Effect of black wolfberry anthocyanin and maltitol on the gelation and microstructural properties of curdlan/gellan gum hybrid gels.

BACKGROUND: Laboratory scale experiments have shown that curdlan and gellan gum gelled together as curdlan/gellan gum (CG) hybrid gels showed better gel properties than the individual curdlan and gellan gum. In this study, CG and black wolfberry anthocyanin (BWA), CG and maltitol (ML) hybrid gels were constructed using CG hybrid gel as matrix. The effects of BWA or ML on the gel properties and microstructure of CG hybrid gels were investigated and a confectionery gel was developed. RESULTS: The presence of BWA increased the storage modulus (G') value of CG at 0.1 Hz, whereas ML had little effect on the G' value of CG. The addition of BWA (5 g L-1 ) and ML (0.3 mol L-1 ) increased the melting and gelling temperatures of CG hybrid gels to 42.4 °C and 34.1 °C and 44.2 °C and 33.2 °C, respectively. Meanwhile, the relaxation time T22 in CG-ML and CG-BWA hybrid gels was reduced to 91.96 and 410.27 ms, indicating the strong binding between BWA and CG, ML and CG. The hydrogen bond interaction between BWA or ML and CG was confirmed by the shift in the hydroxyl stretching vibration peak. Moreover, the microstructures of CG-ML and CG-BWA hybrid gels were denser than that of CG. In addition, confectionery gel containing CG-BWA-ML has good chewing properties. CONCLUSION: These results indicated that the incorporation of BWA or ML could improve the structure of CG hybrid gels and assign a sustainability potential for the development of confectionery gels based on CG complex. © 2024 Society of Chemical Industry.

Formation and characteristics of curcumin-loaded binary gels formed from large yellow croaker (Pseudosciaena crocea) roe protein isolate and gellan gum.

The aim of this study was to investigate the effect of gellan gum (GG) on the cold gelation of large yellow croaker roe protein isolate (pcRPI). The water-holding ability and storage modulus of the pcRPI-GG binary gels increased with the GG concentration, where the storage modulus of the pcRPI-0.2% GG gel was approximately 30.7 times that of the pure pcRPI gel. Compare to the other binary gels, pcRPI-0.2% GG gels exhibited a lower lacunarity and higher junction density, with a denser, more aggregated microstructure. Consequently, curcumin was embedded in pcRPI-0.2% GG gels, and simulated gastrointestinal digestion test results showed that GG addition effectively protected and slowed curcumin release in the gastrointestinal environment. These findings may contribute to elucidating the interaction of pcRPI with GG and demonstrate the potential of binary gels for the embedding and delivery of active substances.

Optimized prime editing efficiently generates heritable mutations in maize.

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.

[Characteristics and Source Apportionment of VOCs and O3 in Shijiazhuang].

To study the composition characteristics and sources of volatile organic compounds (VOCs) in Shijiazhuang City, three national control points were selected to conduct VOCs sampling and analysis from March 2017 to January 2018. The correlation of VOCs through combination with meteorological and ground-level O3 data, and the sources of VOCs were analyzed by positive matrix factorization (PMF). To quantify the pollution period of O3 in summer, its temporal sequence characteristics were studied by wavelet analysis. During the sampling period, the average concentration of ambient total VOCs (TVOCs) was (137.23±64.62) µg·m-3. Haloalkanes were the most dominant VOC compounds, accounting for 31.77% of total VOCs mass, followed by aromatic (30.97%) and oxygenated VOCs (OVOCs, 23.76%). The seasonal variation in VOC concentration followed the trend in winter (187.7 µg·m-3) > autumn (146.8 µg·m-3) > spring (133.24 µg·m-3) > summer (107.1 µg·m-3); the concentration of VOCs shows a trend of increasing gradient from west to east. The O3 concentration correlated negatively with VOCs and NO2, and positively with temperature, sunshine duration, wind speed, and visibility. Changes in meteorological elements were concerned before the occurrence of ozone pollution in summer, especially in 4-5 days in June and 7-8 days during July to August after the occurrence of increasing temperature. Finally six potential sources of VOCs were quantified by the PMF model, including from gasoline emissions (24.78%), diesel vehicle emissions (24.69%), solvent usage (18.64%), the chemical industry (11.87%), regional background (10.84%), and the pharmaceutical industry (9.17%). Ozone formation potential (OFP) contribution of emission sources of gasoline and diesel vehicles (54.98%) was over half of the total contribution. Meanwhile, these findings illustrated that control of vehicle emissions and industrial sources would be an important way to reduce VOCs concentrations and improve air quality in Shijiazhuang.

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.

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.

A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize.

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.

Direct Liquid Phase Exfoliation of Graphite to Produce Few-Layer Graphene by Microfluidization.

Graphene has attracted a great number of attentions due to the excellent physical and chemical properties. For the convenience of investigations and applications, it is crucial to produce the grapheme with high-quality and high-yield by an easy-obtained method. In this research, a promising method is demonstrated to produce a high-concentration few-layer graphene (FLG) dispersion by direct microfluidization in water/surfactant systems. The effects of surfactant selection, chamber pressure and microfluidization cycles on the graphitic material exfoliation efficiency are systematically studied. The FLG concentration and the quality of the as-prepared FLG were determined by a series of characterizations. The graphene dispersions, with an average lateral size of 0.6 µm and a few-layer structure, were stabilized by surfactants at a high concentration of up to 1.7 mg/mL and exhibited a relatively high quality (ID/IG = 0.07-0.56, C/O ~ 19.36) within a processing time of a few hours. This method should facilitate the mass production of high-quality graphene by liquid-phase exfoliation and promote the industrial application of graphene.

Peroneus Longus Tendon Autograft is a Safe and Effective Alternative for Anterior Cruciate Ligament Reconstruction.

The ipsilateral peroneus longus tendon (PLT) was utilized as an autograft for anterior cruciate ligament (ACL) reconstruction of patients with acute ACL rupture and grade III medial collateral ligament (MCL) injury. We investigated the efficacy and safety of this alternative autograft compared with autologous hamstring tendon (HT). Biomechanical testing of the graft options was performed and compared with the native ACL. Thirty-eight patients with acute ACL ruptures and grade III MCL injuries were treated with ACL reconstruction with a doubled autologous PLT or quadrupled autologous HT. Knee stability and function was evaluated clinically with the Lachman test and KT-2000 arthometer as well as subjectively with functional scores. Effects on the donor ankle were evaluated by biomechanical testing. The ultimate tensile strengths of doubled PLT and quadrupled HT were significantly higher than that of the native ACL and the ultimate tensile strength of doubled PLT was comparable with that of quadrupled HT. There were no significant differences in clinical or functional scores between the two groups. There were no significant differences in pre- and postoperative biomechanical testing of the donor ankle. PLT is a suitable alternative autograft for an ACL reconstruction in patients with a concomitant grade III MCL injury without a significant biomechanical disadvantage to the ankle donor site.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

KEY MESSAGE: 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.

MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems.

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.

The Transcription Factor AtDOF4.7 Is Involved in Ethylene- and IDA-Mediated Organ Abscission in Arabidopsis.

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.

Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation.

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.

A CRISPR/Cas9 toolkit for multiplex genome editing in plants.

BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis.

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.

Regulation of stomatal opening by the guard cell expansin AtEXPA1.

Stomatal movement is strictly regulated by various intracellular and extracellular factors in response environmental signals. In our recent study, we found that an Arabidopsis guard cell expressed expansin, AtEXPA1, regulates stomatal opening by altering the structure of the guard cell wall. This addendum proposes a mechanism by which guard cell expansins regulate stomatal movement.

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement.

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

Overexpression of the Arabidopsis α-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus.

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.

Engineering the K+ uptake regulatory pathway by MultiRound Gateway.

In a previous study, we described improved versions of MultiRound Gateway vectors. Here, we engineered a calcineurin B-like (CBL) pathway for potassium (K+) nutrition to demonstrate their effectiveness. Using the two improved entry vectors pL12R34H-Ap and pL34R12-Cm, and through 2-4 rounds of Gateway recombination reactions, we generated five pMDC99-derived binary vectors [pK21 (CIPK23+CBL1), pK29 (CIPK23+CBL9), pK31 (CIPK23+CBL1+AKT1), pK39 (CIPK23+CBL9+AKT1), and pK4 (CIPK23+CBL1+AKT1+CBL9)], in which all four genes have the same pSuper promoter and tNos terminator. pK31, pK39 and pK4 were transformed into Arabidopsis. PCR analysis confirmed that all transgenes usually co-existed in the K31, K39 or K4 transgenic plants, and qRT-PCR analysis indicated that the transgenes were expressed at reasonably high levels. The eight overexpression lines, except K31-1, displayed significantly tolerant phenotypes to low-K+ and low-K+ combined with low-Ca2+ compared to the wild type. Significant differences between the K31, K39 and K4 lines were not observed. These results indicate that the improved MultiRound Gateway vectors efficiently assembled multiple transgenes, which were stably inherited and expressed in transformed plants, even with the same promoter and terminator.

Overexpression of AtDOF4.7, an Arabidopsis DOF family transcription factor, induces floral organ abscission deficiency in Arabidopsis.

After flower pollination, a programmed process called abscission occurs in which unwanted floral organs are actively shed from the main plant body. We found that a member of the DOF (for DNA binding with one finger) transcription factor family, Arabidopsis (Arabidopsis thaliana) DOF4.7, was expressed robustly in the abscission zone. The Arabidopsis 35S::AtDOF4.7 lines with constitutive expression of AtDOF4.7 exhibited an ethylene-independent floral organ abscission deficiency. In these lines, anatomical analyses showed that the formation of the abscission zone was normal. However, dissolution of the middle lamella failed to separate between the cell walls. AtDOF4.7 was identified as a nucleus-localized transcription factor. This protein had both in vitro and in vivo binding activity to typical DOF cis-elements in the promoter of an abscission-related polygalacturonase (PG) gene, PGAZAT. Overexpression of AtDOF4.7 resulted in down-regulation of PGAZAT. AtDOF4.7 interacted with another abscission-related transcription factor, Arabidopsis ZINC FINGER PROTEIN2. Taken together, our results suggest that AtDOF4.7 participates in the control of abscission as part of the transcription complex that directly regulates the expression of cell wall hydrolysis enzymes.