Somatic embryogenesis (SE) serves as an ideal model for studying plant cell totipotency and embryonic development mechanisms, yet the transcriptional and epigenetic regulation underlying this process remains poorly characterized. We found that citrus nucellar polyembryony (a form of apomixis) gene, RWP-RK domain-containing transcription factor (CitRWP) is highly expressed in embryogenic callus with strong SE capacity, and its expression is constantly upregulated during SE induction. Overexpression of CitRWP induced spontaneous somatic embryo formation in a citrus variety that is resistant to SE bypass exogenous inducer. Suppression of CitRWP expression by RNA interference (RNAi) impairs SE capacity. Integrated RNA-seq and Chromatin immunoprecipitation sequencing (ChIP-seq) analyses combined with molecular assays revealed that CitRWP directly binds to the DUO1-ACTIVATED ZINC FINGER 3 (CsDAZ3) promoter and activates its transcription. Overexpression of CsDAZ3 significantly enhanced SE capacity. Comparative analysis of ChIP-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) datasets demonstrated that CitRWP is capable of binding to regions of low chromatin accessibility. CitRWP binds to a condensed chromatin site in the CsDAZ3 promoter and promotes increased local chromatin accessibility at the CsDAZ3 locus. Our findings delineate a novel regulatory framework for citrus SE, highlighting that CitRWP coordinates transcriptional regulation with chromatin remodeling. This research provides insights into plant cell reprogramming and provides pivotal molecular targets for optimizing breeding protocols by modulating SE.

Leaf morphology is a key adaptive and agronomic trait that influences the efficiency of photosynthesis. However, the mechanisms controlling leaf size and shape in Citrus remain unclear. In this study, we demonstrate that CsTCP2 is a key regulator of leaf cell differentiation and maturation. CRISPR-Cas9 knockout of CsTCP2 led to significantly enlarged leaves, primarily due to increases in both cell size and cell number. An integrated analysis of data from RNA-seq and DAP-seq experiments revealed that CsTCP2 coordinates leaf development by directly regulating the expression of multiple genes that encode transcription factors. In cstcp2 mutants, expression of CsTCP2, CsYAB1, and CsIAA14 was downregulated, while CsWOX4, CsGRF5, and CsXTH6 were upregulated. We further demonstrate that CsTCP2 directly binds the CsGRF5 promoter and represses its expression. Our findings establish a CsTCP2-CsGRF5 module that promotes leaf differentiation and maturation, providing key genetic insights for improving citrus leaf traits.

Trichomes develop as outward projections from the epidermal surface. By contrast, as subepidermal secretory cavities, citrus oil glands originate from epidermal cells and develop into specialized hollow structures. During the development of these epidermis-derived structures in vascular plants, transcription factors, such as the homeodomain-leucine zipper and APETALA2/Ethylene responsive factor families, regulate their initiation and morphogenesis. Subsequent biosynthesis of secondary metabolites within these secretory structures is often mediated by jasmonic acid signaling and basic helix-loop-helix proteins, particularly MYC transcription factors. Here, we compare the regulatory mechanisms governing the development of glandular trichomes and secretory cavities, along with the biosynthesis of secondary metabolites. These insights provide the basic knowledge for harnessing these secretory structures as chassis in synthetic biology applications.

Thorns are modified branches that have evolved independently multiple times as defenses against herbivores. We previously identified the TCP transcription factors THORN IDENTITY1 (TI1) and TI2 as key regulators of thorn development in Citrus; however, how these genes are regulated remains unclear. In this study, using comparative transcriptomics, we identified TI3, encoding a SHORT INTERNODES/STYLISH (SHI/STY) family transcription factor that is specifically expressed in thorns. We found that TI3 binds to a previously undefined CTAG core element in the promoters of TI1 and TI2, activating their expression to promote stem cell arrest in the thorn meristem. CRISPR-Cas9-mediated disruption of TI3 function converted thorns into branches. Conversely, the PEBP family protein CsCENTRORADIALIS (CsCEN) represses TI3 expression in the axillary meristem to maintain stem cell activity and promote branch development. Mutations in CsCEN resulted in branch-to-thorn conversions, whereas cscen ti3 double mutants exhibited the ti3 mutant phenotype, supporting the idea that CsCEN regulates TI3 expression. The thorn-specific expression pattern of TI3 homologs across three Rutaceae species suggests that TI3 might have a conserved role in thorn development. Thus, TI3 represents a new regulator of meristem identity, and manipulating its activity is a promising approach for breeding thornless cultivars.

Bacterial diseases like huanglongbing (HLB) and citrus canker severely impact citrus production. HLB, caused by "Candidatus Liberibacter asiaticus" (CLas), leads to tree decline, while citrus canker, caused by Xanthomonas citri pv. citri (Xcc) causes necrotic lesions on leaves and fruit. Many bacterial pathogens secrete effector proteins that suppress host plant immunity and promote pathogenesis through the upregulation of host-encoded susceptibility genes. Xcc uses the type III secretion system to introduce effector proteins such as the transcription factor-like (TAL) effector PthA4 that can directly activate host susceptibility gene expression. In contrast, CLas lacks most bacterial secretion systems and relies predominantly on the Sec secretion system for pathogenesis. While some Sec-secreted proteins have been identified in CLas, their direct role in causing HLB symptoms remains unproven. Several Sugars Will Eventually be Exported Transporter (SWEET) genes, encoding sucrose transporters, are candidate susceptibility genes. Here we investigate the roles of the citrus SWEET10, SWEET12 and SWEET15 genes and show that mutations of SWEET15 resulted in reduced susceptibility to citrus canker in three different citrus cultivars: Carrizo citrange (Citrus sinensis 'Washington' sweet orange × Poncirus trifoliata), 'Limoneria 8A' Lisbon lemon (Citrus limon) and 'Pineapple' sweet orange (C. sinensis). Furthermore, Lisbon lemon plants mutated for SWEET15 also showed reduced CLas titre in infected plants. These results suggest that SWEET15 may act as a broad-spectrum susceptibility gene, and disruption of SWEET15 gene activity could be a viable approach to mitigating bacterial diseases such as citrus canker and HLB in a variety of citrus cultivars.

A common characteristic of thorns, prickles, and spines is a hardened and sharp tip. This feature complicates cultivation and postharvest processing for many crops, yet the molecular mechanisms governing this specific sclerification process remain unclear. By genome editing screening, we identified a MYB family gene named SHORT and SOFT THORN 1 (SST1) that specifically promotes the development of sclerenchyma cells at Citrus thorn tips. CRISPR-Cas9 editing of SST1 results in the formation of short and soft thorns with significantly reduced secondary wall thickening at the tips, while leaving stem growth unaffected. SST1 directly activates NST1 and SND1, driving a hierarchical transcriptional regulatory network for secondary wall biosynthesis. Moreover, SST1 homologs in various species are specifically expressed in thorns, suggesting a potential conserved role. Our results uncover the molecular basis for hard plant protective structures, and modulation of this pathway could be an effective strategy for optimizing cultivation practices.

Seedlessness is a most valuable trait in fruit crops for fresh consumption and processing. The mutations in essential meiosis genes are known to confer sterility and seed abortion in plants. However, defects in meiosis have rarely been reported in fruit crops. Here, we found meiosis defects caused sterility in a seedless citrus bud sport cultivar, with massive unpaired univalents during diakinesis, indicating a disruption in crossover formation. A non-functional CrMER3A-103 bp allele with a 103-bp deletion in the gene body, together with the other non-functional CrMER3a allele with a T deletion in exon, were identified in the seedless cultivar. The CrMER3 protein was undetectable at meiotic prophase I in the seedless cultivar, and knock out of CrMER3 resulted in sterility in precocious Mini-citrus. Therefore, the natural variation in CrMER3 is responsible for sterility and seedlessness in this bud sport cultivar. The CrMER3a allele originated from the primitive wild mandarin and was passed to cultivated mandarins. A Kompetitive Allele-Specific PCR (KASP) marker was developed to identify citrus germplasm with CrMER3a allele and to screen potential sterile and seedless hybrids in citrus cross breeding. Uncovering the natural mutations responsible for meiosis defects in citrus enhances our understanding of mechanisms controlling seedlessness in fruit crops and facilitates breeding of seedless varieties.

The fruit of chestnut rose (Rosa roxburghii Tratt.) contains exceptionally high levels of L-ascorbic acid (AsA) (∼1762 mg/100 g fresh weight), approximately 40-fold higher than those found in sweet orange (Citrus sinensis), which is well known for its high AsA content. However, the molecular mechanisms driving such high accumulation in chestnut rose remain unclear. Here, we report that the genes R. roxburghiiPECTIN METHYLESTERASE (RroxPME), D-GALACTURONATE REDUCTASE (RroxGalUR), and DEHYDROASCORBATE REDUCTASE 2 (RroxDHAR2) play crucial roles in AsA accumulation in chestnut rose fruit. By comparing R. roxburghii with the closely related Rosamultiflora, which has low AsA concentrations, we identified a 545-bp insertion in the promoter of RroxGalUR. We found that TRANSPARENT TESTA GLABRA 2 (RroxTTG2), a well-known key regulator of trichome development, binds to the W-box-containing inserted region of the RroxGalUR promoter as well as the promoters of RroxPME and RroxDHAR2. In contrast, in sweet orange, CsTTG2 can bind only to CsPME. Furthermore, RroxTTG2 retains its conserved role in the regulation of trichome development during early fruit development, suggesting its spatiotemporal specificity in regulating both trichome development and AsA biosynthesis. To evaluate the application value of this pathway in other species, we heterologously expressed RroxTTG2, RroxPME, RroxGalUR, and RroxDHAR2 in lettuce (Lactuca sativa L.), which increased AsA concentrations in the transgenic lines by up to 355% (an increase from approximately 2 to 10 mg/100 g fresh weight). This study provides insights into mechanisms underlying AsA accumulation in chestnut rose and the spatiotemporal transcriptional regulation of AsA biosynthesis and trichome development.

Sweet orange is cultivated worldwide but suffers from various devastating diseases because of its monogenetic background. The elucidation of the origin of a crop facilitates the domestication of new crops that may better cope with new challenges. Here we collected and sequenced 226 citrus accessions and assembled telomere-to-telomere phased diploid genomes of sweet orange and sour orange. On the basis of a high-resolution haplotype-resolved genome analysis, we inferred that sweet orange originated from a sour orange × mandarin cross and confirmed this model using artificial hybridization experiments. We identified defense-related metabolites that potently inhibited the growth of multiple industrially important pathogenic bacteria. We introduced diversity to sweet orange, which showed wide segregation in fruit flavor and disease resistance and produced canker-resistant sweet orange by selecting defense-related metabolites. Our findings elucidate the origin of sweet orange and de novo domesticated disease-resistant sweet oranges, illuminating a strategy for the rapid domestication of perennial crops.

Polyploidization is a common occurrence in the evolutionary history of flowering plants, significantly contributing to their adaptability and diversity. However, the molecular mechanisms behind these adaptive advantages are not well understood. Through comprehensive phenotyping of diploid and tetraploid clones from Citrus and Poncirus genera, we discovered that genome doubling significantly enhances salt stress resilience. Epigenetic and transcriptomic analyses revealed that increased ethylene production in the roots of tetraploid plants was associated with hypomethylation and enhanced chromatin accessibility of the ACO1 gene. This increased ethylene production activates the transcription of reactive oxygen species scavenging genes and stress-related hormone biosynthesis genes. Consequently, tetraploid plants exhibited superior root functionality under salt stress, maintaining improved cytosolic K+/Na+ homeostasis. To genetically validate the link between salt stress resilience and ACO1 expression, we generated overexpression and knockout lines, confirming the central role of ACO1 expression regulation following genome doubling in salt stress resilience. Our work elucidates the molecular mechanisms underlying the role of genome doubling in stress resilience. We also highlight the importance of chromatin dynamics in fine-tuning ethylene gene expression and activating salt stress resilience pathways, offering valuable insights into plant adaptation and crop genome evolution.

Secretory structures in terrestrial plants serve as reservoirs for a variety of secondary metabolites. Among these, the secretory cavity of the Rutaceae family is notable for containing essential oils with a wide range of applications. However, the molecular basis underlying secretory cavity development is unknown. Here, we reveal a molecular framework for Citrus oil gland formation. Using genetic mapping and genome editing, we demonstrated that this process requires LATE MERISTEM IDENTITY1 (LMI1), a key regulator of leaf serration. A conserved GCC box element of the LMI1 promoter recruits DORNROSCHEN-like (DRNL) for transcriptional activation. This DRNL-LMI1 cascade triggers MYC5 activation, facilitating the development of oil glands and the biosynthesis of essential oils. Our findings spotlight cis -regulatory divergence within leaf shape genes, propelling novel functional tissue formation.

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus -related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus -related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol (PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNA-gRNA (inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

KEY MESSAGE:Here, we developed a reliable protocol for the fast and efficient gene-edited Anliu sweet orange plants production. The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90%. In addition, the mutation efficiency of the grafted geneedited sweet orange was significantly improved by short-term heat stress treatments. Thus, the combination strategy of grafting and heat stress treatments provided a reference for the fast and efficient multiplex gene editing of sweet orange.

Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants. In plants, the WUSCHEL-related homeobox (WOX) family of transcription factors function as central organizers of several developmental programs ranging from embryo patterning to meristematic stem-cell maintenance through transcriptional activation and repression mechanisms. The Medicago truncatula STENOFOLIA (STF) gene is a master regulator of leaf-blade lateral development. Here, the crystal structure of the homeodomain (HD) of STF (STF-HD) in complex with its promoter DNA is reported at 2.1 Å resolution. STF-HD binds DNA as a tetramer, enclosing nearly the entire bound DNA surface. The STF-HD tetramer is partially stabilized by docking of the C-terminal tail of one protomer onto a conserved hydrophobic surface on the head of another protomer in a head-to-tail manner. STF-HD specifically binds TGA motifs, although the promoter sequence also contains TAAT motifs. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides DNA binding in the minor groove through basic residues located at its C-terminus. The structural and functional data in planta reported here provide new insights into the DNA-binding mechanisms of plant-specific HDs from the WOX family of transcription factors.

Differential regulation of stem cell activity in shoot meristems contributes to the wide variation in shoot architecture.1-3 In most Citrus species, a thorn meristem and a dormant axillary meristem co-localize at each leaf base, offset from each other in a spiral phyllotactic pattern. We recently identified THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, as necessary for the termination of meristem proliferation and concomitant thorn production in Citrus.4 However, how the dormant axillary meristem at the same leaf axil maintains stem cell activity is still unknown. The phosphatidylethanolamine-binding protein (PEBP)-type transcription factors CENTRORADIALIS (CEN) and TERMINAL FLOWER1 (TFL1) maintain inflorescence meristem indeterminacy in many plant species by antagonizing floral meristem identity regulators.5-9 Here, we show that, in Citrus, Citrus CEN (CsCEN) maintains vegetative axillary meristem indeterminacy by antagonizing TI1. CsCEN is expressed in the axillary meristem, but not in the thorn meristem. Disruption of CsCEN function results in termination of the stem cell activity and conversion of dormant axillary meristems into thorns, although ectopic overexpression of CsCEN represses TI1 expression and converts thorns into dormant buds, a phenotype similar to the ti1 mutant. We further show that CsCEN interacts with Citrus FD (CsFD) to repress TI1 expression. CsCEN activity depends on the function of TI1 and TI2, as mutations in TI1 and TI2 rescue the cscen mutant phenotype. We suggest that the antagonistic roles of CsCEN and TI1 define the pattern of axillary meristem determinacy, which shapes vegetative Citrus tree shoot architecture.

WOX family transcription factors regulate multiple developmental programs. The intermediate clade transcriptional activator WOX9 functions together with the modern clade transcriptional repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. STF and LAM1 are WOX1 orthologs required for leaf blade outgrowth in Medicago truncatula and Nicotiana sylvestris, respectively. Using biochemical methods and genome editing technology, here we show that WOX9 is an abaxial factor and functions antagonistically to STF and LAM1 to regulate leaf blade development. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression and knockout of NsWOX9 in N. sylvestris resulted in a range of severe leaf blade distortions, indicating important role in blade development. Our results indicate that direct repression of WOX9 by WUS clade repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that controlling transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in flowering plants.

Thorns arise from axillary shoot apical meristems that proliferate for a time and then terminally differentiate into a sharp tip. Like other meristems, thorn meristems contain stem cells but, in the case of thorns, these stem cells undergo a programmed cessation of proliferative activity. Using Citrus, we characterize a gene network necessary for thorn development. We identify two Citrus genes, THORN IDENTITY1 (TI1) and THORN IDENTITY2 (TI2), encoding TCP transcription factors, as necessary for stem cell quiescence and thorn identity. Disruption of TI1 and TI2 function results in reactivation of stem cells and concomitant conversion of thorns to branches. Expression of WUSCHEL (WUS) defines the shoot stem cell niche in the apical meristems of many angiosperm species; we show that TI1 binds to the Citrus WUS promoter and negatively regulates its expression to terminate stem cell proliferation. We propose that shifts in the timing and function of components of this gene network can account for the evolution of Citrus thorn identity. Modulating this pathway can significantly alter plant architecture and could be leveraged to improve crop yields.

Base editors (BEs) are RNA-guided CRISPR-Cas-derived genome editing tools that induce single-nucleotide changes. The limitations of current BEs lie in their low precision (especially when multiple target nucleotides of the deaminase are present within the activity window) and their restriction to targets that are in proper distance from the PAM sequence. We have recently developed high-precision cytidine BEs by engineering CDA1 truncations and nCas9 fusions that predominantly edit nucleotide C −18 relative to the PAM sequence NGG. Here, by testing fusions with Cas9 variants that recognize alternative PAMs, we provide a series of high-precision BEs that greatly expand the versatility of base editing. In addition, we obtained BEs that selectively edit C −15 or C −16 . We also show that our high-precision BEs can substantially reduce off-target effect. These improved base editing tools will be widely applicable in basic research, biotechnology and gene therapy.