Search

The importance of polyamines (PAs) in plant growth and development was recognised several decades ago and, since then, their role in cell proliferation, embryogenesis, organogenesis, flowering, fruit development and ripening, etc. has been investigated to a great extent. In more recent years, most of the attention on PAs has been focussed on their functions in biotic and, especially, abiotic stress responses. Exogenous application, transgenic plants over- or under-expressing PA biosynthetic genes, and mutants have been used to unveil their essential contribution to plant tolerance to salinity, drought, chilling, and heavy metal stresses, among others. In parallel, knowledge on their mechanisms of action has increased greatly and it is today evident that PA functions depend upon their ability to (a) bind electrostatically or covalently to numerous compounds thereby modulating membrane, cell wall, nucleic acid, and protein structure and functions, (b) produce hydrogen peroxide via their catabolic pathways, (c) interact with the biosynthetic and signaling pathways of practically all known phyto hormones, and (d) interact with nitric oxide. In this review, a state-of-the-art overview of PA functions in plants and their possible applications in mitigating stress in crop plants is provided. The potential of seed priming with PAs as an economically and environmentally valid approach for enhancing plant tolerance to adverse environmental conditions is discussed and some results from our study on quinoa and salt stress are presented.

Biostimulants improve yield, quality, and stress acclimation in crops. In this work, we tested the possibility of using phenolics-rich extracts from spelt (Triticum dicoccum L.) husks to attenuate the effects of salt stress (100–200 mM NaCl) in maize. Two methanolic extracts were prepared from the soluble-conjugated (SC), and the insoluble-bound (IB) phenolic acid fractions of the spelt husk, and their effects were investigated on several stress-associated biochemical parameters, such as proline, lipid peroxidation, H2O2, GSH levels, and ion content. Results show that SC and IB fractions of husk extracts behaved very differently, no doubt due to their greatly divergent chemical composition, as revealed by both GC-MS and HPLC analyses. The efficacy of treatments in mitigating salt stress was also dose- and timing-dependent. IB, even at the lower concentration tested, was able to recover the performance of stressed plants in terms of growth, photosynthetic pigments content, and levels of salt stress markers. Recovery of shoot growth to control levels and reduction of stress-induced proline accumulation occurred regardless of whether plants were pre-treated or post-treated with IB, whereas only pre-treatment with the higher dose of IB was effective in mitigating oxidative stress. Although in some cases SC and even methanol alone exerted some positive effects, they could also be deleterious whereas IB never was. Overall, results indicate that a polyphenol-containing extract obtained from spelt by-products can behave as biostimulant in maize plants and can mitigate their response to salt stress, by acting on different biochemical targets.

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.

The situation we face today with COVID-19 moves us to reformulate the work of design in favor of the physical and psychological health of people through the study of interactions within the spaces themselves. There are issues that, when observed, become opportunities for analysis and reflection to generate new ways of inhabiting space. This observation forces us to respond as academies in the search for new tools in which professionals are trained to act in favor of present and future solutions. In times of confinement, being obliged to stay at home for a longer time and to reduce interactions in public spaces has consequences of various kinds, leading to the emergence of new needs within the domestic space. This space has had to adapt from one day to the next to fulfill various functions that were not considered original and that now require urgent adaptations. The project Re-signify the Domestic Space in Times of Confinement aims to provide a new approach to the problem of housing in times of confinement from the School of Architecture, Art and Design at the Tecnologico de Monterrey, to train professionals of the XXI century before emergencies such as the one originated by COVID-19. It is intended to re-think and re-signify the domestic space from confinement, as an opportunity to rethink new and different ways of living in the future. The project aims to work in an interdisciplinary way, with students of Industrial Design and Architecture, to address the social challenges of the situation caused by confinement. Micro and autoethnography, virtual ethnography, and person-centered design methods are applied, generating more responsible results, based on conversation and the most direct relationship between the designer, technical knowledge, and practical user knowledge. Here, the person-centered design is understood as a practice of mobilization and response that generates new meanings within the production of spatial imagery. Ethnography and auto-ethnography allow the designer to immerse himself in a particular reality, in people's daily activities, to understand how they operate from a reflective and empathetic position. With micro-ethnographies, it focuses on a particular aspect in a short period of time, placing, in this case, the social problem in a spatial dimension, discovering particular problems, within a global pattern, to through immersion in the reality studied, visualizing an added value based on people's preferences and motivations. This article compiles, compares, and explains the processes and results around the research as the characterization of domestic space in the search for well-being. Thus, it was fundamental to identify the influence of the principal elements within the domestic environment. It was worth noting that the research was carried out under confinement conditions, placing the person and the domestic space experience at the center. The paper concludes with a reflection around the user's personal experience to their own relationship with the domestic space, around the use of new ethnographic tools, and pointing to new questions about the experience of the domestic space, in times of confinement and in the future.

Over a course of 5 weeks, freshmen students of the core curriculum called ‘Creative Studies’ conformed by 8 different academic programs from 2 faculties, developed a research based on discovering insights focusing on cultural and social aspects on a previously selected community into the city of Queretaro, Mexico. The students were enrolled in a new educational model in which they study their first year with an exploration academic core and then they select their major field in order to eventually graduate with a specialization. The students developed a research challenge in which they worked in transdisciplinary teams focusing on classroom exercises and field work alternated to guarantee immersion and experience based on theoretical content taught in the classroom and then applied on field in order to acquire core and transversal competencies. This paper describes the process in the implementation of the challenge-based learning focusing on how the students with different profiles developed their competencies through working on the challenge, guided by the teacher and teaming with their classmates in activities and exercises in order to achieve their goals.

Saponins (SAPs) are a diverse family of plant secondary metabolites and due to their biological activities, SAPs can be utilised as biopesticides and as therapeutic compounds. Given their widespread industrial use, a search for alternative sources of SAPs is a priority. Quinoa ( Chenopodium quinoa Willd) is a valuable food source that is gaining importance worldwide for its nutritional and nutraceutical properties. SAPs from quinoa seed coats could represent a new sustainable source to obtain these compounds in high quantities due to the increasing production and worldwide expansion of the crop. This research aims to characterise saponins of seed coat waste products from six different quinoa varieties for their potential use as a saponin source. Gas chromatography (GC)- and Liquid chromatography (LC)- with mass spectrometry (MS) were applied for qualitative and relative quantitative analysis of saponins. GC–MS led to the identification of three main aglycones, oleanolic acid (Ole), hederagenin (Hed), and a phytolaccagenic acid (Phy), while LC–MS enabled characterization of 24 SAPs with varying sugar moieties. Hed was the most abundant aglycone, followed by Phy and Oledepending on the genotype. Saponin distribution and relative abundances are discussed in the light of genotype provenance and agronomic features. Improved knowledge on the phytochemicals present in quinoa varieties might help in finding valuable and sustainable uses for quinoa SAPs in agroindustry as biopesticides as well as in the production of food and pharmaceuticals.

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.

Playlab, an educational initiative of the School of Architecture, Art and Design of the Tecnologico de Monterrey University, provides a steady academic base for innovation in product co-design adapting to changing social and cultural contexts. Play Lab aims to bring students from different disciplines together to tackle social challenges within a specific local community as a platform for social innovation and moving beyond the classroom into an established social community. The professors deploy a wide arrange of participatory and ethnographic tools from which students choose and implement in the specific community context. Thus the students have to go through a full and profound social and urban research of the chosen context before deciding on a viable collaboration design. Play Lab has had three iterations with different students in 2016, 2017 and 2018. This paper hopes to compile, compare and explain the processes and results of Play Lab while analyzing some variables as a reference. Using a methodology proposed by Hansson et al. (2011) we performed an evaluation of the aforementioned iterations. We believe we have gained clarity regarding future strategies, methods and tools to be further used in Play Lab, our academic alternative for social innovation.

Jasmonic acid (JA) and its methyl ester (MeJA) at concentrations ranging from 0.001 to 10 μM provoked large increases in methylputrescine levels in normal and hairy roots of Hyoscyamus muticus L.; generally, levels of free putrescine and perchloric acid-soluble conjugated putrescine, spermidine and spermine also increased dramatically. More 14C-putrescine was formed when hairy roots were incubated with labelled ornithine than with arginine; conjugated 14C-putrescine was also rapidly formed. In accord with these results, ornithine decarboxylase (EC 4.1.1.17) activity was higher than that of arginine decarboxylase (EC 4.1.1.19), and MeJA enhanced these activities about two- and fourfold, respectively. Although treatment of root cultures with jasmonates enhanced precursor (putrescine, methylputrescine) levels and accumulation of secondary metabolites such as acid-soluble conjugated di-/polyamines, it provoked only modest increases in tropane alkaloid tissue levels.

Many areas of the world are affected simultaneously by salinity and heavy metal pollution. Halophytes are considered as useful candidates in remediation of such soils due to their ability to withstand both osmotic stress and ion toxicity deriving from high salt concentrations. Quinoa (Chenopodium quinoa Willd) is a halophyte with a high resistance to abiotic stresses (drought, salinity, frost), but its capacity to cope with heavy metals has not yet been fully investigated. In this pot experiment, we investigated phytoextraction capacity, effects on nutrient levels (P and Fe), and changes in gene expression in response to application of Cr(III) in quinoa plants grown on saline or non-saline soil. Plants were exposed for three weeks to 500 mg kg−1 soil of Cr(NO3)3·9H2O either in the presence or absence of 150 mM NaCl. Results show that plants were able tolerate this soil concentration of Cr(III); the metal was mainly accumulated in roots where it reached the highest concentration (ca. 2.6 mg g−1 DW) in the presence of NaCl. On saline soil, foliar Na concentration was significantly reduced by Cr(III). Phosphorus translocation to leaves was reduced in the presence of Cr(III), while Fe accumulation was enhanced by treatment with NaCl alone. A real-time RT-qPCR analysis was conducted on genes encoding for sulfate, iron, and phosphate transporters, a phytochelatin, a metallothionein, glutathione synthetase, a dehydrin, Hsp70, and enzymes responsible for the biosynthesis of proline (P5CS), glycine betaine (BADH), tocopherols (TAT), and phenolic compounds (PAL). Cr(III), and especially Cr(III)+NaCl, affected transcript levels of most of the investigated genes, indicating that tolerance to Cr is associated with changes in phosphorus and sulfur allocation, and activation of stress-protective molecules. Moderately saline conditions, in most cases, enhanced this response, suggesting that the halophytism of quinoa could contribute to prime the plants to respond to chromium stress.

Recently, increasing interest has been paid to the biostimulant properties of plant extracts as they have been shown to improve crop yield and quality. Biolchim SpA is a leading company in the production and commercialization of biostimulants. The working approach to the development of innovative products is focused on the research of plant extracts with biostimulant properties. To this aim, Biolchim SpA has created a collection of botanical extracts whose potential use as biostimulants has to be screened according to their biological activity. Bioassays are small-scale tests that assess the biological activity of substances by evaluating their effect on living model organisms. In this study, an array of in vitro (root-tip elongation test) and in vivo (greenhouse growth tests) bioassays was set up and used to test the biostimulant properties of two botanical extracts. Overall results indicate that the array of bioassays is able to screen and differentiate the extracts according to their biological activity. The root-tip elongation bioassay is a first and rapid screening method of their hormone-like properties; the greenhouse growth bioassays provide information on their biostimulant performances in growth conditions similar to the open field and on their possible mode of application. The array of bioassays will be improved by including further in vitro and in vivo tests to fully unravel the biological properties of the extracts. The final goal is to screen the whole collection and classify the extracts according to their potential use as biostimulants. After the screening, prototypes will be formulated by combining different extracts and their efficacy will be tested in open field conditions.

Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the highest TPC and AA under non-saline conditions. Salinity increased TPC in all three landraces, with the strongest increase occurring in R49, and enhanced radical scavenging capacity in R49 and VR. Overall, results show that salinity deeply altered the seed proteome and amino acid profiles and, in general, increased the concentration of bioactive molecules and AA of protein extracts in a genotype-dependent manner.

none 5 no This research was supported by funds from the University of Bologna (RFO2014) to S.B.and from the University of Urbino (RFO 2014) to V.S.. Quinoa (Chenopodium quinoa Willd), an ancient Andean seed crop, exhibits exceptional nutritional properties and resistance to abiotic stress. The species' tolerance to heavy metals has, however, not yet been investigated nor its ability to take up and translocate chromium (Cr). This study aimed to investigate the metabolic adjustments occurring upon exposure of quinoa to several concentrations (0.01–5mM) of CrCl3. Young hydroponically grown plants were used to evaluate Cr uptake, growth, oxidative stress, and other biochemical parameters three and/or seven days after treatment. Leaves accumulated the lowest amounts of Cr, while roots and stems accumulated the most at low and at high metal concentrations, respectively. Fresh weight and photosynthetic pigments were reduced only by the higher Cr(III) doses. Substantially increased lipid peroxidation, hydrogen peroxide, and proline levels were observed only with 5mM Cr(III). Except for a significant decrease at day 7 with 5mM Cr(III), total polyphenols and flavonoids maintained control levels in Cr(III)-treated plants, whereas antioxidant activity increased in a dose-dependent manner. Maximum polyamine accumulation was observed in 1mM CrCl3-treated plants. Even though α- and γ-tocopherols also showed enhanced levels only with the 1mM concentration, tyrosine aminotransferase (TAT, EC 2.6.1.5) activity increased under Cr(III) treatment in a dose- and time-dependent manner. Taken together, results suggest that polyamines, tocopherols, and TAT activity could contribute to tolerance to 1mM Cr(III), but not to the highest concentration that, instead, generated oxidative stress. open Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania

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.

Peach (Prunus persica L. Batsch) was chosen as a model to shed light on the physiological role of jasmonates (JAs) during fruit ripening. To this aim, the effects of methyl jasmonate (MJ, 0.40 mM) and propyl dihydrojasmonate (PDJ, 0.22 mM), applied in planta at different fruit developmental stages, on the time-course of ethylene production and fruit quality traits were evaluated. MJ-induced changes in fruit transcriptome at harvest and the expression profiling of relevant JA-responsive genes were analysed in control and JA-treated fruit. Exogenously applied JAs affected the onset of ripening depending upon the fruit developmental stage, with PDJ being more active than MJ. Both compounds enhanced the transcription of allene oxide synthase (PpAOS1), the first specific enzyme in the biosynthesis of jasmonic acid, and altered the pattern of jasmonic acid accumulation. Microarray transcriptome profiling showed that MJ down-regulated some ripening-related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) and polygalacturonase (PG), and the transcriptional modulator IAA7. MJ also altered the expression of cell wall-related genes, namely pectate lyase (PL) and expansins (EXPs), and up-regulated several stress-related genes, including some of those involved in JA biosynthesis. Time-course expression profiles of PpACO1, PL, PG, PpExp1, and the transcription factor LIM confirmed the array results. Thus, in peach fruit, exogenous JAs led to a ripening delay due to an interference with ripening- and stress/defence-related genes, as reflected in the transcriptome of treated fruit at harvest.

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.

Putrescine (5, 10 and 20 mM), spermidine (0.5, 1 and 2 mM) and aminoethoxyvinylglycine (AVG; 0.32, 0.64 and 1.28 mM) were applied to peach trees (Prunus persica L. Batsch cv Stark Red Gold) under open field conditions. Treatments were performed 28 (polyamines; PAs) and 21 or 7 (AVG) days before harvest at 115 dAFB. Both PAs and AVG reduced ethylene production of fruit, delayed loss of firmness, retained titratable acidity, and prevented the increase in dry matter (DM) and soluble solids concentration (SSC). Fruit drop was consistently reduced by AVG at both application times, and by the highest spermidine concentration. Endogenous PA levels in treated fruit were transiently affected (7 days after treatment), but returned to control values at harvest in both mesocarp and epicarp tissues. Northern analysis of ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO), and of those involved in PA biosynthesis, arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), was performed in mesocarp tissue at harvest. AVG and putrescine reduced the accumulation of ACO and SAMDC message, putrescine also influenced ACS transcript levels, while spermidine did not affect any of these genes. Results are discussed on the basis of the reduction by PAs and AVG of ethylene production, and confirm the capacity of these naturally occurring substances to modulate fruit ripening.

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.

The effect of aminoethoxyvinylglycine (AVG), an inhibitor of 1-aminocyclopropane-1-carboxylate synthase (ACS) activity, on ethylene emission and biosynthetic gene expression, on gene expression and/or activity of polyamine (putrescine, spermidine and spermine) biosynthetic enzymes, and on diamine oxidase (DAO, EC 1.4.3.6) activity was evaluated in tobacco (Nicotiana tabacum L. cv. Samsun) thin layers cultured on a shoot-forming medium (1 μM indol-3-acetic acid (IAA) plus 10 μM benzyladenine (BA)). Northern analyses showed that ACS and 1-aminocyclopropane-1-carboxylate oxidase (ACO) transcripts were present throughout culture with a maximum accumulation on day 7. Besides ethylene emission, AVG (0.5 μM) increasingly reduced ACS and ACO messages. The time course of labelled methionine incorporation into spermidine and spermine, which share with ethylene the common precursor S-adenosylmethionine (SAM), as well as SAM decarboxylase (SAMDC, EC 4.1.1.21) activity and gene expression, were not affected by AVG treatment. On the contrary, labelled putrescine incorporation into the higher polyamines (spermidine and spermine) and into trichloroacetic acid (TCA)-soluble polyamine conjugates was enhanced early in culture (day 2) by the drug. Putrescine biosynthetic enzyme activities, arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), were also increased in AVG-treated explants. Moreover, inhibition of ethylene synthesis by AVG led to a strong reduction in diamine oxidising activity, especially the one associated with a cell wall-enriched fraction. Changes in putrescine biosynthesis, oxidation and flux into higher polyamines are discussed in the light of the rejuvenating effect of AVG.

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.

Previous results indicated that in shoot-forming tobacco thin layers the putative inhibitor of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity methylglyoxal(bis-guanylhydrazone) (MGBG) inhibited meristemoid/primordia formation while enhancing conjugated polyamine accumulation and ethylene biosynthesis, thus showing an inverse relationship between the two phenomena ( Scaramagli et al. 1999). In order to better understand how MGBG causes polyamine overproduction and by using the same model system, we further examined the effect of 0.5 mM MGBG on the incorporation of labelled methionine into spermidine and spermine, on SAMDC transcript levels and activity and on putrescine oxidising (DAO) activity at different times in culture. MGBG-induced ethylene accumulation was also evaluated by gas chromatography (GC). In shoot-forming controls (grown on a medium supplemented with IAA+BA) label was mainly incorporated into free spermidine (82–86% of the total amount incorporated into polyamines) while only 6–12% of radioactivity was recovered in free spermine. Some label was also found in putrescine. In the presence of MGBG, spermidine biosynthetic capacity was enhanced throughout the culture by 28–45% compared with controls. The same occurred for spermine and putrescine. MGBG-enhanced spermidine synthesis was supported by increased SAMDC activity (days 1–2) and higher levels of SAMDC message (days 1–7). On the contrary, the effect of the drug on putrescine oxidation was inhibitory starting from day 2. These results, together with the previous ones, suggest that MGBG, in fact, does not behave as a SAMDC inhibitor in shoot-forming tobacco thin layers and that its negative effect on organised growth is not due to SAMDC inhibition/polyamine depletion.

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).

Summary Aliphatic polyamines, putrescine, spermidine and spermine are ubiquitous in all living organisms. They occur in the free form as cations, but are often conjugated to small molecule like phenolics acids and also to various macromolecules. Polyamines and their biosynthetic enzymes may play an important role in many aspects of plants development including growth, differentiation, senescence and response to differents types of stress. Recent work suggest, but has not proven, a regulatory role of polyamines in these processes. Putrescine accumulation in response to stress, suggests a protective role. By contrast, increased spermidine and spermine synthesis appears essential for DNA replication and cell division.

Chenopodium quinoa Willd. (quinoa) is a halophyte for which some parameters linked to salt tolerance have been investigated separately in different genotypes and under different growth conditions. In this study, several morphological and metabolic responses were analysed in parallel after exposure to salinity. In vitro seed germination was initially delayed by a 150 mM NaCl treatment but eventually reached the same level as the control (0 mM NaCl), whereas seedling root growth was enhanced; both parameters were moderately inhibited (~35–50%) by 300 mM NaCl. In pot grown plants, plant size was reduced by increasing salinity (0–750 mM NaCl). Transpiration and stomatal conductance were decreased at the highest salinity levels tested, consistent with reduced stomatal density and size. The density of epidermal bladder cells (EBCs) on the leaf surface remained unaffected up to 600 mM NaCl. Tissue contents of Na+ and Cl– increased dramatically with salt treatment, but resulted in only a 50% increase in Na+ from 150 to 750 mM NaCl. Internal K+ was unaffected up to 450 mM NaCl but increased at the highest salinity levels tested. Excretion through sequestration into EBCs was limited (generally ≤20%) for all ions. A modest dose-dependent proline accumulation, and concomitant reduction in total polyamines and putrescine efflux occurred in NaCl-treated plants. Results confirm the importance of inorganic ions for osmotic adjustment, the plant’s ability to maintain K+ levels and the involvement of putrescine efflux in maintaining ionic balance under high salinity conditions. Conversely, ion excretion and proline appear to play a minor role. Taken together these results indicate which parameters could be used for future comparison among different genotypes.

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.

The optimal assay conditions and the trend with time in culture (28 days) of arginine decarboxylase (ADE; EC 4.1.1.19), omithine decarboxylase (ODC; EC 4.1.1.17) and diamine oxidase (DAO; EC 1.4.3.6) activities in habituated (H) and normal (N) auxin- and cytokinin-requiring sugar beet callus were compared. Although the response to variations in buffer pH and EDTA and pyridoxal phosphate (PLP) concentrations varied for ADC and ODC activities between the two callus types, pH 8.3, 50 μM PLP and 5 mM EDTA were generally optimal or near-optimal for both H and N callus. In most cases the addition of ornithine or arginine in the ADC and ODC assays, respectively, given to block the interconversion between the two substrates, resulted in lower 14CO2 recovery. DAO activity was very differently affected in H and N callus by the presence of polyvinylpyrrolidone in the extration buffer. However, in both cases, this activity increased with time in culure. ADC activity was always predominant in both cell lines and always higher in N callus. In the latter, ADC activity rose sharply between days 14 and 21 and then leveled off while in H callus it incresed steadily from day 14 onwards. ODC activity was also higher in N callus and peaked sharply on day 21 while in H callus it was not detectable in the second half of the culture period. In both cell lines this activity was low or nil on day 28. 3,4-[14C]-methionine incorporation into ethylene and polyamines was also compared in N and H callus. In the latter, ethylene synthesis was lower and [14C]-spermidine formation higher than in N callus. This is in accord with the significantly higher spermidine titres found in H callus.

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.

An attempt was made to identify some of the hormonal factors that control adventitious root formation in our Prunus avium micropropagation system in order to improve rooting in difficult‐to‐root genotypes. Changes in endogenous contents of free polyamines were determined at intervals during auxin‐induced rooting of shoot cultures. Accumulation of putrescine and spermidine peaked between days 9 and 11. Spermine was only present in traces, Exogenously supplied putrescine or spermine (50‐500 μM), in the presence of optimal or suboptimal levels of indolebutyric acid (IBA), had no effect on rooting percentage or root density, except for spermine at 500 μM. At this external concentration spermine caused a substantial accumulation in both free spermine and putrescine. The use of several inhibitors of polyamine biosynthesis, namely α‐difluoromethylornithine (DFMO), α‐difluoromethylarginine (DFMA), dicyclohexylammonium sulphate (DCHA) and methylglyoxal‐bis‐guanyl‐hydrazone (MGBG) alone or in combination in the 0.1 to 5 μM range, resulted in an inhibition of rooting that was partially reversed by the addition of the corresponding polyamine. Cellular polyamine levels were significantly reduced by DFMO and DFMA but not by DCHA and MGBG, Labeled putrescine incorporation into spermidine increased somewhat in the presence of the ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG). A system based on [3,4‐14C]methionine incorporation was used to measure ethylene synthesis by the in vitro cultured shoots. Label incorporation was drastically reduced by 10 μM AVG and increased 3.5‐fold in the presence of 50 μM IBA with respect to controls (no IBA). Labeled methionine incorporation into spermidine increased to some extent when ethylene synthesis was inhibited by AVG. Adding the ethylene precursor 1‐aminocyclopropane‐l‐carboxylic acid (ACC) to the rooting medium significantly inhibited rooting percentage; AVG caused the formation of a greater number of roots per shoot but delayed their growth. Supplying the shoots with both compounds resulted in an intermediate rooting response, in which both rooting percentage and root density were affected. These results indicate that polyamines may play a significant role at least in some stages of root formation. The polyamine and ethylene biosynthetic pathways seem to be competitive but under our conditions, the enhancement of one pathway when the other was inhibited, was not dramatic. Although IBA promoted ethylene synthesis, AVG, which drastically reduced it, also promoted root formation. Thus, the auxin effect on root induction cannot be directly related to its ability to enhance ethylene synthesis. Copyright © 1990, Wiley Blackwell. All rights reserved

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

Transcript levels and activities of the polyamine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19), ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), as well as free polyamine titres, were analysed throughout the four growth stages S1-S4 leading up to ripening in the mesocarp from peach fruit (Prunus persica L. Batsch cv. Redhaven) grown under field conditions. SAMDC mRNA, which was northern analysed by using a PCR-generated homologous SAMDC probe, and ADC mRNA levels appeared quite stable during fruit development, while ODC transcript accumulation showed a discontinuous trend. The pattern of transcript levels during growth did not correlate with that of the relative enzyme activity, which instead correlated well with free polyamine levels. Both exhibited maximum levels in S1 and a smaller peak in S3. The behaviour of the polyamine biosynthetic machinery is discussed in relation to the different cell growth rates occurring during fruit development.

Studying the interrelationships between the various plant growth regulators is a necessary step for the understanding of the physiological and morphogenic responses in vivo and in vitro. Polyamines (PAs) occupy a particular niche amongst the plant growth regulators in that, although they share some of the features of traditional phytohormones, such as gradients of synthesis/accumulation associated with juvenility, and growth and differentiation processes (Bagni and Torrigiani 1992), other characteristics, namely order of magnitude and lack of specific receptors, differentiate them and suggest similarities with second messengers. Further research on PA-binding proteins (Tassoni et al 1998) may help to clarify this issue.

The effect of various concentrations of aminoethoxyvinylglycine (AVG; 0.32 and 1.28 mM), an ethylene biosynthesis inhibitor, and of the polyamines putrescine (10 mM), spermidine (0.1, 1 and 5 mM) and spermine (2 mM) on peach (Prunus persica L. Batsch cv. Redhaven) fruit ripening was evaluated under field conditions. Treatments were performed 19 (polyamines) and 8 (AVG) days before harvest. Fruit growth (diameter, fresh and dry weight), flesh firmness, soluble solids content and ethylene emission were determined on treated and untreated (controls) fruits. Moreover, endogenous polyamine content and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity were determined to check for a possible competition between polyamines and ethylene for their common precursor S-adenosylmethionine (SAM). Both treatments strongly inhibited ethylene emission and delayed flesh softening. On a biochemical level, AVG and exogenous polyamines both reduced the free-to-conjugate ratio of endogenous polyamines, and transiently altered SAMDC activity. The possible use of these compounds to control fruit ripening is discussed also in the light of their rejuvenating effect on peach fruits.

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.

The results presented in this chapter are mostly based on our own work with Hyoscyamus muticus carried out during the years 1982–1998 in our laboratories in Finland and Italy.

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.

Direct and indirect approaches have demonstrated that polyamines are involved in cell division and in other events connected with growth and morphogenesis in plant tissue and cell cultures and that they affect senescence and stress responses (Bagni, 1989; Galston and Flores, 1991). However since the early report of Bagni (1966) clearly indicated that, in the absence of exogenous hormones and with endogenous hormone concentrations at levels unable to sustain growth, 10 to 100 μM polyamines could support cell division in Helianthus tuberosus tuber expiants, very few reports have demonstrated that a specific morphogenic effect (i.e. embryogenesis or organogenesis) is induced by exogenous polyamines.

Adventitious shoot initiation in cotyledon explants from 6-7-day-old germinated seed of Pinus radiata requires exogenous cytokinin. This requirement was examined in terms of both concentration and duration. Treatments resulting in an insufficient cytokinin supply, particularly during the first 3 days in culture, dramatically reduced organogenesis. Other phytohormones and some other growth-active substances also reduced this cytokinin-induced shoot formation and promoted callus formation. Changes in metabolite pool sizes and in respiratory activity were monitored over the first 21 days in culture, during which meristematic tissue formation occurred along the entire length of the cotyledon. Lipids and free sugars were depleted ca. 10-fold and sixfold, respectively, and there was a steady decline in free amino-N levels. However, the protein-N pool remained relatively large. While there was a continued increase in cotyledon fresh weight, respiration decreased initially and then increased slightly during later...

Biondi, S., Torrigiani, P., Sansovini, A. and Bagni, N. 1988. Inhibition of polyamine biosynthesis by dicyclohexylamine in cultured cotyledons of Pinus radiata. - Physiol. Plant. 72: 471–476. The effect of 1 mAf dicyclohexylamine (DCHA) on the synthesis of spermidine and spermine was examined in excised cotyledons of radiata pine (Pinus radiata D. Don) cultured under shoot-forming (with cytokinin) and non-shoot-forming (minus cytokinin) conditions by incubation with [14C]-putrescine. In control cotyledons incorporation into spermidine showed a peak at day 2 in the presence and at day 5 in the absence of N6-benzyldenine (BA). DCHA-treated cotyledons gave the same labeling pattern, both in the presence and absence of benzyladenine, with a much smaller peak at day 2. The incorporation into spermidine and spermine was insignificant at day 5 and later. The total radioactivity in the trichloroacetic acid supernatant indicated that precursor uptake was strongly reduced by the drug. In addition, the percentage label found in the benzene phase and combined in the 3 polyamines was lower in DCHA-treated cotyledons. Thus, treatment with DCHA not only inhibited the conversion from putrescine to spermidine and spermine, but also reduced its conversion to other benzene-extractable compounds. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity, which furnishes the propylamine group to spermidine and spermine synthases (EC 2.5.1.16 and EC 2.5.1.-), was not significantly affected by DCHA and appeared to be independent of the spermidine and spermine synthase reactions, suggesting that spermine synthesis decreased as a result of substrate depletion. The correlation between morphological development and polyamine biosynthesis is discussed.