Acetylation of transcription factor BpTCP20 by acetyltransferase BpPDCE23 modulates salt tolerance in birch.

Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors function in abiotic stress responses. However, how TCPs confer salt tolerance is unclear. Here, we characterized a TCP transcription factor, BpTCP20, that responds to salt stress in birch (Betula platyphylla Suk). Plants overexpressing BpTCP20 displayed increased salt tolerance, and Bptcp20 knockout mutants displayed reduced salt tolerance relative to the wild-type (WT) birch. BpTCP20 conferred salt tolerance by mediating stomatal closure and reducing reactive oxygen species (ROS) accumulation. Chromatin immunoprecipitation sequencing showed that BpTCP20 binds to NeuroD1, T-box, and two unknown elements (termed TBS1 and TBS2) to regulate target genes. In birch, salt stress led to acetylation of BpTCP20 acetylation at lysine 259. A mutated BpTCP20 variant (abolished for acetylation, termed BpTCP20259) was overexpressed in birch, which led to decreased salt tolerance compared with plants overexpressing BpTCP20. However, BpTCP20259-overexpressing plants still displayed increased salt tolerance relative to untransformed WT plants. BpTCP20259 showed reduced binding to the promoters of target genes and decreased target gene activation, leading to decreased salt tolerance. In addition, we identified dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (BpPDCE23), an acetyltransferase that interacts with and acetylates BpTCP20 to enhance its binding to DNA motifs. Together, these results suggest that BpTCP20 is a transcriptional regulator of salt tolerance, whose activity is modulated by BpPDCE23-mediated acetylation.

BpGRP1 acts downstream of BpmiR396c/BpGRF3 to confer salt tolerance in Betula platyphylla.

Glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, but the function of GRP genes involved in salt stress and the underlying mechanism remain unclear. In this study, we identified BpGRP1 (glycine-rich RNA-binding protein), a Betula platyphylla gene that is induced under salt stress. The physiological and molecular responses to salt tolerance were investigated in both BpGRP1-overexpressing and suppressed conditions. BpGRF3 (growth-regulating factor 3) was identified as a regulatory factor upstream of BpGRP1. We demonstrated that overexpression of BpGRF3 significantly increased the salt tolerance of birch, whereas the grf3-1 mutant exhibited the opposite effect. Further analysis revealed that BpGRF3 and its interaction partner, BpSHMT, function upstream of BpGRP1. We demonstrated that BpmiR396c, as an upstream regulator of BpGRF3, could negatively regulate salt tolerance in birch. Furthermore, we uncovered evidence showing that the BpmiR396c/BpGRF3 regulatory module functions in mediating the salt response by regulating the associated physiological pathways. Our results indicate that BpmiR396c regulates the expression of BpGRF3, which plays a role in salt tolerance by targeting BpGRP1.

Long noncoding RNA from Betula platyphylla, BplncSIR1, confers salt tolerance by regulating BpNAC2 to mediate reactive oxygen species scavenging and stomatal movement.

Long noncoding RNAs (lncRNAs) play an important role in abiotic stress tolerance. However, their function in conferring abiotic stress tolerance is still unclear. Herein, we characterized the function of a salt-responsive nuclear lncRNA (BplncSIR1) from Betula platyphylla (birch). Birch plants overexpressing and knocking out for BplncSIR1 were generated. BplncSIR1 was found to improve salt tolerance by inducing antioxidant activity and stomatal closure, and also accelerate plant growth. Chromatin isolation by RNA purification (ChIRP) combined with RNA sequencing indicated that BplncSIR1 binds to the promoter of BpNAC2 (encoding NAC domain-containing protein 2) to activate its expression. Plants overexpressing and knocking out for BpNAC2 were generated. Consistent with that of BplncSIR1, overexpression of BpNAC2 also accelerated plant growth and conferred salt tolerance. In addition, BpNAC2 binds to different cis-acting elements, such as G-box and 'CCAAT' sequences, to regulate the genes involved in salt tolerance, resulting in reduced ROS accumulation and decreased water loss rate by stomatal closure. Taken together, BplncSIR1 serves as the regulator of BpNAC2 to induce its expression in response to salt stress, and activated BpNAC2 accelerates plant growth and improves salt tolerance. Therefore, BplncSIR1 might be a candidate gene for molecular breeding to cultivate plants with both a high growth rate and improved salt tolerance.

Seasonal dynamics and punctuated carbon sink reduction suggest photosynthetic capacity of boreal silver birch is reduced by the accumulation of hexose.

The 'assimilates inhibition hypothesis' posits that accumulation of nonstructural carbohydrates (NSCs) in leaves reduces leaf net photosynthetic rate, thus internally regulating photosynthesis. Experimental work provides equivocal support mostly under controlled conditions without identifying a particular NSC as involved in the regulation. We combined 3-yr in situ leaf gas exchange observations (natural dynamics) in the upper crown of mature Betula pendula simultaneously with measurements of concentrations of sucrose, hexoses (glucose and fructose), and starch, and similar measurements during several one-day shoot girdling (perturbation dynamics). Leaf water potential and water and nitrogen content were measured to account for their possible contribution to photosynthesis regulation. Leaf photosynthetic capacity (A/Ci) was temporally negatively correlated with NSC accumulation under both natural and perturbation states. For developed leaves, leaf hexose concentration explained A/Ci variation better than environmental variables (temperature history and daylength); the opposite was observed for developing leaves. The weaker correlations between NSCs and A/Ci in developing leaves may reflect their strong internal sink strength for carbohydrates. By contrast, the strong decline in photosynthetic capacity with NSCs accumulation in mature leaves, observed most clearly with hexose, and even more tightly with its constituents, provides support for the role of assimilates in regulating photosynthesis under natural conditions.

Adjustment of storage capacity for non-structural carbohydrates in response to limited water availability in two temperate woody species.

Reserves of non-structural carbohydrates (NSC) stored in living cells are essential for drought tolerance of trees. However, little is known about the phenotypic plasticity of living storage compartments (SC) and their interactions with NSC reserves under changing water availability. Here, we examined adjustments of SC and NSC reserves in stems and roots of seedlings of two temperate tree species, Acer negundo L. and Betula pendula Roth., cultivated under different substrate water availability. We found that relative contents of soluble NSC, starch and total NSC increased with decreasing water availability in stems of both species, and similar tendencies were also observed in roots of A. negundo. In the roots of B. pendula, soluble NSC contents decreased along with the decreasing water availability, possibly due to phloem decoupling or NSC translocation to shoots. Despite the contrast in organ responses, NSC contents (namely starch) positively correlated with proportions of total organ SC. Individual types of SC showed markedly distinct plasticity upon decreasing water availability, suggesting that water availability changes the partitioning of organ storage capacity. We found an increasing contribution of parenchyma-rich bark to the total organ NSC storage capacity under decreasing water availability. However, xylem SC showed substantially greater plasticity than those in bark. Axial storage cells, namely living fibers in A. negundo, responded more sensitively to decreasing water availability than radial parenchyma. Our results demonstrate that drought-induced changes in carbon balance affect the organ storage capacity provided by living cells, whose proportions are sensitively coordinated along with changing NSC reserves.

How tree species have modified the potentially toxic elements distributed in the developed soil-plant system in a post-fire site in highly industrialized region.

The biogeochemical cycles of trace elements are changed by fire as a result of the mineralization of organic matter. Monitoring the accumulation of trace elements in both the environment and the tree biomass during the post-fire (PF) forest ecosystem regeneration process is important for tree species selection for reforestation in ecosystems under anthropogenic pressure. We analyzed the soil concentrations of different groups of potentially toxic elements (PTEs), including beneficial (Al), toxic (Cd, Cr, Pb), and microelements (Cu, Mn, Ni, Zn), and their bioaccumulation in the tree species (Pinus sylvestris, Betula pendula, Alnus glutinosa) biomass introduced after a fire in a forest weakened by long-term emissions of industrial pollutants. The results indicated no direct threat from the PTEs tested at the PF site. The tree species introduced 30 years ago may have modified the biogeochemical cycles of the PTEs through different strategies of bioaccumulation in the belowground and aboveground biomass. Alder had relatively high Al concentrations in the roots and a low translocation factor (TF). Pine and birch had lower Al concentrations in the roots and higher TFs. Foliage concentrations and the TF of Cd increased from alder to pine to birch. However, the highest concentration and bioaccumulation factor of Cd was found in the alder roots. The concentrations of Cr in the foliage and the Cr TFs in the studied species increased from pine to birch to alder. Higher concentrations of Cu and Ni were found in the foliage of birch and alder than of pine. Among the species, birch also had the highest Pb and Zn concentrations in the roots and foliage. We found that different tree species had different patterns of PTE phytostabilization and ways they incorporated these elements into the biological cycle, and these patterns were not dependent on fire disturbance. This suggests that similar patterns might also occur in more polluted soils. Therefore, species-dependent bioaccumulation patterns could also be used to design phytostabilization and remediation treatments for polluted sites under industrial pressure.

Genome-wide identification of the TIFY family reveals JAZ subfamily function in response to hormone treatment in Betula platyphylla.

BACKGROUND: The TIFY family is a plant-specific gene family and plays an important role in plant growth and development. But few reports have been reported on the phylogenetic analysis and gene expression profiling of TIFY family genes in birch (Betula platyphylla). RESULTS: In this study, we characterized TIFY family and identified 12 TIFY genes and using phylogeny and chromosome mapping analysis in birch. TIFY family members were divided into JAZ, ZML, PPD and TIFY subfamilies. Phylogenetic analysis revealed that 12 TIFY genes were clustered into six evolutionary branches. The chromosome distribution showed that 12 TIFY genes were unevenly distributed on 5 chromosomes. Some TIFY family members were derived from gene duplication in birch. We found that six JAZ genes from JAZ subfamily played essential roles in response to Methyl jasmonate (MeJA), the JAZ genes were correlated with COI1 under MeJA. Co-expression and GO enrichment analysis further revealed that JAZ genes were related to hormone. JAZ proteins involved in the ABA and SA pathways. Subcellular localization experiments confirmed that the JAZ proteins were localized in the nucleus. Yeast two-hybrid assay showed that the JAZ proteins may form homologous or heterodimers to regulate hormones. CONCLUSION: Our results provided novel insights into biological function of TIFY family and JAZ subfamily in birch. It provides the theoretical reference for in-depth analysis of plant hormone and molecular breeding design for resistance.

Effects of air humidity and soil moisture on secondary metabolites in the leaves and roots of Betula pendula of different competitive status.

Plant secondary metabolites (PSMs) defend plants against abiotic stresses, including those caused by climate change and against biotic stresses, such as herbivory and competition. There is a trade-off between allocating available carbon to growth and defence in stressful environments. However, our knowledge about trade-off is limited, especially when abiotic and biotic stresses co-occur. We aimed to understand the combined effect of increasing precipitation and humidity, the tree's competitive status, and canopy position on leaf secondary metabolites (LSMs) and fine root secondary metabolites (RSMs) in Betula pendula. We sampled 8-year-old B. pendula trees growing in the free air humidity manipulation (FAHM) experimental site, where treatments included elevated relative air humidity and elevated soil moisture. A high-performance liquid chromatography-quadrupole-time of flight mass spectrometer (HPLC-qTOF-MS) was used to analyse secondary metabolites. Our results showed accumulation of LSM depends on the canopy position and competitive status. Flavonoids (FLA), dihydroxybenzoic acids (HBA), jasmonates (JA) and terpene glucosides (TG) were higher in the upper canopy, and FLA, monoaryl compounds (MAR) and sesquiterpenoids (ST) were higher in dominant trees. The FAHM treatments had a more distinct effect on RSM than on LSM. The RSMs were lower in elevated air humidity and soil moisture conditions than in control conditions. The RSM content depended on the competitive status and was higher in suppressed trees. Our study suggests that young B. pendula will allocate similar amounts of carbon to constitutive chemical leaf defence, but a lower amount to root defence (per fine root biomass) under higher humidity.

Genome-Wide Identification and Characterization of WRKY Transcription Factors in Betula platyphylla Suk. and Their Responses to Abiotic Stresses.

The WRKY transcription factor (TF) family is one the largest plant-specific transcription factor families. It has been proven to play significant roles in multiple plant biological processes, especially stress response. Although many WRKY TFs have been identified in various plant species, WRKYs in white birch (Betula platyphylla Suk.) remain to be studied. Here, we identified a total of 68 BpWRKYs, which could be classified into four main groups. The basic physiochemical properties of these TFs were analyzed using bioinformatics tools, including molecular weight, isoelectric point, chromosome location, and predicted subcellular localization. Most BpWRKYs were predicted to be located in the nucleus. Synteny analysis found 17 syntenic gene pairs among BpWRKYs and 52 syntenic gene pairs between BpWRKYs and AtWRKYs. The cis-acting elements in the promoters of BpWRKYs could be enriched in multiple plant biological processes, including stress response, hormone response, growth and development, and binding sites. Tissue-specific expression analysis using qRT-PCR showed that most BpWRKYs exhibited highest expression levels in the root. After ABA, salt (NaCl), or cold treatment, different BpWRKYs showed different expression patterns at different treatment times. Furthermore, the results of the Y2H assay proved the interaction between BpWRKY17 and a cold-responsive TF, BpCBF7. By transient expression assay, BpWRKY17 and BpWRKY67 were localized in the nucleus, consistent with the previous prediction. Our study hopes to shed light for research on WRKY TFs and plant stress response.

Comparison of Bioactive Secondary Metabolites and Cytotoxicity of Extracts from Inonotus obliquus Isolates from Different Host Species.

Inonotus obliquus, a wood-decaying mushroom, has been used as a health-promoting supplement and nutraceutical for centuries. It is a source of bioactive compounds accumulated in both the conks (pseudosclerotia/sclerotia) and the biomass obtained in vitro. This study aimed to qualitatively and quantitatively analyze the bioelements and selected metabolites produced in mycelial cultures obtained from different host species. The mycochemical potential of mycelial cultures isolated from pseudosclerotia grown in Betula pendula, Alnus glutinosa, and Carpinus betulus was compared. Parent cultures were obtained in two types of medium (malt extract agar substrates without and with birch wood). Experimental cultures were developed in 2 L bioreactors for 10 days. The content of bioelements was determined using FAAS and FAES methods. Organic compounds were estimated using the RP-HPLC-DAD method. The cytotoxicity of the extracts was evaluated in human keratinocytes HaCaT, human skin fibroblasts BJ, human liver cancer HepG2, human melanoma A375, and mouse melanoma B16-F10. The extracts showed the presence of bioelements: sodium, potassium, magnesium, calcium, zinc, manganese, iron, and copper; phenolic acids: p-hydroxybenzoic, caffeic, p-coumaric, and protocatechuic; sterols: lanosterol, ergosterol, ergosterol peroxide; triterpene compounds: betulin, betulinic acid, inotodiol; indole compounds: L-tryptophan, tryptamine, 5-methyltryptamine, melatonin. The content of bioactive substances in the biomass was dependent on both the origin of the host species of the fungus isolate and the type of culture medium. Based on the results of this study, mycelial cultures can be proposed as a potential source of bioactive compounds and are promising naturally derived cytotoxic agents.

The Structural Flexibility of PR-10 Food Allergens.

PR-10 proteins constitute a major cause of food allergic reactions. Birch-pollen-related food allergies are triggered by the immunologic cross-reactivity of IgE antibodies with structurally homologous PR-10 proteins that are present in birch pollen and various food sources. While the three-dimensional structures of PR-10 food allergens have been characterized in detail, only a few experimental studies have addressed the structural flexibility of these proteins. In this study, we analyze the millisecond-timescale structural flexibility of thirteen PR-10 proteins from prevalent plant food sources by NMR relaxation-dispersion spectroscopy, in a comparative manner. We show that all the allergens in this study have inherently flexible protein backbones in solution, yet the extent of the structural flexibility appears to be strikingly protein-specific (but not food-source-specific). Above-average flexibility is present in the two short helices, α1 and α2, which form a V-shaped support for the long C-terminal helix α3, and shape the internal ligand-binding cavity, which is characteristic for PR-10 proteins. An in-depth analysis of the NMR relaxation-dispersion data for the PR-10 allergen from peanut reveals the presence of at least two subglobal conformational transitions on the millisecond timescale, which may be related to the release of bound low-molecular-weight ligands from the internal cavity.

Building a Robust Chromatin Immunoprecipitation Method with Substantially Improved Efficiency.

Chromatin immunoprecipitation (ChIP) is the gold-standard method for detection of interactions between proteins and chromatin and is a powerful tool for identification of epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting in inefficient ChIP enrichment and an active demand for a highly efficient ChIP protocol. In this study, using birch (Betula platyphylla) and Arabidopsis (Arabidopsis thaliana) as the research materials, we identified five factors closely associated with ChIP efficiency, including crosslinking, concentration of chromatin using centrifugal filters, use of a different immunoprecipitation buffer, rescue of DNA with proteinase K, and use of Suc to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, we developed a robust ChIP protocol that achieved a 14-fold improvement in ChIP enrichment for birch and a >6-fold improvement for Arabidopsis compared to the standard ChIP method. As this ChIP method works well in both birch and Arabidopsis, it should also be suitable for other woody and herbaceous species. In addition, this ChIP method enables detection of low-abundance transcription factor-DNA interactions and may extend the application of ChIP in the plant kingdom.

Transcriptome analysis of North American sweet birch (Betula lenta) revealed a higher expression of genes involved in the biosynthesis of secondary metabolites than European silver birch (B. pendula).

The North American Betula lenta L. (sweet birch) has been used for medicinal reasons for centuries by native Americans. Although sophisticated technologies have rapidly been developed, a large information gap has been observed regarding genetic regulators of medicinally important compounds in sweet birch. Very little is known on the different genes involved in secondary metabolic biosynthesis in sweet birch. To gain a deeper insight into genetic factors, we performed a transcriptome analysis of each three biological samples from different independent trees of sweet and European silver birch (B. pendula Roth). This allowed us to precisely quantify the transcripts of about 24,000 expressed genes including 29 prominent candidate genes putatively involved in the biosynthesis of secondary metabolites like terpenoids, and aromatic benzoic acids. A total number of 597 genes were differentially expressed between B. lenta and B. pendula, while 264 and 210 genes showed upregulation in the bark and leaf of B. lenta, respectively. Moreover, we identified 39 transcriptional regulatory elements, involved in secondary metabolite biosynthesis, upregulated in B. lenta. Our study demonstrated the potential of RNA sequencing to identify candidate genes interacting in secondary metabolite biosynthesis in sweet birch. The candidate genes identified in this study could be subjected to genetic engineering to functionally characterize them in sweet birch. This knowledge can be beneficial to the increase of therapeutically important compounds.

Application of amendments for the phytoremediation of a former mine technosol by endemic pioneer species: alder and birch seedlings.

Metal(loid) pollution of soils has important negative effects on the environment and human health. For the rehabilitation of these soils, some eco-innovative strategies, such as phytoremediation, could be chosen. This practice could establish a plant cover to reduce the toxicity of the pollutants and stabilize the soil, preventing soil erosion and water leaching; this technique is called phytoremediation. For this, plants need to be tolerant to the pollutants present; thus, phytoremediation can have better outcomes if endemic species of the polluted area are used. Finally, to further improve phytoremediation success, amendments can be applied to ameliorate soil conditions. Different amendments can be used, such as biochar, a good metal(loid) immobilizer, compost, a nutrient-rich product and iron sulfate, an efficient arsenic immobilizer. These amendments can either be applied alone or combined for further positive effects. In this context, a mesocosm experiment was performed to study the effects of three amendments, biochar, compost and iron sulfate, applied alone or combined to a former mine technosol, on the soil properties and the phytoremediation potential of two endemic species, Alnus sp. and Betula sp. Results showed that the different amendments reduced soil acidity and decreased metal(loid) mobility, thus improving plant growth. Both species were able to grow on the amended technosols, but alder seedlings had a much higher growth compared to birch seedlings. Finally, the combination of compost with biochar and/or iron sulfate and the establishment of endemic alder plants could be a solution to rehabilitate a former mine technosol.

Functional identification of BpMYB21 and BpMYB61 transcription factors responding to MeJA and SA in birch triterpenoid synthesis.

BACKGROUND: Triterpenoids from birch (Betula platyphylla Suk.) exert antitumor and anti-HIV activities. Due to the complexity of plant secondary metabolic pathways, triterpene compounds in plants is not always determined by a single gene; they may be controlled by polygene quantitative traits. Secondary metabolism related to terpenoids involves tissue specificity and localisation of key biosynthetic enzymes. Terpene synthesis is influenced by light, hormones and other signals, as well as upstream transcription factor regulation. RESULTS: Anchor Herein, we identified and characterised two birch MYB transcription factors (TFs) that regulate triterpenoid biosynthesis. BpMYB21 and BpMYB61 are R2R3 TFs that positively and negatively regulate responses to methyl-jasmonate (MeJA) and salicyclic acid (SA), respectively. Expression of BpMYB21 and BpMYB61 was elevated in leaves and stems more than roots during July/August in Harbin, China. BpMYB21 expression was increased by abscisic acid (ABA), MeJA, SA and gibberellins (GAs). BpMYB61 expression in leaves and BpMYB21 expression in stems was reduced by ABA, MeJA and SA, while GAs, ethylene, and injury increased BpMYB61 expression. BpMYB21 was localised in nuclei, while BpMYB61 was detected in cell membranes and nuclei. Promoters for both BpMYB21 (1302 bp) and BpMYB61 (850 bp) were active. BpMYB21 and BpMYB61 were ligated into pYES3, introduced into AnchorINVScl (yeast strain without exogenous genes), INVScl-pYES2-SSAnchorAnchor (transgenic yeast strain harbouring the SS gene from birch), and INVScl-pYES2-SE (transgenic yeast strain harbouring the SE gene from birch), and the squalene content was highest in AnchorINVScl-pYES-MYB21-SS (transgenic yeast strain harbouring SS and MYB21 genes) and INVScl-pYES3-MYB61 (transgenic yeast strain harbouring the MYB61 gene). In BpMYB21 transgenic birch key triterpenoid synthesis genes were up-regulated, and in BpMYB61 transgenic birch AnchorFPS (farnesyl pyrophosphate synthase) and SS (squalene synthase) were up-regulated, but HMGR (3-hydroxy-3-methylglutaryl coenzyme a reductase), BPWAnchor (lupeol synthase), SE (squalene epoxidase) and BPY (b-amyrin synthase) were down-regulated. Both BpMYB21 and BpMYB61 specifically activate SE and BPX (cycloartenol synthase synthesis) promoters. CONCLUSIONS: These findings support further functional characterisation of R2R3-MYB genes, and illuminate the regulatory role of BpMYB21 and BpMYB61 in the synthesis of birch triterpenoids.

Direct Comparison of Leaf Plasmodesma Structure and Function in Relation to Phloem-Loading Type.

The export of photosynthetically produced sugars from leaves depends on plasmodesmatal transport of sugar molecules from mesophyll to phloem. Traditionally, the density of plasmodesmata (PD) along this phloem-loading pathway has been used as a defining feature of different phloem-loading types, with species proposed to have either many or few PD between the phloem and surrounding cells of the leaf. However, quantitative determination of PD density has rarely been performed. Moreover, the structure of PD has not been considered, even though it could impact permeability, and functional data are only available for very few species. Here, a comparison of PD density, structure, and function using data from transmission electron microscopy and live-cell microscopy was conducted for all relevant cell-cell interfaces in leaves of nine species. These species represent the three principal phloem-loading types currently discussed in literature. Results show that relative PD density among the different cell-cell interfaces in one species, but not absolute PD density, is indicative of phloem-loading type. PD density data of single interfaces, even combined with PD diameter and length data, did not correlate with the intercellular diffusion capacity measured by the fluorescence loss in photobleaching method. This means that PD substructure not visible on standard transmission electron micrographs may have a strong influence on permeability. Furthermore, the results support a proposed passive symplasmic loading mechanism in the tree species horse chestnut (Aesculus hippocastanum), white birch (Betula pubescens), orchard apple (Malus domestica), and gray poplar (Populus x canescens) as functional cell coupling and PD structure differed from active symplasmic and apoplasmic phloem-loading species.

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.

Loss of GLK1 transcription factor function reveals new insights in chlorophyll biosynthesis and chloroplast development.

Birch (Betula platyphylla × B. pendula) is an important tree for landscaping due to its attractive white bark and straight trunk. In this study, we characterized a T-DNA yellow-green leaf mutant, yl. We identified six insertion sites (ISs) in the mutant by genome resequencing and found a 40-kb deletion containing BpGLK1 around IS2 on chromosome 2. Complementation experiments with the yl mutant and repression of BpGLK1 in wild-type plants confirmed that BpGLK1 was responsible for the mutated phenotype. Physiological and ultrastructural analyses showed that the leaves of the yl mutant and BpGLK1-repression lines had decreased chlorophyll content and defective chloroplast development compared to the wild-type. Furthermore, the loss function of BpGLK1 also affected photosynthesis in leaves. Transcriptomics, proteomics, and ChIP-PCR analysis revealed that BpGLK1 directly interacted with the promoter of genes related to antenna proteins, chlorophyll biosynthesis, and photosystem subunit synthesis, and regulated their expression. Overall, our research not only provides new insights into the mechanism of chloroplast development and chlorophyll biosynthesis regulated by BpGLK1, but also provides new transgenic birch varieties with various levels of yellowing leaves by repressing BpGLK1 expression.

BplMYB46 from Betula platyphylla Can Form Homodimers and Heterodimers and Is Involved in Salt and Osmotic Stresses.

MYB proteins play important roles in the regulation of plant growth, development, and stress responses. Overexpression of BplMYB46 from Betula platyphylla improved plant salt and osmotic tolerances. In the present study, the interaction of eight avian myeloblastosis viral oncogene homolog (MYB) transcription factors with BplMYB46 was investigated using the yeast two-hybrid system, which showed that BplMYB46 could form homodimers and heterodimers with BplMYB6, BplMYB8, BplMYB11, BplMYB12, and BplMYB13. Relative beta-glucuronidase activity and chromatin immunoprecipitation assays showed that the interaction between BplMYB46 and the five MYBs increased the binding of BplMYB46 to the MYBCORE motif. A subcellular localization study showed that these MYBs were all located in the nucleus. Real-time fluorescence quantitative PCR results indicated that the expressions of BplMYB46 and the five MYB genes could be induced by salt and osmotic stress, and the BplMYB46 and BplMYB13 exhibited the most similar expression patterns. BplMYB46 and BplMYB13 co-overexpression in tobacco using transient transformation technology improved tobacco's tolerance to salt and osmotic stresses compared with overexpressing BplMYB13 or BplMYB46 alone. Taken together, these results demonstrated that BplMYB46 could interact with five other MYBs to form heterodimers that activate the transcription of target genes via an enhanced binding ability to the MYBCORE motif to mediate reactive oxygen species scavenging in response to salt and osmotic stresses.

Cashew Tree Pollen: An Unknown Source of IgE-Reactive Molecules.

Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.