The plant peptide hormone phytosulfokine promotes somatic embryogenesis by maintaining redox homeostasis in Cunninghamia lanceolata.

Somatic embryogenesis (SE) is widely used for studying the mechanisms of embryo development. However, little is known about the underlying mechanisms, especially in woody plants. Previous studies have established an SE system for Chinese fir (Cunninghamia lanceolata), but this system is genotype-dependent, which limits its application in practice. Here, we found that phytosulfokine (PSK), a plant peptide hormone, can not only increase SE efficiency, but also establish SE in recalcitrant genotypes of C. lanceolata. Proembryogenic mass (PEM) browning and determination of hydrogen peroxide (H2 O2 ) content by 2',7'-dichlorofluorescein staining indicated that a reactive oxygen species (ROS) burst occurred rapidly after PEMs were transferred to SE induction medium. Transcriptome analysis and quantitative reverse transcriptase-PCR validation showed that PSK treatment helped to maintain ROS homeostasis by decreasing the activity of peroxidases in early SE induction. This PSK-regulated redox microenvironment might be helpful to induce expression of SE-related genes like WOX2 in early SE induction. Further analyses suggested that PSK promotes SE induction in C. lanceolata partially through decreasing H2 O2 levels, which is necessary but not sufficient for SE induction in recalcitrant genotypes of C. lanceolata. Furthermore, heterologous overexpression of ClPSK in Arabidopsis led to enhanced SE induction and resistance to H2 O2 stress. Taken together, our study reveals a biological function for the plant peptide hormone PSK, extends our knowledge about SE in woody trees, and provides a valuable tool for establishing an efficient and genotype-independent SE system in C. lanceolata and other coniferous trees.

Nitrogen addition affected the root competition in Cunninghamia lanceolata-Phoebe chekiangensis mixed plantation.

Little is known about below-ground competition in mixed-species plantations under increasing nitrogen (N) deposition. This study aims to determine the effects of N addition on root competition in coniferous and broad-leaved species mixed plantations. A pot experiment was conducted using the coniferous species Cunninghamia lanceolata and the broad-leaved species Phoebe chekiangensis planted in mixed plantations with different competition intensities under N addition (0 or 45 kg N ha-1 yr-1). Biomass allocation, root morphology, root growth level, and competitive ability were determined after five months of treatment. Our findings indicated that root interactions in mixed plantations did not influence biomass allocation in either C. lanceolata or P. chekiangensis but promoted growth in C. lanceolata when no N was added. However, N addition decreased biomass accumulation in both species in the mixed plantation and had a negative effect on the root growth of C. lanceolata due to intensified competition. Addition of N increased the relative importance of root predatory competition in P. chekiangensis, and increased the allelopathic competitive advantage in C. lanceolata. This suggests that N addition causes a shift in the root competitive strategy from tolerance to competition. Overall, these findings highlight the significant impact that the addition of N can have on plant interactions in mixed plantations. Our results provide implications for the mechanisms of root competition in response to increasing atmospheric N deposition in mixed plantations.

High nutrient utilization and resorption efficiency promote bamboo expansion and invasion.

Bamboos are fast-growing, aggressively-spreading, and invasive woody clonal species that often encroach upon adjacent tree plantations, forming bamboo-tree mixed plantations. However, the effects of bamboo invasion on leaf carbon (C) assimilation, and nitrogen (N) and phosphorus (P) utilization characteristics remains unclear. We selected four different stands of Pleioblastus amarus invading Chinese fir (Cunninghamia lanceolata) plantations to investigate the concentrations, stoichiometry, and allometric growth relationships of mature and withered leaves of young and old bamboos, analyzing N and P utilization and resorption patterns. The stand type, bamboo age, and their interaction affected the concentrations, stoichiometry and allometric growth patterns of leaf C, N, and P in both old and young bamboos, as well as the N and P resorption efficiency. Bamboo invasion into Chinese fir plantations decreased leaf C, N, and P concentrations, C:N and C:P ratios, N and P resorption efficiency, and allometric growth exponents among leaf C, N, and P, while it only slightly altered N:P ratios. PLS-PM analysis revealed that bamboo invasion negatively impacted leaf C, N, and P concentrations, as well as N and P utilization and resorption. The results indicate that high N and P utilization and resorption efficiency, along with the mutual sharing of C, N, and P among bamboos in interface zones, promote continuous bamboo expansion and invasion. Collectively, these findings highlight the significance of N and P utilization and resorption in bamboo expansion and invasion and provide valuable guidance for the establishment of mixed stands and the ecological management of bamboo forests.

Contrasting impacts of fertilization on topsoil and subsoil greenhouse gas fluxes in a thinned Chinese fir plantation.

Globally, forest soils are considered as important sources and sinks of greenhouse gases (GHGs). However, most studies on forest soil GHG fluxes are confined to the topsoils (above 20 cm soil depths), with only very limited information being available regarding these fluxes in the subsoils (below 20 cm soil depths), especially in managed forests. This limits deeper understanding of the relative contributions of different soil depths to GHG fluxes and global warming potential (GWP). Here, we used a concentration gradient-based method to comprehensively investigate the effects of thinning intensity (15% vs. 35%) and nutrient addition (no fertilizer vs. NPK fertilizers) on soil GHG fluxes from the 0-40 cm soil layers at 10 cm depth intervals in a Chinese fir (Cunninghamia lanceolata) plantation. Results showed that forest soils were the sources of CO2 and N2O, but the sinks of CH4. Soil GHG fluxes decreased with increasing soil depth, with the 0-20 cm soil layers identified as the dominant producers of CO2 and N2O and consumers of CH4. Thinning intensity did not significantly affect soil GHG fluxes. However, fertilization significantly increased CO2 and N2O emissions and CH4 uptake at 0-20 cm soil layers, but decreased them at 20-40 cm soil layers. This is because fertilization alleviated microbial N limitation and decreased water filled pore space (WFPS) in topsoils, while it increased WFPS in subsoils, ultimately suggesting that soil WFPS and N availability (especially NH4+-N) were the predominant regulators of GHG fluxes along soil profiles. Generally, there were positive interactive effects of thinning and fertilization on soil GHG fluxes. Moreover, the 35% thinning intensity without fertilization had the lowest GWP among all treatments. Overall, our results suggest that fertilization may not only cause depth-dependent effects on GHG fluxes within soil profiles, but also impede efforts to mitigate climate change by promoting GHG emissions in managed forest plantations.

Canopy nitrogen addition and understory removal destabilize the microbial community in a subtropical Chinese fir plantation.

Subtropical Chinese fir plantations have been experiencing increased nitrogen deposition and understory management because of human activities. Nevertheless, effect of increased nitrogen deposition and understory removal in the plantations on microbial community stability and the resulting consequences for ecosystem functioning is still unclear. We carried out a 5-year experiment of canopy nitrogen addition (2.5 g N m-2 year-1), understory removal, and their combination to assess their influences on microbial community stability and functional potentials in a subtropical Chinese fir plantation. Nitrogen addition, understory removal, and their combination reduced soil bacterial diversity (OUT richness, Inverse Simpson index, Shannon index, and phylogenetic diversity) by 11-18%, 15-24%, and 19-31%; reduced fungal diversity indexes by 3-5%, 5-6%, and 5-7%, respectively. We found that environmental filtering and interspecific interactions together determined changes in bacterial community stability, while changes in fungal community stability were mainly caused by environmental filtering. Fungi were more stable than bacteria under disturbances, possibly from having a more stable network structure. Furthermore, we found that microbial community stability was linked to changes in microbial community functional potentials. Importantly, we observed synergistic interactions between understory removal and nitrogen addition on bacterial diversity, network structure, and community stability. These findings suggest that understory plants play a significant role in promoting soil microbial community stability in subtropical Chinese fir plantations and help to mitigate the negative impacts of nitrogen addition. Hence, it is crucial to retain understory vegetation as important components of subtropical plantations.

Intraspecific variations in leaf functional traits of Cunninghamia lanceolata provenances.

BACKGROUND: Studies on intra-specific variability in leaf functional traits is important to evaluate adaptation of the species to predicted climate change, and to develop long-term conservation strategy. The main objectives were to investigate the relationship between the functional traits leaves and C, N, P stoichiometry of Chinese fir from different geographical provenances and their relationship with the main environmental factors of provenance. RESULTS: In this study, we measured 12 leaf functional traits on 36-year-old Cunninghamia lanceolata trees from 13 provenances. Analysis of variance (ANOVA) was performed to examine the variability. Redundancy analysis (RA) was computed to examine the relationship between geo-climatic factors of provenance origin and leaf functional traits while Pearson's correlation coefficient was computed to assess inter-trait correlations. The results showed statistically significant differences (P < 0.01) in intraspecific leaf traits among provenances, except leaf P content. The relationships among leaf traits are consistent with the general trend observed in the leaf economic spectrum. Mean annual temperature appeared to be a key factor that influences intraspecific leaf traits variability compared to mean annual precipitation. CONCLUSION: These results provide useful insights about adaptation of leaf trait of Chinese fir in a changing climatic condition. Thus, our findings shed light on the importance of interspecific trait variability in Chinese fir and the potential effect of climate change.

Phytosulfokine contributes to suspension culture of Cunninghamia lanceolata through its impact on redox homeostasis.

BACKGROUND: Suspension culture is widely used in the establishment of efficient plant regeneration systems, as well as in the mass production of plant secondary metabolites. However, the establishment of a suspension culture system of Cunninghamia lanceolata is genotype-dependent given that proembryogenic masses (PEMs) are prone to browning during this process in recalcitrant genotypes. Previously, we reported that the plant peptide hormone phytosulfokine (PSK) can tremendously decrease the hydrogen peroxide (H2O2) level and help to initiate somatic embryogenesis (SE) in recalcitrant C. lanceolata genotypes. However, to date, no studies have revealed whether or how PSK may contribute to the establishment of a suspension culture system in these recalcitrant genotypes. RESULTS: Here, we demonstrated that exogenous application of PSK effectively inhibited PEM browning during suspension culture in a recalcitrant genotype of C. lanceolata. Comparative time-series transcriptome profiling showed that redox homeostasis underwent drastic fluctuations when PEMs were cultured in liquid medium, while additional PSK treatment helped to maintain a relatively stable redox homeostasis. Interestingly, PSK seemed to have a dual effect on peroxidases (PRXs), with PSK simultaneously transcriptionally repressing ROS-producing PRXs and activating ROS-scavenging PRXs. Furthermore, determination of H2O2 and MDA content, as well as cell viability, showed that exogenous PSK treatment inhibited PEM browning and safeguarded PEM suspension culture by decreasing the H2O2 level and increasing PEM activity. CONCLUSIONS: Collectively, these findings provide a valuable tool for the future establishment of large-scale C. lanceolata PEM suspension culture without genotype limitations.

Overexpression of ClWRKY48 from Cunninghamia lanceolata improves Arabidopsis phosphate uptake.

MAIN CONCLUSION: Our findings suggested that ClWRKY48 promoted the expression level of Arabidopsis phosphate transporter genes, enhanced phosphate uptake, and delayed the transition from the vegetative stage to the reproductive phase in Arabidopsis. Phosphorus (P) is an essential mineral for plants that influences their growth and development. ClWRKY48, one of the most highly expressed genes in the leaf, was identified by RT-PCR from Chinese fir [Cunninghamia lanceolata (Lamb.) Hook] (C. lanceolata). Furthermore, when treating C. lanceolata with increasing phosphate (Pi) concentration, the expression level of ClWRKY48 rose in leaves, the trends followed the increasing phosphate concentration treatment. ClWRKY48 is a transcription factor in C. lanceolata, according to the results of a yeast one hybridization experiment. Based on subcellular localization studies, ClWRKY48 is a nuclear-localized protein. Under Pi deficiency conditions, the phosphorus concentration of ClWRKY48 overexpressing Arabidopsis increased by 43.2-51.1% compared to the wild-type. Moreover, under Pi limiting conditions, the phosphate transporter genes AtPHT1;1 (Arabidopsis Phosphate transporter 1;1), AtPHT1;4, and AtPHO1 (Arabidopsis PHOSPHATE 1) were expressed 2.1-2.5, 2.2-2.7, and 6.7-7.3-fold greater than the wild-type in ClWRKY48 transgenic Arabidopsis, respectively. Under Pi-sufficient conditions, the phosphorus concentration and phosphate transporter genes of ClWRKY48 overexpression in Arabidopsis are not significantly different from the wild type. These findings indicated that ClWRKY48 increased phosphate absorption in transgenic Arabidopsis. Furthermore, compared to the wild type, the ClWRKY48 transgenic Arabidopsis not only had a delayed flowering time characteristic but also had lower expression of flowering-related genes AtFT (FLOWERING LOCUS T), AtFUL (FRUITFUL), and AtTSF (TWIN SISTER OF FT). Our findings show that ClWRKY48 enhances phosphate absorption and slows the transition from the vegetative to the reproductive stage in ClWRKY48 transgenic Arabidopsis.

Metagenomics reveals the effects of glyphosate on soil microbial communities and functional profiles of C and P cycling in the competitive vegetation control process of Chinese fir plantation.

Although considerable efforts have been devoted to investigate the behavior of glyphosate on microbiome in various environment, knowledge about the soil microbial community and functional profile in weeds control process of the Chinese fir plantation are limited. In this study, shotgun metagenomic sequencing was used to determine the abundance and diversity of microbial communities and functional genes after foliar application of glyphosate for 1, 2, 3 and 4 months in a Chinese fir plantation. The results showed that glyphosate increased the copy numbers (qPCR) of 16S rRNA gene for 16.9%, improved the bacterial diversity (Shannon index) and complexity of bacterial co-occurrence network, and changed the abundances of some bacterial and fungal taxa, but had no effects on ITS gene copy numbers, fungal Shannon index, and bacterial and fungal communities (PCoA). Glyphosate application significantly decreased the amount of microbial function potentials involved in organic P mineralization for 10.7%, chitin degradation for 13.1%, and CAZy gene families with an exception of PL for 11.5% at the first month, while did not affect the profile of microbial genes response to P and C cycling in longer term. In addition, glyphosate reduced the contents of soil TOC, DOC and NH4+-H for 17.6%, 52.3% and 44.6% respectively, and decreased the starch, soluble sugar, Zn and Fe of Chinese fir leaves for 20.6%, 19.8%, 32.8% and 48.4% respectively. Mantle test, Spearman's correlation, and PLS-PM model revealed the connections among soil properties, tree nutrients, bacterial and fungal communities, and microbial function potentials were influenced by glyphosate. While our findings need to be validated in other filed and mechanistic studies, they may indicate that the foliar application of glyphosate has a potential effect on Chinese fir seedlings, and this effect may contribute to the changes of the bacterial community and soil properties including AN, DON and NH4+-H.

Revealing Further Insights into Astringent Seeds of Chinese Fir by Integrated Metabolomic and Lipidomic Analyses.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) stands as one of the pivotal afforestation tree species and timber resources in southern China. Nevertheless, the occurrence of seed abortion and a notably high proportion of astringent seeds significantly curtail the yield and quality of elite seeds, resulting in substantial economic losses. The development of astringent seeds is accompanied by significant physiological and biochemical alterations. Here, the first combined lipidomic and metabolomic analysis was performed to gain a comprehensive understanding of astringent seed traits. A total of 744 metabolites and 616 lipids were detected, of which 489 differential metabolites and 101 differential lipids were identified. In astringent seeds, most flavonoids and tannins, as well as proline and γ-aminobutyric acid, were more accumulated, along with a notable decrease in lipid unsaturation, indicating oxidative stress in the cells of astringent seeds. Conversely, numerous elemental metabolites were less accumulated, including amino acids and their derivatives, saccharides and alcohols, organic acids and nucleotides and their derivatives. Meanwhile, most lipid subclasses, mainly associated with energy storage (triglyceride and diglyceride) and cell membrane composition (phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine), also exhibited significant reductions. These results reflected a disruption in the cellular system or the occurrence of cell death, causing a reduction in viable cells within astringent seeds. Furthermore, only one lipid subclass, sphingosine phosphate (SoP), was more accumulated in astringent seeds. Additionally, lower accumulation of indole-3-acetic acid and more accumulation of salicylic acid (SA) were also identified in astringent seeds. Both SA and SoP were closely associated with the promotion of programmed cell death in astringent seeds. Collectively, our study revealed significant abnormal changes in phytohormones, lipids and various metabolites in astringent seeds, allowing us to propose a model for the development of astringent seeds in Chinese fir based on existing research and our findings. This work enriches our comprehension of astringent seeds and presents valuable bioindicators for the identification of astringent seeds.

ClNAC100 Is a NAC Transcription Factor of Chinese Fir in Response to Phosphate Starvation.

Phosphate (Pi) deficiency is one of the most limiting factors for Chinese fir growth and production. Moreover, continuous cultivation of Chinese fir for multiple generations led to the reduction of soil nutrients, which hindered the yield of Chinese fir in southern China. Although NAC (NAM, ATAF, and CUC) transcription factors (TFs) play critical roles in plant development and abiotic stress resistance, it is still unclear how they regulate the response of Chinese fir to phosphate (Pi) starvation. Based on Pi-deficient transcriptome data of Chinses fir root, we identified a NAC transcription factor with increased expression under Pi deficiency, which was obtained by PCR and named ClNAC100. RT-qPCR confirmed that the expression of ClNAC100 in the root of Chinese fir was induced by phosphate deficiency and showed a dynamic change with time. It was positively regulated by ABA and negatively regulated by JA, and ClNAC100 was highly expressed in the roots and leaves of Chinese fir. Transcriptional activation assay confirmed that ClNAC100 was a transcriptional activator. The promoter of ClNAC100 was obtained by genome walking, which was predicted to contain a large number of stress, hormone, and growth-related cis-elements. Tobacco infection was used to verify the activity of the promoter, and the core promoter was located between -1519 bp and -589 bp. We identified 18 proteins bound to the ClNAC100 promoter and 5 ClNAC100 interacting proteins by yeast one-hybrid and yeast two-hybrid, respectively. We speculated that AHL and TIFY family transcription factors, calmodulin, and E3 ubiquitin ligase in these proteins might be important phosphorus-related proteins. These results provide a basis for the further study of the regulatory mechanism and pathways of ClNAC100 under Pi starvation.

Genome-Wide Characterization and Gene Expression Analyses of Malate Dehydrogenase (MDH) Genes in Low-Phosphorus Stress Tolerance of Chinese Fir (Cunninghamia lanceolata).

Malate dehydrogenase (MDH) genes play vital roles in developmental control and environmental stress tolerance in sessile plants by modulating the organic acid-malic acid level. However, MDH genes have not yet been characterized in gymnosperm, and their roles in nutrient deficiency are largely unexplored. In this study, 12 MDH genes were identified in Chinese fir (Cunninghamia lanceolata), namely, ClMDH-1, -2, -3, …, and -12. Chinese fir is one of the most abundant commercial timber trees in China, and low phosphorus has limited its growth and production due to the acidic soil of southern China. According to the phylogenetic analysis, MDH genes were classified into five groups, and Group 2 genes (ClMDH-7, -8, -9, and 10) were only found to be present in Chinese fir but not in Arabidopsis thaliana and Populus trichocarpa. In particular, the Group 2 MDHs also had specific functional domains-Ldh_1_N (malidase NAD-binding functional domain) and Ldh_1_C (malate enzyme C-terminal functional domain)-indicating a specific function of ClMDHs in the accumulation of malate. All ClMDH genes contained the conserved MDH gene characteristic functional domains Ldh_1_N and Ldh_1_C, and all ClMDH proteins exhibited similar structures. Twelve ClMDH genes were identified from eight chromosomes, involving fifteen ClMDH homologous gene pairs, each with a Ka/Ks ratio of <1. The analysis of cis-elements, protein interactions, and transcription factor interactions of MDHs showed that the ClMDH gene might play a role in plant growth and development, and in response to stress mechanisms. The results of transcriptome data and qRT-PCR validation based on low-phosphorus stress showed that ClMDH1, ClMDH6, ClMDH7, ClMDH2, ClMDH4, ClMDH5, ClMDH10 and ClMDH11 were upregulated under low-phosphorus stress and played a role in the response of fir to low-phosphorus stress. In conclusion, these findings lay a foundation for further improving the genetic mechanism of the ClMDH gene family in response to low-phosphorus stress, exploring the potential function of this gene, promoting the improvement of fir genetics and breeding, and improving production efficiency.

Transcriptome Profiling of Stem-Differentiating Xylem in Response to Abiotic Stresses Based on Hybrid Sequencing in Cunninghamia lanceolata.

Cunninghamia lanceolata (C. lanceolata) belongs to Gymnospermae, which are fast-growing and have desirable wood properties. However, C. lanceolata's stress resistance is little understood. To unravel the physiological and molecular regulation mechanisms under environmental stresses in the typical gymnosperm species of C. lanceolata, three-year-old plants were exposed to simulated drought stress (polyethylene glycol 8000), salicylic acid, and cold treatment at 4 °C for 8 h, 32 h, and 56 h, respectively. Regarding the physiological traits, we observed a decreased protein content and increased peroxidase upon salicylic acid and polyethylene glycol treatment. Superoxide dismutase activity either decreased or increased at first and then returned to normal under the stresses. Regarding the molecular regulation, we used both nanopore direct RNA sequencing and short-read sequencing to reveal a total of 5646 differentially expressed genes in response to different stresses, of which most had functions in lignin catabolism, pectin catabolism, and xylan metabolism, indicating that the development of stem-differentiating xylem was affected upon stress treatment. Finally, we identified a total of 51 AP2/ERF, 29 NAC, and 37 WRKY transcript factors in C. lanceolata. The expression of most of the NAC TFs increased under cold stress, and the expression of most of the WRKY TFs increased under cold and SA stress. These results revealed the transcriptomics responses in C. lanceolata to short-term stresses under this study's experimental conditions and provide preliminary clues about stem-differentiating xylem changes associated with different stresses.

Rotation age extension synergistically increases ecosystem carbon storage and timber production of Chinese fir plantations in southern China.

Afforestation is an effective method to increase carbon (C) sinks and address climate change. It is crucial to understand how the stand growth affects C sequestration capacity, especially when the trade-offs with timber production from plantations have not been fully examined. We used a chronosequence approach to estimate C storage in Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) plantations (including the trees, understory, litter, and soils) at seven stand ages (3, 8-11, 16, 21, 25, 29, and 32 years). Ecosystem C storage increased nonlinearly from 76.4 to 282.2 t ha-1 with stand age and was fitted with a logistic model that had a maximum C storage and age of 271.9 t ha-1 and 33 years, respectively, to reach 95% of the maximum stored C. The C increment was mainly contributed by an increase in tree biomass, which ranged from 2.8 to 177.7 t ha-1 and comprised 4-64% of the total ecosystem C. Live root C (sum of the stump, coarse, and fine root C) showed a logistic increase from 2.0 to 26.3 t ha-1 with stand age and constituted 2.5-9.3% of ecosystem C. Understory plants and litter represented a small pool (<2% of ecosystem C). The C storage in shrubs and litter slightly increased, while that in herbs decreased as the stands aged. Soil C storage was an important and relatively stable pool, ranging from 69.6 to 130.1 t ha-1. Stand volume was also best fitted with a logistic model with a maximum value of 552.6 m3 ha-1. Additionally, the time needed to reach 95% of the maximum volume was 25 years. Hence, extending the rotation age to over 30 years for Chinese fir plantations could potentially maximize the synergistic benefits of C storage to mitigate climate change and obtain timber products for economic profit.

The interception effect of heavy metals in different types of Chinese fir plantations.

The aim of this research is to investigate the interception effect of heavy metals, such as zinc (Zn), copper (Cu), lead (Pb), arsenic (As), and cadmium (Cd) in different ages of Chinese fir plantations. The study was conducted in 21-year-old (CF21, near mature stand) and 29-year-old (CF29, old stand) pure Chinese fir forest stands (CF) as well as a mixed coniferous and broad-leaved forest stand (MF) in Huitong from November 2016 to October 2017. The study results revealed the variation ranges of interception of Cu, Zn, Cd, As, and Pb by the research forest stands were respectively, 3.436-36.778 mg·m-2·month-1, 7.458-98.763 mg·m-2·month-1, 0.003-0.608 mg·m-2·month-1, 0.094-4.471 mg·m-2·month-1, and 1.251-23.141 mg·m-2·month-1. Compared with the different ages of CF, the average monthly interception of Cu, Zn, Cd, As, and Pb in CF21 were respectively, higher 6.0%, - 3.3%, 59.0%, 1.4%, and 3.4% than those in CF29. However, seasonal changes of that in CF of different ages were those in CF21 > in CF29 in summer and autumn, and those in CF21 < in CF29 in winter and spring. Compared in different forest types, the average monthly interception of Cu, Zn, Cd, As, and Pb in MF was respectively, higher 20.2%, 5.8%, 185.2%, 2.6%, and 12.3% than those in CF. Furthermore, the seasonal flux of heavy metal retention in the forest ecosystems was higher during winter and spring than in summer and in autumn. This study provided an important information regarding the dynamics of heavy metal interception in forest ecosystems.

Drought stress introduces growth, physiological traits and ecological stoichiometry changes in two contrasting Cunninghamia lanceolata cultivars planted in continuous-plantation soils.

BACKGROUND: The decrease in Cunninghamia lanceolata (Lamb.) production on continuously planted soil is an essential problem. In this study, two-year-old seedlings of two cultivars (a normal cultivar, NC, and a super cultivar, SC) were grown in two types of soil (not planted (NP) soil; continuously planted (CP) soil) with three watering regimes, and the interactive effects on plant growth and physiological traits were investigated in a greenhouse experiment. The water contents of the soil in the control (CK) (normal water content), medium water content (MWC) and low water content (LWC) treatments reached 75-80 %, 45-50 % and 20-25 % of the field water capacity, respectively. RESULTS: The results indicated that the CP soil had a negative effect on growth and physiological traits and that the LWC treatment caused even more severe and comprehensive negative effects. In both cultivars, the CP soil significantly decreased the height increment (HI), basal diameter increment (DI), dry matter accumulation (DMA), net photosynthetic rate (Pn), total chlorophyll content (TChl), carotenoid content (Caro) and photosynthetic nitrogen use efficiency (PNUE). Compared to the NP soil, the CP soil also decreased the proline and soluble protein contents, nitrogen use efficiency (NUE) and phosphorus use efficiency (PUE) and increased the nitrogen:phosphorus ratio in roots, stems and leaves. The LWC treatment decreased growth and photosynthesis, changed ecological stoichiometry, induced oxidative stress, promoted water use efficiency and damaged chloroplast ultrastructure. Significant increases in ascorbate peroxidase (APX), peroxidase (POD), soluble protein and proline contents were found in the LWC treatment. Compared with the NC, the SC was more tolerant to the CP soil and water stress, as indicated by the higher levels of DMA, Pn, and WUE. After exposure to the CP soil and watering regimes, the decreases in biomass accumulation and gas exchange were more pronounced. CONCLUSIONS: The combination of drought and CP soil may have detrimental effects on C. lanceolata growth, and low water content enhances the impacts of CP soil stress on C. lanceolata seedlings. The superiority of the SC over the NC is significant in Chinese fir plantation soil. Therefore, continuously planted soil can be utilized to cultivate improved varieties of C. lanceolata and maintain water capacity. This can improve their growth and physiological performance to a certain extent.

Full-length transcriptomic identification of R2R3-MYB family genes related to secondary cell wall development in Cunninghamia lanceolata (Chinese fir).

BACKGROUND: R2R3-MYB is a class of transcription factor crucial in regulating secondary cell wall development during wood formation. The regulation of wood formation in gymnosperm has been understudied due to its large genome size. Using Single-Molecule Real-Time sequencing, we obtained full-length transcriptomic libraries from the developmental stem of Cunninghamia lanceolata, a perennial conifer known as Chinese fir. The R2R3-MYB of C. lanceolata (hereafter named as ClMYB) associated with secondary wall development were identified based on phylogenetic analysis, expression studies and functional study on transgenic line. RESULTS: The evolutionary relationship of 52 ClMYBs with those from Arabidopsis thaliana, Eucalyptus grandis, Populus trichocarpa, Oryza sativa, two gymnosperm species, Pinus taeda, and Picea glauca were established by neighbour-joining phylogenetic analysis. A large number of ClMYBs resided in the woody-expanded subgroups that predominated with the members from woody dicots. In contrast, the woody-preferential subgroup strictly carrying the members of woody dicots contained only one candidate. The results suggest that the woody-expanded subgroup emerges before the gymnosperm/angiosperm split, while most of the woody-preferential subgroups are likely lineage-specific to woody dicots. Nine candidates shared the same subgroups with the A. thaliana orthologs, with known function in regulating secondary wall development. Gene expression analysis inferred that ClMYB1/2/3/4/5/26/27/49/51 might participate in secondary wall development, among which ClMYB1/2/5/26/27/49 were significantly upregulated in the highly lignified compression wood region, reinforcing their regulatory role associated with secondary wall development. ClMYB1 was experimentally proven a transcriptional activator that localised in the nucleus. The overexpression of ClMYB1 in Nicotiana benthamiana resulted in an increased lignin deposition in the stems. The members of subgroup S4, ClMYB3/4/5 shared the ERF-associated amphiphilic repression motif with AtMYB4, which is known to repress the metabolism of phenylpropanoid derived compounds. They also carried a core motif specific to gymnosperm lineage, suggesting divergence of the regulatory process compared to the angiosperms. CONCLUSIONS: This work will enrich the collection of full-length gymnosperm-specific R2R3-MYBs related to stem development and contribute to understanding their evolutionary relationship with angiosperm species.

Half-leaf width symmetric distribution reveals buffering strategy of Cunninghamia lanceolata.

BACKGROUND: Leaf length and width could be a functioning relationship naturally as plant designs. Single-vein leaves have the simplest symmetrical distribution and structural design, which means that fast-growing single-vein species could interpret the scheme more efficiently. The distribution of leaf length and width can be modulated for better adaptation, providing an informative perspective on the various operational strategies in an emergency, while this mechanism is less clear. Here we selected six age groups of Cunninghamia lanceolata pure forests, including saplings, juveniles, mature, and old-growth trees. We pioneered a tapering model to describe half-leaf symmetric distribution with mathematical approximation based on every measured leaf along developmental sequence, and evaluated the ratio of leaf basal part length to total length (called tipping leaf length ratio). RESULTS: The tipping leaf length ratio varied among different tree ages. That means the changes of tipping leaf length ratio and leaf shape are a significant but less-noticed reflection of trees tradeoff strategies at different growth stages. For instance, there exhibited relatively low ratio during sapling and juvenile, then increased with increasing age, showing the highest value in their maturity, and finally decreased on mature to old-growth transition. The tipping leaf length ratio serves as a cost-benefit ratio, thus the subtle changes in the leaf symmetrical distribution within individuals reveal buffering strategy, indicating the selection for efficient design of growth and hydraulic in their developmental sequences. CONCLUSIONS: Our model provides a physical explanation of varied signatures for tree operations in hydraulic buffering through growth stages, and the buffering strategy revealed from leaf distribution morphologically provides evidence on the regulation mechanism of leaf biomechanics, hydraulics and physiologies. Our insight contributes greatly to plant trait modeling, policy and management, and will be of interest to some scientists and policy makers who are involved in climate change, ecology and environment protection, as well as forest ecology and management.

Transcriptome analysis provides insights into the root response of Chinese fir to phosphorus deficiency.

BACKGROUND: Phosphorus is one of the essential elements for plant growth and development, but available phosphorus (Pi) content in many soil types is low. As a fast-growing tree species for timber production, Chinese fir is in great demand of Pi, and the lack of Pi in soil restricts the increase of productivity of Chinese fir plantation. Root morphology and the synthesis and secretion of organic acids play an important role in the uptake of phosphorus, but the molecular mechanisms of Chinese fir root responses to Pi deficiency are largely unexplored. In this study, seedlings of Yang 061 clone were grown under three Pi supply levels (0, 5 and 10 mg·L-1 P) and morphological attributes, organic acid content and enzyme activity were measured. The transcriptome data of Chinese fir root system were obtained and the expression levels of phosphorus responsive genes and organic acid synthesis related genes on citric acid and glyoxylate cycle pathway were determined. RESULTS: We annotated 50,808 Unigenes from the transcriptome of Chinese fir roots. Among differentially expressed genes, seven genes of phosphate transporter family and 17 genes of purple acid phosphatase family were up-regulated by Pi deficiency, two proteins of SPX domain were up-regulated and one was down-regulated. The metabolic pathways of the citric acid and glyoxylate cycle pathway were mapped, and the expression characteristics of the related Unigenes under different phosphorus treatments were analyzed. The genes involved in malic acid and citric acid synthesis were up-regulated, and the activities of the related enzymes were significantly enhanced under long-term Pi stress. The contents of citric acid and malic acid in the roots of Chinese fir increased after 30 days of Pi deficiency. CONCLUSION: Chinese fir roots showed increased expression of genes related with phosphorus starvation, citrate and malate synthesis genes, increased content of organic acids, and enhanced activities of related enzymes under Pi deficiency. The results provide a new insight for revealing the molecular mechanism of adaption to Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots.

Response of soil chemical properties and enzyme activity of four species in the Three Gorges Reservoir area to simulated acid rain.

The chemical composition in the precipitation is constantly changing, thus acid rain type is gradually changing from sulfuric type to mixed type and then nitric type. The influence of the changing acid rain type on the rhizosphere soil of tree species remains unclear. A pot experiment was performed with two-year-old Pinus massoniana, Cunninghamia lanceolate, Cyclobalanpsis glauca and Phyllostachys edulis seedlings with similar growth condition. Simulated acid rain consists of sulfuric(S/N = 5), mixed(S/N = 1) and nitric(S/N = 0.2) acid rain, and each type acid rain diluted to three acid rain intensity: pH = 2.5, 3.5, 4.5. Soil pH, soil organic matter, cation exchange capacity, the exchangeable Na+, K+, Ca2+, Mg2+ and enzyme activity were inhibited by acid rain intensity, while exchangeable Al3+ and H+ were promoted. Mg2+ was most relevant index to the tolerance to acid rain and the correlation degree of soil chemical index was higher than that of enzyme activity. Response of soil chemical properties differed in tree species under different acid rain types. Soil enzyme activity of Pinus massoniana, Cunninghamia lanceolate, and Phyllostachys edulis reached lowest under nitric acid rain, and that of Cyclobalanpsis glauca reached highest. Rhizosphere soil of Cunninghamia lanceolate is tolerant to sulfuric and nitric acid rain, and that of Cyclobalanpsis glauca is tolerant to mixed acid rain.