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Microbial diversity profoundly influences soil ecosystem functions, making it vital to monitor community dynamics to comprehend its structure. Our study focused on six agrosystems in Madeira Archipelago, analysing bacteria, archaea, fungi and AMF through classical microbiology and molecular techniques. Despite distinct edaphoclimatic conditions and management practices, bacterial structures exhibited similarities, with Alphaproteobacteria at 18%-20%, Bacilli at 11%-18% and Clostridia at 9%-14%. The predominance of copiothrophic groups suggested that soil nutrient content was the driver of these communities. Regarding archaea, the communities changed among sites, and it was evident that agrosystems provided niches for methanogens. The Crenarchaeota varied between 15% and 29%, followed by two classes of Euryarchaeota, Methanomicrobia (17%-25%) and Methanococci (4%-32%). Fungal communities showed consistent composition at the class level but had differing diversity indices due to management practices and soil texture. Sordaryomycetes (21%-28%) and Agaricomycetes (15%-23%) were predominant. Conversely, AMF communities appeared to be also influenced by the agrosystem, with Glomus representing over 50% of the community in all agrosystems. These insights into microbial groups' susceptibilities to environmental conditions are crucial for maintaining healthy soil and predicting climate change effects on agrosystems' productivity, resilience and sustainability. Additionally, our findings enable the development of more robust prediction models for agricultural practices., (© 2024 The Authors. Environmental Microbiology Reports published by Applied Microbiology International and John Wiley & Sons Ltd.)

Background: Twenty-two native Crop Wild Relatives (CWR) occur in specific dry environments of Madeira Archipelago, like Desembarcadouro islet in Ponta de São Lourenço and Chão islet in Desertas Islands. Nine of them share the same gene pool with crop species included in Annex I of the International Treaty on Plant Genetic Resources for Food and Agriculture. Amongst them, Beta patula Aiton, an IUCN Critically Endangered species, has been studied in detail for in situ and ex situ conservation. The present paper summarises the information recorded during the Beta patula population assessment. Valuable information on plant communities associated with this species was obtained., New Information: The data provides information of a vegetation census spanning 7 years, from 2014 to 2020, in two uninhabited islets where Beta patula occurs, Desembarcadouro and Chão islets. The collected dataset consists of 1,786 vegetation descriptions, where 31 species were recorded. All generated data have been published and will be used towards the implementation of conservation actions and to establish a middle term management protocol for Beta patula and other CWR in situ conservation in the framework of a genetic reserve. This work is part of a EU LIFE Project, LIFE RECOVER NATURA and was conducted by members of the ISOPlexis Center, University of Madeira., (Humberto Nóbrega, Gregório Freitas, M. A. Zavattieri, Carla Ragonezi, Miguel Â. A. Pinheiro de Carvalho.)

Laser microdissection provides a useful method for isolating specific cell types from complex biological samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus complicating tissue preparation and extraction of macromolecules such as DNA. In this study, we report a method that allows tissue-specific gene amplification. An improved perception of genetic identity of the entire plant can contribute to improved functional marker strategies. Alternative oxidase (AOX) has crucial position for stress-induced responses/adaptation. Daucus carota sequence polymorphisms in AOX were identified, however, never at tissue/cell level. This technology will support studying AOX gene sequences in carrot organs/tissues/cells and specifically exploring differential polymorphisms in root meristem that might be associated to adaptive growth upon all kind of stresses. Details on aspects of tissue preparation, including fixation and embedding procedures, laser capture microdissection, DNA extraction, and amplification, are provided. A combination of laser microdissection and polymerase chain reaction amplification provides a powerful tool for the analysis of AOX gene amplification in methacarn-fixed paraffin-embedded tissues.

Alternative oxidase (AOX) protein is located in the inner mitochondrial membrane and is encoded in the nuclear genome being involved in plant response upon a diversity of environmental stresses and also in normal plant growth and development. Here we report the characterization of the AOX gene family of Hypericum perforatum L. Two AOX genes were identified, both with a structure of four exons (HpAOX1, acc. KU674355 and HpAOX2, acc. KU674356). High variability was found at the N-terminal region of the protein coincident with the high variability identified at the mitochondrial transit peptide. In silico analysis of regulatory elements located at intronic regions identified putative sequences coding for miRNA precursors and trace elements of a transposon. Simple sequence repeats were also identified. Additionally, the mRNA levels for the HpAOX1 and HpAOX2, along with the ones for the HpGAPA (glyceraldehyde-3-phosphate dehydrogenase A subunit) and the HpCAT1 (catalase 1), were evaluated during the post-germinative development. Gene expression analysis was performed by RT-qPCR with accurate data normalization, pointing out HpHYP1 (chamba phenolic oxidative coupling protein 1) and HpH2A (histone 2A) as the most suitable reference genes (RGs) according to GeNorm algorithm. The HpAOX2 transcript demonstrated larger stability during the process with a slight down-regulation in its expression. Contrarily, HpAOX1 and HpGAPA (the corresponding protein is homolog to the chloroplast isoform involved in the photosynthetic carbon assimilation in other plant species) transcripts showed a marked increase, with a similar expression pattern between them, during the post-germinative development. On the other hand, the HpCAT1 (the corresponding protein is homolog to the major H2O2-scavenging enzyme in other plant species) transcripts showed an opposite behavior with a down-regulation during the process. In summary, our findings, although preliminary, highlight the importance to investigate in more detail the participation of AOX genes during the post-germinative development in H. perforatum, in order to explore their functional role in optimizing photosynthesis and in the control of reactive oxygen species (ROS) levels during the process.

Molecular plant breeding usually overlooks the genetic variability that arises from the association of plants with endophytic microorganisms, when looking at agronomic interesting target traits. This source of variability can have crucial effects on the functionality of the organism considered as a whole (the holobiont), and therefore can be selectable in breeding programs. However, seeing the holobiont as a unit for selection and improvement in breeding programs requires novel approaches for genotyping and phenotyping. These should not focus just at the plant level, but also include the associated endophytes and their functional effects on the plant, to make effective desirable trait screenings. The present review intends to draw attention to a new research field on functional hologenomics that if associated with adequate phenotyping tools could greatly increase the efficiency of breeding programs., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This review highlights a four-step rational for the hypothesis that mitochondria play an upstream central role for stress-induced somatic embryogenesis (SE): (1) Initiation of SE is linked to programmed cell death (PCD) (2) Mitochondria are crucially connected to cell death (3) SE is challenged by stress per se (4) Mitochondria are centrally linked to plant stress response and its management. Additionally the review provides a rough perspective for the use of mitochondrial-derived functional marker (FM) candidates to improve SE efficiency. It is proposed to apply SE systems as phenotyping tool for identifying superior genotypes with high general plasticity under severe plant stress conditions.

Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used technique for gene expression analysis. The reliability of this method depends largely on the suitable selection of stable reference genes for accurate data normalization. Hypericum perforatum L. (St. John's wort) is a field growing plant that is frequently exposed to a variety of adverse environmental stresses that can negatively affect its productivity. This widely known medicinal plant with broad pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory, antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with respect to the identification of reference genes suitable for RT-qPCR data normalization. In this study, 11 candidate reference genes were analyzed in H. perforatum plants subjected to cold and heat stresses. The expression stability of these genes was assessed using GeNorm, NormFinder and BestKeeper algorithms. The results revealed that the ranking of stability among the three algorithms showed only minor differences within each treatment. The best-ranked reference genes differed between cold- and heat-treated samples; nevertheless, TUB was the most stable gene in both experimental conditions. GSA and GAPDH were found to be reliable reference genes in cold-treated samples, while GAPDH showed low expression stability in heat-treated samples. 26SrRNA and H2A had the highest stabilities in the heat assay, whereas H2A was less stable in the cold assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress responses and oxidative stress, were used as target genes to validate the reliability of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic analysis for the selection of suitable reference genes for RT-qPCR studies in H. perforatum subjected to temperature stress conditions, but may also provide valuable information about the roles of genes associated with temperature stress responses.

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have beneficial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were collected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporocarps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effectiveness of the techniques used to amplify DNA polymorphic sequences, which enhances the characterization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.