Investigations were performed on growth phase-dependent EcoRII site-specific DNA methylation of the carrot genome during primary culture to elucidate physi
A rapid and efficient micromethod for DNA extraction of Opuntia ficus-indica plants and subsequent RAPD analysis was established. For extraction, 100 mg of the chlorenchyma from cladodes were used. The DNA quantity extracted was significantly higher than for reported macromethods. Up to 100 µg DNA per g fresh weight were achieved. RAPD analysis was performed with 10 ng template DNA by using a kit that contains two polymerases and reduces the pipetting procedure to three steps. The method is being applied on a O. ficus-indica cultivar (Gigante) that we are using for studies on somatic embryogenesis and genetic transformation. Fingerprints obtained demonstrated a very high repeatability of the results and homogeneity of our plant material.
ISHS IV International Congress on Cactus Pear and Cochineal BIOTECHNOLOGICAL STUDIES ON OPUNTIA FICUS-INDICA (L.) MILL.
The alternative oxidase (AOX) gene family is a hot candidate for functional marker development that could help plant breeding on yield stability through more robust plants based on multi-stress tolerance. However, there is missing knowledge on the interplay between...
Genome variation in tissue culture is of importance for commercial use in plant propagation as well as for basic research on plant growth and development. RAPD fingerprinting can be used to trace genetic or epigenetic changes at the genome level. In the present paper, results of RAPD analyses on primary tissue cultures are given with particular attention to the repeatability of the method. The significance of primer binding site competition for the discovery of qualitative and quantitative DNA polymorphism is discussed.
Reprogramming of primary virus-infected cells is the critical step that turns viral attacks harmful to humans by initiating super-spreading at cell, organism and population levels. To develop early anti-viral therapies and proactive administration, it is important to understand the very first steps of this process. Plant somatic embryogenesis (SE) is the earliest and most studied model for de novo programming upon severe stress that, in contrast to virus attacks, promotes individual cell and organism survival. We argued that transcript level profiles of target genes established from in vitro SE induction as reference compared to virus-induced profiles can identify differential virus traits that link to harmful reprogramming. To validate this hypothesis, we selected a standard set of genes named 'ReprogVirus'. This approach was recently applied and published. It resulted in identifying 'CoV-MAC-TED', a complex trait that is promising to support combating SARS-CoV-2-induced cell reprogramming in primary infected nose and mouth cells. In this perspective, we aim to explain the rationale of our scientific approach. We are highlighting relevant background knowledge on SE, emphasize the role of alternative oxidase in plant reprogramming and resilience as a learning tool for designing human virus-defense strategies and, present the list of selected genes. As an outlook, we announce wider data collection in a 'ReprogVirus Platform' to support anti-viral strategy design through common efforts.
In this brief report, we point to virus infection time-dependent transcript levels of polymorphic ASMTL genes in human nasal epithelial cells from seven cell origins. Our observations encourage focused top-down and hypothesis-driven studies in support of more efficient allelic genotyping to identify targets for early resilience prediction.
Recently we identified a major complex trait for early de novo programming (CoV-MAC-TED) in virus-infected human cells by using an interdisciplinary approach that integrates research in plant and human cell systems.[1,2] CoV-MAC-TED included unbalanced reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels connected to increased aerobic fermentation, which was linked to alpha-tubulin–based cell restructuring and control of cell cycle progression. We suggested that adaptive ROS equilibration during the first hours of stress perception might be critical for disease performance; thus, genes involved in ROS equilibration might help target therapeutic tools.[3,4] It is easier to validate traits for resilience in plants than in humans. For validation, we considered well-established unambiguous measures, such as the differential performance of plant varieties in terms of yield height and stability, based on a priori experimentation under multiple field conditions as part of the breeding and variety registration procedures. The genetic capacity for efficient cell reprogramming on isolated and combined stresses can then be studied separately in simple laboratory experiments to explore associations and a posteriori predictability. In this way, plant research can increase the efficiency of knowledge gain for the principal determinants for resilience of holobiont organisms that live under similar environmental conditions, such as water, oxygen, nutrient availability, and temperature.In plants, the small enzyme family of mitochondrial alternative oxidase (AOX) is involved in regulating ROS/RNS homeostasis and equilibration (for example, see Scheibe[5]). Its relevance for adaptive metabolism and stress behavior was widely explored across diverse plant species and environmental threats (see detailed recent reviews in Arnholdt-Schmitt et al,[1] Costa et al,[2] Scheibe,[5] Mohanapriya et al,[6] and Bharadwaj et al[7]). Its beneficial effect in respiration-related deficiencies was also studied in transgene mammals missing this enzyme.[1,2] AOX acts at the level of early cell reprogramming and maintenance. It senses stress levels and coordinates rapid metabolic reorganization for plant plasticity and adaptive plant robustness by permanently optimizing respiration.[1,2,6–9] We hypothesize that plant genotypes, which acclimate more efficiently to changing environmental conditions, have a better chance of maintaining a healthy state that translates into the desired stable higher yield and better food-quality characteristics.[7]Here, we disclose essentials of the novel insights we obtained using two rice plant genotypes, which are known through field experience to differ in multistress (such as that caused by dehydration, cold, abscisic acid application) performance and, especially, in salt tolerance.[10,11] Our results confirm the growing understanding that rapid transcript level changes during early cell reprogramming provide appropriate markers for predicting stress-tolerant phenotypes.[1]Figure 1 presents a simplified scheme that highlights our complex concept (developed and discussed in detail by Bharadwaj et al[7]) by integrating the summarized results for rice achieved during the first 24 h after seedlings were stressed by high-salt (300 mM) treatment compared with a watered control. The watered control demonstrated high transcript levels of both expressed AOX genes, AOX1a and AOX1c, from the first hour of watering in the stress-tolerant rice variety Pokkali. These levels always remained higher than that in the salt-susceptible genotype IR29 at all observed time points (1, 2, 5, 10, and 24 h; not shown, 2–10 h). Under salt treatment, we observed the following.First, there was early downregulation of AOX1a transcript levels in the tolerant variety and recovery of total AOX to control level at 24 h. AOX downregulation indicates an important issue for AOX utility,[12] and the efficiency of downregulation from initially increased levels was shown to be important for the predictability of stress-tolerance performance.[6,7] To the contrary, in the stress-susceptible genotype, the same two AOX genes were transcribed, but no pronounced early response was observed at 1 h. Instead, total AOX transcript accumulation was significantly increased at 24 h after treatment initiation, and upregulated transcription levels were indicated for both transcribed AOX genes.Second, transcript levels of phosphofructokinase (PFK), which represent glycolysis, were found at a higher level in the tolerant genotype during the first hours. This level was linked to higher transcript levels of alcohol dehydrogenase (ADH1 and ADH2), which marks aerobic fermentation. In this context, respiration via the cyclooxygenase (COX) path remained more stable. Nevertheless, during the first 2 h, in both genotypes, a slight increase (nonsignificant) was indicated compared with the control. This seems to show that the cytochrome path began to be overloaded. However, this did not reach significance and was rapidly brought down to control levels, and then to slightly below control at 24 h, which became significant only for the tolerant variety Pokkali. In addition, we suspect that genotype-dependent microbiota interaction could influence the stress-inducible sugar availability for glycolysis or aerobic fermentation.Third, during the first hours of salt treatment, the tolerant variety signaled higher energy depletion, marked by SNRK1 transcript levels, than did the susceptible genotype. This indicates the more efficient energy-dependent cell reorganization in Pokkali.Finally, in both genotypes, differential cell reorganization or restructuration was marked by distinct tubulin transcript level changes. Under salt stress, Pokkali demonstrated increased tubulin transcript accumulation against the water control between 2 and 5 h after salt treatment began, whereas IR29 tubulin transcription remained at the basal level observed at 1 h. Additional analyses indicated that cell proliferation was arrested during the first 24 h after treatment in both genotypes (not shown). This confirmed our recent conclusions from a different experimental system (induction of somatic embryogenesis by severe stress) in which we found that the cell cycle was suppressed during early cell reprogramming.[1,2] Of note, our results show parallel transcript level changes for the two most frequently expressed tubulin genes, alpha- and beta-tubulin (α-Tub and β-Tub) for both genotypes, which we also observed in the water control (not shown). Thus, these observations confirm the approach we applied in Costa et al,[2–4] in which we selected only alpha-tubulin transcript levels as a marker for differential cell reorganization during early cell reprogramming.Overall, this report supports our hypothesis that adaptive ROS equilibration can provide a powerful means of predicting the capacity for resilient performance in plant and human virus cells when viewed during early cell reprogramming at the transcript level in crucial genes and in the context of functional marker-assisted description of energy-dependent cell reorganization and cell cycle regulation. Furthermore, our group studied exhaustively the diversity of polymorphisms in AOX genes related to diverse environments. We suggest that AOX gene diversity in target cells within an individual plant might be a trait that, by itself, allows rapid switching between diverse allelic sequences for acclimation. In parallel, we identified allelic transcript level profiles in the gene ASMTL (N-acetylserotonin methyltransferase-like) in human epithelial nasal cells, which link under viral stress to ROS/RNS balancing. Considering our overall results, we propose that these findings should be explored as promising sources of functional markers that could support prediction of differential disease development.[13,14]Supplemental data are available online with the article.
Recently, we identified early reactive oxygen species (ROS) and reactive nitrogen species (RNS) rebalancing as part of a major complex trait, named CoV-MAC-TED, that characterized host cell response on SARS-CoV-2 infections from two genetic virus variants.[1–3] Transcript level changes of the enzymes acetylserotonin O-methyltransferase-like (ASMTL) and alcohol dehydrogenase 5 (ADH5) were hypothesized to mark relevant adaptive ROS/RNS equilibration early during respiratory infections and preliminary validation in cultured primary target cells supported this view.[2] ASMTL is a paralog of ASMT, which is involved in final melatonin biosynthesis. ADH5, also known as S-nitrosoglutathione reductase, is involved in nitric oxide (NO) homeostasis.[1,2] In parallel to these studies, we released our results on ASMTL and ADH5 transcriptome accumulation from influenza H3N2-infected human nasal epithelial cells (NECs) that originated from seven healthy cell donator origins (D01–D07).[4] We observed that variability of ASMTL and ADH5 transcript levels linked to the timing of virus replication and that this connected to the initiation of the classical immune system response, when marked by interferon regulatory factor 9 (IRF9) transcript level changes. Cells of the donator D02 showed the highest ASMTL transcript levels among all cell origins at 8 hours post-infection (hpi), indicating rapidly unbalanced ROS/RNS that linked to the earliest immune response. On the contrary, transcript levels of ADH5 from D05 indicated early and over times highest NO stress among all seven origins, suggesting unbalanced ROS/RNS in favor of RNS. This response connected to the highest immune response at 24 hpi among all cell origins. In contrast, cells originating from D01 indicated a stably balanced relationship between ROS and RNS marked by ASMTL and ADH5 that associated to the lowest IRF9 response over all time points among the seven cell origins. This equilibrated response of D01 was in accordance with postponed influenza virus replication in relation to all other cell origins.[4] Of note, also under infection by two SARS-CoV-2 variants NECs from one of three donator origins displayed contrasting response to other donator cells as follows: lower transcript levels for enolase (representing glycolysis) and lactate dehydrogenase (representing aerobic fermentation) were observed at 24 hpi for cell cultures from origin D1 that linked to delayed replication of SARS-CoV-2.[3]These overall observations made us optimistic that our approach and marker systems in combination with appropriate primary target human cells could be promising, in general, to early identify differential individual host cell responses on viral infections. Concurrently, we hypothesized that this tool could also help to discriminate individuals through genetic polymorphisms in the relevant functional genes that relate to CoV-MAC-TED (see ReprogVirus gene set in References 1–4).ROS signaling is one of the earliest and relevant cell responses upon abiotic and biotic stresses that transmits any change in environment, including viral attacks, to structures and molecules (e.g., membranes, ion channels [Ca2+], enzymes, microtubulines), which then are crucially determining cell performance. Therefore, we studied polymorphisms in both highlighted marker genes for ROS/RNS balancing, ASMTL, and ADH5. As a first step, we checked the genetic material of the public transcriptome data of these seven donators for NECs.[5] The coding sequence of ADH5 was highly conservative. No sequence nucleotide polymorphisms or other major differences among the seven donor cell origins were encountered. To the contrary, we found several polymorphic sites (sequence nucleotide polymorphisms) in the coding sequence of the ASMTL gene (unpublished data). This might indicate a higher potential for acclimation acquired during evolution.In this brief report, we want to point to transcript level profiles that we observed for one of the polymorphic sites during early virus-induced cell reprogramming. In the upper part of Table 1, we demonstrate the crucial nucleotide for polymorphism in this site (G or A) for influenza H3N2-infected cells from seven donator origins. On the one hand, we observed the following cell origins with the same preferential nucleotide profile over times in this site: cells of D02, D03, and D07 show continuously G at all time points (0, 8, and 24 hpi); D04 and D06 display stably A, such as in the reference gene. On the other hand, we discovered for D01 and D05 unique differential transcript level profiles. Cells from D05 changed at 8 hpi to the preferential transcription of an allele, which shows A instead of G in this polymorphic site. However, at 24 hpi a change in preferential allelic transcription became visible through the reappearance of G instead of A. In contrast, at 8 hpi D01 changed in the same way as observed for D05 to preferential transcription of an allele that shows A instead of the initial G. However, in cells of D01 this allelic site continued longer to be preferentially transcribed and A is still the more frequent nucleotide seen at 24 hpi (boldface at 8 and 24 hpi). Post-infection time-dependent allelic genotyping profiles of the same polymorphic site was also found on infection by two SARS-CoV-2 variants that demonstrated diverse effects on disease severity.[3] ASMTL genotype profiles are shown for NECs from three donators (Table 1, lower part). Overall, these results demonstrate virus- and variant-specific allelic genotype profiles over times when both virus types (influenza H3N2 and SARS-CoV-2) and both SARS-CoV-2 variants are compared within each cell origin.In summary, and despite the restricted number of studied individual cell origins, these observations highlight to our view that efficient allelic genotyping for specific or general virus tolerance and immunophenotyping requires integrative studies on time-dependent individual transcript level changes in genes that were identified as relevant markers for early cell reprogramming, such as recently reported for CoV-MAC-TED.[2–4] Rapid switching between critical gene polymorphisms in critical binding sites can determine metabolic regulation, which in turn can result in differential performance. This was described in plant research for the ROS/RNS balancing gene alternative oxidase and its temperature-dependent interaction with pyruvate.[6] Furthermore, we suggest that high diversity in coding and noncoding sequences of crucial genes for early ROS/RNS balancing should be explored as a trait per se. This trait can support plasticity in the immune response. Consequently, we propose a discussion on a potential paradigm shift toward understanding immunology in a wider sense, which should consider ROS/RNS balancing during stress-induced early cell reprogramming. It is our hope that our rapid communication of these insights might help to avoid spending high amounts of personal and financial resources for less-focused, bottom-up gene polymorphism studies instead of top-down and then hypothesis-driven approaches.[7,8]
A classification scheme based on protein phylogenies and sequence harmony method was used to clarify the taxonomic distribution and evolutionary history of the alternative oxidase (AOX) in angiosperms. A large data set analyses showed that AOX1 and AOX2 subfamilies were distributed into 4 phylogenetic clades: AOX1a–c/1e, AOX1d, AOX2a–c and AOX2d. High diversity in AOX family compositions was found. While the AOX2 subfamily was not detected in monocots, the AOX1 subfamily has expanded (AOX1a–e) in the large majority of these plants. In addition, Poales AOX1b and 1d were orthologous to eudicots AOX1d and then renamed as AOX1d1 and 1d2. AOX1 or AOX2 losses were detected in some eudicot plants. Several AOX2 duplications (AOX2a–c) were identified in eudicot species, mainly in the asterids. The AOX2b originally identified in eudicots in the Fabales order (soybean, cowpea) was divergent from AOX2a–c showing some specific amino acids with AOX1d and then it was renamed as AOX2d. AOX1d and AOX2d seem to be stress-responsive, facultative and mutually exclusive among species suggesting a complementary role with an AOX1(a) in stress conditions. Based on the data collected, we present a model for the evolutionary history of AOX in angiosperms and highlight specific areas where further research would be most beneficial.
