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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. [ABSTRACT FROM AUTHOR]

• The difference between Ar and Af leaf samples was first characterized applying transcriptome and metabolome analysis. • Correlation analysis between metabolites and metabolic enzyme gene expression revealed flavonoid metabolic differences between Ar and Af. • This study screened and obtained key genes potentially involved in the differentially accumulation of flavonoids between Ar leaves and Af leaves. • This study provides an in-depth understanding of differentially flavonoid accumulation between Ar leaves and Af leaves. Anoectochilus roxburghii (Wall.) Lindl (Ar) and Anoectochilus formosanus Hayata (Af) are two species of valuable Chinese herbal medicines that belong to the genus Anoectochilus in Orchidaceae and have often been applied as the Chinese herbal medicine "Jin Xian Lian". However, these species exhibit obvious differences in leaf color, and the difference in their medicinal value remains unclear. In this study, the metabolomes of leaf samples of Ar and Af were analyzed by high-performance liquid chromatography-mass spectrometry (LC–MS) in the positive and negative ion modes. The analysis of the leaves of both species in the positive ion mode identified 4013 metabolites enriched in 106 metabolic pathways, and the analysis in the negative ion mode found 1530 metabolites enriched in 98 metabolic pathways. A differential metabolic profiling analysis of the results obtained in positive ion mode found that 816 differentially accumulated metabolites were enriched in 80 metabolic pathways, whereas 297 differentially accumulated metabolites enriched in 55 metabolic pathways were obtained from the analysis in the negative ion mode. The number of flavonoids was higher than those of alkaloids and terpenoids. A quantitative analysis of the differentially accumulated metabolites showed that the most highly upregulated metabolites (Af/Ar) among the top 20 differentially accumulated metabolites in Af were mainly related to amino acid-related metabolic pathways, whereas the most highly downregulated metabolites (Af/Ar) in Af were mainly associated with flavonoid-related metabolic pathways. In addition, the transcriptome sequencing of Ar and Af leaf samples yielded 40.12 Gb of transcriptomic data. The analysis of differentially expressed genes (DEGs) classified 555 metabolism-related DEGs into 23 metabolism-related KEGG pathways, and the top DEGs were involved in the phenylpropanoid biosynthetic pathways, which included up to 138 DEGs associated with the flavonoid metabolism pathway. In addition, significant differences in the flavonoid-related KEGG pathways were found. A comprehensive analysis of the metabolomes and transcriptomes showed that the genes annotated as chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), caffeoyl-CoA O -methyltransferase (CCOAOMT) and anthocyanidin synthase (ANS) were downregulated in Af leaves compared with Ar leaves, which might account for the downregulation of flavonoid-related metabolites in Af. In summary, both the metabolomic and transcriptomic analyses showed significant differences in flavonoid metabolism between the Ar and Af leaves, and the mechanisms underlying the molecular regulation of these differences were preliminarily analyzed. [ABSTRACT FROM AUTHOR]

Water and nitrogen stresses are major constraints for agricultural and forest productivity. Although the effects of water scarcity or nitrogen stress on plant growth, physiology, and yield have been widely studied, few studies have assessed the combined effects of both stresses. In the present study, we investigated the effects of different nitrogen forms (NO3−N, NH4+-N, and a combination of NO3−N + NH4+-N) on antioxidant enzyme activity, osmotic regulatory substances, and nitrogen assimilation in Chinese fir (Cunninghamia lanceolata) plantlets under drought stress (induced by 10% polyethylene glycol). We found that different N ionic forms had different effects on drought-stressed plantlets. Nitrogen supply greatly increased the activities of superoxide dismutase (SOD), peroxidase (POD) and polyphenol oxidase (PPO) when plantlets were exposed to water stress. The malondialdehyde (MDA) contents significantly decreased under the NH4+ + water stress treatment. The proline (Pr) contents significantly increased in both the NO3−N and NH4+-N + water stress treatment. The nitrate reductase (NR) increased by 7.1% in the NO3− + water stress treatment, and the glutamine synthetase (GS), and the glutamate synthase (GOGAT) activity increased in all the nitrogen + water stress treatments. These results suggested that nitrogen supply could alleviate the adverse effects of drought stress on plants by enhancing antioxidant defense and improving nitrogen assimilation, while the effects on plant tolerance to drought stress varied with nitrogen ionic forms. [ABSTRACT FROM AUTHOR]

• A. roxburghii was first characterized using high-throughput sequencing technologies and metabolomics. • Correlation analysis between metabolites and regulatory genes revealed flavonoid accumulation-related regulatory genes. • This study screened and obtained key genes potentially involved in the accumulation of flavonoids in A. roxburghii. • This study provides an in-depth understanding of the molecular mechanisms underlying flavonoid accumulation in A. roxburghii. Anoectochilus roxburghii , a species used in Chinese herbal medicine, has unique characteristics. This plant has important medicinal and ornamental value and is distributed primarily in China, including Fujian, Zhejiang, Jiangxi, and Guizhou provinces, and in Taiwan. Flavonoids are involved in leaf pigment formation and are major pharmacodynamic substances. However, the molecular mechanisms that regulate accumulation of flavonoids remain unclear, which has significantly limited their application. To elucidate the molecular mechanisms underlying the regulation of flavonoid accumulation in A. roxburghii , root, stem and leaf samples were collected for constructing transcriptomic and metabolomic datasets using RNA sequencing and liquid chromatography-mass spectrometry (LC–MS) techniques. Sequencing of the transcriptomes of the different organs generated 60.2 Gb of data, which were assembled into 186,865 unigenes. Metabolomic analysis resulted in the identification of 10,690 metabolites. Based on the ESI+ mode, 4,010, 4,008 and 4,013 metabolites were annotated in roots, stems, and leaves, respectively; based on the ESI– mode, 1,530, 1,530, and 1,531 metabolites were annotated, respectively. Differential analyses of the transcriptome and flavonoid metabolism of the different organs revealed the greatest significant differences between roots and leaves, followed by differences between stems and leaves; differences between roots and stems were the smallest. According to analysis of differentially expressed genes (DEGs), the secondary metabolism-related DEGs between roots and stems, between roots and leaves, and between leaves and stems were classified into 16, 21, and 19 secondary metabolic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Among these pathways, flavonoid biosynthesis, flavone and flavonol biosynthesis, and isoflavonoid biosynthesis significantly differed in terms of both gene expression and secondary metabolism. Moreover, the key genes involved in flavonoid accumulation were comprehensively analyzed using metabolic and transcriptomic data, and ten transcription factor-encoding genes and fourteen flavonoid biosynthesis genes were identified. Specifically, four of the ten transcription factor-encoding genes appear to activate CHS, CHI, CYP75A and ANR gene expression. The combined approach used in this study provides an in-depth understanding of the molecular regulatory mechanisms underlying flavonoid accumulation in A. roxburghii and is a powerful tool that can uncover valuable information for plant breeders. [ABSTRACT FROM AUTHOR]

Soil respiration and soil organic C content were investigated along a stand development sequence of Phoebe bournei plantations (7, 22, 38 a). The results showed that soil respiration rates of P. bournei plantations with different stand ages are characterized by a similar unimodal humpbacked trend, which peaked in summer. A significant exponential relationship between soil temperature and respiration rate was observed in the P. bournei plantations (P<0.01), while soil respiration rate showed a weak relationship with soil water content (P>0.05). With stand age development, the annual fluxes of soil respiration increased, being 1 117, 1 370 and 1 686 g⋅hm-2 a-1 at 7, 22 and 38 a stand, respectively. The Q10 value (i.e., temperature sensitivity index of soil respiration) also slightly increased with stand age. Soil organic carbon storage increased with stand development, and the mature forest is significantly higher than the middle and young forest (P<0.05). Pearson correlation analysis showed that the annual flux of soil respiration were significantly correlated with the density of surface soil organic C (P<0.01), and the organic C content of surface soil is an important determinant of the annual flux of soil respiration in these forests. [ABSTRACT FROM AUTHOR]

Genetic diversity and variation among 11 populations of Chinese fir from Fujian province and Taiwan were assessed using inter-simple sequence repeat (ISSR) markers to reveal the evolutionary relationship in their distribution range in this report. Analysis of genetic parameters of the different populations showed that populations in Fujian province exhibited a greater level of genetic diversity than did the populations in Taiwan. Compared to Taiwan populations, significant limited gene flow were observed among Fujian populations. An UPGMA cluster analysis showed that the most individuals of Taiwan populations formed a single cluster, whereas 6 discrete clusters were formed by each population from Fujian. All populations were divided into 3 main groups and that all 5 populations from Taiwan were gathered into a subgroup combined with 2 populations, Dehua and Liancheng, formed one of the 3 main groups, which indicated relative stronger relatedness. It is supported by a genetic structure analysis. All those results are suggesting different levels of genetic diversity and variation of Chinese fir between Fujian and Taiwan, and indicating different patterns of evolutionary process and local environmental adaption. [ABSTRACT FROM AUTHOR]

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+. [ABSTRACT FROM AUTHOR]

RNA isolation with RNA in a high quantity is a basic analytical method in plant genetics, molecular biology and related physiological investigations. To understand the genetic and molecular biology of Chinese fir, sufficient high-quality total RNA must be obtained for cDNA library construction and other downstream molecular applications. However, extracting RNA from Chinese fir is difficult and often requires the modification of existing protocols. Chinese fir tissues containing large amounts of polysaccharides and polyphenol compounds and are one of the most difficult plant tissues for RNA isolation. Therefore, we developed a simple method for extracting high-quality RNA from Chinese fir tissues. RNA isolations were performed within two hours, RNA quality was measured for yield and purity. Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing. It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications. Thus, this protocol represents a simple, efficient, and low-cost method. [ABSTRACT FROM AUTHOR]

Pollen grains produce certain metabolites, which can improve or inhibit germination and tube growth. Metabolomic analysis of germinating and growing Chinese fir pollen has not been reported. Therefore, this study aimed to analyse metabolites changes, content and expression in the germinating pollen of Chinese fir. To understand the metabolic differences, two clones from Chinese fir were selected. Metabolomics analyses were performed on three stages (1-, 24- and 48-h) during in vitro pollen germination. The metabolites profiles at different time points were analyzed by using liquid chromatography-mass spectrometry. The results showed that 171 peaks were screened; the corresponding differential metabolites of 121 peaks were classified into nine types of substances. The expression of metabolites showed significant differences across and between clones, and the variation was evident at all germination stages. The expression was obvious at the early stage of germination, which differed clearly from that of the late stage after pollen tube growth. Moreover, the metabolites were mainly enriched in 14 metabolic pathways. Pollen germination and tube growth and metabolites expressions changed per incubation time. Since this work is preliminary, we suggest further investigations to understand the relationship between the differential metabolites and pollen development, and factors affecting pollen germination process. [ABSTRACT FROM AUTHOR]