Publications

ISHS International Symposium on Carrot and Other Apiaceae Characterization of the plastid terminal oxidase gene in carrot-involvement in carotenoids accumulation during storage root development

Olive (Olea europaea L.) trees are mainly propagated by adventitious rooting of semi-hardwood cuttings. However, efficient commercial propagation of valuable olive tree cultivars or landraces by semi-hardwood cuttings can often be restricted by a low rooting capacity. We hypothesize that root induction is a plant cell reaction linked to oxidative stress and that activity of stress-induced alternative oxidase (AOX) is importantly involved in adventitious rooting. To identify AOX as a source for potential functional marker sequences that may assist tree breeding, genetic variability has to be demonstrated that can affect gene regulation. The paper presents an applied, multidisciplinary research approach demonstrating first indications of an important relationship between AOX activity and differential adventitious rooting in semi-hardwood cuttings. Root induction in the easy-to-root Portuguese cultivar 'Cobrançosa' could be significantly reduced by treatment with salicyl-hydroxamic acid, an inhibitor of AOX activity. On the contrary, treatment with H2O2 or pyruvate, both known to induce AOX activity, increased the degree of rooting. Recently, identification of several O. europaea (Oe) AOX gene sequences has been reported from our group. Here we present for the first time partial sequences of OeAOX2. To search for polymorphisms inside of OeAOX genes, partial OeAOX2 sequences from the cultivars 'Galega vulgar', 'Cobrançosa' and 'Picual' were cloned from genomic DNA and cDNA, including exon, intron and 3'-untranslated regions (3'-UTRs) sequences. The data revealed polymorphic sites in several regions of OeAOX2. The 3'-UTR was the most important source for polymorphisms showing 5.7% of variability. Variability in the exon region accounted 3.4 and 2% in the intron. Further, analysis performed at the cDNA from microshoots of 'Galega vulgar' revealed transcript length variation for the 3'-UTR of OeAOX2 ranging between 76 and 301 bp. The identified polymorphisms and 3'-UTR length variation can be explored in future studies for effects on gene regulation and a potential linkage to olive rooting phenotypes in view of marker-assisted plant selection.

Reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization through the use of suitable reference genes. This study aimed selecting robust and reliable reference genes which are constitutively and equally expressed for accurate RT-qPCR normalization analysis in two different experimental systems with carrot. A systematic comparison of 12 selected candidate genes for carrot is presented. These included seven genes commonly used as reference gene: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), 18S ribosomal RNA (18S rRNA), ubiquitin (UBQ), alpha-tubulin (TUA), beta-tubulin (β-TUB) and elongation factor-1alpha (EF-1A). Additionally, other five genes were here presented as potential candidates: other ribosomal RNA (25S and 5.8S rRNA) and ribosomal protein L2 encoding gene (rpL2), the transcriptional initiation factor (TIF1), and the heat shock protein 70 (hsp70). Expression stability was evaluated in: (i) in vivo growth experiment based on carrot tap root secondary growth carried out in growth chambers and (ii) realization phase of somatic embryogenesis. During carrot tap root secondary growth, two reference genes GAPDH and 5.8S rRNA, were stably expressed. In the somatic embryogenesis realization experiment, two ribosomal RNAs were selected as reference genes, the 5.8S and the 25S rRNA. Additionally, the expression profile of the mitochondrial alternative oxidase gene DcAOX1 was conducted in the in vivo growth experiment, to show the impact of reference genes selection. Taken together, our results provide a systematic analysis for the selection of superior reference genes for accurate transcript normalization in carrot, under different experimental conditions. We reinforce the idea that the validation of reference genes for the conditions under study is essential, as emphasized by the expression analysis of DcAOX1 as target gene. These results show that a previous selection of reference genes for each experimental system is crucial to achieve accurate and reliable RT-qPCR gene expression data, avoiding low precision or misleading results.

Alternative oxidase (AOX) has been proposed as a functional marker candidate in a number of events involving cell differentiation, including rooting effici

No abstract is provided for this article.

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 (AFP). Our findings point to DcAOX1 and DcAOX2a as being reasonable candidates for functional marker development related to early cell reprogramming. While the sequence of gDcAOX2a was previously described, we characterize the complete genomic sequence of DcAOX1.

In a perspective entitled ‘From plant survival under severe stress to anti-viral human defense’ we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named ‘ReprogVirus’ was proposed for in vitro -scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the ‘ReprogVirus platform’ was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to ‘RegroVirus’ complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a ma jor c omplex t rait for e arly d e novo programming during SARS-CoV-2 infection, called ‘CoV-MAC-TED’. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target ‘CoV-MAC-TED’ in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that ‘de-stressing’ disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.

Plant alternative oxidase (AOX) is a mitochondrial inner membrane enzyme involved in alternative respiration. The critical importance of the enzyme during acclimation upon stress of plant cells is not fully understood and is still an issue of intensive research and discussion. Recently, a role of AOX was suggested for the ability of plant cells to change easily its fate upon stress. In order to get new insights about AOX involvement in cell reprogramming, quantitative real‐time polymerase chain reaction (PCR) and inhibitor studies were performed during cell redifferentiation and developmental stages of Daucus carota L. somatic embryogenesis. Transcript level analysis shows that D. carota AOX genes ( DcAOX1a and DcAOX2a ) are differentially expressed during somatic embryogenesis. DcAOX1a shows lower expression levels, being mainly down‐regulated, whereas DcAOX2a presented a large up‐regulation during initiation of the realization phase of somatic embryogenesis. However, when globular embryos start to develop, both genes are down‐regulated, being this state transient for DcAOX2a . In addition, parallel studies were performed using salicylhydroxamic acid (SHAM) in order to inhibit AOX activity during the realization phase of somatic embryogenesis. Embryogenic cells growing in the presence of the inhibitor were unable to develop embryogenic structures and its growth rate was diminished. This effect was reversible and concentration dependent. The results obtained contribute to the hypothesis that AOX activity supports metabolic reorganization as an essential part of cell reprogramming and, thus, enables restructuring and de novo cell differentiation.

No abstract is provided for this article.

Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of the inner and outer environment of the plant. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at the systems level. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as germination, root and shoot growth, and formation of adventitious, clonal, and reproductive organs, fruits, and seeds. Fermentative paths can help in acclimation and, to our view, the role of alternative oxidase (AOX) in coordinating complex metabolic and physiological adjustments is underestimated. Cellular levels of sucrose are an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 h after imbibition (HAI) was beneficial to germination. However, parallel upregulated AOX expression was essential to control negative effects by prolonged sucrose treatment. Early downregulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose but suppressed early germination in its presence. The results also suggest that seeds inoculated with arbuscular mycorrhizal fungi (AMF) can buffer sucrose stress during germination to restore normal respiration more efficiently. Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early identifying disease tolerance, predicting plant holobiont behavior, and improving germination. Furthermore, the research strengthens the view that AOX can serve as a powerful functional marker source for seed hologenomes.

Calorespirometric measurements of metabolic heat rates and CO2 emission rates of respiring tissues as functions of temperature enable rapid determination of the temperatures that plants are adapted to without growing them in different environmental temperatures. However, the correct choice of target material for measurements that enable prediction of growth temperature responses is crucial, and needs to be identified in a species- and trait-specific manner. In this study, different carrot materials were tested: a primary culture system proposed as an in vitro test system for carrot yield potential, taproots of young plants, and the root meristem of actively growing plants during secondary root growth. The central root meristem is the most suitable for studying temperature response by calorespirometry for genotype comparison. Calorespirometric methods for predicting genotype-specific temperature responses of crop plant cultivars can be used to predict productivity in environments with differing temperature conditions.

n-Propyl gallate (PG) is a phenolic compound that influences enzymatic processes, mostly involving AOX, PTOX, LOX, POD, and PPO. Here, analyses of different PG concentrations (1, 2.5, and 5 mM) during cowpea seed germination at 16, 32, and 48h showed that 2.5 mM PG partially inhibited seed germination at 16 and/or 32h, but by 48h the germination re-established. Thus, this PG concentration was chosen to study the molecular and biochemical mechanisms linked to the PG inhibitory effects and germination recovery. PG inhibition was related to lower H2O2, higher antioxidant activity, and downregulation of genes linked to cell cycle progression, energy status, and the Krebs cycle at 16 and/or 32h, but these changes were reversed at 48h. In general, genes associated with detoxification, germination-related phytohormones, and NAD(P)H metabolism were highly up-regulated across the time points. AOX1 and Pgb1 were continuously up-regulated along the time points, and linked to NR transcript level increase only at 48h. These findings indicated that AOX and the phytoglobin cycle, both systems involved in NO levels regulation, worked efficiently in germination re-establishment. However, genes other than AOX associated with potential target enzymes of PG, such as LOX, POD, PTOX and PPO (except at 48h), were mostly unchanged or down-regulated. Genes linked to glycolysis (PFK and PK) and acetate synthesis (PDC and ALDH) connected with AOX via NAD(P)+ were up-regulated under PG mainly at 48h. The data are discussed in light of AOX's role in cell reprogramming to reverse PG-induced inhibition of germination in cowpea seeds.

Incorrectly or simply not annotated data is largely increasing in most public databases, undoubtedly caused by the rise in sequence data and the more recent boom of genomic projects. Molecular biologists and bioinformaticists should join efforts to tackle this issue. Practical challenges have been experienced when studying the alternative oxidase (AOX) gene family, and hence the motivation for the present work. Commonly used databases were screened for their capacity to distinguish AOX from the plastid terminal oxidase (also called plastoquinol terminal oxidase; PTOX) and we put forward a simple approach, based on amino acids signatures, that unequivocally distinguishes these gene families. Further, available sequence data on the AOX family in plants was carefully revised to: (1) confirm the classification as AOX and (2) identify to which AOX family member they belong to. We bring forward the urgent need of misannotation awareness and re-annotation of public AOX sequences by highlighting different types of misclassifications and the large under-estimation of data availability.

In this paper photoautotrophic carrot (Daucus carota L.) suspension cultures are described which are able to produce somatic embryos. The development of somatic embryos, however, requires a sucrose supplement. Although an elevation of the CO2 concentration up to 2.3%...