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Several inhibitors of polyamine biosynthesis reduced mycelial growth of the fungus Ophiostoma ulmi (Buism.) Nannf. cultured on malt extract-agar medium. Growth inhibition was particularly evident when expressed in terms of fresh weight rather than colony diameter. Of the different drugs tested, the most effective was difluoromethylarginine (DFMA), while difluoromethylornithine (DFMO) and methylglyoxal(bis)guanyl-hydrazone (MGBG) plus cyclohexylamine (CHA) reduced growth only by 75 and 62% respectively. The growth inhibition by DFMO + DFMA, measured as colony diameter, was apparently reversed by putrescine (PUT), but not when expressed in terms of fresh weight. Spermidine (SPD) was the most abundant polyamine in control cultures, followed by PUT and spermine (SPM). PUT was no longer detectable 8 d after inoculation. On day 10, DFMO and DFMA, alone and in combination, caused a significant reduction in cellular SPD concentrations, while exogenously supplied PUT restored the levels of this polyamine to control values. MGBG + CHA caused a conspicuous accumulation of PUT and an approximately 50% reduction in SPD titres. DFMA, alone or in combination with DFMO and with or without PUT, led to increased cellular levels of SPM. The latter polyamine, but not PUT or SPD, strongly retarded growth when added to the growth medium. As suggested by the effectiveness of DFMA in inhibiting growth, arginine decarboxylase activity was shown to be prevalent over ornithine decarboxylase activity in this fungus.

• Change is reported in the biosynthetic and oxidative activity of hypersensitive (NN) and susceptible (nn) tobacco (Nicotiana tabacum) plants in response to tobacco mosaic virus (TMV). • Mature leaves of nn and NN tobacco were collected over 0–72 h as uninoculated controls or after inoculation with TMV or phosphate buffer (mock-inoculation). The polyamine response to inoculation was analysed by measuring activity and gene expression of S-adenosylmethionine decarboxylase (SAMDC), arginine-(ADC) and ornithine decarboxylases (ODC); incorporation of labelled putrescine; and activity of diamine oxidase (DAO). • In NN leaves SAMDC activity and transcript levels, and DAO activity increased in the TMV-inoculated plants but not in the other treatments; a two-fold increase in DAO activity was seen after 72 h. Both ADC and ODC activity increased in NN leaves at 72 h in TMV-inoculated plants; ADC mRNA increased with activity. The increase in SAMDC mRNA (24 h) preceded the rise in activity (72 h). [3H]putrescine added to NN leaves led to enhanced label recovery and incorporation into spermidine and spermine in TMV-inoculated plants. No significant changes in biosynthetic or oxidative activity occurred in nn plants. • After TMV inoculation, NN, unlike nn, tobacco plants upgrade polyamine synthesis and oxidation; this leads to changes in cellular components which might induce programmed cell death.

In indoor vertical farms, energy consumption represents a bottleneck for both a system’s affordability and environmental footprint. Although switching frequency (sf) represents a crucial factor in determining the efficacy of light emitting diodes (LED) lighting systems in converting electricity into light, the impact of sf is still underexplored. The aim of this work was to investigate the effect of LEDs sf on the productive and qualitative responses of lettuce (Lactuca sativa L.), also considering the resource use efficiency. Plants were grown for 14 days under red and blue LEDs (215 μmol m−2 s−1 and 16/8 h light/dark, with a red:blue ratio of 3) characterized by two different sf for the blue diode, namely high sf (850 kHz) and low sf (293 kHz). A fluorescent light (same light intensity and photoperiod) was included. LED sf did not alter plant morphological parameters, including fresh or dry biomass, leaf number, leaf area, or water use efficiency. A low sf increased the energy use efficiency (EUE) by 40% as compared to high sf. The latter enhanced the leaf antioxidant capacity, as a consequence of increased concentrations of caftaric and chicoric acids, isoquercetin, and luteolin, consistent with the upregulation of a few genes related to the biosynthetic pathway of phenolic compounds (4C3H and DFR). The study highlights that different sf may significantly affect the EUE as well as crop nutritional properties.

To further our understanding of the mechanisms governing salt stress responses and adaptation in halophytes, we explored morphological, metabolic, and gene expression responses to high salinity in quinoa (Chenopodium quinoa Willd). The main objective of this study was to analyze selected responsive genes in a time-course experiment to test for expression kinetics and to compare short-term salt-induced effects at the transcript level between two Chilean landraces belonging to different ecotypes. Quinoa genotypes exhibit a large variability in their responses to salinity, but it is not clear whether this is strictly related to the ecotype to which they belong. We tested this hypothesis by comparing the expression levels of genes involved in growth, ion homeostasis, abscisic acid (ABA) biosynthesis, perception, and conjugate cleavage, polyamine (PA) biosynthesis and oxidation, and proline biosynthesis as well as genes encoding ABA-dependent and −independent transcription factors. Landraces R49 (salares ecotype) and Villarrica (VR, coastal-lowlands ecotype) were analyzed from 0.5 to 120Âh after transfer to saline (300ÂmM NaCl) or non-saline (control) medium. All the genes, except CqSOS1 and CqNHX, were investigated here for the first time in quinoa under salt stress. Transcript levels were determined by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis. Germination, seedling growth, ABA, and PA contents were evaluated in parallel. Even though on saline medium germination was inhibited in VR but not in R49, seedling growth reduction at 120Âh was not substantially different in the two landraces. The ABA biosynthetic enzyme NCED was the most strongly salt-induced gene; ABA content was similarly enhanced (shoots) or unaffected (roots) in both R49 and VR. NaCl treatment also altered transcript levels of some PA metabolic enzymes and the PA profile leading to an enhanced ratio between the higher PAs and putrescine. All other genes also exhibited similar expression profiles in response to salinity in the two landraces especially in roots, while in shoots some differences were observed. Our results provide new information indicating that crucial salt adaptation strategies at the molecular level and in terms of ABA and PA contents are shared by the coastal-lowlands and salares landraces; however, the timing of the onset of transcriptional changes (e.g., NCED, ABF3, and RD22) may reflect genotype-dependent constitutive and/or inducible adaptive strategies.

Phytoremediation is a cost-effective and environment friendly in situ technique for the reclamation of heavy metal-polluted soils. The efficacy of this technique, which relies on tolerant plant species, can be improved by the use of chelating agents. A pot experiment was carried out to evaluate the phytoextraction and phytostabilisation capacities of a white poplar (Populus alba L.) clone named AL35 previously selected for its marked tolerance to copper (Cu) and zinc (Zn). Cuttings were grown on agricultural soil highly contaminated with Cu and Zn, in the presence or not (controls) of a chelant mixture (EDTA/EDDS) known to enhance metal bioavailability and, hence, uptake by plant roots, or the not yet investigated synthetic, highly biodegradable polyaspartic acid (PASP). Both chelant treatments improved the phytostabilisation of Cu and Zn in AL35 plants, whilst the phytoextraction capacity was enhanced only in the case of Cu. Considering that the effectiveness of PASP as phytostabilizer was comparable or better than that of EDTA/EDDS, the low cost of its large-scale chemical synthesis and its biodegradability makes it a good candidate for chelant-enhanced metal phytoextraction from soil while avoiding the toxic side-effects previously described for both EDTA and EDDS.

The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.

† Background and Aims It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant –fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil. † Methods In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal(Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription–PCR. Free and conjugated foliar PA concentrations were determined in parallel. † Results On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil. † Conclusions Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs.

In this work, we have analyzed the changes in the protein expression profile elicited by chromium (Cr) exposure in the freshwater green alga Pseudokirchneriella subcapitata, a well known bio-indicator of water pollution. We tested two experimental conditions, namely 0.2 and 1 ppm of potassium dichromate; this concentration range includes the environmentally-relevant concentrations. Results show that neither concentration of potassium dichromate tested inhibited algal growth. However, the proteomic approach allowed the identification of relevant modifications in protein expression. In fact, among 800 protein spots detected by two-dimensional electrophoresis, 16 Cr-regulated proteins, including predicted and novel ones, were identified using tandem mass spectromic protein analysis. The results demonstrate a Cr-specific action in altering several photosynthetic proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO activase, Light Harvesting Chla/b protein complex, and stress related Chla/b binding protein1. Although Cr toxicity with respect to photosynthesis has been already documented, here we have identified, for the first time, the target proteins of this toxicity. Cr also induced a modulation of some proteins involved in the metabolism of the amino acids glutamine, arginine and methionine. These data are supported by changes in cellular polyamine (PA) accumulation. Present findings provide new insight into the molecular mechanisms underlying Cr toxicity in P. subcapitata.

The gaseous environment inside culture vessels is an important factor affecting growth and differentiation of plant cell and tissue culture. Carbon dioxide (CO2) and ethylene (C2H4), in particular, play important roles. The effect of reduced gas exchange in sealed petri dishes has previously been shown to strongly inhibit adventitious shoot formation in cultured leaf explants of quince ‘BA29’, but has not been investigated in pear. In the present work, the accumulation of CO2 and C2H4 in petri dishes was quantified during the shoot induction and development phases in quince (Cydonia oblonga Mill.) ‘BA29’ and pear (Pyrus communis L.) ‘Conference’ that have a higher and lower regeneration capacity, respectively, on medium supplemented with 4.5 M thidiazuron (TDZ) and 5.4 M -naphthaleneacetic acid (NAA). In order to evaluate the specific role of C2H4 on the shoot-forming process, the effect of treatment with the inhibitor of C2H4 biosynthesis aminoethoxyvinylglycine (AVG) was also evaluated during the shoot induction phase. The results showed that AVG strongly reduced both C2H4 accumulation and the production rate of C2H4, without affecting CO2 accumulation, in both species, whereas it differentially affected regeneration capacity, enhancing it in quince and reducing it in pear. A time-course analysis of free and soluble conjugated polyamine (PA) titres was also performed in control vs. AVG-treated quince and pear explants during the induction phase. Quince explants exhibited a marked peak in free and conjugated spermidine on day-7 in culture, while pear explants did not. AVG did not alter PA levels or the PA profile in either species. The addition of AVG to the induction medium is recommended to improve shoot regeneration in quince 'BA 29' leaf explants in culture systems that prevent optimal gas exchanges. Instead, the genotype-specific low regeneration capacity of pear prevailed over the effect of AVG treatments in our experimental conditions.

It was previously shown that arbuscular mycorrhizal fungi (AMF) exert a significant improvement of growth in a tolerant white poplar (Populus alba L.) clone (AL35) grown on Cu- and Zn-polluted soil via foliar alterations in the levels of defence/stress-related transcripts and molecules. However, nothing is known about the epigenetic changes which occur during tolerance acquisition in response to heavy metals (HMs) in the same mycorrhizal vs. non-mycorrhizal poplar plants. In order to analyse the epigenome in leaves of AL35 plants inoculated or not with AMF and grown in a greenhouse on multimetal polluted or unpolluted soil, the Methylation Sensitive Amplification Polymorphism (MSAP) approach was adopted to detect cytosine DNA methylation. Modest changes in cytosine methylation patterns were detected at first sampling (4 months from planting), whereas extensive alterations (hypomethylation) occurred at second sampling (after 6 months) in mycorrhizal plants grown in the presence of HMs. The sequencing of MSAP fragments led to the identification of genes belonging to several Gene Ontology categories. Seven MSAP fragments, selected on the basis of DNA methylation status in treated vs control AL35 leaves at the end of the experiment, were analysed for their transcript levels by means of qRT-PCR. Gene expression varied in treated samples relative to controls in response to HMs and/or AMF inoculation; in particular, transcripts of genes involved in RNA processing, cell wall and amino acid metabolism were upregulated in the presence of AMF with or without HMs.

Quinoa (Chenopodium quinoa Willd.) is a highly salt-tolerant species subdivided into five ecotypes and exhibiting broad intra-specific differences in tolerance levels. In a greenhouse study, Chilean landraces belonging either to the salares (R49) or coastal lowlands (VI-1, Villarrica) ecotype with contrasting agro-ecological origins were investigated for their responses to high salinity. The effects of two levels of salinity, 100 (T1) and 300 (T2) mM NaCl, on plant growth and on some physiological parameters were measured. Leaf and root Na(+) accumulation differed among landraces. T2 reduced growth and seed yield in all landraces with maximum inhibition relative to controls in R49. Salinity negatively affected chlorophyll and total polyphenol content (TPC) in VI-1 and Villarrica but not R49. Germination on saline or control media of seeds harvested from plants treated or not with NaCl was sometimes different; the best performing landrace was R49 insofar as 45-65% of seeds germinated on 500 mM NaCl-containing medium. In all landraces, average seedling root length declined strongly with increasing NaCl concentration, but roots of R49 were significantly longer than those of VI-1 and Villarrica up to 300 mM NaCl. Salt caused increases in seed TPC relative to controls, but radical scavenging capacity was higher only in seeds from T2 plants of R49. Total SDS-extractable seed proteins were resolved into distinct bands (10-70 kDa) with some evident differences between landraces. Salt-induced changes in protein patterns were landrace-specific. The responses to salinity of the salares landrace are discussed in relation to its better adaptation to an extreme environment.

Neochloris oleoabundans (Chlorophyta) is widely considered one of the most promising microalgae for biotechnological applications. However, the large-scale production of microalgae requires large amounts of water. In this perspective, the possibility of using exhausted growth media for the re-cultivation of N. oleoabundans was investigated in order to simultaneously make the cultivation more economically feasible and environmentally sustainable. Experiments were performed by testing the following media: autotrophic exhausted medium (E+) and mixotrophic exhausted medium after cultivation with glucose (EG+) of N. oleoabundans cells grown in a 20-L photobioreactor (PBR). Both exhausted media were replenished with the same amounts of nitrate and phosphate as the control brackish medium (C). Growth kinetics, nitrate and phosphate consumption, photosynthetic pigments content, photosynthetic efficiency, cell morphology, and lipid production were evaluated. Moreover, the free fatty acid (FFA) composition of exhausted media and the polyamine (PA) concentrations of both algae and media were analyzed in order to test if some molecules, released into the medium, could influence algal growth and metabolism. Results showed that N. oleoabundans can efficiently grow in both exhausted media, if appropriately replenished with the main nutrients (E+ and EG+), especially in E+ and to the same extent as in C medium. Growth promotion of N. oleoabundans was attributed to PAs and alteration of the photosynthetic apparatus to FFAs. Taken together, results show that recycling growth medium is a suitable solution to obtain good N. oleoabundans biomass concentrations, while providing a more sustainable ecological impact on water resources.

S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine, namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on polyamine metabolism, ethylene production and plant morphology was analysed in primary transformants (R0) and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls, all of which were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analysis, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and polyamine titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with polyamines the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and polyamine titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of polyamine conjugation was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.

In the present study callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of different growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthalenacetic acid (NAA), kinetin and 2,4 dichlorophenoxyacetic acid (2,4D), a yellowish and friable callus was obtained from 90% of cotyledon explants, while no callus appeared from other portions of the seedlings. The time course of callus growth and secondary metabolite accumulation was followed after subculturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing NAA and a higher concentration of 2,4-D compared to maintenance medium gave the highest stimulation of callus growth, suggesting that this auxin has a critical role in influencing growth. Addition of an organic nitrogen source such as casein hydrolysate also had a positive effect on growth. HPLC analysis of methanol extractable secondary metabolites from Z. stenophyllum calli grown on different media showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calli grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products. Under the same hormonal conditions, the presence or absence of casein also altered the metabolites produced, both qualitatively and quantitatively. In response to elicitation by methyl jasmonate (MJ), metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium turned out to be very important in determining the response to MJ. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients and elicitors when attemps are made to enhance secondary metabolite accumulation in in vitro cultures.

Corolla life span of undetached flowers of Nicotiana tabacum was divided into stages from the closed corolla (stage 1) through anthesis (stage 5) to death (stage 9). Senescence began around stage 6 in the proximal part, concomitantly with DNA laddering. Nuclear blebbing, DNA laddering, cell wall modification, decline in protein, water, pigment content and membrane integrity were observed during senescence and PCD. Transglutaminase activity was measured as mono- and bis-derivatives of putrescine (mono-PU; bis-PU) and bis-derivatives of spermidine (bis-SD). Bis-derivatives decreased with the progression of senescence, while mono-PU increased during early senescence; derivatives were present in different amounts in the proximal and distal parts of the corolla. In excised flowers, exogenous spermine delayed senescence and PCD, and caused an increase in free and acid-soluble conjugated PA levels. Bis-PU was the most abundant PA-derivative before DNA laddering stage; thereafter, bis-PU generally decreased and mono-PU became the most abundant derivative.

Poplar is a suitable species for phytoremediation, able to tolerate high concentrations of heavy metals (HMs). Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with the roots of most land plants; they improve nutrient uptake and enhance phytoextraction of HMs while alleviating stress in the host plant. This review summarizes previous results from field and greenhouse studies conducted by us and dealing with this topic. In a field trial on a highly Zn- and Cu-contaminated site, differences in plant survival and growth were observed among 168 clones originating from natural populations of Populus alba L. and Populus nigra L. from northern Italy. After two and a half years from planting, the density, activity and metabolic versatility of the culturable fraction of the soil bacteria in the HM-polluted field was higher in the soil close to where larger poplar plants were growing, in spite of comparable HM concentrations recorded in these soils. One well-performing clone of P. alba (AL35), which accumulated a higher concentration of both metals and had high foliar polyamine (PA) levels, was used for further investigation. In a greenhouse study, AL35 cuttings pre-inoculated with AMF (Glomus mosseae or Glomus intraradices) and then transferred to pots containing soil, collected from the HM-polluted site, displayed growth comparable to that of controls grown on unpolluted soil, in spite of higher Cu and Zn accumulation. Such plants also showed an overall up-regulation of metallothionein (MT) and PA biosynthetic genes, together with increased PA levels. A genome-wide transcriptomic (cDNA-AFLP) analysis allowed the identification of a number of genes, mostly belonging to stress-related functional categories of defense and secondary metabolism, that were differentially regulated in mycorrhizal vs. non mycorrhizal plants. A proteomic analysis revealed that, depending on sampling time, changes in protein profiles were differentially affected by AMF and/or HMs. It is concluded that soil-borne microorganisms affect plant performance on HM-polluted soil. In particular, mycorrhizal plants exhibited increased capacity for phytostabilization of HMs, together with improved growth. Their greater stress tolerance may derive from the protective role of PAs, and from the strong modulation in the expression profiles of stress-related genes and proteins.

Treatment of normal and Agrobacterium rhizogenes-transformed root cultures of Hyoscyamus muticus with three different auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and naphthaleneacetic acid (NAA), revealed that the response varied considerably among auxins, between transformed and normal roots, and depending on the parameter. In normal roots all three auxins provoked abundant branching, with IBA and NAA being the most effective at 2.5 and 0.5 μm, respectively, whereas IAA was most effective at low concentrations (0.05 and 0.1 μm). In transformed roots exogenously supplied auxins were generally inhibitory or, at best, without effect on growth and branching. Only 0.01 μm IAA significantly enhanced lateral root number, whereas at the higher concentrations IBA, although inhibitory, was the least effective auxin. In both root types IBA had little effect on primary root growth, but normal roots were more sensitive to IAA and NAA. These results suggest a different sensitivity to auxins of normal and transformed roots since there was no significant difference in endogenous free and conjugated IAA content nor in IAA uptake capacity. Ethylene production and biosynthesis were approximately threefold higher in hairy roots, but production could be stimulated up to tenfold that of control levels in normal roots by supplying NAA or 1-aminocyclopropane-1-carboxylic acid (ACC). Treatment with 2.5 μm NAA, but not IAA or IBA, also enhanced ethylene biosynthesis in normal roots but not in transformed ones. ACC and malonyl-1-aminocyclopropane-1-carboxylic acid accumulated to detectable levels only after treatment with an auxin (NAA).

The phytopathogenic actinomycete Rhodococcus fascians drives its host to form a nutrient-rich niche by secreting a mixture of cytokinins that triggers plant cell division and shoot formation. The discrepancy between the relatively low amount of secreted cytokinins and the severe impact of R. fascians infection on plant development has puzzled researchers for a long time. Polyamine and transcript profiling of wild-type and cytokinin receptor mutant plants revealed that the bacterial cytokinins directly stimulated the biosynthesis of plant putrescine by activating arginine decarboxylase expression. Pharmacological experiments showed that the increased levels of putrescine contributed to the severity of the symptoms. Thus, putrescine functions as a secondary signal that impinges on the cytokinin-activated pathway, amplifying the hormone-induced changes that lead to the formation of a leafy gall. Exogenous putrescine and treatment with polyamine biosynthesis inhibitors combined with transcript and polyamine analyses of wild-type and mutant plants indicated that the direct target of both the bacterial cytokinins and plant putrescine was the expression of D3-type cyclins. Hence, the activated d-type cyclin/retinoblastoma/E2F transcription factor pathway integrates both external and internal hormonal signals, stimulating mitotic cell divisions and inducing pathological plant organogenesis.

Chenopodium quinoa (Willd.) is an Andean plant showing a remarkable tolerance to abiotic stresses. In Chile, quinoa populations display a high degree of genetic distancing, and variable tolerance to salinity. To investigate which tolerance mechanisms might account for these differences, four genotypes from coastal central and southern regions were compared for their growth, physiological, and molecular responses to NaCl at seedling stage. Seeds were sown on agar plates supplemented with 0, 150 or 300mM NaCl. Germination was significantly reduced by NaCl only in accession BO78. Shoot length was reduced by 150mM NaCl in three out of four genotypes, and by over 60% at 300mM (except BO78 which remained more similar to controls). Root length was hardly affected or even enhanced at 150mM in all four genotypes, but inhibited, especially in BO78, by 300mM NaCl. Thus, the root/shoot ratio was differentially affected by salt, with the highest values in PRJ, and the lowest in BO78. Biomass was also less affected in PRJ than in the other accessions, the genotype with the highest increment in proline concentration upon salt treatment. Free putrescine declined dramatically in all genotypes under 300mM NaCl; however (spermidine+spermine)/putrescine ratios were higher in PRJ than BO78. Quantitative RT-PCR analyses of two sodium transporter genes, CqSOS1 and CqNHX, revealed that their expression was differentially induced at the shoot and root level, and between genotypes, by 300mM NaCl. Expression data are discussed in relation to the degree of salt tolerance in the different accessions.

Tocopherols are lipid-soluble compounds synthesised exclusively by photosynthetic organisms. In this study, in vitro callus cultures were established from two plants that are naturally rich in tocopherols, Amaranthus caudatus and Chenopodium quinoa, in order to examine whether callus cultures were able to produce these compounds at levels comparable to those observed in planta. In both species, cotyledon explants produced the best callus induction and, once established, callus cultures were grown under two different hormonal treatments to check for effects of growth and to induce chloroplast differentiation in the cells. A rapid differentiation of chloroplasts occurred only in C. quinoa cell aggregates grown in the presence of benzyladenine, leading to the production of a homogeneous green callus. In both species, only alpha-tocopherol was produced by callus cultures, although levels were much lower than in planta, and the production was not influenced by the hormonal conditions. Interestingly, cell cultures of the two species responded in different ways to methyl jasmonate (MJ). In A. caudatus cultures, treatment with 100 mum MJ increased the production of alpha-tocopherol up to fivefold, and the inductive effect was influenced by the hormonal composition of the medium. This increase in alpha-tocopherol was associated with a proportional increase in tyrosine aminotransferase (TAT) activity, one of the key enzymes involved in tocopherol biosynthesis. By contrast, in C. quinoa cultures, elicitation with MJ did not have any effect, neither on tocopherol production, nor on TAT activity. These results are discussed in relation to chloroplast differentiation and the interplay between jasmonates and phytohormones.

Callus cultures from several species of Passiflora were initiated in vitro, and their capacity to produce four glycosyl flavonoids (orientin, isoorientin, vitexin, isovitexin) was analysed. The aim of the present work was to examine the possible role of UV-B irradiation and elicitation with methyljasmonate (MJ) on the production of these compounds in callus cultures. All the species tested (P incarnata, P. quadrangularis, P edulis) formed friable callus from leaf explants after 4 weeks on medium supplemented with kinetin and 2,4-dichlorophenoxyacefic acid. Among them, R quadrangularis turned out to have a faster growth rate and a more friable texture, and was therefore chosen for experiments with elicitors. In callus cultures only small amounts of isoorientin were found, while the concentration of the other flavonoids was below the detection limit. UV-B irradiation of calluses was able to increase the production of all four glycosyl flavonoids. After a 7-day exposure of cultures to UV-B light, the production of isoorientin reached concentrations similar to those found in fresh leaves from glasshouse-grown plants. Elicitation with methyl jasmonate also enhanced orientin, vitexin and isovitexin concentrations, even though the stimulation was about 6-fold weaker for orientin and vitexin and about 40-fold for isovitexin, than that exerted by UV-B treatment. Callus cultures treated with the UV-B dose which most enhanced flavonoid production showed a higher antioxidant activity compared to untreated calluses, with an increase ranging from 28% to 76%. Results show that the secondary metabolite biosynthetic capacity of Passiflora tissue cultures can be enhanced by appropriate forms of elicitation. (c) 2007 Elsevier B.V All rights reserved.

Poplar is a good candidate for phytoremediation purposes because of its rapid growth, extensive root system, and ease of propagation and transformation; however its tolerance to heavy metals has not been fully investigated yet. In the present work, an in vitro model system with shoot cultures was used to investigate the tolerance to high concentrations of zinc (Zn) of a commercial clone (Villafranca) of Populus alba. Based on chlorophyll content (leaf chlorosis) and the rate of adventitious root formation from shoot cuttings as parameters of damage, 0.5-4mM zinc concentrations were all toxic albeit to different extents. Northern blot and reverse transcriptase (RT)-PCR analyses were used to examine the expression profiles of types 1, 2 and 3 PaMT genes in stems, leaves and roots of plants exposed to Zn treatments. In leaves, MT1 and MT3 mRNA levels were enhanced by Zn, while MT2 transcripts were not affected. The PaMT expression profiles were differentially affected by Zn in an organ-specific manner, and the relationship with Zn concentration and exposure time was rarely linear. The developmental and molecular data reveal that the in vitro model is a sensitive and reliable system to study heavy metal stress responses.

An in vitro model system of micropropagated shoots of the commercial clone ‘Villafranca’ of Populus alba (L.) was used to investigate polyamine (PA) biosynthesis and accumulation in response to high concentrations of zinc (Zn) and copper (Cu). Based on leaf symptoms, rate of adventitious root formation and ethylene production, 0.5–1 mM Zn was transiently toxic while 2–4 mM Zn concentrations were increasingly toxic. Free and conjugated putrescine and spermidine accumulated in a dose–response manner and proportionally to toxicity. The expression profiles of the PA biosynthetic genes were analysed by reverse transcriptase polymerase chain reaction in plants exposed to Zn. In both leaves and stems, PaADC and PaODC transcript levels were early enhanced by all Zn concentrations, while those of PaSAMDC were not. In adventitious roots, free and conjugated PA levels also rose, although their composition and reciprocal ratios were different from those of leaves and no changes in transcript levels of PA biosynthetic genes were detected. Several Cu concentrations (5–500 mM) were tested in shoots showing severe leaf toxicity, strongly impaired adventitious root formation and high ethylene production. No changes in PA levels were detected until end of culture. The different timing of the PA response to Zn and Cu, despite their rapid uptake and translocation already at 24 h, is discussed in relation to the extent of leaf toxicity.

In order to establish whether in vitro model systems are suitable to study the reciprocal relationships between ethylene and polyamines (PAs) in peach fruit, whole detached fruit and fruit explants from “Redhaven” peaches and “Stark Red Gold” nectarines at two different ripening stages were subjected to in vitro treatments with 10mM putrescine (Pu), 1mMspermidine (Sd) or 0.32mM aminoethoxyvinylglycine (AVG) in the presence or in the absence of labelled Pu or methionine. Labelled Pu uptake studies showed that, in the short-term, much more label was recovered in intact nectarines than in peaches. In fact, in the former, ethylene productionwas strongly impaired by Pu and Sd at both stages, while itwas substantially unaffected in the latter. In treated fruit, flesh firmness, soluble solids content and fresh weight were only sporadically affected. Under the same experimental conditions, AVG almost totally inhibited ethylene production although fruit quality was practically unaltered. In explants obtained from fruit at the firmer ripening stage, Pu and Sd did not alter and even enhanced methionine incorporation into ethylene, while in those from softer fruit only Sd was able to counteract ethylene biosynthesis. Also in this case, AVG dramatically reduced ethylene biosynthesis. Short-term treatments of fruit explants showed that only Sd and AVG counteracted ripening. Comparing results from intact fruit and fruit explants indicates that Pu and Sd exert a differential effect on ethylene and fruit quality, depending upon ripening stage and cultivar. © 2006 Elsevier B.V. All rights reserved.

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H2O2-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H2O2 only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H2O2 produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.

Methyl jasmonate (MJ) has been shown to reduce the number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers (TCLs, Biondi et al. – J Exp Bot 52:1-12, 2001). The principal aim of the present study was to determine early cyto-histological events related with this inhibition of shoot formation. To this end, 0.1, 1 and 10 M MJ were applied to TCLs cultured under shoot-forming conditions, and the explants were microscopically analysed during culture. The effect of MJ on the expression of two cell proliferation marker genes, ubi-CEP (ubiquitin carboxyl-extension protein) and top1 (topoisomerase 1), was also examined. Results show that MJ strongly stimulated mitotic activity early in culture (24-48 h) relative to untreated controls, both in superficial and deep layers. Enhanced proliferative growth was still present on days 5-7, in particular in the presence of the lower concentrations of MJ, and confirmed by both ubi-CEP and top1 transcript accumulation. The formation of meristematic cell clusters (MCC) on day 5 was also enhanced by 1 M MJ. Subsequent development of MCC into meristemoids (M) and shoot primordia (S) was, however, reduced by all MJ concentrations in a dose-dependent manner. Treatments with MJ also induced anomalous mitoses, whose presence was precociously (48 h) enhanced by the highest concentration, and later (day 9) by the lowest concentration. Cell expansion was stimulated by MJ, from 24 h up to day 12, especially around and within M and S. The expanded cells displayed thickened and suberized walls; cell wall thickness increased with increasing MJ concentration. Cell expansion and wall thickening/suberization caused alterations in the tunica and stem differentiation of the shoot dome. MJ also inhibited vascular differentiation, and promoted accumulation of phenolics in the vacuoles, especially in the presence of the highest concentration of MJ. All three concentrations of MJ stimulated ethylene biosynthesis, with the strongest effect being exerted by the intermediate one (1 M) at 30 - 48 h in culture. The apparently paradoxical role of MJ which deregulated shoot formation through a stimulation of growth events (i.e., mitotic activity and cell expansion) is discussed, also in relation to its early stimulation of ethylene biosynthesis. Keywords Cell expansion - Ethylene - Methyl jasmonate - Mitotic activity - Shoot formation - Tobacco thin cell layers

In several in vitro systems, treatment with methyl jasmonate (MJ) stimulates polyamine biosynthesis as well as accumulation of acid-soluble and -insoluble conjugated polyamines. This effect is attributed to changes in gene expression and enzyme activity of arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S -adenosylmethionine decarboxylase (SAMDC). In the present study, we used tobacco ( Nicotiana tabacum L. cv. Samsun) leaf discs to investigate the interaction between this MJ-induced response and those triggered by indole-3-acetic acid (IAA), N 6 -benzyladenine (BA) and ethylene. Our results indicate that MJ-induced accumulation of conjugated polyamines is further stimulated by auxin and counteracted by BA. The MJ-induced stimulation of ODC and SAMDC, but not ADC, mRNA levels was diminished by IAA±BA, whereas the corresponding enzyme activities were further enhanced in the presence of hormones. MJ enhanced ethylene production only when combined with hormones, and this trend was reflected in 1-aminocyclopropane-1-carboxylate oxidase transcript levels. Results suggest that the induction by MJ of conjugated polyamine levels, and of the polyamine biosynthetic genes and activities are differentially modulated by hormones, and that ethylene does not seem to be directly involved in this response.

Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonate-mediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general.

The effects of two inhibitors of polyamine (spermidine and spermine) biosynthesis, cyclohexylamine (CHA; 5 and 10 mM) and methylglyoxal(bis-guanylhydrazone) (MGBG; 0.1, 0.5 and 1 mM), on the organogenic response in vegetative bud-forming tobacco (Nicotiana tabacum L. cv. Samsun) thin layer explants were evaluated micro- and macroscopically at different times during culture. The final number of buds formed and the percentage of organogenic explants was significantly reduced by both inhibitors, but much more so by MGBG than CHA. This inhibitory effect was already evident in MGBG-treated explants on day 5, in terms of the number of meristemoids per explant. On the contrary, in the presence of CHA, the number of meristemoids on day 5 was higher than that in the controls. Between days 9 and 13, meristemoid formation slowed down considerably in inhibitor-treated explants compared with controls. On day 13, the number of bud primordia was similar in control and CHA-treated explants, but significantly lower in MGBG-treated explants. This inhibitor also induced peculiar cytohistological events, such as a reduced formation of oval-shaped cell aggregates on the explant surface and more frequent cases of nucleolar extrusion, while CHA led to the appearance of hypertrophic epidermal cells; callus formation at the basal end of the explant and xylogenesis were also affected by the inhibitors. Ethylene biosynthesis, measured as [ C]methionine incorporation, was stimulated 2- (day 2) to 3-fold (15 h) by 0.5 mM MGBG, whereas CHA (10 mM) had little effect and aminoethoxyvinylglycine (AVG; 0.1 μM), an ethylene synthesis inhibitor, was strongly inhibitory. In control explants, the incorporation of labelled methionine into ethylene and spermidine followed an inverse trend up to day 8. In these explants, free putrescine increased 32-fold and spermidine increased about 10-fold between days 0 and 8. Trichloroacetic acid (TCA)-soluble conjugated putrescine also accumulated dramatically during culture. While CHA provoked a decline in spermidine levels, MGBG caused an unexpected increase in free spermidine and spermine titres; however, its most conspicuous effect was on the further enhancement of putrescine conjugate accumulation, while CHA and AVG had the opposite effect. Results are discussed in view of establishing a putative link between MGBG-enhanced ethylene synthesis, increased conjugate titres and inhibition of meristemoid formation.

Treatment of normal and Agrobacterium rhizogenes-transformed root cultures of Hyoscyamus muticus with three different auxins (IAA, IBA, NAA) revealed that the response varied considerably between auxins, between transformed and normal roots and depending on the parameter examined (length of primary root, number and length of lateral roots). While IBA reduced primary root elongation only at the highest concentration tested, the other two auxins were strongly inhibitory at all concentrations in both root lines. All three auxins promoted branching in normal roots (except 2.5 μM IAA) but not in transformed roots where they were generally without effect (IBA) or inhibitory. In normal roots, which produced less ethylene than transformed roots, the auxin stimulation of ethylene formation was more pronounced than in transformed roots. The ethylene precursor ACC and its conjugated form MACC were undetectable in both root lines but accumulated considerably when NAA was supplied. The ethylene synthesis inhibitor AVG had, at high concentrations, a drastic effect on root growth and development while silver thiosulphate, an ethylene antagonist, did not. The latter compound, however, resulted in no or very limited root hair formation in transformed and normal roots, respectively.

The role of ethylene in vegetative bud regeneration was studied in cultured tobacco (Nicotiana tabacum L. cvSamsun) thinlayer expiants. The experimental approach consisted in supplementing the bud-inducing medium with an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), an ethylene antagonist, silver thiosulphate (STS), or an ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA), at various concentrations. The organogenic response was assessed both macroscopically (percentage of bud-forming expiants, final number of buds per expiant) and cytohistologically (number, characteristics, and localisation of meristemoids and bud primordia). The time course of ethylene production during culture was also evaluated. At the end of culture (day 27) all the expiants treated with these compounds had a lower number of buds compared to controls. STS was detrimental to meristemoid initiation at all the concentrations tested. In contrast, 0.5 μM AVG, which strongly inhibited ethylene production, provoked a large increase in the formation of meristemoids early in culture and the appearance of anomalous (“twin”) buds. CEPA reduced meristemoid formation but, at the lower concentrations (1 and 10 μM) speeded up bud emergence. On the whole it mainly favoured disorganised growth and xylogenesis. The results of this work highlight the contrasting effects of ethylene in relation to the two critical stages of the organogenic process, i.e., meristemoid formation and bud primordium development.

Agrobacterium-mediated transformation of white poplar explants was performed using the pMAT-MT construct derived from the ipt-type MAT22 plasmid carrying the PsMTA1 cDNA encoding a pea metallothionein. In vitro shoot regeneration occurred on antibiotic-free medium and transformants were identified by visual selection. Beside the expected abnormal ipt-shooty phenotype, shoots with normal morphology were recovered. Interestingly, an additional phenotype characterised by a remarkable root development was also observed.