Molecular Mechanism of Different Rooting Capacity between Two Clones of Taxodium hybrid 'Zhongshanshan'.

The conifer Taxodium hybrid 'Zhongshanshan' (T. hybrid 'Zhongshanshan') is characterized by rapid growth, strong stress resistance, and high ornamental value and has significant potential for use in afforestation, landscaping, and wood production. The main method of propagating T. hybrid 'Zhongshanshan' is tender branch cutting, but the cutting rooting abilities of different T. hybrid 'Zhongshanshan' clones differ significantly. To explore the causes of rooting ability differences at a molecular level, we analyzed the transcriptome data of cutting base and root tissues of T. hybrid 'Zhongshanshan 149' with a rooting rate of less than 5% and T. hybrid 'Zhongshanshan 118' with rooting rate greater than 60%, at the developmental time points in this study. The results indicated that differentially expressed genes between the two clones were mainly associated with copper ion binding, peroxidase, and oxidoreductase activity, response to oxidative stress, phenylpropanoid and flavonoid biosynthesis, and plant hormone signal transduction, among others. The expression pattern of ThAP2 was different throughout the development of the adventitive roots of the two clone cuttings. Therefore, this gene was selected for further study. It was shown that ThAP2 was a nuclear-localized transcription factor and demonstrated a positive feedback effect on rooting in transgenic Nicotiana benthamiana cuttings. Thus, the results of this study explain the molecular mechanism of cutting rooting and provide candidate gene resources for developing genetic breeding strategies for optimizing superior clones of T. hybrid 'Zhongshanshan'.

Tree species selection for optimizing soil carbon storage: Insights from litter decomposition and bacterial community analysis in coastal ecosystems.

Coastal wetland ecosystems are critical sinks for atmospheric carbon dioxide, playing a vital role in global carbon cycling and climate regulation. The decomposition of leaf litter plays a crucial role in the formation and stability of soil organic carbon (SOC) in these environments. This study investigated the impact of leaf litter decomposition from five tree species (Populus deltoids, Ligustrum lucidum, Taxodium 'Zhongshanshan', Hibiscus hamabo, and Nerium oleander) on SOC dynamics, humus composition, and soil bacterial community structure in a tidal flat. Litterbags were used to monitor the mass loss and changes in litter chemical composition over 270 days. The results revealed significant differences in decomposition rates among the tree species, with Nerium oleander exhibiting the fastest decomposition and Populus deltoids the slowest. Surprisingly, initial litter chemistry did not correlate with decomposition rates; however, changes in lignin and hemicellulose content during decomposition were significantly related to mass loss. Despite its rapid decomposition, Nerium oleander litter resulted in the highest accumulation of SOC, total humus, and humin compared to the other species, challenging the conventional view that slower decomposition leads to greater SOC storage. The soil microbial community structure was significantly influenced by SOC, humus, and litter components, with distinct microbial assemblages associated with each tree species. A random forest model identified key bacterial taxa, predominantly Proteobacteria, as important predictors of SOC content, highlighting the role of bacterial diversity in regulating SOC dynamics. These findings underscore the importance of considering litter quality, decomposition dynamics, and bacterial community composition in strategies aimed at enhancing soil carbon sequestration. This study suggests that selecting tree species with rapidly decomposing litter, such as Nerium oleander, in coastal plantations can be an effective management tool for optimizing soil carbon storage, offering valuable insights for mitigating climate change impacts.

Development of an UV-Resistant Multilayer Film with Enhanced Compatibility between Carboxymethyl Cellulose and Polylactic Acid via Incorporation of Tannin and Ferric Chloride.

Carboxymethyl cellulose (CMC) and polylactic acid (PLA) are recognized for their environmental friendliness. By merging them into a composite film, packaging solutions can be designed with good performance. Nonetheless, the inherent interface disparity between CMC and PLA poses a challenge, and there may be layer separation issues. This study introduces a straightforward approach to mitigate this challenge by incorporating tannin acid and ferric chloride in the fabrication of the CMC-PLA. The interlayer compatibility was improved by the in situ formation of a cohesive interface. The resulting CMC/TA-PLA/Fe multilayer film, devoid of any layer separation, exhibits exceptional mechanical strength, with a tensile strength exceeding 70 MPa, a high contact angle of 105°, and superior thermal stability. Furthermore, the CMC/TA-PLA/Fe film demonstrates remarkable efficacy in blocking ultraviolet light, effectively minimizing the discoloration of various wood surfaces exposed to UV aging.

Extraction and In Vitro Skincare Effect Assessment of Polysaccharides Extract from the Roots of Abelmoschus manihot (L.).

Obtaining high-added value compounds from agricultural waste receives increasing attention, as it can both improve resource utilization efficiency and reduce waste generation. In this study, polysaccharides are extracted from the discarded roots of Abelmoschus manihot (L.) by the high-efficiency ultrasound-assisted extraction (UAE). The optimized condition was determined as solid-liquid ratio SL ratio = 1:20, temperature T = 30 °C and time T = 40 min, achieving an extraction yield of 13.41%. Composition analysis revealed that glucose (Glc, 44.65%), rhamnose (Rha, 26.30%), galacturonic acid (GalA, 12.50%) and galactose (Gal, 9.86%) are the major monosaccharides of the extract. The extract showed a low degree of esterification (DE) value of 40.95%, and its Fourier-transform infrared (FT-IR) spectrum exhibited several characteristic peaks of polysaccharides. Inspired by the wide cosmetic applications of polysaccharides, the skincare effect of the extract was evaluated via the moisture retention, total phenolic content (TPC) quantification, 2,2-Diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging activity, anti-hyaluronidase and anti-elastase activity experiments. The extract solutions demonstrated a 48 h moisture retention rate of 10.75%, which is superior to that of commercially available moisturizer hyaluronic acid (HA). Moreover, both the TPC value of 16.16 mg GAE/g (dw) and DPPH-free radical scavenging activity of 89.20% at the concentration of 2 mg/mL indicated the strong anti-oxidant properties of the extract. Furthermore, the anti-hyaluronidase activity and moderate anti-elastase activity were determined as 72.16% and 42.02%, respectively. In general, in vitro skincare effect experiments suggest moisturizing, anti-oxidant, anti-radical and anti-aging activities of the A. manihot root extract, indicating its potential applications in the cosmetic industry.

Physiological and transcriptional regulation in Taxodium hybrid 'Zhongshanshan' leaves in acclimation to prolonged partial submergence.

MAIN CONCLUSION: Taxodium 703 leaves activate fermentation, amino acids metabolism and ROS detoxification, and reduce TCA cycle and ABA biosynthesis in acclimation to prolonged partial submergence stress. Taxodium hybrid 'Zhongshanshan 703' (T. mucronatum × T. distichum; Taxodium 703) is a highly flooding-tolerant woody plant. To investigate the physiological and transcriptional regulatory mechanisms underlying its leaves in acclimation to long-term flooding, we exposed cuttings of Taxodium 703 to either non-flooding (control) or partial submergence for 2 months. The leaf tissues above (AL) and below (BL) flooding-water were separately harvested. Partial submergence decreased concentrations of chlorophyll (a + b) and dehydroascorbate (DHA) and lactate dehydrogenase (LDH) activity in AL, and reduced biomass, concentrations of succinic acid, fumaric acid and malic acid, and transcript levels of genes involved in tricarboxylic acid (TCA) cycle in BL. Under partial submergence, concentrations of starch, malondialdehyde and abscisic acid (ABA) decreased, and also mRNA levels of nine-cis-epoxycarotenoid dioxygenases that are involved in ABA biosynthesis in AL and BL of Taxodium 703. Partial submergence increased O2- content in AL, and improved concentrations of pyruvate and soluble sugars and activities of LDH and peroxidase in BL. In addition, partial submergence increased concentrations of ethanol, lactate, alanine, γ-aminobutyric acid, total amino acids and ascorbic acid (ASA), and ASA/DHA, activities of alcohol dehydrogenases (ADH) and ascorbate peroxidase, as well as transcript levels of ADH1A, ADH1B and genes involved in alanine biosynthesis and starch degradation in AL and BL of Taxodium 703. Overall, these results suggest that Taxodium 703 leaves activate fermentation, amino acids metabolism and reactive oxygen species detoxification, and maintain a steady supply of sugars, and reduce TCA cycle and ABA biosynthesis in acclimation to prolonged partial submergence stress.

An Integrated Transcriptome and Proteome Analysis Reveals Putative Regulators of Adventitious Root Formation in Taxodium 'Zhongshanshan'.

Adventitious root (AR) formation from cuttings is the primary manner for the commercial vegetative propagation of trees. Cuttings is also the main method for the vegetative reproduction of Taxodium 'Zhongshanshan', while knowledge of the molecular mechanisms regulating the processes is limited. Here, we used mRNA sequencing and an isobaric tag for relative and absolute quantitation-based quantitative proteomic (iTRAQ) analysis to measure changes in gene and protein expression levels during AR formation in Taxodium 'Zhongshanshan'. Three comparison groups were established to represent the three developmental stages in the AR formation process. At the transcript level, 4743 genes showed an expression difference in the comparison groups as detected by RNA sequencing. At the protein level, 4005 proteins differed in their relative abundance levels, as indicated by the quantitative proteomic analysis. A comparison of the transcriptome and proteome data revealed regulatory aspects of metabolism during AR formation and development. In summary, hormonal signal transduction is different at different developmental stages during AR formation. Other factors related to carbohydrate and energy metabolism and protein degradation and some transcription factor activity levels, were also correlated with AR formation. Studying the identified genes and proteins will provide further insights into the molecular mechanisms controlling AR formation.

High-density genetic map construction and quantitative trait loci identification for growth traits in (Taxodium distichum var. distichum × T. mucronatum) × T. mucronatum.

BACKGROUND: 'Zhongshanshan' is the general designation for the superior interspecific hybrid clones of Taxodium species, which is widely grown for economic and ecological purposes in southern China. Growth is the priority objective in 'Zhongshanshan' tree improvement. A high-density linkage map is vital to efficiently identify key quantitative trait loci (QTLs) that affect growth. RESULTS: In total, 403.16 Gb of data, containing 2016,336 paired-end reads, was obtained after preprocessing. The average sequencing depth was 28.49 in T. distichum var. distichum, 25.18 in T. mucronatum, and 11.12 in each progeny. In total, 524,662 high-quality SLAFs were detected, of which 249,619 were polymorphic, and 6166 of the polymorphic markers met the requirements for use in constructing a genetic map. The final map harbored 6156 SLAF markers on 11 linkage groups, and was 1137.86 cM in length, with an average distance of 0.18 cM between adjacent markers. Separate QTL analyses of traits in different years by CIM detected 7 QTLs. While combining multiple-year data, 13 QTLs were detected by ICIM. 5 QTLs were repeatedly detected by the two methods, and among them, 3 significant QTLs (q6-2, q4-2 and q2-1) were detected in at least two traits. Bioinformatic analysis discoveried a gene annotated as a leucine-rich repeat receptor-like kinase gene within q4-2. CONCLUSIONS: This map is the most saturated one constructed in a Taxodiaceae species to date, and would provide useful information for future comparative mapping, genome assembly, and marker-assisted selection.

Effects of cadmium stress on the antioxidant system and chlorophyll fluorescence characteristics of two Taxodium clones.

KEY MESSAGE: The T.118 and T.406 seedlings showed strong adaptability under Cd concentrations ≤ 50 µM. The mechanisms of photoprotection in T.118 and T.406 differed in high-Cd concentrations. To explore the physiological response characteristics of Taxodium hybrids to cadmium (Cd) stress and provide basis for screening of Cd-tolerant species, the hydroponic cultivation of T.118 and T.406 seedlings was conducted to demonstrate the effects of Cd stress on seedling growth, antioxidant system, and chlorophyll fluorescence parameters. After 35 days of Cd stress at a concentration ≤ 50 µM, the dry weight biomass of the two clones did not significantly differ from that of the control. T.406 exhibited a significant increase in POD activity compared to T.118 and maintained high SOD activity after exposure to high concentrations of Cd, whereas MDA levels showed little changes. Under low-Cd stress, chlorophyll content and fluorescence parameters remained stable, especially for T.406. Under high-Cd concentration stress, the above parameters were lower than the control, with a more significant decrease in T.118 than in T.406. The non-photochemical quenching coefficient (NPQ) of both clones increased with increasing Cd concentration. T.118 showed a greater increase than T.406, particularly under high-Cd concentration stress. The T.118 and T.406 seedlings adapted to low-Cd concentration stress by enhancing their antioxidant enzyme activity to maintain the balance of reactive oxygen metabolism and reduce cellular damage. The photochemical activity of mesophyll cells remained high to maintain photosynthetic capacity and normal seedling growth. T.406 showed stronger resistance to Cd than T.118. T.406 prevented photodamage by promoting the photochemical utilization of the excitation energy and maintaining a strong antioxidant stress ability. Enhancement of heat dissipation capability may be the main photoprotection mechanism of T.118.

Differential impacts of polyethylene microplastic and additives on soil nitrogen cycling: A deeper dive into microbial interactions and transformation mechanisms.

The impact of microplastics and their additives on soil nutrient cycling, particularly through microbial mechanisms, remains underexplored. This study investigated the effects of polyethylene microplastics, polyethylene resin, and plastic additives on soil nitrogen content, physicochemical properties, nitrogen cycling functional genes, microbial composition, and nitrogen transformation rates. Results showed that all amendments increased total nitrogen but decreased dissolved total nitrogen. Polyethylene microplastics and additives increased dissolved organic nitrogen, while polyethylene resin reduced it and exhibited higher microbial biomass. Amendments reduced or did not change inorganic nitrogen levels, with additives showing the lowest values. Polyethylene resin favored microbial nitrogen immobilization, while additives were more inhibitory. Amendment type and content significantly interacted with nitrogen cycling genes and microbial composition. Distinct functional microbial biomarkers and network structures were identified for different amendments. Polyethylene microplastics had higher gross ammonification, nitrification, and immobilization rates, followed by polyethylene resin and additives. Nitrogen transformation was driven by multiple functional genes, with Proteobacteria playing a significant role. Soil physicochemical properties affected nitrogen content through transformation rates, with C/N ratio having an indirect effect and water holding capacity directly impacting it. In summary, plastic additives, compared to polyethylene microplastics and resin, are less conducive to nitrogen degradation and microbial immobilization, exert significant effects on microbial community structure, inhibit transformation rates, and ultimately impact nitrogen cycling.

Integrated metabolomics and transcriptomics reveal that HhERF9 positively regulates salt tolerance in Hibiscus hamabo Siebold & Zuccarini.

Hibiscus hamabo Siebold & Zuccarini is one of the few semi-mangrove plants in the genus Hibiscus that can survive in saline-alkali soil and flooded land, but the mechanism underlying its adaptation to salt soil remains unknown. Here, to uncover this unsolved mystery, we characterized the changes in the accumulation of specific metabolites under salt stress in H. hamabo by integrating physiological, metabolic, and transcriptomic data, and found that osmotic adjustment and abscisic acid (ABA) is highly associated with the salt stress response. Further, a weighted gene co-expression network analysis was performed on the root transcriptome data, which identified three key candidate transcription factors responsive to salt stress. Among them, the expression HhERF9 was significantly upregulated under salt stress and ABA treatment and was involved in regulating the expression of genes related to the salt stress response. Further research indicated that HhERF9 enhances the accumulation of proline and soluble sugars by regulating the expression of genes such as NHX2 and P5CS. These findings provide a reference for improving H. hamabo through targeted genetic engineering and lay a theoretical foundation for its future promotion and cultivation in saline-alkali areas.

Integrated small RNA, transcriptome and physiological approaches provide insight into Taxodium hybrid 'Zhongshanshan' roots in acclimation to prolonged flooding.

Although Taxodium hybrid 'Zhongshanshan' 406 (Taxodium mucronatum Tenore × Taxodium distichum; Taxodium 406) is an extremely flooding-tolerant woody plant, the physiological and molecular mechanisms underlying acclimation of its roots to long-term flooding remain largely unknown. Thus, we exposed saplings of Taxodium 406 to either non-flooding (control) or flooding for 2 months. Flooding resulted in reduced root biomass, which is in line with lower concentrations of citrate, α-ketoglutaric acid, fumaric acid, malic acid and adenosine triphosphate (ATP) in Taxodium 406 roots. Flooding led to elevated activities of pyruvate decarboxylase, alcohol dehydrogenase and lactate dehydrogenase, which is consistent with higher lactate concentration in the roots of Taxodium 406. Flooding brought about stimulated activities of superoxide dismutase and catalase and elevated reduced glutathione (GSH) concentration and GSH/oxidized glutathione, which is in agreement with reduced concentrations of O2- and H2O2 in Taxodium 406 roots. The levels of starch, soluble protein, indole-3-acetic acid, gibberellin A4 and jasmonate were decreased, whereas the concentrations of glucose, total non-structural carbohydrates, most amino acids and 1-aminocyclopropane-1-carboxylate (ACC) were improved in the roots of flooding-treated Taxodium 406. Underlying these changes in growth and physiological characteristics, 12,420 mRNAs and 42 miRNAs were significantly differentially expressed, and 886 miRNA-mRNA pairs were identified in the roots of flooding-exposed Taxodium 406. For instance, 1-aminocyclopropane-1-carboxylate synthase 8 (ACS8) was a target of Th-miR162-3p and 1-aminocyclopropane-1-carboxylate oxidase 4 (ACO4) was a target of Th-miR166i, and the downregulation of Th-miR162-3p and Th-miR166i results in the upregulation of ACS8 and ACO4, probably bringing about higher ACC content in flooding-treated roots. Overall, these results indicate that differentially expressed mRNA and miRNAs are involved in regulating tricarboxylic acid cycle, ATP production, fermentation, and metabolism of carbohydrates, amino acids and phytohormones, as well as reactive oxygen species detoxification of Taxodium 406 roots. These processes play pivotal roles in acclimation to flooding stress. These results will improve our understanding of the molecular and physiological bases underlying woody plant flooding acclimation and provide valuable insights into breeding-flooding tolerant trees.

Grafting Causes Physiological Changes and Promotes Adventitious Root Formation in Rejuvenated Soft Shoots of Taxodium hybrid 'Zhongshanshan'.

Taxodium hybrid 'Zhongshanshan' has been widely used as a timber tree in river network areas and coastal regions and is mainly propagated by cuttings. However, when trees age, their capacity to form adventitious roots becomes weaker. We successfully enhanced the rooting ability of shoots in T. hybrid 'Zhongshanshan 302' by their rejuvenation based on grafting. We recorded temporal variation in endogenous auxin, abscisic acid (ABA), gibberellins (GAs), trans-zeatin-riboside (TZR), soluble sugar and H2O2 after root induction. Auxin, soluble sugars and H2O2 levels were higher in rejuvenated shoots than in mature shoots, whereas the opposite was true for ABA and GAs. Notably, indole-3-acetic acid (IAA) and GA3 presented higher contents with more obvious differences in T. hybrid 'Zhongshanshan 302' rejuvenated shoots vs. mature shoots compared with other kinds of auxin and GAs. The evident improvement in the rooting ability of rejuvenated shoots after grafting likely resulted from the differential regulation of plant hormones, carbohydrates and redox signaling. In addition to the physiological basis of improved rooting ability by grafting, this study provided a theoretical basis for the optimization of subsequent propagation techniques in T. hybrid 'Zhongshanshan' and potentially other Taxodium spp.

Transcriptomic and metabolomic analyses unveil the growth advantage mechanism conferred by heterosis of Michelia 'Zhongshanhanxiao'.

Michelia compressa (Maxim.) Sarg. is one of the important timber trees in Taiwan province, P. R. China. Michelia 'Zhongshanhanxiao' is a group of variants found among the progeny of M. compressa that exhibit higher growth rates compared with normal individuals, with a significantly increased stem diameter and height, as well as enlarged leaves and flowers. However, the molecular mechanisms fostering the growth advantage and morphological variations are unknown and deserve further study. Through analysing the transcriptome, metabolome and physiological processes of leaves, we identified remarkable differences in gene expression and metabolic profiles between Michelia 'Zhongshanhanxiao' and both the maternal M. compressa and its normal progeny. These differences were widely associated with a plant-pathogen interaction, phenylpropanoid biosynthesis, cyanoamino acid metabolism, carbon fixation in photosynthetic organisms and plant hormone signal transduction. Additionally, physiological measurements showed that Michelia 'Zhongshanhanxiao' possesses stronger photosynthetic capacity and higher plant hormone content. These results suggest that the heterosis of Michelia 'Zhongshanhanxiao' is regulated by candidates related to cell division, resistance to pathogens and the accumulation of organic compounds. The findings of this study provide crucial information on the molecular mechanisms underlying the growth advantages conferred by heterosis in trees.

Novel molecular markers for Taxodium breeding from the chloroplast genomes of four artificial Taxodium hybrids.

Taxodium "Zhongshanshan" are a group of intraspecific Taxodium hybrids with superparental dominance and high ecological and economic value in southern China. Identifying the parentage of hybrids, especially the male parent, is critically important for genetic studies. However, the large nuclear genomes of members of the genus Taxodium pose a major challenge for the development of molecular markers. Here, we developed novel molecular markers by conducting a comparative analysis of the chloroplast genomes of four artificial Taxodium hybrids and their parents. The lengths of the whole chloroplast genome ranged from 131,942 to 132,128 bp, and the total guanine (GC) content of the chloroplast genomes ranged from 34.6% to 35.81%. A total of 120 unique genes were identified, including 83 protein-coding genes, 33 transfer RNAs, and four ribosomal RNAs. There were 69-71 simple sequence repeats were detected in the four hybrids. Phylogenetic analysis revealed that these hybrids clustered with their paternal parents. Similar findings were obtained by analysis of the GC content of protein-coding genes. Molecular markers were developed using the highly variable regions of the chloroplast genomes, and polymerase chain reaction (PCR) assays revealed that these markers were effective for identifying the male parents of these hybrids. Our findings indicate for the first time that the chloroplast genomes of Taxodium are paternally inherited. Generally, these molecular markers could facilitate breeding and genetic studies of Taxodium.

The genome of hibiscus hamabo reveals its adaptation to saline and waterlogged habitat.

Hibiscus hamabo is a semi-mangrove species with strong tolerance to salt and waterlogging stress. However, the molecular basis and mechanisms that underlie this strong adaptability to harsh environments remain poorly understood. Here, we assembled a high-quality, chromosome-level genome of this semi-mangrove plant and analyzed its transcriptome under different stress treatments to reveal regulatory responses and mechanisms. Our analyses suggested that H. hamabo has undergone two recent successive polyploidy events, a whole-genome duplication followed by a whole-genome triplication, resulting in an unusually large gene number (107 309 genes). Comparison of the H. hamabo genome with that of its close relative Hibiscus cannabinus, which has not experienced a recent WGT, indicated that genes associated with high stress resistance have been preferentially preserved in the H. hamabo genome, suggesting an underlying association between polyploidy and stronger stress resistance. Transcriptomic data indicated that genes in the roots and leaves responded differently to stress. In roots, genes that regulate ion channels involved in biosynthetic and metabolic processes responded quickly to adjust the ion concentration and provide metabolic products to protect root cells, whereas no such rapid response was observed from genes in leaves. Using co-expression networks, potential stress resistance genes were identified for use in future functional investigations. The genome sequence, along with several transcriptome datasets, provide insights into genome evolution and the mechanism of salt and waterlogging tolerance in H. hamabo, suggesting the importance of polyploidization for environmental adaptation.

Identification and functional analysis of LecRLK genes in Taxodium 'Zhongshanshan'.

BACKGROUND: Lectin receptor-like protein kinases (LecRLKs) can transform external stimuli into intracellular signals and play important regulatory roles in plant development and response to environmental stressors. However, research on the LecRLK gene family of conifers has seldom been reported. METHODS: Putative LecRLK genes were identified in the transcriptome of Taxodium 'Zhongshanshan'. The classification, domain structures, subcellular localization prediction, and expression patterns of LecRLK genes, as well as co-expressed genes, were analyzed using bioinformatics methods. Fifteen representative genes were further selected for qRT-PCR analysis in six tissues and under five different environmental stressor conditions. RESULTS: In total, 297 LecRLK genes were identified, including 155 G-type, 140 L-type, and 2 C-type. According to the classification, G-type and L-type LecRLK genes both can be organized into seven groups. The domain architecture of G-type proteins were more complex compared with that of L- and C-type proteins. Conservative motifs were found in G-type and L-type diverse lectin domains. Prediction and transient expression experiments to determine subcellular localization showed that LecRLKs were mainly concentrated in the cell membrane system, and some members were located at multiple sites at the same time. RNA-seq-based transcriptomics analysis suggested functional redundancy and divergence within each group. Unigenes co-expressed with LecRLKs in the transcriptome were found to be enriched in pathways related to signal transduction and environmental adaptation. Furthermore, qRT-PCR analysis of representative genes showed evidence of functional divergence between different groups. CONCLUSIONS: This is the first study to conduct an identification and expression analysis of the LecRLK gene family in Taxodium. These results provide a basis for future studies on the evolution and function of this important gene family in Taxodium.

Molecular cloning and expression analysis of three ThERFs involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products.

As a subfamily of the APETALA 2/ethylene response element binding protein (AP2/EREBP) transcription factor superfamily, the ethylene response factor (ERF) is widely involved in the regulation of growth and response to various abiotic stresses in plants, and has been shown to be the main transcription factor regulating transcription of the genes related to hypoxia and waterlogging stress. In this study, three ThERF genes, with significant differences in expression profile in response to flooding stress, were identified from the transcriptomics data acquired from Taxodium hybrid 'Zhongshanshan 406' (T. mucronatum Tenore × T. distichum (L.) Rich) under waterlogging stress: ThERF15, ThERF39 and ThRAP2.3 (GenBank ID: KY463467, KY463468 and KY463470, respectively).The full-length cDNA of each of the three ERFs was obtained using the RACE (rapid amplification cDNA ends) method, and all three were intron-free. Multiple protein sequence alignments indicated that ThERF15, ThERF39 and ThRAP2.3 proteins all had only one AP2-ERF domain and belonged to the ERF subfamily. A transient gene expression assay demonstrated that ThERF15, ThERF39 and ThRAP2.3 were all localized to the nucleus. Real-time quantitative PCR (qPCR) revealed that the expression of ThERF15, ThERF39 and ThRAP2.3 exhibited significant differences, compared with the control, in response to two levels of flooding treatment (half-flooding or total-submergence) of 'Zhongshanshan 406'. Quantification of ethylene concentration revealed that ethylene was more relevant to the level of expression than the period of flooding treatment. Based on the experimental results above, ThERF15, ThERF39 and ThRAP2.3 were identified as being related to the regulation of downstream flooding- responsive gene expression in 'Zhongshanshan 406'. ThRAP2.3 is most likely to be a key downstream-response ERF gene to respond to the output of the ethylene signal generated by flooding stress.

Cloning and Characterization of ThSHRs and ThSCR Transcription Factors in Taxodium Hybrid 'Zhongshanshan 406'.

Among the GRAS family of transcription factors, SHORT ROOT (SHR) and SCARECROW (SCR) are key regulators of the formation of root tissues. In this study, we isolated and characterized two genes encoding SHR proteins and one gene encoding an SCR protein: ThSHR1 (Accession Number MF045148), ThSHR2 (Accession Number MF045149) and ThSCR (Accession Number MF045152) in the adventitious roots of Taxodium hybrid 'Zhongshanshan'. Gene structure analysis indicated that ThSHR1, ThSHR2 and ThSCR are all intron free. Multiple protein sequence alignments showed that each of the corresponding proteins, ThSHR1, ThSHR2 and ThSCR, contained five well-conserved domains: leucine heptad repeat I (LHRI), the VHIID motif, leucine heptad repeat II (LHR II), the PFYRE motif, and the SAW motif. The phylogenetic analysis indicated that ThSCR was positioned in the SCR clade with the SCR proteins from eight other species, while ThSHR1 and ThSHR2 were positioned in the SHR clade with the SHR proteins from six other species. Temporal expression patterns of these genes were profiled during the process of adventitious root development on stem cuttings. Whereas expression of both ThSHR2 and ThSCR increased up to primary root formation before declining, that of ThSHR1 increased steadily throughout adventitious root formation. Subcellular localization studies in transgenic poplar protoplasts revealed that ThSHR1, ThSHR2 and ThSCR were localized in the nucleus. Collectively, these results suggest that the three genes encode Taxodium GRAS family transcription factors, and the findings contribute to improving our understanding of the expression and function of SHR and SCR during adventitious root production, which may then be manipulated to achieve high rates of asexual propagation of valuable tree species.

Transcriptome analysis of the Taxodium 'Zhongshanshan 405' roots in response to salinity stress.

Taxodium 'Zhongshanshan' is an interspecies hybrid of Taxodium distichum and Taxodium mucronatum, and has been widely planted in southeastern China. It has great ecological and economic potential. However, the scant genomic resources in genus Taxodium have greatly hindered further exploration of its underlying salinity-tolerance mechanism. To understand the genetic basis of its salt tolerance, high-throughput sequencing of mRNA (RNA-Seq) was used to analyze transcriptome changes of 'Zhongshanshan 405' clone roots treated with NaCl stress. After de novo assembly, 70,312 unigenes were achieved, and 41,059 of them were annotated. 9038 differentially expressed genes (DEGs) were identified among the treatments, and 7959 DEGs were found between salt-stressed roots and control, with 489 up-regulated and 570 down-regulated shared by all of the treatments. Genes related to transport, signal transductions as well as undescribed transcripts were among those DEGs in response to salt stress. Gene ontology classification analysis revealed that salt stress-related categories including 'oxidoreductase activity', 'metal ion binding', and 'membrane' were highly enriched among these DEGs. Moreover, the gene expression pattern of 12 unigenes revealed by quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the RNA-Seq data. Our study not only provided the large-scale assessment of transcriptome resources of Taxodium but also guidelines for probing the molecular mechanism underlying 'Zhongshanshan' salt tolerance.

Genetic linkage map construction and QTL mapping of seedling height, basal diameter and crown width of Taxodium 'Zhongshanshan 302' × T. mucronatum.

Taxodium is a genus renowned for its fast growth, good form and tolerance of flooding, salt, alkalinity, disease and strong winds. In this study, a genetic linkage map was constructed using sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers based on an F1 population containing 148 individuals generated from a cross between T. 'Zhongshanshan 302' and T. mucronatum. The map has a total length of 976.5 cM, with a mean distance of 7.0 cM between markers, and contains 34 linkage groups with 179 markers (171 SRAPs and 8 SSRs). Quantitative trait loci (QTLs) affecting growth traits, such as seedling height, basal diameter and crown width, were detected based on the constructed linkage map. Four significant QTLs were identified, three of which, namely qtSH-1 for seedling height, qtBD-1 for basal diameter and qtCW-1 for crown width, were located at 2.659 cM of LG7 with logarithm odds values of 3.72, 3.49 and 3.93, respectively, and explained 24.9, 27.0 and 21.7 % of the total variation of the three grown traits, respectively. Another QTL for crown width (qtCW-2) was detected at 1.0 cM on LG13, with a logarithm of odds value of 3.15, and explained 31.7 % of the total variation of crown width. This is the first report on the construction of a genetic linkage map and QTL analysis in Taxodium, laying the groundwork for the construction of a high-density genetic map and QTL mapping in the genus Taxodium.