Genome-Wide Identification and Analysis of the EIN3/EIL Transcription Factor Gene Family in Doubled Haploid (DH) Poplar.

Ethylene (ET) is an important phytohormone that regulates plant growth, development and stress responses. The ethylene-insensitive3/ethylene-insensitive3-like (EIN3/EIL) transcription factor family, as a key regulator of the ET signal transduction pathway, plays an important role in regulating the expression of ET-responsive genes. Although studies of EIN3/EIL family members have been completed in many species, their role in doubled haploid (DH) poplar derived from another culture of diploid Populus simonii × P. nigra (donor tree, DT) remains ambiguous. In this study, a total of seven EIN3/EIL gene family members in the DH poplar genome were identified. Basic physical and chemical property analyses of these genes were performed, and these proteins were predicted to be localized to the nucleus. According to the phylogenetic relationship, EIN3/EIL genes were divided into two groups, and the genes in the same group had a similar gene structure and conserved motifs. The expression patterns of EIN3/EIL genes in the apical buds of different DH poplar plants were analyzed based on transcriptome data. At the same time, the expression patterns of PsnEIL1, PsnEIN3, PsnEIL4 and PsnEIL5 genes in different tissues of different DH plants were detected via RT-qPCR, including the apical buds, young leaves, functional leaves, xylem, cambium and roots. The findings presented above indicate notable variations in the expression levels of PsnEIL genes across various tissues of distinct DH plants. Finally, the PsnEIL1 gene was overexpressed in DT, and the transgenic plants showed a dwarf phenotype, indicating that the PsnEIL1 gene was involved in regulating the growth and development of poplar. In this study, the EIN3/EIL gene family of DH poplar was analyzed and functionally characterized, which provides a theoretical basis for the future exploration of the EIN3/EIL gene function.

Provenance and family variations in early growth of Manchurian walnut (Juglans mandshurica Maxim.) and selection of superior families.

This study, conducted in China in November 2020, was aimed at exploring the variations in growth traits among different provenances and families as well as to select elite materials of Juglans mandshurica. Thus, seeds of 44 families from six J. mandshurica provenances in Heilongjiang and Jilin provinces were sown in the nursery and then transplanted out in the field. At the age of 5 years, seven growth traits were assessed, and a comprehensive analysis was conducted as well as selection of provenance and families. Analysis of variance revealed statistically significant (P < 0.01) differences in seven growth traits among different provenances and families, thereby justifying the pursuit of further breeding endeavors. The genetic coefficient of variation (GCV) for all traits ranged from 5.44% (branch angle) to 21.95% (tree height) whereas the phenotypic coefficient of variation (PCV) ranged from 13.74% (tapering) to 38.50% (branch number per node), indicating considerable variability across the traits. Further, all the studied traits except stem straightness degree, branch angle and branch number per node, showed high heritability (Tree height, ground diameter, mean crown width and tapering, over 0.7±0.073), indicating that the variation in these traits is primarily driven by genetic factors. Correlation analysis revealed a strong positive correlation (r > 0.8) between tree height and ground diameter (r = 0.86), tree height and mean crown width (r = 0.82), and ground diameter and mean crown width (r = 0.83). This suggests that these relationships can be employed for more precise predictions of the growth and morphological characteristics of trees, as well as the selection of superior materials. There was a strong correlation between temperature factors and growth traits. Based on the comprehensive scores in this study, Sanchazi was selected as elite provenance. Using the top-percentile selection criteria, SC1, SC8, DJC15, and DQ18 were selected as elite families. These selected families exhibit genetic gains of over 10% in tree height, ground diameter and mean crown width, signifying their significant potential in forestry for enhancing timber production and reducing production cycles, thereby contributing to sustainable forest management. In this study, the growth traits of J. mandshurica were found to exhibit stable variation, and there were correlations between these traits. The selected elite provenance and families of J. mandshurica showed faster growth, which is advantageous for the subsequent breeding and promotion of improved J. mandshurica varieties.

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.

Transcriptome-wide m6A methylation in natural yellow leaf of Catalpa fargesii.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA, and involved in various biological processes in plants. However, the distribution features and functions of mRNA m6A methylation have been poorly explored in woody perennial plants. In this study, a new natural variety with yellow-green leaves, named Maiyuanjinqiu, was screened from the seedlings of Catalpa fargesii. Based on the preliminary experiment, the m6A methylation levels in the leaves of Maiyuanjinqiu were significantly higher than those in C. fargesii. Furthermore, a parallel analysis of m6A-seq and RNA-seq was carried out in different leaf color sectors. The result showed that m6A modification were mostly identified around the 3'-untranslated regions (3'-UTR), which was slightly negatively correlated with the mRNA abundance. KEGG and GO analyses showed that m6A methylation genes were associated with photosynthesis, pigments biosynthesis and metabolism, oxidation-reduction and response to stress, etc. The overall increase of m6A methylation levels in yellow-green leaves might be associated with the decreased the expression of RNA demethylase gene CfALKBH5. The silencing of CfALKBH5 caused a chlorotic phenotype and increased m6A methylation level, which further confirmed our hypothesis. Our results suggested that mRNA m6A methylation could be considered as a vital epigenomic mark and contribute to the natural variations in plants.

Genome-Wide Identification and Expression Profiles of C-Repeat Binding Factor Transcription Factors in Betula platyphylla under Abiotic Stress.

CBF (C-repeat binding factor) transcription factor subfamily belongs to AP2/ERF (Apetala 2/ethylene-responsive factor) transcription factor family, known for playing a vital role in plant abiotic stress response. Although some CBF transcription factors have been identified in several species, such as Arabidopsis, tobacco, tomato and poplar, research of CBF focus mainly on model plant Arabidopsis and have not been reported in Betula platyphylla yet. In this study, a total of 20 BpCBF subfamily members were identified. The conserved domains, physicochemical properties, exon-intron gene structure and the structure of conserved protein motifs of BpCBFs were analyzed via bioinformatic tools. The collinearity analysis of CBF genes was performed between Betula platyphylla and Arabidopsis thaliana, Betula platyphylla, and Populus trichocarpa. The cis-acting elements in the promoter region of BpCBFs were identified, which were mainly environmental stress-related and hormone-related element components. In this case, the expression patterns of the 20 BpCBFs upon ABA or salt treatment were investigated. Most of these transcription factors were responsive to ABA or salt stress in different plant tissues. The up-regulation trend upon cold treatment of the six cold-responsive genes validated by qRT-PCR was consistent with the result of RNA-seq. BpCBF7 showed transcription activating activity. This study sheds light on the responses of BpCBFs to abiotic stress and provides a reference for further study of CBF transcription factors in woody plants.

Genome-wide analysis of HSF family and overexpression of PsnHSF21 confers salt tolerance in Populus simonii × P. nigra.

Heat shock transcription factor (HSF) is an important TF that performs a dominant role in plant growth, development, and stress response network. In this study, we identified a total of 30 HSF members from poplar, which are unevenly distributed on 17 chromosomes. The poplar HSF family can be divided into three subfamilies, and the members of the same subfamily share relatively conserved domains and motifs. HSF family members are acidic and hydrophilic proteins that are located in the nucleus and mainly carry out gene expansion through segmental replication. In addition, they have rich collinearity across plant species. Based on RNA-Seq analysis, we explored the expression pattern of PtHSFs under salt stress. Subsequently, we cloned the significantly upregulated PtHSF21 gene and transformed it into Populus simonii × P. nigra. Under salt stress, the transgenic poplar overexpressing PtHSF21 had a better growth state and higher reactive oxygen scavenging ability. A yeast one-hybrid experiment indicated PtHSF21 could improve salt tolerance by specifically binding to the anti-stress cis-acting element HSE. This study comprehensively profiled the fundamental information of poplar HSF family members and their responses to salt stress and specifically verified the biological function of PtHSF21, which provides clues for understanding the molecular mechanism of poplar HSF members in response to salt stress.

Populus D-type cyclin gene PsnCYCD1;1 accelerates cell division and participates in secondary growth of vascular bundles.

Plant growth and development rely heavily on cyclins, which comprise an important class of cell division regulators. D-type cyclins (CYCDs) are responsible for the rate-limiting step of G1 cells. In the plant kingdom, despite the importance of CYCDs in herbaceous plants, there is little knowledge of these proteins in perennial woody plants. Here, the gene of a nucleus-localized cyclin, PsnCYCD1;1, was cloned from Populus simonii × P. nigra. PsnCYCD1;1 was highly expressed in tissues with active cell division, especially the leaf buds, and could be induced by sucrose and phytohormones. Moreover, overexpression of PsnCYCD1;1 in poplar could stimulate cell division, resulting in the generation of small cells and causing severe morphological changes in the vascular bundles, resulting in 'S'-shaped tortuous stems and curled leaves. Furthermore, transcriptomic analysis revealed that endogenous genes related to cell division and vascular cambium development were significantly up-regulated in the transgenic plants. In addition, using yeast two-hybrid and bimolecular fluorescence complementation assays PsnCDKA1, PsnICK3, and PsnICK5 were identified as proteins interacting with PsnCYCD1;1. Our study demonstrates that PsnCYCD1;1 accelerates plant cell division and participates in secondary growth of vascular bundles in poplar.

PeCLH2 Gene Positively Regulate Salt Tolerance in Transgenic Populus alba × Populus glandulosa.

Salt is an important environmental stress factor, which seriously affects the growth, development and distribution of plants. Chlorophyllase plays an important role in stress response. Nevertheless, little is known about the physiological and molecular mechanism of chlorophyll (Chlase, CLH) genes in plants. We cloned PeCLH2 from Populus euphratica and found that PeCLH2 was differentially expressed in different tissues, especially in the leaves of P. euphratica. To further study the role of PeCLH2 in salt tolerance, PeCLH2 overexpression and RNA interference transgenic lines were established in Populus alba × Populus glandulosa and used for salt stress treatment and physiologic indexes studies. Overexpressing lines significantly improved tolerance to salt treatment and reduced reactive oxygen species production. RNA interference lines showed the opposite. Transcriptome analysis was performed on leaves of control and transgenic lines under normal growth conditions and salt stress to predict genes regulated during salt stress. This provides a basis for elucidating the molecular regulation mechanism of PeCLH2 in response to salt stress and improving the tolerance of poplar under salt stress.

Chromosome-level genome assembly of a triploid poplar Populus alba 'Berolinensis'.

Many recent studies have provided significant insights into polyploid breeding, but limited research has been carried out on trees. The genomic information needed to understand growth and response to abiotic stress in polyploidy trees is largely unknown, but has become critical due to the threats to forests imposed by climate change. Populus alba 'Berolinensis,' also known "Yinzhong poplar," is a triploid poplar from northeast China. This hybrid triploid poplar is widely used as a landscape ornamental and in urban forestry owing to its adaptation to adverse environments and faster growth than its parental diploid. It is an artificially synthesized male allotriploid hybrid, with three haploid genomes of P. alba 'Berolinensis' originating from different poplar species, so it is attractive for studying polyploidy genomic mechanisms in heterosis. In this study, we focused on the allelic genomic interactions in P. alba 'Berolinensis,' and generated a high-quality chromosome-level genome assembly consisting of 19 allelic chromosomes. Its three haploid chromosome sets are polymorphic with an average of 25.73 nucleotide polymorphism sites per kilobase. We found that some stress-related genes such as RD22 and LEA7 exhibited sequence differences between different haploid genomes. The genome assembly has been deposited in our polyploid genome online analysis website TreeGenomes (https://www.treegenomes.com). These polyploid genome-related resources will provide a critical foundation for the molecular breeding of P. alba 'Berolinensis' and help us uncover the allopolyploidization effects of heterosis and abiotic stress resistance and traits of polyploidy species in the future.

Identification and Analysis of the CBF Gene Family in Three Species of Acer under Cold Stress.

The C-Repeat Binding Factor (CBF) gene family has been identified and characterized in multiple plant species, and it plays a crucial role in responding to low temperatures. Presently, only a few studies on tree species demonstrate the mechanisms and potential functions of CBFs associated with cold resistance, while our study is a novel report on the multi-aspect differences of CBFs among three tree species, compared to previous studies. In this study, genome-wide identification and analysis of the CBF gene family in Acer truncatum, Acer pseudosieboldianum, and Acer yangbiense were performed. The results revealed that 16 CBF genes (five ApseCBFs, four AcyanCBFs, and seven AtruCBFs) were unevenly distributed across the chromosomes, and most CBF genes were mapped on chromosome 2 (Chr2) and chromosome 11 (Chr11). The analysis of phylogenetic relationships, gene structure, and conserved motif showed that 16 CBF genes could be clustered into three subgroups; they all contained Motif 1 and Motif 5, and most of them only spanned one exon. The cis-acting elements analysis showed that some CBF genes might be involved in hormone and abiotic stress responsiveness. In addition, CBF genes exhibited tissue expression specificity. High expressions of ApseCBF1, ApseCBF3, AtruCBF1, AtruCBF4, AtruCBF6, AtruCBF7, and ApseCBF3, ApseCBF4, ApseCBF5 were detected on exposure to low temperature for 3 h and 24 h. Low expressions of AtruCBF2, AtruCBF6, AtruCBF7 were detected under cold stress for 24 h, and AtruCBF3 and AtruCBF5 were always down-regulated under cold conditions. Taken together, comprehensive analysis will enhance our understanding of the potential functions of the CBF genes on cold resistance, thereby providing a reference for the introduction of Acer species in our country.

Genome-Wide Identification of the ERF Transcription Factor Family for Structure Analysis, Expression Pattern, and Response to Drought Stress in Populus alba × Populus glandulosa.

The Ethylene Responsive Factor (ERF) transcription factor family is important for regulating plant growth and stress responses. Although the expression patterns of ERF family members have been reported in many plant species, their role in Populus alba × Populus glandulosa, an important model plant for forest research, remains unclear. In this study, we identified 209 PagERF transcription factors by analyzing the P. alba × P. glandulosa genome. We analyzed their amino acid sequences, molecular weight, theoretical pI (Isoelectric point), instability index, aliphatic index, grand average of hydropathicity, and subcellular localization. Most PagERFs were predicted to localize in the nucleus, with only a few PagERFs localized in the cytoplasm and nucleus. Phylogenetic analysis divided the PagERF proteins into ten groups, Class I to X, with those belonging to the same group containing similar motifs. Cis-acting elements associated with plant hormones, abiotic stress responses, and MYB binding sites were analyzed in the promoters of PagERF genes. We used transcriptome data to analyze the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa, including axillary buds, young leaves, functional leaves, cambium, xylem, and roots, and the results indicated that PagERF genes are expressed in all tissues of P. alba × P. glandulosa, especially in roots. Quantitative verification results were consistent with transcriptome data. When P. alba × P. glandulosa seedlings were treated with 6% polyethylene glycol 6000 (PEG6000), the results of RT-qRCR showed that nine PagERF genes responded to drought stress in various tissues. This study provides a new perspective on the roles of PagERF family members in regulating plant growth and development, and responses to stress in P. alba × P. glandulosa. Our study provides a theoretical basis for ERF family research in the future.

Genome-Wide Identification and Expression Analysis of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Catalpabungei.

As a plant-specific transcription factor, the SPL gene family plays a critical role in plant growth and development. Although the SPL gene family has been identified in diverse plant species, there have been no genome-wide identification or systematic study reports on the SPL gene family in Catalpa bungei. In this study, we identified 19 putative SPL gene family members in the C. bungei genome. According to the phylogenetic relationship, they can be divided into eight groups, and the genes in the same group have a similar gene structure and conserved motifs. Synteny analysis showed that fragment duplication played an important role in the expansion of the CbuSPL gene family. At the same time, CbuSPL genes have cis-acting elements and functions related to light response, hormone response, growth and development, and stress response. Tissue-specific expression and developmental period-specific expression analysis showed that CbuSPL may be involved in flowering initiation and development, flowering transition, and leaf development. In addition, the ectopic expression of CbuSPL4 in Arabidopsis confirmed that it can promote early flowering and induce the expression of related flowering genes. These systematic research results will lay a foundation for further study on the functional analysis of SPL genes in C. bungei.

Genome-wide identification and expression analysis of the MADS-box gene family during female and male flower development in Juglans mandshurica.

The MADS-box gene family plays a crucial role in multiple developmental processes of plants, especially in floral organ specification and the regulation of fruit development and ripening. Juglans mandshurica is a precious fruit material whose quality and yield are determined by floral organ development. The molecular mechanism of J. mandshurica female and male flower development depending on MADS-box genes remains unclear. In our study, 67 JmMADS genes were identified and unevenly distributed on 15 of 16 J. mandshurica chromosomes. These genes were divided into two types [type I (Mα, Mγ, Mδ) and type II (MIKC)]. The gene structure and motif analyses showed that most genes belonging to the same type had similar gene structures and conserved motifs. The analysis of syntenic relationships showed that MADS-box genes in J. mandshurica, J. sigillata, and J. regia exhibited the highest homology and great collinearity. Analysis of cis-acting elements showed that JmMADS gene promoter regions contained light, stress and hormone response cis-acting elements. The gene expression patterns demonstrated that 30 and 26 JmMADS genes were specifically expressed in the female and male flowers, respectively. In addition, 12 selected genes common to J. mandshurica female and male flowers were significantly upregulated at the mature stage and were used to validate the reliability of the transcriptome data using quantitative real-time PCR. This comprehensive and systematic analysis of J. mandshurica MADS-box genes lays a foundation for future studies on MADS-box gene family functions.

Integrated metabolomic and transcriptomic analyses reveal different metabolite biosynthesis profiles of Juglans mandshurica in shade.

Light is not only a very important source of energy for the normal growth and development of plants, but also a regulator of many development and metabolic processes. The mechanism of plant growth and development under low light conditions is an important scientific question. With the promulgation of the law to stop natural forest cutting, understory regeneration is an important method for artificial forest afforestation. Here, the growth and physiological indexes of Juglans mandshurica, an important hardwood species in Northeast China, were measured under different shade treatments. In addition, transcriptome and metabolome were compared to analyze the molecular mechanism of shade tolerance in J. mandshurica. The results showed that the seedling height of the shade treatment group was significantly higher than that of the control group, and the 50% light (L50) treatment was the highest. Compared with the control group, the contents of gibberellin, abscisic acid, brassinolide, chlorophyll a, and chlorophyll b in all shade treatments were significantly higher. However, the net photosynthetic rate and water use efficiency decreased with increasing shade. Furthermore, the transcriptome identified thousands of differentially expressed genes in three samples. Using enrichment analysis, we found that most of the differentially expressed genes were enriched in photosynthesis, plant hormone signal transduction and chlorophyll synthesis pathways, and the expression levels of many genes encoding transcription factors were also changed. In addition, analysis of differentially accumulated metabolites showed that a total of 470 differential metabolites were identified, and flavonoids were the major differential metabolites of J. mandshurica under light stress. These results improved our understanding of the molecular mechanism and metabolite accumulation under light stress in J. mandshurica.

Comparative Transcriptomic and Metabolic Analyses Reveal the Coordinated Mechanisms in Pinus koraiensis under Different Light Stress Conditions.

Light is one of the most important environmental cues that affects plant development and regulates its behavior. Light stress directly inhibits physiological responses and plant tissue development and even induces mortality in plants. Korean pine (Pinus koraiensis) is an evergreen conifer species widely planted in northeast China that has important economic and ecological value. However, the effects of light stress on the growth and development of Korean pine are still unclear. In this study, the effects of different shading conditions on physiological indices, molecular mechanisms and metabolites of Korean pine were explored. The results showed that auxin, gibberellin and abscisic acid were significantly increased under all shading conditions compared with the control. The contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid also increased as the shading degree increased. Moreover, a total of 8556, 3751 and 6990 differentially expressed genes (DEGs) were found between the control and HS (heavy shade), control and LS (light shade), LS vs. HS, respectively. Notably, most DEGs were assigned to pathways of phytohormone signaling, photosynthesis, carotenoid and flavonoid biosynthesis under light stress. The transcription factors MYB-related, AP2-ERF and bHLH specifically increased expression during light stress. A total of 911 metabolites were identified, and 243 differentially accumulated metabolites (DAMs) were detected, among which flavonoid biosynthesis (naringenin chalcone, dihydrokaempferol and kaempferol) metabolites were significantly different under light stress. These results will provide a theoretical basis for the response of P. koraiensis to different light stresses.

The Manchurian Walnut Genome: Insights into Juglone and Lipid Biosynthesis.

BACKGROUND: Manchurian walnut (Juglans mandshurica Maxim.) is a tree with multiple industrial uses and medicinal properties in the Juglandaceae family (walnuts and hickories). J. mandshurica produces juglone, which is a toxic allelopathic agent and has potential utilization value. Furthermore, the seed of J. mandshurica is rich in various unsaturated fatty acids and has high nutritive value. FINDINGS: Here, we present a high-quality chromosome-scale reference genome assembly and annotation for J. mandshurica (n = 16) with a contig N50 of 21.4 Mb by combining PacBio high-fidelity reads with high-throughput chromosome conformation capture data. The assembled genome has an estimated sequence size of 548.7 Mb and consists of 657 contigs, 623 scaffolds, and 40,453 protein-coding genes. In total, 60.99% of the assembled genome consists of repetitive sequences. Sixteen super-scaffolds corresponding to the 16 chromosomes were assembled, with a scaffold N50 length of 33.7 Mb and a BUSCO complete gene percentage of 98.3%. J. mandshurica displays a close sequence relationship with Juglans cathayensis, with a divergence time of 13.8 million years ago. Combining the high-quality genome, transcriptome, and metabolomics data, we constructed a gene-to-metabolite network and identified 566 core and conserved differentially expressed genes, which may be involved in juglone biosynthesis. Five CYP450 genes were found that may contribute to juglone accumulation. NAC, bZip, NF-YA, and NF-YC are positively correlated with the juglone content. Some candidate regulators (e.g., FUS3, ABI3, LEC2, and WRI1 transcription factors) involved in the regulation of lipid biosynthesis were also identified. CONCLUSIONS: Our genomic data provide new insights into the evolution of the walnut genome and create a new platform for accelerating molecular breeding and improving the comprehensive utilization of these economically important tree species.

Transcriptome Analysis of Populus euphratica under Salt Treatment and PeERF1 Gene Enhances Salt Tolerance in Transgenic Populus alba × Populus glandulosa.

Populus euphratica is mainly distributed in desert environments with dry and hot climate in summer and cold in winter. Compared with other poplars, P. euphratica is more resistant to salt stress. It is critical to investigate the transcriptome and molecular basis of salt tolerance in order to uncover stress-related genes. In this study, salt-tolerant treatment of P. euphratica resulted in an increase in osmo-regulatory substances and recovery of antioxidant enzymes. To improve the mining efficiency of candidate genes, the analysis combining both the transcriptome WGCNA and the former GWAS results was selected, and a range of key regulatory factors with salt resistance were found. The PeERF1 gene was highly connected in the turquoise modules with significant differences in salt stress traits, and the expression levels were significantly different in each treatment. For further functional verification of PeERF1, we obtained stable overexpression and dominant suppression transgenic lines by transforming into Populus alba × Populusglandulosa. The growth and physiological characteristics of the PeERF1 overexpressed plants were better than that of the wild type under salt stress. Transcriptome analysis of leaves of transgenic lines and WT revealed that highly enriched GO terms in DEGs were associated with stress responses, including abiotic stimuli responses, chemical responses, and oxidative stress responses. The result is helpful for in-depth analysis of the salt tolerance mechanism of poplar. This work provides important genes for poplar breeding with salt tolerance.

Ectopic Expression of Poplar PsnCYCD1;1 Reduces Cell Size and Regulates Flower Organ Development in Nicotiana tabacum.

The D-type cyclin (CYCD) gene, as the rate-limiting enzyme in the G1 phase of cell cycle, plays a vital role in the process of plant growth and development. Early studies on plant cyclin mostly focused on herbs, such as Arabidopsis thaliana. The sustainable growth ability of woody plants is a unique characteristic in the study of plant cyclin. Here, the promoter of PsnCYCD1;1 was cloned from poplar by PCR and genetically transformed into tobacco. A strong GUS activity was observed in the areas with vigorous cell division, such as stem tips, lateral buds, and young leaves. The PsnCYCD1;1-GFP fusion expression vector was transformed into tobacco, and the green fluorescence signal was observed in the nucleus. Compared with the control plant, the transgenic tobacco showed significant changes in the flower organs, such as enlargement of sepals, petals, and fruits. Furthermore, the stems of transgenic plants were slightly curved at each stem node, the leaves were curled on the adaxial side, and the fruits were seriously aborted after artificial pollination. Microscopic observation showed that the epidermal cells of petals, leaves, and seed coats of transgenic plants became smaller. The transcriptional levels of endogenous genes, such as NtCYCDs, NtSTM, NtKNAT1, and NtASs, were upregulated by PsnCYCD1;1. Therefore, PsnCYCD1;1 gene played an important role in the regulation of flower organ and stem development, providing new understanding for the functional characterization of CYCD gene and new resources for improving the ornamental value of horticultural plants.

A Bivariate Mapping Model Identifies Major Covariation QTLs for Biomass Allocation Between Leaf and Stem Growth of Catalpa bungei.

Biomass allocation plays a critical role in plant morphological formation and phenotypic plasticity, which greatly impact plant adaptability and competitiveness. While empirical studies on plant biomass allocation have focused on molecular biology and ecology approaches, detailed insight into the genetic basis of biomass allocation between leaf and stem growth is still lacking. Herein, we constructed a bivariate mapping model to identify covariation QTLs governing carbon (C) allocation between the leaves and stem as well as the covariation of traits within and between organs in a full-sib mapping population of C. bungei. A total of 123 covQTLs were detected for 23 trait pairs, including six leaf traits (leaf length, width, area, perimeter, length/width ratio and petiole length) and five stem traits (height, diameter at breast height, wood density, stemwood volume and stemwood biomass). The candidate genes were further identified in tissue-specific gene expression data, which provided insights into the genetic architecture underlying C allocation for traits or organs. The key QTLs related to growth and biomass allocation, which included UVH1, CLPT2, GAD/SPL, COG1 and MTERF4, were characterised and verified via gene function annotation and expression profiling. The integration of a bivariate Quantitative trait locus mapping model and gene expression profiling will enable the elucidation of genetic architecture underlying biomass allocation and covariation growth, in turn providing a theoretical basis for forest molecular marker-assisted breeding with specific C allocation strategies for adaptation to heterogeneous environments.

Molecular Karyotyping on Populus simonii × P. nigra and the Derived Doubled Haploid.

The molecular karyotype could represent the basic genetic make-up in a cell nucleus of an organism or species. A doubled haploid (DH) is a genotype formed from the chromosome doubling of haploid cells. In the present study, molecular karyotype analysis of the poplar hybrid Populus simonii × P. nigra (P. xiaohei) and the derived doubled haploids was carried out with labeled telomeres, rDNA, and two newly repetitive sequences as probes by fluorescence in situ hybridization (FISH). The tandem repeats, pPC349_XHY and pPD284_XHY, with high-sequence homology were used, and the results showed that they presented the colocalized distribution signal in chromosomes. For P. xiaohei, pPD284_XHY produced hybridizations in chromosomes 1, 5, 8, and 9 in the hybrid. The combination of pPD284_XHY, 45S rDNA, and 5S rDNA distinctly distinguished six pairs of chromosomes, and the three pairs of chromosomes showed a significant difference in the hybridization between homologous chromosomes. The repeat probes used produced similar FISH hybridizations in the DH; nevertheless, pPD284_XHY generated an additional hybridization site in the telomere region of chromosome 14. Moreover, two pairs of chromosomes showed differential hybridization distributions between homologous chromosomes. Comparisons of the distinguished chromosomes between hybrid and DH poplar showed that three pairs of chromosomes in the DH presented hybridization patterns that varied from those of the hybrid. The No. 8 chromosome in DH and one of the homologous chromosomes in P. xiaohei shared highly similar FISH patterns, which suggested the possibility of intact or mostly partial transfer of the chromosome between the hybrid and DH. Our study will contribute to understanding the genetic mechanism of chromosomal variation in P. xiaohei and derived DH plants.