Transcriptome sequencing and differential expression analysis of natural and BTH-treated wound healing in potato tubers (Solanum tuberosum L.).

BACKGROUND: Wound healing is a representative phenomenon of potato tubers subjected to mechanical injuries. Our previous results found that benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) promoted the wound healing of potato tubers. However, the molecular mechanism related to inducible wound healing remains unknown. RESULTS: Transcriptomic evaluation of healing tissues from potato tubers at three stages, namely, 0 d (nonhealing), 5 d (wounded tubers healed for 5 d) and 5 d (BTH-treated tubers healed for 5 d) using RNA-Seq and differentially expressed genes (DEGs) analysis showed that more than 515 million high-quality reads were generated and a total of 7665 DEGs were enriched, and 16 of these DEGs were selected by qRT-PCR analysis to further confirm the RNA sequencing data. Gene ontology (GO) enrichment analysis indicated that the most highly DEGs were involved in metabolic and cellular processes, and KEGG enrichment analysis indicated that a large number of DEGs were associated with plant hormones, starch and sugar metabolism, fatty acid metabolism, phenylpropanoid biosynthesis and terpenoid skeleton biosynthesis. Furthermore, a few candidate transcription factors, including MYB, NAC and WRKY, and genes related to Ca2+-mediated signal transduction were also found to be differentially expressed during wound healing. Most of these enriched DEGs were upregulated after BTH treatment. CONCLUSION: This comparative expression profile provided useful resources for studies of the molecular mechanism via these promising candidates involved in natural or elicitor-induced wound healing in potato tubers.

Mechanical wounds expedited starch degradation in the wound tissues of potato tubers.

Starch degradation occurs rapidly in stressed plants, but it is unclear how starch degradation occurs in potato tubers after they incur mechanical wounding. In this study, we found that wounding significantly upregulated the expression levels of StGWD, StAMY, StBAM, and StISA, and decreased the starch content of potato tubers. Meanwhile, wounding markedly upregulated the expression levels of StSUS, StBG, and StINV genes, and increased the content of sucrose, glucose, and fructose. Furthermore, wounding reduced the proportion of small starch granules and increase that of large as well as medium starch granules, in this way enhancing the average size distribution of starch. Initially, the hard surface layer of starch granules was removed by wounding, but the internal channels and other structures were only slightly affected. Taken together, the results show that wounding can accelerate starch degradation by promoting the accumulation of sucrose, glucose, and fructose, and the hydrolysis of starch granules in potato tubers.

Overexpression of StCDPK23 promotes wound healing of potato tubers by regulating StRbohs.

Calcium-dependent protein kinase (CDPK) is a Ca2+ sensor that can phosphorylate and regulate respiratory burst oxidase homolog (Rboh), inducing the production of O2-. However, little is known about how StCDPK23 affects ROS production in the deposition of suberin at potato tuber wounds by regulating StRbohs. In this study, we found that StCDPK23 was induced significantly by the wound in potato tubers, which contains a typical CDPK structure, and was highly homologous to AtCDPK13 in Arabidopsis. Subcellular localization of results showed that StCDPK23 was located in the nucleus and plasma membrane of N. benthamiana epidermis cells. StCDPK23-overexpressing plants and tubers were obtained via Agrobacterium transformation. The expression of StCDPK23 was significantly upregulated in the overexpressing tubers during healing and increased 2.3-fold at 5 d. The expression levels of StRbohs (A-E) were also upregulated in the overexpressing tubers. Among them, StrbohA showed significant expression in the early stage of healing, which was 16.3-fold higher than that of the wild-type tubers at 8 h of healing. Moreover, the overexpressing tubers produced more O2- and H2O2, which are 1.1-fold and 3.5-fold higher than that of the wild-type at 8 h, respectively. More SPP deposition was observed at the wounds of the overexpressing tubers. The thickness of SPP cell layers was 53.2% higher than that of the wild-type after 3 d of the wound. It is suggested that StCDPK23 may participate in the wound healing of potato tubers by regulating Strbohs, which mainly contributes to H2O2 production during healing.

Interference Expression of StMSD Inhibited the Deposition of Suberin and Lignin at Wounds of Potato Tubers by Reducing the Production of H2O2.

Superoxide dismutase (SOD) actively participates in the wound stress of plants. However, whether StMSD mediates the generation of H2O2 and the deposition of suberin polyphenolic and lignin at potato tuber wounds is elusive. In this study, we developed the StMSD interference expression of potato plants and tubers by Agrobacterium tumefaciens-mediated transformation. The StSOD expression showed a marked downregulation in StMSD-interference tubers, especially StCSD2 and StCSD3. The content of O2•- exhibited a noticeable increase together with the inhibition in H2O2 accumulation. Moreover, the gene expression levels of StPAL (phenylalanine ammonia-lyase) and StC4H (cinnamate-4-hydroxylase) were downregulated in StMSD-interference tubers, and less suberin polyphenolic and lignin depositions at the wounds were observed. Taken together, the interference expression of StMSD can result in less suberin polyphenolic and lignin deposition by inhibiting the disproportionation of O2•- to H2O2 and restraining phenylpropanoid metabolism in tubers.

The Interaction Between StCDPK14 and StRbohB Contributes to Benzo-(1, 2, 3)-Thiadiazole-7-Carbothioic Acid S-Methyl Ester-Induced Wound Healing of Potato Tubers by Regulating Reactive Oxygen Species Generation.

Reactive oxygen species (ROS) production is essential for both physiological processes and environmental stress in diverse plants. Previous studies have found that benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH)-inducible ROS were associated with wound healing of potato tubers. Calcium-dependent protein kinases (CDPKs), the important calcium receptors, are known to play a crucial part in plant development and adaptation to abiotic stresses. However, whether CDPK-mediated ROS generation induced by BTH is involved in wound healing is elusive. In this study, we measured Solanum tuberosum CDPKs (StCDPKs) expression using real-time PCR, and it was found that the transcriptional levels of StCDPKs from BTH-treated tissues were significantly induced, among which StCDPK14 presented the most increased level. Subcellular localization results showed that StCDPK14 is located in the nucleus and membrane. The transgenic potato plants and tubers were developed using interference-expression of StCDPK14 by Agrobacterium tumefaciens-mediated transformation. The St respiratory burst oxidase homologs (StRbohs) expression showed a remarkable decrease in StCDPK14 transgenic tubers, notably, H2O2 content and suberin deposition were also significantly declined. To confirm the relationship between StCDPK14 and StRbohB, yeast-two-hybrid and bimolecular fluorescence complementation were used to examine the interaction, and it was shown that StCDPK14 interacted with the specific Ca2 + -binding motif (helix-loop-helix, called EF-hand) of StRbohB N-terminus. The above results unraveled that StCDPK14 functions in ROS generation via interacting with StRbohB during wound healing of potato tubers.

The effect of benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) treatment on regulation of reactive oxygen species metabolism involved in wound healing of potato tubers during postharvest.

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) can improve wound healing of potato tubers; however, how the chemical regulates reactive oxygen species (ROS) generation and scavenging during wound healing is not completely understood. BTH at 100 mg·L-1 regulated changes in ROS generation and scavenging in healing tissues of potato tubers. A higher H2O2 content was presented in healing tissues of potato tubers, while cell membrane permeability and malondialdehyde content declined due to BTH treatment. Additionally, the activities and transcript level of enzymes related with ROS generation, including NADPH oxidase, peroxidase and polyamine oxidase, as well as enzymes involved in ROS scavenging, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, were significantly enhanced by BTH treatment. It is suggested that ROS metabolism might play a crucial role in wound healing of potato tubers mediated by BTH during postharvest.