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.

Genomic Survey of Heat Shock Proteins in Liriodendron chinense Provides Insight into Evolution, Characterization, and Functional Diversities.

Heat shock proteins (HSPs) are conserved molecular chaperones whose main role is to facilitate the regulation of plant growth and stress responses. The HSP gene family has been characterized in most plants and elucidated as generally stress-induced, essential for their cytoprotective roles in cells. However, the HSP gene family has not yet been analyzed in the Liriodendron chinense genome. In current study, 60 HSP genes were identified in the L. chinense genome, including 7 LchiHSP90s, 23 LchiHSP70s, and 30 LchiHSP20s. We investigated the phylogenetic relationships, gene structure and arrangement, gene duplication events, cis-acting elements, 3D-protein structures, protein-protein interaction networks, and temperature stress responses in the identified L. chinense HSP genes. The results of the comparative phylogenetic analysis of HSP families in 32 plant species showed that LchiHSPs are closely related to the Cinnamomum kanehirae HSP gene family. Duplication events analysis showed seven segmental and six tandem duplication events that occurred in the LchiHSP gene family, which we speculated to have played an important role in the LchiHSP gene expansion and evolution. Furthermore, the Ka/Ks analysis indicated that these genes underwent a purifying selection. Analysis in the promoter region evidenced that the promoter region LchiHSPs carry many stress-responsive and hormone-related cis-elements. Investigations in the gene expression patterns of the LchiHSPs using transcriptome data and the qRT-PCR technique indicated that most LchiHSPs were responsive to cold and heat stress. In total, our results provide new insights into understanding the LchiHSP gene family function and their regulatory mechanisms in response to abiotic stresses.

Genome-wide identification of calcineurin B-like protein-interacting protein kinase gene family reveals members participating in abiotic stress in the ornamental woody plant Lagerstroemia indica.

Calcineurin B-like protein-interacting protein kinases (CIPKs) play important roles in plant responses to stress. However, their function in the ornamental woody plant Lagerstroemia indica is remains unclear. In this study, the LiCIPK gene family was analyzed at the whole genome level. A total of 37 LiCIPKs, distributed across 17 chromosomes, were identified. Conserved motif analysis indicated that all LiCIPKs possess a protein kinase motif (S_TKc) and C-terminal regulatory motif (NAF), while seven LiCIPKs lack a protein phosphatase interaction (PPI) motif. 3D structure analysis further revealed that the N-terminal and C-terminal 3D-structure of 27 members are situated near to each other, while 4 members have a looser structure, and 6 members lack intact structures. The intra- and interspecies collinearity analysis, synonymous substitution rate (K s ) peaks of duplicated LiCIPKs, revealed that ∼80% of LiCIPKs were retained by the two whole genome duplication (WGD) events that occurred approximately 56.12-61.16 million year ago (MYA) and 16.24-26.34 MYA ago. The promoter of each LiCIPK contains a number of auxin, abscisic acid, gibberellic acid, salicylic acid, and drought, anaerobic, defense, stress, and wound responsive cis-elements. Of the 21 members that were successfully amplified by qPCR, 18 LiCIPKs exhibited different expression patterns under NaCl, mannitol, PEG8000, and ABA treatments. Given that LiCIPK30, the AtSOS2 ortholog, responded to all four types of stress it was selected for functional verification. LiCIPK30 complements the atsos2 phenotype in vivo. 35S:LiCIPK-overexpressing lines exhibit increased leaf area increment, chlorophyll a and b content, reactive oxygen species scavenging enzyme activity, and expression of ABF3 and RD22, while the degree of membrane lipid oxidation decreases under NaCl treatment compared to WT. The evolutionary history, and potential mechanism by which LiCIPK30 may regulate plant tolerance to salt stress were also discussed. In summary, we identified LiCIPK members involved in abiotic stress and found that LiCIPK30 transgenic Arabidopsis exhibits more salt and osmotic stress tolerance than WT. This research provides a theoretical foundation for further investigation into the function of LiCIPKs, and for mining gene resources to facilitate the cultivation and breeding of new L. indica varieties in coastal saline-alkali soil.

Transcriptome-based analysis reveals that the biosynthesis of anthocyanins is more active than that of flavonols and proanthocyanins in the colorful flowers of Lagerstroemia indica.

Mechanisms associated with the control of flower color in crape myrtle varieties have yet to be sufficiently elucidated, which has tended to hamper the use of modern molecular and genetic strategies in the breeding programs for this plant. The whole transcriptome of four L. indica varieties characterized by different flower colors (white, light purple, deep purplish pink, and strong red) was sequenced, and we performed bioinformatic, quantitative PCR, and co-expression analyses of R2R3 MYB transcription factor and anthocyanin/flavonol pathway genes. We obtained a total of 49,980 transcripts with full-length coding sequences. Both transcriptome and qPCR analyses revealed that anthocyanin/flavonol pathway genes were differentially expressed among the four different flowers types, with the expression of LiPAL, LiCHS, LiCHI, LiDFR, LiANS/LDOX, and LiUFGT being induced in colorful flowers, whereas that of LiF3´5´H, LiFLS, and LiLAR was found to be inhibited. Base on phylogenetic analysis, seven R2R3 MYB transcriptional factors were identified as putative regulators of flower color. The molecular characteristics and co-expression patterns indicated that these MYBs differentially modulate their target genes, with two probably acting as activators, three as repressors, and one contributing to the regulation of vacuolar pH. The findings of this study indicate that the anthocyanin biosynthesis is more active than the flavonol and proanthocyanin in the colorful flowers. These observations provide new genomic information on L. indica and contribute gene resources for the flower color-targeted breeding of crape myrtle.

Maize bHLH55 functions positively in salt tolerance through modulation of AsA biosynthesis by directly regulating GDP-mannose pathway genes.

Ascorbic acid (AsA) is an antioxidant and enzyme co-factor that is vital to plant development and abiotic stress tolerance. However, the regulation mechanisms of AsA biosynthesis in plants remain poorly understood. Here, we report a basic helix-loop-helix 55 (ZmbHLH55) transcription factor that regulates AsA biosynthesis in maize. Analysis of publicly available transcriptomic data revealed that ZmbHLH55 is co-expressed with several genes of the GDP-mannose pathway. Experimental data showed that ZmbHLH55 forms homodimers localized to the cell nuclei, and it exhibits DNA binding and transactivation activity in yeast. Under salt stress conditions, knock down mutant (zmbhlh55) in maize accumulated lower levels of AsA compared with wild type, accompanied by lower antioxidant enzymes activity, shorter root length, and higher malondialdehyde (MDA) level. Gene expression data from the WT and zmbhlh55 mutant, showed that ZmbHLH55 positively regulates the expression of ZmPGI2, ZmGME1, and ZmGLDH, but negatively regulates ZmGMP1 and ZmGGP. Furthermore, ZmbHLH55-overexpressing Arabidopsis, under salt conditions, showed higher AsA levels, increased rates of germination, and elevated antioxidant enzyme activities. In conclusion, these results have identified previously unknown regulation mechanisms for AsA biosynthesis, indicating that ZmbHLH55 may be a potential candidate to enhance plant salt stress tolerance in the future.