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.

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 wheatFADgene family (TaFADs).Results: 68FADgenes were identified from the wheat genome. Based on the phylogenetic analysis, wheatFADsclustered into five subfamilies, includingFAB2,FAD2/FAD6,FAD4,DES/SLD, andFAD3/FAD7/FAD8. TheTaFADswere 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 theTaFADs promoters indicated the role of these genes in plant development and responses to environmental stresses. Likewise, 72 SSRs and 91 miRNAs in 36 and 47TaFADs have been identified. According to RNA-seq data analysis, the highest expression in all developmental stages and tissues was related toTaFAB2.5,TaFAB2.12,TaFAB2.15,TaFAB2.17,TaFAB2.20,TaFAD2.1,TaFAD2.6, andTaFAD2.8genes while the highest expression in response to temperature stress was related toTaFAD2.6,TaFAD2.8,TaFAB2.15,TaFAB2.17, andTaFAB2.20. 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.

Carotenoid cleavage dioxygenases (CCDs) catalyzes the cleavage of various carotenoids into smaller apocarotenoids which are essential for plant growth and development and response to abiotic stresses. CCD family is divided into two subfamilies: 9-cis epoxycarotenoid dioxygenases (NCED) family and CCD family. A better knowledge of carotenoid biosynthesis and degradation could be useful for regulating carotenoid contents. Here, 23 CCD genes were identified from the Populus trichocarpa genome, and their characterizations and expression profiling were validated. The PtCCD members were divided into PtCCD and PtNCED subfamilies. The PtCCD family contained the PtCCD1, 4, 7, and 8 classes. The PtCCDs clustered in the same clade shared similar intron/exon structures and motif compositions and distributions. In addition, the tandem and segmental duplications resulted in the PtCCD gene expansion based on the collinearity analysis. An additional integrated collinearity analysis among poplar, Arabidopsis, rice, and willow revealed the gene pairs between poplar and willow more than that between poplar and rice. Identifying tissue-special expression patterns indicated that PtCCD genes display different expression patterns in leaves, stems, and roots. Abscisic acid (ABA) treatment and abiotic stress suggested that many PtCCD genes are responsive to osmotic stress regarding the comprehensive regulation networks. The genome-wide identification of PtCCD genes may provide the foundation for further exploring the putative regulation mechanism on osmotic stress and benefit poplar molecular breeding.

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.

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.

As an important resource for screening microbial strains capable of conferring stress tolerance in plants, the fungal community associated with the plants grown in stressful environments has received great attention. In this study, high-throughput sequencing was employed to study the rhizosphere fungal community in the reclaimed area (i.e., sites F, H, and T) of the eastern coast of China. Moreover, endophytic fungi from the root of six plant species colonizing the investigated sites were isolated and identified. The differences in soil physicochemical parameters, fungal diversity, and community structure were detected among the sampling sites and between the seasons. Ectomycorrhizal (ECM) fungi (e.g., genera Tuber and Geopora) were dominant at site F, which was characterized by high soil total carbon (SC) and total nitrogen (SN) contents and low soil electrical conductivity (EC) value. Arbuscular mycorrhizal (AM) fungi, including genera Glomus, Rhizophagus, and Entrophospora were dominant at sites H (winter), H (summer), and T (summer), respectively. The positive relationship between the EC value and the abundance of genus Glomus indicated the ability of this AM fungus to protect plants against the salt stress. Endophytic fungi at sites F (Aspergillus and Tetracladium), H (Nigrospora), and T (Nigrospora, Coniochaeta and Zopfiella) were recognized as the biomarkers or keystone taxa, among which only genus Aspergillus was isolated from the plant roots. The aforementioned AM fungi and endophytic fungi could contribute to the promotion of plant growth in the newly reclaimed land.