Plant Biotechnology Journal

The Cas12j‐8 nuclease, derived from the type V CRISPR system, is approximately half the size of Cas9 and recognizes a 5′‐TTN‐3′ protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its editing efficiency remains low in plants. In this study, we rationally engineered both the crRNA and the Cas12j‐8 nuclease. The engineered crRNA and Cas12j‐8 markedly improved genome editing efficiency in plants. When combined, they exhibited robust editing activity in soybean and rice, enabling the editing of target sites that were previously uneditable. Notably, for certain target sequences, the editing activity was comparable to that of SpCas9 when targeting identical sequences, and it outperformed the Cas12j‐2 variant, nCas12j‐2, across all tested targets. Additionally, we developed cytosine base editors based on the engineered crRNA and Cas12j‐8, demonstrating an average increase of 5.36‐ to 6.85‐fold in base‐editing efficiency (C to T) compared with the unengineered system in plants, with no insertions or deletions (indels) observed. Collectively, these findings indicate that the engineered hypercompact CRISPR/Cas12j‐8 system serves as an efficient tool for genome editing mediated by both nuclease cleavage and base editing in plants.

Liquid crystal monomers (LCMs), the integral components in the manufacture of digital displays, have engendered environmental concerns due to extensive utilization and intensive emission. Despite their prevalence and ecotoxicity, the LCM impacts on plant growth and agricultural yield remain inadequately understood. In this study, we investigated the specific response mechanisms of tobacco, a pivotal agricultural crop and model plant, to four representative LCMs (2OdF3B, 5CB, 4PiMeOP, 2BzoCP) through integrative molecular and physiological approaches. The findings reveal specific impacts, with 4PiMeOP exerting the most pronounced effects, followed by 2BzoCP, 5CB, and 2OdF3B. LCM exposure disrupts the photosynthetic apparatus, exacerbating reactive oxygen species (ROS) levels in leaves, which in turn triggers the upregulation of antioxidative enzymes and the synthesis of antioxidant substances. Additionally, LCMs strongly stimulate the expression of genes involved in abscisic acid (ABA) biosynthesis and signalling pathways. The AI‐assisted meta‐analysis implicates ABA as a critical regulator in the tobacco response to LCMs. Notably, exogenous application of ABA alleviates LCM‐induced toxicities, highlighting the pivotal role of ABA in stress amelioration. Our study provides novel insights into the toxicity and tolerance mechanisms of LCMs in plants, shedding light on both their harmful effects on the ecosystems and potential adaptation responses. This is crucial to develop sustainable agricultural systems by reducing the negative environmental impacts caused by emerging organic pollutants.

Transposable elements (TEs) are significant drivers of genome evolution, yet their recent dynamics and impacts within and among species, as well as the roles of host genes and non‐coding RNAs in the transposition process, remain elusive. With advancements in large‐scale pan‐genome sequencing and the development of open data sharing, large‐scale comparative genomics studies have become feasible. Here, we performed complete de novo TE annotations and identified active TEs in 310 plant genome assemblies across 119 species and seven crop populations. Using 811 high‐quality genomes, we detected 13 844 553 TE‐induced structural variants (TE‐SVs), providing unprecedented resolution in delineating recent TE activities. Our integrative analysis revealed a mutual evolutionary relationship between TEs and host genomes. On one hand, host genes and ncRNAs are involved in the transposition process, as evidenced by their colocalization and coactivation with TEs, and may play a role in chromatin regulation. On the other hand, TEs drive genetic innovation by promoting the duplication of host genes and inserting into regulatory regions. Moreover, genes influenced by active TEs are linked to plant growth, nutrient absorption, storage metabolism and environmental adaptation, aiding in crop domestication and adaptation. This TE dynamics atlas not only reveals evolutionary and functional features linked to transposition activity but also highlights the role of TEs in crop domestication and adaptation, paving the way for future exploration of TE‐mediated genome evolution and crop improvement strategies.

Superoxide dismutase (SOD) plays an important role to respond in the defence against damage when tomato leaves are under different types of adversity stresses. This work employed microhyperspectral imaging (MHSI) and visible near‐infrared (Vis–NIR) hyperspectral imaging (HSI) technologies to predict tomato leaf SOD activity. The macroscopic model of SOD activity in tomato leaves was constructed using the convolutional neural network in conjunction with the long and short‐term temporal memory (CNN‐LSTM) technique. Using heterogeneous two‐dimensional correlation spectra (H2D‐COS), the sensitive macroscopic and microscopic absorption peaks connected to tomato leaves' SOD activity were made clear. The combination of CNN‐LSTM algorithm and H2D‐COS analysis was used to research transfer learning between microscopic and macroscopic models based on sensitive wavelengths. The results demonstrated that the CNN‐LSTM model, which was based on the FD preprocessed spectra, had the best performance for the microscopic model, with RC and RP reaching 0.9311 and 0.9075, and RMSEC and RMSEP reaching 0.0109 U/mg and 0.0127 U/mg respectively. There were 10 macroscopic and 10 microscopic significant sensitivity peaks found. The transfer learning was carried out using sensitive wavelengths, and the model performed well with an RP value of 0.7549 and an RMSEP of 0.0725 U/mg. The combined CNN algorithm and H2D‐COS analysis demonstrated the viability of transfer learning across microscopic and macroscopic models for quantitative tomato leaf SOD prediction.

Apple (Malus domestica Borkh.) is one of the most economically valuable fruit crops globally and a key dietary source for various nutrients. However, the levels of ascorbic acid (AsA) and anthocyanin, essential micronutrients for human health, are extremely low in the pulp of commonly cultivated apple varieties. In the present study, the second‐generation hybrid strain of Xinjiang red‐fleshed apple (‘Zihong No. 1’ × ‘Gala’) was used as the test material. The results revealed that AsA content was significantly higher in red‐fleshed apple pulp than in non‐red‐fleshed varieties, and the expression of MdGLDH, a key gene in the D‐mannose/L‐galactose pathway, correlated strongly with AsA levels. Using the promoter of MdGLDH as bait, an R3‐type MYB transcription factor (TF), MdCPC‐like, was identified through yeast one‐hybrid screening. Further analysis revealed that the overexpression of MdCPCL increased the AsA and anthocyanin levels in both callus and fruits, whereas MdCPCL knockdown led to a reduction in their levels. Moreover, the interaction between MdCPCL and the bHLH TF MdILR3‐like was confirmed, forming the MdCPCL‐MdILR3L complex. This complex significantly enhanced the transcription of downstream target genes MdGLDH and MdANS, promoting the synthesis of AsA and anthocyanins. This study contributes to further enrich the anabolic pathways of AsA and anthocyanin in apples and provides a theoretical foundation for the quality breeding of red‐fleshed apple varieties.

The genus Euphorbia, belonging to the family Euphorbiaceae, represents a significant ethnobotanical heritage due to the diverse bioactive properties exhibited. In this study, the phytochemical composition and biological activities of latex and aerial parts of the water extract of Euphorbia gaillardotii were investigated. Phytochemical analyses were performed using gas chromatography‐mass spectrometry and high‐performance liquid chromatography techniques and total antioxidants, phenolics, sugars, organic acids, and aroma components were quantitatively determined. The effects of the test compounds on physicochemical parameters in aqueous media were evaluated by electrical conductivity, dissolved oxygen concentration, and pH measurements. Antimicrobial activity was evaluated on Gram‐positive and Gram‐negative bacteria and yeast strains using the disk diffusion method. The cytotoxic activity on the MCF‐7 human breast cancer cell line was measured spectrophotometrically using the Cell Counting Kit‐8 proliferation/apoptosis detection kit. The results showed that E. gaillardotii latex and aerial parts water extract significantly affected the physicochemical parameters of aqueous media, especially at high concentrations. The test substances displayed antimicrobial activity, with the latex‐impregnated disks demonstrating larger inhibition zones than the aerial parts extract. The results showed that both the latex and extract treatments exhibited concentration‐dependent effects on MCF‐7 cell viability (P < 0.001). Furthermore, in silico docking analyses revealed a robust binding affinity of succinic acid, the most prevalent bioactive compound in the extract, towards the B‐cell lymphoma 2 (Bcl‐2) molecule, with a binding energy of −6.16 kcal/mol. This may be associated with the observed cytotoxicity. These results suggest that E. gaillardotii may be a valuable source for potential pharmacological applications.

Here, we evaluated the role of ethylene in regulating the NaHCO3 stress tolerance of grapevines and clarified the mechanism by which VvERF1B regulates the response to NaHCO3 stress. The exogenous application of ACC and VvACS3 overexpression in grapevines and grape calli revealed that ethylene increased NaHCO3 stress tolerance, and this was accompanied by increased plasma membrane H⁺‐ATPase (PMA) activity. The expression of VvERF1B was strongly induced by ACC, and overexpression of this gene in grapevines conferred increased NaHCO3 stress tolerance and enhanced PMA activity and H⁺ and oxalate secretion. Additionally, the function of VvERF1B was also verified using mutant transgenic grape calli and overexpression in Arabidopsis plants. The expression of VvPMA10 was strongly induced following the overexpression of VvERF1B in grapevine roots, and VvPMA10 was shown to regulate PMA activity, oxalate and H⁺ secretion, and NaHCO3 stress tolerance via its overexpression and mutation in grapevine roots, calli, and/or Arabidopsis. However, VvPMA10 was not a direct target gene of VvERF1B but was directly transactivated by VvMYC2. The function of VvMYC2 was shown to be similar to that of VvPMA10 via its overexpression and mutation in grape calli. Additional experiments revealed that the interaction of VvERF1B with VvMYC2 increased its ability to activate VvPMA10 expression and that VvMYC2 played a role in the VvERF1B‐mediated pathway. Overall, the VvERF1B‐VvMYC2‐VvPMA pathway played a role in regulating ethylene‐induced NaHCO3 stress tolerance in grapevines, and this process contributed to increases in PMA activity and H⁺ and oxalate secretion.

The husk leaf of maize (Zea mays) encases the ear as a modified leaf and plays pivotal roles in protecting the ear from pathogen infection, translocating nutrition for grains and warranting grain yield. However, the natural genetic basis for variation in husk leaf width remains largely unexplored. Here, we performed a genome‐wide association study for maize husk leaf width and identified a 3‐bp InDel (insertion/deletion) in the coding region of the nitrate transporter gene ZmNRT2.5. This polymorphism altered the interaction strength of ZmNRT2.5 with another transporter, ZmNPF5, thereby contributing to variation in husk leaf width. We also isolated loss‐of‐function mutants in ZmNRT2.5, which exhibited a substantial decrease in husk leaf width relative to their controls. We demonstrate that ZmNRT2.5 facilitates the transport of nitrate from husk leaves to maize kernels in plants grown under low‐nitrogen conditions, contributing to the accumulation of proteins in maize seeds. Together, our findings uncovered a key gene controlling maize husk leaf width and nitrate transport from husk leaves to kernels. Identification of the ZmNRT2.5 loci offers direct targets for improving the protein content of maize seeds via molecular‐assisted maize breeding.

With advances in long‐read sequencing and assembly techniques, haplotype‐resolved (phased) genome assemblies are becoming more common, also in the field of plant genomics. Computational tools to effectively explore these phased genomes, particularly for polyploid genomes, are currently limited. Here we describe a new strategy adopting a pangenome approach. To analyse both intra‐ and intergenomic variation in phased genome assemblies, we have made the software package PanTools ploidy‐aware by updating the pangenome graph representation and adding several novel functionalities to assess synteny and gene retention, profile repeats and calculate synonymous and nonsynonymous mutation rates. Using PanTools, we constructed and analysed a pangenome comprising of one diploid and four tetraploid potato cultivars, and a pangenome of five diploid apple species. Both pangenomes show high intra‐ and intergenomic allelic diversity in terms of gene absence/presence, SNPs, indels and larger structural variants. Our findings show that the new functionalities and visualizations are useful to discover introgressions and detect likely misassemblies in phased genomes. PanTools is available at https://git.wur.nl/bioinformatics/pantools.

The green microalga Chlamydomonas reinhardtii is a promising host organism for the production of valuable compounds. Engineering the Chlamydomonas chloroplast genome offers several advantages over the nuclear genome, including targeted gene insertion, lack of silencing mechanisms, potentially higher protein production due to multiple genome copies and natural substrate abundance for metabolic engineering. Tuneable expression systems can be used to minimize competition between heterologous production and host cell viability. However, complex gene regulation and a lack of tight regulatory elements make this a challenge in the Chlamydomonas chloroplast. In this work, we develop two synthetic tuneable systems to control the expression of genes on the chloroplast genome, taking advantage of the properties of the vitamin B12‐responsive METE promoter and a modified thiamine (vitamin B1) riboswitch, along with nucleus‐encoded chloroplast‐targeted regulatory proteins NAC2 and MRL1. We demonstrate the capacity of these systems for robust, fine‐tuned control of several chloroplast transgenes, by addition of nanomolar levels of vitamins. The two systems have been combined in a single strain engineered to avoid effects on photosynthesis and are orthogonal to each other. They were then used to manipulate the production of an industrially relevant diterpenoid, casbene, by introducing and tuning expression of the coding sequence for casbene synthase, as well as regulating the metabolite flux towards casbene precursors, highlighting the utility of these systems for informing metabolic engineering approaches.

Exploring the new elements to re‐design the expression cassette is crucial in synthetic biology. Viruses are one of the most important sources for exploring gene expression elements. In this study, we found that the DNA sequence of the SBG51 deltasatellite from the Sweet potato leaf curl virus (SPLCV) greatly enhanced the gene expression when flanked downstream of the terminator. The SBG51 sequence increased transient GFP gene expression in Nicotiana benthamiana leaves by up to ~6 times and ~10 times compared to the gene expression controlled by the UBQ10 promoter and 35S promoter alone, respectively. The increased GFP gene expression level contributed to the continuous accumulation of GFP protein and GFP fluorescence until 8 days post‐inoculation (dpi). The SBG51 sequence also enhanced the gene expression in the transgenic Arabidopsis plants and maintained the spatio‐temporal pattern of the FLOWERING LOCUS T (FT) and TOO MANY MOUTHS (TMM) promoters. We identified a 123 bp of AT‐rich sequence containing seven “ATAAA” or “TTAAA” elements from the SBG51 DNA, which had the gene expression enhancement effect. Furthermore, the artificial synthetic sequences containing tandem repeated “ATAAA” or “TTAAA” elements were sufficient to increase the gene expression but did not alter the polyadenylation of mRNA, similar to the function of matrix attachment regions (MAR). Additionally, the compact artificial synthetic sequence also had an effect on yeast when the expression cassette was integrated into the genome. We conclude that the geminivirus deltasatellite‐derived sequence and the “ATAAA”/“TTAAA” elements are powerful tools for enhancing gene expression.

Enhanced grain yield and quality traits are everlasting breeding goals. It is therefore of great significance to uncover more genetic resources associated with these two important agronomic traits. Plant MYB family transcription factors play important regulatory roles in diverse biological processes. However, studies on genetic functions of MYB in rice yield and quality are rarely to be reported. Here, we investigated a nucleus‐localized transcription factor OsMYB73 which is preferentially expressed in the early developing pericarp and endosperm. We generated targeted mutagenesis of OsMYB73 in rice, and the mutants had longer grains with obvious white‐belly chalky endosperm appearance phenotype. The mutants displayed various changes in starch physicochemical characteristics and lipid components. Transcriptome sequencing analysis showed that OsMYB73 was chiefly involved in cell wall development and starch metabolism. OsMYB73 mutation affects the expression of genes related to grain size, starch and lipid biosynthesis and auxin biosynthesis. Moreover, inactivation of OsMYB73 triggers broad changes in secondary metabolites. We speculate that rice OsMYB73 and OsNF‐YB1 play synergistic pivotal role in simultaneously as transcription activators to regulate grain filling and storage compounds accumulation to affect endosperm development and grain chalkiness through binding OsISA2, OsLTPL36 and OsYUC11. The study provides important germplasm resources and theoretical basis for genetic improvement of rice yield and quality. In addition, we enriches the potential biological functions of rice MYB family transcription factors.

Epithelial cell adhesion molecule (EpCAM) fused to IgG, IgA and IgM Fc domains was expressed to create IgG, IgA and IgM‐like structures as anti‐cancer vaccines in Nicotiana tabacum. High‐mannose glycan structures were generated by adding a C‐terminal endoplasmic reticulum (ER) retention motif (KDEL) to the Fc domain (FcK) to produce EpCAM‐Fc and EpCAM‐FcK proteins in transgenic plants via Agrobacterium‐mediated transformation. Cross‐fertilization of EpCAM‐Fc (FcK) transgenic plants with Joining chain (J‐chain, J and JK) transgenic plants led to stable expression of large quaternary EpCAM‐IgA Fc (EpCAM‐A) and IgM‐like (EpCAM‐M) proteins. Immunoblotting, SDS–PAGE and ELISA analyses demonstrated that proteins with KDEL had higher expression levels and binding activity to anti‐EpCAM IgGs. IgM showed the strongest binding among the fusion proteins, followed by IgA and IgG. Sera from BALB/c mice immunized with these vaccines produced anti‐EpCAM IgGs. Flow cytometry indicated that the EpCAM‐Fc fusion proteins significantly activated CD8⁺ cytotoxic T cells, CD4⁺ helper T cells and B cells, particularly with EpCAM‐FcKP and EpCAM‐FcP (FcKP) × JP (JKP). The induced anti‐EpCAM IgGs captured human prostate cancer PC‐3 and colorectal cancer SW620 cells. Sera from immunized mice inhibited cancer cell proliferation, migration and invasion; down‐regulated proliferation markers (PCNA, Ki‐67) and epithelial–mesenchymal transition markers (Vimentin); and up‐regulated E‐cadherin. These findings suggest that N. tabacum can produce effective vaccine candidates to induce anti‐cancer immune responses.

Celery (Apium graveolens L.) is an important vegetable crop in the Apiaceae family. It comprises three botanical varieties: common celery with solid and succulent petioles, celeriac or root celery with enlarged and fleshy hypocotyls and smallage or leaf celery with slender, leafy and usually hollow petioles. Here we present a chromosome‐level genome assembly of a celeriac cultivar and a comprehensive genome variation map constructed through resequencing of 177 representative celery accessions. Phylogenetic analysis revealed that smallage from the Mediterranean region represented the most ancient type of cultivated celery. Following initial domestication in this region, artificial selection has primarily aimed at enlarging the hypocotyl, resulting in celeriac, and at solidifying the petiole, leading to common celery. Selective sweep analysis and genome‐wide association study identified several genes associated with hypocotyl expansion and revealed that the hollow/solid petiole trait directly correlated with the presence/absence of a NAC gene. Our study elucidates the complex breeding history of celery and provides valuable genomic resources and molecular insights for future celery improvement and conservation efforts.

Beneficial microorganisms (BMs) promote plant growth and enhance stress resistance. This review summarizes how BMs induce growth promotion by improving nutrient uptake, producing growth‐promoting hormones and stimulating root development. How BMs enhance disease resistance and help protect plants from abiotic stresses has also been explored. Growth‐defense trade‐offs are known to affect the ability of plants to survive under unfavourable conditions. This review discusses studies demonstrating that BMs regulate growth‐defense trade‐offs through microbe‐associated molecular patterns and multiple pathways, including the leucine‐rich repeat receptor‐like kinase pathway, abscisic acid signalling pathway and specific transcriptional factor regulation. This multifaceted relationship underscores the significance of BMs in sustainable agriculture. Finally, the need for integration of artificial intelligence to revolutionize biofertilizer research has been highlighted. This review also elucidates the cutting‐edge advancements and potential of plant‐microbe synergistic microbial agents.

Tomato fruit ripening is a complex developmental process that is important for fruit quality and shelf life. Many factors, including ethylene and several key transcription factors, have been shown to play important roles in the regulation of tomato fruit ripening. However, our understanding of the regulation of tomato fruit ripening is still limited. Here, we describe mut26, an EMS‐induced tomato (Solanum lycopersicum) mutant that exhibits chlorophyll‐deficient phenotypes in various organs, including fruits. Genetic mapping and functional analyses revealed that a single‐nucleotide substitution and a corresponding Pro398–>Ser mis‐sense mutation in SlChlH (GENOMES UNCOUPLED 5, GUN5), which encodes the H subunit of magnesium chelatase, are responsible for the defects in the mut26 strain. Transcript analyses towards the expression of many SlPhANGs revealed that mut26 is defective in plastid retrograde signalling during tomato fruit ripening initiation, namely the transition from mature green to breaker stage. mut26 exhibits delayed progression of fruit ripening characterized by reduced fruit ethylene emission, increased fruit firmness, reduced carotenoid content and delayed plastid conversion from chloroplast to chromoplast. Given that fruit ripening requires signalling from plastids to nucleus, these data support the hypothesis that GUN5‐mediated plastid retrograde signalling promotes tomato fruit ripening. We further showed that the delayed fruit ripening of mut26 is not likely caused by reduced chlorophyll content. Taken together, we identified a new function of SlChlH in the promotion of tomato fruit ripening and ethylene biosynthesis, suggesting that GUN5‐mediated plastid retrograde signalling plays a promotive role in tomato fruit ripening.

Over‐application of potassium (K) fertilizer in fields has a negative impact on the environment. Developing rice varieties with high KUE will reduce fertilizer for sustainable agriculture. However, the genetic basis of KUE in a more diverse and inclusive population remains largely unexplored. Here, we show that the transcription factor OsNAC25 enhances K⁺ uptake and confers high KUE under low K⁺ supply. Disruption of OsNAC25 by CRISPR/Cas9‐mediated mutagenesis led to a considerable loss of K⁺ uptake capacity in rice roots, coupled with reduced K⁺ accumulation in rice and severe plant growth defects under low‐ K⁺ conditions. However, the overexpression of OsNAC25 enhanced K⁺ accumulation by regulating proper K⁺ uptake capacity in rice roots. Further analysis displayed that OsNAC25 can bind to the promoter of OsSLAH3 to repress its transcription in response to low‐ K⁺ stress. Nucleotide diversity analyses suggested that OsNAC25 may be selected during japonica populations' adaptation of low K⁺ tolerance. Natural variation of OsNAC25 might cause differential expression in different haplotype varieties, thus conferring low K⁺ tolerance in the Hap 1 and Hap 4 ‐carrying varieties, and the japonica allele OsNAC25 could enhance low K⁺ tolerance in indica variety, conferring great potential to improve indica low K⁺ tolerance and grain development. Taken together, we have identified a new NAC regulator involved in rice low K⁺ tolerance and grain development, and provide a potential target gene for improving low K⁺ tolerance and grain development in rice.

Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important food crops in the world. CRISPR/Cas12i3, which belongs to the type V‐I Cas system, has attracted extensive attention recently due to its smaller protein size and its less‐restricted canonical ‘TTN’ protospacer adjacent motif (PAM). However, due to its relatively lower editing efficacy in plants and the hexaploidy complex nature of wheat, Cas12i3/Cas12i3‐5M‐mediated genome editing in wheat has not been documented yet. Here, we report the engineering of a robust Cas12i3‐5M‐mediated genome editing system in wheat through the fusion of T5 exonuclease (T5E) in combination with an optimised crRNA expression strategy (Opt). We first showed that fusion of T5E, rather than ExoI, to Cas12i3‐5M increased the gene editing efficiencies by up to 1.34‐fold and 3.87‐fold, compared to Cas12i3‐5M and Cas12i3 in HEK293T cells, respectively. However, its editing efficiency remains low in wheat. We then optimised the crRNA expression strategy and demonstrated that Opt‐T5E‐Cas12i3‐5M could enhance the editing efficiency by 1.20‐ to 1.33‐fold and 4.05‐ to 7.95‐fold in wheat stable lines compared to Opt‐Cas12i3‐5M and Opt‐Cas12i3, respectively, due to progressive 5′‐end resection of the DNA strand at the cleavage site with increased deletion size. The Opt‐T5E‐Cas12i3‐5M enabled an editing efficiency ranging from 60.71% to 90.00% across four endogenous target genes in stable lines of three elite Chinese wheat varieties. Together, the developed robust Opt‐T5E‐Cas12i3‐5M system enriches wheat genome editing toolkits for either biological research or genetic improvement and may be extended to other important polyploidy crop species.