Genome-Wide Analysis and Expression Profiling of the Glutathione Peroxidase-like Enzyme Gene Family in Solanum tuberosum.

Glutathione peroxidase-like enzyme is an important enzymatic antioxidant in plants. It is involved in scavenging reactive oxygen species, which can effectively prevent oxidative damage and improve resistance. GPXL has been studied in many plants but has not been reported in potatoes, the world's fourth-largest food crop. This study identified eight StGPXL genes in potatoes for the first time through genome-wide bioinformatics analysis and further studied the expression patterns of these genes using qRT-PCR. The results showed that the expression of StGPXL1 was significantly upregulated under high-temperature stress, indicating its involvement in potato defense against high-temperature stress, while the expression levels of StGPXL4 and StGPXL5 were significantly downregulated. The expression of StGPXL1, StGPXL2, StGPXL3, and StGPXL6 was significantly upregulated under drought stress, indicating their involvement in potato defense against drought stress. After MeJA hormone treatment, the expression level of StGPXL6 was significantly upregulated, indicating its involvement in the chemical defense mechanism of potatoes. The expression of all StGPXL genes is inhibited under biotic stress, which indicates that GPXL is a multifunctional gene family, which may endow plants with resistance to various stresses. This study will help deepen the understanding of the function of the potato GPXL gene family, provide comprehensive information for the further analysis of the molecular function of the potato GPXL gene family as well as a theoretical basis for potato molecular breeding.

Cardamonin Presents in Vivo Activity against Schistosoma mansoni and Inhibits Potato Apyrase.

Schistosomiasis, a neglected tropical disease caused by Schistosoma species, harms over 250 million people in several countries. The treatment is achieved with only one drug, praziquantel. Cardamonin, a natural chalcone with in vitro schistosomicidal activity, has not been in vivo evaluated against Schistosoma. In this work, we evaluated the in vivo schistosomicidal activities of cardamonin against Schistosoma mansoni worms and conducted enzymatic apyrase inhibition assay, as well as molecular docking analysis of cardamonin against potato apyrase, S. mansoni NTPDase 1 and S. mansoni NTPDase 2. In a mouse model of schistosomiasis, the oral treatment with cardamonin (400 mg/kg) showed efficacy against S. mansoni, decreasing the total worm load in 46.8 % and reducing in 54.5 % the number of eggs in mice. Cardamonin achieved a significant inhibition of the apyrase activity and the three-dimensional structure of the potato apyrase, obtained by homology modeling, showed that cardamonin may interact mainly through hydrogen bonds. Molecular docking studies corroborate with the action of cardamonin in binding and inhibiting both potato apyrase and S. mansoni NTPDases.

Genome-wide identification, evolution, and transcript profiling of Aldehyde dehydrogenase superfamily in potato during development stages and stress conditions.

The Aldehyde dehydrogenase (ALDH) superfamily comprises a group of enzymes involved in the scavenging of toxic aldehyde molecules by converting them into their corresponding non-toxic carboxylic acids. A genome-wide study in potato identified a total of 22 ALDH genes grouped into ten families that are presented unevenly throughout all the 12 chromosomes. Based on the evolutionary analysis of ALDH proteins from different plant species, ALDH2 and ALDH3 were found to be the most abundant families in the plant, while ALDH18 was found to be the most distantly related one. Gene expression analysis revealed that the expression of StALDH genes is highly tissue-specific and divergent in various abiotic, biotic, and hormonal treatments. Structural modelling and functional analysis of selected StALDH members revealed conservancy in their secondary structures and cofactor binding sites. Taken together, our findings provide comprehensive information on the ALDH gene family in potato that will help in developing a framework for further functional studies.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

HD2-type histone deacetylases: unique regulators of plant development and stress responses.

Plants have developed sophisticated and complex epigenetic regulation-based mechanisms to maintain stable growth and development under diverse environmental conditions. Histone deacetylases (HDACs) are important epigenetic regulators in eukaryotes that are involved in the deacetylation of lysine residues of histone H3 and H4 proteins. Plants have developed a unique HDAC family, HD2, in addition to the RPD3 and Sir2 families, which are also present in other eukaryotes. HD2s are well conserved plant-specific HDACs, which were first identified as nucleolar phosphoproteins in maize. The HD2 family plays important roles not only in fundamental developmental processes, including seed germination, root and leaf development, floral transition, and seed development but also in regulating plant responses to biotic and abiotic stresses. Some of the HD2 members coordinate with each other to function. The HD2 family proteins also show functional association with RPD3-type HDACs and other transcription factors as a part of repression complexes in gene regulatory networks involved in environmental stress responses. This review aims to analyse and summarise recent research progress in the HD2 family, and to describe their role in plant growth and development and in response to different environmental stresses.

The Cysteine-Rich Peptide Snakin-2 Negatively Regulates Tubers Sprouting through Modulating Lignin Biosynthesis and H2O2 Accumulation in Potato.

Potato tuber dormancy is critical for the post-harvest quality. Snakin/Gibberellic Acid Stimulated in Arabidopsis (GASA) family genes are involved in the plants' defense against pathogens and in growth and development, but the effect of Snakin-2 (SN2) on tuber dormancy and sprouting is largely unknown. In this study, a transgenic approach was applied to manipulate the expression level of SN2 in tubers, and it demonstrated that StSN2 significantly controlled tuber sprouting, and silencing StSN2 resulted in a release of dormancy and overexpressing tubers showed a longer dormant period than that of the control. Further analyses revealed that the decrease expression level accelerated skin cracking and water loss. Metabolite analyses revealed that StSN2 significantly down-regulated the accumulation of lignin precursors in the periderm, and the change of lignin content was documented, a finding which was consistent with the precursors' level. Subsequently, proteomics found that cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (COMT) and peroxidase (Prx), the key proteins for lignin synthesis, were significantly up-regulated in silencing lines, and gene expression and enzyme activity analyses also supported this effect. Interestingly, we found that StSN2 physically interacts with three peroxidases catalyzing the oxidation and polymerization of lignin. In addition, SN2 altered the hydrogen peroxide (H2O2) content and the activities of superoxide dismutase (SOD) and catalase (CAT). These results suggest that StSN2 negatively regulates lignin biosynthesis and H2O2 accumulation, and ultimately inhibits the sprouting of potato tubers.

Potato apyrase reduces granulomatous area and increases presence of multinucleated giant cells in murine schistosomiasis.

Granulomas are inflammatory tissue responses directed to a set of antigens. Trapped Schistosoma mansoni eggs promote productive granulomas in the tissues, and they are the main damage caused by schistosomiasis. Some S. mansoni antigenic proteins may have a direct involvement in the resolution of the granulomatous response. The ATP diphosphohydrolases isoforms of this parasite are immunogenic, expressed in all phases of the parasite life cycle and secreted by eggs and adult worms. Potato apyrase is a vegetable protein that cross-reactive with parasite ATP diphosphohydrolases isoforms. In this study, the vegetable protein was purified, before being inoculated in C57BL/6 mice that were later infected with cercariae. Sixty days after infection, adult worms were recovered, antibodies and cytokines were measured, and morphological granuloma alterations evaluated. Immunization of the animals induced significant levels of IgG and IgG1 antibodies and IFN-γ, IL-10 and IL-5 cytokines, but not IL-13, suggesting that potato apyrase is an immunoregulatory protein. Supporting this hypothesis, it was found that liver damage associated with schistosomiasis was mitigated, reducing the size of the areas affected by granuloma to 35% and increasing the presence of multinucleated giant cells in this environment. In conclusion, potato apyrase was found to be effective immunomodulatory antigen for murine schistosomiasis.

In vivo tumor growth inhibition by Solanum tuberosum aspartic protease 3 (StAP3) treatment.

Solanum tuberosum aspartic Proteases (StAPs) show selective plasma membrane permeabilization, inducing cytotoxicity of cancer cells versus normal cells in vitro. Herein, we aimed to evaluate both StAP3 systemic toxicity and antitumoral activity against human melanoma in vivo. The toxicity of a single high dose of StAP3 (10 µg/g body weight, intraperitoneally) was assessed in a Balb/c mice model. Subcutaneous A375 human melanoma xenografts in athymic nude (nu/nu) mice were induced. Once tumors developed (mean larger dimension = 3.8 ± 0.09 mm), mice were StAP3-treated (6 µg/g body weight, subcutaneously under the tumor at a single dose). For both models, controls were treated with physiologic saline solution. StAP3-treated mice showed a significant inhibition of tumor growth (p < 0.05) compared with controls. No signs of toxicity were detected in StAP3-treated mice in both models. These results suggest the potential of these plant proteases as anticancer agents.

The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase.

Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.

Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato.

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.

Comparison of root tolerance to drought and aphid (Myzus persicae Sulzer) resistance among different potato (Solanum tuberosum L.) cultivars.

This study was conducted to determine the root system architecture and biochemical responses of three potato (Solanum tuberosum L.) cultivars to drought and aphid (Myzus persicae Sulzer) infestation under greenhouse conditions. A factorial experiment comprising three potato cultivars (Qingshu 9, Longshu 3, and Atlantic), two levels of water (Well watered and drought) application and aphid infestation (Aphids and no aphids) was conducted. The results show that drought stress and aphid infestation significantly increased the root-projected area, root surface area, number of root tips, and number of root forks of all cultivars, relative to their corresponding control plants. The least root projected area, root surface area, number of root tips, and number of root forks occurred on DXY under both drought and aphid infestation. Nevertheless, the greatest root projected area, root surface area, number of root tips and number of root forks occurred on QS9 plants. Moreover, increased SOD, CAT, and POD activities were observed across all cultivars, under drought and aphid stress. The highest SOD, POD, and CAT activities occurred in QS9; under drought and aphid stress, while the least SOD, POD, and CAT activities was observed in DXY. The Atlantic cultivar, which possesses a root system sensitive to water deficit, demonstrated greater resistance to aphid infestation under well-watered and drought-stressed conditions. Conversely, Qingshu 9, which possesses a root system tolerant to water deficit, was highly susceptible to aphids. This study shows that the root architectural and biochemical traits that enhance potato tolerance to drought do not necessarily correlate to a plant's tolerance to aphids.

Bioconversion of Stevioside to Rebaudioside E Using Glycosyltransferase UGTSL2.

Rebaudioside E, one of the minor components of steviol glycosides, was first isolated and identified from Stevia rebaudiana in 1977. It is a high-intensity sweetener that tastes about 150-200 times sweeter than sucrose and is also a precursor for biosynthesis of rebaudioside D and rebaudioside M, the next-generation Stevia sweeteners. In this work, new unknown steviol glycosides were enzymatically synthesized from stevioside by coupling UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Rebaudioside E was speculated to be the main product of glucosylation of the Glc(ß1→C-19) residue of stevioside along with the formation of a (ß1→2) linkage based on the analysis of the regioselectivity and stereoselectivity of UGTSL2, and verified afterwards by LC-MS/MS with standard. In a 20-ml bioconversion reaction of 20 g/l stevioside by UGTSL2 and StSUS1, 15.92 g/l rebaudioside E was produced for 24 h.

Influence of Artificial Inoculation with Pseudomonas fluorescens on Enzymatic Browning Reactions of Fresh-cut Potatoes.

We initially correlated fluorescent pseudomonads and severity of enzymatic browning on fresh-cut potatoes. Subsequently, we determined the influence of inoculation with Pseudomonas fluorescens following its isolation from the brown tissues on the browning response on fresh-cut potatoes. Bacterial counts on potato slices were higher on browning tissues than on non-browning tissues. P. fluorescens that has been isolated only from the severely browning tissues developed brown discoloration on surface tissues when inoculated onto potato slices. When potato slices were initially inoculated with 103 colony-forming unit (CFU) per mL of P. fluorescens and then stored at 5ºC, bacterial counts, polyphenol oxidase (PPO) activity, phenolic content, and browning severity increased after 3 days of storage. We observed plant PPO derived from potatoes and bacterial PPO released by P. fluorescens and dictated that the plant PPO contributed to browning reactions because only the plant PPO was activated at pH 6-7 that lies in potato tissues. The PPO1 gene that contributed to browning on potatoes was expressed prominently in potato tissues following inoculation with P. fluorescens. These results indicated that P. fluorescens enhanced browning of fresh-cut potatoes by inducing the plant PPO gene, plant PPO activity, and accumulation of phenolics as a biocontrol agent.

StMAPK3 controls oxidase activity, photosynthesis and stomatal aperture under salinity and osmosis stress in potato.

Mitogen-activated protein kinase 3 (MAPK3) is involved in plant growth and development, as well as response to adverse stress. Here we aimed to explore the role of StMAPK3 in response to salt and osmosis stress. Polyethylene glycol (PEG) (5% and 10%) and mannitol (40 mM and 80 mM) were used to induce osmosis stress. To induce salinity stress, potato plant was cultured with NaCl (40 mM and 80 mM). StMAPK3 overexpression and RNA interference-mediated StMAPK3 knockdown were constructed to explore the role of StMAPK3 in potato growth, stomatal aperture size, activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), and contents of H2O2, proline and malonaldehyde (MDA). Meanwhile, we detected transpiration, net photosynthesis, stomatal conductance, and water use efficiency. Subcellular location of StMAPK3 protein was also detected. PEG, mannitol and NaCl treatments induced the accumulation of StMAPK3 mRNA in potato plants. StMAPK3 protein was located on the membrane and nucleus. Abnormal expression of StMAPK3 changed potato phenotypes, enzyme activity of SOD, CAT and POD, as well as H2O2, proline and MDA contents under osmosis and salinity stress. Photosynthesis and stomatal aperture were regulated by StMAPK3 in potato treated by PEG, mannitol and NaCl. Modulation of potato phenotypes and physiological activity indicates StMAPK3 as a regulator of osmosis and salinity tolerance.

Potato E3 ubiquitin ligase PUB27 negatively regulates drought tolerance by mediating stomatal movement.

The ubiquitin-proteasome system (UPS) is one of the main ways of eukaryotic protein degradation and post-translational modification. It has proven as an essential process for plants to respond to abiotic stresses. Plant U-box (PUB) protein acts as a ubiquitin ligase, which recognizes and ubiquitinates the target proteins. Many PUBs have been involved in water stress in Arabidopsis and rice, but similar comprehensive studies in potato remained limited. In this study, the overexpressed and interfered transgenic potato plants of StPUB27 were obtained and their performances were evaluated under osmotic stress. The result showed that overexpression of StPUB27 accelerated the dehydration of detached leaves companied with greater stomatal conductance, while the down-regulated StPUB27 expression by RNA interference (RNAi) showed a smaller stomatal conductance and a lower rate of water loss in detached leaves, thus showing higher tolerance to osmotic stress. In addition, no significant changes in the proline content were observed between StPUB27 overexpressed and RNAi potato plants. The result demonstrated that potato E3 ubiquitin ligase PUB27 may negatively regulate drought tolerance by mediating stomatal conductance.

Structural analysis and reaction mechanism of the disproportionating enzyme (D-enzyme) from potato.

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D-enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α-1, 4-glucan. D-enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D-enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26-meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D-enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D-enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D-enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.

Screening of plant derived chalcones on the inhibition of potato apyrase: Potential protein biotechnological applications in health.

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

Aldehyde dehydrogenase plays crucial roles in response to lower temperature stress in Solanum tuberosum and Nicotiana benthamiana.

The aim of this study is to elucidate the role of ALDH2B7a during the response to lower temperature in Solanum tuberosum. This gene was found to have altered intragenic DNA methylation status in our previous reports. A total of 18 orthologs of StALDH2B7a were identified in the S. tuberosum genome, which were then divided into 8 aldehyde dehydrogenase (ALDH) subfamilies. The methylation statuses of four intragenic cytosine sites in intron 5 and exon 6 of genomic StALDH2B7a were altered by lower temperature stress, resulting in changes in the expression of StALDH2B7a. Silencing of NbALDH2C4, a homolog of StALDH2B7a in Nicotiana benthamiana, resulted in plants which were sensitive to lower temperature and accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA). These data suggested that the expression of StALDH2B7a was upregulated by alteration of its intragenic cytosine methylation status during lower temperature stress, and additional StALDH2B7a enzymes scavenged excess aldehydes resulting from ROS in a response to cold stress in potato. Our study expands the understanding of the mechanisms involved in plant responses to lower temperature, and provides a new gene source to improve potato tolerance to cold stress in northern China, where lower temperature is one of the key limiting factors for crop production.

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants.

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols. MsHPL, StHPL, and CsHPL converted the 13-hydroperoxides of linoleic (13-HPOD) and α-linolenic acids (13-HPOT) primarily to the chain cleavage products. The minor by-products of 13-HPOD and 13-HPOT conversions by these enzymes were the oxiranyl carbinols, 11-hydroxy-12,13-epoxy-9-octadecenoic and 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. At the same time, all enzymes studied converted 9-hydroperoxides into corresponding oxiranyl carbinols with HPL by-products. Thus, the results showed the additional epoxyalcohol synthase activity of studied CYP74B enzymes. The 13-HPOD conversion reliably resulted in smaller yields of the HPL products and bigger yields of the epoxyalcohols compared to the 13-HPOT transformation. Overall, the results show the dualistic HPL/EAS behaviour of studied CYP74B enzymes, depending on hydroperoxide isomerism and unsaturation.

Reactive oxygen species induce cyanide-resistant respiration in potato infected by Erwinia carotovora subsp. Carotovora.

Studies have shown that pathogenic bacteria infections induce the overproduction of reactive oxygen species (ROS) in plants. Cyanide-resistant respiration, an energy-dissipating pathway in plants, has also been induced by a pathogenic bacteria infection. However, it is unknown whether the induction of cyanide-resistant respiration under the pathogenic bacteria infection was caused by ROS. In this study, two pathogenic Erwinia strains were used to infect potato tuber, and membrane lipid peroxidation levels and the cyanide-resistant respiration capacity were determined. In addition, StAOX expression and regulation by ROS in potato tuber were analyzed. Moreover, the role of the Ca2+ pathway in regulating cyanide-resistant respiration was determined. The results showed that ROS induced cyanide-resistant respiration in potato tuber infected by Erwinia. Cyanide-resistant respiration inhibited the production of H2O2. Intracellular Ca2+ regulated the expression of calcium-dependent protein kinase (StCDPK1, StCDPK4, and StCDPK5) in potato, which indirectly controlled intracellular ROS levels. These results indicate that Ca2+ metabolism is involved in ROS-induced cyanide-resistant respiration.