Sugar metabolism and 14-3-3 protein genes expression induced by arbuscular mycorrhizal fungi and phosphorus addition to response drought stress in Populus cathayana.

Sugar, as a nutrient exchange substance between arbuscular mycorrhizal (AM) fungi and host plants, plays an important role in the abiotic stress response of mycorrhizal plants. This experiment aimed to study the effects of AM fungi and phosphorus (P) addition on the sugar metabolism and 14-3-3 gene expression of Populus cathayana under drought stress. The results showed that drought affects the process of sugar metabolism by increasing the activities of amylase and invertase, resulting in the decrease of starch content in leaves and roots and the accumulation of soluble sugars (including reducing sugar and sucrose) in roots. Under drought stress, the activity or content of sucrose synthetase, sucrose phosphate synthase, acid invertase, alkaline invertase, reducing sugar, soluble sugar, sucrose, and starch in the root showed the best mycorrhizal effect at the 100 mg P level. The expression levels of the 14-3-3 genes (PcGRF10 and PcGRF11) were significantly increased by mycorrhizal induction under drought stress. These levels were positively correlated with SS, SPS, sucrose, and starch phosphorylase in leaves, as well as with almost all sugar metabolism indicators in roots. However, they were negatively correlated with starch content in both leaves and roots. Sugar metabolism and 14-3-3 protein gene expression were induced by AM fungi and P addition in response to drought stress. The 14-3-3 genes induced by AM fungi may be involved in participating in osmotic regulation during drought stress. This study provides a new idea for the mechanism of sugar metabolism of mycorrhizal plants in arid regions.

Expression of the Populus Orthologues of AtYY1, YIN and YANG Activates the Floral Identity Genes AGAMOUS and SEPALLATA3 Accelerating Floral Transition in Arabidopsis thaliana.

YIN YANG 1 (YY1) encodes a dual-function transcription factor, evolutionary conserved between the animal and plant kingdom. In Arabidopsis thaliana, AtYY1 is a negative regulator of ABA responses and floral transition. Here, we report the cloning and functional characterization of the two AtYY1 paralogs, YIN and YANG (also named PtYY1a and PtYY1b) from Populus (Populus trichocarpa). Although the duplication of YY1 occurred early during the evolution of the Salicaceae, YIN and YANG are highly conserved in the willow tree family. In the majority of Populus tissues, YIN was more strongly expressed than YANG. Subcellular analysis showed that YIN-GFP and YANG-GFP are mainly localized in the nuclei of Arabidopsis. Stable and constitutive expression of YIN and YANG resulted in curled leaves and accelerated floral transition of Arabidopsis plants, which was accompanied by high expression of the floral identity genes AGAMOUS (AG) and SEPELLATA3 (SEP3) known to promote leaf curling and early flowering. Furthermore, the expression of YIN and YANG had similar effects as AtYY1 overexpression to seed germination and root growth in Arabidopsis. Our results suggest that YIN and YANG are functional orthologues of the dual-function transcription factor AtYY1 with similar roles in plant development conserved between Arabidopsis and Populus.

Genome-wide analysis of R2R3-MYB transcription factors reveals their differential responses to drought stress and ABA treatment in desert poplar (Populus euphratica).

The R2R3-MYB transcription factors are widely involved in the regulation of plant growth, biotic and abiotic stress responses. Meanwhile, seed germination, which is stimulated by internal and external environments, is a critical stage in the plant life cycle. However, the identification, characterization, and expression profiling of the Populus euphratica R2R3-MYB family in drought response during seed germination have been unknown. Our study attempted to identify and characterize the R2R3-MYB genes in P. euphratica (PeR2R3-MYBs) and explore how R2R3-MYBs trigger the drought and abscisic acid (ABA) response mechanism in its seedlings. Based on the analysis of comparative genomics, 174 PeR2R3-MYBs were identified and expanded driven by whole genome duplication or segment duplication events. The analysis of Ka/Ks ratios showed that, in contrast to most PeR2R3-MYBs, the other PeR2R3-MYBs were subjected to positive selection in P. euphratica. Further, the expression data of PeR2R3-MYBs under drought stress and ABA treatment, together with available functional data for Arabidopsis thaliana MYB genes, supported the hypothesis that PeR2R3-MYBs involved in response to drought are dependent or independent on ABA signaling pathway during seed germination, especially PeR2R3-MYBs with MYB binding sites (MBS) cis-element and/or tandem duplication. This study is the first report on the genome-wide analysis of PeR2R3-MYBs, as well as the other two Salicaceae species. The duplication events and differential expressions of PeR2R3-MYBs play important roles in enhancing the adaptation to drought desert environment. Our results provide a reference for prospective functional studies of R2R3-MYBs of poplars and lay the foundation for new breeding strategies to improve the drought tolerance of P. euphratica.

Poplar leaf abscission through induced chlorophyll breakdown by Mg-dechelatase.

Chlorophyll breakdown is observed during senescence. The first step in chlorophyll breakdown is the removal of central Mg by Mg-dechelatase. This reaction is the rate-limiting step in the chlorophyll breakdown pathway. We evaluated the effect of induced chlorophyll breakdown on abscission through the removal of Mg by Mg-dechelatase. Poplar transformants carrying the dexamethasone-inducible Mg-dechelatase gene were prepared using the Arabidopsis Stay-Green1 cDNA. When leaves were treated with dexamethasone, chlorophyll was degraded, photosynthetic capacity was reduced, and an abscission zone was formed, resulting in leaf abscission. In addition, ethylene, which plays an important role during senescence, was produced in this process. Thus, chlorophyll breakdown induces the phenotype in the same way as commonly observed during leaf senescence. This study suggests a physiological role of chlorophyll breakdown in the leaf abscission of deciduous trees. Furthermore, this study shows that the dexamethasone-inducible gene expression system is an available option for deciduous tree studies.

Phosphate (Pi) stress-responsive transcription factors PdeWRKY6 and PdeWRKY65 regulate the expression of PdePHT1;9 to modulate tissue Pi concentration in poplar.

Phosphorus (P) is an important nutrient for plants. Here, we identify a WRKY transcription factor (TF) in poplar (Populus deltoides × Populus euramericana) (PdeWRKY65) that modulates tissue phosphate (Pi) concentrations in poplar. PdeWRKY65 overexpression (OE) transgenic lines showed reduced shoot Pi concentrations under both low and normal Pi availabilities, while PdeWRKY65 reduced expression (RE) lines showed the opposite phenotype. A gene encoding a Pi transporter (PHT), PdePHT1;9, was identified as the direct downstream target of PdeWRKY65 by RNA sequencing (RNA-Seq). The negative regulation of PdePHT1;9 expression by PdeWRKY65 was confirmed by DNA-protein interaction assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), co-expression of the promoters of PdePHT1;9 and PdeWRKY65 in tobacco (Nicotiana benthamiana) leaves, and chromatin immunoprecipitation-quantitative PCR. A second WRKY TF, PdeWRKY6, was subsequently identified and confirmed to positively regulate the expression of PdePHT1;9 by DNA-protein interaction assays. PdePHT1;9 and PdeWRKY6 OE and RE poplar transgenic lines were used to confirm their positive regulation of shoot Pi concentrations, under both normal and low Pi availabilities. No interaction between PdeWRKY6 and PdeWRKY65 was observed at the DNA or protein levels. Collectively, these data suggest that the low Pi-responsive TFs PdeWRKY6 and PdeWRKY65 independently regulate the expression of PHT1;9 to modulate tissue Pi concentrations in poplar.

QTL mapping of drought-related traits in the hybrids of Populus deltoides 'Danhong'×Populus simonii 'Tongliao1'.

BACKGROUND: Poplar trees provide a large amount of wood material, but many parts of the world are arid or semi-arid areas because of insufficient annual precipitation, which seriously affects the growth of poplar trees. Populus simonii 'Tongliao1' shows strong tolerance to stress environments, and Populus deltoides 'Danhong' shows a stronger growth rate in a suitable environment. To identify drought tolerance-related QTLs and genes, an F1 population derived from the cross between the 'Danhong' and 'Tongliao 1' Populus was assessed under drought stress. RESULTS: We measured drought-related traits such as the relative height growth, relative diameter growth, leaf senescence number, specific leaf area, and leaf relative water content in the population under control and drought environments. The results showed that drought stress reduced the plant height relative growth, ground diameter relative growth, specific leaf area and leaf relative water content and increased the number of leaf drops. A total of 208 QTLs were identified by QTL mapping analysis, and they consisted of 92, 63 and 53 QTLs under control, drought stress treatment and drought index conditions, respectively. A molecular identification marker for drought tolerance, np2841, which was associated with a QTL (qDLRWC-LG10-1) for relative leaf water content, was initially developed. We mined 187 candidate genes for QTL regions of five traits under a drought environment. The reference genome annotation for Populus trichocarpa and a homologous gene analysis of Arabidopsis thaliana identified two candidate genes, Potri.003G171300 and Potri.012G123900, with significant functions in response to drought stress. We identified five key regulatory genes (Potri.006G273500, Potri.007G111500, Potri.007G111600, Potri.007G111700, and Potri.007G111800) related to drought tolerance through the poplar coexpression network. CONCLUSION: In this study, our results indicate that the QTLs can effectively enhance the drought tolerance of poplar. It is a step closer towards unravelling the genetic basis of poplar drought tolerance-related traits, and to providing validated candidate genes and molecular markers for future genetic improvement.

Two high hierarchical regulators, PuMYB40 and PuWRKY75, control the low phosphorus driven adventitious root formation in Populus ussuriensis.

Adventitious rooting is an essential biological process in the vegetative propagation of economically important horticultural and forest tree species. It enables utilization of the elite genotypes in breeding programmes and production. Promotion of adventitious root (AR) formation has been associated with starvation of inorganic phosphate and some factors involved in low phosphorus (LP) signalling. However, the regulatory mechanism underlying LP-mediated AR formation remains largely elusive. We established an efficient experimental system that guaranteed AR formation through short-term LP treatment in Populus ussuriensis. We then generated a time-course RNA-seq data set to recognize key regulatory genes and regulatory cascades positively regulating AR formation through data analysis and gene network construction, which were followed by experimental validation and characterization. We constructed a multilayered hierarchical gene regulatory network, from which PuMYB40, a typical R2R3-type MYB transcription factor (TF), and its interactive partner, PuWRKY75, as well as their direct targets, PuLRP1 and PuERF003, were identified to function upstream of the known adventitious rooting genes. These regulatory genes were functionally characterized and proved their roles in promoting AR formation in P. ussuriensis. In conclusion, our study unveiled a new hierarchical regulatory network that promoted AR formation in P. ussuriensis, which was activated by short-term LP stimulus and primarily governed by PuMYB40 and PuWRKY75.

Comprehensive analysis of the BES1 gene family and its expression under abiotic stress and hormone treatment in Populus trichocarpa.

The BRI1 EMS SUPPRESSOR 1/BRASSINAZOLE RESISTANT 1 (BES1/BZR1) plays a vital role in plant growth and development and stress responses, but there are few studies on poplar BES1 genes. In this study, we identified 14 BES1 genes in the Populus trichocarpa genome and analyzed the expression under hormone treatment and abiotic stress. The PtrBES1 genes were classified into seven subgroups (I-VII) through phylogenetic analysis. All the paralogous gene pairs were shown to be subjected to expansion by segment duplication and purification selection during the PtrBES1 family evolution. Promoter cis-element analysis showed that the PtrBES1 promoter contains stress related cis-elements including ABRE-motif, MBS and TC-rich elements. Quantitative real time reverse transcription PCR (RT-qPCR) analysis showed that the PtrBES1 genes were upregulated upon NaCl, Polyethylene glycol 6000 (PEG6000) stress as well as the major stress hormone abscisic acid (ABA) treatment. Under the three treatments, PtrBES1-7 showed high expression levels in leaves and roots. Physiological experiments showed that the overexpression PtrBES1-7 line could enhance tolerance to drought stress in P. trichocarpa by improving the ability to scavenge ROS (reactive oxygen species). This is specifically reflected in the fact that the overexpression line contains less ROS (O2- and H2O2) and more antioxidant enzymes (1.42 times SOD and 1.5 times POD) than the control line. The preliminary results of this study provided a solid basis for the future functional studies of the BES1 gene family in P. trichocarpa.

Excellent Anti-lung Cancer Activity of Populus nigra and Phylogenetic Analysis.

Lung cancer has the highest incidence rate among malignant tumors all over the world, and it is also the leading cause of death. In this present research, we aimed to evaluate the anti-cancer activity of the Populus nigra extract against the lung cancer and study the genome evolution of the Populus nigra. Firstly, the inhibitory activity of the Populus nigra extract on the NCI-H292 lung cancer cell viability was determined with Cell Counting Kit-8 (CCK-8) assay. The trans-well assay was conducted and the influence of the Populus nigra extract on the NCI-H292 lung cancer cell migration and invasion ability was determined. In addition to this, the chloroplast (cp) genome of Populus nigra was sequenced with high-throughput Illumina pair-end sequencing, which was a classical useful model for genome evolution assessment. The CCK-8 and trans-well assay indicated the Populus nigra extract exhibited excellent inhibitory activity on the NCI-H292 lung cancer cell viability, migration and invasion ability. The circular cp genome of the Populus nigra was 156,354 bp in size, including a large single-copy (LSC) region of 84,528 bp and a small single-copy (SC) region of 16,564 bp, which were separated by two inverted repeat (IR) regions (38,612 bp each). A total of 132 genes were predicted, including 8 ribosomal RNAs (rRNAs), 37 transfer RNAs (tRNAs), and 90 protein-coding genes (PCGs). Furthermore, phylogenetic analysis revealed that Populus nigra has the closest relationship with Populus alba var. pyramidalis. In addition to Populus alba var. pyramidalis, Populus adenopoda and Populus tomentosa are also has closely relationship with Populus nigra.

How Cysteine Protease Gene PtCP5 Affects Seed Germination by Mobilizing Storage Proteins in Populus trichocarpa.

In higher plants, seed storage proteins are deposited in protein storage vacuoles (PSVs) and degraded by protease, especially cysteine proteases, as a source of nitrogen for seed germination. In this study, a cathepsin B-like cysteine protease PtCP5, which is important for seed germination and pollen development, was first cloned in Populus trichocarpa. The GUS staining of the ProPtCP5-GUS reporter line showed that PtCP5 is expressed in the roots, stems, leaves, flowers, siliques and seeds of Arabidopsis. We reveal that PtCP5 is present in plasma membrane and co-localizes with the plasma membrane marker REM1.3. Both seed germination and early seedling development are slower in OX-PtCP5 transgenic Arabidopsis when compared with the wild-type. Further analysis revealed that, when stained with toluidine blue, the observed storage protein accumulation was lower in OX-PtCP5 than in the wild-type. Our results also show that the number of abnormal pollen grains is higher and the germination rate of pollen is lower in OX-PtCP5 than in the wild-type. These results indicate that PtCP5 is an important factor in mobilizing storage proteins and that the proper expression of PtCP5 is necessary for both pollen and seed maturation and germination. This study sheds further light on the biological functions of cysteine proteases and provides further reference for seed development research on woody plants.

Anti-osteosarcoma Biological Activity Evaluation and Complete Chloroplast Genome Sequencing of Populus yunnanensis.

Recently, the Populus yunnanensis extract has drawn the attention of most researchers, because of their anti-cancer activity. In this present research, the anti-cancer activity of the Populus yunnanensis extract was measured with Cell Counting Kit-8 (CCK-8) detection kit on the cancer cells. Then, the inhibitory activity of the Populus yunnanensis extract on the migration and invasion ability of the cancer cells was also determined in this present research with trans-well assay. Subsequently, to reveal the evolutionary genome evolution evaluation of the Populus yunnanensis and other Populus species, the high-throughput Illumina pair-end sequencing was performed and the chloroplast (cp) genome of Populus yunnanensis was determined, and the phylogenetic analysis was finished as wells. The results of the CCK-8 assay indicated that the Populus yunnanensis extract showed inhibitory effect on the cancer cell viability. Besides, the migration and invasion ability of the cancer cell was also reduced by the Populus yunnanensis extract. The complete chloroplast genome sequence results revealed that the Populus yunnanensis has a 156,505 bp circular cp genome. The phylogenetic analysis further revealed that the Populus yunnanensis has closely relationship with Populus simonii.

Anti-oral Cancer Biological Activity Evaluation and Chloroplast Genome Analyses of Populus euphratica.

In this research, the anti-cancer activity of the Populus euphratica extract was evaluated with Cell Counting Kit-8 (CCK-8) assay. The inhibitory activity of the Populus euphratica extract on the activation levels of VEGF signaling pathway in the cancer cells was measured with real time RT-PCR. Next, the high-throughput Illumina pair-end sequencing was performed to detect the chloroplast (cp) genome of Populus euphratica for genome evolution assessment. The CCK-8 results indicated that the extract of Populus euphratica exhibited the significantly suppression effect on the viability of the cancer cells, and the data of the real time RT-PCR showed the activation levels of VEGF signaling pathway in the cancer cells was also reduced obviously by the Populus euphratica extract. The circular cp genome of the Populus euphratica is 157,806 bp, encoding 131 genes, containing 8 Ribosomal RNA genes (rRNAs), 37 Transfer RNA genes (tRNAs) and 86 Protein coding genes (PCGs). And the results of the phylogenetic analysis indicated that the Populus euphratica. Furthermore, phylogenetic analysis revealed that Populus euphratica has the closest relationship with Populus pruinosa. In addition to Populus pruinosa, Populus ilicifolia also has closely relationship with Populus euphratica. These three species could be clustered on the same clade.

[Bioinformatics analysis, prokaryotic expression and biological activity of HSP70 domain-containing proteins derived from pollen of Populus deltoides].

Objective To study the bioinformatics characteristics of HSP70 domain proteins derived from pollen of Populus deltoides (P. deltoides), optimize the prokaryotic expression methods, and identify the biological activity of these proteins. Methods Physicochemical characteristics of three kinds of HSP70 domain-containing proteins were analyzed by bioinformatics software. The T/B cell epitopes of these proteins were predicted by Immune Epitope Database and Analysis Resource (IEDB). According to the amino acid sequence provided by Uniprot database, their nucleotide sequences were synthesized and cloned into pET28a(+) plasmid for prokaryotic expression. Protein expression was detected by SDS-PAGE, then the expressed products were purified by nickel column and identified by Western blotting. The protein concentration was measured by protein quantitative kit. Then the three proteins were used as antigens to prepare mouse asthma models, and the concentration of serum total IgE antibody was determined by ELISA. Results The bioinformatics analysis showed that the relative molecular mass (Mr) of B9N9W6, B9GX02 and A0A2K2AYN8 were 71 900, 94 600 and 75 200, respectively. The 13 T-cell epitopes and 14 B-cell epitopes were identified in the three proteins which had high hydrophilia and stability. SDS-PAGE analysis revealed that the genes encoding the three proteins were expressed with three specific bands of approximately Mr 72 000, 95 000 and 75 000, respectively. Western blotting showed the specific bands at the corresponding sites. ELISA showed that the IgE level in the extract group and the A0A2K2AYN8 group were higher than that in the PBS group. Compared with the A0A2K2AYN8 group, the IgE concentration in the B9N9W6 group and B9GX02 group increased significantly. Conclusion The soluble HSP70 domain-containing proteins A0A2K2AYN8, B9GX02 and B9N9W6 derived from pollen of P. deltoides can be expressed as well as purified, and have the biological activity of producing IgE antibodies.

Identification and Characterization of the APX Gene Family and Its Expression Pattern under Phytohormone Treatment and Abiotic Stress in Populus trichocarpa.

Ascorbate peroxidase (APX) is a member of class I of the heme-containing peroxidase family. The enzyme plays important roles in scavenging reactive oxygen species for protection against oxidative damage and maintaining normal plant growth and development, as well as in biotic stress responses. In this study, we identified 11 APX genes in the Populus trichocarpa genome using bioinformatic methods. Phylogenetic analysis revealed that the PtrAPX proteins were classifiable into three clades and the members of each clade shared similar gene structures and motifs. The PtrAPX genes were distributed on six chromosomes and four segmental-duplicated gene pairs were identified. Promoter cis-elements analysis showed that the majority of PtrAPX genes contained a variety of phytohormone- and abiotic stress-related cis-elements. Tissue-specific expression profiles indicated that the PtrAPX genes primarily function in roots and leaves. Real-time quantitative PCR (RT-qPCR) analysis indicated that PtrAPX transcription was induced in response to drought, salinity, high ammonium concentration, and exogenous abscisic acid treatment. These results provide important information on the phylogenetic relationships and functions of the APX gene family in P. trichocarpa.

Wood properties and transcriptional responses of poplar hybrids in mixed cropping with the nitrogen-fixing species Robinia pseudoacacia.

Cultivation of fast-growing tree species is often confined to marginal land. Mixed cropping with Robinia pseudoacacia, a legume tree species that forms a symbiosis with N2-fixing bacteria, has been proposed to be a measure to improve soil fertility and to achieve beneficial effects on the cocultivated tree species. The goal of our study was to examine the influence of a Robinia mixture on wood chemistry, anatomy and gene expression in poplar. We hypothesized that annual wood growth is stimulated in species mixtures due to the positive effects of Robinia on nitrogen availability and complementary resource use. Alternatively, we hypothesized that competition, especially for water, has negative effects on the wood growth of poplar. We used two commercial biomass clones, Hybride 275 (H275, Populus trichocarpa × Populus maximowiczii) and Max1 (Populus nigra × P. maximowiczii), which were planted at two locations with contrasting soil fertility in monoculture or mixed plots with Robinia to investigate the annual wood increment, wood nitrogen and δ13C, wood anatomy (length, cell wall thickness, lumina and frequencies of fibers and vessels) and transcriptional profiles in the developing xylem of 4-year-old stems. In a mixture with Robinia, the annual stem increment was reduced, nitrogen in wood was enhanced, δ13C in wood was decreased, vessel and fiber frequencies were increased and fiber lengths and fiber lumina were decreased. Transcriptional profiles showed stronger differences between the genotypes and sites than between mono and mixed cultivation. The transcriptional abundances of only one gene (the putative nitrate transporter, NRT1.2) and one gene ontology term ('immune system process') were significantly enriched in wood-forming tissues in response to the mixture, irrespective of the poplar genotype and growth location. Weighted gene coexpression network analyses extracted gene modules that linked wood nitrogen mainly to vessel traits and wood δ13C with fiber traits. Collectively, molecular and anatomical changes in poplar wood suggest beneficial effects on the water and N supply in response to the mixture with Robinia. These alterations may render poplars less drought-susceptible. However, these benefits are accompanied by a reduced wood increment, emphasizing that other critical factors, presumably light competition or allelopathic effects, overrule a potential growth stimulation.

Molecular and immunochemical characterization of Pop n 2: A new allergen of Populus nigra pollen.

BACKGROUND: Pollen is one of the most common allergens that cause respiratory allergies worldwide. Pollen grains from poplars have been reported as important sources of pollinosis in many countries. OBJECTIVE: The aim of the present study was to determine the molecular and immunochemical characterization of Pop n 2, a novel allergen of Populus nigra (P nigra) pollen extract. METHODS: In this study, the pollen extract of P nigra was analysed by SDS-PAGE, and the allergenic profile was determined by IgE immunoblotting and specific ELISA using the sera of twenty allergic patients. The coding sequence of Pop n 2 was cloned and expressed in the Escherichia coli BL21 (DE3) using plasmid the pET-21b (+). Finally, the expressed recombinant Pop n 2 was purified by affinity chromatography. RESULTS: Pop n 2 belongs to the profilin family with a molecular weight of approximately 14 kDa. Pop n 2 is the most IgE-reactive protein (about 65%) in the P nigra pollen extract. The cDNA sequencing results indicated an open reading frame 396 bp that encodes 131 amino acid residues. The results of ELISA and Immunoblotting assays showed that recombinant Pop n 2 could react with the IgE antibody in patients' sera, like its natural counterpart. CONCLUSION: Our data revealed that Pop n 2 is a significant allergen in the P nigra pollen extract. Moreover, we observed that the recombinant Pop n 2 produced by the pET-21b (+) vector in the E colisystem acts as its natural counterpart.

Construction of a breeding parent population of Populus tomentosa based on SSR genetic distance analysis.

Parent selection is the core of hybrid breeding. The breeding strategy involving the parental identification of superior open-pollinated progeny of Populous tomentosa germplasm resources can significantly improve the efficiency of parental matching. However, due to some factors such as loose powdering time and pollen competitiveness, the offspring derived from open-pollination families which do not undergo completely random mating. Although hybrid combinations based on the male identification method have a high combining ability, this method cannot easily cover the mating combinations of all male and female specimens in the germplasm bank. In addition, the performance of superior plants in open-pollinated families also affects the selection result. If the trait performance value is higher than the population average, then the special combining ability of the reconstructed hybrid combination may be overestimated. Obtaining a solution to the above problems is of great significance for improving the efficiency and accuracy of selecting hybrid parents of P. tomentosa. In this study, 24 pairs of SSR (Simple Sequence Repeats) molecular markers were used to analyze the genetic differentiation of P. tomentosa germplasm resources. The results showed that the genetic variation of the P. tomentosa population was derived from individuals within the provenance, indicating that high genetic diversity is preserved in provenances. The correlation analysis showed that there was a significant positive correlation between the special combining ability of planting height and diameter at breast height (dbh) of the 34 full-sib progeny population and the genetic distance between the parents. Then, the genetic distance between 18 female plants with high fertility and 68 male plants with large pollen quantity was analyzed using this correlation. Fifteen female parents and 12 male parents were screened out, and 52 hybrid combinations with high specific combining ability for growth traits were predicted. Furthermore, for the male parent identification of superior individual plants, we constructed the breeding parent population including 10 female parents and 5 male parents, generating 14 hybrid combinations with potentially high combining ability. The results of the hybridization test showed that the specific combining ability of plant height and dbh was significantly higher than the controlled pollination. Moreover, genetic distance and paternal identification can be used to rapidly and efficiently construct hybrid parent combinations and breeding parent populations.

Efficient evaluation of a gene containment system for poplar through early flowering induction.

KEY MESSAGE: The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.

Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance.

Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (gs ) can be ascribed to changes in guard cells functioning in amphistomateous leaves. gs was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where gs was at the highest. In contrast, genes encoding H+ -ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca2+ -vacuolar antiporters, K+ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.

Biotechnological mechanism for improving plant remobilization of phosphorus during leaf senescence.

Phosphorus enrichment of aquatic ecosystems through diffuse source pollution is an ongoing issue worldwide. A potential solution lies in the use of fast-growing, multipurpose feedstocks, such as trees, to limit the flow of phosphorus into riparian areas through luxury consumption. However, the perennial nature of trees and their use of leaves as storage organs for excess phosphorus may reduce the effectiveness of contaminant removal during periods of leaf abscission. In an attempt to improve phosphorus remobilization during autumnal senescence, transgenic hybrid poplar P39 (Populus alba × Populus grandidentata) and Arabidopsis thaliana harbouring a constitutively expressed low-affinity potato phosphate transporter (35S::StPht1-1) were generated using Agrobacterium-mediated transformation. For both species, the highest expressing 35S::StPht1-1 lines were grown alongside wild-type plants and subjected to increasing phosphate applications. StPht1-1 expression in A. thaliana led to a reduction in biomass when grown under high-phosphate conditions and had no effect on phosphate remobilization during senescence. In contrast, StPht1-1 constitutive expression in P39 resulted in increased leaf phosphate content in the highest expressing transgenic line and minimal to no effect on P resorption efficiency. Surprisingly, sulphate resorption showed the greatest improvement in all three transgenic poplar lines, displaying a 31%-37% increase in resorption efficiency. These results highlight the complexity of nutrient resorption mechanisms in plants.