CRISPR/Cas genome editing in plants: mechanisms, applications, and overcoming bottlenecks.

The CRISPR/Cas systems have emerged as transformative tools for precisely manipulating plant genomes and enhancement. It has provided unparalleled applications from modifying the plant genomes to resistant enhancement. This review manuscript summarises the mechanism, application, and current challenges in the CRISPR/Cas genome editing technology. It addresses the molecular mechanisms of different Cas genes, elucidating their applications in various plants through crop improvement, disease resistance, and trait improvement. The advent of the CRISPR/Cas systems has enabled researchers to precisely modify plant genomes through gene knockouts, knock-ins, and gene expression modulation. Despite these successes, the CRISPR/Cas technology faces challenges, including off-target effects, Cas toxicity, and efficiency. In this manuscript, we also discuss these challenges and outline ongoing strategies employed to overcome these challenges, including the development of novel CRISPR/Cas variants with improved specificity and specific delivery methods for different plant species. The manuscript will conclude by addressing the future perspectives of the CRISPR/Cas technology in plants. Although this review manuscript is not conclusive, it aims to provide immense insights into the current state and future potential of CRISPR/Cas in sustainable and secure plant production.

Integrative transcriptome and metabolome analysis reveals the discrepancy in the accumulation of active ingredients between Lycium barbarum cultivars.

MAIN CONCLUSION: The combined analysis of transcriptome and metabolome provided molecular insight into the dynamics of multiple active ingredients biosynthesis and accumulation across different cultivars of Lycium barbarum. Lycium barbarum L. has a high concentration of active ingredients and is well known in traditional Chinese herbal medicine for its therapeutic properties. However, there are many Lycium barbarum cultivars, and the content of active components varies, resulting in inconsistent quality between Lycium barbarum cultivars. At present, few research has been conducted to reveal the difference in active ingredient content among different cultivars of Lycium barbarum at the molecular level. Therefore, the transcriptome of 'Ningqi No.1' and 'Qixin No.1' during the three development stages (G, T, and M) was constructed in this study. A total of 797,570,278 clean reads were obtained. Between the two types of wolfberries, a total of 469, 2394, and 1531 differentially expressed genes (DEGs) were obtained in the 'G1 vs. G10,' 'T1 vs. T10,' and 'M1 vs. M10,' respectively, and were annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. Using these transcriptome data, most DEGs related to the metabolism of the active ingredients in 'Ningqi No.1' and 'Qixin No.1' were identified. Moreover, a widely targeted metabolome analysis of the metabolites of 'Ningqi 1' and 'Qixin 1' fruits at the maturity stage revealed 1,135 differentially expressed metabolites (DEMs) in 'M1 vs. M10,' and many DEMs were associated with active ingredients such as flavonoids, alkaloids, terpenoids, and so on. We further quantified the flavonoid, lignin, and carotenoid contents of the two Lycium barbarum cultivars during the three developmental stages. The present outcome provided molecular insight into the dynamics of multiple active ingredients biosynthesis and accumulation across different cultivars of Lycium barbarum, which would provide the basic data for the formation of Lycium barbarum fruit quality and the breeding of outstanding strains.

Transcriptional deciphering of the metabolic pathways associated with the bioactive ingredients of wolfberry species with different quality characteristics.

BACKGROUND: Wolfberry is rich in carotenoids, flavonoids, vitamins, alkaloids, betaines and other bioactive ingredients. For over 2,000 years, wolfberry has been used in China as a medicinal and edible plant resource. Nevertheless, the content of bioactive ingredients varies by cultivars, resulting in uneven quality across wolfberry cultivars and species. To date, research has revealed little about the underlying molecular mechanism of the metabolism of flavonoids, carotenoids, and other bioactive ingredients in wolfberry. RESULTS: In this context, the transcriptomes of the Lycium barbarum L. cultivar 'Ningqi No. 1' and Lycium chinense Miller were compared during the fruit maturity stage using the Illumina NovaSeq 6000 sequencing platform, and subsequently, the changes of the gene expression profiles in two types of wolfberries were analysed. In total, 256,228,924 clean reads were obtained, and 8817 differentially expressed genes (DEGs) were identified, then assembled by Basic Local Alignment Search Tool (BLAST) similarity searches and annotated using Gene Ontology (GO), Clusters of Orthologous Groups of proteins (KOG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). By combining these transcriptome data with data from the PubMed database, 36 DEGs related to the metabolism of bioactive ingredients and implicated in the metabolic pathway of carotenoids, flavonoids, terpenoids, alkaloids, vitamins, etc., were identified. In addition, among the 9 differentially expressed transcription factors, LbAPL, LbPHL11 and LbKAN4 have raised concerns. The protein physicochemical properties, structure prediction and phylogenetic analysis indicated that LbAPL and LbPHL11 may be good candidate genes involved in regulating the flavonoid metabolism pathway in wolfberry. CONCLUSIONS: This study provides preliminary evidence for the differences in bioactive ingredient content at the transcription level among different wolfberry species, as well as a research and theoretical basis for the screening, cloning and functional analysis of key genes involved in the metabolism of bioactive ingredients in wolfberry.

Technological Development and Application of Plant Genetic Transformation.

Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting desirable genetic characteristics. Research on plant genetic transformation technology can promote the functional analysis of plant genes, the utilization of excellent traits, and precise breeding. Various technologies of genetic transformation have been continuously discovered and developed for convenient manipulation and high efficiency, mainly involving the delivery of exogenous genes and regeneration of transformed plants. Here, currently developed genetic transformation technologies were expounded and compared. Agrobacterium-mediated gene delivery methods are commonly used as direct genetic transformation, as well as external force-mediated ways such as particle bombardment, electroporation, silicon carbide whiskers, and pollen tubes as indirect ones. The regeneration of transformed plants usually involves the de novo organogenesis or somatic embryogenesis pathway of the explants. Ectopic expression of morphogenetic transcription factors (Bbm, Wus2, and GRF-GIF) can significantly improve plant regeneration efficiency and enable the transformation of some hard-to-transform plant genotypes. Meanwhile, some limitations in these gene transfer methods were compared including genotype dependence, low transformation efficiency, and plant tissue damage, and recently developed flexible approaches for plant genotype transformation are discussed regarding how gene delivery and regeneration strategies can be optimized to overcome species and genotype dependence. This review summarizes the principles of various techniques for plant genetic transformation and discusses their application scope and limiting factors, which can provide a reference for plant transgenic breeding.

Identification and characteristics of SnRK genes and cold stress-induced expression profiles in Liriodendron chinense.

BACKGROUND: The sucrose non-fermenting 1 (SNF1)-related protein kinases (SnRKs) play a vivid role in regulating plant metabolism and stress response, providing a pathway for regulation between metabolism and stress signals. Conducting identification and stress response studies on SnRKs in plants contributes to the development of strategies for tree species that are more tolerant to stress conditions. RESULTS: In the present study, a total of 30 LcSnRKs were identified in Liriodendron chinense (L. chinense) genome, which was distributed across 15 chromosomes and 4 scaffolds. It could be divided into three subfamilies: SnRK1, SnRK2, and SnRK3 based on phylogenetic analysis and domain types. The LcSnRK of the three subfamilies shared the same Ser/Thr kinase structure in gene structure and motif composition, while the functional domains, except for the kinase domain, showed significant differences. A total of 13 collinear gene pairs were detected in L. chinense and Arabidopsis thaliana (A. thaliana), and 18 pairs were detected in L. chinense and rice, suggesting that the LcSnRK family genes may be evolutionarily more closely related to rice. Cis-regulation element analysis showed that LcSnRKs were LTR and TC-rich, which could respond to different environmental stresses. Furthermore, the expression patterns of LcSnRKs are different at different times under low-temperature stress. LcSnRK1s expression tended to be down-regulated under low-temperature stress. The expression of LcSnRK2s tended to be up-regulated under low-temperature stress. The expression trend of LcSnRK3s under low-temperature stress was mainly up-or down-regulated. CONCLUSION: The results of this study will provide valuable information for the functional identification of the LcSnRK gene in the future.

Systematic Characterization of GATA Transcription Factors in Liriodendron chinense and Functional Validation in Abiotic Stresses.

The Liriodendron chinense in the Magnoliaceae family is an endangered tree species useful for its socio-economic and ecological benefits. Abiotic stresses (cold, heat, and drought stress), among other factors, affect its growth, development, and distribution. However, GATA transcription factors (TFs) respond to various abiotic stresses and play a significant role in plant acclimatization to abiotic stresses. To determine the function of GATA TFs in L. chinense, we investigated the GATA genes in the genome of L. chinense. In this study, a total of 18 GATA genes were identified, which were randomly distributed on 12 of the total 17 chromosomes. These GATA genes clustered together in four separate groups based on their phylogenetic relationships, gene structures, and domain conservation arrangements. Detailed interspecies phylogenetic analyses of the GATA gene family demonstrated a conservation of the GATAs and a probable diversification that prompted gene diversification in plant species. In addition, the LcGATA gene family was shown to be evolutionarily closer to that of O. sativa, giving an insight into the possible LcGATA gene functions. Investigations of LcGATA gene duplication showed four gene duplicate pairs by the segmental duplication event, and these genes were a result of strong purified selection. Analysis of the cis-regulatory elements demonstrated a significant representation of the abiotic stress elements in the promoter regions of the LcGATA genes. Additional gene expressions through transcriptome and qPCR analyses revealed a significant upregulation of LcGATA17, and LcGATA18 in various stresses, including heat, cold, and drought stress in all time points analyzed. We concluded that the LcGATA genes play a pivotal role in regulating abiotic stress in L. chinense. In summary, our results provide new insights into understanding of the LcGATA gene family and their regulatory functions during abiotic stresses.

Transcriptional and Post-Translational Regulation of Plant bHLH Transcription Factors during the Response to Environmental Stresses.

Over the past decades, extensive research has been conducted to identify and characterize various plant transcription factors involved in abiotic stress responses. Therefore, numerous efforts have been made to improve plant stress tolerance by engineering these transcription factor genes. The plant basic Helix-Loop-Helix (bHLH) transcription factor family represents one of the most prominent gene families and contains a bHLH motif that is highly conserved in eukaryotic organisms. By binding to specific positions in promoters, they activate or repress the transcription of specific response genes and thus affect multiple variables in plant physiology such as the response to abiotic stresses, which include drought, climatic variations, mineral deficiencies, excessive salinity, and water stress. The regulation of bHLH transcription factors is crucial to better control their activity. On the one hand, they are regulated at the transcriptional level by other upstream components; on the other hand, they undergo various modifications such as ubiquitination, phosphorylation, and glycosylation at the post-translational level. Modified bHLH transcription factors can form a complex regulatory network to regulate the expression of stress response genes and thus determine the activation of physiological and metabolic reactions. This review article focuses on the structural characteristics, classification, function, and regulatory mechanism of bHLH transcription factor expression at the transcriptional and post-translational levels during their responses to various abiotic stress conditions.