of two herbivore species from two feeding guilds, M. sexta larvae and Myzus persicae nicotianae, did not differ between wild-type and IRSys plants, demonstrating that varying endogenous systemin levels do not alter the direct defenses of S. nigrum. Field experiments with wild-type and IRSys plants and the flea beetle Epitrix pubescens supported these glasshouse data. That levels of oral secretion-elicited jasmonic acid did not differ between wild-type and IRSys plants suggests that systemin is unlikely to mediate jasmonate signaling in S. nigrum as it does in tomato. We conclude that the tomato-homologous polypeptide does not mediate direct defense responses in S. nigrum.

The technology of obtaining the tobacco plants possessing the hereditary changes in the photosynthesis pigments accumulation during development using exogenous DNA has been elaborated. The plants possessing changes in proportion of chlorophylls content in the leave tissues, inherited elevated carotenoid content and altered proportion of carotene and xanthophylls, luteine and violaxanthine have been obtained by action of the salt-tolerant nightshade (Solanum nigrum L.) DNA and the DNAs of pCAMVNEO, pTi8628 plasmids on the tobacco cultivar Krupnolystny 20 (Large-leaf) germinating seeds. These plants have simultaneously the useful features--accelerated development, early blooming phenotype and higher productivity. Possible mechanisms emphasized such inherited biochemical changes have been discussed.

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.

Previous studies have shown that suspension-cultured cells of Solanum genotypes with various polygenic resistances to Phytophthora infestans differed in activities of early oxidative processes in response to culture filtrate (CF) from this pathogen. These studies have now been extended by analysing production of reactive oxygen species (ROS), lipid peroxidation and Lipoxygenase (LOX, E.C.1.13.11.12) activity induced by CF in detached leaves of S. tuberosum cv Bzura and clone H-8105, polygenically resistant and susceptible, respectively, as well as S. nigrum, nonhost, completely resistant. The relative increase in the ROS production was higher in the susceptible clone H-8105 than in both resistant genotypes. Lipid peroxidation increased only in the nonhost S. nigrum. An increase in lipid peroxidation in S. nigrum leaves coincided with enhanced LOX activity. In both S. tuberosum genotypes, significant increases in LOX activity were delayed and unaccompanied by changes in the level of lipid peroxidation. LOX activity attained a higher level in both of the resistant genotypes than in the susceptible one. The present results suggest that the involvement of both ROS production and LOX activity in the defense strategy in Solanum species/P. infestans interactions.

Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum, we describe: (i) an Agrobacterium-based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions. Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants. Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

In breeding for resistance to late blight, ( Phytophthora infestans Mont. de Bary), an economically important disease affecting potatoes, the search for new sources of durable resistance includes the non-host wild Solanum species. The aim of this work was to evaluate the resistance to P. infestans in the somatic hybrids between S. nigrum L. and diploid potato clone ZEL-1136. Sixteen somatic hybrids, their fusion parents, and three standard potato cultivars were screened for resistance to P. infestans in two types of tests-on whole plants and detached leaves-with two highly aggressive and virulent isolates of P. infestans, US8 and MP322. In the whole plant assay, the foliage of the somatic hybrids showed no symptoms of infection, while the foliage of the potato fusion parent and the standard cultivars was infected with P. infestans. In the detached leaflet assay, the breaking-down of resistance of the S. nigrum L. parent and the variable response of individual hybrid clones were noted. Nine S. nigrum L. (+) ZEL-1136 hybrids showed a resistance that was significantly higher than that of S. nigrum, while six clones expressed a resistance to P. infestans similar to that of S. nigrum. The results confirm the effective transfer of late blight resistance of S. nigrum into its somatic hybrids with potato.

Somatic hybrids between the cultivated potato diploid hybrid clone, ZEL-1136, and hexaploid non-tuber-bearing wild species Solanum nigrum L. exhibiting resistance to Phytophthora infestans were regenerated after PEG-mediated fusion of mesophyll protoplasts. The objective was to transfer the late-blight resistance genes from the wild species into plants of the cultivated potato clone. From a total of 59 regenerants, 40 clones survived and have been maintained in vitro on hormone-free MS/2 medium. Thirty-two somatic hybrids were identified by their intermediate morphology (leaves of nigrum type and flowers of tuberosum type) and verified by flow cytometry and random amplified polymorphic DNA (RAPD) patterns. The RAPD analysis of nuclear DNA confirmed the hybrid nature of 29 clones. Flow cytometry revealed a wide range of ploidy in the generated hybrids, from nearly the tetra- to decaploid level. Most of the hybrid clones were stable in vitro, grew vigorously in soil, and set flowers and parthenocarpic berries. However, all of the flowering hybrids were male-sterile. Nine hybrid clones produced tuber-like structures in soil. The most vigorous flowering somatic hybrids were selected for assessment of the late-blight resistance.