CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions.

The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities for plant breeding, it also raises serious biosafety concerns that require careful consideration, including potential off-target effects and the unintended transfer of modified genes to other organisms. This paper highlights strategies to mitigate biosafety risks and explores innovative plant gene editing detection methods. Our review investigates the international biosafety guidelines for gene-edited crops, analyzing their broad implications for agricultural and biotechnology research and advancement. We hope to provide illuminating and refined perspectives for industry practitioners and policymakers by evaluating CRISPR genome enhancement in plants.

Characteristics and function of the pathogenesis-related protein 1 gene family in poplar.

The pathogen-associated protein 1 (PR1) plays an important role in plant response to biotic and abiotic stresses. In this study, 17 PtPR1 genes were identified in Populus trichocarpa genome. The 17 PtPR1 genes were distributed on 7 chromosomes, and divided into A, B subfamilies by evolutionary tree analysis. RTqPCR analysis showed that the PtPR1 gene family showed different degrees of response to drought stress. PtPR1 genes showed changes in expression in response to fungal pathogen Septotinia populiperda or insect attacks (Nausinoe geometralis, Hyphantria cunea). Also, we found that subfamily B of PtPR1 may play an important role in response to biotic stress. We identified a new resistance gene PtPR1A. Overexpression of PtPR1A in Arabidopsis thaliana significantly enhanced the resistance to Pseudomonas syringae, while overexpression of PtPR1A in poplar significantly enhanced the resistance to S. populiperda. The present study investigates the expression pattern of the PtPR1 genes under biotic and abiotic stresses, and it found that the characteristics of the PtPR1 genes diverged, which provided a theoretical basis for the further study of the PtPR1 genes in the plant defense response.

Regulation of plant epigenetic memory in response to cold and heat stress: towards climate resilient agriculture.

Plants have evolved to adapt and grow in hot and cold climatic conditions. Some also adapt to daily and seasonal temperature changes. Epigenetic modifications play an important role in regulating plant tolerance under such conditions. DNA methylation and post-translational modifications of histone proteins influence gene expression during plant developmental stages and under stress conditions, including cold and heat stress. While short-term modifications are common, some modifications may persist and result in stress memory that can be inherited by subsequent generations. Understanding the mechanisms of epigenomes responding to stress and the factors that trigger stress memory is crucial for developing climate-resilient agriculture, but such an integrated view is currently limited. This review focuses on the plant epigenetic stress memory during cold and heat stress. It also discusses the potential of machine learning to modify stress memory through epigenetics to develop climate-resilient crops.

CRISPR-mediated genome editing in poplar issued by efficient transformation.

Background: CRISPR has been increasingly used for plant genetic improvements because of its high efficiency and precision. Recently, the authors have reported the possibility of homology-directed repair (HDR) using CRISPR/Cas9 through woody plants such as poplar. HDR often replaces nucleotides with one donor DNA template (DDT), including homologous sequences. Methods: CRISPR-Cas9 was recruited, and three variables, Agrobacteria inoculator concentration, pDDT/pgRNA ratio, and homologous arm length, were designed to integrate nptII and 2XCamV 35S into the MKK2 promoter zone. Results: Here, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by the precise integration of 2XcamV 35S and nptII, improving biochemical and phenotypic properties. Our findings confirmed that Agrobacterium inoculator OD600 = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased MKK2 expression. Conclusion: Efficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.

Poplar CCR4-associated factor PtCAF1I is necessary for poplar development and defense response.

Poplar is one of the most widely used tree species in afforestation projects. CCR4 associated factor 1 (CAF1) is a major member of CCR4-NOT and plays an important role in eukaryotic mRNA deadenylation. However, its role in poplar remains unclear. In this study, the full-length cDNA of the PtCAF1I gene was cloned from the poplar by screening the highly expressed PtCAF1I gene in the identified PtCAF1 gene family by poplar sterilization. PtCAF1I was localized in the nucleus. Through sequence alignment, it was found that the PtCAF1I sequence contains three motifs and is highly similar to the CAF1 protein sequence of other species. In the quantitative expression analysis of tissues, the expression of PtCAF1I in different tissues of Populus trichocarpa, 'Nanlin895', and Shanxinyang was not much different. In addition, the analysis of the expression of the PtCAF1I gene under different stress treatments showed that PtCAF1I responded to abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), NaCl, PEG6000, hydrogen peroxide (H2O2) and cold stress to different degrees. To study the potential biological functions of PtCAF1I, 6 transgenic lines were obtained through transformation using an Agrobacterium tumefaciens infection system. The transcriptome sequencing results showed that DEGs were mainly concentrated in pathways of phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, carbon metabolism, and carotenoid biosynthesis. Compared with WT poplar, the contents of cellulose, hemicellulose, lignin, total sugar, and flavonoids, and the cell wall thickness of PtCAF1I overexpression poplars were significantly higher. Under Septotinia populiperda treatment, transgenic poplars clearly exhibited certain disease resistance. Meanwhile, upregulation of the expression of JA and SA pathway-related genes also contributed to improving the disease tolerance of transgenic poplar. In conclusion, our results suggest that PtCAF1I plays an important role in the growth and development of poplars and their resistance to pathogens.

Evaluation of the Ecological Environment Affected by Cry1Ah1 in Poplar.

Populus is a genus of globally significant plantation trees used widely in industrial and agricultural production. Poplars are easily damaged by Micromelalopha troglodyta and Hyphantria cunea, resulting in decreasing quality. Bt toxin-encoded by the Cry gene has been widely adopted in poplar breeding because of its strong insect resistance. There is still no comprehensive and sufficient information about the effects of Cry1Ah1-modified (CM) poplars on the ecological environment. Here, we sampled the rhizosphere soils of field-grown CM and non-transgenic (NT) poplars and applied 16S rRNA and internal transcribed spacer amplicon Illumina MiSeq sequencing to determine the bacterial community associated with the CM and NT poplars. Based on the high-throughput sequencing of samples, we found that the predominant taxa included Proteobacteria (about 40% of the total bacteria), Acidobacteria (about 20% of the total bacteria), and Actinobacteria (about 20% of the total bacteria) collected from the natural rhizosphere of NT and CM poplars. In addition, studies on the microbial diversity of poplar showed that Cry1Ah1 expression has no significant influence on rhizosphere soil alkaline nitrogen, but significantly affects soil phosphorus, soil microbial biomass nitrogen, and carbon. The results exhibited a similar bacterial community structure between CM varieties affected by the expression of Cry1Ah1 and non-transgenic poplars. In addition, Cry1Ah1 expression revealed no significant influence on the composition of rhizosphere microbiomes. These results broadly reflect the effect of the Bt toxin-encoded by Cry1Ah1 on the ecology and environment and provide a clear path for researchers to continue research in this field in the future.

Systematic identification and functional characterization of the CFEM proteins in poplar fungus Marssonina brunnea.

Proteins containing Common in Fungal Extracellular Membrane (CFEM) domains uniquely exist in fungi and play significant roles in their whole life history. In this study, a total of 11 MbCFEM proteins were identified from Marssonina brunnea f. sp. multigermtubi (MULT), a hemibiotrophic pathogenic fungus on poplars that causes severe leaf diseases. Phylogenic analysis showed that the 11 proteins (MbCFEM1-11) were divided into three clades based on the trans-membrane domain and the CFEM domain. Sequence alignment and WebLogo analysis of CFEM domains verified the amino acids conservatism therein. All of them possess eight cysteines except MbCFEM4 and MbCFEM11, which lack two cysteines each. Six MbCFEM proteins with a signal peptide and without trans-membrane domain were considered as candidate effectors for further functional analysis. Three-dimensional (3D) models of their CFEM domains presented a helical-basket structure homologous to the crucial virulence factor Csa2 of Candida albicans. Afterward, four (MbCFEM1, 6, 8, and 9) out of six candidate effectors were successfully cloned and a yeast signal sequence trap (YSST) assay confirmed their secretion activity. Pathogen challenge assays demonstrated that the transient expression of four candidate MbCFEM effectors in Nicotiana benthamiana promoted Fusarium proliferatum infection, respectively. In an N. benthamiana heterogeneous expression system, MbCFEM1, MbCFEM6, and MbCFEM9 appeared to suppress both BAX/INF1-triggered PCD, whereas MbCFEM8 could only defeat BAX-triggered PCD. Additionally, subcellular localization analysis indicated that the four candidate MbCFEM effectors accumulate in the cell membrane, nucleus, chloroplast, and cytosolic bodies. These results demonstrate that MbCFEM1, MbCFEM6, MbCFEM8, and MbCFEM9 are effectors of M. brunnea and provide valuable targets for further dissection of the molecular mechanisms underlying the poplar-M. brunnea interaction.

Different color regulation mechanism in willow barks determined using integrated metabolomics and transcriptomics analyses.

BACKGROUND: The rich yellow-orange to vividly deep red bark of willow (Salix spp.) branches have high ornamental and economic value. However, the mechanism underlying the regulation of willow branch color remains unknown. Therefore, we performed metabolomics and transcriptomics analyses of purple, green, and red willow barks to elucidating the mechanisms regulating color development. RESULTS: Seven anthocyanins were isolated; pelargonidin, petunidin 3-O-rutinoside, and cyanin chloride were the most abundant in red bark, whereas pelargonin chloride was most abundant in purple bark. The green bark contained the highest level of malvidin; however, the malvidin level was not significantly higher than in the red bark. The purple bark contained the largest amount of canthaxanthin, a carotenoid pigment. The integrated pathways of flavonoid biosynthesis, carotenoid biosynthesis, and porphyrin and chlorophyll metabolism were constructed for the willow barks. Among the three barks, the expression of the structural genes ANS, ANR, and BZ1, which are involved in anthocyanin synthesis, was the highest in red bark, likely causing anthocyanin accumulation. The expression of CrtZ, which participates in the carotenoid pathway, was the highest in purple bark, likely leading to canthaxanthin accumulation. The high expression of DVR, POR, and CRD1 may be associated with green pigment synthesis in the chlorophyll biosynthesis pathway. CONCLUSIONS: Purple bark color is co-regulated by anthocyanins and carotenoids, whereas red bark is characterized by anthocyanin accumulation and chlorophyll degradation. The green pigment is regulated by maintaining chlorophyll synthesis. BZ1 and CrtZ are candidate genes regulating anthocyanin and canthaxanthin accumulation in red and purple barks respectively. Collectively, our results may facilitate the genetic breeding and cultivation of colorful willows with improved color and luster.

The whole-genome assembly of an endangered Salicaceae species: Chosenia arbutifolia (Pall.) A. Skv.

BACKGROUND: As a fast-growing tree species, Chosenia arbutifolia has a unique but controversial taxonomic status in the family Salicaceae. Despite its importance as an industrial material, in ecological protection, and in landscaping, C. arbutifolia is seriously endangered in Northeast China because of artificial destruction and its low reproductive capability. RESULTS: To clarify its phylogenetic relationships with other Salicaceae species, we assembled a high-quality chromosome-level genome of C. arbutifolia using PacBio High-Fidelity reads and Hi-C sequencing data, with a total size of 338.93 Mb and contig N50 of 1.68 Mb. Repetitive sequences, which accounted for 42.34% of the assembly length, were identified. In total, 33,229 protein-coding genes and 11,474 small noncoding RNAs were predicted. Phylogenetic analysis suggested that C. arbutifolia and poplars diverged approximately 15.3 million years ago, and a large interchromosomal recombination between C. arbutifolia and other Salicaceae species was discovered. CONCLUSIONS: Our study provides insights into the genome architecture and systematic evolution of C. arbutifolia, as well as comprehensive information for germplasm protection and future functional genomic studies.

Isoprenoid biosynthesis regulation in poplars by methylerythritol phosphate and mevalonic acid pathways.

It is critical to develop plant isoprenoid production when dealing with human-demanded industries such as flavoring, aroma, pigment, pharmaceuticals, and biomass used for biofuels. The methylerythritol phosphate (MEP) and mevalonic acid (MVA) plant pathways contribute to the dynamic production of isoprenoid compounds. Still, the cross-talk between MVA and MEP in isoprenoid biosynthesis is not quite recognized. Regarding the rate-limiting steps in the MEP pathway through catalyzing 1-deoxy-D-xylulose5-phosphate synthase and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) and also the rate-limiting step in the MVA pathway through catalyzing 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the characterization and function of HMGR from Populus trichocarpa (PtHMGR) were analyzed. The results indicated that PtHMGR overexpressors (OEs) displayed various MEP and MVA-related gene expressions compared to NT poplars. The overexpression of PtDXR upregulated MEP-related genes and downregulated MVA-related genes. The overexpression of PtDXR and PtHMGR affected the isoprenoid production involved in both MVA and MEP pathways. Here, results illustrated that the PtHMGR and PtDXR play significant roles in regulating MEP and MVA-related genes and derived isoprenoids. This study clarifies cross-talk between MVA and MEP pathways. It demonstrates the key functions of HMGR and DXR in this cross-talk, which significantly contribute to regulate isoprenoid biosynthesis in poplars.

Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9.

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.

Characterization, expression, and functional analysis of the pathogenesis-related gene PtDIR11 in transgenic poplar.

Lignins and lignans are important for plant resistance to pathogens. Dirigent (DIR) proteins control the regio- and stereo-selectivity of coniferyl alcohol in lignan and lignin biosynthesis. DIR genes have been implicated in defense-related responses in several plant species, but their role in poplar immunity is unclear. We cloned PtDIR11 from Populus trichocarpa; we found that overexpression of PtDIR11 in poplar improved the lignan biosynthesis and enhanced the resistance of poplar to Septotis populiperda. PtDIR11 has a typical DIR domain; it belongs to the DIR-b/d family and is expressed in the cell membrane. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis showed that PtDIR11 expression was highest in stems, followed by leaves and roots. Furthermore, PtDIR11 expression was induced by S. populiperda, salicylic acid (SA), jasmonate (JA), and ethylene (ET) stresses. The recombinant PtDIR11 protein inhibited the growth of S. populiperda in vitro. Overexpressing (OE) PtDIR11 in "Nanlin 895" poplar enhanced growth. The OE lines exhibited minimal changes in lignin content, but their total lignan and flavonoid contents were significantly greater than in the wild-type (WT) lines. Overexpression of PtDIR11 affected multiple biological pathways of poplar, such as phenylpropanoid biosynthesis. The methanol extracts of OE-PtDIR11 lines showed greater anti-S. populiperda activity than did lignin extracts from the WT lines. Furthermore, OE-PtDIR11 lines upregulated genes that were related to phenylpropanoid biosynthesis and genes associated with the JA and ET signal transduction pathways; it downregulated genes that were related to SA signal transduction compared with the WT line under S. populiperda stress. Therefore, the OE transgenic plants analysis revealed that PtDIR11 is a good candidate gene for breeding of disease resistant poplar.

A Method to Reduce off-Targets in CRISPR/Cas9 System in Plants.

One of the strategies to reduce the off-target mutations in CRISPR/Cas9 system is to use the temperature-independent gene transformation method. Mesoporous silica nanoparticles (MSNs)-gene delivery system is temperature-independent; thus, it can transfer the interesting plasmid (pDNA) to the target plant at different temperatures, including 37 °C. Due to the high activity of SpCas9 at 37 °C compared to lower temperatures, on-target mutagenesis increases at 37 °C. Therefore, we describe the synthesis of the functionalized MSNs with the particle size of less than 40 nm, binding pDNA to the MSNs, and transferring of the pDNA-MSNs into the target plants.

Plant Secondary Metabolites with an Overview of Populus.

Populus trees meet continuous difficulties from the environment through their life cycle. To warrant their durability and generation, Populus trees exhibit various types of defenses, including the production of secondary metabolites. Syntheses derived from the shikimate-phenylpropanoid pathway are a varied and plentiful class of secondary metabolites manufactured in Populus. Amongst other main classes of secondary metabolites in Populus are fatty acid and terpenoid-derivatives. Many of the secondary metabolites made by Populus trees have been functionally described. Any others have been associated with particular ecological or biological processes, such as resistance against pests and microbial pathogens or acclimatization to abiotic stresses. Still, the functions of many Populus secondary metabolites are incompletely understood. Furthermore, many secondary metabolites have therapeutic effects, leading to more studies of secondary metabolites and their biosynthesis. This paper reviews the biosynthetic pathways and therapeutic impacts of secondary metabolites in Populus using a genomics approach. Compared with bacteria, fewer known pathways produce secondary metabolites in Populus despite P. trichocarpa having had its genome sequenced.

Genome-Wide and Comprehensive Analysis of the Multiple Stress-Related CAF1 (CCR4-Associated Factor 1) Family and Its Expression in Poplar.

Poplar is one of the most widely used tree in afforestation projects. However, it is susceptible to abiotic and biotic stress. CCR4-associated factor 1 (CAF1) is a major member of CCR4-NOT, and it is mainly involved in transcriptional regulation and mRNA degradation in eukaryotes. However, there are no studies on the molecular phylogeny and expression of the CAF1 gene in poplar. In this study, a total of 19 PtCAF1 genes were identified in the Populus trichocarpa genome. Phylogenetic analysis of the PtCAF1 gene family was performed with two closely related species (Arabidopsis thaliana and Oryza sativa) to investigate the evolution of the PtCAF1 gene. The tissue expression of the PtCAF1 gene showed that 19 PtCAF1 genes were present in different tissues of poplar. Additionally, the analysis of the expression of the PtCAF1 gene showed that the CAF1 family was up-regulated to various degrees under biotic and abiotic stresses and participated in the poplar stress response. The results of our study provide a deeper understanding of the structure and function of the PtCAF1 gene and may contribute to our understanding of the molecular basis of stress tolerance in poplar.

Genome-Wide Evolution and Comparative Analysis of Superoxide Dismutase Gene Family in Cucurbitaceae and Expression Analysis of Lagenaria siceraria Under Multiple Abiotic Stresses.

Superoxide dismutase (SOD) is an important enzyme that serves as the first line of defense in the plant antioxidant system and removes reactive oxygen species (ROS) under adverse conditions. The SOD protein family is widely distributed in the plant kingdom and plays a significant role in plant growth and development. However, the comprehensive analysis of the SOD gene family has not been conducted in Cucurbitaceae. Subsequently, 43 SOD genes were identified from Cucurbitaceae species [Citrullus lanatus (watermelon), Cucurbita pepo (zucchini), Cucumis sativus (cucumber), Lagenaria siceraria (bottle gourd), Cucumis melo (melon)]. According to evolutionary analysis, SOD genes were divided into eight subfamilies (I, II, III, IV, V, VI, VII, VIII). The gene structure analysis exhibited that the SOD gene family had comparatively preserved exon/intron assembly and motif as well. Phylogenetic and structural analysis revealed the functional divergence of Cucurbitaceae SOD gene family. Furthermore, microRNAs 6 miRNAs were predicted targeting 3 LsiSOD genes. Gene ontology annotation outcomes confirm the role of LsiSODs under different stress stimuli, cellular oxidant detoxification processes, metal ion binding activities, SOD activity, and different cellular components. Promoter regions of the SOD family revealed that most cis-elements were involved in plant development, stress response, and plant hormones. Evaluation of the gene expression showed that most SOD genes were expressed in different tissues (root, flower, fruit, stem, and leaf). Finally, the expression profiles of eight LsiSOD genes analyzed by qRT-PCR suggested that these genetic reserves responded to drought, saline, heat, and cold stress. These findings laid the foundation for further study of the role of the SOD gene family in Cucurbitaceae. Also, they provided the potential for its use in the genetic improvement of Cucurbitaceae.

Identification, evolution, expression, and docking studies of fatty acid desaturase genes in wheat (Triticum aestivum L.).

BACKGROUNDS: Fatty acid desaturases (FADs) introduce a double bond into the fatty acids acyl chain resulting in unsaturated fatty acids that have essential roles in plant development and response to biotic and abiotic stresses. Wheat germ oil, one of the important by-products of wheat, can be a good alternative for edible oils with clinical advantages due to the high amount of unsaturated fatty acids. Therefore, we performed a genome-wide analysis of the wheat FAD gene family (TaFADs). RESULTS: 68 FAD genes were identified from the wheat genome. Based on the phylogenetic analysis, wheat FADs clustered into five subfamilies, including FAB2, FAD2/FAD6, FAD4, DES/SLD, and FAD3/FAD7/FAD8. The TaFADs were distributed on chromosomes 2A-7B with 0 to 10 introns. The Ka/Ks ratio was less than one for most of the duplicated pair genes revealed that the function of the genes had been maintained during the evolution. Several cis-acting elements related to hormones and stresses in the TaFADs promoters indicated the role of these genes in plant development and responses to environmental stresses. Likewise, 72 SSRs and 91 miRNAs in 36 and 47 TaFADs have been identified. According to RNA-seq data analysis, the highest expression in all developmental stages and tissues was related to TaFAB2.5, TaFAB2.12, TaFAB2.15, TaFAB2.17, TaFAB2.20, TaFAD2.1, TaFAD2.6, and TaFAD2.8 genes while the highest expression in response to temperature stress was related to TaFAD2.6, TaFAD2.8, TaFAB2.15, TaFAB2.17, and TaFAB2.20. Furthermore, docking simulations revealed several residues in the active site of TaFAD2.6 and TaFAD2.8 in close contact with the docked oleic acid that could be useful in future site-directed mutagenesis studies to increase the catalytic efficiency of them and subsequently improve agronomic quality and tolerance of wheat against environmental stresses. CONCLUSIONS: This study provides comprehensive information that can lead to the detection of candidate genes for wheat genetic modification.

Characterization, Expression Profiling, and Functional Analysis of PtDef, a Defensin-Encoding Gene From Populus trichocarpa.

PtDef cloned from Populus trichocarpa contained eight cysteine domains specific to defensins. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis showed that PtDef was expressed in all tissues tested, with lower expression in leaves and higher expression in petioles, stems, and roots. Purified fused PtDef inhibited Aspergillus niger, Alternaria Nees, Mucor corymbifer, Marssonina populi, Rhizopus sp., and Neurospora crassa. PtDef also inhibited the growth of Escherichia coli by triggering autolysis. PtDef overexpression in Nanlin895 poplar (Populus × euramericana cv. Nanlin895) enhanced the level of resistance to Septotinia populiperda. qRT-PCR analysis also showed that the expression of 13 genes related to salicylic acid (SA) and jasmonic acid (JA) signal transduction differed between transgenic and wild-type (WT) poplars before and after inoculation, and that PR1-1 (12-72 h), NPR1-2, TGA1, and MYC2-1 expression was higher in transgenic poplars than in WT. During the hypersensitivity response (HR), large amounts of H2O2 were produced by the poplar lines, particularly 12-24 h after inoculation; the rate and magnitude of the H2O2 concentration increase were greater in transgenic lines than in WT. Overall, our findings suggest that PtDef, a defensin-encoding gene of P. trichocarpa, could be used for genetic engineering of woody plants for enhanced disease resistance.

Overexpression of PtDefensin enhances resistance to Septotis populiperda in transgenic poplar.

Plant defensins have been implicated in the plant defense system, but their role in poplar immunity is still unclear. In the present study, we present evidence that PtDefensin, a putative plant defensin, participates in the defense of poplar plants against Septotis populiperda infection. After the construction of recombinant plasmid PET-32a-PtDefensin, PtDefensin protein was expressed in Escherichia coli strain BL21 (DE3) and purified through Ni-IDA resin affinity chromatography. The Trx-PtDefensin fusion protein displayed no cytotoxic activity against RAW264.7 cells but had cytotoxic activity against E. coli K12D31 cells. Analyses of PtDefensin transcript abundance showed that the expression levels of PtDefensin responded to abiotic and biotic stresses. Overexpression of PtDefensin in 'Nanlin 895' poplars (Populus × euramericana cv 'Nanlin895') increased resistance to Septotis populiperda, coupled with upregulation of MYC2 (basic helix-loop-helix (bHLH) transcription factor) related to jasmonic acid (JA) signal transduction pathways and downregulation of Jasmonate-zim domain (JAZ), an inhibitor in the JA signal transduction pathway. We speculate that systemic acquired resistance (SAR) was activated in non-transgenic poplars after S. populiperda incubation, and that induced systemic resistance (ISR) was activated more obviously in transgenic poplars after S. populiperda incubation. Hence, overexpression of PtDefensin may improve the resistance of poplar plants to pathogens.

Overexpression of PtHMGR enhances drought and salt tolerance of poplar.

BACKGROUND AND AIMS: Soil salinization and aridification are swiftly engulfing the limited land resources on which humans depend, restricting agricultural production. Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is important in the biosynthesis of terpenoids, which are involved in plant growth, development and responses to environmental stresses. This study aimed to provide guidance for producing salt- and drought-resistant poplar. METHODS: A protein expression system was used to obtain PtHMGR protein, and high-performance liquid chromatography was used to detect the activity of PtHMGR protein in vitro. In addition, a simplified version of the leaf infection method was used for transformation of 'Nanlin895' poplar (Populus×euramericana). qRT-PCR was used to identify expression levels of genes. KEY RESULTS: PtHMGR catalysed a reaction involving HMG-CoA and NADPH to form mevalonate. Overexpression of PtHMGR in Populus × euramericana 'Nanlin895' improved drought and salinity tolerance. In the presence of NaCl and PEG6000, the rates of rooting and survival of PtHMGR-overexpressing poplars were higher than those of wild-type poplars. The transgenic lines also exhibited higher proline content and peroxidase and superoxide dismutase activities, and a lower malondialdehyde level under osmotic stress. In addition, the expression of genes related to reactive oxygen species (ROS) scavenging and formation was altered by osmotic stress. Moreover, the effect of osmotic stress on transcript levels of stress-related genes differed between the transgenic and wild-type poplars. CONCLUSION: PtHMGR catalysed a reaction involving HMG-CoA and NADPH to form mevalonate in vitro. Overexpression of PtHMGR promoted root development, increased the expression of ROS scavenging-related genes, decreased the expression of ROS formation-related genes, and increased the activity of antioxidant enzymes in transgenic poplars, enhancing their tolerance of osmotic stress. In addition, overexpression of PtHMGR increased expression of the stress-related genes KIN1, COR15 and AAO3 and decreased that of ABI, MYB, MYC2 and RD22, enhancing the stress resistance of poplar.