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.

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.

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.

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.

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.

Strength and size of phosphorus-rich patches determine the foraging strategy of Neyraudia reynaudiana.

BACKGROUND: Under natural conditions, soil nutrients are heterogeneously distributed, and plants have developed adaptation strategies to efficiently forage patchily distributed nutrient. Most previous studies examined either patch strength or patch size separately and focused mainly on root morphological plasticity (increased root proliferation in nutrient-rich patch), thus the effects of both patch strength and size on morphological and physiological plasticity are not well understood. In this study, we examined the foraging strategy of Neyraudia reynaudiana (Kunth) Keng ex Hithc, a pioneer grass colonizing degraded sites, with respect to patch strength and size in heterogeneously distributed phosphorus (P), and how foraging patchily distributed P affects total plant biomass production. Plants were grown in sand-culture pots divided into ½, », 1/6 compartments and full size and supplied with 0 + 0/30, 0 + 7.5/30 and 7.5 + 0/30 mg P/kg dry soil as KH2PO4 or 0 + 15/15, 0 + 18.5/ 18.5, 7.5 + 15/15 mg kg - 1 in the homogenous treatment. The first amount was the P concentration in the central region, and that the second amount was the P concentration in the outer parts of the pot. RESULTS: After 3 months of growth under experimental conditions, significantly (p < 0.05) high root elongation, root surface area, root volume and average root diameter was observed in large patches with high patch strength. Roots absorbed significantly more P in P-replete than P-deficient patches. Whole plant biomass production was significantly higher in larger patches with high patch strength than small patches and homogeneous P distribution. CONCLUSION: The result demonstrates that root morphological and physiological plasticity are important adaptive strategies for foraging patchily distributed P and the former is largely determined by patch strength and size. The results also establish that foraging patchily distributed P resulted in increased total plant biomass production compared to homogeneous P distribution.

Dynamics of major air pollutants from crop residue burning in mainland China, 2000-2014.

Based on satellite image data and China's Statistical Yearbooks (2000 to 2014), we estimated the total mass of crop residue burned, and the proportion of residue burned in the field vs. indoors as domestic fuel. The total emissions of various pollutants from the burning of crop residue were estimated for 2000-2014 using the emission factor method. The results indicate that the total amount of crop residue and average burned mass were 8690.9Tg and 4914.6Tg, respectively. The total amount of emitted pollutants including CO2, CO, NOx, VOCs, PM2.5, OC (organic carbon), EC (element carbon) and TC (total carbon) were 4212.4-8440.9Tg, 192.8-579.4Tg, 4.8-19.4Tg, 18.6-61.3Tg, 18.8-49.7Tg, 6.7-31.3Tg, 2.3-4.7Tg, and 8.5-34.1Tg, respectively. The emissions of pollutants released from crop residue burning were found to be spatially variable, with the burning of crop residue mainly occurring in Northeast, North and South China. In addition, pollutant emissions per unit area (10 km × 10 km) were mostly concentrated in the central and eastern regions of China. Emissions of CO2, NOx, VOCs, OC and TC were mainly from rice straw burning, while burning of corn and wheat residues contributed most to emissions of CO, PM2.5 and EC. The increased ratio of PM2.5 emissions from crop residue burning to the total emitted from industry during the study period is attributed to the implementation of strict emissions management policies in Chinese industry. This study also provides baseline data for assessment of the regional atmospheric environment.

Bioaccumulation and detoxification mechanisms for lead uptake identified in Rhus chinensis Mill. seedlings.

A greenhouse experiment was conducted to assay the bioaccumulation and tolerance characteristics of Rhus chinensis Mill. to lead (Pb). The effects of exposing R. chinensis Mill seedlings to increasing Pb concentrations (0, 250, 500, 100mgkg-1) in the soil were assessed by measuring Pb accumulation, subcellular distribution, ultrastructure, photosynthetic characteristics, antioxidative enzyme activity, malondialdehyde content, and phytochelatin content. The majority of Pb taken up by R. chinensis Mill was associated with the cell wall fraction in the roots, where the absorption of Ca increased to maintain cell wall stability, and Pb deposits were found in the intercellular space or in the cell wall structures. In leaves, Pb was primarily stored in the cell wall, while it was compartmentalized into the vacuolar structures in the stem. Pb concentrations adversely affected the morphology of Rhus chinensis Mill cellular substructures. Furthermore, increased Peroxidase (POD) and catalase (CAT) activity was observed in plants grown in Pb-amended soil, and this may have led to reduced ROS to maintain the function of the membrane. Changes in phytochelatin levels (PCs) that were observed in Pb treated plants suggest that PCs formed complexes with Pb in the cytoplasm to reduce Pb2+ toxicity in the metabolically active cellular compartment. This mechanism may allow for the plant to accumulate higher concentrations of toxic Pb and survive for a longer period of time. Our study provides a better understanding of how Rhus chinensis Mill detoxifies Pb.

Lead tolerance mechanism in Conyza canadensis: subcellular distribution, ultrastructure, antioxidative defense system, and phytochelatins.

We used hydroponic experiments to examine the effects of different concentrations of lead (Pb) on the performance of the Pb-tolerable plant Conyza canadensis. In these experiments, most of the Pb was accumulated in the roots; there was very little Pb accumulated in stems and leaves. C. canadensis is able to take up significant amounts of Pb whilst greatly restricting its transportation to specific parts of the aboveground biomass. High Pb concentrations inhibited plant growth, increased membrane permeability, elevated antioxidant enzyme activity in roots, and caused a significant increase in root H2O2 and malondialdehyde content. Analysis of Pb content at the subcellular level showed that most Pb was associated with the cell wall fraction, followed by the nucleus-rich fraction, and with a minority present in the mitochondrial and soluble fractions. Furthermore, transmission electron microscopy and energy dispersive X-ray analysis of root cells revealed that the cell wall and intercellular space in C. canadensis roots are the main locations of Pb accumulation. Additionally, high Pb concentrations adversely affected the cellular structure of C. canadensis roots. The increased enzyme activity suggests that the antioxidant system may play an important role in eliminating or alleviating Pb toxicity in C. canadensis roots. However, the levels of non-protein sulfhydryl compounds, glutathione, and phytochelatin did not significantly change in either the roots or leaves under Pb-contaminated treatments. Our results provide strong evidence that cell walls restrict Pb uptake into the root and act as an important barrier protecting root cells, while demonstrating that antioxidant enzyme levels are correlated with Pb exposure. These findings demonstrate the roles played by these detoxification mechanisms in supporting Pb tolerance in C. canadensis.

Effect of arbuscular mycorrhizal symbiosis on growth and biochemical characteristics of Chinese fir (Cunninghamia lanceolata) seedlings under low phosphorus environment.

Background: The continuous establishment of Chinese fir (Cunninghamia lanceolata) plantations across multiple generations has led to the limited impact of soil phosphorus (P) on tree growth. This challenge poses a significant obstacle in maintaining the sustainable management of Chinese fir. Methods: To investigate the effects of Arbuscular mycorrhizal fungi (AMF) on the growth and physiological characteristics of Chinese fir under different P supply treatments. We conducted an indoor pot simulation experiment in the greenhouse of the Forestry College of Fujian Agriculture and Forestry University with one-and-half-year-old seedlings of Chinese fir from March 2019 to June 2019, with the two P level treatment groups included a normal P supply treatment (1.0 mmol L-1 KH2PO4, P1) and a no P supply treatment (0 mmol L-1 KH2PO4, P0). P0 and P1 were inoculated with Funneliformis mosseae (F.m) or Rhizophagus intraradices (R.i) or not inoculated with AMF treatment. The AMF colonization rate in the root system, seedling height (SH), root collar diameter (RCD) growth, chlorophyll (Chl) photosynthetic characteristics, enzyme activities, and endogenous hormone contents of Chinese fir were estimated. Results: The results showed that the colonization rate of F.m in the roots of Chinese fir seedlings was the highest at P0, up to 85.14%, which was 1.66 times that of P1. Under P0 and P1 treatment, root inoculation with either F.m or R.i promoted SH growth, the SH of R.i treatment was 1.38 times and 1.05 times that of F.m treatment, respectively. In the P1 treatment, root inoculation with either F.m or R.i inhibited RCD growth. R.i inhibited RCD growth more aggressively than F.m. In the P0 treatment, root inoculation with F.m and R.i reduced the inhibitory effect of phosphorus deficiency on RCD. At this time, there was no significant difference in RCD between F.m, R.i and CK treatments (p < 0.05). AMF inoculation increased Fm, Fv, Fv/Fm, and Fv/Fo during the chlorophyll fluorescence response in the tested Chinese fir seedlings. Under the two phosphorus supply levels, the trend of Fv and Fm of Chinese fir seedlings in different treatment groups was F.m > R.i > CK. Under P0 treatment, The values of Fv were 235.86, 221.86 and 147.71, respectively. The values of Fm were 287.57, 275.71 and 201.57, respectively. It increased the antioxidant enzyme activity and reduced the leaf's malondialdehyde (MDA) content to a certain extent. Conclusion: It is concluded that AMF can enhance the photosynthetic capacity of the host, regulate the distribution of endogenous hormones in plants, and promote plant growth by increasing the activity of antioxidant enzymes. When the P supply is insufficient, AMF is more helpful to plants, and R.i is more effective than F.m in alleviating P starvation stress in Chinese fir.

Dynamics of Non-Structural Carbohydrates Release in Chinese Fir Topsoil and Canopy Litter at Different Altitudes.

Non-structural carbohydrates (NSCs) are labile components in forest litter that can be released quickly at the early stage of litter decomposition and accelerate the metabolic turnover of soil microorganisms, which is essential for the formation of forest soil organic matter. Therefore, understanding the NSCs response mechanisms to forest litter at different altitudes is critical for understanding nutrient cycling in the forest soil under climate change conditions. In this study, we used the net bag decomposition method to observe the dynamics of NSCs release in Chinese fir topsoil and canopy litter at four altitudes for 360 days based on the climatic zone characteristics distributed vertically along the elevation of Wuyi Mountain. The release of NSCs in Chinese fir litter rise gradually with height increases during the decomposition. The difference of the cumulative release percentage of soluble sugar between different altitudes is more significant than that of starch. The response of the NSC content in different treatment groups at four altitudes are different. The release of NSCs in the leaf canopy litter is higher than that in the leaf topsoil litter. On the contrary, the release of NSCs in the mixture of leaf and twig topsoil litter is higher than that in the mixture of leaf and twig canopy litter. Taken together, this study is of great significance for a comprehensive understanding of the effect of climate change on NSCs during the decomposition of Chinese fir litter.

Environmental Adaptability and Energy Investment Strategy of Different Cunninghamia lanceolata Clones Based on Leaf Calorific Value and Construction Cost Characteristics.

The calorific value and construction cost of leaves reflect the utilization strategy of plants for environmental resources. Their genetic characteristics and leaf functional traits as well as climate change affect the calorific values. This study explores the differences in energy investment strategies and the response characteristics of energy utilization in leaves to climate change among nine clones of Chinese fir (Cunninghamia lanceolata). Considering the objectives, the differences in the energy utilization strategies were analyzed by determining the leaf nutrients, specific leaf area, and leaf calorific value and by calculating the construction cost. The results showed a significant difference in the ash-free calorific value and construction cost of leaves among different Chinese fir clones (p < 0.05). There were also significant differences in leaf carbon (C) content, leaf nitrogen (N) content, specific leaf area, and ash content. The correlation analysis showed that leaves' ash-free calorific value and construction cost were positively correlated with the C content. Principal component analysis (PCA) showed that P2 is inclined to the "fast investment return" energy investment strategy, while L27 is inclined to the "slow investment return" energy investment strategy. Redundancy analysis (RDA) indicates that the monthly average temperature strongly correlates positively with leaf construction cost, N content, and specific leaf area. The monthly average precipitation positively impacts the ash-free calorific value and construction cost of leaves. In conclusion, there are obvious differences in energy investment strategies among different Chinese fir clones. When temperature and precipitation change, Chinese fir leaves can adjust their energy investment to adapt to environmental changes. In the future, attention should be paid to the impact of climate change-related aspects on the growth and development of Chinese fir plantations.

Application of a novel strong promoter from Chinese fir (Cunninghamia lanceolate) in the CRISPR/Cas mediated genome editing of its protoplasts and transgenesis of rice and poplar.

Novel constitutive promoters are essential for plant biotechnology. Although in angiosperms, a number of promoters were applied in monocots or dicots genetic engineering, only a few promoters were used in gymnosperm. Here we identified two strong promoters (Cula11 and Cula08) from Chinese fir (C. lanceolate) by screening the transcriptomic data and preliminary promoter activity assays in tobacco. By using the newly established Chinese fir protoplast transient expression technology that enables in vivo molecular biology studies in its homologous system, we compared the activities of Cula11 and Cula08 with that of the commonly used promoters in genetic engineering of monocots or dicots, such as CaM35S, CmYLCV, and ZmUbi, and our results revealed that Cula11 and Cula08 promoters have stronger activities in Chinese fir protoplasts. Furthermore, the vector containing Cas gene driven by Cula11 promoter and sgRNA driven by the newly isolated CulaU6b polyIII promoters were introduced into Chinese fir protoplasts, and CRISPR/Cas mediated gene knock-out event was successfully achieved. More importantly, compared with the commonly used promoters in the genetic engineering in angiosperms, Cula11 promoter has much stronger activity than CaM35S promoter in transgenic poplar, and ZmUbi promoter in transgenic rice, respectively, indicating its potential application in poplar and rice genetic engineering. Overall, the novel putative constitutive gene promoters reported here will have great potential application in gymnosperm and angiosperm biotechnology, and the transient gene expression system established here will serve as a useful tool for the molecular and genetic analyses of Chinese fir genes.

Propagation Methods Decide Root Architecture of Chinese Fir: Evidence from Tissue Culturing, Rooted Cutting and Seed Germination.

The root is the main organ of a plant for absorbing resources and whose spatial distribution characteristics play an important role in the survival of seedlings after afforestation. Chinese fir (Cunninghamia lanceolata) is one of China's most important plantation species. To clarify the effects of propagation methods on root growth and spatial distribution characteristics of Chinese fir trees, sampled trees cultivated by seed germination, tissue culture, and asexual cutting of Chinese fir were taken as the research objects. The root morphology, geometric configuration, and spatial distribution characteristics of different trees were analyzed. The influence of geometric root morphology on its spatial distribution pattern was explored by correlation analysis, and the resource acquisition characteristics reflected by the roots of Chinese fir trees with different propagation methods are discussed. The main results showed that the root mean diameter (1.56 mm, 0.95 mm, and 0.97 mm), root volume (2.98 m3, 10.25 m3, and 4.07 m3), root tip count (397, 522, and 440), main root branch angle (61°, 50° and 32°) and other geometric configurations of Chinese fir under seed germination, tissue culture and rooted cutting respectively, were significantly different, which resulted in different distribution characteristics of roots in space. Chinese fir seed germination had noticeable axial roots, and the growth advantage was obvious in the vertical direction. A fishtail branch structure (TI = 0.87) was constructed. The shallow root distribution of tissue culture and rooted cutting was obvious, and belonged to the fork branch structure (TI = 0.71 and 0.74, respectively). There was a tradeoff in the spatial growth of the root system of Chinese fir trees with different propagation methods to absorb nutrients from heterogeneous soil patches. A negative correlation was present between the root system and root amplitude. There was an opposite spatial growth trend of Chinese fir trees with different propagation methods in the vertical or horizontal direction. In conclusion, selecting suitable propagation methods to cultivate Chinese fir trees is beneficial to root development and the "ideal" configuration formation of resource acquisition to improve the survival rate of Chinese fir afforestation.

Transcriptome Level Analysis of Genes of Exogenous Ethylene Applied under Phosphorus Stress in Chinese Fir.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is a widely grown gymnosperm in China. Phosphorus (P) is an indispensable nutrient for the growth of Chinese fir. Inorganic phosphate (Pi) deficiency exists in soils of many Chinese fir planting area regions, and the trees themselves have limited efficiency in utilizing P from the soil. Ethylene is important in regulation responses to nutrient deficiencies. However, little is known about how ethylene signals participate in Pi stress in Chinese fir. A total of six different treatments were performed to reveal the transcript levels of Chinese fir under Pi, ethephon (an ethylene-releasing compound), and CoCl2 (cobalt chloride, an ethylene biosynthesis inhibitor) treatments. We assembled a full-length reference transcriptome containing 22,243 unigenes as a reference for UMI RNA-seq (Digital RNA-seq). There were 586 Differentially Expressed Genes (DEGs) in the Pi starvation (NP) group, while DEGs from additional ethephon or CoCl2 in NP were 708 and 292, respectively. Among the DEGs in each treatment, there were 83 TFs in these treatment groups. MYB (v-myb avian myeloblastosis viral oncogene homolog) family was the most abundant transcription factors (TFs). Three ERF (Ethylene response factor) family genes were identified when only ethylene content was imposed as a variable. Enrichment analysis indicated that the ascorbate and aldarate metabolism pathway plays a key role in resistance to Pi deficiency. This study provides insights for further elucidating the regulatory mechanism of Pi deficiency in Chinese fir.

The impact of genetic modified Ma bamboo on soil microbiome.

Evaluating the potential alteration of microbial communities is a vital step for biosafety of genetic modified plants. Recently, we have produced genetic modified Ma bamboo with increased cold and drought tolerance by anthocyanin accumulation. In this work, we aim to study the potential effects on microbial communities in rhizosphere soils during the cultivation of genetic modified bamboo. Rhizosphere and surrounding soil were collected at 3-month post-transplant. The amplicon (16S rDNA and ITS1) were sequenced for analysis of bacterial and fungal communities. Multiple software and database (Picrust2, FAPROTAX and FUNGulid) were applied to predict and compare the microbial functions involving basic metabolisms, nitrogen usage and presence of plant pathogens. There were no substantial change of the structure and abundance of rhizosphere soil microbial communities between genetic modified and wild type bamboo. For the surrounding soil, the bacterial biota α-diversity increased (chao1: 1,001 ± 80-1,276 ± 84, observed species: 787 ± 52-1,194 ± 137, PD whole tree: 75 ± 4-117 ± 18) and fungal biota α-diversity decreased (chao1: 187 ± 18-145 ± 10) in samples of genetic modified bamboo compared to those of wild type bamboo. The microbiota predicted functions did not change or had no negative alteration between genetic modified and wild type bamboo, in both rhizosphere and surrounding soils. As a conclusion, the growth of genetic modified bamboo had no substantial change on rhizosphere soil microbial communities, while minor alteration on bamboo surrounding soil microbial communities with no harmful effects. Moreover, the genetic modified bamboo had no negative effect on the predicted functions of microbiota in soil.

Effect of Glomus intraradices on root morphology, biomass production and phosphorous use efficiency of Chinese fir seedlings under low phosphorus stress.

Introduction: Available phosphorus (P) scarcity in the highly weathered soils of the subtropical forests in southern China is a serious concern. To ensure whether inoculation of arbuscular mycorrhizal fungi (AMF) with Chinese fir (Cunninghamia lanceolata) under low P stress conditions could promote its growth and P utilization capacity, an indoor pot simulation experiment was carried out with the different P supply treatments and Chinese fir seedlings as the tested material. Methods: The experiment had two P supply treatments, no P supply (P0, 0 mmol·L-1 KH2PO4) and normal P supply (P1, 1.0 mmol·L-1 KH2PO4). The seedling in each P supply treatment was inoculated with Glomus intraradices (Gi), a widespread species of AMF in the natural environment, and with no AMF inoculation as a control treatment (CK). The Gi infection rate in the root system, root cortex tissue dissolution rate, root morphological indexes and biomass, whole plant P use efficiency, and root P use efficiency of Chinese fir were determined under different treatment conditions. Results and Discussion: The results showed that P0 treatment significantly increased the Gi infection rate (p< 0.05). After inoculating AMF with different P supply treatments, the root cortex tissue dissolution rate was considerably enhanced. In contrast, the Chinese fir's root length and surface area were reduced; however, the root volume did not change significantly. The average root diameter in the P0 treatment and inoculated with AMF was significantly more prominent than in the uninoculated treatment (p< 0.05). The root biomass and root-to-shoot ratio at different P supply treatments were significantly higher in the Gi infection treatment than in the CK group. Under different P supply treatments, root inoculation with Gi promoted root P use efficiency and whole plant P use efficiency. In conclusion, low P stress condition promoted the colonization of AMF in the root system, increased the dissolution of root cortex tissue, root volume, and the average diameter, and promoted root biomass accumulation and P use efficiency.

Comprehensive Transcriptome Analysis of Stem-Differentiating Xylem Upon Compression Stress in Cunninghamia Lanceolata.

Compression wood (CW) in gymnosperm brings great difficulties to wood industry using wood as raw materials since CW presents special wood structure and have different physical and chemical properties from those of normal wood (NW). Chinese fir (Cunninghamia lanceolata) is widely distributed in China. However, global transcriptome profiling of coding and long non-coding RNA in response to compression stress has not been reported in the gymnosperm species. In this study, we revealed that CW in Chinese fir exhibited distinct morphology and cytology properties compared with those of NW, including high lignin content, thick and round tracheid cells. Furthermore, we combined both PacBio long-read SMRT sequencing (Iso-Seq) and Illumina short-read RNA-Seq to reveal the transcriptome in stem-differentiating xylem (SDX) under different time points (2, 26, and 74 h) upon compression stress in NW, CW, and OW (opposite wood), respectively. Iso-Seq was successfully assembled into 41,253 de-novo full-length transcriptome reference (average length 2,245 bp). Moreover, there were striking differences in expression upon compression stress, which were involved 13 and 7 key enzyme genes in the lignin and cellulose synthesis, respectively. Especially, we revealed 11 secondary growth-related transcription factors show differential expression under compression stress, which was further validated by qRT-PCR. Finally, the correlation between 6,533 differentially expressed coding genes and 372 differentially expressed long non-coding RNAs (lncRNAs) indicates that these lncRNAs may affect cell wall biogenesis and xyloglucan metabolism. In conclusion, our results provided comprehensive cytology properties and full-length transcriptome profiling of wood species upon compression stress. Especially we explored candidate genes, including both coding and long non-coding genes, and provided a theoretical basis for further research on the formation mechanism of CW in gymnosperm Chinese fir.

Spatiotemporal distribution of canopy litter and nutrient resorption in a chronosequence of different development stages of Cunninghamia lanceolata in southeast China.

Canopy litter is an important component of coarse woody debris (CWD), which affects nutrient and carbon cycling in forest ecosystems. For marcescent plant species (characterized by dead branches and leaves remaining in the canopy for several years before abscission), nutrient resorption from senescing leaves is an important nutrient conservation strategy. However, investigating the ecological function of canopy litter is challenging due to its limited accessibility and also the heterogeneous canopy microclimate in terms of light transmission, temperature and moisture. We studied the spatiotemporal distribution of canopy litter mass and seasonal dynamics of leaf nutrients and nutrient resorption during senescence in the canopy along a chronosequence of Chinese fir [Cunninghamia lanceolata (Lamb.) Hook] plantations in southeast China. The dry mass weight of dead branches and dead leaves in the canopy significantly increased with stand stage (14.6, 14.2, and 17.4 t ha-1 for young, middle-aged, and mature stands respectively), accounting for high proportions of total aboveground litter of 85.7%, 79.1% and 80.0%, respectively, along with annual litterfall production (2.44, 3.75, and 4.34 t ha-1, respectively). The canopy height distribution of dead branches and leaves also increased with stand age, ranging from 0 to 4 m in young stands, 3-8 m in middle-aged stands, to 4-10 m in mature stands. The seasonal pattern of canopy litter mass was the inverse of litterfall production: canopy litter mass peaked, while litterfall production was lowest in winter. Mean N, P, K, and Mg nutrient resorption efficiencies across stands at each stage were 53.8-58.9%, 64.0-68.9%, 85.0-90.2%, and 46.5-56.6%, respectively, while Ca was not retranslocated from senescing leaves. In summary, Chinese fir plantations retain large amounts of dead branches and leaves in the canopy from which at least ~50% of the nutrients N, P, K and Mg are recycled, representing an important nutrient conservation strategy that has evolved to adapt to nutrient-limited habitats. Canopy litter therefore plays an important role in these forest plantation ecosystems and should be protected instead of being removed from the canopy to the forest floor.