Screening of cold-tolerant mutants
In the study, tetraploid aseptic plantlets and diploid aseptic plantlets (as the control) were treated with L-Hyp. After 7 days of 10 mmol/L L-Hyp treatments, the browning phenomenon was found to appear at the edge of the leaves in the tetraploid and diploid plantlets. After 14 days, some of the leaves in diploid treated with 10 mmol/L L-Hyp browned, stopped growing or began to fall off. On the other hand, only a few leaves of tetraploid plantlets fell off, and some of the leaf tip edges appeared brown, and the whole plants were still green.
As shown in Table 2 and Table 3, the survival rates of tetraploid plantlets treated with L-hyp were higher than those of diploid plantlets. After 30 days of treatment, the symptoms of freezing injury in tetraploid plantlets were lighter than those of diploidy.
After 30 days of treatment, the survival rates of tetraploid and diploid plntlets were 100% in MS medium with 0 mmol/L L-Hyp. In MS medium with 2 mmol/L L-Hyp, the survival rate of tetraploid plantlets was 100%, and a small number of diploid plantlets died, with a survival rate of 86.87%. In MS medium with 4 mmol/L L-Hyp, the tetraploid plantlets began to die, with a survival rate of 96.67%, while the survival rate of diploid plantlets was only 33.33%. In MS medium with 6 mmol/L L-Hyp, the survival rate of tetraploid plants was 83.33%, but only a small number of diploid plants survived (the survival rate was only 20%). In MS medium with 10 mmol/L L-Hyp, all diploid plants died,but the survival rate of tetraploid plants was still 53.33%. The survival rate of tetraploid plantlets was significantly higher than that of diploid plantlets (Table 1).
According to the experimental results, it was concluded that the semi-lethal concentration of L-Hyp in tetraploid plants was 8 mmol/L. With the concentration increase of L-Hyp, the survival rate of diploidy decreased significantly, while the survival rate of tetraploidy decreased gradually. And the survival rates of tetraploid plants at high concentrations were significantly higher those of diploid plants. The results showed that the tolerance of tetraploid plants to L-Hyp was stronger than that of diploidy.
After 30 days of treatment, they were transferred to a normal medium, and the changes of fresh weight before and after treatment were recorded (Table 2). Under the concentration of L-Hyp of 0 mmol/L, the increased rate of tetraploidy the fresh weight was 110.79%, and the increase rate of diploidy fresh weight was 114.13%. Under the condition of no L-Hyp stress, diploidy grew faster and increased fresh weight more than tetraploidy. In MS medium with 2 mmol/L L-Hyp, the increased rate of fresh weight of tetraploid and diploid was 104.14% and 93.63%, respectively. The increased rate of fresh weight of tetraploid was higher than that of diploidy, and some diploidy died. Under the stress of 4 mmol/L L-Hyp, the increase rate of fresh weight of tetraploidy was 101.94%, while that of diploid was 66.24%. Under the stress of 8 mmol/L L-Hyp, the increase rate of diploid fresh weight was only 10.28%, and that of tetraploidy was 96.83%. Under the stress of 10 mmol/L L-Hyp, all the diploidy died, no fresh weight increased, and the rate of increase of tetraploidy fresh weight was 75.67%.
The seedling heights of tetraploidy and diploidy were measured before and 30 days after treatment (Table 3). The results showed that the height increase rate of diploidy (152.94%) was higher than that of tetraploidy (140.12%) without L-Hyp treatment. Under the stress with 4 mmol/L L-Hyp, the height of diploid plants increased only slightly, and that of tetraploid plants increased, while under the stress of 6 mmol/L L-Hyp, the diploid plants basically stopped growing. Under the stress of 8 mmol/L L-Hyp, the height of diploid plantlets did not increase and the height of tetraploid plants increased slightly. Under the stress with 10 mmol/L L-Hyp, all the diploid plants died, so there was no seedling height data, and the growth of tetraploid plants basically stopped (Table 4). The results showed that the growth rate of tetraploid plants treated with L-Hyp at different concentrations was higher than that of diploid plants.
Effect of low-temperature treatment on yellow kiwifruit
As shown in Figure 1, the freezing injury symptoms of tetraploid plantlets were less than that of diploid plantlets. The survival rates of tetraploid and diploid plantlets at 0 °C for 3 h were 88.89% and 17.78% respectively (Table 5). At 0 °C for 12 h, the survival rate of tetraploid plantlets was 42.22%, and the survival rate of diploidy was only 2.22%. After low-temperature stress, the tetraploid and diploid plantlets were transferred to normal MS medium and placed at room temperature for observation. Seven days later, the control plantlets grew slowly, the leaves wilted, and dead tip and edge of the leaves appeared. However, most of the tetraploid plantlets grew normally, the degree of chilling injury was obviously alleviated, and even no chilling injury occurred. Therefore, the cold tolerance of tetraploid plantlets was higher than that of diploid plantlets.
Results of transcriptome analysis
Results of sequence data filtering
The original data of 6 tetraploid samples were shown in Table 6. To improve the accuracy of the analysis results of the transcriptome data, the sequence data were filtered before the data analysis, and the filtered data were shown in Table 6. The sequence data of sample transcriptome was of high quality.
Detection of DEGs
There were 1630 DEGs between control group and treatment group. By analyzing the DEGs between control group and treatment group, it was found that 619 DEGs were up-regulated and 1011 DEGs were down-regulated in the treatment group compared with the control group (Table 7).
Functional classification and enrichment analysis of DEGs
According to the results of differential gene detection, the DEGs were classified and enriched in the GO database. GO enrichment analysis could be divided into three main functions including molecular function, cellular component, and biological process. A total of 1025 DEGs were classified into 44 functional categories. There were 10 functional classifications of molecular function, 13 functional classifications of cellular component and 21 functional classifications of biological process (Figure 2).
Functional classification and enrichment analysis of KEGG pathway
A total of 410 differentially expressed genes were involved in 89 metabolic pathways in control group and treatment group.The top five pathways enriching DEGs were plant hormone signal transduction (including 25 DEGs), phenylpropanoid biosynthesis24 DEGs), plant-pathogen interaction (20 DEGs), carbon metabolism (14 DEGs), and cysteine and methionine metabolism (13 DEGs). Among them, plant hormone signal transduction was the pathway with the largest number of DEGs annotated to this pathway, which belonged to environmental information processing in five branches.
The number of up-regulated DEGs enriched by phenylpropanoid biosynthesis was 16 in the first three pathways of the KEGG metabolic pathway, and 14 DEGs were enriched in plant-pathogen interaction. A total of 11 DEGs were enriched by carbon metabolism. The top three pathways of down-regulation were plant hormone signal transduction, enrichment of 19 DEGs, phenylpropanoid biosynthesis, enrichment of eight DEGs, and six DEGs were enriched in carotenoid biosynthesis.
Screening and analysis of cold tolerance related genes
In this study, the largest number of DEGs was annotated to plant hormone signal transduction pathway. Many cold-tolerance related studies have found that most of the DEGs were enriched in this pathway. Signal transduction pathway plays a key role in the response of plants to low temperature (Janská et al., 2010). Therefore, the DEGs enriched in this pathway might be related to cold tolerance, so the DEGs in this pathway were analyzed in detail. The pathway map (KO04075) was shown in Figure 3.
There were 25 DEGs in the plant hormone signal transduction pathway, of which six were up-regulated. They were CEY00_Acc07445 (disease-related leaf protein), CEY00_Acc03316 (abscisic acid receptor PYL), CEY00_Acc10294 (indole-3-acetic acid-amide synthetase), CEY00_Acc13130 (bZIP transcription factor family protein), CEY00_Acc33627 (TIFY protein) and CEY00_Acc21162 (pathogenesis related protein); 19 DEGs were down-regulated, including CEY00_Acc07128 (auxin-induced protein), CEY00_Acc19743 (CICLE hypothetical protein), CEY00_Acc23775 (containing Cyclin_N domain protein) and CEY00_Acc07415 (indole-3-acetic acid-amide synthase). The genes involved in this pathway were shown in Table 8.
Abscisic acid affects plant cold tolerance and gene expression regulation and can improve plant cold tolerance. Abscisic acid signal transduction was due to the interaction between abscisic acid receptors in the process (Hubbard et al., 2010). Abscisic acid receptor PYL was one of the receptors, and CEY00_Acc03316 was annotated as abscisic acid receptor PYL, so it could be inferred that CEY00_Acc03316 may be a cold tolerance gene. The overexpression of bZIP transcription factor family proteins under stress was reported to improve the cold tolerance of plants (Chang et al., 2017; Ma et al., 2018). Therefore, CEY00_Acc13130 gene may be a cold tolerance gene. TIFY protein was induced and expressed at low temperature, and its gene family might be involved in low-temperature response and adaptation (Ye et al., 2009; Wang et al., 2017). Therefore, it could be inferred that CEY00_Acc33627 gene may be a cold tolerance gene.
Starch and sucrose metabolic pathway was found to be related to cold tolerance (Li et al., 2011, 2018). So the DEGs of starch and sucrose metabolism pathway were analyzed (Figure 4). There were eight DEGs in starch and sucrose metabolism pathway, of which six were CEY00_Acc26744 (alpha-trehalose phosphate synthase), CEY00_Acc28966 (beta-amylase), CEY00_Acc16695 (beta-amylase), CEY00_Acc14271 (beta-glucosides GH1 family), CEY00_Acc16756 (trehalose phosphatase) and CEY00_Acc08918 (beta-amylase). There were two down-regulated DEGs, CEY00_Acc17108 (beta-glucosidase) and CEY00_Acc04508 (alpha-amylase) (Table 9).
Trehalose-6-phosphate synthase can protect biological cell membrane and protein from damage, and is widely used in transgenic experiments with plants for the improvement of cold tolerance of plants (Kim et al., 2017). CEY00_Acc26744 and CEY00_Acc16756 were annotated as alpha-trehalose-phosphate synthase genes and inferred as cold-resistant genes. Maltose, which is produced by starch decomposition catalyzed by beta-amylase, may help protect the electron transport chain and protein in the stress environment. Cold tolerance of plants could be regulated by soluble sugar (Tarkowski and Van den Ende, 2015). Beta-amylase might reduce cold stress injury by increasing the content of sugars and enhance the cold tolerance of plants (Lv et al., 2017). Hence, unigenes of CEY00_Acc28966 (beta-amylase), CEY00_Acc16695 (beta-amylase) and CEY00_Acc08918 (beta-amylase) were inferred as cold-resistant genes.
RT-qPCR validation
The results RT-qPCR analysis of selected DEGs showed that the expression levels of all the genes selected were consistent with the transcriptome results (Figure 5). The findings suggested that there were statistically significant differences between the expression levels of these genes in low-temperature treated and control plantlets.