Chitooligosaccharide accelerated wound healing in potato tubers by promoting the deposition of suberin polyphenols and lignin at wounds.

Chitooligosaccharide (COS) is a low molecular weight product of chitosan degradation. Although COS induces plant resistance by activating phenylpropanoid metabolism, there are few reports on whether COS accelerates wound healing in potato tubers by promoting the deposition of phenolic acids and lignin monomers at wounds. The results showed that COS activated phenylalanine ammonialyase and cinnamate 4-hydroxylase and promoted the synthesis of cinnamic, caffeic, p-coumaric, ferulic acids, total phenolics and flavonoids. COS activated 4-coumaric acid coenzyme A ligase and cinnamyl alcohol dehydrogenase and promoted the synthesis of sinapyl, coniferyl and cinnamyl alcohols. COS also increased H2O2 levels and peroxidase activity and accelerated the deposition of suberin polyphenols and lignin on wounds. In addition, COS reduced weight loss and inhibited lesion expansion in tubers inoculated with Fusarium sulfureum. Taken together, COS accelerated wound healing in potato tubers by inducing phenylpropanoid metabolism and accelerating the deposition of suberin polyphenols and lignin at wounds.

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.

Meyerozyma guilliermondii promoted the deposition of GSH type lignin by activating the biosynthesis and polymerization of monolignols at the wounds of potato tubers.

Lignin is a crucial component in the wound tissue of tubers. The biocontrol yeast Meyerozyma guilliermondii increased the activities of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coenzyme coenzyme A ligase, and cinnamyl alcohol dehydrogenase, and elevated the levels of coniferyl, sinapyl, and p-coumaryl alcohol. The yeast also enhanced the activities of peroxidase and laccase, as well as the content of hydrogen peroxide. The lignin promoted by the yeast was identified as guaiacyl-syringyl-p-hydroxyphenyl type using Fourier transform infrared spectroscopy and two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance. Furthermore, a larger signal area for G2, G5, G'6, S2, 6, and S'2, 6 units was observed in the treated tubers, and the G'2 and G6 units were only detected in the treated tuber. Taken together, M. guilliermondii could promote deposition of guaiacyl-syringyl-p-hydroxyphenyl type lignin by activating the biosynthesis and polymerization of monolignols at the wounds of potato tubers.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

AIMS: Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. METHODS AND RESULTS: A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46-fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. CONCLUSIONS: AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. SIGNIFICANCE AND IMPACT OF STUDY: Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

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.

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.

Effects of nitric oxide treatment on lignin biosynthesis and texture properties at wound sites of muskmelons.

Lignin is an important component of the healing tissue in fruits. In this study, we treated muskmelon (Cucumis melo L. cv. "Manao") fruit with exogenous nitric oxide (NO) donor sodium nitroprusside (SNP) to observe and analyze its effect on lignin synthesis and accumulation during healing. Results showed that SNP treatment enhanced the contents of endogenous NO and H2O2, increased the activities of phenylalanine ammonia lyase, cinnamate 4 hydroxylase, cinnamyl alcohol dehydrogenase, and peroxidase, and raised the contents of sinapyl alcohol, coniferyl alcohol, coumaryl alcohol, and lignin. SNP augmented the hardness of the healing tissue and decreased its resilience, springiness, and cohesiveness. In addition, SNP treatment effectively reduced the weight loss and disease index of wounded muskmelons. All these results suggest that lignin metabolism mediated by NO play a crucial role in wound healing of muskmelons.

Mechanism of Ca2+-mediated NOX modulated in ROS metabolism induced by T-2 toxin in potato tuber.

T-2 toxin at low concentrations can induce ROS accumulation and modulate host resistance in plants. NOX plays crucial roles in ROS production and is regulated by Ca2+via direct binding to EF-hand motifs. In this study, the effect of EGTA (Ca2+ chelating agent) on the expression and enzymatic activity of NOX, as well as the activities and corresponding gene expressions involved in ROS metabolism and cell membrane integrity, were investigated in treated slices. Results indicated that EGTA treatment significantly affected gene expression and activity of NOX, and reduced ROS accumulation and cell membrane integrity and the enzymatic activities and gene expression involved in ROS metabolism when exposed to treatment. The addition of exogenous Ca2+ restored the initial relative transcript abundance, ROS accumulation and their activities. Results suggest that Ca2+ affected by EGTA plays a crucial role in NOX activity regulation, ultimately affecting ROS metabolism in slices induced by T-2 toxin.

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.

Effect of benzothiadiazole treatment on improving the mitochondrial energy metabolism involved in induced resistance of apple fruit during postharvest storage.

The effects of benzothiadiazole (BTH) on Penicillium expansum development, mitochondria energy metabolism, and changes in the number and structure of mitochondria in apple fruit were investigated after the fruit were immersed in 100 mg L-1 BTH for 10 min and then stored at 22 °C. The results indicated that BTH treatment significantly decreased the lesion diameter of fruit challenged with P. expansum; further, treatment enhanced the activities of mitochondrial respiratory metabolism-related enzymes, such as succinate dehydrogenase, cytochrome oxidase, H+-ATPase and Ca2+-ATPase, along with high ATP level and energy status in apple fruit during storage. Moreover, transmission electron microscopy results indicated that BTH treatment was beneficial for maintaining the number and structure of mitochondria during storage. The results suggested that BTH treatment enhanced ATP levels via mitochondrial energy metabolism, which might contribute to the induced resistance in apple fruit during storage.

Cation-Stress-Responsive Transcription Factors SltA and CrzA Regulate Morphogenetic Processes and Pathogenicity of Colletotrichum gloeosporioides.

Growth of Colletotrichum gloeosporioides in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of Aspergillus nidulans to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of A. nidulans SltA and CrzA were identified in C. gloeosporioides. The C. gloeosporioides CrzA homolog is a 682-amino acid protein, which contains a C2H2 zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The C. gloeosporioides SltA homolog encodes a 775-amino acid protein with strong similarity to A. nidulans SltA and Trichoderma reesei ACE1, and highest conservation in the three zinc-finger regions with almost no changes compared to ACE1 sequences. Knockout of C. gloeosporioides crzA (ΔcrzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calciu006D-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of C. gloeosporioides sltA (ΔsltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ΔcrzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca2+ supplementation. Inhibition of appressorium formation in ΔsltA mutants was accompanied by downregulation of the MAP kinase pmk1 and carnitine acetyl transferase (cat1), genes involved in appressorium formation and colonization, which was restored by Ca2+ supplementation. Furthermore, exposure of C. gloeosporioides ΔcrzA or ΔsltA mutants to cations such as Na+, K+ and Li+ at concentrations that the wild type C. gloeosporioides is not affected had further adverse morphogenetic effects on C. gloeosporioides which were partially or fully restored by Ca2+. Overall results suggest that both genes modulating alkali cation homeostasis have significant morphogenetic effects that reduce C. gloeosporioides colonization.

De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment.

BACKGROUND: The mango belongs to the genus Mangifera, consisting of numerous tropical fruiting trees in the flowering plant family, Anacardiaceae. Postharvest treatment by hot water brushing (HWB) for 15-20 s was introduced commercially to improve fruit quality and reduce postharvest disease. This treatment enabled successful storage for 3-4 weeks at 12°C, with improved color and reduced disease development, but it enhanced lenticel discoloration on the fruit peel. We investigated global gene expression induced in fruit peel by HWB treatment, and identified key genes involved in mechanisms potentially associated with fruit resistance to pathogens, peel color improvement, and development of lenticel discoloration; this might explain the fruit's phenotypic responses. RESULTS: The mango transcriptome assembly was created and characterized by application of RNA-seq to fruit-peel samples. RNA-seq-based gene-expression profiling identified three main groups of genes associated with HWB treatment: 1) genes involved with biotic and abiotic stress responses and pathogen-defense mechanisms, which were highly expressed; 2) genes associated with chlorophyll degradation and photosynthesis, which showed transient and low expression; and 3) genes involved with sugar and flavonoid metabolism, which were highly expressed. CONCLUSIONS: We describe a new transcriptome of mango fruit peel of cultivar Shelly. The existence of three main groups of genes that were differentially expressed following HWB treatment suggests a molecular basis for the biochemical and physiological consequences of the postharvest HWB treatment, including resistance to pathogens, improved color development, and occurrence of lenticel discoloration.