Nanoplastic toxicity induces metabolic shifts in Populus × euramericana cv. '74/76' revealed by multi-omics analysis.

There is increasing global concern regarding the pervasive issue of plastic pollution. We investigated the response of Populus × euramericana cv. '74/76' to nanoplastic toxicity via phenotypic, microanatomical, physiological, transcriptomic, and metabolomic approaches. Polystyrene nanoplastics (PS-NPs) were distributed throughout the test plants after the application of PS-NPs. Nanoplastics principally accumulated in the roots; minimal fractions were translocated to the leaves. In leaves, however, PS-NPs easily penetrated membranes and became concentrated in chloroplasts, causing thylakoid disintegration and chlorophyll degradation. Finally, oxidant damage from the influx of PS-NPs led to diminished photosynthesis, stunted growth, and etiolation and/or wilting. By integrating dual-omics data, we found that plants could counteract mild PS-NP-induced oxidative stress through the antioxidant enzyme system without initiating secondary metabolic defense mechanisms. In contrast, severe PS-NP treatments promoted a shift in metabolic pattern from primary metabolism to secondary metabolic defense mechanisms, an effect that was particularly pronounced during the upregulation of flavonoid biosynthesis. Our findings provide a useful framework from which to further clarify the roles of key biochemical pathways in plant responses to nanoplastic toxicity. Our work also supports the development of effective strategies to mitigate the environmental risks of nanoplastics by biologically immobilizing them in contaminated lands.

Effect of T-DNA Integration on Growth of Transgenic Populus × euramericana cv. Neva Underlying Field Stands.

Multigene cotransformation has been widely used in the study of genetic improvement in crops and trees. However, little is known about the unintended effects and causes of multigene cotransformation in poplars. To gain insight into the unintended effects of T-DNA integration during multigene cotransformation in field stands, here, three lines (A1-A3) of Populus × euramericana cv. Neva (PEN) carrying Cry1Ac-Cry3A-BADH genes and three lines (B1-B3) of PEN carrying Cry1Ac-Cry3A-NTHK1 genes were used as research objects, with non-transgenic PEN as the control. Experimental stands were established at three common gardens in three locations and next generation sequencing (NGS) was used to identify the insertion sites of exogenous genes in six transgenic lines. We compared the growth data of the transgenic and control lines for four consecutive years. The results demonstrated that the tree height and diameter at breast height (DBH) of transgenic lines were significantly lower than those of the control, and the adaptability of transgenic lines in different locations varied significantly. The genotype and the experimental environment showed an interaction effect. A total of seven insertion sites were detected in the six transgenic lines, with B3 having a double-site insertion and the other lines having single copies. There are four insertion sites in the gene region and three insertion sites in the intergenic region. Analysis of the bases near the insertion sites showed that AT content was higher than the average chromosome content in four of the seven insertion sites within 1000 bp. Transcriptome analysis suggested that the differential expression of genes related to plant hormone transduction and lignin synthesis might be responsible for the slow development of plant height and DBH in transgenic lines. This study provides an integrated analysis of the unintended effects of transgenic poplar, which will benefit the safety assessment and reasonable application of genetically modified trees.

Progress in the understanding of WRKY transcription factors in woody plants.

The WRKY transcription factor (TF) family, named for its iconic WRKY domain, is among the largest and most functionally diverse TF families in higher plants. WRKY TFs typically interact with the W-box of the target gene promoter to activate or inhibit the expression of downstream genes; these TFs are involved in the regulation of various physiological responses. Analyses of WRKY TFs in numerous woody plant species have revealed that WRKY family members are broadly involved in plant growth and development, as well as responses to biotic and abiotic stresses. Here, we review the origin, distribution, structure, and classification of WRKY TFs, along with their mechanisms of action, the regulatory networks in which they are involved, and their biological functions in woody plants. We consider methods currently used to investigate WRKY TFs in woody plants, discuss outstanding problems, and propose several new research directions. Our objective is to understand the current progress in this field and provide new perspectives to accelerate the pace of research that enable greater exploration of the biological functions of WRKY TFs.

Complete chloroplast genomes and comparative analysis of Ligustrum species.

In this study, we assembled and annotated the chloroplast (cp) genomes of four Ligustrum species, L. sinense, L. obtusifolium, L. vicaryi, and L. ovalifolium 'Aureum'. Including six other published Ligustrum species, we compared various characteristics such as gene structure, sequence alignment, codon preference, and nucleic acid diversity, and performed positive-selection genes screening and phylogenetic analysis. The results showed that the cp genome of Ligustrum was 162,185-166,800 bp in length, with a circular tetrad structure, including a large single-copy region (86,885-90,106 bp), a small single-copy region (11,446-11,499 bp), and a pair of IRa and IRb sequences with the same coding but in opposite directions (31,608-32,624 bp). This structure is similar to the cp genomes of most angiosperms. We found 132-137 genes in the cp genome of Ligustrum, including 89-90 protein-coding genes, 35-39 tRNAs, and 8 rRNAs. The GC content was 37.93-38.06% and varied among regions, with the IR region having the highest content. The single-nucleotide (A/T)n was dominant in simple-sequence repeats of the Ligustrum cp genome, with an obvious A/T preference. Six hotspot regions were identified from multiple sequence alignment of Ligustrum; the ycf1 gene region and the clpP1 exon region can be used as potential DNA barcodes for the identification and phylogeny of the genus Ligustrum. Branch-site model and Bayes empirical Bayes (BEB) analysis showed that four protein-coding genes (accD, clpP, ycf1, and ycf2) were positively selected, and BEB analysis showed that accD and rpl20 had positively selected sites. A phylogenetic tree of Oleaceae species was constructed based on the whole cp genomes, and the results were consistent with the traditional taxonomic results. The phylogenetic results showed that genus Ligustrum is most closely related to genus Syringa. Our study provides important genetic information to support further investigations of the phylogenetic development and adaptive evolution of Ligustrum species.

Complete chloroplast genome structure of four Ulmus species and Hemiptelea davidii and comparative analysis within Ulmaceae species.

In this study, the chloroplast (cp) genomes of Hemiptelea davidii, Ulmus parvifolia, Ulmus lamellosa, Ulmus castaneifolia, and Ulmus pumila 'zhonghuajinye' were spliced, assembled and annotated using the Illumina HiSeq PE150 sequencing platform, and then compared to the cp genomes of other Ulmus and Ulmaceae species. The results indicated that the cp genomes of the five sequenced species showed a typical tetrad structure with full lengths ranging from 159,113 to 160,388 bp. The large single copy (LSC), inverted repeat (IR), and small single copy (SSC) lengths were in the range of 87,736-88,466 bp, 26,317-26,622 bp and 18,485-19,024 bp, respectively. A total of 130-131 genes were annotated, including 85-86 protein-coding genes, 37 tRNA genes and eight rRNA genes. The GC contents of the five species were similar, ranging from 35.30 to 35.62%. Besides, the GC content was different in different region and the GC content in IR region was the highest. A total of 64-133 single sequence repeat (SSR) loci were identified among all 21 Ulmaceae species. The (A)n and (T)n types of mononucleotide were highest in number, and the lengths were primarily distributed in 10-12 bp, with a clear AT preference. A branch-site model and a Bayes Empirical Bayes analysis indicated that the rps15 and rbcL had the positive selection sites. Besides, the analysis of mVISTA and sliding windows got a lot of hotspots such as trnH/psbA, rps16/trnQ, trnS/trnG, trnG/trnR and rpl32/trnL, which could be utilized as potential markers for the species identification and phylogeny reconstruction within Ulmus in the further studies. Moreover, the evolutionary tree of Ulmaceae species based on common protein genes, whole cp genome sequences and common genes in IR region of the 23 Ulmaceae species were constructed using the ML method. The results showed that these Ulmaceae species were divided into two branches, one that included Ulmus, Zelkova and Hemiptelea, among which Hemiptelea was the first to differentiate and one that included Celtis, Trema, Pteroceltis, Gironniera and Aphananthe. Besides, these variations found in this study could be used for the classification, identification and phylogenetic study of Ulmus species. Our study provided important genetic information to support further investigations into the phylogenetic development and adaptive evolution of Ulmus and Ulmaceae species.

Effects of three regeneration methods on the growth and bacterial community diversity of Populus × euramericana.

To study the effects of different regeneration methods on the growth and bacterial community diversity of Populus × euramericana cv. '74/76' (poplar 107), we investigated the growth of poplar 107 trees under three regeneration methods in 2017 and 2020, and sequenced the 16S rDNA V5-V7 regions in stem endophytic, root endophytic, and rhizosphere soil bacteria present in samples from the three regeneration methods using the Illumina high-throughput sequencing platform. The growth analysis showed that stump grafting regeneration (ST) and stump sprouting regeneration (SP) presented similar tree height and diameter at breast height (DBH), which were significantly lower by planted seedling regeneration (CK). The high-throughput sequencing results showed that the rhizosphere soil bacteria appeared to be significantly more diverse and rich than the root and stem endophytic bacteria. Cluster analysis showed that the similarity of bacterial community structure among the rhizosphere soil, root, and stem was small. Thus, the three sample types showed significant differences in bacteria. While comparing the two years, 2020 was significantly more diverse and rich than 2017. With the increase in stand age, the abundance of Proteobacteria increased and the abundance of Acidobacteria decreased. Among the three regeneration methods, ST significantly increased the diversity of stem endophytic bacteria. Chthoniobacter was enriched in SP, which promoted the decomposition of organic matter, and more plant growth promoting rhizobacteria (PGPR) were accumulated in the rhizosphere of SP and ST. The composition of the bacterial community was similar in the three regeneration methods, but the community composition was different. Regeneration and transformation of poplar plantations can be better carried out by stump grafting and stump sprouting.

Integrated physiological and transcriptomic analyses reveal the molecular mechanism behind the response to cultivation in Quercus mongolica.

Quercus mongolica, a common tree species for building and landscaping in northern China, has great commercial and ecological value. The seedlings of Q. mongolica grow poorly and develop chlorosis when introduced from high-altitude mountains to low-altitude plains. Effective cultivation measures are key to improving the quality of seedlings. To investigate the complex responses of Q. mongolica to different cultivation measures, we compared the adaptability of 3-year-old Q. mongolica seedlings to pruning (P), irrigation (W), and fertilization [F (nitro compound fertilizer with 16N-16P-16K)]. Physiological measurements and transcriptome sequencing were performed on leaves collected under the P treatments (control, cutting, removal of all lateral branches, and removal of base branches to one-third of seedling height), the W treatments (0, 1, 2, 3, 4, or 5 times in sequence), and the F treatments (0, 2, 4, and 6 g/plant). Analyses of the physiological data showed that P was more effective than W or F for activating intracellular antioxidant systems. By contrast, W and F were more beneficial than P for inducing the accumulation of soluble sugar. OPLS-DA identified superoxide dismutase, malondialdehyde, and peroxidase as critical physiological indices for the three cultivation measures. Transcriptome analyses revealed 1,012 differentially expressed genes (DEGs) in the P treatment, 1,035 DEGs in the W treatment, and 1,175 DEGs in the F treatment; these DEGs were mainly enriched in Gene Ontology terms related to the stress response and signal transduction. Weighted gene coexpression network analyses indicated that specific gene modules were significantly correlated with MDA (one module) and soluble sugar (four modules). Functional annotation of the hub genes differentially expressed in MDA and soluble sugar-related modules revealed that Q. mongolica responded and adapted to different cultivation measures by altering signal transduction, hormone levels, reactive oxygen species, metabolism, and transcription factors. The hub genes HOP3, CIPK11, WRKY22, and BHLH35 in the coexpression networks may played a central role in responses to the cultivation practices. These results reveal the mechanism behind the response of Q. mongolica to different cultivation measures at the physiological and molecular levels and provide insight into the response of plants to cultivation measures.

Correlation Analysis of the Bacterial Community and Wood Properties of Populus × euramericana cv. "74/76" Wet Heartwood.

Wetwood disease of poplar limits the processing and manufacturing of poplar, and the pathogenic bacteria of wet heartwood are poorly known. We used high-throughput sequencing methods to analyze the bacterial community of the heartwood, sapwood, root tissue, and rhizosphere soil of Populus × euramericana cv. "74/76" (poplar 107) in wetwood trees and healthy trees to explore the cause of poplar wetwood disease. Bacterial diversity and community structure were analyzed, and the correlation between wood properties and bacterial relative abundance was analyzed to explore their relationship. Two alpha-diversity indices of endophytic bacteria in the heartwood of wetwood trees were significantly (p < 0.05) lower than that in the heartwood of healthy trees, and the community structure between the two types of trees was significantly different. No significant differences in the alpha-diversity indices nor community structure were observed in the sapwood, root tissue, or rhizosphere bacterial community of diseased and healthy trees. The distribution of dominant bacteria genus in the heartwood of diseased and healthy trees differed. Proteiniphilum, Actinotalea, and Methanobacterium were the dominant genera in diseased trees' heartwood. Proteiniphilum, Dysgonomonas, and Bacteroides were the dominant genera in healthy trees' heartwood. The relative abundance of Proteiniphilum, Actinotalea, and Methanobacterium was significantly higher in the heartwood of wetwood trees than those of healthy trees. A db-RDA analysis found that these three bacterial genera were positively correlated with the rate of wet heartwood. These three bacterial genera may be the main pathogens causing poplar wetwood disease.

Exogenous Gene Expression and Insect Resistance in Dual Bt Toxin Populus × euramericana 'Neva' Transgenic Plants.

Bacillus thuringiensis (Bt) insecticidal protein genes are important tools in efforts to develop insect resistance in poplar. In this study, the Cry1Ac and Cry3A Bt toxin genes were simultaneously transformed into the poplar variety Populus × euramericana 'Neva' by Agrobacterium-mediated transformation to explore the exogenous gene expression and insect resistance, and to examine the effects of Bt toxin on the growth and development of Anoplophora glabripennis larvae after feeding on the transgenic plant. Integration and expression of the transgenes were determined by molecular analyses and the insect resistance of transgenic lines was evaluated in feeding experiments. Sixteen transgenic dual Bt toxin genes Populus × euramericana 'Neva' lines were obtained. The dual Bt toxin genes were expressed at both the transcriptional and translational levels; however, Cry3A protein levels were much higher than those of Cry1Ac. Some of the transgenic lines exhibited high resistance to the first instar larvae of Hyphantria cunea and Micromelalopha troglodyta, and the first and second instar larvae and adults of Plagiodera versicolora. Six transgenic lines inhibited the growth and development of A. glabripennis larvae. The differences in the transcriptomes of A. glabripennis larvae fed transgenic lines or non-transgenic control by RNA-seq analyses were determined to reveal the mechanism by which Bt toxin regulates the growth and development of longicorn beetle larvae. The expression of genes related to Bt prototoxin activation, digestive enzymes, binding receptors, and detoxification and protective enzymes showed significant changes in A. glabripennis larvae fed Bt toxin, indicating that the larvae responded by regulating the expression of genes related to their growth and development. This study lay a theoretical foundation for developing resistance to A. glabripennis in poplar, and provide a foundation for exploring the mechanism of Bt toxin action on Cerambycidae insects.

Corrigendum: Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.

[This corrects the article DOI: 10.3389/fpls.2020.01123.].

Expression of Multiple Exogenous Insect Resistance and Salt Tolerance Genes in Populus nigra L.

Four exogenous genes, Cry3A, Cry1Ac, mtlD, and BADH, were inserted into the p1870 vector to obtain multigenic transgenic Populus nigra L. with improved insect resistance and salt tolerance. During vector construction, different promoters were used for each gene, the AtADH 5'-UTR enhancer was added between the Cry1Ac promoter and the target gene, and the matrix attachment region (MAR, GenBank: U67919.1) structure was added at both ends of the vector. It was then successfully transferred into the genome of European black poplar by Agrobacterium-mediated leaf disk transformation, and a total of 28 transgenic lines were obtained by kanamycin screening. Five events with the highest insect resistance were selected based on preliminary tests: nos. 1, 7, 9, 12, and 17. PCR, real-time PCR, and enzyme-linked immunosorbent assays (ELISA) were used to detect the expression of exogenous genes and to analyze the Bt protein toxin levels in transgenic lines from June to October. PCR results showed that all four genes were successfully introduced into the five selected lines. Fluorescence quantitative PCR showed no significant differences in the transcript abundance of the four exogenous genes between different lines. A Bt protein toxin assay showed that the Cry3A protein toxin content was significantly higher than the Cry1Ac protein toxin content by approximately three orders of magnitude. Levels of the two toxins were negatively correlated. Over the course of the growing season, Cry1Ac content raised and varied between 0.46 and 18.41 ng·g-1. Cry3A content decreased over the same time period and varied between 2642.75 and 15775.22 ng·g-1. Indoor insect feeding assay showed that the transgenic lines had high insect resistance, with mortality rates of 1-2-year-old Hyphantria cunea larvae reaching more than 80%, and those of Plagiodera versicolora larvae and nymphs reaching 100%. No. 17 and no. 12 lines had better insect resistance to Lepidoptera and Coleoptera pests. There was no clear improvement in salt tolerance of the transgenic lines, but comprehensive evaluation of 11 salt tolerance indicators showed that lines no. 17 and no. 7 had certain degrees of salt tolerance.

[Responses of photosynthetic physiology and sap flow to the introduction of external dye in Populus ×euramericana cv. '74/76']. / 107æ¨å ‰åˆç”Ÿç†ä¸Žæ ‘å¹²æ¶²æµå¯¹å¤–æºæŸ“æ¶²å¯¼å ¥çš„å“åº”.

The exogenous liquid introduction technology is an effective way to produce the value-added poplar wood with excellent pattern color. This technology was used to add the various concentrated active red dyeing solution (0.2%, 0.4% and 0.6%) into target trees of six-year-old 107 poplar (Populus ×euramericana cv. '74/76'). The photosynthetic gas exchange parameter and sap flow rate were measured by Li-6400 photosynthetic instrument and TDP stem flowmeter, respectively. We analyzed the relationship between photosynthetic parameters, sap flow rate and dye absorption, and the effects of exogenous dye solution on the photosynthetic physiology and sap flow characteristics. The results showed that exogenous dyeing solution significantly inhibited flow rate of poplar trunks. The 0.2% concentrated liquid was far less effective than others (0.4% and 0.6%). The net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (Tr) of poplars treated with different concentrated dyeing liquids were significantly lower than the control poplar. The intercellular carbon dioxide concentration (Ci) decreased first and then increased. The inhibitory effects of 0.4% and 0.2% concentrated dyeing solutions on photosynthesis were stronger than that of 0.6%. Dye absorption decreased with increasing dye concentration. The maximum liquid flow rate, Pn, gs and Tr were significantly negatively correlated with the dye content. The contents of chlorophyll (a+b), chlorophyll a and chlorophyll b in exogenous dyeing solution treatments were significantly lower than those of the control at the later stage. The concentration of dyeing solution and introduction time determined the amount of dye absorption. The dye solution 0.4%, which was introduced for three days, could ensure the appropriate dye absorption and reduce the inhibitory effect on the physiological activities of the poplar.

The Reason for Growth Inhibition of Ulmus pumila 'Jinye': Lower Resistance and Abnormal Development of Chloroplasts Slow Down the Accumulation of Energy.

Ulmus pumila 'Jinye', the colorful leaf mutant of Ulmus pumila L., is widely used in landscaping. In common with most leaf color mutants, U. pumila 'Jinye' exhibits growth inhibition. In this study, U. pumila L. and U. pumila 'Jinye' were used to elucidate the reasons for growth inhibition at the physiological, cellular microstructural, and transcriptional levels. The results showed that the pigment (chlorophyll a, chlorophyll b, and carotenoids) content of U. pumila L. was higher than that of U. pumila 'Jinye', whereas U. pumila 'Jinye' had a higher proportion of carotenoids, which may be the cause of the yellow leaves. Examination of the cell microstructure and RNA sequencing analysis showed that the leaf color and growth inhibition were mainly due to the following reasons: first, there were differences in the structure of the thylakoid grana layer. U. pumila L. has a normal chloroplast structure and clear thylakoid grana slice layer structure, with ordered and compact thylakoids. However, U. pumila 'Jinye' exhibited the grana lamella stacking failures and fewer thylakoid grana slice layers. As the pigment carrier and the key location for photosynthesis, the close stacking of thylakoid grana could combine more chlorophyll and promote efficient electron transfer promoting the photosynthesis reaction. In addition, U. pumila 'Jinye' had a lower capacity for light energy absorption, transformation, and transportation, carbon dioxide (CO2) fixation, lipopolysaccharide biosynthesis, auxin synthesis, and protein transport. The genes related to respiration and starch consumption were higher than those of U. pumila L., which indicated less energy accumulation caused the growth inhibition of U. pumila 'Jinye'. Finally, compared with U. pumila 'Jinye', the transcription of genes related to stress resistance all showed an upward trend in U. pumila L. That is to say, U. pumila L. had a greater ability to resist adversity, which could maintain the stability of the intracellular environment and maintain normal progress of physiological metabolism. However, U. pumila 'Jinye' was more susceptible to changes in the external environment, which affected normal physiological metabolism. This study provides evidence for the main cause of growth inhibition in U. pumila 'Jinye', information for future cultivation, and information on the mutation mechanism for the breeding of colored leaf trees.

Comparative analysis of chloroplast genomes of five Robinia species: Genome comparative and evolution analysis.

The black locust (Robinia pseudoacacia) is widely distributed, and has strong drought resistance and salt tolerance. These characteristics make it the best type of tree for landscaping and resource conservation in China. In this study, the chloroplast genomes of five black locusts were identified de novo and the evolutionary relationship among these black locusts and their taxonomic statuses in leguminous plants were determined. The chloroplast structures of the five black locusts were typical and had four parts, including two single copy regions (large and small single copy sections) and a pair of inverted repeats (IRs). Genome sizes were between 155,364 bp and 155,655 bp; the genome of R. pseudoacacia var. decaisneana was the smallest, while that of R. pseudoacacia var. tortuosa was the largest. The genomes contained 124-130 protein-coding genes; R. pseudoacacia var. tortuosa had the fewest, while R. hispida and R. pseudoacacia var. decaisneana had the most. In this study, eight to ten genes from chloroplast genomes contained introns. Nine genes from the chloroplast genomes of R. pseudoacacia and R. pseudoacacia f. unifolia contained introns that had lost the trnL-CAA gene via evolution, while eight chloroplast genes of R. pseudoacacia var. tortuosa contained introns that had lost the trnL-CAA and psaA genes. Among them, the rpoC1 gene had the longest introns at 2828 bp, and rps12+ had the smallest introns at only 533 bp. There were various amplification phenomena in the IR region among the five black locusts. Most of the protein-coding genes of the five black locusts had a high degree of codon preference. To determine the phylogenetic positions of the five black locusts, we conducted a systematic evolutionary analysis using common protein-coding genes in chloroplast sequences from 34 species of leguminous plants and 12 other species. The results showed that the relationship between Robinia and Acacia ligulata was the most distant among those of the leguminous plants, and the relationship between Robinia and Lotus japonicus was the closest. The chloroplast protein-coding genes in different black locusts were relatively conservative by evolutionary selection pressure analysis standards. These results are important for our understanding of their photosynthetic mechanisms and evolution, and the transgenic engineering of their chloroplasts.

The Relationship between Insect Resistance and Tree Age of Transgenic Triploid Populus tomentosa Plants.

To explore the stability of insect resistance during the development of transgenic insect-resistant trees, this study investigated how insect resistance changes as transgenic trees age. We selected 19 transgenic insect-resistant triploid Populus tomentosa lines as plant material. The presence of exogenous genes and Cry1Ac protein expression were verified using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. The toxicity for Clostera anachoreta and Lymantria dispar was evaluated by feeding fresh leaves to first instar larvae after the trees were planted in the field for 2 years and after the sixth year. Results of PCR showed that the exogenous genes had a long-term presence in the poplar genome. ELISA analyses showed significant differences existed on the 6-year-old transgenic lines. The insect-feeding experiment demonstrated significant differences in the mortality rates of C. anachoreta and L. dispar among different transgenic lines. The average corrected mortality rates of C. anachoreta and L. dispar ranged from 5.6-98.7% to 35.4-7.2% respectively. The larval mortality rates differed significantly between the lines at different ages. Up to 52.6% of 1-year-old transgenic lines and 42.1% of 2-year-old transgenic lines caused C. anachoreta larval mortality rates to exceed 80%, whereas only 26.3% of the 6-year-old transgenic lines. The mortality rates of L. dispar exhibited the same trend: 89.5% of 1-year-old transgenic lines and 84.2% of 2-year-old transgenic lines caused L. dispar larval mortality rates to exceed 80%; this number decreased to 63.2% for the 6-year-old plants. The proportion of 6-year-old trees with over 80% larval mortality rates was clearly lower than that of the younger trees. The death distribution of C. anachoreta in different developmental stages also showed the larvae that fed on the leaves of 1-year-old trees were killed mostly during L1 and L2 stages, whereas the proportion of larvae that died in L3 and L4 stages was significantly increased when fed on leaves of 6-year-old trees. Results of correlation analysis showed there was a significant correlation between the larvae mortality rates of trees at different ages, as well as between Cry1Ac protein contents and larvae mortality rates of 6-year-old trees.

The first complete chloroplast genome sequences of Ulmus species by de novo sequencing: Genome comparative and taxonomic position analysis.

Elm (Ulmus) has a long history of use as a high-quality heavy hardwood famous for its resistance to drought, cold, and salt. It grows in temperate, warm temperate, and subtropical regions. This is the first report of Ulmaceae chloroplast genomes by de novo sequencing. The Ulmus chloroplast genomes exhibited a typical quadripartite structure with two single-copy regions (long single copy [LSC] and short single copy [SSC] sections) separated by a pair of inverted repeats (IRs). The lengths of the chloroplast genomes from five Ulmus ranged from 158,953 to 159,453 bp, with the largest observed in Ulmus davidiana and the smallest in Ulmus laciniata. The genomes contained 137-145 protein-coding genes, of which Ulmus davidiana var. japonica and U. davidiana had the most and U. pumila had the fewest. The five Ulmus species exhibited different evolutionary routes, as some genes had been lost. In total, 18 genes contained introns, 13 of which (trnL-TAA+, trnL-TAA-, rpoC1-, rpl2-, ndhA-, ycf1, rps12-, rps12+, trnA-TGC+, trnA-TGC-, trnV-TAC-, trnI-GAT+, and trnI-GAT) were shared among all five species. The intron of ycf1 was the longest (5,675bp) while that of trnF-AAA was the smallest (53bp). All Ulmus species except U. davidiana exhibited the same degree of amplification in the IR region. To determine the phylogenetic positions of the Ulmus species, we performed phylogenetic analyses using common protein-coding genes in chloroplast sequences of 42 other species published in NCBI. The cluster results showed the closest plants to Ulmaceae were Moraceae and Cannabaceae, followed by Rosaceae. Ulmaceae and Moraceae both belonged to Urticales, and the chloroplast genome clustering results were consistent with their traditional taxonomy. The results strongly supported the position of Ulmaceae as a member of the order Urticales. In addition, we found a potential error in the traditional taxonomies of U. davidiana and U. davidiana var. japonica, which should be confirmed with a further analysis of their nuclear genomes. This study is the first report on Ulmus chloroplast genomes, which has significance for understanding photosynthesis, evolution, and chloroplast transgenic engineering.

Effects of the sequence and orientation of an expression cassette in tobacco transformed by dual Bt genes.

This study investigated the effects of the sequence arrangement and orientation of a target gene expression cassette in vectors on expression levels to determine the optimal combination for highly efficient multi-gene expression. Five plant transformation vectors were constructed using dual Bt genes, Cry1Ac and Cry3A, which differed in the sequence arrangement and orientation of the target gene expression cassette. Through an Agrobacterium-mediated method, 5 vectors were used for the genetic transformation of tobacco to obtain transgenic lines. Fluorescence quantitative PCR showed that the target genes were expressed at the transcriptional level, which did not differ significantly among the different vectors. However, an enzyme-linked immunosorbent assay showed that there were significant differences in the toxin expression levels of the different vectors. In vectors N12 and N19, the Cry1Ac gene, located upstream, showed lower average expression than the Cry3A gene, located downstream. Similarly, in vectors N13 and N18, the Cry3A gene, located upstream, had lower expression than the downstream Cry1Ac gene. For vector N21, with the expression cassette containing the Cry1Ac gene located upstream in a trans-arrangement and that of the Cry3A gene located downstream in a cis-arrangement, the Cry1Ac and Cry3A toxin levels were the highest, at 7.41 and 13.24µg·g-1, respectively. The insect resistance of transgenic lines transformed by the different vectors was related to the Bt toxin level. Resistance to H. armigera, Lepidoptera, and Cry1Ac toxin level were positively correlated; resistance to A. germari larvae, Coleoptera, and Cry3A toxin content were also positively correlated. This study showed that the sequence arrangement of 2 expression cassettes with target genes may be the key to the target gene expression. Two expression cassettes in the same orientation had little influence on gene expression; however, when the 2 expression cassettes were in the reverse arrangement, the expression of both of the target genes was promoted to a certain extent.

[Influence of temperature on insect-resistance of transgenic hybrid poplar 741].

The study showed that the insect-resistance of transgenic hybrid poplar 741 was closely related with temperature. The total mortality and accumulative mortality of Gypsy moth (Lmantriadispar L.) larvae increased with increasing temperature, and the sensitivity of larvae to the clones of transgenic hybrid poplar 741 and temperature decreased gradually with the increasing instars. Temperature had a promotion to the development of larvae fed on CK. When the temperature was high, the development of larvae quickened, the development period shortened, and the weight increasing rate and the frass increasing quantity quickened, but the development rate of larvae fed on transgenic hybrid poplar 741 was not obvious with varied temperature. The regressive equation of temperature and Clostera anachoreta (Fabricius) mortality during 1998-2000 year indicated that high resistant and medium resistant clones' temperature-insect mortality curve presented an obvious regressive relation.