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Chinese fir (Cunninghamia lanceolata) is the most cultivated timber species in China, with a plantation area of 11 million ha. Due to its extensive geographical distribution, drought stress caused by the spatial and seasonal heterogeneity of precipitation has limited its survival and productivity. To facilitate the breeding of drought-tolerant clones and understand the inter-response mechanisms to drought stress, we screened two drought-tolerant (DT) clones and evaluated their differences in physiological and molecular response to drought. The results showed that the No. 228 clone (high-DT ability) had higher antioxidant enzyme abilities than the No. 026 clone (low-DT ability) under drought stress, e.g., peroxidase (POD), polyphenol oxidase (PPO), superoxide dismutase (SOD), and catalase (CAT). Transcriptome analyses revealed that 6637 genes and 1168 genes were up-regulated in No. 228 and No. 026 under drought stress, respectively, when compared to the control (CK). The genes may participate in response to drought-stimulated signal transduction, water/oxygen-containing compound synthesis, photosynthesis, and transmembrane transport functions. Particularly, under drought stress, 14,213 up-regulated and differentially expressed genes (DEGs) were observed in the No. 228 clone compared with the No. 026 clone, and 4274 up-regulated genes were differentially expressed (15-fold difference). These significant DEGs were involved in plant hormone signal transduction, flavonoid biosynthesis, peroxisomes, and other key pathways related to drought. Interestingly, under drought stress, two Chitinases (ClCHIs) and four POD genes (ClPERs) were induced to express in No. 228, which was consistent with the higher antioxidant enzyme activities in No. 228. A heat map of 49 DEGs revealed that dehydrin family genes, ion binding/transmembrane proteins, auxin receptor proteins, and ethylene-responsive transcription factors were significantly up-regulated under drought stress. The results can enhance our understanding of drought tolerance mechanisms and provide a guideline for screening DT genes and breeding drought-tolerant Chinese fir clones. [ABSTRACT FROM AUTHOR]

Research Highlights: The present study firstly reported the metabolic dynamics of astringent seed, a special type of abortion in Chinese fir, during the astringent material stages. The results provide a reference for further study on its occurrence mechanism and enrich the understanding of the plant seed developmental physiology. Background and Objectives: Astringent seed is a type of abortive phenomenon in Chinese fir, which significantly reduces the yield and quality of elite seeds for its high-incidence and indistinguishableness in seed orchard. Embryo defects can be observed in the astringent seed, accompanied with rapid accumulation of secondary metabolites. However, types of those metabolites in astringent seed, dynamic changes during seed growth process, and different accumulative characteristics compared to germinable seed have not been explained. Materials and Methods: Astringent and germinable seed samples were collected at four stages aim to determine the differences in their metabolic patterns. A liquid chromatography-mass spectrometry (LC-MS) detection was used to generate the raw metabolic peaks. Bioinformatics statistical strategies were used to further investigation. Results: A total of 421 metabolites were screened and 112 metabolites were identified as the different expressive metabolites including 68 up-regulated and 44 down-regulated metabolites. Those different expressive metabolites were grouped into 26 classes. Flavone, flavonol, and amino acid derivatives compounds were the most varied metabolites. Four subcategories which could represent the diverse basic expressive patterns or accumulative activity in different sample groups were further clustered. Moreover, pathways related to biosynthesis/degradation/metabolism of flavonoid-like compounds, amino acid/nucleotides derivatives, zeatin, and IAA were clearly enriched. Conclusions: Significant metabolic differences were observed across and between astringent and germinable seeds 105 d after pollination. Massive accumulation of flavonoids-like compounds, significant reduction of amino acids/nucleotides and their derivatives, and the abnormal expression of phytohormones, lipids and other secondary metabolites are the main metabolic characteristics in astringent seeds. [ABSTRACT FROM AUTHOR]

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]

In vitro pollen germination provides a novel approach and strategy to accelerate genetic improvement of tree breeding. Studies about pollen germination and tube growth of Chinese fir are limited. Therefore, this study aimed to investigate the effects of sucrose, boric acid, pH, and time of incubation on pollen germination and tube growth. Pollen from 9 clones were selected. In vitro germination was performed in basic media as control, and in different concentrations of sucrose (0, 10 and 15%), boric acid (0.01, 0.1 and 0.2%), and pH levels (4.5, 5 and 7). Pollen germination rates and tube growth were recorded periodically at 1, 12, 24, and 48 h. The results showed that sucrose imposes significant effects on pollen germination and tube growth. The effects are most obvious at concentration of 15%. Boric acid significantly promoted germination and tube growth. The promotion was most notable in lower concentration of 0.01%. The media adjusted to pH 7.0 boosted the germination and pollen tube growth. The optimum time of incubation was 24 and 48 h for pollen germination and tube growth, respectively. Sucrose, pH, and time of incubation were positively correlated, whereas boric acid negatively correlated with pollen germination and tube growth. This study provided experimental evidences for selecting viable pollens for Chinese fir breeding. [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]

To guide the cultivation of superior Chinese fir plantlets, we designed an L16(4)4 orthogonal experiment to determine how leaf age and exogenous hormones influence key growth processes in leaf cuttings. Hormone concentration and treatment duration significantly affected leaf cuttings in all three age categories; 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA), and treatment time exerted the strongest effects on callus initiation rates. Additionally, NAA had the largest effect on the rooting rate across all cuttings, and all three hormones significantly influenced the bud germination rate. Based on our experimental results, expected optimal treatments for callus initiation were 10 mg∙L−1 indole-3-butyric acid (IBA) for 10 min, 30 mg∙L−1 NAA for 15 min, and 10 mg∙L−1 NAA plus 30 mg∙L−1 IBA for 10 min. For the rooting rate, the expected optimal treatment was 50 mg∙L−1 NAA and 40 mg∙L−1 IBA for 5–20 min. Finally, for bud germination, optimal treatments were 20 min of immersion in water, 30 mg∙L−1 6-BA plus 50 mg∙L−1 NAA for 15 min, and 30 mg∙L−1 6-BA for 5 min. Plantlet formation only occurred in the