Morphological, physiological, and transcriptional responses to low nitrogen stress in Populus deltoides Marsh. clones with contrasting nitrogen use efficiency.

BACKGROUND: Nitrogen (N) is one of the main factors limiting the wood yield in poplar cultivation. Understanding the molecular mechanism of N utilization could play a guiding role in improving the nitrogen use efficiency (NUE) of poplar. RESULTS: In this study, three N-efficient genotypes (A1-A3) and three N-inefficient genotypes (C1-C3) of Populus deltoides were cultured under low N stress (5 µM NH4NO3) and normal N supply (750 µM NH4NO3). The dry matter mass, leaf morphology, and chlorophyll content of both genotypes decreased under N starvation. The low nitrogen adaptation coefficients of the leaves and stems biomass of group A were significantly higher than those of group C (p < 0.05). Interestingly, N starvation induced fine root growth in group A, but not in group C. Next, a detailed time-course analysis of enzyme activities and gene expression in leaves identified 2062 specifically differentially expressed genes (DEGs) in group A and 1118 in group C. Moreover, the sensitivity to N starvation of group A was weak, and DEGs related to hormone signal transduction and stimulus response played an important role in the low N response this group. Weighted gene co-expression network analysis identified genes related to membranes, catalytic activity, enzymatic activity, and response to stresses that might be critical for poplar's adaption to N starvation and these genes participated in the negative regulation of various biological processes. Finally, ten influential hub genes and twelve transcription factors were identified in the response to N starvation. Among them, four hub genes were related to programmed cell death and the defense response, and PodelWRKY18, with high connectivity, was involved in plant signal transduction. The expression of hub genes increased gradually with the extension of low N stress time, and the expression changes in group A were more obvious than those in group C. CONCLUSIONS: Under N starvation, group A showed stronger adaptability and better NUE than group C in terms of morphology and physiology. The discovery of hub genes and transcription factors might provide new information for the analysis of the molecular mechanism of NUE and its improvement in poplar.

Genetic diversity and population structure of black cottonwood (Populus deltoides) revealed using simple sequence repeat markers.

BACKGROUND: Black cottonwood (Populus deltoides) is one of the keystone forest tree species, and has become the main breeding parents in poplar hybrid breeding. However, the genetic diversity and population structure of the introduced resources are not fully understood. RESULTS: In the present study, five loci containing null alleles were excluded and 15 pairs of SSR (simple sequence repeat) primers were used to analyze the genetic diversity and population structure of 384 individuals from six provenances (Missouri, Iowa, Washington, Louisiana, and Tennessee (USA), and Quebec in Canada) of P. deltoides. Ultimately, 108 alleles (Na) were detected; the expected heterozygosity (He) per locus ranged from 0.070 to 0.905, and the average polymorphic information content (PIC) was 0.535. The provenance 'Was' had a relatively low genetic diversity, while 'Que', 'Lou', and 'Ten' provenances had high genetic diversity, with Shannon's information index (I) above 1.0. The mean coefficient of genetic differentiation (Fst) and gene flow (Nm) were 0.129 and 1.931, respectively. Analysis of molecular variance (AMOVA) showed that 84.88% of the genetic variation originated from individuals. Based on principal coordinate analysis (PCoA) and STRUCTURE cluster analysis, individuals distributed in the Mississippi River Basin were roughly classified as one group, while those distributed in the St. Lawrence River Basin and Columbia River Basin were classified as another group. The cluster analysis based on the population level showed that provenance 'Iow' had a small gene flow and high degree of genetic differentiation compared with the other provenances, and was classified into one group. There was a significant relationship between genetic distance and geographical distance. CONCLUSIONS: P. deltoides resources have high genetic diversity and there is a moderate level of genetic differentiation among provenances. Geographical isolation and natural conditions may be the main factors causing genetic differences among individuals. Individuals reflecting population genetic information can be selected to build a core germplasm bank. Meanwhile, the results could provide theoretical support for the scientific management and efficient utilization of P. deltoides genetic resources, and promote the development of molecular marker-assisted breeding of poplar.

Pln24NT: a web resource for plant 24-nt siRNA producing loci.

ABSTRACT: In plants, 24 nucleotide small interfering RNAs (24-nt siRNAs) account for a large percentage of the total siRNA pool, and they play an important role in guiding plant-specific RNA-directed DNA methylation (RdDM), which transcriptionally silences transposon elements, transgenes, repetitive sequences and some endogenous genes. Several loci in plant genomes produce clusters of 24-nt RNAs, and these loci are receiving increasing attention from the research community. However, at present there is no bioinformatics resource dedicated to 24-nt siRNA loci and their derived 24-nt siRNAs. Thus, in this study, Pln24NT, a freely available web resource, was created to centralize 24-nt siRNA loci and 24-nt siRNA information, including fundamental locus information, expression profiles and annotation of transposon elements, from next-generation sequencing (NGS) data for 10 popular plant species. An intuitive web interface was also developed for convenient searching and browsing, and analytical tools were included to help users flexibly analyze their own siRNA NGS data. Pln24NT will help the plant research community to discover and characterize 24-nt siRNAs, and may prove useful for studying the roles of siRNA in RNA-directed DNA methylation in plants. AVAILABILITY AND IMPLEMENTATION: http://bioinformatics.caf.ac.cn/Pln24NT . CONTACT: suxh@caf.ac.cn. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PoplarGene: poplar gene network and resource for mining functional information for genes from woody plants.

Poplar is not only an important resource for the production of paper, timber and other wood-based products, but it has also emerged as an ideal model system for studying woody plants. To better understand the biological processes underlying various traits in poplar, e.g., wood development, a comprehensive functional gene interaction network is highly needed. Here, we constructed a genome-wide functional gene network for poplar (covering ~70% of the 41,335 poplar genes) and created the network web service PoplarGene, offering comprehensive functional interactions and extensive poplar gene functional annotations. PoplarGene incorporates two network-based gene prioritization algorithms, neighborhood-based prioritization and context-based prioritization, which can be used to perform gene prioritization in a complementary manner. Furthermore, the co-functional information in PoplarGene can be applied to other woody plant proteomes with high efficiency via orthology transfer. In addition to poplar gene sequences, the webserver also accepts Arabidopsis reference gene as input to guide the search for novel candidate functional genes in PoplarGene. We believe that PoplarGene (http://bioinformatics.caf.ac.cn/PoplarGene and http://124.127.201.25/PoplarGene) will greatly benefit the research community, facilitating studies of poplar and other woody plants.

Disposable competitive-type immunoassay for determination of aflatoxin B1 via detection of copper ions released from Cu-apatite.

A disposable electrochemical immunosensor was developed for detection of aflatoxin B1 (AFB1) based on stripping voltammetric detection of copper ions released from Cu-apatite. AFB1 antibody (Ab) was firstly fixed on the gold nanoparticle (Au NPs) modified screen-printed carbon electrode (SPCE). AFB1-bovine serum albumin (AFB1-BSA) conjugate was labeled with Cu-apatite, and then competed with AFB1 for binding to the Ab. Copper ions were released from Cu-apatite through acidolysis and stripping voltammetry signal of the copper ions was used for the detection. The Cu-apatite increased the amount of loaded copper ions, and the anodic stripping strategy performed in the micro electrolytic cell of the SPCE simplified the detection procedure and further amplified the electrochemical signal. This immunosensor could detect AFB1 over a wide concentration range from 0.001 to 100ng mL(-1) with a detection limit of 0.2pg mL(-1). The low cost, high sensitive, rapid and accurate method may find widely potential application in the detection of other toxic or harmful substances.

Genome-Wide Analysis of the Fasciclin-Like Arabinogalactan Protein Gene Family Reveals Differential Expression Patterns, Localization, and Salt Stress Response in Populus.

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) involved in plant growth, development and response to abiotic stress. Although many studies have been performed to identify molecular functions of individual family members, little information is available on genome-wide identification and characterization of FLAs in the genus Populus. Based on genome-wide analysis, we have identified 35 Populus FLAs which were distributed on 16 chromosomes and phylogenetically clustered into four major groups. Gene structure and motif composition were relatively conserved in each group. All the members contained N-terminal signal peptide, 23 of which included predicted glycosylphosphatidylinositol (GPI) modification sites and were anchored to plasma membranes. Subcellular localization analysis showed that PtrFLA2/20/26 were localized in cell membrane and cytoplasm of protoplasts from Populus stem-differentiating xylem. The Ka/Ks ratios showed that purifying selection has played a leading role in the long-term evolutionary period which greatly maintained the function of this family. The expression profiles showed that 32 PtrFLAs were differentially expressed in four tissues at four seasons based on publicly available microarray data. 18 FLAs were further verified with qRT-PCR in different tissues, which indicated that PtrFLA1/2/3/7/11/12/20/21/22/24/26/30 were significantly expressed in male and female flowers, suggesting close correlations with the reproductive development. In addition, PtrFLA1/9/10/11/17/21/23/24/26/28 were highly expressed in the stems and differentiating xylem, which may be involved in stem development. To determine salt response of FLAs, qRT-PCR was performed to analyze the expression of 18 genes under salinity stress across two time points. Results demonstrated that all the 18 FLAs were expressed in root tissues; especially, PtrFLA2/12/20/21/24/30 were significantly induced at different time points. In summary, this study may lay the foundation for further investigating the biological functions of FLA genes in Populus trichocarpa.

Transcriptome sequencing of transgenic poplar (Populus × euramericana 'Guariento') expressing multiple resistance genes.

BACKGROUND: Transgenic poplar (Populus × euramericana 'Guariento') plants harboring five exogenous, stress-related genes exhibit increased tolerance to multiple stresses including drought, salt, waterlogging, and insect feeding, but the complex mechanisms underlying stress tolerance in these plants have not been elucidated. Here, we analyzed the differences in the transcriptomes of the transgenic poplar line D5-20 and the non-transgenic line D5-0 using high-throughput transcriptome sequencing techniques and elucidated the functions of the differentially expressed genes using various functional annotation methods. RESULTS: We generated 11.80 Gb of sequencing data containing 63, 430, 901 sequences, with an average length of 200 bp. The processed sequences were mapped to reference genome sequences of Populus trichocarpa. An average of 62.30% and 61.48% sequences could be aligned with the reference genomes for D5-20 and D5-0, respectively. We detected 11,352 (D5-20) and 11,372 expressed genes (D5-0), 7,624 (56.61%; D5-20) and 7,453 (65.54%; D5-0) of which could be functionally annotated. A total of 782 differentially expressed genes in D5-20 were identified compared with D5-0, including 628 up-regulated and 154 down-regulated genes. In addition, 196 genes with putative functions related to stress responses were also annotated. Gene Ontology (GO) analysis revealed that 346 differentially expressed genes are mainly involved in 67 biological functions, such as DNA binding and nucleus. KEGG annotation revealed that 36 genes (21 up-regulated and 15 down-regulated) were enriched in 51 biological pathways, 9 of which are linked to glucose metabolism. KOG functional classification revealed that 475 genes were enriched in 23 types of KOG functions. CONCLUSION: These results suggest that the transferred exogenous genes altered the expression of stress (biotic and abiotic) response genes, which were distributed in different metabolic pathways and were linked to some extent. Our results provide a theoretic basis for investigating the functional mechanisms of exogenous genes in transgenic plants.

Analysis of the leaf methylomes of parents and their hybrids provides new insight into hybrid vigor in Populus deltoides.

BACKGROUND: Plants with heterosis/hybrid vigor perform better than their parents in many traits. However, the biological mechanisms underlying heterosis remain unclear. To investigate the significance of DNA methylation to heterosis, a comprehensive analysis of whole-genome DNA methylome profiles of Populus deltoides cl.'55/65' and '10/17' parental lines and their intraspecific F1 hybrids lines was performed using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing. RESULTS: Here, a total of 486.27 million reads were mapped to the reference genome of Populus trichocarpa, with an average unique mapping rate of 57.8%. The parents with similar genetic background had distinct DNA methylation levels. F1 hybrids with hybrid vigor possessed non-additive DNA methylation level (their levels were higher than mid-parent values). The DNA methylation levels in promoter and repetitive sequences and transposable element of better-parent F1 hybrids and parents and lower-parent F1 hybrids were different. Compared with the maternal parent, better-parent F1 hybrids had fewer hypermethylated genes and more hypomethylated ones. Compared with the paternal parent and lower-parent L1, better-parent F1 hybrids had more hypermethylated genes and fewer hypomethylated ones. The differentially methylated genes between better-parent F1 hybrids, the parents and lower-parent F1 hybrids were enriched in the categories metabolic processes, response to stress, binding, and catalytic activity, development, and involved in hormone biosynthesis, signaling pathway. CONCLUSIONS: The methylation patterns of the parents both partially and dynamically passed onto their hybrids, and F1 hybrids has a non-additive mathylation level. A multidimensional process is involved in the formation of heterosis.

Expression and molecular evolution of two DREB1 genes in black poplar (Populus nigra).

Environmental stresses such as low temperature, drought, and high salinity significantly affect plant growth and yield. As selective forces, these adverse factors play essential roles in shaping phenotypic variation in plant populations. Black poplar (Populus nigra) is an economically and ecologically important forest tree species with widely distributed populations and is thus suitable for experiments detecting evolutionary footprints left by stress. Here, we performed expression and evolutionary analysis of two duplicated DREB A1-subgroup (DREB1) genes, PnDREB68 and PnDREB69, encoding transcription factors that are involved in stress responses. The two genes showed partially overlapping but distinct expression patterns in response to stresses. These genes were strongly and rapidly induced by cold stress in leaves, stems, and roots. In leaf tissue, dehydration stress induced the expression of PnDREB68 but not PnDREB69. PnDREB69 displayed more rapid responses and longer expression durations than PnDREB68 under salt and ABA stress, respectively. Based on single nucleotide polymorphism (SNP) analysis, we found significant population genetic differentiation, with a greater FST value (0.09189) for PnDREB69 than for PnDREB68 (0.07743). Nucleotide diversity analysis revealed a two-fold higher πT for PnDREB68 than for PnDREB69 (0.00563 vs. 0.00243), reflecting strong purifying selection acting on the former. The results suggest that positive selection acted on PnDREB69, as evidenced by neutral testing using Tajima's D statistic. The distinct selective forces to which each of the genes was subjected may be associated with expression divergence. Linkage disequilibrium (LD) was low for the sequenced region, with a higher level for PnDREB68 than for PnDREB69. Additionally, analysis of the relationship among carbon isotope ratios, SNP classes and gene expression, together with motif and domain analysis, suggested that 14 polymorphisms within the two genes may be candidates for an association study of important traits such as water use efficiency/drought tolerance in black poplar.

Expression of multiple resistance genes enhances tolerance to environmental stressors in transgenic poplar (Populus × euramericana 'Guariento').

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana 'Guariento') harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.

Patterns of DNA sequence variation at candidate gene loci in black poplar (Populus nigra L.) as revealed by single nucleotide polymorphisms.

Black poplar (Populus nigra L.) is an economically and ecologically important tree species and an ideal organism for studies of genetic variation. In the present work, we use a candidate gene approach to infer the patterns of DNA variation in natural populations of this species. A total of 312 single nucleotide polymorphisms (SNPs) are found among 8,056 bp sequenced from nine drought-adaptation and photosynthesis-related gene loci. The median SNP frequency is one site per 26 bp. The average nucleotide diversity is calculated to be theta(W) = 0.01074 and pi(T) = 0.00702, higher values than those observed in P. tremula, P. trichocarpa and most conifer species. Tests of neutrality for each gene reveal a general excess of low-frequency mutations, a greater number of haplotypes than expected and an excess of high-frequency derived variants in P. nigra, which is consistent with previous findings that genetic hitchhiking has occurred in this species. Linkage disequilibrium is low, decaying rapidly from 0.45 to 0.20 or less within a distance of 300 bp, although the declines of r(2) are variable among different loci. This is similar to the rate of decay reported in most other tree species. Our dataset is expected to enhance understanding of how evolutionary forces shape genetic variation, and it will contribute to molecular breeding in black poplar.

[Research progress of single nucleotide polymorphisms in forest trees.].

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in many organisms. Forest is an important plant group and key component of the terrestrial plant ecosystem. As a new marker system, SNP has been applied in genetic and breeding studies in some tree species of genera Pinus, Populus, Pseudotsuga, Eucalyptus, and Picea, and the related genetic information, such as nucleotide diversity, linkage disequilibrium (LD) and population structure has been generated. These studies are mainly based on sequencing and analysis of candidate genes. SNP-based association genetics analysis or LD mapping has become a useful tool for dissection of complex traits in forest trees. Association studies in Eucalyptus spp. and Pinus taeda L. found that some SNP sites in various genes were associated with distinct wood property traits. Meanwhile, estimation of genetic parameters has revealed the evolutionary and ecological significance at different extents in several tree species. Intensive applications of SNP approach are expected to greatly accelerate the development of research on forest genetics and breeding.

Identification of expressed sequence tags in an alkali grass (Puccinellia tenuiflora) cDNA library.

Alkali grass (Puccinellia tenuiflora), a monocotyledonous halophyte, can serve as a model of salt tolerance in monocotyledon crops. To elucidate the molecular events associated with salt tolerance in alkali grass, we generated a directional cDNA library from leaves treated with the alkali salt, NaHCO3. Large-scale sequencing of the cDNA library identified 2942 ESTs representing 2366 non-redundant transcripts. These have been deposited in the dbEST division of GenBank. BLASTX evaluation indicated that 1274 of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially gramineae species. The other 1092 ESTs, which showed little if any homology to known sequences, were considered novel. Based on the encoded proteins, the 1274 identified ESTs fell into 12 functional categories, of which four were abundant, namely metabolism (18.84%), transcription (12.48%), unclassified (11.22%) and cell rescue/defense (9.66%). The 162 unique transcripts corresponding to possible salt-related genes were also identified. This study provides an overview of gene expression in NaHCO3-stressed alkali grass, as well as useful information for further investigation of salt resistance in plants.