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Anthocyanins produce different-colored pigments in plant organs, which provide ornamental value. Thus, this study was conducted to understand the mechanism of anthocyanin synthesis in ornamental plants. Phoebe bournei, a Chinese specialty tree, has high ornamental and economic value due to its rich leaf color and diverse metabolic products. Here, the metabolic data and gene expression of red P. bournei leaves at the three developmental stages were evaluated to elucidate the color-production mechanism in the red-leaved P. bournei species. First, metabolomic analysis identified 34 anthocyanin metabolites showing high levels of cyanidin-3-O-glucoside (cya-3-O-glu) in the S1 stage, which may suggest that it is a characteristic metabolite associated with the red coloration of the leaves. Second, transcriptome analysis showed that 94 structural genes were involved in anthocyanin biosynthesis, especially flavanone 3′-hydroxy-lase (PbF3′H), and were significantly correlated with the cya-3-O-glu level. Third, K-means clustering analysis and phylogenetic analyses identified PbbHLH1 and PbbHLH2, which shared the same expression pattern as most structural genes, indicating that these two PbbHLH genes may be regulators of anthocyanin biosynthesis in P. bournei. Finally, overexpression of PbbHLH1 and PbbHLH2 in Nicotiana tabacum leaves triggered anthocyanin accumulation. These findings provide a basis for cultivating P. bournei varieties that have high ornamental value.

The introduction of broadleaf species into Cunninghamia lanceolata monoculture plantations could be a sustainable silvicultural practice for restoring soil fertility. We hypothesize that the addition of Phoebe bournei tree species will change soil physicochemical factors and microbial composition. Soil physicochemical factors were determined. Bacterial composition and N-cycling functional genes were investigated by Illumina MiSeq sequencing and qPCR, respectively, and by using a co-occurrence network to explore the connections between the relative abundance of bacteria and soil physicochemical parameters. The introduction of P. bournei into C. lanceolata plantations increased available nutrients in the soil, decreased toxicity (Al3+ level), and slightly increased the soil bacterial diversity. The relative abundance of the total Acidobacteria community, as well as the most abundant subgroups (1, 2, and 3), were significantly higher in the monospecific stands. Different Acidobacteria subgroups behaved differently, i.e., Gp2 correlated negatively with soil nutrients and positively with the Al3+ level, while Gp17 showed the opposite behavior. Quantification of seven nitrogen cycling genes revealed that the nifH, nrfA, and nosZ abundances were higher in the mixed stands, while the nirK abundance was higher in the monospecific plantations. Co-occurrence network analyses showed a strong correlation between soil properties and the bacterial community. P. bournei introduction into C. lanceolata plantations improved soil nutrients and affected the soil bacterial community. The higher nitrogen nifH and nrfA abundance in the mixed stands indicated a higher nitrogen fixation and dissimilatory nitrate reduction potential. Acidobacterial subgroups showed the opposite behavior to P. bournei introduction and could be used as indicators of soil nutrient changes.

Phoebe sheareri is a valuable tree species known as "Golden Nanmu" and is one of the most important protected tree species in China. However, natural populations are decreasing because of climate change and anthropogenic factors. To evaluate the genetic diversity and structure of remnant populations and the impacts of climate change on the distribution of potential suitable habitats, we conducted a field investigation and sampled 21 P. sheareri natural populations to evaluate their genetic diversity and structure using simple sequence repeat (SSR) molecular markers. Then, we predicted the distribution of suitable P. sheareri habitats across China under future scenarios (RCP 2.6 and RCP 8.5) and periods (2050 and 2070) using multivariate modeling methods-the MaxEnt model. The results showed a medium level of genetic diversity and low inbreeding in the 21 P. sheareri natural populations, and genetic differentiation among populations was significant, with 21.2 % genetic variation among populations. The remnant populations of P. sheareri were grouped into four genetic clusters based on genetic structure; five environmental variables involving four temperature variables and precipitation seasonality (Bio12) might determine the distribution of P. sheareri populations. In the future, the suitable habitats of P. sheareri are manifested as northward migration, and the highly suitable habitats are expected to increase. Our results highlight the importance of conservation units in situ, giving priority to populations with higher genetic diversity (e.g., TMS, FJS, and THY populations); sampling strategies for ex situ conservation, breeding and reforestation should consider climate change, especially Bio1 (annual mean temperature) and Bio12 (annual precipitation). Overall, this study may provide useful genetic information for strategies for the protection, management, and utilization of P. sheareri.

Phoebe bournei, a plant species endemic to China, is a precious timber tree and widely used in landscaping. This tree contains numerous secondary metabolites, underscoring its potential economic value. However, studies on this species, including molecular genetic research, remain limited. In this study, both a somatic embryogenesis (SE) technical system and Agrobacterium-mediated genetic transformation were successfully employed in P. bournei for the first time. The SE technical system was constructed using immature embryos as original material. The primary embryo and embryonic callus induction rates were 30.66% and 41.67%, respectively. The highest rate of embryonic callus proliferation was 3.84. The maximum maturity coefficient and germination rate were 53.44/g and 39%, respectively. Agrobacterium-mediated genetic transformation was performed using the SE technical system, and the highest transformation rate was 11.24%. The results presented here are the first to demonstrate an efficient approach to achieve numerous P. bournei plantlets, which serves as the basis for artificial cultivation and resource conservation. Furthermore, the genetic transformation platform constructed in this study will facilitate assessment of gene function and molecular regulation.

Chinese fir (Cunninghamia lanceolata) is an important coniferous species that accounts for 20–30% of the total commercial timber production in China. Though traditional breeding of Chinese fir has achieved remarkable success, molecular-assisted breeding has made little progress due to limited availability of genomic information. In this study, a survey of Chinese fir genome was performed using the Illumina HiSeq Xten sequencing platform. K-mer analysis indicated that Chinese fir has a large genome of approximately 11.6 Gb with 74.89% repetitive elements and is highly heterozygous. Meanwhile, its genome size was estimated to be 13.2 Gb using flow cytometry. A total of 778.02 Gb clean reads were assembled into 10,982,272 scaffolds with an N50 of 1.57 kb. In total, 362,193 SSR loci were detected with a frequency of 13.18 kb. Dinucleotide repeats were the most abundant (up to 73.6% of the total SSRs), followed by trinucleotide and tetranucleotide repeats. Forty-six polymorphic pairs were developed, and 298 alleles were successfully amplified from 199 Chinese fir clones. The average PIC value was 0.53, indicating that the identified genomic SSR (gSSR) markers have a high degree of polymorphism. In addition, these breeding resources were divided into three groups, and a limited gene flow existed among these inferred groups.

Terpenoids, including aromatic volatile monoterpenoids and sesquiterpenoids, function in defense against pathogens and herbivores. Phoebe trees are remarkable for their scented wood and decay resistance. Unlike other Lauraceae species investigated to date, Phoebe species predominantly accumulate sesquiterpenoids instead of monoterpenoids. Limited genomic data restrict the elucidation of terpenoid variation and functions. Here, we present a chromosome-scale genome assembly of a Lauraceae tree, Phoebe bournei, and identify 72 full-length terpene synthase (TPS) genes. Genome-level comparison shows pervasive lineage-specific duplication and contraction of TPS subfamilies, which have contributed to the extreme terpenoid variation within Lauraceae species. Although the TPS-a and TPS-b subfamilies were both expanded via tandem duplication in P. bournei, more TPS-a copies were retained and constitutively expressed, whereas more TPS-b copies were lost. The TPS-a genes on chromosome 8 functionally diverged to synthesize eight highly accumulated sesquiterpenes in P. bournei. The essential oil of P. bournei and its main component, β-caryophyllene, exhibited antifungal activities against the three most widespread canker pathogens of trees. The TPS-a and TPS-b subfamilies have experienced contrasting fates over the evolution of P. bournei. The abundant sesquiterpenoids produced by TPS-a proteins contribute to the excellent pathogen resistance of P. bournei trees. Overall, this study sheds light on the evolution and adaptation of terpenoids in Lauraceae and provides valuable resources for boosting plant immunity against pathogens in various trees and crops.

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.

Bamboo is one of the most important non-timber forest resources worldwide. It has considerable economic value and unique flowering characteristics. The long juvenile phase in bamboo and unpredictable flowering time limit breeding and genetic improvement and seriously affect the productivity and application of bamboo forests. Members of SQUA-like subfamily genes play an essential role in controlling flowering time and floral organ identity. A comprehensive study was conducted to explain the functions of five SQUA-like subfamily genes in Phyllostachys edulis. Expression analysis revealed that all PeSQUAs have higher transcript levels in the reproductive period than in the juvenile phase. However, PeSQUAs showed divergent expression patterns during inflorescence development. The protein–protein interaction (PPI) patterns among PeSQUAs and other MADS-box members were analyzed by yeast two-hybrid (Y2H) experiments. Consistent with amino acid sequence similarity and phylogenetic analysis, the PPI patterns clustered into two groups. PeMADS2, 13, and 41 interacted with multiple PeMADS proteins, whereas PeMADS3 and 28 hardly interacted with other proteins. Based on our results, PeSQUA might possess different functions by forming protein complexes with other MADS-box proteins at different flowering stages. Furthermore, we chose PeMADS2 for functional analysis. Ectopic expression of PeMADS2 in Arabidopsis and rice caused early flowering, and abnormal phenotype was observed in transgenic Arabidopsis lines. RNA-seq analysis indicated that PeMADS2 integrated multiple pathways regulating floral transition to trigger early flowering time in rice. This function might be due to the interaction between PeMADS2 and homologous in rice. Therefore, we concluded that the five SQUA-like genes showed functional conservation and divergence based on sequence differences and were involved in floral transitions by forming protein complexes in P. edulis. The MADS-box protein complex model obtained in the current study will provide crucial insights into the molecular mechanisms of bamboo’s unique flowering characteristics.

Bamboo is one of the most important non-timber forest resources worldwide. It has considerable economic value and unique flowering characteristics. The long juvenile phase in bamboo and unpredictable flowering time limit breeding and genetic improvement and seriously affect the productivity and application of bamboo forests. Members of SQUA-like subfamily genes play an essential role in controlling flowering time and floral organ identity. A comprehensive study was conducted to explain the functions of five SQUA-like subfamily genes in Phyllostachys edulis. Expression analysis revealed that all PeSQUAs have higher transcript levels in the reproductive period than in the juvenile phase. However, PeSQUAs showed divergent expression patterns during inflorescence development. The protein–protein interaction (PPI) patterns among PeSQUAs and other MADS-box members were analyzed by yeast two-hybrid (Y2H) experiments. Consistent with amino acid sequence similarity and phylogenetic analysis, the PPI patterns clustered into two groups. PeMADS2, 13, and 41 interacted with multiple PeMADS proteins, whereas PeMADS3 and 28 hardly interacted with other proteins. Based on our results, PeSQUA might possess different functions by forming protein complexes with other MADS-box proteins at different flowering stages. Furthermore, we chose PeMADS2 for functional analysis. Ectopic expression of PeMADS2 in Arabidopsis and rice caused early flowering, and abnormal phenotype was observed in transgenic Arabidopsis lines. RNA-seq analysis indicated that PeMADS2 integrated multiple pathways regulating floral transition to trigger early flowering time in rice. This function might be due to the interaction between PeMADS2 and homologous in rice. Therefore, we concluded that the five SQUA-like genes showed functional conservation and divergence based on sequence differences and were involved in floral transitions by forming protein complexes in P. edulis. The MADS-box protein complex model obtained in the current study will provide crucial insights into the molecular mechanisms of bamboo’s unique flowering characteristics.

Inorganic phosphate (Pi) is often lacking in natural and agro-climatic environments, which impedes the growth of economically important woody species. Plants have developed strategies to cope with low Pi (LP) availability. MicroRNAs (miRNAs) play important roles in responses to abiotic stresses, including nutrition stress, by regulating target gene expression. However, the miRNA-mediated regulation of these adaptive responses and their underlying coordinating signals are still poorly understood in forestry trees such as Betula luminifera. Transcriptomic libraries, small RNA (sRNA) libraries, and a mixed degradome cDNA library of B. luminifera roots and shoots treated under LP and normal conditions (CK) were constructed and sequenced using next-generation deep sequencing. A comprehensive B. luminifera transcriptome derived from its roots and shoots was constructed, and a total of 76,899 unigenes were generated. Analysis of the transcriptome identified 8,095 and 5,584 differentially expressed genes in roots and shoots, respectively, under LP conditions. sRNA sequencing analyses indicated that 66 and 60 miRNAs were differentially expressed in roots and shoots, respectively, under LP conditions. A total of 109 and 112 miRNA–target pairs were further validated in the roots and shoots, respectively, using degradome sequencing. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differential miRNA targets indicated that the “ascorbate and aldarate metabolism” pathway responded to LP, suggesting miRNA-target pairs might participating in the removing of reactive oxidative species under LP stress. Moreover, a putative network of miRNA–target interactions involved in responses to LP stress in B. luminifera is proposed. Taken together, these findings provide useful information to decipher miRNA functions and establish a framework for exploring P signaling networks regulated by miRNAs in B. luminifera and other woody plants. It may provide new insights into the genetic engineering of high use efficiency of Pi in forestry trees.

Background: Phoebe sheareri (Lauraceae) is a valuable and endemic tree species in China, with limited large natural communities remnant. Genetic diversity and differentiation analysis are essential to manage their conservation and utilization. To provide a conservation and utilization strategy of P. sheareri based on sound genetic diversity and differentiation data.Results: We found medium level of genetic diversity and low inbreeding. Nei's gene diversity and Shannon’s information index value showed medium genetic diversity in nature populations of P. sheareri, which was higher than other two Phoebe species. AMOVA showed the genetic differentiation among populations was significantly, and 21.2% of genetic variation was among populations. Bayesian clustering, obtained with STRUCTURE, grouped the populations into four genetic clusters, whereas UPGMA analysis distinguished three main groups approximately in line with the geographic area of occurrence.Conclusion: Based on the study results, the establishment of gene conservation units must be considered in nature conserves in order to protect the genetic diversity of the species, and the proposal of sampling strategies for ex situ conservation and reforestation.

In the present study, a simple and fast method for simultaneous detection of zearalenone, citrinin, and ochratoxin A utilising capillary zone electrophoresis with an ultraviolet detector was developed. The optimised approach was validated and applied using pepper samples. The proposed method yielded satisfactory linearity between the signal and the mycotoxin concentration in the range of 1.5-150 μg/kg for zearalenone, 4.5-150 μg/kg for citrinin, and 0.8-150 μg/kg for ochratoxin A. The limits of detection for these mycotoxins ranged from 0.3 to 1.5 μg/kg. The corresponding intra- and inter-day precisions were less than 3.5 % and 4.1 %, respectively. Moreover, the matrix effect was also assessed and the result was compared using the capillary zone electrophoresis and high-performance liquid chromatography methods. The developed approach could be used for simultaneous detection of zearalenone, citrinin, and ochratoxin A in pepper.

To understand how historical and environmental factors have shaped genetic diversity and spatial genetic patterns of tree species, we characterized the genetic diversity and the genetic structure of a coniferous species, Cryptomeria japonica, which is distributed in Japan (two taxonomic groups) and some parts of southeastern China (C. japonica var. sinensis). We investigated population samples for three groups using high-throughput SNP genotyping, sequencing of multiple genes, and then estimated level of genetic diversity, the genetic differentiation, and the divergence time between the three groups. We also investigated the natural distribution during LGM using species distribution modeling. The genetic variation in the Chinese population, var. sinensis, was lower compared to Japanese populations but it harbored some unique genotypes. The species distribution modeling showed that the Chinese population has decreased since Last Glacial Maximum (LGM) because of lower precipitation than present time, but in Japan the favored environment have remained available for this species even in the LGM. Estimated divergence times of the three groups mostly correspond with the time since the Last Glacial. Our data supports the hypothesis that the glacial period, particularly limitations associated with precipitation, strongly influenced the distribution of C. japonica populations. Our findings suggest that climate change plays an important role in speciation processes of C. japonica.

Background MADS-box genes encode a large family of transcription factors that play significant roles in plant growth and development. Bamboo is an important non-timber forest product worldwide, but previous studies on the moso bamboo (Phyllostachys edulis) MADS-box gene family were not accurate nor sufficiently detailed. Results Here, a complete genome-wide identification and characterization of the MADS-box genes in moso bamboo was conducted. There was an unusual lack of type-I MADS-box genes in the bamboo genome database (http://202.127.18.221/bamboo/index.php), and some of the PeMADS sequences are fragmented and/or inaccurate. We performed several bioinformatics techniques to obtain more precise sequences using transcriptome assembly. In total, 42 MADS-box genes, including six new type-I MADS-box genes, were identified in bamboo, and their structures, phylogenetic relationships, predicted conserved motifs and promoter cis-elements were systematically investigated. An expression analysis of the bamboo MADS-box genes in floral organs and leaves revealed that several key members are involved in bamboo inflorescence development, like their orthologous genes in Oryza. The ectopic overexpression of one MADS-box gene, PeMADS5, in Arabidopsis triggered an earlier flowering time and the development of an aberrant flower phenotype, suggesting that PeMADS5 acts as a floral activator and is involved in bamboo flowering. Conclusion We produced the most comprehensive information on MADS-box genes in moso bamboo. Additionally, a critical PeMADS gene (PeMADS5) responsible for the transition from vegetative to reproductive growth was identified and shown to be related to bamboo floral development. Electronic supplementary material The online version of this article (10.1186/s12870-018-1394-2) contains supplementary material, which is available to authorized users.

We identified a set of ecotype-specific cold-responsive genes, providing candidate genes for enhancing cold resistance in Phoebe bournei (Hemsl.) Yang and other woody plants. Phoebe bournei (Hemsl.) Yang is an indigenous, endangered, precious timber and ornamental tree in China. Cold stress greatly affects the survival of Phoebe species. To elucidate the molecular mechanisms of cold response and identify candidate genes for improving cold resistance, we used RNA-Seq and qRT-PCR to compare the transcriptomic profiles of two ecotypes of P. bournei, cold-resistant Wuyuan (WY) and cold-sensitive Wuping (WP), under cold stress. A total of 3970 and 4433 genes were differentially expressed in WY and WP, respectively, with 2030 differentially expressed genes (DEGs) were specific to WY. Specifically, the categories “defense response”, “secondary metabolites”, and “ABC transporters” were enriched among WY-specific DEGs. Several transcription factor genes might involve in cold stress response, including RAP2-3, NAC2, NAC43, WRKY47, and WRKY51, were induced only in WY, and CBF3, ZAT10, MYB308, and WER exhibited greater cold stress-induced expression changes in WY compared with WP, which may partially explain the distinct transcriptomic changes induced by cold stress between the two ecotypes. Greater fold changes in the expression of starch metabolism-related genes such as BAM1/3 and lower fold changes in the expression of DPE2 were detected in WY, which was consistent with its greater abundance of total soluble sugars. The stronger transcriptional differentiation of related genes under cold stress in WY may explain its greater cold resistance compared with the WP ecotype, providing important genetic resources for improving cold-resistant characteristics in P. bournei. In addition, two interaction networks were constructed, resulting in identification of a series of hub genes, which facilitates functional dissection of the molecular mechanisms involved in the response to cold stress of P. bournei and extends our ability to improve cold resistance in this valued species.

This paper proposes a miRNA-mediated regulatory network of heat stress response in a tree, Betula luminifera. Plants are sessile organisms that must gauge stressful conditions to ensure survival and reproductive success. In nature, plants often encounter chronic or recurring abiotic stresses, such as heat stress, although the strategies to cope with high temperature are poorly understood in woody plants. In this study, we investigated the potential roles of microRNAs (miRNAs) in heat stress response in Betula luminifera. Using next-generation deep sequencing, we identified a total of 206 miRNAs, comprising 115 known and 84 novel miRNAs, and seven potential miRNA candidates. Of these miRNAs, 98 (57 known and 41 novel miRNAs) were differentially expressed under heat stress. A total of 101 miRNA-target pairs consisting of 44 miRNAs and 71 target genes were verified by degradome sequencing. Target annotation suggested that these miRNAs might be important in the regulation of specific stress-responsive genes, including many transcription factors and a series of genes encoding important enzymes of functional proteins targeted by miRNAs. Further functional analysis suggested that these target genes were related to protein synthesis, transport and turnover, cell wall organization, protein folding, antioxidation, defense response, and modulation of attenuated plant growth and development under heat stress. The expression patterns of 15 randomly selected miRNAs derived from quantitative reverse transcription PCR had similar trends as those derived from high-throughput sequencing, as well as the expression patterns of six target genes under heat stress. These results improve our understanding of the miRNA-mediated regulatory network of heat stress response in B. luminifera, which will contribute to improving heat stress-resistant breeding in woody plants.

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.

Glycosyltransferase (GT) plays a pivotal role in cell wall biosynthesis in plants. Galacturonosyltransferase-like (GATL) genes, belonging to the GT8 family, have been proven to be involved in pectin and/or xylan biosynthesis of the cell wall. Here, we identified eight GATL genes from the Eucalyptus grandis genome and characterized the gene structure and chromosomal location. The genes were found to be distributed across five chromosomes, including two pairs in block duplication regions. None of the EgrGATL genes contained introns. And, with the exception of EgrGATL8, the remainder of the EgrGATL proteins possessed the three classic motifs characteristic of all GATL proteins. Expression analysis in the different tissues showed that EgrGATL1, EgrGATL4, and EgrGATL8 were highly expressed in xylem and phloem; EgrGATL6 exhibited the highest expression in leaves, and in phloem and leaves, EgrGATL2 and EgrGATL3 both exhibited very low expression. However, the abiotic stress response of plants can be affected by changes in the components and structure of the cell wall. The expression patterns of EgrGALTs under low-temperature, high-temperature, drought, salinity, and abscisic acid (ABA) treatments were assessed by qRT-PCR. The results showed that most of the EgrGATL genes could be induced under low temperature, and some were even able to increase their expression level under high temperature. Under drought conditions, the expression levels of most of the genes initially increased and then decreased. Similar expression patterns were observed in leaves under treatment with NaCl and ABA. Our results provide fundamental information towards the functional dissection of EgrGATL genes and their potential involvement in improving plant abiotic stress tolerance.

BackgroundBetula luminiferaH. Winkler, which is widely distributed in southern China, is an economically important broadleaf tree species. However, little genomic information ofB.luminiferais available, and little is known about the molecular mechanisms of wood formation in this species. Meanwhile, few efforts have focused on investigating the early transcriptional changes during tension wood formation in woody plants.ResultsA reference transcriptome dataset was first generated containing 45,700 Unigenes, and 35,135 (76.9%) Unigenes were annotated by a BLAST similarity search against four public databases. Then, based on an anatomical investigation, the global gene expression changes during the early stages of tension wood formation were analyzed. Gene expression profiling showed that a total of 13,273 Unigenes were differentially regulated during the early stages of tension wood formation. Most genes involved in cellulose and lignin biosynthesis were highlighted to reveal their biological importance in tension wood formation. In addition, the transcription levels of many genes involved in the auxin response pathway were significantly changed during the early stages of tension wood formation. Furthermore, 18 TFs co-expressed with key enzymes of cellulose synthesis were identified.ConclusionsOur results revealed the transcriptional changes associated with TW formation and identified potential key genes in the regulation of this process. These results will help to dissect the molecular mechanism of wood formation and provide key candidate genes for marker-assisted selection inB. luminifera.

Phoebe chekiangensis is a uniquely precious timber and ornamental tree species of Southern China that enjoys nationwide protection as an endangered species. In the present study, we identified a total of 22,985 SSR loci based on transcriptome sequencing data, and 41 polymorphic markers were chosen to analyze the genetic diversity of seven natural populations including 190 individuals. Results showed that a moderate level of genetic diversity was in this species. A UPGMA dendrogram revealed that the seven populations clustered into four categories, and the 190 individuals could be divided into five groups with the aid of STRUCTURE 2.3.1; the clustering tendency revealed using two distinct algorithms was similar. The genetic variation was mainly within populations, and there was frequent gene flow among populations. On Mantel testing, genetic distance was positively and significantly correlated with geographic distance. In terms of conservation, the WY, YS, SY, and HZ populations, which exhibited the highest genetic diversity, should be the prime targets of both in situ and ex situ conservation efforts. Populations TS, YJ, and LC, with fewer individuals and lower genetic diversity, are susceptible to anthropogenic interference and should be carefully protected in situ . Also, ex situ conservation and propagation are required.

Cryptromeria fortunei is one of the main forest plantation species in the subtropical high altitude areas in China. In this paper we collected 49 C. fortunei germplasm resources and provides a study of the utility of freely available C. japonica EST resources for the development of markers necessary for genetic diversity analyses of C. fortunei. By screening 24,299 EST sequences from C. japonica with SSR Finder, we identified 2384 ESTs carrying 2783 SSR motifs. We successfully obtained 364 (15 %) primers from 2419 putative SSR loci. Of the 80 candidate SSR markers tested, 70 (87.5 %) yielded stable and clear PCR products. With those primers, the genetic diversity of 49 C. fortunei we collected was studied. The results showed that 18 primers yield polymorphism within these accessions. These 18 primers generated 48 scorable SSR loci and the average number of polymorphic SSR loci per primer was 2.7. The PIC value varied from 0.375 to 0.8101, with the average of 0.4780. The Shannon index is 0.5718, and the value of the observed number of alleles and effective number of alleles are 1.9167 and 1.7289, respectively. The genetic coefficient of these 49 accessions varied from 0.28 to 0.87. According to the genetic distances, a cluster tree was constructed. At genetic coefficient of 0.60, these 49 accessions can group into 3: group I contains only FJ-laizhou accession, and group II contains 2 accessions from FJ-layang, and the other one group contains mixed accessions. At genetic coefficient of 0.68, the former group II was constructed into 7 subgroups, with the first 3 subgroups contain 16 accessions in which 11 (69 %) are from Fujian province, and the later 4 subgroup contain 31 accessions in which 20 (65 %) were from Zhejiang province.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is an excellent fast-growing timber species and has significant value in the forestry industry. In order to increase the nitrogen and phosphorus absorption and utilization in Chinese fir, shared differentially expressed genes under low nitrogen and phosphorus stress were screened in this study. Seedling of Chinese fir clone X6 was cultivated in aeroponic system with 3 treatments, namely, low nitrogen (LN), low phosphorus (LP) and a control check (with nitrogen and phosphorus sufficient, CK). After 4 months of treatment, the roots from the LN, LP and CK groups were collected and transcriptome sequencing was done by LC Sciences (USA) using an Illumina platform. When comparing the LN stress group with the CK group, 977 SDGEs were detected, 264 of which had KEGG annotations; 931 SDGEs were detected when comparing the LP stress group with the CK group, of which 189 had KEGG annotations; 297 SDGEs were detected in both the LN stress and LP stress groups, 78 of which had KEGG annotations representing 98 metabolic pathways. Among the 78 selected SDEGs that were differentially expressed under both LN and LP stress conditions, Twenty-one SDEGs were selected based on the metabolic pathways that nitrogen and phosphorus are involved in, these genes are PNR, PSBA, EGLC, GLC, END, BGLU, AMY, A1E, PAL, GOGAT, NIR1, NIR2, C4M, PAL, PRDX6, POX, CCR, CCoAOMT, FDH, CHS and ANR. These genes can potentially be used in breeding to improve both nitrogen and phosphorus utilization efficiency in Chinese fir.

Cellulose, a significant proportion of the plant cell wall, is the main determining factor of the wood qualities of a timber tree. Cellulose synthase (CESA), an essential catalyst for cellulose biosynthesis, is encoded by the CesA gene family. We isolated eight full-length CesA cDNAs from Betula luminifera on the basis of transcriptome sequencing. The predicted proteins comprised 985–1,103 amino acids, sharing 85–92 % identity with the corresponding PtiCESAs from Populus trichocarpa. Each protein contained typical motifs of plant CESA proteins. Phylogenetic analysis showed that BlCESA1, BlCESA3 and BlCESA4 belonged to three clades linked to secondary cell wall synthesis, and BlCESA2, BlCESA5, BlCESA6, BlCESA7 and BlCESA8 belonged to three clades associated with primary cell wall synthesis. Quantitative reverse transcription PCR and in situ mRNA hybridization indicated that transcripts of BlCesA1, BlCesA3 and BlCesA4 were most abundant in the xylem of the lignified stem and had similar expression patterns when treated with hormones and sucrose. Thus, the proteins encoded by these three genes might be subunits of the secondary wall CesA complex. However, the expression patterns of BlCesA1, BlCesA3 and BlCesA4 were different in response to mechanical stress. These three genes showed much higher expression levels in the tension wood than in the control, and they showed different expression patterns in the opposite wood, suggesting that these genes play different roles during secondary wall formation. BlCesA2, BlCesA5, BlCesA6 and BlCesA7 were predominantly expressed in the leaf and phloem. BlCesA8, which is most similar to BlCesA6, was strongly expressed in the leaf, phloem and cambium. Interestingly, these two genes showed distinct expression patterns when treated with hormones, sucrose and bending. Thus, in addition to biosynthesis of the primary cell wall, BlCesA8 might have other functions.

microRNAs (miRNAs) play vital roles in plants regulating a panoply of biological processes, such as development, hormone signaling, and stress response, by inhibiting target genes at the post-transcriptional level. However, the roles of miRNAs in Betula luminifera remain elusive. To mine for B. luminifera miRNAs and targets, we used a deep sequencing approach to analyze the sRNAs and degradome sequencing of mixed samples, including roots, stems, and leaves. A total of 114 known miRNAs or miRNA*s from 44 families, and 24 novel miRNAs and 17 miRNA*s plus 15 plausible miRNA candidates were identified, of which 36 known miRNAs, 29 miRNA*s, and all novel miRNAs had precursor sequences. Additionally, 49 targets for 19 known miRNA families and seven miRNA*s, and seven targets for novel miRNAs were identified using a high-throughput degradome-sequencing approach. The conserved miRNA targets were mainly transcription factors, whereas the miRNA* targets were mainly protein-coding genes, with preferential propensity to functional enzymes. A Gene Ontology analysis showed that the predicted targets were classified into 62 biological processes, 20 cellular components, and 28 molecular functions, respectively. We found two different targets for miR396a* and miR396c*, and the target changed when the miR156d precursor cleavage site was shifted toward the 5′-end by two nucleotides, indicating the diverse regulatory roles of MIRNA genes. Furthermore, three targets identified by degradome sequencing were validated further through 5′ rapid amplification of cDNA ends. The expression patterns of the randomly selected miRNAs varied among different tissues. miR164 expression was induced under nitrogen starvation, with tissue-specific expression patterns, and was negatively correlated with the NAC1 target gene in roots and leaves, but not in stems. This study is a transcriptome-based analysis of miRNAs and the degradome, providing useful information to explore the functions of miRNAs and their target genes in B. luminifera.

Background Chinese fir (Cunninghamia lanceolata) is an important timber species that accounts for 20–30% of the total commercial timber production in China. However, the available genomic information of Chinese fir is limited, and this severely encumbers functional genomic analysis and molecular breeding in Chinese fir. Recently, major advances in transcriptome sequencing have provided fast and cost-effective approaches to generate large expression datasets that have proven to be powerful tools to profile the transcriptomes of non-model organisms with undetermined genomes. Results In this study, the transcriptomes of nine tissues from Chinese fir were analyzed using the Illumina HiSeq™ 2000 sequencing platform. Approximately 40 million paired-end reads were obtained, generating 3.62 gigabase pairs of sequencing data. These reads were assembled into 83,248 unique sequences (i.e. Unigenes) with an average length of 449 bp, amounting to 37.40 Mb. A total of 73,779 Unigenes were supported by more than 5 reads, 42,663 (57.83%) had homologs in the NCBI non-redundant and Swiss-Prot protein databases, corresponding to 27,224 unique protein entries. Of these Unigenes, 16,750 were assigned to Gene Ontology classes, and 14,877 were clustered into orthologous groups. A total of 21,689 (29.40%) were mapped to 119 pathways by BLAST comparison against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The majority of the genes encoding the enzymes in the biosynthetic pathways of cellulose and lignin were identified in the Unigene dataset by targeted searches of their annotations. And a number of candidate Chinese fir genes in the two metabolic pathways were discovered firstly. Eighteen genes related to cellulose and lignin biosynthesis were cloned for experimental validating of transcriptome data. Overall 49 Unigenes, covering different regions of these selected genes, were found by alignment. Their expression patterns in different tissues were analyzed by qRT-PCR to explore their putative functions. Conclusions A substantial fraction of transcript sequences was obtained from the deep sequencing of Chinese fir. The assembled Unigene dataset was used to discover candidate genes of cellulose and lignin biosynthesis. This transcriptome dataset will provide a comprehensive sequence resource for molecular genetics research of C. lanceolata.

To lay a theoretical foundation for studies on strategies for improvement of Magnolia officinalis and select superior gemplasm resources to meet the demand for modernization, industrialization and internationalization of Chinese medicine.Seeds of Magnolia officinalis from 13 main habitats of 7 provinces were collected and strewn in a place of Jingning County, Zhejiang. At the age of seven, 195 samples were collected from the same height of the trunk of 15 individual trees of each provenance, and assayed for effective ingredients with HPLC.Differences in the content of phenols were significant among the seed sources and even greater among individuals within a seed source.3 seed sources viz. Wufeng, Hefeng and Enshi of Hubei were obviously superior to other seed sources on account of high contents of magnolol, honokoiol and total phenols. Extension and application of these 3 seed sources is an effective path leading to quality improvement of Magnolia officinalis. With great differences in the content of phenols existing among individuals within each source, there is a big gap between production of medicinal materials by merely using superior seed sources of Magnolia officinalis and the demand of stable and controllable quality for modernization and internationalization of Chinese medicine. But the great difference has laid a material foundation and brought about a great potential for genetic improvement of Magnolia officinalis. Therefore, the superior individuals within a superior seed source are an excellent material for the breeding of Magnolia officinalis.

Small RNAs (sRNAs), as a key component of molecular biology, play essential roles in plant development, hormone signaling, and stress response. However, little is known about the relationships among sRNAs, hormone signaling, and dormancy regulation in gymnosperm embryos. To investigate the roles of sRNAs in embryo dormancy maintenance and release in Larix leptolepis, we deciphered the endogenous "sRNAome" in dormant and germinated embryos. High-throughput sequencing of sRNA libraries showed that dormant embryos exhibited a length bias toward 24-nt while germinated embryos showed a bias toward 21-nt lengths. This might be associated with distinct levels of RNA-dependent RNA polymerase2 (RDR2) and/or RDR6, which is regulated by hormones. Proportions of miRNAs to nonredundant and redundant sRNAs were higher in germinated embryos than in dormant embryos, while the ratio of unknown sRNAs was higher in dormant embryos than in germinated embryos. We identified a total of 160 conserved miRNAs from 38 families, 3 novel miRNAs, and 16 plausible miRNA candidates, of which many were upregulated in germinated embryos relative to dormant embryos. These findings indicate that larches and possibly other gymnosperms have complex mechanisms of gene regulation involving miRNAs and other sRNAs operating transcriptionally and posttranscriptionally during embryo dormancy and germination. We propose that abscisic acid modulates embryo dormancy and germination at least in part through regulation of the expression level of sRNA-biogenesis genes, thus changing the sRNA components.