- Understanding the mechanism underlying cold resistance in kiwifruit is important for breeding cold resistance.. - We found that the content of soluble sucrose and the activity of β -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. - 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. - 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.. - Keywords: Actinidia arguta, Cold resistance, BSR-Seq, Single-molecule real-time sequencing, Cold resistance genes. - Low temperature drastically influences plant development, productivity and geographic distribution. - low-temperature stress [1]. - Therefore, it is important to enhance cold resistance to minimize the economic loss from low temperature injury. - Kiwifruit has been domesti- cated only in the past 100 years, and it has abundant wild resources, which contain excellent cold resistance traits, such as Actinidia arguta, which was found to withstand − 38 °C in our previous study [2]. - However, the lack of a comprehensive low temperature transcriptome, unex- plored cold resistance genes and low temperature. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article Lin et al. - signaling hinder our full understanding of cold resistance in kiwifruit. - Therefore, identifying cold resistance genes in A. - arguta is a method for cold resistance breeding and im- proving kiwifruit cold resistance.. - Cold acclimation is one of the most important mechanisms against low temperature stress in winter. - The response of plants to low temperature is a highly complex process involving multiple levels of regulation [3]. - The role of the starch metabolism path- way in plants under low temperature stress has been widely studied, and sugars accumulate rapidly in plants under low temperature [9]. - Currently, single-molecular sequencing technologies provide an op- portunity to thoroughly investigate the molecular mech- anisms of the kiwifruit response to low temperature.. - Therefore, studies of the genetic mechanism underlying the freezing tolerance trait of this species are still needed. - The development of these candidate genes will be the focus of future research, and these results will facilitate the study of the molecular mechanism of freezing tolerance in kiwifruit.. - Low temperature treatment and evaluation of cold resistance. - 1), and the REL ranged from 42 to 97% (Table S1). - The cold-resistant trait in the populations showed the phenomenon of the superparent. - The average LT50 of the 20 higher and lower cold-resistant populations were − 30.52 °C and − 13.97 °C. - β -Amylase activity and total soluble sugar content β-amylase activity and total soluble sugar content were measured in F1 populations in the tolerant and sensitive pools. - The β-amylase activity was higher in the tolerant pool than in the sensitive pool, and the average β- amylase activity in the sensitive resistant and tolerant populations was 12.2 U/mg and 19.15 U/mg, respectively.. - were generated in the tolerant pool and raw reads and clean reads (47.38 G clean bases) with a Q 30 value of 91.38% were generated in the sensi- tive pool by the Illumina HiSeq 2000 platform.. - Table 1 LT50 of populations in the tolerant pool and sensitive pool. - Lin et al. - Annotation against the NR database showed that unigenes in the PacBio transcriptome were identical to Vitis vinifera (25.6. - while the uni- genes in the Illumina transcriptome were identical to Vitis vinifera (36.6. - After low temperature treatment,. - 2 The activity of beta-amylase and the content of soluble sugar in shoots of populations. - Table 2 Summary of the transcriptome data from the PacBio Sequel platform. - 3 Analysis of the length distribution of CDS and consensus reads, lncRNA number, and distribution of SSR motifs. - B: Clustering analysis of the DEGs. - BLAST analysis and GO term annotation were per- formed to improve our understanding of the functions of these specifically regulated genes. - Seven unigenes related to starch and sucrose metabolism were upregulated by low temperature treatment, namely, AGPase, granule-bound starch synthase, sucrose synthase (SUS), 1,4-alpha-glucan-branch enzyme (GBE), alpha-1,4 glucan phosphorylase, beta-amylase (BAM), glucan water dikinase (GWD), and neutral-alpha-glucosidase and dis- proportionating enzyme 2 (Table S4).. - To verify the reliability of the cold responsive gene ex- pression profiles for DEGs, 27 DEGs that contained 18 up-regulated (ADP-Glc, GWD, BAM, EBF, Proline rich protein, SUS, Ca 2+ transporting ATPase, DPE2, BSL3,. - The tolerant pool, sensitive pool and the three randomly selected populations were used as templates. - 6, the fold change values obtained by qRT-PCR were highly consistent with those based on BSR-Seq data for all of the selected cold responsive genes, despite the difference in the absolute fold change between the two methods. - Therefore, some alleles originating from the tolerant pool were preferentially induced to be expressed under low temperature.. - arguta is a deciduous fruit tree, and it is a specie that has a higher cold resistance than other Actinidia species.. - A study on the cold resistance of A. - arguta was signifi- cant for understanding the mechanism of cold resistance in Actinidia. - 5 Enrichment of differentially expressed genes in the KEGG pathway. - combined transcriptome analysis of NGS and SMRT se- quencing and investigated the mechanism in response to low temperature.. - and the Arabidopsis HyPRP gene protects the cells during. - In this study, the higher expression of PRP genes, leading to proline accumulation, may be because the increase in proline added mechanical strength to the cell wall and stabilized the structure of organs under low temperature stress [34, 35].. - it is a member of the CBF regulon and plays an important role in cold tolerance by modulating the levels of soluble sugars acting as osmolytes or antioxidants [40]. - In our results, the activity of β-amylase was higher in the toler- ant pool, and the bam gene expression in populations was largely consistent with the activation of the enzyme, suggesting that both the expression and activity of β- amylase are up-regulated by low temperature, which is in agreement with an earlier report [41].. - The sugar content was higher in the tolerance pool than in the sensitive pool, implying that tolerant populations produced more soluble sugars to resist cold and sugar reserves between nonsoluble and soluble sugar forms when faced with cold temperatures.. - In our study, DnaJ and HSP70 were up-regulated in the tolerant pool. - In this study, EBF1/2 was upregulated in the tolerance pool, implying that A. - arguta could increase cold resistance by increasing the expression of EBF to regulate the CBF gene. - All of the genes were up-regulated. - in the sensitive pool. - In our study, in the sensitive pool, the higher expression of 14–3-3 and the phenotype of sensitive cold resistance led us to speculate that 14–3-3 negatively regulates freezing tolerance in A. - In addition, we found that the transcription factor CHY was up-regulated in the sensitive pool, CHY could respond to low temperature in pineapples, and A. - First, most of the genes coding for the regulatory proteins, such as the CBFs, exhibited a transient and temporary change under abiotic stresses. - At our low temperature (with 8 h), the mRNA abundance of the regulatory genes may be indistinguishable. - Second, woody plants differ from herbal plants (such as Arabi- dopsis) when faced with low temperature stress due to. - their natural variation in the length of the growth cycle [49]. - Third, there were nonsignificant differences in the tolerance and sensitive pools under a long-term, low- temperature treatment. - 30 °C low-temperature treatment were identified. - Low- temperature treatment increased the content of total sol- uble sugar and the activity of β-amylase in the cold- tolerant pool. - Most of the DEGs were enriched in starch and sugar metabolism and their regulatory pathway. - We concluded that starch degradation and soluble sugar synthesis are important for cold resistance in kiwifruit.. - and the cross was finished at Zhengzhou Fruit Research Institute. - Low temperature treatment. - Fifty populations with the highest REL and 50 pop- ulations with the lowest REL were selected to evaluate the detailed cold resistance and treated at − 15 °C. - REL (sensitive pool) were chosen, and the shoots were treated with − 30 °C for 8 h and then used for measure- ment of β-amylase activity, total Soluble Sugar content and BSR-Seq.. - After low-temperature treatment, the shoots without buds were cut into 1–2-mm-thick slices. - Then, 0.2 g of the slices was incubated in 30 ml of double-distilled water for 2 h with shaking at 200 rpm at room temperature. - The data were analyzed by Dun- can’s multiple range tests in the ANOVA program of SPSS (IBM SPSS 22), using P <. - Forty populations and ‘Kuilv’ male RNA in the dor- mancy shoots were extracted using an RNA Isolation Kit (HUAYUEYANG, China). - The concentration and quality of the extracted RNAs were assessed using the Nano- Drop spectrophotometer. - Tolerance and sensitive pools were generated after − 30 °C low temperature treatment for BSR-Seq analysis.. - The Illumina library was prepared according to the protocol described in the next-generation transcriptome article [52].. - Seven databases (NR, Swiss-Prot, GO, NT, KOG, Pfam, KEGG) were selected to map the nonredundant tran- script sequences and obtain the annotation information of the transcript with e-values of 1e − 5 against a total of seven databases [53–58].. - The expression levels of the unigenes were expressed as frag- ments per kilobase of transcript per million mapped read (FPKM) values to eliminate the influences of gene length and sequencing quality difference on the estimated gene expression [61].. - Samples were ground as described in the materials. - β - actin in the kiwifruit was considered the control gene for normalization. - The raw reads of the BSR-Seq data in this study have been deposited in the NCBI SRA database under accession number SRR12668267, SRR12668268.. - Advances in research on cold resistance in kiwifruit. - Proteomic and physiological assessment of stress sensitive and tolerant variety of tomato treated with brassinosteroids and hydrogen peroxide under low-temperature stress. - pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. - Draft genome of the kiwifruit Actinidia chinensis. - SMRT sequencing of the full-length transcriptome of the Sunda pangolin (Manis javanica). - The Arabidopsis 14-3-3 protein RARE COLD INDUCIBLE 1A links low-temperature response and ethylene biosynthesis to regulate freezing tolerance and cold acclimation. - Divergence in the transcriptional landscape between low temperature and freeze shock in cultivated grapevine (Vitis vinifera). - intercultivar differences in the gene expression and activity of key enzymes. - Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S: Ethylene signaling negatively regulates freezing tolerance by repressing expression of the CBF and type-AARR genes in Arabidopsis. - PIF3 is a negative regulator of the CBF pathway and freezing tolerance in Arabidopsis. - Identification and characterization of low temperature stress responsive genes in Poncirus trifoliata by suppression subtractive hybridization. - Pathway analysis of the transcriptome and metabolome of salt sensitive. - KEGG for linking genomes to life and the environment
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