Germination of Pisum sativum L. Seeds Is Associated with the Alternative Respiratory Pathway.

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.

Dynamic Regulation of Grapevine's microRNAs in Response to Mycorrhizal Symbiosis and High Temperature.

MicroRNAs (miRNAs) are non-coding small RNAs that play crucial roles in plant development and stress responses and can regulate plant interactions with beneficial soil microorganisms such as arbuscular mycorrhizal fungi (AMF). To determine if root inoculation with distinct AMF species affected miRNA expression in grapevines subjected to high temperatures, RNA-seq was conducted in leaves of grapevines inoculated with either Rhizoglomus irregulare or Funneliformis mosseae and exposed to a high-temperature treatment (HTT) of 40 °C for 4 h per day for one week. Our results showed that mycorrhizal inoculation resulted in a better plant physiological response to HTT. Amongst the 195 identified miRNAs, 83 were considered isomiRs, suggesting that isomiRs can be biologically functional in plants. The number of differentially expressed miRNAs between temperatures was higher in mycorrhizal (28) than in non-inoculated plants (17). Several miR396 family members, which target homeobox-leucine zipper proteins, were only upregulated by HTT in mycorrhizal plants. Predicted targets of HTT-induced miRNAs in mycorrhizal plants queried to STRING DB formed networks for Cox complex, and growth and stress-related transcription factors such as SQUAMOSA promoter-binding-like-proteins, homeobox-leucine zipper proteins and auxin receptors. A further cluster related to DNA polymerase was found in R. irregulare inoculated plants. The results presented herein provide new insights into miRNA regulation in mycorrhizal grapevines under heat stress and can be the basis for functional studies of plant-AMF-stress interactions.

TaqMan Probes for Plant Species Identification and Quantification in Food and Feed Traceability.

In the last few years, the traceability and labeling of processed food and feeds have gained increasing importance due to the impact that mislabeling and product fraud may have on human/animal health or on the quality of final products, such as milk, cheese, and meat, as a consequence of animal dietary. The presence of contaminants or possible frauds due to the use of alternative plant materials in food and feeds can greatly impact the economy; therefore, they are becoming important targets for product certification by competent institutional services. This is especially relevant when complex matrixes are considered, in which the visual identification of the different components is quite difficult or even impossible. Despite the existence of mandatory traceability requirements for the analysis of feed/food composition addressed by European Community regulations, the labels do not always provide a sufficient guarantee about the ingredients and additive composition of those products. In this sense, the development of new methodologies that aim to assess the traceability of feed and food complex matrixes is crucial. In this chapter, a general protocol is presented for the establishment of quantitative real-time PCR-based techniques based on TaqMan assays applied to feed/food traceability, with a special focus on applications in the areas of food and feed security (e.g., for the detection of plant species involved in allergenic reactions), fraud detection (e.g., genetically modified organisms), and certification (e.g., protected denomination of origin).

Understanding the Role of PIN Auxin Carrier Genes under Biotic and Abiotic Stresses in Olea europaea L.

The PIN-FORMED (PIN) proteins represent the most important polar auxin transporters in plants. Here, we characterized the PIN gene family in two olive genotypes, the Olea europaea subsp. europaea var. sylvestris and the var. europaea (cv. 'Farga'). Twelve and 17 PIN genes were identified for vars. sylvestris and europaea, respectively, being distributed across 6 subfamilies. Genes encoding canonical OePINs consist of six exons, while genes encoding non-canonical OePINs are composed of five exons, with implications at protein specificities and functionality. A copia-LTR retrotransposon located in intron 4 of OePIN2b of var. europaea and the exaptation of partial sequences of that element as exons of the OePIN2b of var. sylvestris reveals such kind of event as a driving force in the olive PIN evolution. RNA-seq data showed that members from the subfamilies 1, 2, and 3 responded to abiotic and biotic stress factors. Co-expression of OePINs with genes involved in stress signaling and oxidative stress homeostasis were identified. This study highlights the importance of PIN genes on stress responses, contributing for a holistic understanding of the role of auxins in plants.

Exploring the Applicability of Calorespirometry to Assess Seed Metabolic Stability Upon Temperature Stress Conditions-Pisum sativum L. Used as a Case Study.

The availability of phenotyping tools to assist breeding programs in the selection of high-quality crop seeds is of obvious interest with consequences for both seed producers and consumers. Seed germination involves the activation of several metabolic pathways, such as cellular respiration to provide the required ATP and reducing power. This work tested the applicability of calorespirometry, the simultaneous measurement of heat and CO2 rates, as a phenotyping tool to assess seed respiratory properties as a function of temperature. The effect of temperature on seed germination was evaluated after 16 h of seed imbibition by calorespirometric experiments performed in isothermal mode at 15, 20, 25, and 28°C on the seeds of three cultivars of peas (Pisum sativum L.) commonly used in conventional agriculture (cvs. 'Rondo', 'Torta de Quebrar', and 'Maravilha d'América'). Significant differences in metabolic heat rate and CO2 production rate (R CO2 ) as well as in the temperature responses of these parameters were found among the three cultivars. A seed germination trial was conducted during the 6 days of imbibition to evaluate the predictive power of the parameters derived from the calorespirometric measurements. The germination trial showed that the optimal germination temperature was 20°C and low germination rates were observed at extreme temperatures (15 or 28°C). The cv. 'Torta de Quebrar' showed significantly higher germination in comparison with the other two cultivars at all three temperatures. In comparison with the other two cultivars, 'Torta de Quebrar' has the lowest metabolic heat and CO2 rates and the smallest temperature dependence of these measured parameters. Additionally, 'Torta de Quebrar' has the lowest values of growth rate and carbon use efficiency calculated from the measured variables. These data suggest that calorespirometry is a useful tool for phenotyping physiologic efficiency at different temperatures during early germination stages, and can determine the seeds with the highest resilience to temperature variation, in this case 'Torta de Quebrar'.

Carrot AOX2a Transcript Profile Responds to Growth and Chilling Exposure.

Alternative oxidase (AOX) is a key enzyme of the alternative respiration, known to be involved in plant development and in response to various stresses. To verify the role of DcAOX1 and DcAOX2a genes in carrot tap root growth and in response to cold stress, their expression was analyzed in two experiments: during root growth for 13 weeks and in response to a cold challenge trial of 7 days, in both cases using different carrot cultivars. Carrot root growth is initially characterized by an increase in length, followed by a strong increase in weight. DcAOX2a presented the highest expression levels during the initial stages of root growth for all cultivars, but DcAOX1 showed no particular trend in expression. Cold stress had a negative impact on root growth, and generally up-regulated DcAOX2a with no consistent effect on DcAOX1. The identification of cis-acting regulatory elements (CAREs) located at the promoters of both genes showed putative sequences involved in cold stress responsiveness, as well as growth. However, DcAOX2a promoter presented more CAREs related to hormonal pathways, including abscisic acid and gibberellins synthesis, than DcAOX1. These results point to a dual role of DcAOX2a on carrot tap root secondary growth and cold stress response.

Erratum: Velada et al. Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA. Biology 2021, 10, 209.

The author wishes to make an erratum to the published version of the paper [...].

Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA.

Higher plants are composed of different tissue and cell types. Distinct cells host different biochemical and physiological processes which is reflected in differences in gene expression profiles, protein and metabolite levels. When omics are to be carried out, the information provided by a specific cell type can be diluted and/or masked when using a mixture of distinct cells. Thus, studies performed at the cell- and tissue-type level are gaining increasing interest. Laser microdissection (LM) technology has been used to isolate specific tissue and cell types. However, this technology faces some challenges depending on the plant species and tissue type under analysis. Here, we show for the first time a LM protocol that proved to be efficient for harvesting specific tissue types (phloem, cortex and epidermis) from olive stem nodal segments and obtaining RNA of high quality. This is important for future transcriptomic studies to identify rooting-competent cells. Here, nodal segments were flash-frozen in liquid nitrogen-cooled isopentane and cryosectioned. Albeit the lack of any fixatives used to preserve samples' anatomy, cryosectioned sections showed tissues with high morphological integrity which was comparable with that obtained with the paraffin-embedding method. Cells from the phloem, cortex and epidermis could be easily distinguished and efficiently harvested by LM. Total RNA isolated from these tissues exhibited high quality with RNA Quality Numbers (determined by a Fragment Analyzer System) ranging between 8.1 and 9.9. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types of olive stems and obtaining high-quality RNA.

Response of Mycorrhizal 'Touriga Nacional' Variety Grapevines to High Temperatures Measured by Calorespirometry and Near-Infrared Spectroscopy.

Heat stress negatively affects several physiological and biochemical processes in grapevine plants. In this work, two new methods, calorespirometry, which has been used to determine temperature adaptation in plants, and near-infrared (NIR) spectroscopy, which has been used to determine several grapevine-related traits and to discriminate among varieties, were tested to evaluate grapevine response to high temperatures. 'Touriga Nacional' variety grapevines, inoculated or not with Rhizoglomus irregulare or Funneliformis mosseae, were used in this study. Calorespirometric parameters and NIR spectra, as well as other parameters commonly used to assess heat injury in plants, were measured before and after high temperature exposure. Growth rate and substrate carbon conversion efficiency, calculated from calorespirometric measurements, and stomatal conductance, were the most sensitive parameters for discriminating among high temperature responses of control and inoculated grapevines. The results revealed that, although this vine variety can adapt its physiology to temperatures up to 40 °C, inoculation with R. irregulare could additionally help to sustain its growth, especially after heat shocks. Therefore, the combination of calorespirometry together with gas exchange measurements is a promising strategy for screening grapevine heat tolerance under controlled conditions and has high potential to be implemented in initial phases of plant breeding programs.

Somatic Embryogenesis from Mature Embryos of Olea europaea L. cv. 'Galega Vulgar' and Long-Term Management of Calli Morphogenic Capacity.

Several olive cultivars, characterized by high-quality olive oil show agronomical issues such as excessive vigor, high susceptibility to biotic and abiotic stresses, and low propagation ability. They are strong candidates for breeding based on new technologies to improve their performance in a short period of time. For this reason, the first step is developing efficient somatic embryogenesis (SE) protocols. Somatic embryogenesis in olive is highly genotype-dependent for both adult tissues and mature embryos as initial explants, requiring the development of specific protocols for each genotype. Trials using cotyledons and radicles as initial explants, isolated from ripe seeds from the Portuguese olive cv. 'Galega vulgar', gave more than 95% calli development. Radicles proved to be the most responsive tissue for SE induction, with an average of 2 embryos per callus after callus transfer to expression medium, and 14 embryos per callus after subculture on the olive cyclic embryogenesis medium (ECO). Embryogenic competence could be recovered after several subcultures on ECO medium that maintained cyclic embryogenesis for an indeterminate period of time. Embryo conversion and plant acclimatization were also attained with high success rates. Media management for cyclic embryogenesis maintenance is of general importance for SE protocols in any olive genotype. Somatic embryogenesis was thus attained for the first time in embryo-derived explants of cv. 'Galega vulgar'.

Expression Profile of PIN-Formed Auxin Efflux Carrier Genes during IBA-Induced In Vitro Adventitious Rooting in Olea europaea L.

Exogenous auxins supplementation plays a central role in the formation of adventitious roots (AR) for several plant species. However, the molecular mechanisms underlying the process of adventitious rooting are still not completely understood and many plants with economic value, including several olive cultivars, exhibit a recalcitrant behavior towards cutting propagation, which limits its availability in plant nurseries. PIN-formed proteins are auxin efflux transporters that have been widely characterized in several plant species due to their involvement in many developmental processes including root formation. The present study profiled the expression of the OePIN1a-c, OePIN2b, OePIN3a-c, OePIN5a-c, OePIN6, and OePIN8 gene members during indole-3-butyric acid (IBA)-induced in vitro adventitious rooting using the olive cultivar 'Galega vulgar'. Gene expression analysis by quantitative real time PCR (RT-qPCR) showed drastic downregulation of most transcripts, just a few hours after explant inoculation, in both nontreated and IBA-treated microcuttings, albeit gene downregulation was less pronounced in IBA-treated stems. In contrast, OePIN2b showed a distinct expression pattern being upregulated in both conditions, and OePIN5b was highly upregulated in IBA-induced stems. All transcripts, except OePIN8, showed different expression profiles between nontreated and IBA-treated explants throughout the rooting experiment. Additionally, high levels of reactive oxygen species (ROS) were observed soon after explant preparation, decreasing a few hours after inoculation. Altogether, the results suggest that wounding-related ROS production, associated with explant preparation for rooting, may have an impact on auxin transport and distribution via changes in OePIN gene expression. Moreover, the application of exogenous auxin may modulate auxin homeostasis through regulation of those genes, leading to auxin redistribution throughout the stem-base tissue, which may ultimately play an important role in AR formation.

AOX1-Subfamily Gene Members in Olea europaea cv. "Galega Vulgar"-Gene Characterization and Expression of Transcripts during IBA-Induced in Vitro Adventitious Rooting.

Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar "Galega vulgar". The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars.

A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples.

Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and species-specific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.

Interlaboratory Comparison of Methods Determining the Botanical Composition of Animal Feed.

A consortium of European enterprises and research institutions has been engaged in the Feed-Code Project with the aim of addressing the requirements stated in European Union Regulation No. 767/2009, concerning market placement and use of feed of known and ascertained botanical composition. Accordingly, an interlaboratory trial was set up to compare the performance of different assays based either on optical microscope or DNA analysis for the qualitative and quantitative identification of the composition of compound animal feeds. A tubulin-based polymorphism method, on which the Feed-Code platform was developed, provided the most accurate results. The present study highlights the need for the performance of ring trials for the determination of the botanical composition of animal feeds and raises an alarm on the actual status of analytical inaccuracy.

Alternative Oxidase Gene Family in Hypericum perforatum L.: Characterization and Expression at the Post-germinative Phase.

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.

Stress-Induced Accumulation of DcAOX1 and DcAOX2a Transcripts Coincides with Critical Time Point for Structural Biomass Prediction in Carrot Primary Cultures (Daucus carota L.).

Stress-adaptive cell plasticity in target tissues and cells for plant biomass growth is important for yield stability. In vitro systems with reproducible cell plasticity can help to identify relevant metabolic and molecular events during early cell reprogramming. In carrot, regulation of the central root meristem is a critical target for yield-determining secondary growth. Calorespirometry, a tool previously identified as promising for predictive growth phenotyping has been applied to measure the respiration rate in carrot meristem. In a carrot primary culture system (PCS), this tool allowed identifying an early peak related with structural biomass formation during lag phase of growth, around the 4th day of culture. In the present study, we report a dynamic and correlated expression of carrot AOX genes (DcAOX1 and DcAOX2a) during PCS lag phase and during exponential growth. Both genes showed an increase in transcript levels until 36 h after explant inoculation, and a subsequent down-regulation, before the initiation of exponential growth. In PCS growing at two different temperatures (21°C and 28°C), DcAOX1 was also found to be more expressed in the highest temperature. DcAOX genes' were further explored in a plant pot experiment in response to chilling, which confirmed the early AOX transcript increase prior to the induction of a specific anti-freezing gene. Our findings point to DcAOX1 and DcAOX2a as being reasonable candidates for functional marker development related to early cell reprogramming. While the genomic sequence of DcAOX2a was previously described, we characterize here the complete genomic sequence of DcAOX1.

Do Mitochondria Play a Central Role in Stress-Induced Somatic Embryogenesis?

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.

Intra and Inter-Spore Variability in Rhizophagus irregularis AOX Gene.

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF symbiosis improves nutrient uptake and buffers the plant against a diversity of stresses. Rhizophagus irregularis is one of the most widespread AMF species in the world, and its application in agricultural systems for yield improvement has increased over the last years. Still, from the inoculum production perspective, a lack of consistency of inoculum quality is referred to, which partially may be due to a high genetic variability of the fungus. The alternative oxidase (AOX) is an enzyme of the alternative respiratory chain already described in different taxa, including various fungi, which decreases the damage caused by oxidative stress. Nevertheless, virtually nothing is known on the involvement of AMF AOX on symbiosis establishment, as well on the existence of AOX variability that could affect AMF effectiveness and consequently plant performance. Here, we report the isolation and characterisation of the AOX gene of R. irregularis (RiAOX), and show that it is highly expressed during early phases of the symbiosis with plant roots. Phylogenetic analysis clustered RiAOX sequence with ancient fungi, and multiple sequence alignment revealed the lack of several regulatory motifs which are present in plant AOX. The analysis of RiAOX polymorphisms in single spores of three different isolates showed a reduced variability in one spore relatively to a group of spores. A high number of polymorphisms occurred in introns; nevertheless, some putative amino acid changes resulting from non-synonymous variants were found, offering a basis for selective pressure to occur within the populations. Given the AOX relatedness with stress responses, differences in gene variants amongst R. irregularis isolates are likely to be related with its origin and environmental constraints and might have a potential impact on inoculum production.

Reference genes selection and normalization of oxidative stress responsive genes upon different temperature stress conditions in Hypericum perforatum L.

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.