Guozheng Qin

Induction of defense responses against Alternaria rot by different elicitors in harvested pear fruit

Pear fruit (Pyrus pyrifolia L. cv. Yali) treated by different elicitors, such as salicylic acid (SA), oxalic acid, calcium chloride, and antagonistic yeast Cryptococcus laurentii, were investigated to determine the induction of defense responses. The possible mechanism by which elicitors induced the resistance of pear fruit against postharvest disease was also evaluated. The results indicated that all the elicitors could significantly enhance defense-related enzyme activities, such as β-1,3-glucanase, phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activity, and reduce the disease incidence caused by Alternaria alternata in pear fruit (P=0.05). Among these different elicitors, SA treatment showed the best result in inducing the defense responses and reducing the decay in pear fruit.

Oxidative damage of mitochondrial proteins contributes to fruit senescence: a redox proteomics analysis.

Oxidative damage to mitochondria caused by reactive oxygen species (ROS) has been implicated in the process of senescence as well as a number of senescence-related disorders in a variety of organisms. Whereas mitochondrial DNA was shown to be oxidatively modified during cellular senescence, mitochondrial protein oxidation is not well-understood. With the use of high-resolution, two-dimensional gel electrophoresis coupled with immunoblotting, we show here that protein carbonylation, a widely used marker of protein oxidation, increased in mitochondria during the senescence of peach fruit. Specific mitochondrial proteins including outer membrane transporter (voltage-dependent anion-selective channel, VDAC), tricarboxylic acid cycle enzymes (malate dehydrogenase and aconitase), and antioxidant proteins (manganese superoxide dismutase, MnSOD) were found as the targets. The oxidative modification was concomitant with a change of VDAC function and loss of catalytic activity of malate dehydrogenase and MnSOD, which in turn facilitated the release of superoxide radicals in mitochondria. Reduction of ROS content by lowering the environmental temperature prevented the accumulation of protein carbonylation in mitochondria and retarded fruit senescence, whereas treatment of fruit with H2O2 had the opposite effect. Our data suggest that oxidative damage of specific mitochondrial proteins may be responsible for impairment of mitochondrial function, thus, leading to fruit senescence. Proteomics analysis of mitochondrial redox proteins provides considerable information on the molecular mechanisms involved in the progression of fruit senescence.

Unraveling the regulatory network of the MADS box transcription factor RIN in fruit ripening

The MADS box transcription factor RIN is a global regulator of fruit ripening. However, the direct targets modulated by RIN and the mechanisms underlying the transcriptional regulation remain largely unknown. Here we identified 41 protein spots representing 35 individual genes as potential targets of RIN by comparative proteomic analysis of a rin mutant in tomato fruits. Gene expression analysis showed that the mRNA level of 26 genes correlated well with the protein level. After examining the promoter regions of the candidate genes, a variable number of RIN binding sites were found. Five genes (E8, TomloxC, PNAE, PGK and ADH2) were identified as novel direct targets of RIN by chromatin immunoprecipitation. The results of a gel mobility shift assay confirmed the direct binding of RIN to the promoters of these genes. Of the direct target genes, TomloxC and ADH2, which encode lipoxygenase (LOX) and alcohol dehydrogenase, respectively, are critical for the production of characteristic tomato aromas derived from LOX pathway. Further study indicated that RIN also directly regulates the expression of HPL, which encodes hydroperoxide lyase, another rate-limiting enzyme in the LOX pathway. Loss of function of RIN causes de-regulation of the LOX pathway, leading to a specific defect in the generation of aroma compounds derived from this pathway. These results indicate that RIN modulates aroma formation by direct and rigorous regulation of expression of genes in the LOX pathway. Taken together, our findings suggest that the regulatory effect of RIN on fruit ripening is achieved by targeting specific molecular pathways.

Crucial Role of Antioxidant Proteins and Hydrolytic Enzymes in Pathogenicity of Penicillium expansum Analysis Based on Proteomics Approach

Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase.

Reactive oxygen species involved in regulating fruit senescence and fungal pathogenicity.

Senescence is a vital aspect of fruit life cycles, and directly affects fruit quality and resistance to pathogens. Reactive oxygen species (ROS), as the primary mediators of oxidative damage in plants, are involved in senescence. Mitochondria are the main ROS and free radical source. Oxidative damage to mitochondrial proteins caused by ROS is implicated in the process of senescence, and a number of senescence-related disorders in a variety of organisms. However, the specific sites of ROS generation in mitochondria remain largely unknown. Recent discoveries have ascertained that fruit senescence is greatly related to ROS and incidental oxidative damage of mitochondrial protein. Special mitochondrial proteins involved in fruit senescence have been identified as the targets of ROS. We focus in discussion on our recent advances in exploring the mechanisms of how ROS regulate fruit senescence and fungal pathogenicity.

Inhibitory effect of boron against Botrytis cinerea on table grapes and its possible mechanisms of action

Boron, an essential plant micronutrient, was effective in the form of potassium tetraborate for control of postharvest gray mold caused by Botrytis cinerea on table grapes stored at room temperature or at 0 degrees C. The inhibition of fruit decay was closely correlated with boron concentrations and partially influenced by pH value of the solution. Boron strongly inhibited spore germination, germ tube elongation, and mycelial spread of B. cinerea in the culture medium. Application of boron at 1% caused the appearance of abnormal spores (disrupted) in some cases. By using propidium iodide fluorescent staining, loss of membrane integrity in B. cinerea was observed after boron treatment. Furthermore, boron led to the leakage of cellular constituents (soluble proteins and carbohydrates) from hyphae of B. cinerea. These data suggest that the mechanisms by which boron decreased gray mold decay of table grapes may be directly related to the disruption effect of boron on cell membrane of the fungal pathogen that resulted in the breakdown of the cell membrane and loss of cytoplasmic materials from the hyphae.

Proteomic analysis of changes in mitochondrial protein expression during fruit senescence.

Fruit senescence has been reported to be an oxidative phenomenon, but the detailed mechanisms by which ROS regulate this process remain largely unknown. Here we show that senescence process of apple fruit was concomitant with the dynamic alterations in the mitochondrial proteome. Mitochondrial proteins involved in tricarboxylic acid cycle, electron transport chain, carbon metabolism, and stress response were found to be differentially expressed during fruit senescence. Alleviating oxidative stress by lowering the ambient oxygen concentration noticeably decreased the number of changed proteins and delayed fruit senescence, indicating the involvement of ROS in this process. To further investigate the regulatory effect of ROS on senescence process, we analyzed the mitochondrial proteome variations upon exposure to high oxygen (100%), which induces oxidative stress and accelerates fruit senescence. High oxygen treatment led to a further identification of differentially expressed proteins such as mitochondrial manganese superoxide dismutase, an antioxidant scavenging superoxide radicals produced in the mitochondria. Activity of manganese superoxide dismutase was reduced after high oxygen exposure, accompanied by an increase in oxidative protein carbonylation (damaged proteins). These data suggest that ROS may regulate fruit senescence by changing expression profiles of specific mitochondrial proteins and impairing the biological function of these proteins.

Functions of defense-related proteins and dehydrogenases in resistance response induced by salicylic acid in sweet cherry fruits at different maturity stages.

We report here a comparative analysis of sweet cherry (Prunus avium) fruits proteome induced by salicylic acid (SA) at different maturity stages. The results demonstrated that SA enhanced the resistance of sweet cherry fruits against Penicillium expansum, resulting in lower disease incidences and smaller lesion diameters, especially at earlier maturity stage. Based on proteomics analysis, 13 and 28 proteins were identified after SA treatment at earlier (A) and later (B) maturity stage, respectively. Seven antioxidant proteins and three pathogenesis related‐proteins were identified at both A and B stages, while five heat shock proteins and four dehydrogenases were only detected at B stage. SA treatment also stimulated higher transcript levels of peroxidase, but repressed that of catalase. Moreover, some proteins regulated by SA at B maturity stage were identified as enzymes involved in glycolysis and tricarboxylic acid cycle. These findings indicated that younger sweet cherry fruits showed stronger resistance against pathogen invasion after SA treatment. It further indicated that antioxidant proteins were involved in the resistance response of fruits at every maturity stage, while heat shock proteins and dehydrogenases might potentially act as factors only at later maturity stages.

Exploring Pathogenic Mechanisms of Botrytis cinerea Secretome under Different Ambient pH Based on Comparative Proteomic Analysis

Botrytis cinerea causes gray mold rot on over 200 plant species worldwide, resulting in great economic loss every year. Cooperation of proteins secreted by B. cinerea plays an important role in its successful infection to host plants. The ambient pH, as one of the most important environmental parameters, can regulate expression of secreted proteins in various fungal pathogens. In the present study, we mainly investigated the effect of ambient pH on secretome of B. cinerea strain B05.10 with a comparative proteomic method based on 2-DE. Distinct differences in secretome of B. cinerea were found between pH 4 and 6 treatments, and 47 differential spots, corresponding to 21 unique proteins, were identified using MALDI-TOF/TOF. At pH 4, more proteins related to proteolysis were induced, whereas most of up-accumulated proteins were cell wall degrading enzymes at pH 6. Analysis of gene expression using quantitative real-time PCR suggests that production of most of these proteins was regulated at the level of transcription. These findings indicate that B. cinerea can adjust protein profile of secretome responding to different ambient pH values and provide evidence to deeply understand the complicated infecting mechanisms of B. cinerea on a wide range of plant hosts.

Effect of microbial biocontrol agents on alleviating oxidative damage of peach fruit subjected to fungal pathogen

Levels of protein carbonylation in peach fruits inoculated with four antagonistic yeasts (Pichia membranaefaciens, Cryptococcus laurentii, Candida guilliermondii and Rhodotorula glutinis) were significantly reduced in response to reactive oxygen species (ROS) caused by Monilinia fructicola. In control fruit without yeast treatments, proteins carbonylation obviously increased after inoculation with M. fructicola, ranging from molecular mass 20 to 120 kDa. However, in yeast-treated fruits, no proteins carbonylation was detected at 1 d, only a small quantity of carbonylation ranging from 28.5 to 45 kDa was found at 2 d. Antagonistic yeasts significantly stimulated the activities of chitinase, beta-1,3-glucanase, catalase (CAT), peroxidase (POD) and the expressions of relevant genes during all storage periods. These results suggest that yeast treatments may be related to alleviating proteins carbonylation and mitigating pathogen-induced oxidative damage, which result in decrease of fruit decay and imply that antioxidant defense response may be involved in the mechanisms of microbial biocontrol agents against fungal pathogen.