The ETHYLENE INSENSITIVE3-LIKE (EIL) family is one of the most important transcription factor (TF) families in plants and is involved in diverse plant physiological and biochemical processes. In this study, ten EIL transcription factors (CsEILs) in sweet orange were systematically characterized via whole-genome analysis. The CsEIL genes were unevenly distributed across the four sweet orange chromosomes. Putative cis-acting regulatory elements (CREs) associated with CsEIL were found to be involved in plant development, as well as responses to biotic and abiotic stress. Notably, quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that CsEIL genes were widely expressed in different organs of sweet orange and responded to both high and low temperature, NaCl treatment, and to ethylene-dependent induction of transcription, while eight additionally responded to Xanthomonas citri pv. Citri (Xcc) infection, which causes citrus canker. Among these, CsEIL2, CsEIL5 and CsEIL10 showed pronounced upregulation. Moreover, nine genes exhibited differential expression in response to Candidatus Liberibacter asiaticus (CLas) infection, which causes Citrus Huanglongbing (HLB). The genome-wide characterization and expression profile analysis of CsEIL genes provide insights into the potential functions of the CsEIL family in disease resistance.

Seedless is a highly valued commercial characteristic in the citrus industry, both for fresh consumption and for processed markets. In this study, the ‘Succari Sweet Orange’ (WT) and its seedless mutant ‘Juxiangyuan’ (MT), which originated from a bud mutation, were selected to study the formation of a citrus seedless phenotype. Microscopic analysis of MT’s floral organs, including anther and ovary cross-sections, provides insights into its seedless phenotype compared to the original seeded cultivar. Additionally, pollen features, viability, and in vitro germination were examined to determine the cause of seedlessness. MT exhibited significant developmental deformities in both male and female gametes, with pollen grain analysis indicating a high rate of deformity (41.48%), low viability (9.59%), and minimal in vitro germination (9.56%). Hybridization experiments were conducted to assess male and female sterility and pollen incompatibility. Both WT and MT exhibited parthenocarpic development. Notably, MT fruit produced with an average of 3.51 seeds pollinated to WT, despite severe pollen abortion of MT. MT, however, produced seedless fruit through self-breeding or cross-breeding with other varieties, demonstrating stable female sterility. Despite reduced pollen quantity and viability in the seedless mutant ‘Juxiangyuan’, its seedlessness primarily stems from female sterility. This study contributed to a deeper understanding of seedless formation in ‘Juxiangyuan’ and provided valuable information relevant to its commercial cultivation.

Global climate change is leading to more frequent extreme cold events, underscoring the need to study citrus cold tolerance to support breeding and enable potential northward expansion of citrus cultivation. In this study, the ‘Miyagawa’ wild type and its cold-tolerant mutant were selected for systematic comparison across cold-resistant phenotypes, leaf tissue structure, physiological and biochemical characteristics, and Cor8 gene expression. The mutant exhibited 50% lower relative conductivity and malondialdehyde (MDA) content under −6 °C stress compared to the wild type, indicating reduced membrane damage. Antioxidant enzyme activities were significantly higher in the mutant: superoxide dismutase (SOD) activity increased by 10–30%, peroxidase (POD) by 28%, and catalase (CAT) by up to 2-fold. Proline content was 57% higher in the mutant at peak levels, supporting stronger osmotic regulation. Moreover, Cor8 gene expression in the mutant was up to 2.98 times higher than in the wild type during natural overwintering. These findings confirm that the ‘Miyagawa’ mutant possesses distinct physiological, anatomical, and molecular advantages for low-temperature adaptation and provides valuable germplasm for breeding cold-tolerant citrus varieties.

The global citrus industry is under severe threat from devastating diseases, including Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), and Citrus tristeza disease, caused by Citrus tristeza virus (CTV). Establishing efficient pathogen elimination techniques is crucial for developing virus-free citrus nursery systems and helps facilitate the prevention and control of devastating diseases. This study systematically evaluated the multiple effects of hot water treatment and plant growth regulator application on bud germination, pathogen elimination, antioxidant enzyme activities, and CsWUS expression in citrus scion. The results showed that moderate hot-water treatment at 40 °C for 5 min promoted bud germination, while higher temperatures or extended durations were inhibitory. Both 6-benzylaminopurine (6-BA) and melatonin (MT) significantly enhanced germination, with 50 mg/L 6-BA and 100 μM MT being most effective. All treatments significantly enhanced pathogen elimination and increased the activities of antioxidant enzymes SOD, POD, and CAT, with melatonin concentrations of 100 μM and above reducing CLas and CTV titers by over 93.64% and 96.07%. Furthermore, hot-water treatment and MT, but not 6-BA, upregulated the expression of the virus-resistance-related gene CsWUS. A combined treatment of hot water and MT did not yield synergistic benefits. In conclusion, melatonin treatment, particularly at 100 μM, optimally balances high bud germination with effective pathogen elimination, likely through activating antioxidant defenses and CsWUS-mediated immunity. This study provides a practical and efficient strategy for citrus pathogen-free seedling production.

Cation/proton antiporters (CAXs) are key membrane transporters involved in plant development and stress adaptation. In this study, five CsCAX genes (CsCAX1–CsCAX5) were identified from the Citrus sinensis genome through comprehensive bioinformatic analysis. All CsCAX proteins are hydrophobic with 10–11 transmembrane domains, predominantly composed of α-helices and random coils, and are localized to either the plasma membrane or vacuole. Phylogenetic analysis classified them into two subfamilies (IA and IB). Promoter prediction identified stress- and hormone-responsive cis-elements (e.g., LTR, SA, and ABA), implying transcriptional regulation under environmental cues. Tissue-specific expression profiling revealed the highest CsCAX3 transcript abundance in stems and leaves. CsCAX3 was upregulated upon Xanthomonas citri subsp. citri (Xcc) infection but downregulated by Candidatus Liberibacter asiaticus (CLas). Subcellular localization confirmed CsCAX3 targeting to the plasma membrane. Functional verification showed that CsCAX3 overexpression increased susceptibility to Xcc, accompanied by a six-fold rise in bacterial load and substantial repression of antioxidant genes (CAT2, POD). Furthermore, transient overexpression of CsCAX3 in tobacco (Nicotiana benthamiana) will eliminate reactive oxygen species (ROS) accumulation. These results indicate that CsCAX3 negatively affects disease resistance by reducing sensitivity to ROS-mediated defense responses. Overall, this study elucidates the structural and functional characteristics of the CsCAX gene family and provides new insights into ROS-mediated immune regulation in citrus.

Cold stress is a critical abiotic factor that severely limits plant growth and agricultural productivity in subtropical regions. Poncirus trifoliata exhibits exceptional cold hardiness and is widely used as a rootstock in Citrus. However, the key genes and mechanisms conferring this resilience remain largely unexplored. Here, we characterized PtCOR8, a cold-induced gene isolated from P. trifoliata. Phylogenetic and subcellular localization analyses confirmed that PtCOR8 encodes a plasma membrane-localized protein belonging to the WCOR413 family. Functional validation revealed that heterologous overexpression of PtCOR8 in tomato significantly enhanced cold tolerance, concomitant with reduced malondialdehyde (MDA) content, elevated peroxidase (POD) activity, and upregulation of cold-responsive genes (e.g., CIN8). Notably, expression profiling of COR8 in 16 citrus accessions under natural overwintering conditions indicated a strong positive correlation between its expression level and cold tolerance of different genotypes. Transgenic tomato plants with PtCOR8 driven by its native promoter also presented enhanced cold tolerance, confirming that the native promoter is sufficient to drive functional expression under cold stress in the tomato system. Through promoter deletion and β-glucuronidase (GUS) staining experiments, the ATCT motif was further identified as a cis-acting element capable of mediating cold-induced promoter activity. Our findings uncover a dual-layered mechanism in which the PtCOR8 protein alleviates membrane lipid peroxidation and oxidative damage, while its transcription level is precisely modulated by a novel promoter regulatory mechanism, thereby improving freezing tolerance. This study provides important genetic insights and a valuable gene resource for cold-resistant citrus breeding.