Maurizio Badiani

Calabrian pine (Pinus nigra subsp. laricio) is an ecologically and economically important Mediterranean conifer increasingly exposed to the pine processionary moth (PPM; Thaumetopoea pityocampa). We investigated temporal differences in needle terpene metabolism between saplings maintained in a control enclosure and saplings exposed to prolonged PPM infestation in a separate, otherwise identical enclosure. Three-year-old saplings underwent semi-natural infestation for 195 days, and needles were sampled at 0, 21, 84, and 195 days post-infestation. Solvent-extractable monoterpenoids and diterpene resin acids (DRAs) were quantified through gas chromatography–mass spectrometry, while the transcript abundances of mono- and diterpene synthase (MTPS and DTPS, respectively) genes were analyzed through quantitative real-time PCR. During the whole experimental period, the terpenoid blend remained unchanged, qualitatively speaking, in both control and PPM-infested saplings, whereas the two experimental groups showed marked time-dependent quantitative differences among each other in terms of individual compound and transcript abundances. In PPM-infested plants, β-pinene, α-pinene, α-terpineol, and (−)-β-phellandrene accumulated mainly at 84 and 195 days, with changes in their putatively associated Pnl MTPS transcripts accompanying or preceding metabolite accumulation. Conversely, δ-3-carene and Pnl MTPS3 transcript abundance declined strongly. Abietane-type DRAs and dehydroabietic acid accumulated at later stages, together with higher Pnl DTPS1 transcript abundance. Multivariate analyses showed earlier divergence of TPS transcript profiles than metabolite profiles. Thus, Calabrian pine saplings infested by PPM exhibit selective, temporally structured reprogramming of mono- and diterpenoid pathways, rather than a generalized terpenoid induction.

In the present work, a subset extracted from a core collection of common beans (Phaseolus vulgaris L.) landrace accessions from the Lazio region in central Italy was used to identify the most suitable drought-tolerant or -susceptible genotypes. By applying several morpho-physiological and agronomic selection criteria recommended by the available literature, we conducted a pre-screening experiment under controlled conditions on a primary group of 24 landraces. These landraces were chosen to represent the diversity in the Lazio region in terms of geographical provenance, elevation, landform, growth habit, customary water management in the field, and native gene pool. Pre-screening under controlled conditions allowed us to identify two subsets of landraces: one exhibiting the most drought-tolerance and one showing the most susceptibility to drought. These two subsets were then tested in field trials using two water treatments, i.e., normal irrigation versus no irrigation. Such field experiments were simultaneously conducted at two sites within the Lazio region, deliberately chosen to maximize their differences in terms of pedo-climatic conditions. This notwithstanding, the core findings from the two separate field experiments were remarkably consistent and coherent among each other, highlighting a notable degree of variability within the group of the tested landraces. In general, the morpho-physiological traits considered were found to be less responsive to water shortage than yield parameters. A statistically significant Landrace × Treatment interaction was found for almost all the yield parameters considered, suggesting that certain genotypes are more susceptible than others to water shortage. By taking into account the concept of “yield stability”, i.e., the ability to maintain comparatively high yields even under conditions of water scarcity, certain common bean landraces were found to be the most promising, in terms of drought tolerance. Two genotype pairs, in particular, could be of interest for studying the morpho-physiological and molecular mechanisms underlying drought tolerance in common bean, as well as for identifying quantitative trait loci associated with water scarcity, which could be beneficially employed in breeding programs. The results reported here also suggest that pre-screening under laboratory conditions, followed by targeted field trials, can constitute a reliable, efficient, and resourceful combined approach, in which morpho-physiological traits measured on juvenile plants might play a role in predicting drought tolerance at the agronomic level.

Background: Drought, N deficiency and herbivory are considered the most important stressors caused by climate change in the agro- and eco-systems and varied in space and time shaping highly dynamic and heterogeneous stressful environments. This study aims to evaluate the tomato morpho-physiological and metabolic responses to combined abiotic and herbivory at different within-plant spatial levels and temporal scales. Methods: Leaf-level morphological, gas exchange traits and volatile organic compounds (VOCs) profiles were measured in tomato plants exposed to N deficiency and drought, Tuta absoluta larvae and their combination. Additive, synergistic or antagonistic effects of the single stress when combined were also evaluated. Morpho-physiological traits and VOCs profile were also measured on leaves located at three different positions along the shoot axes. Results: The combination of the abiotic and biotic stress has been more harmful than single stress with antagonistic and synergistic but non-additive effects for the morpho-physiological and VOCs tomato responses, respectively. Combined stress also determined a high within-plant phenotypic plasticity of the morpho-physiological responses. Conclusions: These results suggested that the combined stress in tomato determined a “new stress state” and a higher within-plant phenotypic plasticity which could permit an efficient use of the growth and defense resources in the heterogeneous and multiple stressful environmental conditions.

Background: Drought, N deficiency and herbivory are considered the most important stressors caused by climate change in the agro- and eco-systems and varied in space and time shaping a highly dynamic and heterogeneous stressful environments. This study aims to evaluate the to-mato morpho-physiological and metabolic responses to combined abiotic and herbivory at dif-ferent within-plant spatial levels and temporal scales. Methods: Leaf-level morphological, gas exchange traits and VOC profiles were measured in to-mato plants exposed to N deficiency and drought, T. absoluta larvae and their combination. Ad-ditive, synergistic or antagonistic effects of the single stress when combined were also evaluat-ed. Morpho-physiological traits and VOC profile were also measured on leaves located at three different positions along the shoot axes. Results: The combination of the abiotic and biotic stress has been more harmful than single stress with antagonistic and synergistic but non-additive effects for the morpho-physiological and VOC tomato responses, respectively. Combined stress also determined a high within-plant phenotypic plasticity of the morpho-physiological responses. Conclusions: These results suggest that the combined stress in tomato determined a “new stress state” and a higher within-plant phenotypic plasticity which could permit an efficient use of the growth and defence resources in the heterogeneous and multiple stressful environmental condi-tions.

In the present study, we carried out a quantitative analysis of the monoterpenes composition in different tissues of the non-model conifer Pinus nigra J.F. Arnold subsp. laricio Palib. ex Maire (P. laricio, in short). All the P. laricio tissues examined showed the presence of the same fourteen monoterpenes, among which the most abundant were β-phellandrene, α-pinene, and β-pinene, whose distribution was markedly tissue-specific. In parallel, from the same plant tissues, we isolated seven full-length cDNA transcripts coding for as many monoterpene synthases, each of which was found to be attributable to one of the seven phylogenetic groups in which the d1-clade of the canonical classification of plants’ terpene synthases can be subdivided. The amino acid sequences deduced from the above cDNA transcripts allowed to predict their putative involvement in the biosynthesis of five of the monoterpenes identified. Transcripts profiling revealed a differential gene expression across the different tissues examined, and was found to be consistent with the corresponding metabolites profiles. The genomic organization of the seven isolated monoterpene synthase genes was also determined.

A quali-quantitative analysis of diterpenoid composition in tissues obtained from different organs of Pinus nigra subsp. laricio (Poiret) Maire (Calabrian pine) was carried out. Diterpene resin acids were the most abundant diterpenoids across all the examined tissues. The same nine diterpene resin acids were always found, with the abietane type prevailing on the pimarane type, although their quantitative distribution was found to be remarkably tissue-specific. The scrutiny of the available literature revealed species specificity as well. A phylogeny-based approach allowed us to isolate four cDNAs coding for diterpene synthases in Calabrian pine, each of which belonging to one of the four groups into which the d3 clade of the plants’ terpene synthases family can be divided. The deduced amino acid sequences allowed predicting that both monofunctional and bifunctional diterpene synthases are involved in the biosynthesis of diterpene resin acids in Calabrian pine. Transcript profiling revealed differential expression across the different tissues and was found to be consistent with the corresponding diterpenoid profiles. The isolation of the complete genomic sequences and the determination of their exon/intron structures allowed us to place the diterpene synthase genes from Calabrian pine on the background of current ideas on the functional evolution of diterpene synthases in Gymnosperms.

Terpenoids make up the biggest and most diversified class of chemical substances discovered in plants, encompassing over 40,000 individual compounds. In conifers, the production of terpenoids, either as oleoresin or emitted as volatile compounds, play an important role in the physical and chemical defence responses against pathogens and herbivores. In the present work, we examined, for the first time to the best of our knowledge, the terpenic defensive relations of Calabrian pine (Pinus nigra subsp. laricio (Poiret) Maire), facing the attack of the pine processionary moth (Thaumetopoea pityocampa (Denis and Schiffermüller, 1775)), brought about in the open on adult plant individuals growing at two distinct forest sites. Among the volatile terpenoids emitted from pine needles, bornyl acetate [(4,7,7-trimethyl-3-bicyclo[2.2.1]heptanyl) acetate] was the most frequently and selectively associated with the infestation, increasing during the period of most intense trophic activity of the caterpillars (defoliation), and decreasing thereafter. Although further work is needed to clarify whether the observed response reflects defence reactions and/or they are involved in communication among the infested plants and their biotic environment, the present results boost the currently growing interest in the isolation and characterization of plant secondary metabolites that can be used to control pests, pathogens, and weeds.

In the biosynthesis of terpenoids, the ample catalytic versatility of terpene synthases (TPS) allows the formation of thousands of different molecules. A steadily increasing number of sequenced plant genomes invariably show that the TPS gene family is medium to large in size, comprising from 30 to 100 functional members. In conifers, TPSs belonging to the gymnosperm-specific TPS-d subfamily produce a complex mixture of mono-, sesqui-, and diterpenoid specialized metabolites, which are found in volatile emissions and oleoresin secretions. Such substances are involved in the defence against pathogens and herbivores and can help to protect against abiotic stress. Oleoresin terpenoids can be also profitably used in a number of different fields, from traditional and modern medicine to fine chemicals, fragrances, and flavours, and, in the last years, in biorefinery too. In the present work, after summarizing the current views on the biosynthesis and biological functions of terpenoids, recent advances on the evolution and functional diversification of plant TPSs are reviewed, with a focus on gymnosperms. In such context, an extensive characterization and phylogeny of all the known TPSs from different Pinus species is reported, which, for such genus, can be seen as the first effort to explore the evolutionary history of the large family of TPS genes involved in specialized metabolism. Finally, an approach is described in which the phylogeny of TPSs in Pinus spp. has been exploited to isolate for the first time mono-TPS sequences from Pinus nigra subsp. laricio, an ecologically important endemic pine in the Mediterranean area.

Background: To limit the impact of the downy mildew disease of grapevine and reduce the need to recur to chemical treatments, an effective strategy might be recovering adaptive resistance traits in both cultivated and wildV. viniferagermplasm.Considering that stilbenes represent the most important class of phytoalexins in theVitaceae, the constitutive expression and transcriptional activation of all the functional members of the stilbene synthase gene family were analysed in a group of nine grapevine genotypes following artificial infection with the oomycetePlasmopara viticola, the causal agent of the disease. In addition, in the same genotypes we analyzed the expression of genes encoding for two transcription factors involved in the transcriptional regulation of the stilbene synthase genes, namelyVvMYB14andVvMYB15, and of genes encoding for chalcone synthases.Results: Downy mildew incidence and severity ranged from nihil to high in the grapevine genotypes considered, being low to moderate in a subgroup ofV. viniferagenotypes. The constitutive expression of the stilbene synthase genes as well as the extent of their transcriptional activation followingP. viticolainoculation appeared to be inversely related to the proneness to develop disease symptoms upon infection. In a specular manner, followingP. viticolainoculation all the chalcone synthase genes were up-regulated in the susceptible grapevine genotypes and down-regulated in the resistant ones. The infection brought byP. viticolaappeared to elicit a co-ordinated and sequential transcriptional activation of distinct stilbene synthase genes subsets, each of which may be regulated by a distinct and specific MYB transcription factor.Conclusions: The present results suggest that the induction of stilbene biosynthesis may contribute to the basal immunity against the downy mildew of grapevine, thus representing an adaptive resistance trait to recover, in both cultivated and wildV. viniferagermplasm. During the early stages ofP. viticolainfection, an antagonistic interaction between flavonol and stilbene biosynthesis might occur, whose outcome might determine the subsequent extent of disease symptoms. Further studies are needed to decipher the possible regulatory mechanisms involved in the antagonistic crosstalk between these two metabolic pathways in resistant and susceptible genotypes in response toP. viticola.

Seedlings of durum wheat [Triticum turgidum subsp. durum (Desf.) Husn] were exposed to zinc nutrition and to ozone (O₃) in a factorial combination: adequate (+Zn treatment) or no Zn (−Zn) in the nutrient solution, followed by exposure to either ozone-free air (filtered air, FA) or to 150 nL L⁻¹ ozone (O₃) for 4 h. Although omitting Zn from the nutrient solution failed to impose a genuine Zn deficiency, −Zn*FA durum wheat seedlings showed a typical deficiency behaviour, i.e. Zn mobilisation from root to shoot. Such inter-organ Zn redistribution, however, did not occur in −Zn*O₃ plants. Exposure to each stress singly decreased the activity and the protein amount of foliar plasma membrane H⁺-ATPase, but not stress combination, which even increased the H⁺-ATPase expression with respect to control. In the −Zn*O₃ plants, moreover, the foliar activities of the plasma membrane-bound NAD(P)H-dependent superoxide synthase and of Cu,Zn-superoxide dismutase, and the transcripts abundance of the luminal binding protein and of the protein disulphide isomerase, were also stimulated. It is proposed that, even in the absence of actual Zn starvation, the perception of deficiency conditions could trigger changes in redox homoeostasis at the plasma membrane level, helpful in compensating an O₃-dependent oxidative damage.