The association analysis of DNA methylation and transcriptomics identified BpCYCD3;2 as a participant in influencing cell division in autotetraploid birch (Betula pendula) leaves.

Polyploidization plays a crucial role in plant breeding and genetic improvement. Although the phenomenon of polyploidization affecting the area and number of plant epidermal pavement cells is well described, the underlying mechanism behind this phenomenon is still largely unknown. In this study, we found that the leaves of autotetraploid birch (Betula pendula) stopped cell division earlier and had a larger cell area. In addition, compared to diploids, tetraploids have a smaller stomatal density and fewer stomatal numbers. Genome-wide DNA methylation analysis revealed no significant difference in global DNA methylation levels between diploids and tetraploids. A total of 9154 differential methylation regions (DMRs) were identified between diploids and tetraploids, with CHH-type DMRs accounting for 91.73% of all types of DMRs. Further research has found that there are a total of 2105 differentially methylated genes (DMEGs) with CHH-type DMRs in birch. The GO functional enrichment results of DMEGs showed that differentially methylated genes were mainly involved in terms such as cellular process and metabolic process. The analysis of differentially methylated genes and differentially expressed genes suggests that hyper-methylation in the promoter region may inhibit the gene expression level of BpCYCD3;2 in tetraploids. To investigate the function of BpCYCD3;2 in birch, we obtained overexpression and repressed expression lines of BpCYCD3;2 through genetic transformation. The morphogenesis of both BpCYCD3;2-OE and BpCYCD3;2-RE lines was not affected. However, low expression of BpCYCD3;2 can lead to inhibition of cell division in leaves, and this inhibition of cell proliferation can be compensated for by an increase in cell size. Additionally, we found that the number and density of stomata in the BpCYCD3;2-RE lines were significantly reduced, consistent with the tetraploid. These data indicate that changes in cell division ability and stomatal changes in tetraploid birch can be partially attributed to low expression of the BpCYCD3;2 gene, which may be related to hyper-methylation in its promoter region. These results will provide new insights into the mechanism by which polyploidization affects plant development.

The cold-responsive C-repeat binding factors in Betula platyphylla Suk. positively regulate cold tolerance.

Cold stress is one of the most destructive abiotic stresses limiting plant growth and development. CBF (C-repeat binding factor) transcription factors and their roles in cold response have been identified in Arabidopsis as well as several other plant species. However, the biological functions and related molecular mechanisms of CBFs in birch (Betula platyphylla Suk.) remain undetermined. In this study, five cold-responsive BpCBF genes, BpCBF1, BpCBF2, BpCBF7, BpCBF10 and BpCBF12 were cloned. Via protoplast transformation, BpCBF7 was found to be localized in nucleus. The result of yeast one hybrid assay validated the binding of BpCBF7 to the CRT/DRE (C-repeat/dehydration responsive element) elements in the promoter of BpERF1.1 gene. By overexpressing and repressing BpCBFs in birch plants, it was proven that BpCBFs play positive roles in the cold tolerance. At the metabolic level, BpCBFs OE lines had lower ROS accumulation, as well as higher activities of antioxidant enzymes (SOD, POD and CAT) and higher accumulation of protective substances (soluble sugar, soluble protein and proline). Via yeast one hybrid and co-transformation of effector and reporter vectors assay, it was proven that BpCBF7 can regulate the expression of BpERF5 and BpZAT10 genes by directly binding to their promoters. An interacting protein of BpCBF7, BpWRKY17, was identified by yeast two hybrid library sequencing and the interaction was validated with in vivo methods. These results indicates that BpCBFs can increase the cold tolerance of birch plants, partly by gene regulation and protein interaction. This study provides a reference for the research on CBF transcription factors and genetic improvement of forest trees upon abiotic stresses.

Responses of stomatal density and carbon isotope composition of sugar maple and yellow birch foliage to N, P and CaSiO3 fertilization.

Stomatal density, stomatal length and carbon isotope composition can all provide insights into environmental controls on photosynthesis and transpiration. Stomatal measurements can be time-consuming; it is therefore wise to consider efficient sampling schemes. Knowing the variance partitioning at different measurement levels (i.e., among stands, plots, trees, leaves and within leaves) can aid in making informed decisions around where to focus sampling effort. In this study, we explored the effects of nitrogen (N), phosphorus (P) and calcium silicate (CaSiO3) addition on stomatal density, length and carbon isotope composition (δ13C) of sugar maple (Acer saccharum Marsh.) and yellow birch (Betula alleghaniensis Britton). We observed a positive but small (8%) increase in stomatal density with P addition and an increase in δ13C with N and CaSiO3 addition in sugar maple, but we did not observe effects of nutrient addition on these characteristics in yellow birch. Variability was highest within leaves and among trees for stomatal density and highest among stomata for stomatal length. To reduce variability and increase chances of detecting treatment differences in stomatal density and length, future protocols should consider pretreatment and repeated measurements of trees over time or measure more trees per plot, increase the number of leaf impressions or standardize their locations, measure more stomata per image and ensure consistent light availability.

Bp-miR408a participates in osmotic and salt stress responses by regulating BpBCP1 in Betula platyphylla.

The microRNAs, which are small RNAs of 18-25 nt in length, act as key regulatory factors in posttranscriptional gene expression during plant growth and development. However, little is known about their regulatory roles in response to stressful environments in birch (Betula platyphylla). Here, we characterized and further explored miRNAs from osmotic- and salt-stressed birch. Our analysis revealed a total of 190 microRNA (miRNA) sequences, which were classified into 180 conserved miRNAs and 10 predicted novel miRNAs based on sequence homology. Furthermore, we identified Bp-miR408a under osmotic and salt stress and elucidated its role in osmotic and salt stress responses in birch. Notably, under osmotic and salt stress, Bp-miR408a contributed to osmotic and salt tolerance sensitivity by mediating various physiological changes, such as increases in reactive oxygen species accumulation, osmoregulatory substance contents and Na+ accumulation. Additionally, molecular analysis provided evidence of the in vivo targeting of BpBCP1 (blue copper protein) transcripts by Bp-miR408a. The overexpression of BpBCP1 in birch enhanced osmotic and salt tolerance by increasing the antioxidant enzyme activity, maintaining cellular ion homeostasis and decreasing lipid peroxidation and cell death. Thus, we reveal a Bp-miR408a-BpBCP1 regulatory module that mediates osmotic and salt stress responses in birch.

Pollen production of downy birch (Betula pubescens Ehrh.) along an altitudinal gradient in the European Alps.

High-altitude environments are highly susceptible to the effects of climate change. Thus, it is crucial to examine and understand the behaviour of specific plant traits along altitudinal gradients, which offer a real-life laboratory for analysing future impacts of climate change. The available information on how pollen production varies at different altitudes in mountainous areas is limited. In this study, we investigated pollen production of 17 birch (Betula pubescens Ehrh.) individuals along an altitudinal gradient in the European Alps. We sampled catkins at nine locations in the years 2020-2021 and monitored air temperatures. We investigated how birch pollen, flowers and inflorescences are produced in relation to thermal factors at various elevations. We found that mean pollen production of Betula pubescens Ehrh. varied between 0.4 and 8.3 million pollen grains per catkin. We did not observe any significant relationships between the studied reproductive metrics and altitude. However, minimum temperature of the previous summer was found to be significantly correlated to pollen (rs = 0.504, p = 0.039), flower (rs = 0.613, p = 0.009) and catkin (rs = 0.642, p = 0.005) production per volume unit of crown. Therefore, we suggest that temperature variability even at such small scales is very important for studying the response related to pollen production.

Genomic signals of local adaptation and hybridization in Asian white birch.

Disentangling the numerous processes that affect patterns of genome-wide diversity in widespread tree species has important implications for taxonomy, conservation, and forestry. Here, we investigate the population genomic structure of Asian white birch (Betula platyphylla) in China and seek to explain it in terms of hybridization, demography and adaptation. We generate whole genome sequence data from 83 individuals across the species range in China. Combining this with an existing data set for 79 European and Russian white birches, we show a clear distinction between B. pendula and B. platyphylla, which have sometimes been lumped taxonomically. Genomic diversity of B. platyphylla in north-western China and Central Russia is affected greatly by hybridization with B. pendula. Excluding these hybridized populations, B. platyphylla in China has a linear distribution from north-eastern to south-western China, along the edge of the inland mountainous region. Within this distribution, three genetic clusters are found, which we model as long diverged with subsequent episodes of gene flow. Patterns of covariation between allele frequencies and environmental variables in B. platyphylla suggest the role of natural selection in the distribution of diversity at 7609 SNPs of which 3767 were significantly differentiated among the genetic clusters. The putative adaptive SNPs are distributed throughout the genome and span 1633 genic regions. Of these genic regions, 87 were previously identified as candidates for selective sweeps in Eurasian B. pendula. We use the 7609 environmentally associated SNPs to estimate the risk of nonadaptedness for each sequenced B. platyphylla individual under a scenario of future climate change, highlighting areas where populations may be under future threat from rising temperatures.

Separating the effects of air and soil temperature on silver birch. Part II. The relation of physiology and leaf anatomy to growth dynamics.

The aboveground parts of boreal forest trees grow earlier in the growing season, the roots mostly later. The idea was to examine whether root growth followed soil temperature, or whether shoot growth also demanded most resources in the early growing season (soil temperature vs internal sink strengths for resources). The linkage between air and soil temperature was broken by switching the soil temperature. We aimed here to identify the direct effects of different soil temperature patterns on physiology, leaf anatomy and their interactions, and how they relate to the control of the growth dynamics of silver birch (Betula pendula Roth). Sixteen 2-year-old seedlings were grown in a controlled environment for two 14-week simulated growing seasons (GS1, GS2). An 8-week dormancy period interposed the GSs. In GS2, soil temperature treatments were applied: constant 10 °C (Cool), constant 18 °C (Warm), early growing season at 10 °C switched to 18 °C later (Early Cool Late Warm) and 18 °C followed by 10 °C (Early Warm Late Cool) were applied during GS2. The switch from cool to warm enhanced the water status, net photosynthesis, chlorophyll content index, effective yield of photosystem II (ΔF/Fm') and leaf expansion of the seedlings. Warm treatment increased the stomatal number per leaf. In contrast, soil cooling increased glandular trichomes. This investment in increasing the chemical defense potential may be associated with the decreased growth in cool soil. Non-structural carbohydrates were accumulated in leaves at a low soil temperature showing that growth was more hindered than net photosynthesis. Leaf anatomy differed between the first and second leaf flush of silver birch, which may promote tree fitness in the prevailing growing conditions. The interaction of birch structure and function changes with soil temperature, which can further reflect to ecosystem functioning.

Impacts of elevation on plant traits and volatile organic compound emissions in deciduous tundra shrubs.

The northernmost regions of our planet experience twice the rate of climate warming compared to the global average. Despite the currently low air temperatures, tundra shrubs are known to exhibit high leaf temperatures and are increasing in height due to warming, but it is unclear how the increase in height will affect the leaf temperature. To study how temperature, soil moisture, and changes in light availability influence the physiology and emissions of climate-relevant volatile organic compounds (VOCs), we conducted a study on two common deciduous tundra shrubs, Salix glauca (separating males and females for potential effects of plant sex) and Betula glandulosa, at two elevations in South Greenland. Low-elevation Salix shrubs were 45% taller, but had 37% lower rates of net CO2 assimilation and 63% lower rates of isoprene emission compared to high-elevation shrubs. Betula shrubs showed 40% higher stomatal conductance and 24% higher glandular trichome density, in the low-elevation valley, compared to those from the high-elevation mountain slope. Betula green leaf volatile emissions were 235% higher at high elevation compared to low elevation. Male Salix showed a distinct VOC blend and emitted 55% more oxygenated VOCs, compared to females, possibly due to plant defense mechanisms. In our light response curves, isoprene emissions increased linearly with light intensity, potentially indicating adaptation to strong light. Leaf temperature decreased with increasing Salix height, at 4 °C m-1, which can have implications for plant physiology. However, no similar relationship was observed for B. glandulosa. Our results highlight that tundra shrub traits and VOC emissions are sensitive to temperature and light, but that local variations in soil moisture strongly interact with temperature and light responses. Our results suggest that effects of climate warming, alone, poorly predict the actual plant responses in tundra vegetation.

Functional roles of the birch BpRAV1 transcription factor in salt and osmotic stress response.

RAV (Related to ABI3/VP1) transcription factors play vital roles in regulating plant response to abiotic stresses; however, the regulatory mechanisms underlying stress response are still poorly understood for most of the RAVgenes. In this study, a novel gene BpRAV1 was cloned from white birch (Betula platyphylla). BpRAV1 protein is localized in the nucleus and serves as a transcriptional activator. The expression of BpRAV1 was induced by salt and osmotic stress treatments. BpRAV1-overexpression birch seedlings exhibited dramatically less ROS accumulation and reduced cell death in response to salt and osmotic stresses. BpRAV1 can specifically bind to the known RAV1A element. In addition, a novel cis-acting element (termed RBS1) bound by BpRAV1 was identified by transcription factor (TF)- centered Y1H assay. BpRAV1 activated the RAV1A and RBS1 elements to induce the expression of SOD and POD genes, resulting in increased SOD and POD activities to enhance ROS scavenging ability, thus improving salt and osmotic stress tolerance. These results indicate that BpRAV1 is a positive regulator governing abiotic stress response.

Herbivory and warming interact in opposing patterns of covariation between arctic shrub species at large and local scales.

A major challenge in predicting species' distributional responses to climate change involves resolving interactions between abiotic and biotic factors in structuring ecological communities. This challenge reflects the classical conceptualization of species' regional distributions as simultaneously constrained by climatic conditions, while by necessity emerging from local biotic interactions. A ubiquitous pattern in nature illustrates this dichotomy: potentially competing species covary positively at large scales but negatively at local scales. Recent theory poses a resolution to this conundrum by predicting roles of both abiotic and biotic factors in covariation of species at both scales, but empirical tests have lagged such developments. We conducted a 15-y warming and herbivore-exclusion experiment to investigate drivers of opposing patterns of covariation between two codominant arctic shrub species at large and local scales. Climatic conditions and biotic exploitation mediated both positive covariation between these species at the landscape scale and negative covariation between them locally. Furthermore, covariation between the two species conferred resilience in ecosystem carbon uptake. This study thus lends empirical support to developing theoretical solutions to a long-standing ecological puzzle, while highlighting its relevance to understanding community compositional responses to climate change.

Effect of Drought and Heavy Metal Contamination on Growth and Photosynthesis of Silver Birch Trees Growing on Post-Industrial Heaps.

Silver birch trees (Betula pendula Roth) are a pioneering species in post-industrial habitats, and have been associated with an expansive breeding strategy and low habitat requirements. We conducted ecophysiological and dendroclimatological studies to check whether there are any features of which the modification enables birch trees to colonise extreme habitats successfully. We characterised the efficiency of the photosynthetic apparatus, the gas exchange, the content of pigments in leaves, and the growth (leaf thickness and tree-ring width) of birch trees on a post-coal mine heap, a post-smelter heap, and a reference site. Birch growth was limited mainly by temperature and water availability during summer, and the leaves of the birch growing on post-industrial heaps were significantly thicker than the reference leaves. Moreover, birch trees growing on heaps were characterised by a significantly higher content of flavonols and anthocyanins in leaves and higher non-photochemical quenching. In addition, birches growing on the post-coal mine heap accumulated a concentration of Mn in their leaves, which is highly toxic for most plant species. Increasing the thickness of leaves, and the content of flavonols and anthocyanins, as well as efficient non-photochemical quenching seem to be important features that improve the colonization of extreme habitats by birches.

Constant ratio of Cc to Ci under various CO2 concentrations and light intensities, and during progressive drought, in seedlings of Japanese white birch.

Constant mesophyll conductance (gm), and two-resistance gm model (involved in resistances of cell wall and chloroplast), where gm reaches maximum under higher CO2 concentrations, cannot describe the phenomenon that gm decreases with increasing intercellular CO2 concentration (Ci) under relatively higher CO2 concentrations. Yin et al. (2020) proposed a gm model, according to which the ratio of chloroplastic CO2 concentration (Cc) to Ci is constant in the two-resistance gm model, which can describe the decreasing gm with increasing Ci. In the present study, we investigated the relationship between Cc and Ci in leaves of Japanese white birch by using simultaneous measurements of gas exchange and chlorophyll fluorescence under various CO2 concentrations, light intensities, and during progressive drought. Across the range of ambient CO2 from 50 to 1000 µmol mol-1, and light intensities of 50 to 2000 µmol m-2 s-1, measured under well irrigation, the ratio of Cc to Ci kept constant. During the progressive drought, overestimated Ci due to stomatal patchiness and/or cuticular transpiration was empirically corrected (threshold: stomatal conductance < 0.08 mol H2O m-2 s-1) from the A/Ci response measured under adequate irrigation. The ratio of Cc to Ci during progressive drought (predawn leaf potential reached ≈ - 2 MPa) also remained constant irrespective of soil drying rate in various pot sizes. The present study suggests the involvement of some physiologically regulative mechanisms to keep Cc:Ci ratio constant, which might act on gm in addition to the physical interaction of diffusive resistances in the cell components.

The effect of tree age, daily sap volume and date of sap collection on the content of minerals and heavy metals in silver birch (Betula pendula Roth) tree sap.

The aim of this study was to determine the effect of the age of trees, daily sap volume as well as the term of tapping birch sap collected in the forest environment on the content of selected minerals (zinc, copper and manganese) and heavy metals (lead, nickel, chromium and cadmium). The study was performed on material taken from two stands (aged 34 and 84 years) in a moist broadleaved forest habitat with a dominant share of silver birch (Betula pendula Roth). The research results confirmed the presence of both nutritional essential minerals and hazardous heavy metals in the birch sap. At the same time, the content of minerals and heavy metals was found to be very variable and the differences between their concentrations, recorded on the same day of collecting in several trees of the same age group, can be even several dozen times higher. Depending on the examined elements, the factors influencing their content vary. The age of the trees determines only the manganese content; daily sap volume significantly affects the content of manganese and copper, and date of collection differentiates the content of zinc, lead, nickel and cadmium. The results may be interesting in the context of developing procedures for collecting birch sap for the purpose of obtaining raw material with beneficial nutritional values and a high level of health safety. For this reason, our recommendation for guaranteeing the health safety and high nutritional value of birch sap is to combine batches of raw material taken from as many trees as possible, and at the same time to publicize the fact that collecting birch sap from just one single tree may result in a raw material that is both dangerous and has no nutritional benefits.

Overestimation of the effect of climatic warming on spring phenology due to misrepresentation of chilling.

Spring warming substantially advances leaf unfolding and flowering time for perennials. Winter warming, however, decreases chilling accumulation (CA), which increases the heat requirement (HR) and acts to delay spring phenology. Whether or not this negative CA-HR relationship is correctly interpreted in ecosystem models remains unknown. Using leaf unfolding and flowering data for 30 perennials in Europe, here we show that more than half (7 of 12) of current chilling models are invalid since they show a positive CA-HR relationship. The possible reason is that they overlook the effect of freezing temperature on dormancy release. Overestimation of the advance in spring phenology by the end of this century by these invalid chilling models could be as large as 7.6 and 20.0 days under RCPs 4.5 and 8.5, respectively. Our results highlight the need for a better representation of chilling for the correct understanding of spring phenological responses to future climate change.

Betula platyphylla BpHOX2 transcription factor binds to different cis-acting elements and confers osmotic tolerance.

The homeodomain-leucine zipper (HD-Zip) proteins play crucial roles in plant developmental and environmental responses. However, how they mediate gene expression to facilitate abiotic stress tolerance remains unknown. In the present study, we characterized BpHOX2 (encoding a HD-Zip I family protein) from birch (Betula platyphylla). BpHOX2 is predominately expressed in mature stems and leaves, but expressed at a low level in apical buds and roots, suggesting that it has tissue-specific characteristics. BpHOX2 expression was highly induced by osmotic and salt, but only slightly induced by abscisic acid. Overexpression of BpHOX2 markedly improved osmotic tolerance, while knockdown of BpHOX2 increased sensitivity to osmotic stress. BpHOX2 could induce the expression of pyrroline-5-carboxylate synthase, peroxidase, and superoxide dismutase genes to improve proline levels and the reactive oxygen species scavenging capability. Chromatin immunoprecipitation sequencing combined with RNA sequencing showed that BpHOX2 could bind to at least four cis-acting elements, including dehydration-responsive element "RCCGAC", Myb-p binding box "CCWACC," and two novel cis-acting elements with the sequences of "AAGAAG" and "TACGTG" (termed HBS1 and HBS2, respectively) to regulate gene expression. Our results suggested that BpHOX2 is a transcription factor that binds to different cis-acting elements to regulate gene expression, ultimately improving osmotic tolerance in birch.

Response of BpBZR genes to abiotic stress and hormone treatment in Betula platyphylla.

Brassinazole-resistant (BZR) transcription factors have important roles in the brassinosteroid (BR) signalling pathway and are widely involved in plant growth and abiotic stress processes. However, there are few studies on the functions and regulatory mechanisms of BZR TFs in birch. In this study, 5 BZR genes were identified from birch. The qRT-PCR results showed that the expression levels of most BpBZRs were significantly downregulated and/or upregulated in at least one organ following NaCl and PEG stress or ABA, GA3 and JA treatments. In particular, BpBZR1 expression was changed in all three organs after exposure to NaCl stress at all time points, indicating that this gene may be involved in salt stress. The BpBZR1 transcription factor was shown to have transcriptional activation activity in a yeast two-hybrid assay. Through a transient transformation system, we found that overexpression of BpBZR1 in birch resulted in lower H2O2 and MDA accumulation, higher SOD and POD activities and maintained a higher photosynthetic intensity and a lower chlorophyll degradation rate than those of the control plants under salt stress. These results preliminarily showed that overexpression of the BpBZR1 gene increased the tolerance of birch to salt stress.

Overexpression of an AP2/ERF family gene, BpERF13, in birch enhances cold tolerance through upregulating CBF genes and mitigating reactive oxygen species.

The AP2/ERF (APETALA2/ethylene-responsive factor) family of transcription factors (TF) is involved in regulating biotic and abiotic stress responses in plants. To explore the role of AP2/ERFs in cold tolerance in woody plants, BpERF13 was cloned and characterized in Betula platyphylla (white birch), a species primarily found in Asia in temperate and boreal climates. Based on phylogenetic analysis, BpERF13 is a member of the IXb subfamily of ERFs. Using qRT-PCR, we found that BpERF13 was differentially expressed in different tissues, and its expression could be induced by cold treatment (4 °C). BpERF13 protein, fused with GFP, was exclusively localized to nuclei. To further assess the role of BpERF13 in cold tolerance, BpERF13 overexpression (OE) transgenic lines were generated in B. platyphylla and used for cold stress treatment and biochemical/physiological studies. BpERF13 overexpression lines had significantly increased tolerance to subfreezing treatment and reduced reactive oxygen species. Using a TF-centered yeast one-hybrid (Y1H) experimental system, we showed that BpERF13 could bind to LTRECOREATCOR15 and MYBCORE cis-elements to activate a reporter gene. ChIP-seq and ChIP-PCR experiments further demonstrated that BpERF13 bound to these cis-elements when present in the 5' proximal regions of superoxide dismutase (SOD), peroxidase (POD), and C-repeat-binding factor (CBF) genes. qRT-PCR was employed to examine the expression levels of these genes in response to cold stress; SOD, POD, and CBF genes were significantly upregulated in BpERF13 transgenic lines compared to wild-type plants in response to cold stress. These results indicate that the transcription factor BpERF13 regulates physiological processes underlying cold tolerance in woody plants.

Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions.

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

A 5700 year-old human genome and oral microbiome from chewed birch pitch.

The rise of ancient genomics has revolutionised our understanding of human prehistory but this work depends on the availability of suitable samples. Here we present a complete ancient human genome and oral microbiome sequenced from a 5700 year-old piece of chewed birch pitch from Denmark. We sequence the human genome to an average depth of 2.3× and find that the individual who chewed the pitch was female and that she was genetically more closely related to western hunter-gatherers from mainland Europe than hunter-gatherers from central Scandinavia. We also find that she likely had dark skin, dark brown hair and blue eyes. In addition, we identify DNA fragments from several bacterial and viral taxa, including Epstein-Barr virus, as well as animal and plant DNA, which may have derived from a recent meal. The results highlight the potential of chewed birch pitch as a source of ancient DNA.

Characterization and T-DNA insertion sites identification of a multiple-branches mutant br in Betula platyphylla × Betula pendula.

BACKGROUND: Plant architecture, which is mostly determined by shoot branching, plays an important role in plant growth and development. Thus, it is essential to explore the regulatory molecular mechanism of branching patterns based on the economic and ecological importance. In our previous work, a multiple-branches birch mutant br was identified from 19 CINNAMOYL-COENZYME A REDUCTASE 1 (CCR1)-overexpressed transgenic lines, and the expression patterns of differentially expressed genes in br were analyzed. In this study, we further explored some other characteristics of br, including plant architecture, wood properties, photosynthetic characteristics, and IAA and Zeatin contents. Meanwhile, the T-DNA insertion sites caused by the insertion of exogenous BpCCR1 in br were identified to explain the causes of the mutation phenotypes. RESULTS: The mutant br exhibited slower growth, more abundant and weaker branches, and lower wood basic density and lignin content than BpCCR1 transgenic line (OE2) and wild type (WT). Compared to WT and OE2, br had high stomatal conductance (Gs), transpiration rate (Tr), but a low non-photochemical quenching coefficient (NPQ) and chlorophyll content. In addition, br displayed an equal IAA and Zeatin content ratio of main branches' apical buds to lateral branches' apical buds and high ratio of Zeatin to IAA content. Two T-DNA insertion sites caused by the insertion of exogenous BpCCR1 in br genome were found. On one site, chromosome 2 (Chr2), no known gene was detected on the flanking sequence. The other site was on Chr5, with an insertion of 388 bp T-DNA sequence, resulting in deletion of 107 bp 5' untranslated region (UTR) and 264 bp coding sequence (CDS) on CORONATINE INSENSITIVE 1 (BpCOII). In comparison with OE2 and WT, BpCOI1 was down-regulated in br, and the sensitivity of br to Methyl Jasmonate (MeJA) was abnormal. CONCLUSIONS: Plant architecture, wood properties, photosynthetic characteristics, and IAA and Zeatin contents in main and lateral branches' apical buds changed in br over the study's time period. One T-DNA insertion was identified on the first exon of BpCOI1, which resulted in the reduction of BpCOI1 expression and abnormal perception to MeJA in br. These mutation phenotypes might be associated with a partial loss of BpCOI1 in birch.