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.

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.

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.

Development of selectable marker-free transgenic potato plants expressing cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say).

BACKGROUND: Elimination of selectable marker genes (SMGs) is important for the safe assessment and commercial use of transgenic plants. The destructive and invasive Colorado potato beetle (CPB) poses a serious threat to potato production. In response to this need, selectable marker-free transgenic potato lines expressing cry3A were developed to control the damage and spread of CPB. RESULTS: We simultaneously introduced cry3A and npt II genes harboured in different plasmids into the potato genome using the Agrobacterium-mediated cotransformation method. Four selectable marker-free transgenic potato (CT) lines expressing cry3A were developed by self-crossing segregation and molecular analyses, including Southern blot, western blot and enzyme-linked immunosorbent assay (ELISA) assays. CT lines were used in a resistance bioassay against CPB in the laboratory and field. In the laboratory, CT lines exhibited high resistance to CPB, and 100% mortality of first-instar larvae occurred 6 days after infestation. In the field, untransformed plant leaves were almost entirely consumed, with an average of 155 larvae present per plant 25 days after inoculation. However, CT lines showed no damage symptoms, with approximately 2.5 larvae surviving per plant. CONCLUSION: We successfully eliminated SMGs from the transgenic potato lines expressing cry3A in order to decrease CPB damage, control the spread of this pest eastwards and alleviate the concern regarding the safe assessment of regulatory requirements. © 2015 Society of Chemical Industry.