The DOF transcription factors regulating plant bud dormancy were novelly identified. A representative member, PlOBP4, was first experimentally verified to promote bud elongation. Herbaceous peony (Paeonia lactiflora) is a world-renowned ornamental flower mainly cultivated in temperate regions. However, insufficient winter chilling accumulation may limit bud endodormancy release (BER) due to climate change or when grown at lower latitudes or elevations. Understanding the regulatory mechanisms of endodormancy release will help develop new cultivars adapted to warm winter conditions, advancing the application of herbaceous peony in subtropical and even tropical areas. The role of DNA-BINDING WITH ONE FINGER (DOF) TF family members in the regulation of bud dormancy remains limited. In this study, a bioinformatics analysis of the DOF family in P. lactiflora 'Hang Baishao' (a subtropical low-chilling-requirement cultivar) was performed using the full-length transcriptome sequencing data collected during a whole winter and early spring. A total of 15 DOF family members were identified and were phylogenetically classified into four subgroups. Expression profiles of some PlDOFs were correlated with bud endodormancy acquisition, BER, release of ecodormancy, and inducing break. A PlOBP4 with a 906bp coding sequence, which is highly expressed in the BER stage, was identified and cloned. Subcellular localization confirmed that PlOBP4 was nuclear-localized. Silencing PlOBP4 in buds during the endodormancy induction period significantly reduced bud length after sprouting, indicating that PlOBP4 may promote bud elongation in P. lactiflora 'Hang Baishao'. This relatively rare study in regulating bud dormancy/budbreak enriches the knowledge regarding the role of DOF TFs and may further aid in breeding cultivars with short endodormancy duration (low chilling requirement) and facilitate the cultivation of herbaceous peony in subtropical or even tropical regions.

This study elucidates endoplasmic reticulum (ER) stress response mechanisms in Brassica campestris at the gene level using high-throughput transcriptome analysis. The productivity of Brassica campestris is severely limited by adverse environmental conditions that trigger ER stress and disrupt cellular homeostasis. However, the molecular mechanisms underlying ER stress responses in B. campestris remain poorly understood. In this study, tunicamycin (TM) and tauroursodeoxycholic acid (TUDCA; TU) were applied to induce and alleviate ER stress, respectively, followed by transcriptome profiling through high-throughput RNA sequencing. A total of 11,728 differentially expressed genes (DEGs) were identified across distinct pairwise comparisons. These DEGs were significantly enriched in GO terms related to oxidative stress response, protein processing in the ER, unfolded protein binding, and protein folding, as well as activated pathways associated with ROS signaling, calcium signaling, and flavonoid biosynthesis. Hierarchical clustering analysis grouped these DEGs into five clusters, of which C1 showed higher expression associated with TM and TU, C2 showed downregulation during ER stress, and C3 showed higher expression in TM treatment. Among DEGs, 351 transcription factors (TFs) were identified under ER stress, with the most abundant families being bZIP (57), NAC (83), MYB (113), HSF (26), and WRKY (72). Weighted gene co-expression network analysis (WGCNA) further identified three co-expression modules (green, brown, and turquoise) comprising 60 key genes, including four unique genes (BraC03g015140, BraC05g046930, BraC08g032210, and BraC09g046280), and 56 hub genes overlapping with DEGs. The identified 60 candidate genes are involved in protein quality control, chaperone activity, ubiquitin-mediated degradation, redox regulation, vesicle trafficking, and metabolic adjustment. Therefore, this study provides a comprehensive transcriptional landscape of ER stress responses in B. campestris, identifies key regulatory genes for functional validation, and offers valuable insights into improving stress resilience in Brassica crops.

Key messageBiolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes.The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00299-026-03741-9.

Starch branching enzymes (BEs) play crucial roles in determining amylopectin structure, starch components, and ultimately, starch properties and crop quality. This review begins by outlining starch biosynthesis and its relationship with starch properties and rice qualities. We summarized the current understanding of the tertiary structure, catalytic mechanism, and key functional sites of BEs, encompassing catalytic residues, substrate-binding sites, and phosphorylation sites. The regulating BEIIb for modulating amylopectin chain length distribution, starch crystalline lamellar structure, starch granule morphology, starch gelatinization resistance, and starch resistance to enzymatic hydrolysis is reviewed. The regulatory impacts of BEIIb deficiency (via frameshift mutation or expression downregulation) and amino acid substitution on rice qualities are critically discussed. Finally, we proposed some future research directions, including: high-throughput screening and identification methods for BEIIb allelic mutants exhibiting a transparency endosperm, utilizing base editing technology to create novel elite BEIIb alleles, pyramiding multiple genes to develop novel rice germplasm rich in resistant starch while maintaining elite grain quality, especially for appearance quality and eating and cooking quality, andintegrating AI and machine learning to predict regulation effects. This review not only enriches the theoretical framework concerning BEIIb-mediated regulation of starch components and rice qualities but also provides some specific molecular targets within BEIIb for targeted quality improvement strategies.

Our results highlight the key role of NaMYC2a/b in regulating trypsin protease inhibitor activity after insect feeding by controlling the expression of NaPI, NaKTI2, and NaPI-like. When attacked by insect herbivores, Nicotiana attenuata plants activate jasmonate (JA) signaling, and increase trypsin protease inhibitor (TPI) activities by switching on the transcription of various protease inhibitor (PI) genes, such as NaPI and NaKTI2. However, how PI genes are regulated during insect feeding remains unclear. Here we identified a new PI, NaPI-like, that confers Spodoptera litura resistance. NaPI-like shares low sequence identity to NaPI. However, its expression could be specifically and highly induced by S. litura oral secretion (OS). TPI activity was increased by NaPI-like overexpression, and was reduced by silencing NaPI-like. Accordingly, the generalist S. litura performed better in NaPI-like-silenced plants. Further study revealed that the expression levels of NaPI, NaPI-like, and NaKTI2 were all strongly elicited by treatment with methyl JA or wounding plus S. litura oral secretion (W + OS) in wild-type (WT) plants. However, they were induced to a much lesser extent in JA-deficient irAOC plants with W + OS treatment, suggesting that these three PI genes are regulated by JA signaling. Finally, we demonstrated that co-silencing NaMYC2a and NaMYC2b significantly reduced TPI activity, resulting in decreased insect resistance in N. attenuata. EMSA and Dual-LUC assays revealed that NaMYC2a/b regulates the expression of NaPI and NaKTI directly, and NaPI-like indirectly. Our results highlight the diversity of PI genes elicited by insect, and the key role of NaMYC2a/b in regulating TPI activity after insect feeding by controlling NaPI, NaPI-like, and NaKTI2 expression. Thus, our data provide new insights into the regulation of PIs during insect feeding. Our results highlight the key role of NaMYC2a/b in regulating trypsin protease inhibitor activity after insect feeding by controlling the expression of NaPI, NaKTI2, and NaPI-like.

NtRPP13, a CNL-type gene suppressed by Ralstonia solanacearum, mediates a positive regulation of hypersensitive response and phytohormone-related defense genes for bacterial wilt resistance in tobacco. Bacterial wilt, caused by Ralstonia solanacearum, is a devastating soil-borne disease that infects hundreds of plant species worldwide. To date, while effective control strategies for this pathogen remain limited, exploring resistant genes is particularly important in disease-resistant breeding. Nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins are key participants in effector-triggered immunity in plants. This study identified a novel NBS-LRR resistance gene, NtRPP13, in tobacco, which exhibited downregulation in roots of a susceptible tobacco cultivar upon R. solanacearum infection. The NtRPP13 protein contained a typical coiled-coil (CC) domain at its N-terminus and was classified into the CC-NBS-LRR category. Subcellular localization analysis revealed that NtRPP13 localizes to the plasma membrane. Additionally, exposure to phytohormones-including abscisic acid, auxin and gibberellic acid, and abiotic stressors such as drought and cold altered NtRPP13 expression. This could be attribute to the corresponding cis-acting elements in the NtRPP13 promoter. Transient overexpression of NtRPP13 triggered a hypersensitive response (HR) in Nicotiana benthamiana, while stable overexpression in transgenic tobacco plants significantly enhanced resistance to R. solanacearum, with varying resistance levels observed between different transgenic lines. Moreover, following inoculation with R. solanacearum, the transgenic plants exhibited marked upregulation of some key defense-related marker genes associated with the HR, salicylic acid (SA), jasmonic acid (JA), and ethylene signaling pathways, along with significantly elevated levels of JA and SA, compared to wild-type controls. These findings suggest that NtRPP13 contributes to tobacco defense against R. solanacearum by mediating crosstalk between multiple signaling pathways.

BjZIP1 is a plasma membrane-localized protein. Overexpression of BjZIP1 in yeast, Arabidopsis thaliana, and Brassica juncea hairy roots confirmed its role in promoting Cd/Zn uptake. Heavy metal contamination in agricultural soils significantly threatens global food security. While Brassica juncea is recognized as a promising hyperaccumulator for phytoremediation, the specific transporters mediating its metal uptake remain largely unexplored. Here, we identify BjZIP1, a plasma membrane-localized protein that functions as a transporterfor cadmium (Cd) and zinc (Zn) uptake in B. juncea. Heterologous expression of BjZIP1 in Cd-sensitive yeast mutant (Δyap1) increased intracellular accumulation of Cd and Zn by 17.8% and 25.0%, respectively, and consequently enhanced metal sensitivity. In Arabidopsis thaliana, BjZIP1 overexpression lines accumulated 31.1-58.3% more Cd and 1.68-2.48-fold higher Zn in roots under metal stress, which was accompanied by a growth inhibition phenotype. Crucially, BjZIP1-overexpressing B. juncea hairy roots accumulated 1.05-1.30-fold more Cd and 1.42-1.92-fold more Zn, alongside a concomitant exacerbation of cellular damage under Cd exposure. Collectively, our results establish BjZIP1 as a plasma membrane transporter responsible for Cd and Zn uptake in B. juncea, thereby providing a promising molecular target for genetic enhancement of phytoremediation efficiency.

Transcriptomic analysis revealed that GmERF1 expression is induced by Phytophthora sojae and positively regulates soybean resistance to P. sojae via salicylic acid signal transduction pathway. Soybean (Glycine max) root rot caused by Phytophthora sojae is a major disease constraining the global soybean industry. Therefore, improving crop resistance to this pathogen remains a key objective in breeding efforts. However, the mechanisms by which soybeans respond to P. sojae infection, as well as the specific regulatory networks of key transcription factors (TFs), remain to be elucidated. Here, we report that Ethylene Response Factor 1 (GmERF1), encoding an AP2/ERF transcription factor, exhibits significant differences in expression between resistant and susceptible soybean cultivars. Molecular evaluation and disease resistance analysis show that GmERF1 could improve soybean resistance to P. sojae. Further transcriptomic analysis and quantitative analysis of salicylic acid (SA) signal transduction genes indicate that GmERF1 could positively regulate the expression of Non-expressor of Pathogenesis-Related genes 1 (GmNPR1), TGACG sequence-specific binding factor (GmTGA) and Pathogenesis-Related gene 1 (GmPR1). Taken together, these results suggest that GmERF1 positively regulates soybean resistance to P. sojae by enhancing SA signaling, providing novel insights into soybean resistance to Phytophthora root rot.

The receptor-like kinase SlLRR-RLK94 enhances tomato resistance to Phytophthora infestans by effecting pathogenesis-related (PR) gene expression, reactive oxygen species (ROS) homeostasis, and the phenylpropanoid biosynthesis pathway. Late blight, caused by Phytophthora infestans (P. infestans), is one of the most devastating diseases affecting tomato yield and quality. Receptor-like kinases (RLKs) are essential for plants to sense various signaling molecules and to trigger early immune responses. However, most Leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest RLK subfamily, have unknown functions in tomato resistance to P. infestans. Here, we identified 209 LRR-RLK family members in tomato, and clustered them into 14 subfamilies based on phylogenetic analysis. Transcriptome analysis revealed that SlLRR-RLK94 (belonging to the XI subfamily) showed the strongest response to P. infestans infection. Using virus-induced gene silencing and overexpression in tomato, we demonstrated that SlLRR-RLK94 positively regulates tomato resistance to P. infestans. In addition, SlLRR-RLK94 also regulates the expression of PR genes, ROS-scavenging genes, and antioxidant enzyme activity. Integrated transcriptomic and metabolomic analyses suggested that SlLRR-RLK94 mediates tomato resistance to P. infestans, possibly by effecting the phenylpropanoid biosynthesis pathway. These findings establish SlLRR-RLK94 as a key factor facilitating tomato defense signaling and offer a valuable genetic basis for its potential application in crop breeding.

Replacing wheat chromosome 1D with its relative Leymus mollis chromosome 1Ns facilitates the incorporation of storage protein subunits, thereby improving the grain quality of wheat. Wild relatives of wheat serve as valuable gene pools for enhancing genetic diversity of wheat. Leymus mollis Trin. (L. mollis, 2n = 4x = 28, NsNsXmXm) exhibits multiple advantageous traits including disease resistance, stress tolerance, and high grain quality, rendering it a promising genetic resource for wheat improvement via distant hybridization. In this study, 82 wheat-L. mollis derivatives were assessed for grain quality. The superior line WM24 was further analyzed using biochemical, molecular, and cytogenetic methods. Seed storage protein electrophoresis revealed that improved quality of WM24 stems from introducing high-molecular-weight glutenin subunits and gliadins from L. mollis. Genomic in situ hybridization (GISH) confirmed that the chromosomal composition of WM24 was 2n = 42 = 21 II, including a pair of homologous chromosomes from Ns genome of L. mollis. The combination of molecular markers and sequential FISH-GISH indicated that WM24 carries two 1Ns chromosomes substituting for wheat chromosome 1D. SNP array analysis showed predominantly deletion (NA) genotypes of SNPs at 1D loci, corroborating the substitution event. These findings demonstrate that the introducing of L. mollis chromatin positively influences wheat grain quality. WM24 is a wheat-L. mollis 1Ns (1D) substitution line with higher protein content and sedimentation value due to altered storage protein profiles, resulting in enhanced quality traits. The development of these derivatives provides valuable germplasms for wheat quality breeding and exploration of novel exogenous quality-related genes.