Cloning and overexpression of PeWRKY31 from Populus × euramericana enhances salt and biological tolerance in transgenic Nicotiana.

BACKGROUND: As important forest tree species, biological stress and soil salinization are important factors that restrict the growth of Populus × euramericana. WRKYs are important transcription factors in plants that can regulate plant responses to biotic and abiotic stresses. In this study, PeWRKY31 was isolated from Populus × euramericana, and its bioinformation, salt resistance and insect resistance were analyzed. This study aims to provide guidance for producing salt-resistant and insect-resistant poplars. RESULTS: PeWRKY31 has a predicted open reading frame (ORF) of 1842 bp that encodes 613 amino acids. The predicted protein is the unstable, acidic, and hydrophilic protein with a molecular weight of 66.34 kDa, and it has numerous potential phosphorylation sites, chiefly on serines and threonines. PeWRKY31 is a zinc-finger C2H2 type-II WRKY TF that is closely related to WRKY TFs of Populus tomentosa, and localizes to the nucleus. A PeWRKY31 overexpression vector was constructed and transformed into Nicotiana tabacum L. Overexpression of PeWRKY31 improved the salt tolerance and insect resistance of the transgenic tobacco. Transcriptome sequencing and KEGG enrichment analysis showed the elevated expression of genes related to glutathione metabolism, plant hormone signal transduction, and MAPK signaling pathways, the functions of which were important in plant salt tolerance and insect resistance in the overexpressing tobacco line. CONCLUSIONS: PeWRKY31 was isolated from Populus × euramericana. Overexpression of PeWRKY31 improved the resistance of transgenic plant to salt stress and pest stress. The study provides references for the generation of stress-resistant lines with potentially great economic benefit.

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.

A 5-year field study showed no apparent effect of the Bt transgenic 741 poplar on the arthropod community and soil bacterial diversity.

China is currently the only country that has commercialized genetically engineered tree species, and this has attracted worldwide attention. As a perennial tree species, transgenic poplar has a long growth cycle and needs to be tested for long-term ecological risks. The main purpose of this study was to explore the ecological safety of perennial transgenic poplars in arthropod community, physical and chemical properties of soil, gene flow, and soil microbial diversity. The study found transgenic poplars could effectively inhibit the number of pests. Moreover, transgenic poplar 741 did not affect the stability of the arthropod community. Studies on the microbial diversity of poplar showed that transgenic poplars did not affect the physical and chemical properties of the soil and the soil microbial community structure. Furthermore, the microbial community structure was obviously affected by location and season. The results showed that a 5-year-old transgenic 741 poplar did not pose an ecological risk, and did not affect the microbial community structure or functional diversity. This study provides a reference for the ecological security evaluation of transgenic poplars, and provides a theoretical basis for promoting the commercialization of transgenic poplars.

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.

Centrosomal Nlp is an oncogenic protein that is gene-amplified in human tumors and causes spontaneous tumorigenesis in transgenic mice.

Disruption of mitotic events contributes greatly to genomic instability and results in mutator phenotypes. Indeed, abnormalities of mitotic components are closely associated with malignant transformation and tumorigenesis. Here we show that ninein-like protein (Nlp), a recently identified BRCA1-associated centrosomal protein involved in microtubule nucleation and spindle formation, is an oncogenic protein. Nlp was found to be overexpressed in approximately 80% of human breast and lung carcinomas analyzed. In human lung cancers, this deregulated expression was associated with NLP gene amplification. Further analysis revealed that Nlp exhibited strong oncogenic properties; for example, it conferred to NIH3T3 rodent fibroblasts the capacity for anchorage-independent growth in vitro and tumor formation in nude mice. Consistent with these data, transgenic mice overexpressing Nlp displayed spontaneous tumorigenesis in the breast, ovary, and testicle within 60 weeks. In addition, Nlp overexpression induced more rapid onset of radiation-induced lymphoma. Furthermore, mouse embryonic fibroblasts (MEFs) derived from Nlp transgenic mice showed centrosome amplification, suggesting that Nlp overexpression mimics BRCA1 loss. These findings demonstrate that Nlp abnormalities may contribute to genomic instability and tumorigenesis and suggest that Nlp might serve as a potential biomarker for clinical diagnosis and therapeutic target.