Plants are continually challenged by abiotic stressors, including drought, salinity, and extreme temperatures, which can adversely affect their growth and development. The plasma membrane, acting as a pivotal interface between the cell and its environment, is particularly susceptible to such stresses. This review focuses on current understanding of how abiotic stresses affect plasma membrane integrity in plants. The review also explores the critical roles of plasma membrane proteins and lipids under stress conditions, highlighting signal transduction pathways that the plasma membrane initiates to mitigate abiotic stress. By consolidating these findings, this review provides a comprehensive overview that is pivotal for advancing the development of stress-tolerant plant varieties. The insights gained from this synthesis are expected to contribute significantly to the enhancement of plant resilience in the face of environmental challenges.

Yellowhorn (Xanthoceras sorbifolium) is an excellent economic tree species in northern China, but its main distribution area often suffers from drought stress, which seriously affects its yield and the expansion of suitable growth area. The increase of ABA content and wax accumulation in plants are both measures to cope with drought stress, but the interaction mechanism between ABA and wax is currently unclear. We found that the high wax variety ‘Zhongshi 4’ has higher ABA level compared with the low-wax variety, and the XsMYB30 transcription factor which can positively regulate leaf wax synthesis was also up-regulated in the high-wax variety. Therefore, in this study, we analyzed the relationship between ABA and XsMYB30 in the wax accumulation of yellowhorn. It was found that ABA synthesis inhibitor reduced the deposition of wax on the leaves of yellowhorn. Moreover, overexpression of XsMYB30 increased the ABA contents, affecting the stomatal aperture in response to drought. Biological analysis confirmed that XsMYB30 could directly bind to the promoter of XsNCED3 and promote its expression. We further showed that the inhibition of the ABA level resulted in the reduced wax accumulation and weakened drought resistance in XsMYB30 overexpression lines. Our research indicates that ABA plays an important role in XsMYB30-mediated cuticular wax accumulation and drought resistance, and provides new insights into the underlying mechanism between ABA and wax.

Cas12 nucleases, such as Cas12a, Cas12i, and Cas12f, are genome-editing tools that possess several unique attributes. However, the potential of various Cas12 variants for multiplex genome editing in Arabidopsis (Arabidopsis thaliana) remains insufficiently characterized. In this report, we systematically evaluated 18 additional targets and demonstrated that the LbCas12a variant carrying D156R and E795L mutations exhibits minimal target bias. Furthermore, we achieved an editing efficiency of at least 73.8% (45/61) in generating T1 homozygous sextuple mutants, with more than half of these mutants exhibiting a complete seed germination arrest phenotype. Comparative analysis of 7 LbCas12a variants revealed that the optimization of nuclear localization sequences, rather than codon usage, is fundamental for improved editing efficiency, and that the E795L mutation had synergistic effects with other mutations in highly efficient LbCas12a variants. Further investigation into 1 Cas12i3 and 2 AsCas12f variants showed that the Cas12i3 variant also exhibits sufficiently high editing efficiency in Arabidopsis, although additional refinements were required to mitigate its target bias. Collectively, in this study, we developed the most efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) tool for multiplex genome editing in Arabidopsis, as demonstrated by the highly efficient generation of never-germinating seeds harboring mutations in 6 clade A type 2C protein phosphatase genes.

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop breeding. Four new types of prime editors (PEs) named PE6a-d were recently generated using evolved and engineered reverse transcriptase (RT) variants from three different sources. In this study, we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice (Oryza sativa) plants. PE6c, with an evolved and engineered RT variant from the yeast Tf1 retrotransposon, yielded the highest prime-editing efficiency. The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes. We also demonstrated the feasibility of using two RT modules to improve prime-editing efficiency. Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants. In addition, our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

Cas12a (Cpf1), a Class 2 Type V CRISPR/Cas nuclease, has several unique attributes for genome editing and may provide a valuable alternative to Cas9. However, a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad application. In this report, we generated two variants, ttAsCas12 Ultra and ttLbCas12a Ultra harboring three (E174R, M537R, and F870L) or two (D156R and E795L) mutations, respectively, by combining the mutations from the temperature-tolerant variants ttAsCas12a (E174R) and ttLbCas12a (D156R), and those from the highly active variants AsCas12a Ultra (M537R and F870L) and LbCas12a Ultra (E795L). We compared editing efficiencies of the five resulting Cas12a variants (LbCas12a, ttLbCas12a, ttLbCas12a Ultra, AsCas12a Ultra, and ttAsCas12 Ultra) at six target sites of four genes in Arabidopsis (Arabidopsis thaliana). The variant ttLbCas12a Ultra, harboring the D156R and E795L mutations, exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22 °C. In addition, optimization of ttLbCas12a Ultra, by varying nuclear localization signal sequences and codon usage, further greatly improved editing efficiency. Collectively, our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis.

Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering prime-editing guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA-evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

Dear Editor, Prime editing is a novel and universal CRISPR-Cas-derived pre-cise genome-editing technology and has great potentials for ap-plications in basic plant research and crop molecular breeding(Anzalone et al.,2019).Although low efficiency has restrained the original prime editors(PEs)from being used as a routine tool for precise genome editing in plants,an iterative update of the PEs is removing this obstacle(Lin et al.,2021;Xu et al.,2022).

Prime editing is a universal and very promising precise genome editing technology. However, optimization of prime editor (PE) from different aspects remains vital for its use as a routine tool in plant basic research and crop molecular breeding. In this report, we tested MS2-based prime editor (MS2PE). We fused the M-MLV reverse transcriptase (RT) gene variant to the MS2 RNA binding protein gene, MCP , and allowed the MCP-RT fusion gene to co-express with the SpCas9 nickase gene, SpCas9H840A , and various engineered pegRNAs harboring MS2 RNA (MS2pegR). Compared with control PEs, MS2PEs significantly enhanced editing efficiency at four of six targets in rice protoplasts, and achieved 1.2∼10.1-fold increase in editing efficiency at five of six targets in transgenic rice lines. Furthermore, we tested total 22 different MS2pegR scaffolds, 3 RT variants or genes, 2 MCP variants, and various combinations of the Cas9 nickase, RT, and MCP modules. Our results demonstrated an alternative strategy for enhancing prime editing.

Prime editing is a universal and very promising precise genome editing technology. However, optimization of prime editor (PE) from different aspects remains vital for its use as a routine tool in plant basic research and crop molecular breeding. In this report, we tested MS2-based prime editor (MS2PE). We fused the M-MLV reverse transcriptase (RT) gene variant to the MS2 RNA binding protein gene, MCP, and allowed the MCP-RT fusion gene to co-express with the SpCas9 nickase gene, SpCas9H840A, and various engineered pegRNAs harboring MS2 RNA (MS2pegR). Compared with control PEs, MS2PEs significantly enhanced editing efficiency at four of six targets in rice protoplasts, and achieved 1.2~10.1-fold increase in editing efficiency at five of six targets in transgenic rice lines. Furthermore, we tested total 22 different MS2pegR scaffolds, 3 RT variants or genes, 2 MCP variants, and various combinations of the Cas9 nickase, RT, and MCP modules. Our results demonstrated a new strategy for more efficient prime editing and provide a platform for further directed evolution of PEs.

Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2 . Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

Aquaporins play essential roles in growth and development including stem elongation in plants. Tonoplast aquaporin AtTIP5;1 has been proposed to positively regulate hypocotyl elongation under high concentrations of boron (high-B) in Arabidopsis thaliana (L.) Heynh. However, the mechanism underlying this process remains unanswered. Here, we show that paclobatrazol, an inhibitor of GA biosynthesis, significantly suppressed the hypocotyl cell elongation of wild-type (WT) seedlings, and more strongly suppressed that of AtTIP5;1 overexpressors under high-B stress. Two AtTIP5;1 null mutants displayed arrested elongation of cells in the upper part of hypocotyls compared with the WT in the presence of high-B or GA3. Moreover, paclobatrazol treatment completely inhibited the increases in AtTIP5;1 transcripts induced by high-B, whereas GA3 application upregulated AtTIP5;1 expression in the WT. In addition, treatment with high-B remarkably elevated the expression levels of GA3ox1, GA20ox1 and GA20ox2 - key biosynthesis genes of GAs - in WT seedlings. The GA3 and GA4 content also increased in WT seedlings grown in MS medium containing high-B. Additionally, application of high-B failed to enhance AtTIP5;1 expression in the double mutant rga-24-gai-t6 of DELLA genes. Together, these results suggest that AtTIP5;1 is an essential downstream target of GAs. High-B induces the accumulation of GAs, which activates AtTIP5;1 through modulation of the DELLA proteins Repressor of ga1-3 and GA-insensitive, further promoting hypocotyl elongation in A. thaliana.

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We present evidence that the frequency of off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants is significantly higher than expected. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. We demonstrate that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that the tools optimized in this report will be useful in improving genome editing specificity in plants and other organisms.

Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.

Organ abscission is an important plant developmental process that occurs in response to environmental stress or pathogens. In Arabidopsis, ligand signals, such as ethylene or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), can regulate organ abscission. Previously, we reported that overexpression of AtDOF4.7, a transcription factor gene, directly suppresses the expression of the abscission-related gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 2 (ADPG2), resulting in a deficiency of floral organ abscission. However, the relationship between AtDOF4.7 and abscission pathways still needs to be investigated. In this study, we showed that ethylene regulates the expression of AtDOF4.7, and the peptide ligand, IDA negatively regulates AtDOF4.7 at the transcriptional level. Genetic evidence indicates that AtDOF4.7 and IDA are involved in a common pathway, and a MAPK cascade can phosphorylate AtDOF4.7 in vitro. Further in vivo data suggest that AtDOF4.7 protein levels may be regulated by this phosphorylation. Collectively, our results indicate that ethylene regulates AtDOF4.7 that is involved in the IDA-mediated floral organ abscission pathway.

微胶囊是具有聚合物壁壳的微型容器或包装物.用涂层薄膜或壳材料敷涂微小的固体颗粒、液滴或气泡,就可以得到微胶囊.目前微胶囊技术中常用的壁材主要有天然高分子材料、半合成高分子材料、全合成高分子材料.而聚吡咯作为一种具有优异的导电性、无毒性以及稳定性的全合成高分子材料壁材得到了研究者的广泛关注.综述了近几年来聚吡咯胶囊的不同合成方法的研究状况及其应用进展,主要包括聚吡咯的结构、聚吡咯胶囊的各种合成方法和机理以及聚吡咯胶囊的应用前景.

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

High reduced viscosity meta-polybenzimidazole (mPBI) as well as high-strength fibers of this polymer were fabricated via solution polycondensation and wet spinning, and the content of phosphorus pentoxide (P2O5) in the solvent, the reaction period and the draw ratio were altered. The results indicate that the reduced viscosity of the polymer reached a maximum of 2.75 dL g−1 with 82 wt% P2O5 in the solvent, and the reaction period was 8 h. The polymer displayed good solubility and excellent thermal stability. At a coagulation bath concentration, polymer reduced viscosity and stretch ratio of 61.5 wt%, 2.75 dL g−1 and 1.87, respectively, mPBI fibers that had relatively smooth surfaces and almost circular cross sections were formed (minimum diameter of 138 μm and maximum breaking strength of 306 MPa). In addition, the fibers exhibited good dimensional and chemical stability.

该文研究了时滞对一个带Neumann边值的捕食者-食饵的反应扩散系统的影响.通过对特征根的分析,讨论了非负平衡解的稳定性和Hopf分支的存在性.应用规范型方法和中心流形理论,文章讨论了Hopf分支周期解的稳定性和分支方向.