Transcriptomic Revelation of Phenolic Compounds Involved in Aluminum Toxicity Responses in Roots of Cunninghamia lanceolata (Lamb.) Hook.

: Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is one of the most important coniferous evergreen tree species in South China due to its desirable attributes of fast growth and production of strong and hardy wood. However, the yield of Chinese fir is often inhibited by aluminum (Al) toxicity in acidic soils of South China. Understanding the molecular mechanisms of Chinese fir root responses to Al toxicity might help to further increase its productivity. Here we used the Illumina Hiseq4000 platform to carry out transcriptome analysis of Chinese fir roots subjected to Al toxicity conditions. A total of 88.88 Gb of clean data was generated from 12 samples and assembled into 105,732 distinct unigenes. The average length and N50 length of these unigenes were 839 bp and 1411 bp, respectively. Among them, 58362 unigenes were annotated through searches of five public databases (Nr: NCBI non-redundant protein sequences, Swiss-Prot: A manually annotated and reviewed protein sequence database, GO: Gene Ontology, KOG/COG: Clusters of Orthologous Groups of proteins, and KEGG: the Kyoto Encyclopedia of Genes and Genomes database), which led to association of unigenes with 44 GO terms. Plus, 1615 transcription factors (TFs) were functionally classified. Then, differentially expressed genes (DEGs, |log2(fold change)| ≥ 1 and FDR ≤ 0.05) were identified in comparisons labelled TC1 (CK-72 h/CK-1 h) and TC2 (Al-72 h/Al-1 h). A large number of TC2 DEGs group were identified, with most being down-regulated under Al stress, while TC1 DEGs were primarily up-regulated. Combining GO, KEGG, and MapMan pathway analysis indicated that many DEGs are involved in primary metabolism, including cell wall metabolism and lipid metabolism, while other DEGs are associated with signaling pathways and secondary metabolism, including flavonoids and phenylpropanoids metabolism. Furthermore, TFs identified in TC1 and TC2 DEGs represented 21 and 40 transcription factor families, respectively. Among them, expression of bHLH, C2H2, ERF, bZIP, GRAS, and MYB TFs changed considerably under Al stress, which suggests that these TFs might play crucial roles in Chinese fir root responses to Al toxicity. These differentially expressed TFs might act in concert with flavonoid and phenylpropanoid pathway genes in fulfilling of key roles in Chinese fir roots responding to Al toxicity.

Effects of sulfuric, nitric, and mixed acid rain on Chinese fir sapling growth in Southern China.

The influence of acid rain on plant growth includes direct effects on foliage as well as indirect soil-mediated effects that cause a reduction in root growth. In addition, the concentration of NO3- in acid rain increases along with the rapid growth of nitrogen deposition. In this study, we investigated the impact of simulated acid rain with different SO42-/NO3- (S/N) ratios, which were 1:0, 5:1, 1:1, 1:5 and 0:1, on Chinese fir sapling growth from March 2015 to April 2016. Results showed that Chinese fir sapling height growth rate (HGR) and basal diameter growth rate (DGR) decreased as acid rain pH decreased, and also decreased as the percentage of NO3- increased in acid rain. Acid rain pH significantly decreased the Chlorophyll a (Chla) and Chlorophyll b (Chlb) content, and Chla and Chlb contents with acid rain S/N 1:5 were significantly lower than those with S/N 1:0 at pH 2.5. The chlorophyll fluorescence parameters, maximal efficiency of Photosystem II photochemistry (Fv/Fm) and non-photochemical quenching coefficient (NPQ), with most acid rain treatments were significantly lower than those with CK treatments. Root activities first increased and then decreased as acid rain pH decreased, when acid rain S/N ratios were 1:1, 1:5 and 0:1. Redundancy discriminant analysis (RDA) showed that the Chinese fir DGR and HGR had positive correlations with Chla, Chlb, Fv/Fm ratio, root activity, catalase and superoxide dismutase activities in roots under the stress of acid rain with different pH and S/N ratios. The structural equation modelling (SEM) results showed that acid rain NO3- concentration and pH had stronger direct effects on Chinese fir sapling HGR and DGR, and the direct effects of acid rain NO3- concentration and pH on HGR were lower than those on DGR. Our results suggest that the ratio of SO42- to NO3- in acid rain is an important factor which could affect the sustainable development of monoculture Chinese fir plantations in southern China.

Effects of short-term warming and nitrogen addition on the quantity and quality of dissolved organic matter in a subtropical Cunninghamia lanceolata plantation.

Increasing temperature and nitrogen (N) deposition are two large-scale changes projected to occur over the coming decades. The effects of these changes on dissolved organic matter (DOM) are largely unknown. This study aimed to assess the effects of warming and N addition on the quantity and quality of DOM from a subtropical Cunninghamia lanceolata plantation. Between 2014 and 2016, soil solutions were collected from 0-15, 15-30, and 30-60 cm depths by using a negative pressure sampling method. The quantity and quality of DOM were measured under six different treatments. The spectra showed that the DOM of the forest soil solution mainly consisted of aromatic protein-like components, microbial degradation products, and negligible amounts of humic-like substances. Warming, N addition, and warming + N addition significantly inhibited the concentration of dissolved organic carbon (DOC) in the surface (0-15 cm) soil solution. Our results suggested that warming reduced the amount of DOM originating from microbes. The decrease in protein and carboxylic acid contents was mostly attributed to the reduction of DOC following N addition. The warming + N addition treatment showed an interactive effect rather than an additive effect. Thus, short-term warming and warming + N addition decreased the quantity of DOM and facilitated the migration of nutrients to deeper soils. Further, N addition increased the complexity of the DOM structure. Hence, the loss of soil nutrients and the rational application of N need to be considered in order to prevent the accumulation of N compounds in soil.

Optimization of hormone combinations for root growth and bud germination in Chinese fir (Cunninghamia lanceolata) clone leaf cuttings.

In order to ascertain the optimal hormone combination for Chinese fir (Cunninghamia lanceolata) leaf cuttings, an L16(4)4 orthogonal test of 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA) (0, 10, 30, or 50 mg · L-1 of each exogenous hormone) immersion for 5, 10, 15, or 20 min was conducted. Callus initiation rate and rooting promotion rate were mainly affected by treatment time, root length increase by 6-BA concentration, and bud germination rate and plantlet formation rate by NAA concentration. The expected optimal combination for callus initiation rate was 50 mg · L-1 6-BA + 0 mg · L-1 NAA + 30 mg · L-1 IBA + 10 min; for rooting promotion rate, it was 0-10 mg · L-1 6-BA + 10 mg · L-1 NAA + 30 mg · L-1 IBA + 20 min; for bud germination rate, it was 50 mg · L-1 6-BA + 0-10 mg · L-1 NAA + 0-30 mg · L-1 IBA + 20 min; and for seedling formation rate for No. 4, it was 10 mg · L-1 6-BA + 10 mg · L-1 NAA + 0 mg · L-1 IBA + 20 min. Light microscopy image analysis revealed that a cluster of primordial cells was produced 40 days after cutting, and mastoid cells developed into peninsula cells in calli that were cultured for 50 days.

Somatic Embryogenesis of Immature Cunninghamia lanceolata (Lamb.) Hook Zygotic Embryos.

Two efficient somatic embryogenesis systems were developed in Chinese fir, the most important conifer for industrial wood production in China. Three development stages (cleavage polyembryony, dominant embryo, and precotyledon) of immature embryos derived from 25 genotypes of open-pollinated mother trees were used as initial explants. Cleavage polyembryony-stage embryos with a 12.44% induction rate was the most embryogenic response stage. The highest frequency of embryogenic callus (13.86%) induction was obtained from DCR medium supplemented with 1.5 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.3 mg L-1 kinetin (KN). An average of 53.33 early somatic embryos were produced from approximately 0.2 g (fresh weight) embryogenic callus after 2 weeks of incubation on medium supplemented with 50 µmol L-1 abscisic acid (ABA) and 100 g L-1 polyethylene glycol (PEG) 6000. About 53% dominant embryos have an embryogenic response after a 6-week cultivation on medium supplemented with 1.0-2.0 mg L-1 benzyladenine (BA), 0.2 mg L-1 naphthylacetic acid (NAA) or 2,4-D, and 0.004 mg L-1 thidiazuron (TDZ). After three successive transfer cultures on medium supplemented with 1.5 mg L-1 BA, 0.2 mg L-1 NAA, and 0.004 mg L-1 TDZ, 4.49-16.51% of the embryos developed into somatic embryos.

Transcriptome and Degradome Sequencing Reveals Dormancy Mechanisms of Cunninghamia lanceolata Seeds.

Seeds with physiological dormancy usually experience primary and secondary dormancy in the nature; however, little is known about the differential regulation of primary and secondary dormancy. We combined multiple approaches to investigate cytological changes, hormonal levels, and gene expression dynamics in Cunninghamia lanceolata seeds during primary dormancy release and secondary dormancy induction. Light microscopy and transmission electron microscopy revealed that protein bodies in the embryo cells coalesced during primary dormancy release and then separated during secondary dormancy induction. Transcriptomic profiling demonstrated that expression of genes negatively regulating gibberellic acid (GA) sensitivity reduced specifically during primary dormancy release, whereas the expression of genes positively regulating abscisic acid (ABA) biosynthesis increased during secondary dormancy induction. Parallel analysis of RNA ends revealed uncapped transcripts for ∼55% of all unigenes. A negative correlation between fold changes in expression levels of uncapped versus capped mRNAs was observed during primary dormancy release. However, this correlation was loose during secondary dormancy induction. Our analyses suggest that the reversible changes in cytology and gene expression during dormancy release and induction are related to ABA/GA balance. Moreover, mRNA degradation functions as a critical posttranscriptional regulator during primary dormancy release. These findings provide a mechanistic framework for understanding physiological dormancy in seeds.

Physiological and proteomic analysis reveals the different responses of Cunninghamia lanceolata seedlings to nitrogen and phosphorus additions.

UNLABELLED: Both nitrogen (N) and phosphorus (P) additions in soils can increase tree photosynthetic rate (Pn), biomass accumulation and further increase primary production of plantation. However, the improved photosynthetic ability is varied from the added nutrient types and the mechanisms are sophisticated. In this study, an iTRAQ-based quantitative proteome combined with physiological analysis of Chinese fir (Cunninghamia lanceolata) leaves was performed to determine the common and different responses on photosynthetic process to the N and P additions. The results showed that, either N or P added in soils significantly increased Pn, but N addition had more positive effects than P addition in improving photosynthetic ability. Physiologically, N addition caused more in improving photosynthetic rate than P addition, which attributes to higher leaf N and chlorophyll contents, enlarged chloroplast size and more number of thylakoids. Proteomic data revealed that the increased Pn to N and P additions may attribute to the increased abundance of proteins involved in carbon fixation and RuBP regeneration during the light-independent reactions. However, N addition increased the abundance of photosystem II related proteins and P addition increased the abundance of photosystem I related proteins. Additionally, proteomic data also gave some clues on the different metabolic processes caused by N and P additions on glycolysis and TCA cycle, which were potentially related to higher growth and developmental rates of C. lanceolata. Therefore, this study provides new insights into the different photosynthesis and metabolic processes of Chinese fir in response to N and P additions. BIOLOGICAL SIGNIFICANCE: Fertilization is an important management measure to improve timber yield and primary production of Cunninghamia lanceolata, which is the largest planted coniferous species in southeast China. Nitrogen (N) and phosphorus (P) additions into soils can improve tree photosynthesis, and further increase plantation production. However, the mechanism of N and P additions in improving photosynthesis is still unclearly. In this study, a physiological measurement combined with proteomic analysis was performed on a controlled experiment in the greenhouse. These results improve understanding of the essentially photosynthetic activity and metabolic process of C. lanceolata responding to N and P fertilization.

Ion Flux in Roots of Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook) under Aluminum Stress.

Chinese fir is a tall, fast-growing species that is unique to southern China. In Chinese fir plantations, successive plantings have led to a decline in soil fertility, and aluminum toxicity is thought to be one of the main reasons for this decline. In this study, Non-invasive Micro-test Technology was used to study the effect of aluminum stress on the absorption of 4 different ions in the roots of the Chinese fir clone FS01. The results are as follows: with increased aluminum concentration and longer periods of aluminum stress, the H+ ion flow gradually changed from influx into efflux; there was a large variation in the K+ efflux, which gradually decreased with increasing duration of aluminum stress; and 1 h of aluminum stress uniformly resulted in Ca2+ influx, but it changed from influx to efflux after a longer period of aluminum stress. Changes in the different concentrations of aluminum had the largest influence on Mg2+.

Cell synchronization and isolation of chromosomes from Chinese fir root tips for flow cytometric analysis.

OBJECTIVES: To optimize treatment parameters to synchronize the cell cycle of Chinese fir root tip meristem cells. RESULTS: Optimal mitotic indices were achieved by treating seedlings with 1.25 mM hydroxyurea for 10 h, incubating for 7 h without chemical treatment to release the hydroxyurea block, and then treating emerging roots with 0.05 % colchicine for 16 h. The mitotic index of synchronized Chinese fir root tips was over 30 %. Suspensions of intact chromosomes were prepared using a simple slicing procedure. CONCLUSIONS: The chromosome preparations were suitable for flow cytometric characterization and sorting. The chromosome peaks of the observed flow karyotype resembled the predicted flow karyotype calculated from the Chinese fir chromosome size.

Effects of increased nitrogen deposition and rotation length on long-term productivity of Cunninghamia lanceolata plantation in southern China.

Cunninghamia lanceolata (Lamb.) Hook. has been widely planted in subtropical China to meet increasing timber demands, leading to short-rotation practices that deplete soil nutrients. However, increased nitrogen (N) deposition offsets soil N depletion. While long-term experimental data investigating the coupled effects related to short rotation practices and increasing N deposition are scarce, applying model simulations may yield insights. In this study, the CenW3.1 model was validated and parameterized using data from pure C. lanceolata plantations. The model was then used to simulate various changes in long-term productivity. Results indicated that responses of productivity of C. lanceolata plantation to increased N deposition were more related to stand age than N addition, depending on the proportion and age of growing forests. Our results have also shown a rapid peak in growth and N dynamics. The peak is reached sooner and is higher under higher level of N deposition. Short rotation lengths had a greater effect on productivity and N dynamics than high N deposition levels. Productivity and N dynamics decreased as the rotation length decreased. Total productivity levels suggest that a 30-year rotation length maximizes productivity at the 4.9 kg N ha(-1) year(-1) deposition level. For a specific rotation length, higher N deposition levels resulted in greater overall ecosystem C and N storage, but this positive correlation tendency gradually slowed down with increasing N deposition levels. More pronounced differences in N deposition levels occurred as rotation length decreased. To sustain C. lanceolata plantation productivity without offsite detrimental N effects, the appropriate rotation length is about 20-30 years for N deposition levels below 50 kg N ha(-1) year(-1) and about 15-20 years for N deposition levels above 50 kg N ha(-1) year(-1). These results highlight the importance of assessing N effects on carbon management and the long-term productivity of forest ecosystems.

[Effect of vanillin and P-hydroxybenzoic acid on physiological characteristics of Chinese fir seedlings].

Effects of vanillin and P-hydroxybenzoic acid at different concentrations on physiological characteristic of Chinese fir seedlings were studied by potted experiment. The results showed that 10 mmol.L-1 and 1 mmol.L-1 of two kinds of phenolics significantly reduced the content of chlorophyll, rate of photosynthesis and root activity, and that the higher the concentration of vanillin and P-hydroxybenzoic acid, the more the physiological activities was inhibited. By treatment with 1 and 10 mmol.L-1 vanillin, Chinese fir seedlings reduced its photosynthesis rate 25.1% and 37.0%, transpiration rate 20.3% and 37.0%, stomata conductance 33.7% and 46.8% and root activity 51.6% and 78.8%, respectively. The results suggested that vanillin and P-hydroxybenzoic acid accumulated in the soil by continuous cropping of Chinese fir may have some allelopathic effect on the seedlings of Chinese fir and the effect is one of the factors leading to the low productivity of continuously cropped Chinese fir forest.