Birgit Arnholdt-Schmitt

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.

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. Johns 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.

Can functional hologenomics aid tackling current challenges in plant breeding

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.

Calorespirometry, oxygen isotope analysis and functional-marker-assisted selection (‘CalOxy-FMAS’) for genotype screening: A novel concept and tool kit for predicting stable plant growth performance and functional marker identification

We propose a novel concept and tool kit for predictive phenotyping. The proposed technology measures respiration properties as functions of growth conditions to identify genotypes with higher plasticity via homeostasis and adaptive morphophysiology. Combining calorespirometry, oxygen isotope analysis and functional-marker-assisted selection (CalOxy-FMAS) for genotype screening will enable predicting the genetic potential for stable plant growth performance. Application of this novel tool kit can help identify genotypes with controlled homeostasis in changing environments and optimized growth performance. Simultaneously, it will allow a better balance in breeding for high yields and quality characteristics. Applying CalOxy-FMAS can efficiently narrow the pool of genotypes to be screened for final phenotyping in the field.

Characterization of Hypericum perforatum L. Plants from Various Accessions by RAPD Fingerprinting

SUMMARY RAPD fingerprints of individual plants of Hypericum perforatum from different accessions are discussed in this paper. From a wild population, 10 offspring were investigated by genome analyses. The results obtained revealed that the collection included significantly polymorphic genotypes. Whereas 7 of the individuals displayed identical fingerprints, obviously representing the dominant genotype of this accession, a deviating but also identical genotype was shown by 2 plants and a third genotype was represented by another plant. Additionally, genome characterization of progenies from individual plants of 4 further accessions were performed. The results show that most of the fingerprints from each accession indicated an identical mode of reproduction for H perforatum. Nevertheless, non-identical progenies could be discovered as a minor event by RAPD analyses.

Calorespirometry as a tool for studying temperature response in carrot (Daucus carota L.)

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.

In silico identification of alternative oxidase 2 (AOX2) in monocots: A new evolutionary scenario

We identified AOX2 genes in monocot species from Lemnoideae (Spirodela polyrhiza, Lemna gibba and Landoltia punctata), Pothoideae (Anthurium andraeanum and Anthurium amnicola) and Monsteroideae (Epipremnum aureum) subfamilies within the Araceae, an early-diverging monocot family. These findings highlight the presence of AOX2 in the most ancient monocot ancestor and also that at least partial loss of this gene occurred during speciation events within several monocot orders. The presence of AOX2 in monocot species challenges (1) new understanding of the evolutionary history of the AOX gene family in angiosperms and (2) drives experimental and bioinformatics efforts to explore functional relevance of the two AOX gene family members for plant growth and development. Knowledge gain in this field will impact running strategies on AOX-derived functional marker candidate development for plant breeding.

Functional marker development is challenged by the ubiquity of endophytes-a practical perspective.

Functional markers (FMs) are supposed to assist in diagnosis, disease treatment and turning plant and animal breeding more efficient. However, efficient FM application is challenged through current insights in the multi-organism nature of life. This letter aims to raise awareness for re-thinking concepts for FM development in plant breeding and proposes a novel perspective.

Misannotation Awareness: A Tale of Two Gene-Groups

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.

Phenotyping carrot (Daucus carota L.) for yield-determining temperature response by calorespirometry.

AbstractMain conclusionCalorespirometric measurements proved to be useful for phenotyping temperature response in terms of optimum temperatures for growth and low temperature limits for growth respiration in diverse carrot genotypes.n High and low-temperature tolerance is an important trait in many breeding programs, but to date, improvement strategies have had limited success. Developing new, cost efficient and reliable screening tools to identify and select the most tolerant crop plant genotypes is necessary to assist plant breeding on cold and heat tolerance, and calorespirometry is proposed for this. Calorespirometry is a technique to simultaneously measure metabolic heat rates and CO2 emission rates of respiring tissues and can be used as a rapid method to determine how changes in the environment (e.g., temperature) influence plant growth. The main aim of this work was, therefore, to test the usefulness of calorespirometry as a phenotyping tool for carrot taproot growth in response to temperature. Calorespirometric measurements in the carrot taproot meristems of plants from eight carrot inbred lines allowed identification of optimum and minimum temperatures for growth of plants and to distinguish between phenotypes based on those characteristics. The technique proved to be useful for predicting yield-determining temperature responses in diverse carrot genotypes. Preliminary screening of new crop plant genotypes with calorespirometry based on their temperature adaptation and acclimation capability could make the screening process much less laborious by allowing selection of genotypes presenting the best growth performance under particular biotic or abiotic conditions before field tests.