Genome-wide molecular evolution analysis of the GRF and GIF gene families in Plantae (Archaeplastida).

BACKGROUND: Plant growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) interact with each other and collectively have important regulatory roles in plant growth, development, and stress responses. Therefore, it is of great significance to explore the systematic evolution of GRF and GIF gene families. However, our knowledge and understanding of the role of GRF and GIF genes during plant evolution has been fragmentary. RESULTS: In this study, a large number of genomic and transcriptomic datasets of algae, mosses, ferns, gymnosperms and angiosperms were used to systematically analyze the evolution of GRF and GIF genes during the evolution of plants. The results showed that GRF gene first appeared in the charophyte Klebsormidium nitens, whereas the GIF genes originated relatively early, and these two gene families were mainly expanded by segmental duplication events after plant terrestrialization. During the process of evolution, the protein sequences and functions of GRF and GIF family genes are relatively conservative. As cooperative partner, GRF and GIF genes contain the similar types of cis-acting elements in their promoter regions, which enables them to have similar transcriptional response patterns, and both show higher levels of expression in reproductive organs and tissues and organs with strong capacity for cell division. Based on protein-protein interaction analysis and verification, we found that the GRF-GIF protein partnership began to be established in pteridophytes and is highly conserved across different terrestrial plants. CONCLUSIONS: These results provide a foundation for further exploration of the molecular evolution and biological functions of GRF and GIF genes.

The relationship between atmospheric particulate matter, leaf surface microstructure, and the phyllosphere microbial diversity of Ulmus L.

BACKGROUND: Plants can retain atmospheric particulate matter (PM) through their unique foliar microstructures, which has a profound impact on the phyllosphere microbial communities. Yet, the underlying mechanisms linking atmospheric particulate matter (PM) retention by foliar microstructures to variations in the phyllosphere microbial communities remain a mystery. In this study, we conducted a field experiment with ten Ulmus lines. A series of analytical techniques, including scanning electron microscopy, atomic force microscopy, and high-throughput amplicon sequencing, were applied to examine the relationship between foliar surface microstructures, PM retention, and phyllosphere microbial diversity of Ulmus L. RESULTS: We characterized the leaf microstructures across the ten Ulmus lines. Chun exhibited a highly undulated abaxial surface and dense stomatal distribution. Langya and Xingshan possessed dense abaxial trichomes, while Lieye, Zuiweng, and Daguo had sparsely distributed, short abaxial trichomes. Duomai, Qingyun, and Lang were characterized by sparse stomata and flat abaxial surfaces, whereas Jinye had sparsely distributed but extensive stomata. The mean leaf retention values for total suspended particulate (TSP), PM2.5, PM2.5-10, PM10-100, and PM> 100 were 135.76, 6.60, 20.10, 90.98, and 13.08 µg·cm- 2, respectively. Trichomes substantially contributed to PM2.5 retention, while larger undulations enhanced PM2.5-10 retention, as evidenced by positive correlations between PM2.5 and abaxial trichome density and between PM2.5-10 and the adaxial raw microroughness values. Phyllosphere microbial diversity patterns varied among lines, with bacteria dominated by Sediminibacterium and fungi by Mycosphaerella, Alternaria, and Cladosporium. Redundancy analysis confirmed that dense leaf trichomes facilitated the capture of PM2.5-associated fungi, while bacteria were less impacted by PM and struggled to adhere to leaf microstructures. Long and dense trichomes provided ideal microhabitats for retaining PM-borne microbes, as evidenced by positive feedback loops between PM2.5, trichome characteristics, and the relative abundances of microorganisms like Trichoderma and Aspergillus. CONCLUSIONS: Based on our findings, a three-factor network profile was constructed, which provides a foundation for further exploration into how different plants retain PM through foliar microstructures, thereby impacting phyllosphere microbial communities.

GRP78 promotes bone metastasis of prostate cancer by regulating bone microenvironment through Sonic hedgehog signaling.

Bone metastasis is the leading cause of tumor-related deaths in patients with prostate cancer (PCa). The interactions between PCa and the bone microenvironment form a vicious cycle. However, the complex molecular mechanism by which PCa regulates the bone microenvironment remains unclear. To determine the role of glucose-regulated protein (GRP78) in bone metastasis and growth, we established intracardiac injection and tibial injection models, and performed their histological staining. To assess the effect of GRP78 on the differentiation of osteoblasts and osteoclasts, we performed cell co-culture, enzyme-linked immunosorbent assay, alizarin red staining, and tartrate-resistant acid phosphatase staining. We found that GRP78 is upregulated in PCa tissues and that its upregulation is associated with PCa progression in patients. Functional experiments showed that GRP78 overexpression in PCa cells considerably promotes bone metastasis and induces bone microstructure changes. Silencing GRP78 substantially inhibits the migration and invasion of PCa cells in vitro and bone metastasis and tumor growth in vivo. Mechanistically, GRP78 promotes the migration and invasion of PCa cells via the Sonic hedgehog (Shh) signaling pathway. Cell co-culture showed that GRP78 promotes the differentiation of osteoblasts and osteoclasts through Shh signaling. Our findings suggest that tumor-bone matrix interactions owing to GRP78-activated paracrine Shh signaling by PCa cells regulate the differentiation of osteoblasts and osteoclasts. This process promotes bone metastasis and the proliferation of PCa cells in the bone microenvironment. Targeting the GRP78/Shh axis can serve as a therapeutic strategy to prevent bone metastasis and improve the quality of life of patients with PCa.

Research Progress and Biosynthetic Mechanisms of Nutritional Compounds Obtained from Various Organs During the Developmental Stages of a Medicinal Plant (Chinese Jujube).

The fruit of the jujube tree is high in nutrients and has various health benefits. China is a major producer of jujube, and it is now cultivated all around the world. Numerous studies have demonstrated the nutritional value and potential health advantages of bioactive compounds found in the jujube tree. Furthermore, the jujube tree has a remarkable 7000-year agricultural history. The jujube plant has developed a rich gene pool, making it a valuable resource for germplasm. Different studies have focused on the developmental stages of jujube fruits to identify the optimal time for harvest and to assess the changes in their bioactive natural compounds or products during the process of development but the molecular mechanism underlying the production of bioactive natural products in Z. jujuba is still poorly understood. Moreover, the potential differential expressed genes (DEGs) identified as responsible for the synthesis of these compounds should be further functionally verified. It has been noticed that the contents of total flavonoids, total phenolic, and vitamin C increase significantly during the ripening process, while the contents of soluble sugars and organic acids decrease gradually. In this review, we have also scrutinized the challenges that hinder the utilization of jujube fruit resources and suggested potential areas for further research. As such, our review serves as a valuable resource for the future development of jujube-based nutritional compounds and the incorporation of their nutritional elements into the functional foods industry.

Analysis of the RNA Editing Sites and Orthologous Gene Function of Transcriptome and Chloroplast Genomes in the Evolution of Five Deutzia Species.

In this study, the chloroplast genomes and transcriptomes of five Deutzia genus species were sequenced, characterized, combined, and analyzed. A phylogenetic tree was constructed, including 32 other chloroplast genome sequences of Hydrangeoideae species. The results showed that the five Deutzia chloroplast genomes were typical circular genomes 156,860-157,025 bp in length, with 37.58-37.6% GC content. Repeat analysis showed that the Deutzia species had 41-45 scattered repeats and 199-201 simple sequence repeats. Comparative genomic and pi analyses indicated that the genomes are conservative and that the gene structures are stable. According to the phylogenetic tree, Deutzia species appear to be closely related to Kirengeshoma palmata and Philadelphus. By combining chloroplast genomic and transcriptomic analyses, 29-31 RNA editing events and 163-194 orthologous genes were identified. The ndh, rpo, rps, and atp genes had the most editing sites, and all RNA editing events were of the C-to-U type. Most of the orthologous genes were annotated to the chloroplast, mitochondria, and nucleus, with functions including energy production and conversion, translation, and protein transport. Genes related to the biosynthesis of monoterpenoids and flavonoids were also identified from the transcriptome of Deutzia spp. Our results will contribute to further studies of the genomic information and potential uses of the Deutzia spp.

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.

Metabolomics and Transcriptomics Analyses Reveals the Molecular Regulatory Mechanisms of Walnut (Juglans regia L.) Embryos in Response to Shade Treatment.

The walnut is an important nut that has numerous uses worldwide. However, due to dwarf and close plantation methods as well as continuous cloudy or rainy days that occur during periods of walnut oil accumulation, the walnut fruit exhibits varying degrees of stress under low-light conditions. However, the effects of shade on metabolites and genes in walnut embryos remain unclear in the literature. The purpose of this study is to investigate the lipid biosynthesis process that occurs in walnut embryos under shade treatment via the use of metabolomics and transcriptomics analyses. The results indicate that the oil content decreases significantly under shaded conditions, while the protein content increases significantly. The expression levels of fatty acid desaturase 2 (FAD2) and stearoyl-ACP-desaturase (SAD) involved in the lipid biosynthesis mechanism were significantly reduced in the shaded group, which resulted in reductions in oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids. The reduced oil content was consistent with the downregulation of genes associated with the lipid biosynthesis mechanism. In the amino acid biosynthesis process, the upregulated cysteine synthase (cscK) and anthranilate synthase beta subunit 2 (trpG) genes promoted the accumulation of L-aspartic acid and L-citrulline. The increase in protein content was consistent with the upregulation of genes related to amino acid biosynthesis. Thus, our study provides new insights into the regulatory mechanisms of shade underlying overall walnut fruit quality.

Whole-genome resequencing using next-generation and Nanopore sequencing for molecular characterization of T-DNA integration in transgenic poplar 741.

BACKGROUND: The molecular characterization information of T-DNA integration is not only required by public risk assessors and regulators, but is also closely related to the expression of exogenous and endogenous genes. At present, with the development of sequencing technology, whole-genome resequencing has become an attractive approach to identify unknown genetically modified events and characterise T-DNA integration events. RESULTS: In this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905-9,283,937 bp on chromosome 3 (Chr03) and 10,868,777-10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29. CONCLUSIONS: This study provides molecular characterization information of T-DNA integration in transgenic poplar 741 line Pb29, which contribute to safety supervision and further extensive commercial planting of transgenic poplar 741.

Combined transcriptome and proteome analysis provides insights into anthocyanin accumulation in the leaves of red-leaved poplars.

KEY MESSAGE: Anthocyanin was highly accumulated in the leaves of red-leaved poplars; Many structural genes involved in anthocyanin synthesis were significantly up-regulated in 'Quanhong' and 'Xuanhong'; TTG2, HYH, and HY5 may be directly involved in the regulation of anthocyanin synthesis in both red-leaved poplars. The red-leaved poplar cultivars 'Quanhong' and 'Xuanhong' are bud mutations of Populus deltoides cv. 'Zhonglin 2025'. These cultivars are valued for their beautiful shape, lack of flying catkins, and ornamental leaf colors. However, the understanding of the molecular mechanism of anthocyanin accumulation in the leaves of red-leaved poplars is still unclear. Here, we profiled the changes of pigment content, transcriptome and proteome expression in the leaves of three poplar cultivars and the results showed that the ratios of anthocyanin to total chlorophyll in both red-leaved poplars were higher than that in 'Zhonglin 2025', indicating that the anthocyanin was highly accumulated in the leaves of red-leaved poplars. Based on the results of combined transcriptome and proteome analysis, 15 and 11 differentially expressed genes/proteins involved in anthocyanin synthesis were screened in 'Quanhong' and 'Xuanhong', respectively, indicating that the two red-leaved poplar cultivars have slightly different patterns of regulating anthocyanin biosynthesis. Among the 120 transcription factors, 3 (HY5, HYH, and TTG2), may be directly involved in the regulation of anthocyanin synthesis in both red-leaved poplars. This study screens the candidate genes involved in anthocyanin accumulation in the leaves of red-leaved poplars and lays a foundation for further exploring the molecular mechanism of leaf red coloration in red-leaved poplars.

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.

Hypoxia reduces the osteogenic differentiation of peripheral blood mesenchymal stem cells by upregulating Notch-1 expression.

Purpose: Mesenchymal stem cells (MSCs) seeded on biocompatible scaffolds have therapeutic potential for bone defect repair. However, MSCs can be affected by hypoxia and nutritional deficiency due to a lack of blood vessels in the scaffolds. Here, we explored the effects of hypoxia on MSC differentiation to clarify these mechanisms. Methods: Peripheral blood mesenchymal stem cells (PBMSCs) were cultured in small individual chambers with oxygen concentrations of 1%, 9%, and 21%. Cell proliferation was evaluated by Cell Counting Kit 8 assays, and cell survival was determined using live/dead assays. Scratch assays were performed to evaluate cell migration. Ca2+ deposition/mineralization experiments, reverse transcription quantitative real-time polymerase chain reaction, and Western blotting were performed to assess the osteogenic differentiation of cells. Notch1 expression was downregulated by lentivirus-transfected PBMSCs to observe the effects of Notch1 knockdown on osteogenic gene and protein expression. Results: PBMSCs exposed to hypoxia (1% O2) demonstrated accelerated proliferation, increased migration, and reduced survival in the absence of serum. Although 9% oxygen promoted osteogenic differentiation, the osteogenic differentiation of PBMSCs was significantly reduced by 1% O2, and this effect was associated with increased Notch1 expression. Reducing Notch1 expression using small interfering RNA significantly restored the osteogenic differentiation of PBMSCs. Conclusions: Hypoxia accelerated proliferation, increased migration, and reduced PBMSC differentiation into osteoblasts by increasing Notch1 expression. These findings may contribute to the development of appropriate cell culture or in vivo transplantation conditions to maintain the full osteogenic potential of PBMSCs.

Effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of MC3T3-E1 cells.

In physiological and pathological environments, the concentration of oxygen around osteoblasts varies widely. No studies have systematically evaluated the effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of osteoblasts. In this study, we cultured the osteoblast precursor cell line MC3T3-E1 in small individual chambers with oxygen concentrations of 1%, 3%, 6%, 9%, and 21%. Cell proliferation was evaluated by the proliferation index test and EdU staining. To test cell survival, a live/dead assay was performed. A tablet scratch assay was performed to detect the migratory ability of the cells. Bone nodule formation experiments and immunofluorescence and Western blotting analyses of osteogenic-related proteins were performed to assess the osteogenic differentiation of the cells. We found that the proliferation and osteogenic differentiation ability of MC3T3-E1 cells in different oxygen concentrations were both approximately bell-shaped curves and that the optimal oxygen concentrations were approximately 6% and 9%, respectively. The live/dead assay showed that the survival of MC3T3-E1 cells in different oxygen concentrations was affected by the amount of serum. The tablet scratch experiment showed that there was greater cell migration with oxygen concentrations of 1%, 3%, and 21% than with oxygen concentrations of 6% and 9%. Our results have significant reference value for the intervention of the pathological processes involving osteoblasts, such as fracture, osteoporosis, and some vascular diseases. These results also have an important guiding role for the new scientific idea that osteoblasts can function as treatment cells to repair bone defects.

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.

Structural and Comparative Analysis of the Complete Chloroplast Genome of Pyrus hopeiensis-"Wild Plants with a Tiny Population"-and Three Other Pyrus Species.

Pyrus hopeiensis is a valuable wild resource of Pyrus in the Rosaceae. Due to its limited distribution and population decline, it has been listed as one of the "wild plants with a tiny population" in China. To date, few studies have been conducted on P. hopeiensis. This paper offers a systematic review of P. hopeiensis, providing a basis for the conservation and restoration of P. hopeiensis resources. In this study, the chloroplast genomes of two different genotypes of P. hopeiensis, P. ussuriensis Maxin. cv. Jingbaili, P. communis L. cv. Early Red Comice, and P. betulifolia were sequenced, compared and analyzed. The two P. hopeiensis genotypes showed a typical tetrad chloroplast genome, including a pair of inverted repeats encoding the same but opposite direction sequences, a large single copy (LSC) region, and a small single copy (SSC) region. The length of the chloroplast genome of P. hopeiensis HB-1 was 159,935 bp, 46 bp longer than that of the chloroplast genome of P. hopeiensis HB-2. The lengths of the SSC and IR regions of the two Pyrus genotypes were identical, with the only difference present in the LSC region. The GC content was only 0.02% higher in P. hopeiensis HB-1. The structure and size of the chloroplast genome, the gene species, gene number, and GC content of P. hopeiensis were similar to those of the other three Pyrus species. The IR boundary of the two genotypes of P. hopeiensis showed a similar degree of expansion. To determine the evolutionary history of P. hopeiensis within the genus Pyrus and the Rosaceae, 57 common protein-coding genes from 36 Rosaceae species were analyzed. The phylogenetic tree showed a close relationship between the genera Pyrus and Malus, and the relationship between P. hopeiensis HB-1 and P. hopeiensis HB-2 was the closest.

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.

Genome-wide identification of the Pyrus R2R3-MYB gene family and PhMYB62 regulation analysis in Pyrus hopeiensis flowers at low temperature.

The R2R3-MYB gene family play an important role in plant growth, development and stress responses. In this study, a total of 122 PcoR2R3-MYB genes were identified and grouped into 26 clades in pear. And these PcoMYBs were unevenly distributed among 17 chromosomes. The sequence characteristics, conversed motifs, exon/intron structures, classification, duplication events and cis-acting elements were also investigated. The gene duplication events showed that segmental duplication may play key roles in expansion of the PcoMYB gene family. Pyrus hopeiensis, which is a valuable wild resource, has strong cold resistance. An integrative analyses of miRNA and mRNA showed that PhMYB62 was involved in regulating low-temperature stress in P. hopeiensis flower organs. Subcellular localization analysis showed that PhMYB62 protein was specifically localized to the nucleus. The result of DAP-seq showed that PhMYB62 responded to low-temperature stress in P. hopeiensis by regulating TFs, which were associated with plant stress resistance, and POD, GAUT12, AUX28 and CHS genes. Subsequently, yeast one-hybrid verified that PhMYB62 could bind and activate the promoter of POD gene. The current study would provide a comprehensive information for further functional research on the stress-responsive R2R3-MYB gene candidates in pear, and may help to identify the genes associated with cold resistance for the cultivation of cold-resistant pear varieties.

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.

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.

Conjoint Analysis of Genome-Wide lncRNA and mRNA Expression during the Salicylic Acid Response in Populus × euramericana.

Long noncoding RNAs (lncRNAs) participate in a wide range of biological processes, but lncRNAs in plants remain largely unknown; in particular, we lack a systematic identification of plant lncRNAs involved in hormone responses. To explore the molecular mechanism of the response of poplar to salicylic acid (SA), the changes in protective enzymes, which are closely related to plant resistance induced by exogenous SA, were studied, and the expression of mRNA and lncRNA were determined by high-throughput RNA sequencing. The results showed that the activities of phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), in the leaves of Populus × euramericana, were significantly increased by exogenous SA application. High-throughput RNA sequencing showed that 26,366 genes and 5690 lncRNAs were detected under the different treatment conditions: SA and H2O application. Among these, 606 genes and 49 lncRNAs were differentially expressed. According to target prediction, lncRNAs and target genes involved in light response, stress response, plant disease resistance, and growth and development, were differentially expressed in SA-treated leaves. Interaction analysis showed that lncRNA-mRNA interactions, following exogenous SA, were involved in the response of poplar leaves to the external environment. Our study provides a comprehensive view of Populus × euramericana lncRNAs and offers insights into the potential functions and regulatory interactions of SA-responsive lncRNAs, thus forming the foundation for future functional analysis of SA-responsive lncRNAs in Populus × euramericana.

Identification of Differentially Expressed lncRNAs in Response to Blue Light and Expression Pattern Analysis of Populus tomentosa Hybrid Poplar 741.

Poplar is an important shelterbelt, timber stand, and city tree species that has been the focus of forestry research. The regulatory role of the long non-coding RNA molecule (lncRNA; length > 200 nt) has been a research hotspot in plants. In this study, seedlings of 741 poplar were irradiated with LED blue and white light, and the Illumina HiSeq 2000 sequencing platform was used to identify lncRNAs. |logFC| > 1 and p < 0.05 were considered to indicate differentially expressed lncRNAs, and nine differentially expressed lncRNAs were screened, the target genes of which were predicted, and three functionally annotated target genes were obtained. The differentially expressed lncRNAs were identified as miRNA targets. Six lncRNAs were determined to be target sites for twelve mRNAs in six miRNA families. LncRNAs and their target genes, including lncRNA MSTRG.20413.1-ptc-miR396e-5p-GRF9, were verified using quantitative real-time polymerase chain reaction analysis, and the expression patterns were analyzed. The analysis showed that the ptc-miR396e-5p expression was downregulated, while lncRNA MSTRG.20413.1 and GRF9 expression was upregulated, after blue light exposure. These results indicate that lncRNAs interact with miRNAs to regulate gene expression and affect plant growth and development.