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 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.

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.

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.

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.

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.

Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants.

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

Overexpression of PtDXS Enhances Stress Resistance in Poplars.

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) is the rate-limiting enzyme in the plastidial methylerythritol phosphate pathway (MEP). In this study, PtDXS (XM_024607716.1) was isolated from Populus trichocarpa. A bioinformatics analysis revealed that PtDXS had high homology with the DXSs of other plant species. PtDXS expression differed among plant tissues and was highest in young leaves and lowest in roots. The recombinant protein was produced in Escherichia coli BL21 (DE3), purified, and its activity evaluated. The purified protein was capable of catalyzing the formation of 1-deoxy-d-xylulose-5-phosphate (DXP) from glyceraldehyde-3-phosphate and pyruvate. A functional color assay in E. coli harboring pAC-BETA indicated that PtDXS encodes a functional protein involved in the biosynthesis of isoprenoid precursors. The treatment of P. trichocarpa seedlings with 200 µM abscisic acid (ABA), 200 mM NaCl, 10% polyethylene glycol6000, and 2 mM H2O2 resulted in increased expression of PtDXS. The ABA and gibberellic acid contents of the transgenic lines (Poplar Nanlin 895) were higher than wild types, suggesting that DXS is important in terpenoid biosynthesis. Overexpression of PtDXS enhanced resistance to S. populiperda infection. Furthermore, the transgenic lines showed decreased feeding by Micromelalopha troglodyta, supporting the notion that PtDXS is a key enzyme in terpenoid biosynthesis.

Expression and characterization of the antimicrobial peptide ABP-dHC-cecropin A in the methylotrophic yeast Pichia pastoris.

ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris, we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The SacI-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4-20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein.

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy.

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.

Transformation of a Thermostable G-Quadruplex Structure into DNA Duplex Driven by Reverse Gyrase.

Reverse gyrase is a topoisomerase that can introduce positive supercoils to its substrate DNA. It is demonstrated in our studies that a highly thermal stable G-quadruplex structure in a mini-plasmid DNA was transformed into its duplex conformation after a treatment with reverse gyrase. The structural difference of the topoisomers were verified and analyzed by gel electrophoresis, atomic force microscopy examination, and endonuclease digestion assays. All evidence suggested that the overwinding structure of positive supercoil could provide a driven force to disintegrate G-quadruplex and reform duplex. The results of our studies could suggest that hyperthermophiles might use reverse gyrase to manipulate the disintegration of non-B DNA structures and safekeep their genomic information.

High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli.

The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve the expression level of ABP-dHC-cecropin A in E. coli, tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors (n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins (n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A.

RNA-directed DNA methylation in plants.

In plants, many small interfering RNAs (siRNAs) direct de novo methylation by DNA methyltransferase. DNA methylation typically occurs by RNA-directed DNA methylation (RdDM), which directs transcriptional gene silencing of transposons and endogenous transgenes. RdDM is driven by non-coding RNAs (ncRNAs) produced by DNA-dependent RNA polymerases IV and V (PolIV and PolV). The production of siRNAs is initiated by PolIV and ncRNAs produced by PolIV are precursors of 24-nucleotide siRNAs. In contrast, ncRNAs produced by PolV are involved in scaffolding RNAs. In this review, we summarize recent studies of RdDM. In particular, we focus on the mechanisms involved in chromatin remodeling by PolIV and PolV.

Responses of Populus trichocarpa galactinol synthase genes to abiotic stresses.

Galactinol synthase (GolS; EC 2.4.1.123) is a member of the glycosyltransferase eight family that catalyzes the first step in the biosynthesis pathway of the raffinose family of oligosaccharides (RFOs). The accumulation of RFOs in response to abiotic stress indicates a role for RFOs in stress adaptation. To obtain information on the roles of RFOs in abiotic stress adaptation in trees, we investigated the expression patterns of nine Populus trichocarpa GolS (PtrGolS) genes with special reference to stress responses. PtrGolS genes were differentially expressed in different organs, and the expressions of PtrGolS4 and PtrGolS6 were relatively high in all tested organs. The expression levels of all PtrGolS genes, except PtrGolS9, changed in response to abiotic stress in gene- and stress-type-specific manners. Moreover, short- and long-term stress treatments revealed that induction of PtrGolS by salt stress is obvious only in the early period of treatment (within 24 h), whereas water-deficit stress treatments continued to upregulate PtrGolS gene expression after two days of treatment, in addition to induction within 24 h of treatment. Consistent with these expression patterns, the galactinol content in leaves increased after four days of drought stress, but not under salt stress. Our findings suggest divergent roles for PtrGolS genes in abiotic stress responses in poplars.

Overexpression of PtSOS2 Enhances Salt Tolerance in Transgenic Poplars.

Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we cloned and identified three salt overly sensitive 2 (SOS2) genes in the woody plant Populus trichocarpa, designated as PtSOS2.1, PtSOS2.2, and PtSOS2.3, which were transformed into hybrid poplar clone T89 (Populus tremula× Populus tremuloides Michx clone T89) mediated by Agrobacterium tumefaciens. Southern and northern blot analyses verified that the three genes integrated into the plant genome, and were expressed at a stable transcription level. Meanwhile, overexpression of all three PtSOS2 genes did not retard the growth of plants under normal conditions. Instead, it promoted growth in both agar-medium and soil conditions in response to salinity stress. Under salt stress, overexpression of PtSOS2.1, PtSOS2.2, and PtSOS2.3 increased the concentrations of proline and photosynthetic pigments, and the relative water content (RWC), and the activity of antioxidant enzymes, and decreased the malondialdehyde (MDA) concentrations in transgenic lines compared to the control. These results suggest that overexpression of PtSOS2 plays a significant role in improving the salt tolerance of poplars, reducing the damage to membrane structures, and enhancing osmotic adjustment and antioxidative enzyme regulation under salt stress.

An efficient Agrobacterium-mediated transformation system for poplar.

Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone "Nanlin895" (Populus deltoides×P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.

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.

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.

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.

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.