Comparative transcriptome analysis of multiple organs reveals cold stress responsiveness in two contrasting loquat cultivars

【Objective】Loquat (Eriobotrya japonica Lindl.) is an important evergreen fruit crop native to subtropical regions of China. It flowers in the autumn and winter, during which its flowers and young fruits are particularly susceptible to freezing stress. Most research on the mechanisms underlying freezing stress in loquat has concentrated on individual organs; however, the synergistic effects of various organs, such as flowers, leaves, and young fruits, on cold resistance remain largely unexplored. Notably, the cold sensitivity of loquat flowers and young fruits is considerably greater than that of leaves, suggesting that the mechanisms of cold resistance may involve the differential regulation of inter-organ substance transport, signal transduction, and metabolic pathways. To gain a deeper understanding of the molecular mechanisms associated with cold resistance in different organs, we conducted a transcriptome analysis, comparing the cold-tolerant cultivar Yonglu with the cold- sensitive cultivar Ruantiaobaisha. 【Methods】Branches of Yonglu and Ruantiaobaisha with leaves, flowers and young fruits were collected and treated in a low-temperature illumination incubator and maintained at -4 ℃ with 16/8 h (day/ night) photoperiod. Control branches were grown in a separate chamber at 25 ℃ under the same photoperiod. After 24 h, leaves, flowers and young fruits were collected from both treated and control groups. A portion of the samples was utilized to determine the relative electrical conductivity (REC), while the remaining samples were preserved for RNA sequencing. Additionally, a field investigation of frost damage was conducted on the two cultivars following the low-temperature exposure, specifically from February 15 to March 10, 2024, during which the freezing rate was calculated.【Results】Significant differences in cold resistance were observed between the two cultivars. The flowers and young fruits of Ruantiaobaisha exhibited severe symptoms of freezing stress, with freezing rates of 72.1% and 92.1%, respectively. In contrast, Yonglu displayed only slight freezing symptoms, with freezing rates of 31.4% and 45.2%. The values of REC corroborated these observations. After exposure to freezing stress, the REC of the flowers and fruits of the Ruantiaobaisha variety increased by 69.4% and 80.0%, respectively, which were 30.7% and 35.4% higher than those of Yonglu, indicating significant differences. Transcriptome analysis identified a total of 18 547 differentially expressed genes (DEGs) across 12 samples, among which 116 genes were shared by the leaves, flowers, and young fruits of Yonglu after cold stress. The 116 co-expressed genes were annotated to various signaling pathways, including abscisic acid-activated pathways, ethylene signaling pathways, salicylic acid response pathways, calcium ion- binding proteins, phosphorelay signal transduction systems, protein serine/threonine phosphatase activities, proline-rich receptor-like protein kinases, and disease resistance proteins. Notably, the abscisic acid-activated signaling, calcium- binding protein CML23, KRP1, and sorbitol dehydrogenase genes were significantly induced by cold stress in the leaves, flowers, and fruits of Yonglu, exhibiting markedly up-regulated expression levels. In contrast, in Ruantiaobaisha, these genes were only induced by cold stress in the leaves, with expression levels either down-regulated or showing no significant change in the flowers and young fruits. Furthermore, the expression levels of genes regulating stomatal movement decreased significantly in the flowers, leaves, and fruits of Yonglu, whereas the opposite trend was observed in Ruantiaobaisha. The top enriched Gene Ontology (GO) terms (P＜0.05) of common DEGs exhibited a similar ranking across the two cultivars; however, the shared GO terms varied among different organs. For instance, in the Yonglu variety, the shared GO terms in leaves, flowers, and fruits included‘response to chitin’‘, regulation of defense response’, and‘response to wounding’within the biological process category. In contrast, the Ruantiaobaisha variety showed that only the‘karrikin response’was concurrently enriched in all three tissues. KEGG pathway analysis revealed that 17 and 24 significantly enriched pathways (P＜0.05) were identified for all DEGs in Ruantiaobaisha and Yonglu, respectively. 11 common enriched pathways were shared between both cultivars, with‘plant hormone signal transduction’ exhibiting the highest number of enriched DEGs, while‘plant- pathogen interaction’and‘phenylpropanoid biosynthesis’also ranked highly. Further analysis of the DEGs indicated that 133 DEGs involved in plant hormone signal transduction were identified in response to freezing treatments across both cultivars. Among these, 38 DEGs were shared between the two loquat cultivars, while 52 and 48 DEGs were unique to Ruantiaobaisha and Yonglu, respectively. The DEGs were annotated to hormone signal transduction pathways, including auxin, ethylene, gibberellin, jasmonic acid, abscisic acid, twocomponent response regulator proteins, and brassinosteroid. Notable differences in expression patterns of common DEGs were observed between the two cultivars in response to cold treatments. The expression levels of abscisic acid signaling genes PP2C.2 and PP2C.3, as well as genes regulating stomatal movement, were significantly down-regulated in the leaves, flowers, and fruits of the cold-tolerant cultivar. Genes involved in jasmonic acid, gibberellin, ethylene, and auxin signaling pathways exhibited varying degrees of up- regulation or down- regulation. Transcription factors AP2/ERF, MYB, NAC, bHLH and bZIP showed cold- responsive trait in Yonglu. The accuracy of the RNA- seq derived transcript expression data was validated by analyzing the expression patterns of 12 DEGs using qRT-PCR. 【Conclusion】Compared to Ruantiaobaisha, Yonglu exhibited significantly greater tolerance to freezing stress, which was associated with modifications in general resistance/stress pathways across leaf, flower, and fruit. Future studies should focus on specific genes involved in Ca2 + signaling, hormone signal transduction and their downstream transcription factors to enhance our understanding of the processes underlying cold tolerance.