A structural variation in the promoter of the leucoanthocyanidin reductase gene AaLAR1 enhances freezing tolerance by modulating proanthocyanidin accumulation in kiwifruit (Actinidia arguta).

Proanthocyanidins (PAs) are important metabolites that enhance freezing tolerance of plants. Actinidia arguta, especially freezing-tolerant germplasms, accumulate abundant PAs in dormant shoots and thereby enhance freezing tolerance, but the underlying mechanism is unknown. In this study, we used two A. arguta with contrasting cold-resistant phenotypes, KL and RB, to explore the mechanisms in response to cold tolerance. We determined that a leucoanthocyanidin reductase gene (AaLAR1) was more highly expressed in freezing-tolerant KL than in freezing-sensitive RB. Moreover, overexpressing AaLAR1 in kiwifruit promoted PAs biosynthesis and enhanced cold tolerance. The AaLAR1 promoters of various A. arguta germplasms differ due to the presence of a 60-bp deletion in cold-tolerant genotypes that forms a functional binding site for MYC-type transcription factor. Yeast one-hybrid and two-hybrid, dual-luciferase reporter, bimolecular fluorescence complementation and coimmunoprecipitation assays indicated that the AaMYC2a binds to the MYC-core cis-element in the AaLAR1 promoter with the assistance of AaMYB5a, thereby promoting PAs accumulation in the shoots of cold-tolerant kiwifruit. We conclude that the variation in the AaLAR1 promoter and the AaMYC2a-AaMYB5a-AaLAR1 module shape freezing tolerance in A. arguta. The identification of a key structural variation in the AaLAR1 promoter offers a new target for resistance breeding of kiwifruit.

Screening and identification of photoresponse factors in kiwifruit (Actinidia arguta) development.

BACKGROUND: Light is essential for kiwifruit development, in which photoresponse factors contributes greatly to the quality formation. 'Light sensitive hypocotyls, also known as light-dependent short hypocotyls' (LSH) gene family can participate in fruit development as photoresponse factor. However, the key LSH gene that determine kiwifruit development remains unclear. This study aim to screen and identify the key gene AaLSH9 in A. arguta. MATERIALS AND METHODS: Genome-wide identification of the LSH gene family was used to analyse LSH genes in kiwifruit. Homologous cloning was used to confirm the sequence of candidate LSH genes. qRT-PCR and cluster analysis of expression pattern were used to screen the key AaLSH9 gene. Subcellular localization of AaLSH9 in tobacco leaves and overexpression of AaLSH9 in Arabidopsis thaliana hy5 mutant plants were used to define the acting place in cell and identify molecular function, respectively. RESULTS: We identified 15 LSH genes, which were divided into two sub-families namely A and B. Domain analysis of A and B showed that they contained different domain organizations, which possibly played key roles in the evolution process. Three LSH genes, AaLSH2, AaLSH9, and AaLSH11, were successfully isolated from Actinidia arguta. The expression pattern and cluster analysis of these three AaLSH genes suggested AaLSH9 might be a key photoresponse gene participating in fruit development in A. arguta. Subcellular localization showed AaLSH9 protein was located in the nucleus. The overexpression of AaLSH9 gene in Arabidopsis thaliana hy5 mutant plants partially complemented the long hypocotyls of hy5 mutant, implying AaLSH9 played a key role as photoresponse factor in cells. In addition, the seed coat color of A. thaliana over-expressing AaLSH9 became lighter than the wide type A.thaliana. Finally, AaCOP1 was confirmed as photoresponse factor to participate in developmental process by stable transgenic A. thaliana. CONCLUSIONS: AaLSH9 can be involved in kiwifruit (A. arguta) development as key photoresponse factor. Our results not only identified the photoresponse factors AaLSH9 and AaCOP1 but also provided insights into their key role in fruit quality improvement in the process of light response.

Insight into melting point differences of dinitroimidazoles and dinitropyrazoles from the perspective of intermolecular interactions.

Dinitroimidazole (DNI) and dinitropyrazole (DNP), along with their congeners, possess similar molecular structures but exhibit distinct melting points. To analyse and elucidate the fundamental reasons for property differences from the perspective of intermolecular interactions, we proposed a simplified approach named binding energy in clusters (BEC) in computing weak interactions within complex crystal systems. Based on the results of the symmetry-adapted perturbation theory (SAPT) calculations, an approximate estimation of the melting point range can be derived by taking into account the cumulative effect (energy of electrostatic, dispersion and induction terms) and repulsive effect (energy of exchange term) values. We have also proposed a formula for calculating the specific melting point, which indicates that stronger intermolecular interactions have a major impact on the melting point, while the distribution of weak interactions also affects the melting point. This work would provide an effective reference for molecular design and structure-performance analysis.

Transcriptome-Wide Identification and Functional Characterization of CIPK Gene Family Members in Actinidia valvata under Salt Stress.

Fruit plants are severely constrained by salt stress in the soil due to their sessile nature. Ca2+ sensors, which are known as CBL-interacting protein kinases (CIPKs), transmit abiotic stress signals to plants. Therefore, it is imperative to investigate the molecular regulatory role of CIPKs underlying salt stress tolerance in kiwifruit. In the current study, we have identified 42 CIPK genes from Actinidia. valvata (A.valvata). All the AvCIPKs were divided into four different phylogenetic groups. Moreover, these genes showed different conserved motifs. The expression pattern analysis showed that AvCIPK11 was specifically highly expressed under salt stress. The overexpression of AvCIPK11 in 'Hongyang' (a salt sensitive commercial cultivar from Actinidia chinensis) enhanced salt tolerance by maintaining K+/Na+ homeostasis in the leaf and positively improving the activity of POD. In addition, the salt-related genes AcCBL1 and AcNHX1 had higher expression in overexpression lines. Collectively, our study suggested that AvCIPK11 is involved in the positive regulation of salt tolerance in kiwifruit.

A High-K+ Affinity Transporter (HKT) from Actinidia valvata Is Involved in Salt Tolerance in Kiwifruit.

Ion transport is crucial for salt tolerance in plants. Under salt stress, the high-affinity K+ transporter (HKT) family is mainly responsible for the long-distance transport of salt ions which help to reduce the deleterious effects of high concentrations of ions accumulated within plants. Kiwifruit is well known for its susceptibility to salt stress. Therefore, a current study was designed to decipher the molecular regulatory role of kiwifruit HKT members in the face of salt stress. The transcriptome data from Actinidia valvata revealed that salt stress significantly induced the expression of AvHKT1. A multiple sequence alignment analysis indicated that the AvHKT1 protein contains three conserved amino acid sites for the HKT family. According to subcellular localization analysis, the protein was primarily present in the cell membrane and nucleus. Additionally, we tested the AvHKT1 overexpression in 'Hongyang' kiwifruit, and the results showed that the transgenic lines exhibited less leaf damage and improved plant growth compared to the control plants. The transgenic lines displayed significantly higher SPAD and Fv/Fm values than the control plants. The MDA contents of transgenic lines were also lower than that of the control plants. Furthermore, the transgenic lines accumulated lower Na+ and K+ contents, proving this protein involvement in the transport of Na+ and K+ and classification as a type II HKT transporter. Further research showed that the peroxidase (POD) activity in the transgenic lines was significantly higher, indicating that the salt-induced overexpression of AvHKT1 also scavenged POD. The promoter of AvHKT1 contained phytohormone and abiotic stress-responsive cis-elements. In a nutshell, AvHKT1 improved kiwifruit tolerance to salinity by facilitating ion transport under salt stress conditions.

Transcriptional Analysis on Resistant and Susceptible Kiwifruit Genotypes Activating Different Plant-Immunity Processes against Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa), a bacterial pathogen, is a severe threat to kiwifruit production. To elucidate the species-specific interaction between Psa and kiwifruit, transcriptomic-profiles analyses were conducted, under Psa-infected treatment and mock-inoculated control, on shoots of resistant Maohua (MH) and susceptible Hongyang (HY) kiwifruit varieties. The plant hormone-signal transduction and plant-pathogen interaction were significantly enriched in HY compared with MH. However, the starch and sucrose metabolism, antigen processing and presentation, phagosome, and galactose metabolism were significantly enriched in MH compared with HY. Interestingly, the MAP2 in the pathogen/microbe-associated molecular patterns (PAMPs)-triggered immunity (PTI) was significantly up-regulated in MH. The genes RAR1, SUGT1, and HSP90A in the effector-triggered immunity (ETI), and the NPR1 and TGA genes involved in the salicylic acid signaling pathway as regulatory roles of ETI, were significantly up-regulated in HY. Other important genes, such as the CCRs involved in phenylpropanoid biosynthesis, were highly expressed in MH, but some genes in the Ca2+ internal flow or involved in the reactive oxygen metabolism were obviously expressed in HY. These results suggested that the PTI and cell walls involved in defense mechanisms were significant in MH against Psa infection, while the ETI was notable in HY against Psa infection. This study will help to understand kiwifruit bacterial canker disease and provide important theoretical support in kiwifruit breeding.

Full-Length Transcriptome and RNA-Seq Analyses Reveal the Mechanisms Underlying Waterlogging Tolerance in Kiwifruit (Actinidia valvata).

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.

Characterization and Identification of a Ripening-Related Gene AaPG18 in Actinidia arguta.

Actinidia arguta (A. arguta) is a kind of climacteric fruit that quickly softens and limits fruit shelf-life and commercial value. Therefore, it is of great significance to develop kiwifruit genotypes with an extended shelf-life of fruit. However, the ripening and softening mechanisms remain unclear in A. arguta. Here, we demonstrated that a key polygalacturonase (PG)-encoding gene AaPG18 was involved in A. arguta ripening through the degradation of the cell wall. Fruits were harvested at three developmental stages (S1, S2, and S3) for high-throughput transcriptome sequencing, based on which two candidate transcripts c109562_g1 and c111961_g1 were screened. The genome-wide identification of the PG gene family assigned c109562_g1 and c111961_g1 to correspond to AaPG4 and AaPG18, respectively. The expression profiles of candidate genes at six preharvest stages of fruit showed significantly higher expression levels of AaPG18 than AaPG4, indicating AaPG18 might be a key gene during fruit ripening processes. The subcellular localization displayed AaPG18 was located at the cytoplasmic membrane. The transient overexpression of AaPG18 in strawberry and the following morphological observation suggested AaPG18 played a key role in maintaining the stability of cell morphology. The homologous transient transformation in A. arguta "RB-4" proved the crucial function of AaPG18 in fruit ripening processes by causing the rapid redness of the fruit, which was an indicator of fruit maturity. All in all, our results identified AaPG18 as a key candidate gene involved in cell wall degeneration, which provides a basis for the subsequent exploration of the molecular mechanisms underlying the ripening and softening of A. arguta fruit.

Overexpression of AcEXPA23 Promotes Lateral Root Development in Kiwifruit.

Kiwifruit is loved by consumers for its unique taste and rich vitamin C content. Kiwifruit are very sensitive to adverse soil environments owing to fleshy and shallow roots, which limits the uptake of water and nutrients into the root system, resulting in low yield and poor fruit quality. Lateral roots are the key organs for plants to absorb water and nutrients. Improving water and fertilizer use efficiency by promoting lateral root development is a feasible method to improve yield and quality. Expansin proteins plays a major role in lateral root growth; hence, it is important to identify expansin protein family members, screen key genes, and explore gene function in root development. In this study, 41 expansin genes were identified based on the genome of kiwifruit ('Hongyang', Actinidia chinensis). By clustering with the Arabidopsis thaliana expansin protein family, the 41 AcExpansin proteins were divided into four subfamilies. The AcExpansin protein family was further analysed by bioinformatics methods and was shown to be evolutionarily diverse and conserved at the DNA and protein levels. Based on previous transcriptome data and quantitative real-time PCR assays, we screened the candidate gene AcEXPA23. Overexpression of AcEXPA23 in kiwifruit increased the number of kiwifruit lateral roots.

BSR-Seq analysis provides insights into the cold stress response of Actinidia arguta F1 populations.

BACKGROUND: Freezing injury, which is an important abiotic stress in horticultural crops, influences the growth and development and the production area of kiwifruit (Actinidia Lind1). Among Actinidia species, Actinidia arguta has excellent cold resistance, but knowledge relevant to molecular mechanisms is still limited. Understanding the mechanism underlying cold resistance in kiwifruit is important for breeding cold resistance. RESULTS: In our study, a population resulting from the cross of A. arguta 'Ruby-3' × 'Kuilv' male was generated for kiwifruit hardiness study, and 20 cold-tolerant and 20 cold-sensitive populations were selected from 492 populations according to their LT50. Then, we performed bulked segregant RNA-seq combined with single-molecule real-time sequencing to identify differentially expressed genes that provide cold hardiness. We found that the content of soluble sucrose and the activity of ß-amylase were higher in the cold-tolerant population than in the cold-sensitive population. Upon - 30 °C low-temperature treatment, 126 differentially expressed genes were identify; the expression of 59 genes was up-regulated and that of 67 genes was down-regulated between the tolerant and sensitive pools, respectively. KEGG pathway analysis showed that the DEGs were primarily related to starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism. Ten major key enzyme-encoding genes and two regulatory genes were up-regulated in the tolerant pool, and regulatory genes of the CBF pathway were found to be differentially expressed. In particular, a 14-3-3 gene was down-regulated and an EBF gene was up-regulated. To validate the BSR-Seq results, 24 DEGs were assessed via qRT-PCR, and the results were consistent with those obtained by BSR-Seq. CONCLUSION: Our research provides valuable insights into the mechanism related to cold resistance in Actinidia and identified potential genes that are important for cold resistance in kiwifruit.