Transgenic and gene-editing technologies are essential for gene functional analysis and crop improvement. However, the pleiotropic effects and unknown mechanisms of morphogenic genes have hindered their broader application. In this study, we employed the one-step de novo shoot organogenesis (DNSO) method, and demonstrated that overexpression of the morphogenic gene Arabidopsis thanalia GROWTH-REGULATING FACTOR 5 (AtGRF5) significantly enhanced genetic transformation efficiency in cucurbit crops by promoting callus proliferation and increasing dense cells during regeneration. High-resolution time-series transcriptomics and single-cell RNA sequencing revealed that AtGRF5 overexpression induced auxin-related genes and expanded stem cell populations during cucumber DNSO. Using DNA-affinity purification sequencing (DAP-seq) in combination with spatiotemporal differential gene expression analysis, we identified CsIAA19 as a key downstream target of AtGRF5, with its modulation playing a pivotal role in regeneration. Rescuing CsIAA19 in AtGRF5-overexpressing explant reversed the enhanced callus proliferation and regeneration. To address growth defects caused by AtGRF5 overexpression, we developed an abscisic acid-inducible AtGRF5 expression system, significantly improving transformation and gene-editing efficiency across diverse genotypes while minimizing pleiotropic effects. In summary, this research provides mechanistic insights into AtGRF5-mediated transformation and offers a practical solution to overcome challenges in cucurbit crop genetic modification.

Due to its tropical origins, rice (Oryza sativa) is susceptible to cold stress, which poses severe threats to production. OsNAC5, a NAC-type transcription factor, participates in the cold stress response of rice, but the detailed mechanisms remain poorly understood. Here, we demonstrate that OsNAC5 positively regulates cold tolerance at germination and in seedlings by directly activating the expression of ABSCISIC ACID INSENSITIVE 5 (OsABI5). Haplotype analysis indicated that single nucleotide polymorphisms in a NAC-binding site in the OsABI5 promoter are strongly associated with cold tolerance. OsNAC5 also enhanced OsABI5 stability, thus regulating the expression of cold-responsive (COR) genes, enabling fine-tuned control of OsABI5 action for rapid, precise plant responses to cold stress. DNA affinity purification sequencing coupled with transcriptome deep sequencing identified several OsABI5 target genes involved in COR expression, including DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 1A (OsDREB1A), OsMYB20, and PEROXIDASE 70 (OsPRX70). In vivo and in vitro analyses suggested that OsABI5 positively regulates COR gene transcription, with marked COR upregulation in OsNAC5-overexpressing lines and downregulation in osnac5 and/or osabi5 knockout mutants. This study extends our understanding of cold tolerance regulation via OsNAC5 through the OsABI5-CORs transcription module, which may be used to ameliorate cold tolerance in rice via advanced breeding.

Synthetic microbial communities (SynComs) are a promising tool for making full use of the beneficial functions imparted by whole bacterial consortia. However, the complexity of reconstructed SynComs often limits their application in sustainable agriculture. Furthermore, inter-strain interactions are often neglected during SynCom construction. Here, we propose a strategy for constructing a simplified and functional SynCom (sfSynCom) by using elite helper strains that significantly improve the beneficial functions of the core symbiotic strain, here Bradyrhizobium elkanii BXYD3, to sustain the growth of soybean (Glycine max). We first identified helper strains that significantly promote nodulation and nitrogen fixation in soybean mediated by BXYD3. Two of these helper strains assigned to the Pantoea taxon produce acyl homoserine lactones, which significantly enhanced the colonization and infection of soybean by BXYD3. Finally, we constructed a sfSynCom from these core and helper strains. This sfSynCom based on the core-helper strategy was more effective at promoting nodulation than inoculation with BXYD3 alone and achieved effects comparable to those of a complex elite SynCom previously constructed on the basis of potential beneficial functions between microbes and plants alone. Our results suggest that considering interactions between strains as well as those between strains and the host plant might allow construction of sfSynComs.

Reactive oxygen species (ROS) plays critical roles in modulating plant growth and stress response and its homeostasis is fine tuned using multiple peroxidases. H₂O₂, a major kind of ROS, is removed rapidly and directly using three catalases, CAT1, CAT2, and CAT3, in Arabidopsis. Although the activity regulations of catalases have been well studied, their degradation pathway is less clear. Here, we report that CAT2 and CAT3 protein abundance was partially controlled using the 26S proteasome. To further identify candidate proteins that modulate the stability of CAT2, we performed yeast-two-hybrid screening and recovered several clones encoding a protein with RING and vWA domains, CIRP1 (CAT2 Interacting RING Protein 1). Drought and oxidative stress downregulated CIRP1 transcripts. CIRP1 harbored E3 ubiquitination activity and accelerated the degradation of CAT2 and CAT3 by direct interaction and ubiquitination. The cirp1 mutants exhibited stronger drought and oxidative stress tolerance, which was opposite to the cat2 and cat3 mutants. Genetic analysis revealed that CIRP1 acts upstream of CAT2 and CAT3 to negatively regulate drought and oxidative stress tolerance. The increased drought and oxidative stress tolerance of the cirp1 mutants was due to enhanced catalase (CAT) activities and alleviated ROS levels. Our data revealed that the CIRP1–CAT2/CAT3 module plays a vital role in alleviating ROS levels and balancing growth and stress responses in Arabidopsis.

Calcium oscillations are induced by different stresses. Calcium-dependent protein kinases (CDPKs/CPKs) are one major group of the plant calcium decoders that are involved in various processes including drought response. Some CPKs are calcium-independent. Here, we identified ZmCPK2 as a negative regulator of drought resistance by screening an overexpression transgenic maize pool. We found that ZmCPK2 does not bind calcium, and its activity is mainly inhibited during short term abscisic acid (ABA) treatment, and dynamically changed in prolonged treatment. Interestingly, ZmCPK2 interacts with and is inhibited by calcium-dependent ZmCPK17, a positive regulator of drought resistance, which is activated by ABA. ZmCPK17 could prevent the nuclear localization of ZmCPK2 through phosphorylation of ZmCPK2T60. ZmCPK2 interacts with and phosphorylates and activates ZmYAB15, a negative transcriptional factor for drought resistance. Our results suggest that drought stress-induced Ca²⁺ can be decoded directly by ZmCPK17 that inhibits ZmCPK2, thereby promoting plant adaptation to water deficit.

The phytohormone jasmonates (JAs) regulate plant growth and defense responses. The reproductive organs of flowers are devastated by insect herbivores. However, the molecular mechanisms of floral defense remain largely unknown. Here, we found that the Arabidopsis JA receptor CORONATINE INSENSITIVE1 (COI1) and its substrates JA ZIM-domain (JAZ) repressors, and the mediator subunit MEDIATOR25-based MED25–MYC–MYB (MMM) complexes, including MYC2/3/4/5 and MYB28/29/76, mediated floral defense against the insects Helicoverpa armigera, Spodoptera exigua, and Spodoptera frugiperda. The flower-specific IIIa bHLH factors ABORTED MICROSPORES (AMS) and DYSFUNCTIONAL TAPETUM 1 (DYT1) were JAZ-interaction proteins. They interacted with members of the MMM complexes, inhibited the transcriptional activity of MYC2 and MYB28, and repressed floral defense against insects. AMS and DYT1 recruited the flower-specific MYB21/24, and these MYBs interacted with members of MMM complexes, inhibited the MYC2–MYB28 function, and suppressed floral defense against insects. Our study revealed that the JA–COI1–JAZ–MMM pathway mediated flower defense, and the AMS/DYT1–MYB21/24 module antagonized the MMM complexes to repress floral defense against insects.

Besides playing a crucial role in plant immunity via the nonexpressor of pathogenesis-related (NPR) proteins, increasing evidence shows that salicylic acid (SA) can also regulate plant root growth. However, the transcriptional regulatory network controlling this SA response in plant roots is still unclear. Here, we found that NPR1 and WRKY45, the central regulators of SA response in rice leaves, control only a reduced sector of the root SA signaling network. We demonstrated that SA attenuates root growth via a novel NPR1/WRKY45-independent pathway. Furthermore, using regulatory network analysis and mutant characterization, we identified a set of new NPR1/WRKY45-independent regulators that conservedly modulate the root development and root-associated microbiota composition in both Oryza sativa (monocot) and Arabidopsis thaliana (dicot) in response to SA. Our results established the SA signaling as a central element regulating plant root functions under ecologically relevant conditions. These results provide new insights to understand how regulatory networks control plant responses to abiotic and biotic stresses.

Saline–alkaline soils are a major environmental problem that limit plant growth and crop productivity. Plasma membrane H⁺-ATPases and the salt overly sensitive (SOS) signaling pathway play important roles in plant responses to saline–alkali stress. However, little is known about the functional genes and mechanisms regulating the transcription of H⁺-ATPases and SOS pathway genes under saline–alkali stress. In the present study, we identified that the plant AT-rich sequence and zinc-binding (TaPLATZ2) transcription factor are involved in wheat response to saline–alkali stress by directly suppressing the expression of TaHA2/TaSOS3. The knockdown of TaPLATZ2 enhances salt and alkali stress tolerance, while overexpression of TaPLATZ2 leads to salt and alkali stress sensitivity in wheat. In addition, TaWRKY55 directly upregulated the expression of TaPLATZ2 during saline–alkali stress. Through knockdown and overexpression of TaWRKY55 in wheat, TaWRKY55 was shown to negatively modulate salt and alkali stress tolerance. Genetic analyses confirmed that TaPLATZ2 functions downstream of TaWRKY55 in response to salt and alkaline stresses. These findings provide a TaWRKY55–TaPLATZ2–TaHA2/TaSOS3 regulatory module that regulates wheat responses to saline–alkali stress.

Panicle exsertion is one of the crucial agronomic traits in rice (Oryza sativa). Shortening of panicle exsertion often leads to panicle enclosure and severely reduces seed production. Gibberellin (GA) plays important roles in regulating panicle exsertion. However, the underlying mechanism and the relative regulatory network remain elusive. Here, we characterized the oswrky78 mutant showing severe panicle enclosure, and found that the defect of oswrky78 is caused by decreased bioactive GA contents. Biochemical analysis demonstrates that OsWRKY78 can directly activate GA biosynthesis and indirectly suppress GA metabolism. Moreover, we found OsWRKY78 can interact with and be phosphorylated by mitogen-activated protein kinase (MAPK) kinase OsMAPK6, and this phosphorylation can enhance OsWRKY78 stability and is necessary for its biological function. Taken together, these results not only reveal the critical function of OsWRKY78, but also reveal its mechanism via mediating crosstalk between MAPK and the GA signaling pathway in regulating panicle exsertion.

Lodging reduces grain yield and quality in cereal crops. Lodging resistance is affected by the strength of the culm, which is influenced by the culm diameter, culm wall thickness, and cell wall composition. To explore the genetic architecture of culm diameter in rice (Oryza sativa), we conducted a genome-wide association study (GWAS). We identified STRONG CULM 2 (STRONG2), which encodes the mannan synthase CSLA5, and showed that plants that overexpressed this gene had increased culm diameter and improved lodging resistance. STRONG2 appears to increase the levels of cell wall components, such as mannose and cellulose, thereby enhancing sclerenchyma development in stems. SNP14931253 in the STRONG2 promoter contributes to variation in STRONG2 expression in natural germplasms and the transcription factor MYB61 directly activates STRONG2 expression. Furthermore, STRONG2 overexpressing plants produced significantly more grains per panicle and heavier grains than the wild-type plants. These results demonstrate that the MYB61–STRONG2 module positively regulates culm diameter and lodging resistance, information that could guide breeding efforts for improved yield in rice.

In C₃ plants, photorespiration is an energy expensive pathway that competes with photosynthetic CO₂ assimilation and releases CO₂ into the atmosphere, potentially reducing C₃ plant productivity by 20%-50%. Consequently, reducing the flux through photorespiration has been recognized as a major way to improve C₃ crop photosynthetic carbon fixation and productivity. While current research efforts in engineering photorespiration are mainly based on the modification of chloroplast glycolate metabolic steps, only limited studies have explored optimizations in other photorespiratory metabolic steps. Here, we engineered an imGS bypass within the rice mitochondria to bypass the photorespiratory glycine toward glycine betaine, thereby, improving the photosynthetic carbon fixation in rice. The imGS transgenic rice plants exhibited significant accumulation of glycine betaine, reduced photorespiration, and elevated photosynthesis and photosynthate levels. Additionally, the introduction of imGS bypass into rice leads to an increase in the number of branches and grains per panicle which may be related to cytokinin and gibberellin signaling pathways. Taken together, these results suggest diverting mitochondrial glycine from photorespiration toward glycine betaine synthesis can effectively enhance carbon fixation and panicle architecture in rice, offering a promising strategy for developing functional mitochondrial photorespiratory bypasses with the potential to enhance plant productivity.

Cultivating high-yield wheat under limited water resources is crucial for sustainable agriculture in semiarid regions. Amid water scarcity, plants activate drought response signaling, yet the delicate balance between drought tolerance and development remains unclear. Through genome-wide association studies and transcriptome profiling, we identified a wheat atypical basic helix-loop-helix (bHLH) transcription factor (TF), TabHLH27-A1, as a promising quantitative trait locus candidate for both relative root dry weight and spikelet number per spike in wheat. TabHLH27-A1/B1/D1 knock-out reduced wheat drought tolerance, yield, and water use efficiency (WUE). TabHLH27-A1 exhibited rapid induction with polyethylene glycol (PEG) treatment, gradually declining over days. It activated stress response genes such as TaCBL8-B1 and TaCPI2-A1 while inhibiting root growth genes like TaSH15-B1 and TaWRKY70-B1 under short-term PEG stimulus. The distinct transcriptional regulation of TabHLH27-A1 involved diverse interacting factors such as TaABI3-D1 and TabZIP62-D1. Natural variations of TabHLH27-A1 influence its transcriptional responses to drought stress, with TabHLH27-A1^Hap-II associated with stronger drought tolerance, larger root system, more spikelets, and higher WUE in wheat. Significantly, the excellent TabHLH27-A1^Hap-II was selected during the breeding process in China, and introgression of TabHLH27-A1^Hap-II allele improved drought tolerance and grain yield, especially under water-limited conditions. Our study highlights TabHLH27-A1's role in balancing root growth and drought tolerance, providing a genetic manipulation locus for enhancing WUE in wheat.

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in plant immunity by transducing external signals inside plant cells. Here, we defined a wheat MAPK cascade, composed of the mitogen-activated protein kinase kinase (MAPKK) TaMKK2 and its downstream MAPK TaMAPK6, which phosphorylates the core immune regulator TaSGT1 (suppressor of G2 allele of Skp1), resulting in enhanced nuclear entry of TaSGT1, thereby conferring resistance against the devastating wheat pathogen Puccinia striiformis f. sp. tritici (Pst). Hence, we identified a TaMKK2-TaMAPK6-TaSGT1 signaling cascade that contributes to wheat stripe rust resistance. During infection, Pst secrets a haustorium-associated secreted protein 215 (HASP215), that targets TaMKK2 and interferes with the interaction of TaMKK2 with TaMAPK6 to suppress TaMAPK6 phosphorylation and activation, thereby leading to reduced capacity of TaMAPK6 to phosphorylate TaSGT1. Consequently, inhibition of TaMAPK6-mediated TaSGT1 phosphorylation resulted in decreased nuclear translocation of TaSGT1 and suppressed plant immunity. Our work elucidates the positive function of TaMKK2-TaMAPK6 cascade in wheat immunity by regulating the immune component TaSGT1, and its regulation by the rust effector HASP215, providing new insights into the MAPK cascade on crop immunity and the pathogenicity mechanism of obligate biotrophic fungus.

Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross-pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a-DUO1 module down-regulated the expression of YUC2/ YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a- DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.

Coniferous forests are under severe threat of the rapid anthropogenic climate warming. Abies (firs), the fourth-largest conifer genus, is a keystone component of the boreal and temperate dark-coniferous forests and harbors a remarkably large number of relict taxa. However, the uncertainty of the phylogenetic and biogeographic history of Abies significantly impedes our prediction of future dynamics and efficient conservation of firs. In this study, using 1,533 nuclear genes generated from transcriptome sequencing and a complete sampling of all widely recognized species, we have successfully reconstructed a robust phylogeny of global firs, in which four clades are strongly supported and all intersectional relationships are resolved, although phylogenetic discordance caused mainly by incomplete lineage sorting and hybridization was detected. Molecular dating and ancestral area reconstruction suggest a Northern Hemisphere high-latitude origin of Abies during the Late Cretaceous, but all extant firs diversified during the Miocene to the Pleistocene, and multiple continental and intercontinental dispersals took place in response to the late Neogene climate cooling and orogenic movements. Notably, four critically endangered firs endemic to subtropical mountains of China, including A. beshanzuensis, A. ziyuanensis, A. fanjingshanensis and A. yuanbaoshanensis from east to west, have different origins and evolutionary histories. Moreover, three hotspots of species richness, including western North America, central Japan, and the Hengduan Mountains, were identified in Abies. Elevation and precipitation, particularly precipitation of the coldest quarter, are the most significant environmental factors driving the global distribution pattern of fir species diversity. Some morphological traits are evolutionarily constrained, and those linked to elevational variation (e.g., purple cone) and cold resistance (e.g., pubescent branch and resinous bud) may have contributed to the diversification of global firs. Our study sheds new light on the spatiotemporal evolution of global firs, which will be of great help to forest management and species conservation in a warming world.

Rice stripe mosaic virus (RSMV) is an emerging pathogen which significantly reduces rice yields in the southern region of China. It is transmitted by the leafhopper Recilia dorsalis, which overwinters in rice fields. Our field investigations revealed that RSMV infection causes delayed rice heading, resulting in a large number of green diseased plants remaining in winter rice fields. This creates a favorable environment for leafhoppers and viruses to overwinter, potentially contributing to the rapid spread and epidemic of the disease. Next, we explored the mechanism by which RSMV manipulates the developmental processes of the rice plant. A rice heading‐related E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), was found to be hijacked by the RSMV‐encoded P6. The impairment of HAF1 function affects the ubiquitination and degradation of downstream proteins, HEADING DATE 1 and EARLY FLOWERING3, leading to a delay in rice heading. Our results provide new insights into the development regulation‐based molecular interactions between virus and plant, and highlights the importance of understanding virus‐vector‐plant tripartite interactions for effective disease management strategies.

Pineapple is the third most crucial tropical fruit worldwide and available in five varieties. Genomes of different pineapple varieties have been released to date; however, none of them are complete, with all exhibiting substantial gaps and representing only two of the five pineapple varieties. This significantly hinders the advancement of pineapple breeding efforts. In this study, we sequenced the genomes of three varieties: a wild pineapple variety, a fiber pineapple variety, and a globally cultivated edible pineapple variety. We constructed the first gap-free reference genome (Ref) for pineapple. By consolidating multiple sources of evidence and manually revising each gene structure annotation, we identified 26,656 protein-coding genes. The BUSCO evaluation indicated a completeness of 99.2%, demonstrating the high quality of the gene structure annotations in this genome. Utilizing these resources, we identified 7,209 structural variations across the three varieties. Approximately 30.8% of pineapple genes were located within ±5 kb of structural variations, including 30 genes associated with anthocyanin synthesis. Further analysis and functional experiments demonstrated that the high expression of AcMYB528 aligns with the accumulation of anthocyanins in the leaves, both of which may be affected by a 1.9-kb insertion fragment. In addition, we developed the Ananas Genome Database, which offers data browsing, retrieval, analysis, and download functions. The construction of this database addresses the lack of pineapple genome resource databases. In summary, we acquired a seamless pineapple reference genome with high-quality gene structure annotations, providing a solid foundation for pineapple genomics and a valuable reference for pineapple breeding.