Actinidia valvata possesses waterlogging tolerance; however, the mechanisms underlying this trait are poorly characterized. Here, we performed a transcriptome analysis by combining single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing and investigated the physiological responses of the roots of KR5 (A. valvata, a tolerant genotype) after 0, 12, 24 and 72 h of waterlogging stress. KR5 roots responded to waterlogging stress mainly via carbohydrate and free amino acids metabolism and reactive oxygen species (ROS) scavenging pathways. Trehalose-6-phosphate synthase (TPS) activity, alcohol dehydrogenase (ADH) activity and the total free amino acid content increased significantly under waterlogging stress. The nicotinamide adenine dinucleotide-dependent glutamate synthase/alanine aminotransferase (NADH-GOGAT/AlaAT) cycle was correlated with alanine accumulation. Levels of genes encoding peroxidase (POD) and catalase (CAT) decreased and enzyme activity increased under waterlogging stress. Members of the LATERAL ORGAN BOUNDARIES (LOB), AP2/ERF-ERF, Trihelix and C3H transcription factor families were identified as potential regulators of the transcriptional response. Several hub genes were identified as key factors in the response to waterlogging stress by a weighted gene co-expression network analysis (WGCNA). Our results provide insights into the factors contributing to waterlogging tolerance in kiwifruit, providing a basis for further studies of interspecific differences in an important plant trait and for molecular breeding.

Rootstocks from Actinidia valvata are much more tolerant to waterlogging stress than those from Actinidia deliciosa, which are commonly used in kiwifruit production. To date, the tolerance mechanism of A. valvata rootstocks’ adaptation to waterlogging stress has not been well explored. In this study, the responses of KR5 (A. valvata) and ‘Hayward’ (A. deliciosa) to waterlogging stress were compared. Results showed that KR5 plants performed much better than ‘Hayward’ during waterlogging by exhibiting higher net photosynthetic rates in leaves, more rapid formation of adventitious roots at the base of stems, and less severe damage to the main root system. In addition to morphological adaptations, metabolic responses of roots including sufficient sucrose reserves, modulated adjustment of fermentative enzymes, avoidance of excess lactic acid and ethanol accumulation, and promoted accumulation of total amino acids all possibly rendered KR5 plants more tolerant to waterlogging stress compared to ‘Hayward’ plants. Lysine contents of roots under waterlogging stress were increased in ‘Hayward’ and decreased in KR5 compared with their corresponding controls. Overall, our results revealed the morphological and metabolic adaptations of two kiwifruit rootstocks to waterlogging stress, which may be responsible for their genotypic difference in waterlogging tolerance.

Kiwifruit is commonly sensitive to waterlogging stress, and grafting onto a waterlogging-tolerant rootstock is an efficient strategy for enhancing the waterlogging tolerance of kiwifruit plants. KR5 (Actinidia valvata) is more tolerant to waterlogging than ‘Hayward’ (A. deliciosa) and is a potential resistant rootstock for kiwifruit production. Here, we focused on evaluating the performance of the waterlogging-sensitive kiwifruit scion cultivar ‘Zhongmi 2′ when grafted onto KR5 (referred to as ZM2/KR5) and Hayward (referred to as ZM2/HWD) rootstocks, respectively, under waterlogging stress. The results showed ‘Zhongmi 2′ performed much better when grafted onto KR5 than when grafted onto ‘Hayward’, exhibiting higher photosynthetic efficiency and reduced reactive oxygen species (ROS) damage. Furthermore, the roots of ZM2/KR5 plants showed greater root activity and energy supply, lower ROS damage, and more stable osmotic adjustment ability than the roots of ZM2/HWD plants under waterlogging stress. In addition, we detected the expression of six key genes involved in the kiwifruit waterlogging response mechanism, and these genes were remarkably induced in the ZM2/KR5 roots but not in the ZM2/HWD roots under waterlogging stress. Moreover, principal component analysis (PCA) further demonstrated the differences in the physiological responses of the ZM2/KR5 and ZM2/HWD plants under waterlogging stress. These results demonstrated that the KR5 rootstock can improve the waterlogging tolerance of grafted kiwi plants by regulating physiological and biochemical metabolism and molecular responses.

Kiwifruit vines are generally sensitive to waterlogging stress. So far, molecular responses of different kiwifruit genotypes for waterlogging stress are less well-explored. In this study, using RNA-sequencing, we examined transcriptional regulation in the roots of a waterlogging-tolerant genotype KR5 (Actinidia valvata), and a sensitive genotype ‘Hayward’ (Actinidia deliciosa) subjected to 0, 12, 24, and 72 h of waterlogging. Compared with 0 h, transcriptional adjustments of these two genotypes occurred as early as 12 h and became notably pronounced 72 h after waterlogging. Waterlogging stress for 72 h promoted the expression of genes involved in ethylene biosynthesis, sucrose and hexose transport, anaerobic fermentation, nitrate reduction, alanine accumulation, and reactive oxygen scavenging in both genotypes. The differential regulation of genes encoding 9-cis-epoxycarotenoid dioxygenase, phosphoglucomutase, alanine-glyoxylate transaminase, and other enzymes pointed to their diverse strategies upon waterlogging in these two genotypes. In addition, more sucrose and trehalose contents, as well as a higher activity of alcohol dehydrogenase and manganese superoxide dismutases were stimulated in KR5 roots after 72h of waterlogging than that in ‘Hayward’. Overall, our results provided more insights into the molecular basis of the waterlogging response in kiwifruit.

Actinidia valvata, a promising rootstock for kiwifruit cultivation, demonstrates superior waterlogging tolerance compared with commercial cultivars. Lateral organ boundaries domain (LBD) transcription factors (TFs) are known to be pivotal in plant responses to abiotic stress. Nevertheless, the characterization of the LBD family under waterlogging stress in A. valvata remains limited. In this study, 26 AvLBD genes were identified from a transcriptome dataset, with the majority classified into phylogenetic Class II. Under waterlogging stress, transcript accumulation of most AvLBD41 members, particularly AvLBD41_11 and AvLBD41_7, was markedly increased in roots. Bimolecular fluorescence complementation (BiFC) assays indicated that AvLBD41_7 heterodimerizes with both the AP2/ERF activator AvERF75 and the trihelix repressor AvHRA1, whereas AvLBD41_11 only interacts with AvERF75. Neither AvLBD41 isoform interacts with AvERF73, thereby defining distinct components of a waterlogging-responsive module. Yeast-based assays revealed an absence of transactivation activity for AvLBD41_7, and transient expression analyses confirmed its exclusive nuclear localization. The promoters of both AvLBD41_11 and AvLBD41_7 harbor numerous cis-elements responsive to hormones and abiotic stresses. An AvLBD41_7-derived PCR marker could be used to distinguish A. valvata from A. deliciosa accessions. Collectively, these findings provide a comprehensive functional annotation of the LBD gene family in A. valvata and establish AvLBD41_7 as a potential molecular target for future kiwifruit breeding programs aimed at waterlogging resilience.