Carla António’s research while affiliated with University of Lisbon and other places

Aim: The objective of the work was to assess the effect of biostimulation with selected plant growth-promoting bacteria on growth and metabolite profile of Salicornia europaea. Methods and results: S. europaea seeds were inoculated with different combinations of plant growth-promoting bacteria Brevibacterium casei EB3, Pseudomonas oryzihabitans RL18 and Bacillus aryabhattai SP20. Plants germinated from inoculated seeds were grown either in laboratory conditions or in a saline crop field. Fresh and dry weight were determined at the end of the experiment, for biomass quantification. The microbiological quality of fresh shoots for human consumption as salad greens was assessed, and the persistence of the inoculated strains in the plant rhizosphere was confirmed by next-generation sequencing (Illumina) of the 16S rDNA gene. The primary metabolite profile of biostimulated plants was characterized by GC-TOF-MS.In laboratory conditions, inoculation with the two strains B. casei EB3 and P. oryzihabitans RL18 caused the most significant increase in biomass production (fresh and dry weight), and caused a shift in the central metabolic pathways of inoculated plants towards amino acid biosynthesis. In the field experiment, no significant biostimulation effect was detected with any of the tested inoculants. Seed inoculation had no significant effect on the microbiological quality of the edible parts. The persistence of inoculants was confirmed in both experiments. Conclusions: Manipulation of the plant microbiome can trigger primary metabolic reconfiguration and modulate the plant metabolism while promoting plant growth.

Wild rocket is a popular baby leaf-type crop in the Brassicaceae family, associated with an healthy Mediterranean diet style, grown in Southern Europe, the Middle East and in South-East Asia. However, the high susceptibility of wild rocket to the severe foliar downy mildew (DM) disease caused by the oomycete Hyaloperonospora sp. represents an increasing threat in rocket crop production and quality. The severe legislation adopted in Europe on the use and registration of new pesticides, and the consequent environmental concerns, imply the need to develop resistant varieties less dependent on chemicals. To better understand how central metabolism is affected by this pathogen, six accessions of wild rocket Diplotaxis tenuifolia from different origins and resistance responses to DM (i.e., resistant (R), partially-resistant (PR) and susceptible (S)) were selected for GC-TOF-MS primary metabolite profiling. The metabolic differences in these accessions can result from their direct or indirect involvement in resistance mechanisms. This information will be used for selection of new and improved varieties in future breeding programs.

The pinewood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of the pine wilt disease (PWD) and represents one of the major threats to conifer forests. The detection of the PWN in Portugal, associated with Pinus pinaster, increased the concern of its spread to European forests. Despite its susceptibility to PWD, genetic variability found among P. pinaster populations has been associated with heritable PWD resistance. Understanding the mechanisms underlying tree resistance constitutes a valuable resource for breeding programs toward more resilient forest plantations. This study investigated changes in anatomy, chlorophyll a fluorescence (ChlF), and primary metabolism in susceptible and resistant P. pinaster half-sib plants, after PWN inoculation. Susceptible plants showed a general shutdown of central metabolism, osmolyte accumulation, photosynthetic inhibition, and a decrease in the plant water status. The ChlF transient rise (OJIP curve) revealed the appearance of L- and K-bands, indicators of environmental stress. In contrast, resistant plants revealed a regulated defense response and were able to restrict PWN migration and cellular damage. Furthermore, the accumulation of γ-aminobutyric acid (GABA) and succinate suggested a role of these metabolites in PWD resistance and the possible activation of the GABA shunt. Altogether, these results provide new insights to the role of primary metabolism in PWD resistance and in the selection of resistant phenotypes for disease mitigation.

The reasons underlying the differential tolerance of Actinidia spp. to the pandemic pathogen Pseudomonas syringae pv. actinidiae (Psa) have not yet been elucidated. We hypothesised that differential plant-defence strategies linked to transcriptome regulation, phytohormones, and primary metabolism might be key, and that A. chinensis susceptibility results from an inefficient activation of defensive mechanisms and metabolic impairments shortly following infection. Here, 48 hours post-inoculation bacterial density was 10-fold higher in A. chinensis var. deliciosa than in A. arguta, accompanied by significant increases in glutamine, ornithine, jasmonic acid (JA) and salicylic acid (SA) (up to 3.2-fold). A. arguta showed decreased abscisic acid (ABA) (0.7-fold), no changes in primary metabolites, and 20 defence-related genes that were only differentially expressed in this species. These include GLOX1, FOX1, SN2 and RBOHA, which may contribute to its higher tolerance. Results suggest that A. chinensis' higher susceptibility to Psa is due to an inefficient activation of plant defences, with the involvement of ABA, JA and SA, leading to impairments in primary metabolism, particularly the ammonia assimilation cycle. A schematic overview on the interaction between Psa and genotypes with distinct tolerance is provided, highlighting the key transcriptomic and metabolomic aspects contributing to the different plant phenotypes after infection. This article is protected by copyright. All rights reserved.

Actinorhizal plants have been regarded as promising species in the current climate change context due to their high tolerance to a multitude of abiotic stresses. While combined salt-heat stress effects have been studied in crop species, their impact on the model actinorhizal plant, Casuarina glauca, has not yet been fully addressed. The effect of single salt (400 mM NaCl) and heat (control at 26/22 °C, supra optimal temperatures at 35/22 °C and 45/22 °C day/night) conditions on C. glauca branchlets was characterised at the physiological level, and stress-induced metabolite changes were characterised by mass spectrometry-based metabolomics. C. glauca could withstand single salt and heat conditions. However, the harshest stress condition (400 mM NaCl, 45 °C) revealed photosynthetic impairments due to mesophyll and membrane permeability limitations as well as major stress-specific differential responses in C and N metabolism. The increased activity of enzymatic ROS scavengers was, however, revealed to be sufficient to control the plant oxidative status. Although C. glauca could tolerate single salt and heat stresses, their negative interaction enhanced the effects of salt stress. Results demonstrated that C. glauca responses to combined salt-heat stress could be explained as a sum of the responses from each single applied stress.

Climate change scenarios pose major threats to many crops worldwide, including coffee. We explored the primary metabolite responses in two Coffea genotypes, C. canephora cv. Conilon Clone 153 and C. arabica cv. Icatu, grown at normal (aCO2) or elevated (eCO2) CO2 concentrations of 380 or 700 ppm, respectively, under well-watered (WW), moderate (MWD), or severe (SWD) water deficit conditions, in order to assess coffee responses to drought and how eCO2 can influence such responses. Primary metabolites were analyzed with a gas chromatography time-of-flight mass spectrometry metabolomics platform (GC-TOF-MS). A total of 48 primary metabolites were identified in both genotypes (23 amino acids and derivatives, 10 organic acids, 11 sugars, and 4 other metabolites), with differences recorded in both genotypes. Increased metabolite levels were observed in CL153 plants under single and combined conditions of aCO2 and drought (MWD and SWD), as opposed to the observed decreased levels under eCO2 in both drought conditions. In contrast, Icatu showed minor differences under MWD, and increased levels (especially amino acids) only under SWD at both CO2 concentration conditions, although with a tendency towards greater increases under eCO2. Altogether, CL153 demonstrated large impact under MWD, and seemed not to benefit from eCO2 in either MWD and SWD, in contrast with Icatu.

The pinewood nematode (PWN) is the causal agent of pine wilt disease, a pathology that affects conifer forests, mainly Pinus spp. PWN infection can induce the expression of phytohormone-related genes; however, changes at the early phytohormone level have not yet been explored. Phytohormones are low-abundance metabolites, and thus, difficult to quantify. Moreover, most methodologies focus mainly on Arabidopsis or crop species. This work aimed to validate a fast (run time 6.6 min) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) analytical method to quantify 14 phytohormones in Pinus pinaster stem tissues. This method was further applied to evaluate, for the first time, early phytohormone changes in susceptible and resistant phenotypes of P. pinaster 24, 48 and 72 h after inoculation (HAI) with PWN. A significant increase in salicylic acid (SA, 48 and 72 HAI) and jasmonic acid methyl ester (JA-ME, 72 HAI) was observed in susceptible phenotypes. Results indicate that the higher susceptibility of P. pinaster to PWN infection might result from an inefficient trigger of hypersensitive responses, with the involvement of JA and SA pathways. This work provides an important update in forest research, and adds to the current knowledge of Pinus spp. defence responses to PWN infection.