The alternative oxidase (AOX) is a ubiquinol oxidase with a crucial role in the mitochondrial alternative respiratory pathway, which is associated with various processes in plants. In this study, the activity of AOX in pea seed germination was determined in two pea cultivars, ‘Maravilha d’América’ (MA) and ‘Torta de Quebrar’ (TQ), during a germination trial using cytochrome oxidase (COX) and AOX inhibitors [rotenone (RT) and salicylic hydroxamic acid (SHAM), respectively]. Calorespirometry was used to assess respiratory changes during germination. In both cultivars, SHAM had a greater inhibitory effect on germination than RT, demonstrating the involvement of AOX in germination. Although calorespirometry did not provide direct information on the involvement of the alternative pathway in seed germination, this methodology was valuable for distinguishing cultivars. To gain deeper insights into the role of AOX in seed germination, the AOX gene family was characterized, and the gene expression pattern was evaluated. Three PsAOX members were identified—PsAOX1, PsAOX2a and PsAOX2b—and their expression revealed a marked genotype effect. This study emphasizes the importance of AOX in seed germination, contributing to the understanding of the role of the alternative respiratory pathway in plants.

Metabolites represent the end product of gene expression, protein interaction and other regulatory mechanisms. The metabolome reflects a biological system’s response to genetic and environmental changes, providing a more accurate description of plants’ phenotype than the transcriptome or the proteome. Grapevine (Vitis vinifera L.), established for the production of wine grapes, table grapes, and raisins, holds immense agronomical and economic significance not only in the Mediterranean region but worldwide. As all plants, grapevines face the adverse impact of biotic and abiotic stresses that negatively affect multiple stages of grape and wine industry, including plant and berry development pre- and post-harvest, fresh grapes processing and consequently wine quality. In the present review we highlight the applicability of metabolome analysis in the understanding of the mechanisms involved in grapevine response and acclimatization upon the main biotic and abiotic constrains. The metabolome of induced morphogenic processes such as adventitious rooting and somatic embryogenesis is also explored, as it adds knowledge on the physiological and molecular phenomena occurring in the explants used, and on the successfully propagation of grapevines with desired traits. Finally, the microbiome-induced metabolites in grapevine are discussed in view of beneficial applications derived from the plant symbioses.

Somatic embryogenesis (SE) involves the formation of embryo-like structures from somatic cells without fertilization and is widely used for clonal propagation and genetic transformation. However, in olive (Olea europaea sp. europaea), SE remains challenging due to the recalcitrant behavior of adult tissues when used as initial explants. Bioactive molecules released into the culture medium (conditioned medium, CM) by embryogenic cultures have been identified as modulators of the SE response. However, their potential role in enhancing SE efficiency in olive and overcoming tissue recalcitrance remains largely unexplored. To investigate the role of these biomolecules in olive SE, a protocol was established using SE cultures of cv. ‘Galega Vulgar’. Proteins and metabolites were separated by filtration, concentrated through lyophilization, and precipitated using three methods: Acetone, TCA/Acetone, and Methanol/Chloroform. The efficiency of these methods was evaluated through total protein quantification and via SDS-PAGE electrophoresis. LC-MS/MS was employed to analyze secretome composition using the TCA/Acetone precipitation method. Additionally, metabolite profiles were analyzed using ¹H NMR spectroscopy. The results led to the identification of 1096 (526 protein groups) Olea europaea proteins, including well-known SE biomarkers such as kinases and peroxidases. NMR spectroscopy identified several metabolites secreted into the medium or resulting from the metabolic activity of secreted enzymes, confirming the applicability of the procedure. Although extracting secreted biomolecules from the culture medium presents significant challenges, the protocol established in this study successfully enabled the isolation and identification of both proteins and metabolites, revealing a valuable workflow for future in-depth analyses of secreted biomolecules in olive SE.

High temperatures significantly impact grapevine growth and development and lead to severe losses in grape quality and production. To minimize the impact of those environmental constraints, the application of biostimulants (BSts) has emerged as one of the most interesting strategies. BSts application derived from cactus species has been described as a successful approach to enhance tolerance to biotic and abiotic stresses. In this study, an aqueous extract prepared from the cladodes of Opuntia ficus-indica was applied through foliar spraying to grapevine plants (Vitis vinifera L.) ’Aragonez’ already under heat stress. The effect of the extract application on protecting grapevine plants against heat stress was assessed in an experiment running during 15 days after extract application by determining several physiological parameters and detecting the changes in the whole proteome profile by comparing non-treated and extract-treated samples. Results show that physiological parameters directly related to photosynthesis showed a positive effect of the extract in mitigating heat stress in grapevines. Proteomic analysis indicated that the extract significantly upregulated proteins associated with photosynthesis and stress responses. This study provides new insights about the effect of O. ficus-indica extract in grapevines, offering a valuable strategy for future applications under field conditions.

Somatic embryogenesis (SE) is a powerful biotechnological tool for large-scale clonal plant propagation. However, most woody species exhibit a recalcitrant response. Olea europaea L., a highly valuable tree crop, is among these recalcitrant species. Unravelling the molecular mechanisms underlying SE efficiency in O. europaea is, therefore, essential. Access to embryogenic lines with contrasting capacities for somatic embryo differentiation is a key requirement for such studies. Mature fruits of olive cultivars ‘Galega Vulgar’ and ‘Arbequina’ were collected from open-pollinated orchard-grown plants, and various explants taken from zygotic embryos were used to establish SE. A differentiated response was observed both within each cultivar and between cultivars, with cv. ‘Arbequina’ showing the highest embryogenic induction, particularly when radicles were used. Secondary SE was successfully established for both high- and low-efficiency lines, providing valuable material for future molecular studies. Somatic embryo conversion into plants, a key indicator of embryo quality, was successfully achieved in both cultivars. Flow cytometry analysis revealed a high degree of chromosomal stability. This study presents a reliable procedure to obtain and maintain distinct embryogenic responses in O. europaea, identifying lines with both high and low embryogenic efficiency that can serve as model systems for future molecular investigations.

Organic agriculture, recognized as a more sustainable agricultural system, strongly depends on the use of highly resilient genotypes. Seed resilience, with increased tolerance to germination that provide vigorous seedlings under environmental stresses, currently represents one of the most important agronomical traits. Seed germination involves the activation of several metabolic pathways, including cellular respiration. Alternative oxidase (AOX), a key enzyme in the alternative respiratory pathway, plays a crucial role in regulating cell reprogramming by controlling metabolic transitions related to the cellular redox state and the variable carbon balance. The involvement of the alternative respiratory pathway during germination was explored by analysis of PsAOX gene/protein expression. Seeds of four Pisum sativum L. cultivars (‘Respect-1′, ‘S134′, ‘G78′, and ‘S91′) were imbibed in sterile tap water for 16 h and metabolic parameters were measured by calorespirometry (heat and CO₂ emission rates) in a multi-cell differential scanning calorimeter in isothermal mode at 25 °C. The involvement of PsAOX was evaluated by transcript quantification (PsAOX1, PsAOX2a, and PsAOX2b) through RT-qPCR, and by analyzing PsAOX expression through Western blot. The results demonstrate that cv. ‘S91′, characterized by a low germination rate, exhibited the lowest metabolic heat and CO₂ emission rate. However, contrary to expectations, PsAOX transcript accumulation and PsAOX protein expression were significantly higher for ‘S91′ than for the other cultivars. These results indicate that higher levels of PsAOX (transcript and protein) could be linked to lower metabolic rates for embryo growth when seed germination is compromised.