Patrizia Torrigiani

Polyamines: A new class of growth substances

This review on polyamines attempts to present evidence in favour of their role as plant growth substances. Many results suggest that polyamines are implicated in a number of growth and developmental processes in plants, ranging from cell division, embryogenesis, senescence and flowering. However, due to their cationic nature and low molecular weight, polyamines also display effects similar to those of inorganic cations that will be only marginally considered in this short review Finally, their changes in response to stress phenomena and their utilization for alkaloid synthesis are not dealt with and the reader should refer to recent reviews covering these aspects in considerable detail (Bachrach and Heimer, 1989; Flores et al., 1990).

Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression

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.

Clonal differences in survival capacity, copper and zinc accumulation, and correlation with leaf polyamine levels in poplar: a large-scale field trial on heavily polluted soil.

Three ex situ collections of poplar clones from natural populations of Populus alba and P. nigra growing in northern Italy were assessed for their genetic dissimilarity (GD) by means of amplified fragment length polymorphism (AFLP). The high GD evidenced within populations was exploited for screening 168 clones in a field trial on heavy metal-polluted soil. After one growth season, clonal differences in plant survival and growth were observed. On the basis of performance, six clones were singled out, and used to evaluate copper and zinc accumulation in different organs. Clonal differences in metal concentrations were most evident for leaves and stems; one clone of P. alba (AL35) had a distinctly higher concentration of both metals in the roots. Leaf polyamine (putrescine, spermidine, spermine) profiles correlated with tissue metal concentrations, depending on the clone, plant organ and metal. In particular, the high metal-accumulating clone AL35 exhibited a dramatically higher concentration of free and conjugated putrescine. Overall, the results indicate that, given the high GD of Populus even within populations, it is possible to identify genotypes best suited for soil clean-up, and useful also for investigating physiological markers associated with high metal accumulation/tolerance.

Jasmonate-induced transcriptional changes suggest a negative interference with the ripening syndrome in peach fruit

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.

Evidence for the Subcellular Localization of Polyamines and their Biosynthetic Enzymes in Plant Cells

Summary In the present work ornithine-, arginine- and S-adenosylmethionine decarboxylase activities were found in chloroplasts of Pinus radiata D. Don cotyledons and in isolated mitochondria from activated slices of Helianthus tuberosus L. tubers. Buffer molarity and pH optima were determined for each enzyme. In organelles different ratios between free polyamine contents were found with respect to the entire cell. Most of the S-adenosylmethionine decarboxylase activity was found in the 26,000 g supernatant of both plants where arginine and ornithine decarboxylase activities were markedly lower. Arginase activity was also detected in mitochondria. The different ratios between the enzyme activities in the various subcellular fractions of both systems may be an indication that these are effectively compartmentalized.

Morpho-funcional Gradients in Superficial and Deep Tissues along Tobacco Stem: Polyamine Levels, Biosynthesis and Oxidation, and Organogenesis in vitro

Summary In order to determine how polyamine metabolism operates and where it is localized in mature or developing tissues, superficial explants (two cell layers: epidemis and subepidermis) and deep tissue (pith) explants were excised at different levels (7th to 22nd internode) along the stem of tobacco ( Nicotiana tabacum L.) plants in the vegetative stage. They were grouped in 5 stocks (sits 1–5) and subjected to: histological observations, determinations of free and conjugated polyamine titers and related biosynthetic (arginine and ornithine decarboxylases, ADC and ODC, respectively) and oxidative (diamine oxidase, DAO) activities, and in vitro culture. Cell area increased basipetally along the stem both in superficial and pith explants; lignification occurred in the basal pith parenchyma. ADC and ODC activities decreased basipetally, predominated in the supernatant rather than the particulate fraction and in superficial tissues rather than deep tissues. No DAO activity was detectable under physiological conditions. Free polyamines were more concentrated in the upper sites in superficial explants and in the lower sites in pith explants while conjugated ones, more abundant in superficial layers, did not show any particular trend along the stem. The morphogenic response in pith explants decreased basipetally up to sites 3–4. The mean number of vegetative shoots was higher in hormone-supplemented (anomalous shoots) than in hormone-free (normal shoots) medium and sites 2 and 5 produced more shoots per explant than the others. Most superficial explants did not exhibit any response in vitro and some of them produced callus or were swollen; the presence of hormones in the culture medium induced shoot formation on a small percentage of explants. The mean number of shoots per superficial explant, lower than in pith explants, was not significantly different along the stem.

Polyamines and Morphogenesis in Normal and Transgenic Plant Cultures

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.

Root histogenesis from tobacco thin cell layers

SummaryInternode stem expiants ofNicotiana tabacum cv. Samsun, consisting of eight cell layers: epidermis, subepidermal chlorenchyma, collenchyma and cortical parenchyma (i.e., thin cell layers), were cultured under conditions inducing rhizogenesis. The aim was to investigate the histological sequence of adventitious root formation in this system. The earliest cytological events in culture (12 h) were nucleolar extrusions and amitotic nuclear divisions. Though not restricted to a specific cell layer, the two phenomena were more frequent in the subepidermal chlorenchyma, and characterized the first phases (12-96 h) of cell proliferation mainly occurring in this layer. Amitoses were followed by the formation of thin walls within the original cells, resulting in the formation of intracellular clusters. These subepidermal clusters were separated by enlarged cells of the parent tissue, whose nuclei showed nucleolar extrusion. At day 3 the first mitoses were observed in cells having abundant starch inclusions. Amitotic divisions also continued, but less frequently. The increasing frequency of mitoses in the subepidermal chlorenchyma (day 4), as well as in the two underlying collenchymatous layers, contributed to the growth of the superficial clusters, in which small clumps of meristematic cells were formed; these, later (day 9), gave rise to root domes. The 5th cell layer remained undivided for a relatively long time (two weeks). The 6th and 7th layers proliferated mitotically later (from day 8 onwards) than the superficial layers and formed root domes following the same histological sequence. Wound callus, generated by the innermost layer, increased markedly in the last two weeks of culture and concomitantly formed vascular clumps surrounded by meristematic layers; these produced root primordia which were frequently anomalous (day 26–27). Regardless of its origin (i.e., superficial or deep layers of the expiant, or wound callus cells), root tip formation was always preceded by the differentiation of a sheath of starch-containing cells, from which the root cap developed.

Polyamine titer and biosynthetic enzymes during tuber formation ofHelianthus tuberosus

During the formation ofHelianthus tuberosus tubers the activities of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC), examined in medullary parenchyma cells, increase with the increase in weight of the tuber. The ornithine decarboxylase (ODC) activity is about 100-fold less with respect to ADC activity, and it was detected only during the deceleration phase of the growth curve. Spermidine and spermine content are strictly related to the SAMDC activity and tuber growth. The increase of ADC and SAMDC activity is directly related to cell extension and increase in weight. The limited area of cell division in parenchyma tissue found during the first stage of tuber formation could justify the low ODC activity. The data suggest that ADC affects mainly growth processes, while ODC seems to be preferentially related to cell division.

Exogenous spermidine, arsenic and β-aminobutyric acid modulate tobacco resistance to tobacco mosaic virus, and affect local and systemic glucosylsalicylic acid levels and arginine decarboxylase gene expression in tobacco leaves.

The polyamine spermidine and the metalloid arsenic increased resistance responses in the well-known pathosystem NN tobacco/tobacco mosaic virus (TMV). Both the hypersensitive response to TMV in a leaf disk model system (inoculated disks floating in the 0.1mM treatments) and systemic acquired resistance (SAR) in whole plants were significantly affected. In the latter case, 1mM foliar sprays of spermidine and arsenic were as effective as TMV and dl-beta-aminobutyric acid (BABA), both taken as positive controls, in improving the plants response to subsequent challenge inoculation with TMV. Moreover, this phenotypic response was correlated with changes in the endogenous concentration of the SAR-related molecule salicylic acid and in transcript levels of some pathogenesis/stress-related genes (pathogenesis-related proteins PR-1a and PR-2 and arginine decarboxylase (ADC)). Concentrations of free salicylic acid and of 2-O-beta-d-glucosylsalicylic acid and mRNA amount of PR-1a, PR-2 and ADC were analyzed in plants treated with either spermidine or arsenic, and compared with those from untreated plants and from positive (TMV-inoculated or BABA-treated) controls. Conjugated salicylic acid content and ADC transcripts were found to significantly increase, at both the local and systemic levels, relative to untreated controls.