Induction Of Dormancy Research Articles

Functional variants of DOG1 control seed chilling responses and variation in seasonal life-history strategies in Arabidopsis thaliana.

The seasonal timing of seed germination determines a plant's realized environmental niche, and is important for adaptation to climate. The timing of seasonal germination depends on patterns of seed dormancy release or induction by cold and interacts with flowering-time variation to construct different seasonal life histories. To characterize the genetic basis and climatic associations of natural variation in seed chilling responses and associated life-history syndromes, we selected 559 fully sequenced accessions of the model annual species Arabidopsis thaliana from across a wide climate range and scored each for seed germination across a range of 13 cold stratification treatments, as well as the timing of flowering and senescence. Germination strategies varied continuously along 2 major axes: 1) Overall germination fraction and 2) induction vs. release of dormancy by cold. Natural variation in seed responses to chilling was correlated with flowering time and senescence to create a range of seasonal life-history syndromes. Genome-wide association identified several loci associated with natural variation in seed chilling responses, including a known functional polymorphism in the self-binding domain of the candidate gene DOG1. A phylogeny of DOG1 haplotypes revealed ancient divergence of these functional variants associated with periods of Pleistocene climate change, and Gradient Forest analysis showed that allele turnover of candidate SNPs was significantly associated with climate gradients. These results provide evidence that A. thaliana's germination niche and correlated life-history syndromes are shaped by past climate cycles, as well as local adaptation to contemporary climate.

A simple and convenient method for determination of entrance into dormancy in woody plants

BACKGROUND: Bud dormancy is a quantitative condition that is gradually acquired and lost. Better and more convenient methods for assessment of the time of dormancy entrance of woody plants are highly needed. OBJECTIVE: To demonstrate a simple and convenient method for determination of dormancy in woody plants. METHODS: We employed a seasonal series of soft tipping of vigorously growing annual shoots and used the loss of ability of subtending lateral buds to break and grow as a measure of entrance into dormancy. RESULTS: There was a gradual decline in the ability of the buds to burst and grow during the month of July and early August, culminating with a complete loss of this ability. This coincided with the known time of growth cessation and dormancy induction in shoots of intact plants and occurred in the berry shrubs raspberry and black currant and the forest tree silver birch. CONCLUSIONS: The decline and loss of ability of the buds to grow during late summer is a direct expression of the entrance of buds into the state of endodormancy, rendering the tipping method a simple and convenient method for precise determination of the time of entrance into dormancy in woody plants.

Warm winter temperature induced changes in the dormant buds of ‘Dangshansuli’ pear (Pyrus bretschneideri Rehd.)

Winter dormancy is one of the crucial ways leading to the survival of deciduous fruit trees in the temperate and boreal zones. In order to study the effects of higher winter temperatures, such as caused by global warming, on bud dormancy and related hormone abundance in ‘pear, a greenhouse was set up to enclose adult trees of the pear in field, the temperature inside the greenhouse was maintained about 3.0°C higher than that in the open field. Our results indicated that, greenhouse grown pear trees resulted in about 7-day delay of dormancy induction, 4–5 days ahead of dormancy breaking and much quickly into deep dormancy compared to control trees. Higher temperature led to significant changes of indole-3-acetic acid (IAA), gibberellin acid (GA3), zeatinriboside (ZR), and abscisic acid (ABA) contents as well as lower ratios of ZR/GA3, ZR/IAA, ABA/GA3, and ABA/IAA in flower buds but not vegetative buds, suggesting that higher winter temperature did not favour flower bud dormancy breaking. Over the dormancy period, ABA content, but not IAA, GA3 and ZR, in flower bud increased alone with the decreased temperature and peaked at the deep dormancy stage, suggesting a great role of ABA in flower bud dormancy formation. The data suggested that higher winter temperature has negative effects on pear flower bud dormancy.

A 14q32.31 Genomic-Imprinted DLK1-DIO3 microrna promotes Leukemogenesis By Inducing Stem Cell Quiescence and Inhibiting NK Cell Anti-Cancer Immunity

Leukemia emergence, maintenance, relapse and/or progression are causally linked to the presence of drug-resistant leukemia-initiating cells and impaired natural killer (NK) cell anti-tumor immune-response. Bone marrow microenvironment (BMM)- and/or leukemia-derived signals induce aberrant non-coding RNA expression and inhibit protein phosphatase 2A (PP2A) tumor suppressor activity. PP2A loss-of-function is essential for NK cell activity and leukemic but not normal stem and progenitor cell proliferation and survival. The human MIR300 gene is an intergenic miRNA that belongs to the 14q32.31 DLK1-DIO3 genomic-imprinted tumor suppressor miRNA cluster B. MIR300 was found involved in loss-of heterozygosity, inhibited in several tumor types with high mitotic index and during epithelial-to-mesenchymal transition (EMT), and associated with a cancer stem cell phenotype. By using primary cells from Philadelphia-positive (Ph+) chronic myelogenous leukemia (CML) in chronic (CP) and blastic (BC) phase, and complex karyotype (CK) acute myeloid leukemia (AML) patients, as paradigmatic examples of stem cell-derived neoplasms characterized by constitutive expression of oncogenic kinases, PP2A loss-of-function, altered microRNA expression and impaired NK cell proliferation and cytotoxicity, we found that MIR300 is a cell context-independent tumor suppressor with anti-proliferative and PP2A-dependent pro-apoptotic activities which are sequentially activated in a MIR300 dose-dependent manner through inhibition of CCND2/CDK6 and SET (PP2A inhibitor), respectively. To prevent PP2A-induced apoptosis, MIR300 is inhibited by oncogenic signals in CD34+CML (CP and BC) and CK-AML progenitors. Conversely, tumor-naïve BMM-induced C/EBPbeta-mediated signals (hypoxia and MSC exosomes) markedly upregulate MIR300 expression in primary CML and AML CD34+CFSEmax leukemic stem (LSC) and CD56+CD3-NK cells to induce/maintain quiescence (increased CD34+leukemic blasts in G0) and impair immune-response (suppression of NK cell proliferation and cytotoxic activity toward CD34+ leukemic blasts and CFSEmaxCD34+ CML-BC quiescent LSCs), respectively. Inhibition of MIR300 expression/activity rescues NK cell proliferation and anti-tumor cytotoxicity and prevented MSC- and hypoxia-induced growth-suppression of CD34+leukemic blasts by inhibiting degradation of MIR300 targets (e.g. SET, CCND2). We found that CML and AML LSCs escape MIR300-induced PP2A-mediated apoptosis through the hypoxia- and tumor-dependent TGFb1-FoxM1-mediated upregulation of TUG1 lncRNA. TUG1 is an oncogenic lncRNA described as a MIR300sponge and found upregulated in solid tumors, in which it has strong diagnostic, prognostic and therapeutic relevance and is associated with cancer stem cell maintenance and EMT. In quiescent CML and AML LSCs, TUG1 uncouples and limits MIR300 tumor suppressor functions to cytostasis by maintaining unbound MIR300 at levels sufficient to inhibit CCND2 and CDK6 but not SET expression. Exposure to clinically-relevant CpG-modified oligonucleotides modulating MIR300levels and/or inhibiting TUG1 MIR300-sponging activity, restores NK cell proliferation and cytotoxic activity, and suppresses human leukemic but not normal hematopoiesis by eradicating nearly all (> 95% reduction) CFSEmaxCD34+ and CD45+CD34+CD38-CD90+ LSCs and CD34+leukemic CML (CP and BC) and CK-AML blasts in vitro (CFCs, LTC-IC, and CFSEmaxCD34+cell tracking) and/or in NRG-SGM3 PDX mouse models of acute and chronic myeloid leukemias. Altogether, this work highlights the therapeutic importance of altering MIR300 expression in anti-LSC and NK cell-based approaches for myeloid leukemias, and indicates that tumor-naïve BMM-induced MIR300 tumor suppressor anti-proliferative and PP2A-activating functions may support leukemogenesis by promoting the formation and initial expansion of the quiescent LSC pool through the induction of LSC dormancy and inhibition of quiescent LSC killing by cytokine-activated NK cells, respectively. (G.S. and R.T. equally contributed to this work) Disclosures Stagno: BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Deininger:TRM: Consultancy; Sangoma: Consultancy; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Ascentage Pharma: Consultancy, Honoraria; Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Fusion Pharma: Consultancy; Adelphi: Consultancy. Milojkovic:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Apperley:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

Studying and modelling winter dormancy in olive trees

The abundance of scientific papers dealing with olive reproductive phenology contrasts with the scarce information available in relation to the winter dormant state of olive vegetative structures. In this study, three experiments with young olive trees were performed in Southern Spain, aiming to provide insight into some features of the winter rest period in this evergreen species. Experiment 1 evaluated the environmental cues triggering dormancy induction by measuring leaf appearance rates in trees subjected to different conditions of temperature and daylength over the course of the 2012 autumn. In Experiment 2, several sets of plants were placed into a greenhouse at different dates along the 2013/2014 winter, testing the ability of dormant plants to resume growth upon the return of favourable temperatures. Finally, Experiment 3 was carried out during the autumns of 2016 and 2017 in two locations, and was devoted to assess differences between five cultivars in the onset of dormancy under natural conditions. Our findings revealed that dormancy induction is not controlled by photoperiod, but by low temperatures. The subsequent winter rest state seems to be easily reversed after 1–2 weeks of exposure to warm conditions, irrespective of the initial date of exposure. With regard to cultivar variability, differences in the timing of growth cessation was found to be rather small. Finally, two simple models for predicting the onset of dormancy based on the accumulation of a certain amount of chilling (either considering or not a reversal of chilling by warm temperatures) are presented. Calibration and validation was performed with independent datasets from Experiments 1, 2 and 3. Validation tests highlighted the reliability of both models in reproducing the date of growth cessation.

Changes in phytohormone content and associated gene expression throughout the stages of pear (Pyrus pyrifolia Nakai) dormancy.

To elucidate the role of phytohormones during bud dormancy progression in the Japanese pear (Pyrus pyrifolia Nakai), we investigated changes in phytohormone levels of indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA) and trans-zeatin (tZ). Using ultra-performance liquid chromatography/mass spectrometry/mass spectrometry, we monitored phytohormone levels in the buds of field-grown and potted trees that were artificially heated to modify the timing of dormancy and flowering (spring flush) progression. We also analyzed the expression of GA- and ABA-metabolic genes during dormancy. Indole acetic acid and tZ levels were low during dormancy and increased toward the flowering stage. Gibberellic acid levels were maintained at relatively high concentrations during the dormancy induction stage, then decreased before slightly increasing prior to flowering. The low GA concentration in potted trees compared with field-grown trees indicated that GA functions in regulating tree vigor. Abscisic acid levels increased from the dormancy induction stage, peaked near endodormancy release and steadily decreased before increasing again before the flowering stage. The ABA peak levels did not always coincide with endodormancy release, but peak height correlated with flowering uniformity, suggesting that a decline in ABA concentration was not necessary for resumption of growth but the abundance of ABA might be associated with dormancy depth. From monitoring the expression of genes related to GA and ABA metabolism, we inferred that phytohormone metabolism changed significantly during dormancy, even though the levels of bioactive molecules were consistently low. Phytohormones regulate dormancy progression not only upon the reception of internal signals but also upon sensing ambient conditions.

The temperature and length for the release of primary and induction of secondary physiological dormancy in Korean pine (Pinus koraiensis Sieb. et Zucc.) seeds

Primary physiological dormancy and secondary physiological dormancy of Korean pine seeds restrict the regeneration of broad-leaved Korean pine (Pinus koraiensis) mixed forest. Dry and imbibed seeds were stratified at 1 °C and 5 °C for 1, 2, 4 and 6 months. Germination percentage, mean germination time (MGT) and germination rate index (GRI) were measured to determine the optimal low temperature and its duration for the release of primary physiological dormancy. Once primary physiological dormancy was released through cold stratification, seeds were stored in an environment in which the temperature progressively increased from 5 to 25 °C. After one month of storage at each storage temperature, the germination percentage, MGT and GRI were measured to determine the threshold temperature for the induction of secondary physiological dormancy. Both dry and imbibed seeds not only exhibited a high germination percentage (approximately 80%) but also germinated rapidly (MGT and GRI were 17 days and 2.36, respectively) after 6 months of storage at either 1 °C or 5 °C. The germination percentage of cold stratified seeds gradually decreased from 78% (5 °C) to 72% (10 °C), 55% (15 °C), 10% (20 °C) and 8% (25 °C). The results of this study suggest that stratifying seeds at 1 °C or 5 °C for 6 months releases primary physiological dormancy. The induction of secondary physiological dormancy occurs at temperatures above 15 °C.

Roles of abscisic acid and gibberellins in maintaining primary and secondary dormancy of Korean pine seeds

Primary dormancy of seeds of Korean pine (Pinus koraiensis Sieb. et Zucc.) after dispersal in the autumn and the induction of secondary dormancy the first summer following seed dispersal limit the regeneration of mixed broadleaved Korean pine forests in Northeast China. This study was to determine how changes in the levels of abscisic acid (ABA) and gibberellic acid (GA) maintain primary and secondary dormancy of Korean pine seeds under germination conditions. We transferred seeds with one of five primary dormancy states or three secondary dormancy states to germination conditions and measured changes in the levels of ABA, GA1+3 (GA1 and GA3) and GA4+7 (GA4 and GA7) in the seed coat, megagametophyte and embryo during incubation. Seed coat ABA levels in primary dormant seeds (PDS) and ABA levels in various parts of secondary dormant seeds (SDS) gradually declined during incubation but were still higher than in seeds for which dormancy was progressively released. GA4+7 and GA1+3 levels in embryos greatly decreased 35% and 24%, respectively, during incubation of SDS, and thus, the ratio of ABA to GA4+7 in embryos and megagametophytes significantly increased. The ratio of ABA to GA1+3 in various parts of SDS increased slightly during incubation. In contrast, in seeds for which secondary dormancy was already released, GA4+7 and GA1+3 levels in the embryo, GA4+7/ABA ratio in the embryo and seed coat, and the GA1+3/ABA in the embryo and megagametophyte significantly increased during incubation. There was no trend in the changes in the levels of ABA, GA4+7 or GA1+3 in embryos and megagametophytes of PDS or the levels of GA4+7 or GA1+3 in megagametophytes of SDS during incubation. The results suggest that high ABA levels in the seed coat maintain primary dormancy of Korean pine seeds. Maintenance of secondary dormancy involves a reduction of GA4+7, GA1+3, GA4+7/ABA, and GA1+3/ABA and the retention of high ABA levels.

Echinochloa crus-galli seed physiological dormancy and germination responses to hypoxic floodwaters.

Hypoxic floodwaters can seriously damage seedlings. Seed dormancy could be an effective trait to avoid lethal underwater germination. This research aimed to discover novel adaptive dormancy responses to hypoxic floodwaters in seeds of Echinochloa crus-galli, a noxious weed from rice fields and lowland croplands. Echinochloa crus-galli dormant seeds were subjected to a series of sequential treatments. Seeds were: (i) submerged under hypoxic floodwater (simulated with hypoxic flasks) at different temperatures for 15 or 30days, and germination tested under drained conditions while exposing seeds to dormancy-breaking signals (alternating temperatures, nitrate (KNO3 ), light); or (ii) exposed to dormancy-breaking signals during hypoxic submergence, and germination monitored during incubation and after transfer to drained conditions. Echinochloa crus-galli seed primary dormancy was attenuated under hypoxic submergence but to a lesser extent than under drained conditions. Hypoxic floodwater did not reinforced dormancy but hindered secondary dormancy induction in warm temperatures. Seeds did not germinate under hypoxic submergence even when subjected to dormancy-breaking signals; however, these signals broke dormancy in seeds submerged under normoxic water. Seeds submerged in hypoxic water could sense light through phytochrome signals and germinated when normoxic conditions were regained. Hypoxic floodwaters interfere with E.crus-galli seed seasonal dormancy changes. Dormancy-breaking signals are overridden during hypoxic floods, drastically decreasing underwater germination. In addition, results indicate that a fraction of E.crus-galli seeds perceive dormancy-breaking signals under hypoxic water and germinate immediately after aerobic conditions are regained, a hazardous yet less competitive environment for establishment.

Temperature mediates secondary dormancy in resting cysts of Pyrodinium bahamense (Dinophyceae).

High-biomass blooms of the toxic dinoflagellate Pyrodinium bahamense occur most summers in Tampa Bay, Florida, USA, posing a recurring threat to ecosystem health. Like many dinoflagellates, P. bahamense forms immobile resting cysts that can be deposited on the seafloor-creating a seed bank that can retain the organism within the ecosystem and initiate future blooms when cysts germinate. In this study, we examined changes in the dormancy status of cysts collected from Tampa Bay and applied lessons from plant ecology to explore dormancy controls. Pyrodinium bahamense cysts incubated immediately after field collection displayed a seasonal pattern in dormancy and germination that matched the pattern of cell abundance in the water column. Newly deposited (surface) cysts and older (buried) cysts exhibited similar germination patterns, suggesting that a common mechanism regulates dormancy expression in new and mature cysts. Extended cool- and warm-temperature conditioning of field-collected cysts altered the cycle of dormancy compared with that of cysts in nature, with the duration of cool temperature exposure being the best predictor of when cysts emerged from dormancy. Extended warm conditioning, on the other hand, elicited a return to dormancy, or secondary dormancy, in nondormant cysts. These results directly demonstrate environmental induction of secondary dormancy in dinoflagellates-a mechanism common and thoroughly documented in higher plants with seasonal growth cycles. Our findings support the hypothesis that a seasonal cycle in cyst germination drives P. bahamense bloom periodicity in Tampa Bay and point to environmentally induced secondary dormancy as an important regulatory factor of that cycle.

A transcriptome analysis reveals a role for the indole GLS-linked auxin biosynthesis in secondary dormancy in rapeseed (Brassica napus L.)

BackgroundBrassica napus L. has little or no primary dormancy, but exhibits great variation in secondary dormancy. Secondary dormancy potential in oilseed rape can lead to the emergence of volunteer plants that cause genetic contamination, reduced quality and biosafety issues. However, the mechanisms underlying secondary dormancy are poorly understood. In this study, cultivars Huaiyou-WSD-H2 (H) and Huaiyou-SSD-V1 (V), which exhibit low (approximately 5%) and high (approximately 95%) secondary dormancy rate, respectively, were identified. Four samples, before (Hb and Vb) and after (Ha and Va) secondary dormancy induction by polyethylene glycol (PEG), were collected to identify the candidate genes involved in secondary dormancy via comparative transcriptome profile analysis.ResultsA total of 998 differentially expressed genes (DEGs), which are mainly involved in secondary metabolism, transcriptional regulation, protein modification and signaling pathways, were then detected. Among these DEGs, the expression levels of those involved in the sulfur-rich indole glucosinolate (GLS)-linked auxin biosynthesis pathway were markedly upregulated in the dormant seeds (Va), which were validated by qRT-PCR and subsequently confirmed via detection of altered concentrations of indole-3-acetic acid (IAA), IAA conjugates and precursors. Furthermore, exogenous IAA applications to cultivar H enhanced secondary dormancy.ConclusionThis study first (to our knowledge) elucidated that indole GLS-linked auxin biosynthesis is enhanced during secondary dormancy induced by PEG, which provides valuable information concerning secondary dormancy and expands the current understanding of the role of auxin in rapeseed.

Revisiting hypoxia therapies for tuberculosis.

The spectre of the coming post-antibiotic age demands novel therapies for infectious diseases. Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the single deadliest infection throughout human history. M. tuberculosis has acquired antibiotic resistance at an alarming rate with some strains reported as being totally drug resistant. Host-directed therapies (HDTs) attempt to overcome the evolution of antibiotic resistance by targeting relatively immutable host processes. Here, I hypothesise the induction of hypoxia via anti-angiogenic therapy will be an efficacious HDT against TB. I argue that anti-angiogenic therapy is a modernisation of industrial revolution era sanatoria treatment for TB, and present a view of the TB granuloma as a 'bacterial tumour' that can be treated with anti-angiogenic therapies to reduce bacterial burden and spare host immunopathology. I suggest two complementary modes of action, induction of bacterial dormancy and activation of host hypoxia-induced factor (HIF)-mediated immunity, and define the experimental tools necessary to test this hypothesis.

The Hepatic Microenvironment and TRAIL-R2 Impact Outgrowth of Liver Metastases in Pancreatic Cancer after Surgical Resection.

Most patients with pancreatic ductal adenocarcinoma (PDAC) undergoing curative resection relapse within months, often with liver metastases. The hepatic microenvironment determines induction and reversal of dormancy during metastasis. Both tumor growth and metastasis depend on the Tumor necrosis factor (TNF)-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2). This study investigated the interplay of TRAIL-R2 and the hepatic microenvironment in liver metastases formation and the impact of surgical resection. Although TRAIL-R2-knockdown (PancTu-I shTR2) decreased local relapses and number of macroscopic liver metastases after primary tumor resection in an orthotopic PDAC model, the number of micrometastases was increased. Moreover, abdominal surgery induced liver inflammation involving activation of hepatic stellate cells (HSCs) into hepatic myofibroblasts (HMFs). In coculture with HSCs, proliferation of PancTu-I shTR2 cells was significantly lower compared to PancTu-I shCtrl cells, an effect still observed after switching coculture from HSC to HMF, mimicking surgery-mediated liver inflammation and enhancing cell proliferation. CXCL-8/IL-8 blockade diminished HSC-mediated growth inhibition in PancTu-I shTR2 cells, while Vascular Endothelial Growth Factor (VEGF) neutralization decreased HMF-mediated proliferation. Overall, this study points to an important role of TRAIL-R2 in PDAC cells in the interplay with the hepatic microenvironment during metastasis. Resection of primary PDAC seems to induce liver inflammation, which might contribute to outgrowth of liver metastases.

Aquaporins influence seed dormancy and germination in response to stress.

Aquaporins influence water flow in plants, yet little is known of their involvement in the water‐driven process of seed germination. We therefore investigated their role in seeds in the laboratory and under field and global warming conditions. We mapped the expression of tonoplast intrinsic proteins (TIPs) during dormancy cycling and during germination under normal and water stress conditions. We found that the two key tonoplast aquaporins, TIP3;1 and TIP3;2, which have previously been implicated in water or solute transport, respectively, act antagonistically to modulate the response to abscisic acid, with TIP3;1 being a positive and TIP3;2 a negative regulator. A third isoform, TIP4;1, which is normally expressed upon completion of germination, was found to play an earlier role during water stress. Seed TIPs also contribute to the regulation of depth of primary dormancy and differences in the induction of secondary dormancy during dormancy cycling. Protein and gene expression during annual cycling under field conditions and a global warming scenario further illustrate this role. We propose that the different responses of the seed TIP contribute to mechanisms that influence dormancy status and the timing of germination under variable soil conditions.

Identification, Structural and Functional Characterization of Dormancy Regulator Genes in Apricot (Prunus armeniaca L.).

In the present study, we identified and characterized the apricot (Prunus armeniaca L.) homologs of three dormancy-related genes, namely the ParCBF1 (C-repeat binding factor), ParDAM5 (dormancy-associated MADS-BOX) and ParDAM6 genes. All highly conserved structural motifs and the 3D model of the DNA-binding domain indicate an unimpaired DNA-binding ability of ParCBF1. A phylogenetic analysis showed that ParCBF1 was most likely homologous to Prunus mume and Prunus dulcis CBF1. ParDAM5 also contained all characteristic domains of the type II (MIKCC) subfamily of MADS-box transcription factors. The homology modeling of protein domains and a phylogenetic analysis of ParDAM5 suggest its functional integrity. The amino acid positions or small motifs that are diagnostic characteristics of DAM5 and DAM6 were determined. For ParDAM6, only a small part of the cDNA was sequenced, which was sufficient for the quantification of gene expression. The expression of ParCBF1 showed close association with decreasing ambient temperatures in autumn and winter. The expression levels of ParDAM5 and ParDAM6 changed according to CBF1 expression rates and the fulfillment of cultivar chilling requirements (CR). The concomitant decrease of gene expression with endodormancy release is consistent with a role of ParDAM5 and ParDAM6 genes in dormancy induction and maintenance. Cultivars with higher CR and delayed flowering time showed higher expression levels of ParDAM5 and ParDAM6 toward the end of endodormancy. Differences in the timing of anther developmental stages between early- and late-flowering cultivars and two dormant seasons confirmed the genetically and environmentally controlled mechanisms of dormancy release in apricot generative buds. These results support that the newly identified apricot gene homologs have a crucial role in dormancy-associated physiological mechanisms.

Comparative reproductive dormancy differentiation in European black scavenger flies (Diptera: Sepsidae).

Seasonality is a key environmental factor that regularly promotes life history adaptation. Insects invading cold-temperate climates need to overwinter in a dormant state. We compared the role of temperature and photoperiod in dormancy induction in the laboratory, as well as winter survival and reproduction in the field and the laboratory, of 5 widespread European dung fly species (Diptera: Sepsidae) to investigate their extent of ecological differentiation and thermal adaptation. Unexpectedly, cold temperature is the primary environmental factor inducing winter dormancy, with short photoperiod playing an additional role mainly in species common at high altitudes and latitudes (Sepsis cynipsea, neocynipsea, fulgens), but not in those species also thriving in southern Europe (thoracica, punctum). All species hibernate as adults rather than juveniles. S. thoracica had very low adult winter survivorship under both (benign) laboratory and (harsh) field conditions, suggesting flexible quiescence rather than genetically fixed winter diapause, restricting their distribution towards the pole. All other species appear well suited for surviving cold, Nordic winters. Females born early in the season reproduce before winter while late-born females reproduce after winter, fulgens transitioning earliest before winter and thoracica and punctum latest; a bet-hedging strategy of reproduction during both seasons occurs rarely but is possible physiologically. Fertility patterns indicate that females can store sperm over winter. Winter dormancy induction mechanisms of European sepsids are congruent with their geographic distribution, co-defining their thermal niches. Flexible adult winter quiescence appears the easiest route for insects spreading towards the poles to evolve the necessary overwinter survival.

Transcriptomics of cytokinin and auxin metabolism and signaling genes during seed maturation in dormant and non-dormant wheat genotypes

To gain insights into the roles of cytokinin (CK) and auxin in regulating dormancy during seed maturation in wheat, we examined changes in the levels of CK and indole-3-acetic acid (IAA) and expression patterns of their metabolism and signaling genes in embryonic and endospermic tissues of dormant and non-dormant genotypes. Seed maturation was associated with a decrease in the levels of isopentenyladenine in both tissues mainly via repression of the CK biosynthetic TaLOG genes. Differential embryonic trans-zeatin content and expression patterns of the CK related genes including TacZOG, TaGLU and TaARR12 between maturing seeds of the two genotypes implicate CK in the control of seed dormancy induction and maintenance. Seed maturation induced a decrease of IAA level in both tissues irrespective of genotype, and this appeared to be mediated by repression of specific IAA biosynthesis, transport and IAA-conjugate hydrolysis genes. The differential embryonic IAA content and expression pattern of the IAA biosynthetic gene TaAO during the early stage of seed maturation between the two genotypes imply the role of IAA in dormancy induction. It appears from our data that the expression of specific auxin signaling genes including TaRUB, TaAXR and TaARF mediate the role of auxin signaling in dormancy induction and maintenance during seed maturation in wheat.

Spring Is Coming: Genetic Analyses of the Bud Break Date Locus Reveal Candidate Genes From the Cold Perception Pathway to Dormancy Release in Apple (Malus × domestica Borkh.).

Chilling requirement (CR) for bud dormancy completion determines the time of bud break in apple (Malus × domestica Borkh.). The molecular control of bud dormancy is highly heritable, suggesting a strong genetic control of the trait. An available Infinium II SNP platform for genotyping containing 8,788 single nucleotide polymorphic markers was employed, and linkage maps were constructed in a F1 cross from the low CR M13/91 and the moderate CR cv. Fred Hough. These maps were used to identify quantitative trait loci (QTL) for bud break date as a trait related to dormancy release. A major QTL for bud break was detected at the beginning of linkage group 9 (LG9). This QTL remained stable during seven seasons in two different growing sites. To increase mapping efficiency in detecting contributing genes underlying this QTL, 182 additional SNP markers located at the locus for bud break were used. Combining linkage mapping and structural characterization of the region, the high proportion of the phenotypic variance in the trait explained by the QTL is related to the coincident positioning of Arabidopsis orthologs for ICE1, FLC, and PRE1 protein-coding genes. The proximity of these genes from the most explanatory markers of this QTL for bud break suggests potential genetic additive effects, reinforcing the hypothesis of inter-dependent mechanisms controlling dormancy induction and release in apple trees.

Abstract A144: The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine

Abstract Melanoma (MM) cells leave primary tumors early and evolve at different sites in parallel. However, timing in human MM is unknown. Lymphatic dissemination is fast shortly after the dermal invasion, but metastases may occur late. Such delay in metastases formation suggests possible host defense mechanism, or particular nature of non-actively proliferative (dormant) MM stem cell (MSC) population. T lymphocytes may also induce tumor dormancy. The proposed mechanism involves a cytostatic mode of tumor silencing resulting from the suppression of both neoplastic cell proliferation and cell cycle progression. Significant progress in the development of novel advanced MM therapies was made. However, not all patients benefit from the therapy, developing late metastases decades after treatment ends. Moreover, using the immunotherapy-based model of MM dormancy in mouse, vaccination against MM prevented tumor growth but failed to prevent tumor cell dissemination or elimination of all MM cells. We have developed therapeutic gene modified allogenic MM vaccine (AGI-101H) that has been tested since 1997 and resulted in long-term survival of a substantial fraction of patients. Genetic modification of vaccine cells to express designer cytokine cDNA encoding Hyper-IL-6 protein (comprising of IL-6 linked with a soluble IL-6 receptor) has altered AGI-101H cells phenotype towards MSC-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). We have found significant induction of anti-MSCs response in immunized patients by a generation of functionally active cytotoxic CD8+T-cells and antibodies specific for ALDH1A1 in circulation. However, the AGI-101H mode of action is complex: besides inducing a direct anticancer response to MSC population, long-term vaccination may induce MM dormancy through T-cell mediated immunity. The above hypothesis is based on clinical observations of AHI-101H treated patient who was progression-free for many years, but injury induced metastases formation in the bruise. This patient developed skin MM in 1999. In 2002 in transit metastases appeared (resected). The patient was enrolled into AGI-101H program. In 2003 metastases in liver (resected), 2004 metastasis in ovary (resected). 11 years (2004-2015) no progression. In Nov. 2015 she had bicycle accident, trauma, and MM metastasis presenting with a bruise (resected). The treatment continues with no progression. The aim was to determine the molecular mechanisms of peripheral T-cell-mediated induction of MM dormancy induced by vaccination. Therefore, T-cell mRNA expression profiling of long-term surviving patients was carried out. PBMCs were isolated from treated patients (18), untreated MM patients (13) and healthy controls (8). Untouched T-cells were separated by magnetic beads and total RNA isolated. The transcriptome profiling was performed with Affymetrix HG U219 microarray (19,285 markers). Differential gene expression (DGE) analysis between all groups was conducted and validated with RT-qPCR and FACS. The transcriptomic results were further analyzed with Gene Set Enrichment Analysis (GSEA) tool. DGE analyses comparing AGI-101H vaccinated (AV) and nonimmunized (C) patients revealed 538 differentially expressed genes (DEGs), with 373 downregulated and 165 upregulated in AV (adj p < 0.05). Among these 538 markers, the expression of 14 (with |log2FC| > 1) is now being validated with RT-qPCR and FACS. Also, GSEA analysis revealed significant enrichment of “TNFa_signaling_via_NFkB”, “TGFb_signaling” and “G2/M_checkpoint” hallmark processes (MSigDB Hallmark gene sets) in AGI-101H vaccinated patients, that indicates considerable activation of antitumor response and prevention of conversion of MM dormanT-cells into proliferative metastases. Transcriptome profiling of peripheral T-cells in long-surviving MM patients immunized with AGI-101H suggests activation of the molecular mechanism that sustains MM cells in the non-actively proliferative dormant state. Citation Format: Andrzej A. Mackiewicz, Partycja Czerwinska, Marcin Rucinski, Katarzyna Gryska, Iga Grzadzielewska, Anna Jaworska, Jacek Mackiewicz. The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A144.

CRISPR: a new principle of genome engineering linked to conceptual shifts in evolutionary biology

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity have become a household name among biologists and even the general public thanks to the unprecedented success of the new generation of genome editing tools utilizing Cas proteins. However, the fundamental biological features of CRISPR-Cas are of no lesser interest and have major impacts on our understanding of the evolution of antivirus defense, host-parasite coevolution, self versus non-self discrimination and mechanisms of adaptation. CRISPR-Cas systems present the best known case in point for Lamarckian evolution, i.e. generation of heritable, adaptive genomic changes in response to encounters with external factors, in this case, foreign nucleic acids. CRISPR-Cas systems employ multiple mechanisms of self versus non-self discrimination but, as is the case with immune systems in general, are nevertheless costly because autoimmunity cannot be eliminated completely. In addition to the autoimmunity, the fitness cost of CRISPR-Cas systems appears to be determined by their inhibitory effect on horizontal gene transfer, curtailing evolutionary innovation. Hence the dynamic evolution of CRISPR-Cas loci that are frequently lost and (re)acquired by archaea and bacteria. Another fundamental biological feature of CRISPR-Cas is its intimate connection with programmed cell death and dormancy induction in microbes. In this and, possibly, other immune systems, active immune response appears to be coupled to a different form of defense, namely, “altruistic” shutdown of cellular functions resulting in protection of neighboring cells. Finally, analysis of the evolutionary connections of Cas proteins reveals multiple contributions of mobile genetic elements (MGE) to the origin of various components of CRISPR-Cas systems, furthermore, different biological systems that function by genome manipulation appear to have evolved convergently from unrelated MGE. The shared features of adaptive defense systems and MGE, namely the ability to recognize and cleave unique sites in genomes, make them ideal candidates for genome editing and engineering tools.