Plants have evolved a sophisticated chemical defense network to counteract pathogens, with phenolamides and salicylic acid (SA) playing pivotal roles in the immune response. However, the synergistic regulatory mechanisms of their biosynthesis remain to be explored. Here, we identified a biosynthetic gene cluster on chromosome 2 (BGC2) associated with the biosynthesis of phenolamide and SA, wherein the key component SlEPS1 exhibits dual catalytic functions for the synthesis of phenolamides and SA. Overexpression of the key component SlEPS1 of BGC2 in tomato enhanced resistance to the bacterial pathogen Pst DC3000, whereas knockout plants were more susceptible. Exogenous applications of SA and phenolamides revealed that these two compounds act synergistically to enhance plant resistance. Notably, during tomato domestication, a disease-resistant allele of SlEPS1, SlEPS1^HapB, was subject to negative selection, leading to a reduction in phenolamide and SA levels and compromised disease resistance in modern varieties. Moreover, the SlMYB78 directly regulates the BGC2 gene cluster to enhance phenolamide and SA biosynthesis, modulating resistance to Pst DC3000. Our study employed multi-omics approaches to describe the synergistic regulation of phenolamide and SA biosynthesis, offering new insights into the complexity of plant immune-related metabolism.

Deep learning-based genomic prediction (DL-based GP) has shown promising performance compared to traditional GP methods in plant breeding, particularly in handling large, complex multi-omics data sets. However, the effective development and widespread adoption of DL-based GP still face substantial challenges, including the need for large, high-quality data sets, inconsistencies in performance benchmarking, and the integration of environmental factors. Here, we summarize the key obstacles impeding the development of DL-based GP models and propose future developing directions, such as modular approaches, data augmentation, and advanced attention mechanisms.

Pollen hydration, germination, and tube growth are vital processes for the successful fertilization of flowering plants. These processes involve complex signaling pathways. Reactive oxygen species (ROS) generated in apoplast involves signaling for the cell wall expansion during tube growth, however molecular regulators are less known. We identified pollen-specific receptor-like kinase (OsPRK) family genes from rice (Oryza sativa), which have conserved leucine-rich repeat (LRR) and kinase domains. To understand the function of these genes, we produced single and triple mutations for OsPRK1, OsPRK2, and OsPRK3 using the clustered regularly interspaced palindromic repeats (CRISPR/Cas9) system. Among these mutants, triple knockout (KO) lines (osprk1/2/3) exhibited the male-sterile phenotype with normal vegetative growth and floret formation. Through cytological analysis, we confirmed that the reduced seed fertility was due to defects in pollen hydration and germination with low ROS accumulation. This defect of pollen germination was partially recovered by treatment with exogenous H₂O₂. We also confirmed that OsPRKs could interact with the LRR extension protein. Our results suggest that rice PRKs redundantly play a role in ROS signaling for pollen hydration and germination, and fertility can be controlled by exogenous application.

Silicon (Si) plays a crucial role in plant growth, development, and stress tolerance. However, in some consumable plant products, such as fruits, Si deposition leads to the formation of a white powdery layer known as bloom, which diminishes glossiness and consumer appeal. Despite its significance, the genetic basis of bloom formation remains largely unexplored. Here, we identified a unique cucumber backbone parent line exhibiting bloomless fruit, which was designated bloomless cucumber 1 (bec1). Map-based cloning of the bec1 locus revealed that BEC1, harboring a natural C-to-T variation at the 754th base of its coding region, is a strong candidate gene for the bloomless trait. Functional validation through gene-editing mutants and BEC1::BEC1-GFP transgenic lines confirmed that BEC1, encoding a Si efflux transporter, is responsible for bloom formation. Mutation of BEC1 impaired Si uptake, thereby preventing the deposition of Si on the surface of glandular trichomes and resulting in bloomless fruits. Additionally, Si deficiency in BEC1 mutants compromised resistance to Corynespora cassiicola and chilling stress. Interestingly, grafting bec1 scions onto bloom rootstocks restored the Si accumulation and stress resistance, while maintaining bloomless phenotype. Overall, our findings elucidate the role of BEC1 in bloom formation and provide a valuable genetic target for breeding bloomless cucumber with enhanced stress resilience.

Sex chromosomes have evolved independently in numerous lineages across the Tree of Life, in both diploid-dominant species, including many animals and plants, and the less studied haploid-dominant plants and algae. Strict genetic sex determination ensures that individuals reproduce by outcrossing. However, species with separate sexes (termed dioecy in diploid plants, and dioicy in haploid plants) may sometimes evolve different sex systems, and become monoicous, with the ability to self-fertilize. Here, we studied dioicy-monoicy transitions in the ancient liverwort haploid-dominant plant lineage, using three telomere-to-telomere gapless chromosome-scale reference genome assemblies from the Ricciaceae group of Marchantiales. Ancestral liverworts are believed to have been dioicous, with U and V chromosomes (chromosome 9) determining femaleness and maleness, respectively. We confirm the finding that monoicy in Ricciocarpos natans evolved from a dioicous ancestor, and most ancestrally U chromosomal genes have been retained on autosomes in this species. We also describe evidence suggesting the possible re-evolution of dioicy in the genus Riccia, with probable de novo establishment of a sex chromosome from an autosome (chromosome 5), and further translocations of genes from the new sex chromosome to autosomes. Our results also indicated that micro-chromosomes are consistent genomic features, and may have evolved independently from sex chromosomes in Ricciocarpos and Riccia lineages.

Maize (Zea mays L.) growth and yield are severely limited by drought stress worldwide. Stomata play crucial roles in transpiration and gas exchange and are thus essential for improving plant water-use efficiency (WUE) to help plants deal with the threat of drought. In this study, we characterized the maize dsd1 (decreased stomatal density 1) mutant, which showed defects in stomatal development, including guard mother cell differentiation, subsidiary cell formation and guard cell maturation. DSD1 encodes the basic helix-loop-helix transcription factor INDUCER OF CBF EXPRESSION b (ZmICEb) and is a homolog of ICE1 in Arabidopsis (Arabidopsis thaliana). DSD1/ZmICEb is expressed in stomatal file cells throughout stomatal development and plays a conserved role in stomatal development across maize and Arabidopsis. Mutations in DSD1/ZmICEb dramatically improved drought tolerance and WUE in maize and reduced yield losses under drought conditions. Therefore, DSD1/ZmICEb represents a promising candidate target gene for the genetic improvement of drought tolerance in maize by manipulating stomatal density.

Diverse heterotic groups have been developed in China over several decades, but their genomic divergences have not been systematically studied after improvement. In this study, we performed Maize6H-60K array of 5,822 maize accessions and whole-genome re-sequencing of 150 inbred lines collected in China. Using multiple population structure analysis methods, we established a genetic boundary used to categorize heterotic groups and germplasm resources. We identified three chloroplast–cytoplasmic types that evolved during adaptation to diverse climatic environments in maize through phylogenetic and haplotype analyses. Comparative analyses revealed obvious genetic differences between heterotic groups and germplasm resources at both the chloroplast and nuclear genome levels, especially in the unique heterotic groups HG1 and HG2, which exhibited distinct regionality and genetic uniqueness. The divergent differentiation of heterotic groups from germplasm resources was driven by differential selection in specific genomic regions. Genome-wide selective sweep analysis identified core selected regions and candidate selected genes associated with traits between heterotic groups, highlighting that stress response- and plant defense-related genes were selected for environmental adaptation across a broad latitudinal range in China. Meanwhile, a genome-wide association study analysis provided evidence that core selected genes served as an important candidate gene pool with a potential role in genetic improvement. Gene exchanges among heterotic groups, which avoided the predominant heterotic patterns as much as possible, occurred to achieve population improvement during modern maize breeding. This study provides insights into the population differentiation and genetic characteristics of heterotic groups, which will facilitate the utilization of germplasm resources, the creation of novel maize germplasm, and the optimization of heterotic patterns during future maize breeding in China.

Successful reproduction depends on the stable germination and growth of the pollen tubes (PT). However, the molecular mechanisms involved in rice PT growth and development remain largely unknown. In a previous study, microarray transcriptome analysis identified 627 genes preferentially expressed in the tricellular and germinating pollen of rice (i.e., Oryza sativa ssp. japonica). To elucidate key genes involved in the gene transfer process facilitated by male gametophytes, we systematically screened T-DNA lines containing disrupted sequences that corresponded to these 627 genes and analyzed the genotypes of heterozygote progeny from 107 T-DNA-indexed lines covering 105 genes. We found that 42 lines exhibited a distorted segregation ratio among the wild-type (WT), heterozygote (HT), and homozygote (HM) genotypes, which deviated from the expected Mendelian ratio of 1:2:1 (WT:HT:HM). Further characterization using CRISPR/Cas9 mutants revealed that knockout mutants of certain genes that exhibited segregation distortion in the T-DNA insertion region were completely sterile. Moreover, even when T-DNA insertion lines followed Mendelian segregation patterns, sterility could be induced by simultaneously mutating functionally redundant genes, thereby overcoming genetic compensation. Interestingly, although some T-DNA insertion lines exhibited segregation ratios approximating 1:1:0, the corresponding CRISPR/Cas9 mutants produced homozygous seeds and showed partial sterility. Partial sterility suggests that despite mutant pollen grains being less competitive than WT pollen, they retain their fertilization potential under relaxed competition from WT pollen. Beyond mutant-based analysis, transcriptomic profiling of sterile mutant lines provided additional insight into the regulatory relationship between key germination regulators and the 105 target genes studied here. Overall, this study demonstrates the effectiveness of a multi-pronged strategy to accelerate the identification of defective phenotypes using mutant studies and provides valuable genetic resources for inducing novel male sterility in rice.

Avena barbata, a wild oat species within the genus Avena, is a widely used model for studying plant ecological adaptation due to its strong environmental adaptability and disease resistance, serving as a valuable genetic resource for oat improvement. Here, we phased the high-quality chromosome-level genome assembly of A. barbata (6.88 Gb, contig N50 = 53.74 Mb) into A (3.57 Gb with 47,687 genes) and B (3.31 Gb with 46,029 genes) subgenomes. Comparative genomics and phylogenomic analyses clarified the evolutionary relationships and trajectories of A, B, C and D subgenomes in Avena. We inferred that the A subgenome donor of A. barbata was Avena hirtula, while the B subgenome donor was probably an extinct diploid species closely related to Avena wiestii. Genome evolution analysis revealed the dynamic transposable element (TE) content and subgenome divergence, as well as extensive structure variations across A, B, C, and D subgenomes in Avena. Population genetic analysis of 211 A. barbata accessions from distinct ecotypes identified several candidate genes related to environmental adaptability and drought resistance. Our study provides a comprehensive genetic resource for exploring the genetic basis underlying the strong environmental adaptability of A. barbata and the molecular identification of important agronomic traits for oat breeding.

The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement. CRISPR/Cas12i3, which belongs to the Class II Type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and less restricted canonical “TTN” protospacer adjacent motif (PAM). However, due to its relatively lower editing efficiency, Cas12i3-mediated multiplex gene editing has not yet been documented in plants. Here, we fused four 5′ exonucleases (Exo) including T5E, UL12, PapE, ME15 to the N terminal of an optimized Cas12i3 variant (Cas12i3-5M), respectively, and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines. We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M, respectively. Notably, the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%–98.61% across the six tested endogenous genes. We further demonstrated that, although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing, UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three, four, five or six genes with efficiencies of 82.76%, 61.36%, 52.94%, and 51.06% in rice stable lines, respectively. Together, we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice. The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.

NONEXPRESSER OF PATHOGENESIS-RELATED GENES 1 (NPR1), the receptor for salicylic acid (SA), plays a central role in the SA-mediated basal antiviral responses. Recent studies have shown that two different plant RNA viruses encode proteins that suppress such antiviral responses by inhibiting its SUMOylation and inducing its degradation, respectively. However, it is unclear whether targeting NPR1 is a general phenomenon in viruses and whether viruses have novel strategies to inhibit NPR1. In the present study, we report that two different positive-sense single-stranded RNA (+ssRNA) viruses, namely, alfalfa mosaic virus (AMV) and potato virus X (PVX); one negative-sense single-stranded RNA (−ssRNA) virus (calla lily chlorotic spot virus, CCSV); and one single-stranded DNA virus (beet severe curly-top virus, BSCTV) that also encode one or more proteins that interact with NPR1. In addition, we found that the AMV-encoded coat protein (CP) can induce NPR1 degradation by recruiting S-phase kinase-associated protein 1 (Skp1), a key component of the Skp1/cullin1/F-box (SCF) E3 ligase. In contrast, the BSCTV-encoded V2 protein inhibits NPR1 function, probably by affecting its nucleocytoplasmic distribution via the nuclear export factor ALY. Taken together, these data suggest that NPR1 is one of the central hubs in the molecular arms race between plants and viruses and that different viruses have independently evolved different strategies to target NPR1 and disrupt its function.

Although the frequency of ancient hybridization across the Tree of Life is greater than previously thought, little work has been devoted to uncovering the extent, timeline, and geographic and ecological context of ancient hybridization. Using an expansive new dataset of nuclear and chloroplast DNA sequences, we conducted a multifaceted phylogenomic investigation to identify ancient reticulation in the early evolution of oaks (Quercus). We document extensive nuclear gene tree and cytonuclear discordance among major lineages of Quercus and relatives in Quercoideae. Our analyses recovered clear signatures of gene flow against a backdrop of rampant incomplete lineage sorting, with gene flow most prevalent among major lineages of Quercus and relatives in Quercoideae during their initial radiation, dated to the Early-Middle Eocene. Ancestral reconstructions including fossils suggest ancestors of Castanea + Castanopsis, Lithocarpus, and the Old World oak clade probably co-occurred in North America and Eurasia, while the ancestors of Chrysolepis, Notholithocarpus, and the New World oak clade co-occurred in North America, offering ample opportunity for hybridization in each region. Our study shows that hybridization—perhaps in the form of ancient syngameons like those seen today—has been a common and important process throughout the evolutionary history of oaks and their relatives. Concomitantly, this study provides a methodological framework for detecting ancient hybridization in other groups.

Plants, algae and photosynthetic bacteria convert light into chemical energy by means of photosynthesis, thus providing food and energy for most organisms on Earth. Photosynthetic pigments, including chlorophylls (Chls) and carotenoids, are essential components that absorb the light energy necessary to drive electron transport in photosynthesis. The biosynthesis of Chl shares several steps in common with the biosynthesis of other tetrapyrroles, including siroheme, heme and phycobilins. Given that many tetrapyrrole precursors possess photo-oxidative properties that are deleterious to macromolecules and can lead to cell death, tetrapyrrole biosynthesis (TBS) requires stringent regulation under various developmental and environmental conditions. Thanks to decades of research on model plants and algae, we now have a deeper understanding of the regulatory mechanisms that underlie Chl synthesis, including (i) the many factors that control the activity and stability of TBS enzymes, (ii) the transcriptional and post-translational regulation of the TBS pathway, and (iii) the complex roles of tetrapyrrole-mediated retrograde signaling from chloroplasts to the cytoplasm and the nucleus. Based on these new findings, Chls and their derivatives will find broad applications in synthetic biology and agriculture in the future.

Both the filling and development of grain are key processes determining agriculture production and reproductive growth in rice. The processes of grain filling and endosperm development are crucial for the accumulation of major storage compounds in rice grains. This requires extensive remobilization of carbon reserves from source to sink and the precise regulation of sucrose-to-starch conversion. Both the developmental sequence of the panicle and environmental signals influence the carbon flow between the leaves, leaf sheath, stem, and spikelets during grain filling. This, in turn, affects endosperm development and the production of storage compounds. In this review, we synthesize recent insight into grain development in rice, focusing on the dynamic changes in phytohormones and how their homeostasis integrates developmental and environmental cues to control grain filling in the developing panicle. We also highlight recent advances in the genetic control of carbohydrate remobilization and the transcriptional regulatory networks governing carbohydrate metabolism and grain development in rice. The asynchronous initiation and imbalance in grain filling limit the full yield potential of cereal crops. The “superior/inferior spikelets” serve as a model system for understanding the regulatory mechanisms underlying grain filling and development. Systematic research on carbohydrate flow and phytohormone crosstalk could enhance our understanding of optimizing yield production in cereal crops. Additionally, a thorough analysis of key genetic regulatory mechanisms can offer a genetic foundation and targets for precisely adjusting grain filling traits, ultimately aiding in the development of high-yield crop varieties.

Plants have evolved a remarkable ability to sense and respond to changes in photoperiod, allowing adjustments to their growth and development based on seasonal and environmental cues. The floral transition is a pivotal stage in plant growth and development, signifying a shift from vegetative to reproductive growth. CONSTANS (CO), a central photoperiodic response factor conserved in various plants, mediates day-length signals to control the floral transition, although its mechanisms of action vary among plants with different day-length requirements. In addition, recent studies have uncovered roles for CO in organ development and stress responses. These pleiotropic roles in model plants and crops make CO a potentially fruitful target for molecular breeding aimed at modifying crop agronomic traits. This review systematically traces research on CO, from its discovery and functional studies to the exploration of its regulatory mechanisms and newly discovered functions, providing important insight into the roles of CO and laying a foundation for future research.

The past decade has witnessed rapid developments in gene discovery, biological big data (BBD), artificial intelligence (AI)-aided technologies, and molecular breeding. These advancements are expected to accelerate crop breeding under the pressure of increasing demands for food. Here, we first summarize current breeding methods and discuss the need for new ways to support breeding efforts. Then, we review how to combine BBD and AI technologies for genetic dissection, exploring functional genes, predicting regulatory elements and functional domains, and phenotypic prediction. Finally, we propose the concept of intelligent precision design breeding (IPDB) driven by AI technology and offer ideas about how to implement IPDB. We hope that IPDB will enhance the predictability, efficiency, and cost of crop breeding compared with current technologies. As an example of IPDB, we explore the possibilities offered by CropGPT, which combines biological techniques, bioinformatics, and breeding art from breeders, and presents an open, shareable, and cooperative breeding system. IPDB provides integrated services and communication platforms for biologists, bioinformatics experts, germplasm resource specialists, breeders, dealers, and farmers, and should be well suited for future breeding.

Land plants use diverse hormones to coordinate their growth, development and responses against biotic and abiotic stresses. Salicylic acid (SA) is an essential hormone in plant immunity, with its levels and signaling tightly regulated to ensure a balanced immune output. Over the past three decades, molecular genetic analyses performed primarily in Arabidopsis have elucidated the biosynthesis and signal transduction pathways of key plant hormones, including abscisic acid, jasmonic acid, ethylene, auxin, cytokinin, brassinosteroids, and gibberellin. Crosstalk between different hormones has become a major focus in plant biology with the goal of obtaining a full picture of the plant hormone signaling network. This review highlights the roles of SA in plant immunity and summarizes our current understanding of the pairwise interactions of SA with other major plant hormones. The complexity of these interactions is discussed, with the hope of stimulating research to address existing knowledge gaps in hormone crosstalk, particularly in the context of balancing plant growth and defense.

The rhizosphere plays a crucial role in plant growth and resilience to biotic and abiotic stresses, highlighting the complex communication between plants and their dynamic rhizosphere environment. Plants produce a wide range of signaling molecules that facilitate communication with various rhizosphere factors, yet our understanding of these mechanisms remains elusive. In addition to protein-coding genes, increasing evidence underscores the critical role of microRNAs (miRNAs), a class of non-coding single-stranded RNA molecules, in regulating plant growth, development, and responses to rhizosphere stresses under diverse biotic and abiotic factors. In this review, we explore the crosstalk between miRNAs and their target mRNAs, which influence the development of key plant structures shaped by the belowground environment. Moving forward, more focused studies are needed to clarify the functions and expression patterns of miRNAs, to uncover the common regulatory mechanisms that mediate plant tolerance to rhizosphere dynamics. Beyond that, we propose that using artificial miRNAs and manipulating the expression of miRNAs and their targets through overexpression or knockout/knockdown approaches could effectively investigate their roles in plant responses to rhizosphere stresses, offering significant potential for advancing crop engineering.

Small peptides (SPs) are pivotal signaling molecules that play essential roles in the precise regulation of plant growth, development, and stress responses. Recent advancements in sequencing technologies, bioinformatics approaches, and biochemical and molecular techniques have significantly enhanced the accuracy of SP identification, unveiling their diverse biological functions in plants. This review provides a comprehensive overview of the characteristics and methodologies for identifying SPs in plants. It highlights recent discoveries regarding the biological roles and signaling pathways of SPs in regulating plant growth, development, and plant–microbial interactions, as well as their contributions to plant resilience under various environmental stresses, including abiotic stress, nutrient deficiencies, and biotic challenges. Additionally, we discuss current insights into the potential applications of SPs and outline future research directions aimed at leveraging these molecules to enhance plant adaptation to environmental challenges. By integrating recent findings, this review lays a foundation for advancing the understanding and utilization of SPs to improve plant resilience and productivity.

The modern cultivated tomato (Solanum lycopersicum) was domesticated from Solanum pimpinellifolium native to the Andes Mountains of South America through a “two-step domestication” process. It was introduced to Europe in the 16th century and later widely cultivated worldwide. Since the late 19th century, breeders, guided by modern genetics, breeding science, and statistical theory, have improved tomatoes into an important fruit and vegetable crop that serves both fresh consumption and processing needs, satisfying diverse consumer demands. Over the past three decades, advancements in modern crop molecular breeding technologies, represented by molecular marker technology, genome sequencing, and genome editing, have significantly transformed tomato breeding paradigms. This article reviews the research progress in the field of tomato molecular breeding, encompassing genome sequencing of germplasm resources, the identification of functional genes for agronomic traits, and the development of key molecular breeding technologies. Based on these advancements, we also discuss the major challenges and perspectives in this field.

Ethylene treatment promotes orange coloration in the flavedo of Satsuma mandarin (Citrus unshiu Marc.) fruit, but the corresponding regulatory mechanism is still largely unknown. In this study, we identified a C2H2-type zinc-finger transcription factor, CitZAT4, the expression of which was markedly induced by ethylene. CitZAT4 directly binds to the CitPSY promoter and activates its expression, thereby promoting carotenoid biosynthesis. Transient expression in Satsuma mandarin fruit and stable transformation of citrus calli showed that overexpressing of CitZAT4 inhibited CitLCYE expression, thus inhibiting α-branch yellow carotenoid (lutein) biosynthesis. CitZAT4 overexpression also enhanced the transcript levels of CitLCYB, CitHYD, and CitNCED2, promoting β-branch orange carotenoid accumulation. Molecular biochemical assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR), and luciferase (LUC) assays, demonstrated that CitZAT4 directly binds to the promoters of its target genes and regulates their expression. An ethylene response factor, CitERF061, which is induced by ethylene signaling, was found to directly bound to the CitZAT4 promoter and induced its expression, thus positively regulating CitZAT4-mediated orange coloration in citrus fruit. Together, our findings reveal that a CitZAT4-mediated transcriptional cascade is driven by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism in promoting orange coloration in the flavedo of Satsuma mandarin fruit. The molecular regulatory mechanism revealed here represents a significant step toward developing strategies for improving the quality and economic efficiency of citrus crops.

Fruit taste quality is greatly influenced by the content of soluble sugars, which are predominantly stored in the vacuolar lumen. However, the accumulation and regulation mechanisms of sugars in most fruits remain unclear. Recently, we established the citrus fruit vacuole proteome and discovered the major transporters localized in the vacuole membrane. Here, we demonstrated that the expression of tonoplast sugar transporter 2 (CsTST2) is closely associated with sugar accumulation during sweet orange (Citrus sinensis) ripening. It was further demonstrated that CsTST2 had the function of transporting hexose and sucrose into the vacuole. Overexpression of CsTST2 resulted in an elevation of sugar content in citrus juice sac, calli, and tomato fruit, whereas the downregulation of its expression led to the reduction in sugar levels. CsTST2 was identified as interacting with CsCIPK23, which binds to the upstream calcium signal sensor protein CsCBL1. The phosphorylation of the three serine residues (Ser277, Ser337, and Ser354) in the loop region of CsTST2 by CsCIPK23 is crucial for maintaining the sugar transport activity of CsTST2. Additionally, the expression of CsCIPK23 is positively correlated with sugar content. Genetic evidence further confirmed that calcium and CsCIPK23-mediated increase in sugar accumulation depends on CsTST2 and its phosphorylation level. These findings not only unveil the functional mechanism of CsTST2 in sugar accumulation, but also explore a vital calcium signal regulation module of CsCBL1/CIPK23 for citrus sweetness quality.