Kai Xu

Kiwifruit ( Actinidia spp.) rootstocks play a crucial role in enhancing environmental adaptability, yet the lack of efficient genetic transformation systems has limited functional genomic studies on stress resistance mechanisms. Here, we established a rapid and cost-effective Agrobacterium rhizogenes -mediated hairy root transformation system for the widely used rootstock Actinidia valvata , bypassing the need for tissue culture. This system achieved transgenic root production within 3–5 weeks and demonstrated broad applicability in both Myrica rubra and Actinidia chinensis . Utilizing GFP as a viability marker, we observed that 25 µM Cd² ⁺ markedly impaired root cell physiology within 2 h, preceding visible phenotypic changes. Transcriptomic profiling of Cd² ⁺ -treated roots identified extensive differential gene expression, with enrichment in key pathways such as “Plant hormone signal transduction” and “MAPK signaling pathway.” Hormonal quantification further revealed significant alterations in IAA, IBA, ABA, JA, SA, and GA levels under Cd² ⁺ stress. Our integrative multi-omics approach, combining transcriptomics, hormone profiling, and physiological validation, provides a comprehensive framework for decoding genotype-to-phenotype relationships in woody horticultural species. The established transformation platform not only enables efficient functional gene verification in kiwifruit roots but also provides a reliable platform for dissecting root-specific cadmium stress responses in Ericales woody species.

Alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is a destructive fungal disease affecting citrus production worldwide. Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes constitute a major class of plant immune receptors; however, their genome-wide characteristics and potential association with Alternaria brown spot resistance loci in citrus remain poorly understood. In this study, we performed a comprehensive genome-wide identification and comparative analysis of NBS-LRR genes across representative citrus species. A total of 417 and 326 NBS-LRR genes were identified in Citrus reticulata and Citrus clementina, respectively, and were classified into NL, CNL, TNL, and RNL subfamilies based on domain architecture. Phylogenetic reconstruction, gene structure analysis, conserved motif composition, chromosomal distribution, synteny relationships, and promoter cis-element profiling collectively revealed considerable structural variation and lineage-specific expansion of the NBS-LRR gene family in citrus genomes. By integrating previously reported quantitative trait locus (QTL) data for Alternaria brown spot, we identified several NBS-LRR genes located within a resistance-associated genomic interval on chromosome 3. Among these, a candidate gene, designated LRR2, exhibited differential transcriptional responses upon pathogen inoculation and displayed distinct sequence variations between citrus genotypes. Structural modeling and molecular docking analyses suggested potential binding interfaces between LRR2 and multiple host-selective toxins, although the biological relevance of these interactions requires further experimental validation. Subcellular localization assays in Nicotiana benthamiana showed that LRR2 is distributed in both the nucleus and cytoplasm. Notably, transient overexpression of LRR2 triggered hypersensitive response-like cell death and H₂O₂ accumulation. Collectively, this study provides a comprehensive overview of the citrus NBS-LRR gene family and presents a multifaceted characterization of a QTL-anchored candidate gene. These findings establish a genomic and molecular framework for further functional investigations of citrus–Alternaria interactions.

Pear (Pyrus L.) is a significant commercial fruit globally, with diverse species exhibiting variations in their flowering periods due to environmental factors. CONSTANS-like (COL) genes, known from previous studies in Arabidopsis, are key regulators of flowering time by sensing photoperiod. However, the evolutionary history and functions of COL genes in different pear species remain unclear. In this study, we identified a total of 79 COL genes in different pear species, including 12 COL genes in Pyrus bretschneideri ‘DangshanSuli’, 9 in Pyrus ussuriensis × hybrid ‘Zhongai 1’, 11 in Pyrus communis ‘Bartlett’, 13 in Pyrus betulifolia, 18 in Pyrus pyrifolia ‘Cuiguan’, 16 in Pyrus pyrifolia ‘Nijisseiki’. Analysis of gene structure, phylogenetic tree, and multiple sequences provided valuable insights into the fundamental understanding of COL genes in pear. The impact of selection pressure on the PbrCOLs in Chinese white pear was assessed using Ka/Ks, revealing that the evolution rate of PbrCOLs was influenced by purification selection factors. The study also revealed different tissue-specific expression patterns of PbrCOLs under varying light quality. Real-time quantitative PCR revealed that under natural light conditions, the expression patterns of PbrCOL2, PbrCOL3, and PbrCOL4 are similar to previous studies on CONSTANS gene in Arabidopsis, with increased expression levels during the day and decreased levels at night. However, PbrCOL1, PbrCOL6, and PbrCOL9 exhibit different expression patterns, with decreased expression levels both during the day and at night. After red light treatment, high expression of PbrCOL3 and PbrCOL4 was observed at night, while the expression patterns of the other four genes did not show significant changes. Following blue light treatment, the expression peaks of PbrCOL1 and PbrCOL6 occurred during the night, showing opposite expression patterns compared to the study in Arabidopsis. The overexpression of PbrCOL3 significantly increase the chlorophyll content in pear seedlings, and its expression significantly affected the expression of other key flowering-related genes. Also, overexpression of PbrCOL3 resulted in a late-flowering phenotype in Arabidopsis. These findings indicate diverse responsive mechanisms and functions of PbrCOL genes on flowering time in pear. In conclusion, this study established a foundation for a deeper understanding of the specific roles of PbrCOLs in regulating the reproductive development of pear, particularly in the context of the photoperiodic flowering process.

The genetic transformation of plants has provided fundamental insights into plant biology. However, the genetic transformation systems for most horticultural plants remain incomplete. Genome editing has significantly contributed to the improvement of crop traits, but it heavily relies on effective genetic transformation. Currently, reducing costs and improving the efficiency of genetic transformation are crucial for promoting the widespread application of genome editing in plants. Here, we review the advances in plant genetic transformation research, performing analysis of three methods for plant gene function analysis that bypass tissue culture: Agrobacterium rhizogenes-mediated root genetic transformation, developmental regulators (DRs)-mediated genetic transformation, and virus-mediated genome editing. We analyzed transformation efficiency in strawberry and citrus using the A. rhizogenes infiltration method, employing GFP to label different subcellular locations to investigate the morphology of microfilaments, nuclei, and peroxisomes in strawberry cells. Sequence analysis revealed that a series of developmental regulators critical for enhancing genetic transformation efficiency in specific species are highly conserved across different plant species. Additionally, we successfully edited the endogenous Pds gene in Cas9-overexpressing transgenic tobacco using TRV and CLBV containing the gRNA module. These three methods offer the benefits of being cost-effective and time-efficient, providing valuable technical insights for the application of plant genome editing.

Bayberry is the most economically significant fruit within the Myricaceae family, having high nutritional and medicinal value. The AP2/ERF family is a class of transcription factors found mainly in plants. However, the bayberry AP2/ERF gene family has not previously been studied. In this study, 113 members of the bayberry AP2/ERF gene family were identified. According to the phylogenetic tree, the members of this group are divided into three subfamilies, namely AP2, ERF, and DREB. The gene structure and conserved motifs were analyzed. Chromosome localization showed that 95 genes were unevenly distributed on 8 chromosomes and 18 genes were located on the skeleton. Gene collinearity analysis of the bayberry AP2/ERF gene family showed 12 segmental duplication events, involving 21 AP2/ERFs. In addition, we further investigated the evolutionary relationship of the AP2/ERF gene family between bayberry and six other species. It was found that bayberry was most closely related to Populus trichocarpa and Malus pumila, with 153 and 141 homologous gene pairs, respectively. Cis-acting elements indicated that AP2/ERFs were related to phytohormone responses, light response, abiotic and biotic stress tolerance. Transcriptomic data showed that the expression pattern of AP2/ERF gene was different in bayberry space electric field treatment and at different stages of fruit development. The results of GO annotation revealed the biological processes, cellular component and molecular function that the AP2/ERF genes were involved. And KEGG enrichment analysis indicated that these genes were mainly clustered in genetic information processing and metabolism pathways. The AP2/ERF gene was identified in the genome of bayberry, and its structure, conserved motif, and phylogenetic relationship were analyzed. These findings of this study serve as a reference for the genome-wide identification of the AP2/ERF gene family in other species and groundwork for future research on the function of AP2/ERF genes in bayberry.

Loquat (Eriobotrya japonica) is a subtropical evergreen fruit tree with high economic benefits and nutritional value. AP2/ERF (APETALA2/ethylene responsive factor) are a wide range of transcription factors found in plants, which play an important role in regulating plant development and biological processes. However, the systematic identification and characterization of AP2/ERF genes in loquat remains unclear. In the present research, a total of 189 loquat AP2/ERF members were identified. According to the phylogenetic tree, these members were divided into three subfamilies, including AP2 (42), ERF (96), and DREB (51). Except for three genes located on the skeleton, most of the loquat AP2/ERF genes were unevenly distributed across all chromosomes. The collinearity results showed that 175 duplicate gene pairs were found in the AP2/ERF gene family of loquat. Ka/Ks results indicated that these genes underwent purification selection pressure. The syntenic analysis of AP2/ERF genes between Arabidopsis, pomegranate, peach, and loquat provided valuable clues for the potential evolution of AP2/ERF family in loquat. Cis-element analysis showed that most of the AP2/ERF genes in loquat had multiple cis-elements related to stress response, plant hormone signal transduction, and plant growth and development. Transcriptomic data indicated that the expression patterns of AP2/ERF genes exhibited diversity in different loquat cultivars during fruit development and ripening. The results of this study displayed the first comprehensive analysis of AP2/ERF genes in loquat, which provides a solid foundation for future research on the function of AP2/ERF genes in loquat.