Fig. 1 Representatives of whole (A) and fresh-cut (B) Z. latifolia after 3 days of storage at 25 ℃
WANG Weihua1, JIANG Li1, CHEN Wei2, WANG Libin1, LUO Haibo3,*, YU Zhifang1,*
(1. College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China;2. College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China;3. Faculty of Food Science, Zhejiang Pharmaceutical College, Ningbo 315100, China)
Abstracts: In order to explore the molecular mechanism by which fresh-cut handling accelerates the senescence and quality deterioration of Zizania latifolia, the dynamic proteome changes of whole (control) and fresh-cut Z. latifolia during storage at room temperature (25 ℃) for 0, 3 and 5 days were investigated by using two-dimensional electrophoresis(2-DE). Approximately 660 protein spots were detected on the gels. Totally 39 spots showed a significant (P < 0.05)change in protein abundance based on 2.0-fold difference of which 35 were successfully identified by matrix-assisted laser desorption/ ionization time of flight mass spectrometry compared to the whole Z. latifolia. They could be categorized into seven functional groups, including signal transduction (8.6%), metabolism (22.9%), cell structure (8.6%), stress response and defense (31.4%), protein synthesis (11.4%), senescence (5.7%), and unclear functional proteins (11.4%). During storage,the expressions of 14 spots increased while the other 21 were down-regulated in the whole stem. Meanwhile, six spots(plant basic secretory protein, adenosine kinase, C2 domain, class III peroxidases, LbH_gamma_CA_like, and small Rasrelated GTP-binding protein), which were down-regulated in whole Z. latifolia, were up-regulated in fresh-cut Z. latifolia during storage, suggesting that these proteins may play important roles in the response to wounding stress. Also, fresh-cut significantly promoted the tendency for 14 up-regulated and other 15 down-regulated proteins. These results imply that the promotion of senescence and quality deterioration of Z. latifolia after fresh-cut processing may be due to the production and transduction of wounding signals, the acceleration of carbohydrate and nucleotide catabolism, the disorder of energy homeostasis and the enhancement of free radical damage, as well as the degradation of cell structure.
Keywords:Zizania latifolia; fresh-cut; senescence; quality deterioration; proteomics
Zizania latifolia (Griseb.) Turcz. ex Stapf, botanically different from the three species (Z. aquatica, Z. palustris and Z. texana) grown in North America, is a perennial aquatic grass belonging to tribe Oryzeae of the grass family(Poaceae) in China[1]. Z. latifolia grows along the littorals of freshwater lakes, streams, marshes and pools[2]. It is usually parasitized by the smut fungus (Ustilago esculenta P. Henn.),which stimulates enlargement of the culms of the grass[3-4].The enlarged infected culms have been eaten as a vegetable in China since the l0thcentury, commonly called Jiaobai,Jiaosun, Gusun, Gushou or Gaosun[5]. Because of its highly nutritional and economic value, the vegetable has been widely cultivated in rice fields of South China.
Z. latifolia is very suitable to be processed into fresh-cut product. It is usually cut into cubes, slices or shreds before marketing[6]. However, fresh-cut operations may accelerate the senescence and quality deterioration triggering cut surface browning, tissue lignification, shriveling, microbial contamination, off-flavor, respiratory rate, cellulose and lignin contents increase, which would reduce the shelf life of fresh-cut Z. latifolia to 2-3 days at room temperature[6].Therefore, clarification of the mechanisms involved in senescence and quality deterioration is of great importance to extend the shelf life of fresh-cut products.
Previously, the physiological and biochemical responses in Z. latifolia after fresh-cut operation have been investigated.In general, fresh-cut processing operations destroy the integrity of Z. latifolia, which in turn promote a faster physiological deterioration and biochemical changes such as respiratory activity, ethylene production, reactive oxygen species and malondialdehyde accumulation,lignification and antioxidant-related enzymes changes etc., thus accelerating the rate of senescence and quality deterioration[7]. Nevertheless, these researches mainly focused on wounding stress-induced changes at physiological and biochemical levels.
Fresh-cut products, which are subjected to mechanical damage by cutting operations, are still living organisms.Proteins play crucial roles in the structure and function of all living cells together with nucleic acids, carbohydrates and lipids, forming the biochemical basis of life[8]. Currently,proteomic approaches are being extensively used in fruits and vegetables to gain understanding on the mechanisms related to ripening and regulation[9], senescence[10], stress response[6]and preservation[11-12]. Our previous studies showed that the senescence of postharvest Z. latifolia during storage at 1 ℃ may be a consequence of multi-action,comprising the regulation of material metabolism, change of energy metabolism pathway, decline of reactive oxygen scavenging capacity and degradation of cell structure[10]. In addition, we analyzed the changes in the protein profile of Z. latifolia during refrigerated (1 ℃) storage. Results showed that 25 proteins differentially expressed and identified,with their functions mainly concentrated on cell structure,stress response and defense, and senescence[6]. However, the molecular mechanism of senescence and quality deterioration in fresh-cut Z. latifolia still not well understood. Also, there is limited information available on the wounding-responsive proteins occurring in the vegetable tissues after fresh-cut operation during storage at room temperature.
The objective of this study was to evaluate the changes in protein profile between whole and fresh-cut Z. latifolia during storage at room temperature (25 ℃) for exploring the possible molecular mechanism that fresh-cut operation accelerates senescence and quality deterioration of Z. latifolia.
Fresh Z. latifolia at mature stage were hand-harvested from a commercial farmland in Yixing, Jiangsu, China.
Ethanol, hydrochloric acid, sucrose, mannitol, glycine,L-cysteine, tris-aminomethane, ethylenediaminetetraacetic acid, bovine serum albumin, polyvinylpyrrolidone, Coomassie brilliant blue G-250 and bromophenol blue Nanjing Shoude Testing Devices and Materials Co., Ltd. (China); Sodium dodecyl sulfate, trichloroacetic acid, ammonium persulfate,N,N,N’,N’-tetramethyldiethylamine, trypsin Promega Corporation (USA); Urea, thiourea, 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate, dithiothreitol,iodoacetamide and IPG buffer Sigma-Aldrich Co. (USA).
SPX-320 intelligent biochemical incubator Ningbo Jiangnan Instrument Factory (China); KQ-300DB ultrasonic cleaning machine Kunshan Ultrasonic Instrument Co.,Ltd. (China); FRESCO 17 micro high-speed refrigerated centrifuge Thermo Fisher Scientific Inc. (USA); AJ-30i high-speed refrigerated centrifuge Beckman Instruments Inc. (USA); Versdoc 3000 scanner Bio-Rad Laboratories Inc. (USA); TripleTOF 5600 system AB Sciex Inc. (USA).
1.3.1 Sample preparation
18 kg Z. latifolia with uniform size and the absence of visible defects, were selected, rinsed with tap water, and then randomly divided into two groups. For the first group, about 5 cm were removed from each end of the stems after peeling,then the remainder (mean length (5.00 ± 0.26) cm) were cut into slices of 3-5 mm thickness with a sharp stainless steel knife. The second group without cutting operation was acted as the control. The slices and the whole stem were then packaged into polyethylene plastic bags (thickness: 15 μm,size: 40 cm × 60 cm) with the mass of 400 g (fresh-cut Z. latifolia) and 1 kg (control), respectively. 9 bags per treatment were then stored at 25 ℃ for 0, 3 and 5 d,respectively. Samples (3 bags per treatment) were taken at different storage time, respectively, frozen in liquid nitrogen,and then stored at -20 ℃ until further analysis. For the control, the samples were peeled and removed 5 cm from each end of the stems before being frozen in liquid nitrogen.
1.3.2 Protein extraction and 2-DE analysis
Protein extraction and two-dimensional electrophoresis(2-DE) were carried out as described earlier[6]. Three biological replicates were performed for each storage time.
1.3.3 Image analysis
The gels were scanned using a Versdoc 3000 scanner at 300 dpi and saved as the GSC image files. Image analysis of each gel was performed using the PDQuest 2-DE analysis software (Version 8.0.1).
1.3.4 Tryptic digestion spots
Spots showing statistically significant changes and above a 2-fold threshold were carefully excised from the gels and then digested with trypsin according to the method described by Luo Haibo et al[6].
1.3.5 Identification of proteins by MALDI-TOF/TOF and database search
Protein identification was performed with matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF/TOF). The functional classification of the identified proteins was annotated using the database at http://uniprot.org/uniprot. For protein accessions identified by MASCOT that had no functional annotation, a function was assigned via BLASTP searches of the corresponding protein accessions.
During storage, the symptoms of senescence and quality deterioration in both whole and fresh-cut Z. latifolia were gradually severe including shell etiolation, surface browning,and tissue hollowness in whole Z. latifolia as well as cutting surface browning, and tissue lignification in fresh-cut Z. latifolia (Fig. 1).
Fig. 1 Representatives of whole (A) and fresh-cut (B) Z. latifolia after 3 days of storage at 25 ℃
The proteins from whole and fresh-cut Z. latifolia during storage were extracted and separated by 2-DE. The 2-DE maps were obtained using isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels (17 cm, pH 4-7)followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels before staining with Coomassie brilliant blue G250(Fig. 2). The 2-DE gels were matched and analyzed by PDQuest 2-DE analysis software (Version 8.0.1).Approximately 660 protein spots were detected on the gels.Quantitative image analysis of three replicates of each sample revealed that 39 protein spots showed a more than 2.0-fold increase/decrease in protein abundance in at least one storage time point compared to the whole Z. latifolia of corresponding time point. The specific distribution in Fig. 3 and the magnified views of some differentially expressed proteins were shown in Fig. 4.
Fig. 2 Representative 2-DE gels of whole and fresh-cut Z. latifolia during storage at 25 ℃
Fig. 3 2-DE reference map of Z. latifolia in Fig. 2B1
Fig. 4 Five times magnified views of some differentially abundant proteins marked in Fig. 2
35 out of 39 spots which significantly changes(P ＜ 0.05), were successfully identified and could then be classified into seven functional categories listed in Fig. 5,including signal transduction (8.6%), metabolism (22.9%),cell structure (8.6%), stress response and defense (31.4%),protein synthesis (11.4%), senescence (5.7%), and unclear functional proteins (11.4%). Cluster analysis was carried out on differentially expressed proteins from day 0 to day 5 (Fig. 6),revealing a dynamic proteome profile shift of Z. latifolia at different time points during storage at 25 ℃.
Fig. 5 Functional distribution of identified proteins expressed differentially covering seven protein groups categorized according to their putative functions
As shown in Table 1, 14 out of the 35 differentially expressed and identified proteins were up-regulated while the others were down-regulated during the storage of whole Z. latifolia. For the up-regulated proteins, spot 14(fructokinases), spot 17 (glycosyl hydrolases family 17), spot 19 (2,3-bisphosphoglycerate-independent phosphoglycerate mutase) and spot 26 (diphosphonucleotide phosphatase 1)were related to metabolism; spot 1 (profilin) to cell structure;spot 3 (β-1,3-glucanase precursor), spot 4 (glucan endo-1,3-beta-glucosidase), spot 5 (β-1,3-glucanase precursor), spot 12 (universal stress protein family), spot 21 (thaumatin-like protein isoform 2), spot 22 (glutathione S-transferase) and spot 28 (β-1,3-glucanase) were related to stress response and defense; spot 16 (cysteine protease) and spot 23 (papain-like cysteine proteinase) were related to senescence.
Fig. 6 Cluster analysis of whole and fresh-cut Z. latifolia during storage at 25 ℃
For the down-regulated ones, spot 8 (14-3-3-like protein), spot 10 (C2 domain) and spot 38 (small Ras-related GTP-binding protein) were related to signal transduction;spot 6 (adenosine kinase), spot 20 (transketolase), spot 30(enolase) and spot 37 (isocitrate dehydrogenase NADP+)were related to metabolism; spot 18 (actin) and spot 27 (actin depolymerisation factor/cofilin-like domains) were related to cell structure; spot 2 (plant basic secretory protein), spot 24(class III peroxidases), spot 33 (dehydroascorbate reductase)and spot 34 (chitinase class I) were related to stress response and defense; spot 11 (glycine-rich RNA-binding protein),spot 13 (putative chaperonin 21 precursor), spot 15(putative ribosomal protein S12) and spot 31 (RNA recognition motif) were related to protein synthesis;furthermore, the functions of spot 9 (DREPP plasma membrane polypeptide), spot 29 (WD40 domain), spot 35 (LbH_gamma_CA_like) and spot 36 (ricinB_lectin_2)were still unclarified.
Table1 Differentially expressed proteins of whole and fresh-cut Z. latifolia during storage at 25 ℃
a. Spot numbers refer to Fig. 3; b. Functional classification.
On the other hand, when compared to control tissue, the responses of 29 proteins during storage were enhanced after fresh-cut operations. However, the expressions of spot 2, spot 6, spot 10, spot 24, spot 35,and spot 38, which decreased during storage of whole Z. latifolia, were up-regulated after fresh-cut operation,indicating that they might play vital roles in response to wounding stress.
During senescence and quality deterioration of Z. latifolia, a variety of physiological and biochemical responses occurred, in association with the changes in the protein profile, which could be influenced by a variety of internal or external factors. In this study, changes in abundance 35 proteins between whole and freshcut Z. latifolia during storage were observed based on 2.0-fold difference. They are suggested to play roles in signal transduction, carbohydrate metabolism, nucleotide metabolism, cell structure, stress response and defense,protein synthesis, as well as senescence.
Signal transduction is the process which begins with the binding of specific ligands to receptors located at the surface of the plasma membrane, thus causing an intracellular metabolic change[13]. Three proteins (spot 8, spot 10,spot 38) involved in signal transduction were identified.14-3-3-like proteins (spot 8) are important regulators of signal transduction pathways through binding to multiple functionally diverse signaling proteins such as kinases,phosphatases and transmembrane receptors[14-15]. C2 domains(spot 10) are usually Ca2+-dependent membrane-targeting module found in many cellular proteins, functions in membrane trafficking or signal transduction[16]. Small Rasrelated GTP-binding protein (spot 38) has diverse biological functions, such as nuclear transport, DNA replication, spindle formation during mitosis and cell division[17]. All of them decreased during the storage of whole Z. latifolia; however,spot 10 and spot 38 were up-regulated by fresh-cut operation,suggesting that signal transduction was enhanced.
An important strategy for plant to adapt different abitic/biotic stresses is through the regulation of physiological metabolism[18]. Two proteins involved in nucleotide metabolism were identified: adenosine kinase (AK, spot 6) is involved in the purine salvage pathway and plays a key role in the prevention of adenosine accumulation and supports the metabolic cycling of adenosine[19], while diphosphonucleotide phosphatase 1 (DPP, spot 26) catalyzes the hydrolysis of a wide range of activated phosphoric acid mono- and di-esters and anhydrides at pH in the range of 5 to 6[20]. In the present study, the expressions of both AK and DPP were up-regulated by fresh-cut operation at 3rdand/or 5thday of storage,indicating that nucleotide catabolism was accelerated.
Fructokinases (FRKs, spot 14), glycosyl hydrolases family 17 (GHF, spot 17), 2,3-bisphosphoglycerateindependent phosphoglycerate mutase (BIPM, spot 19),transketolase (TK, spot 20), enolase (spot 30) and isocitrate dehydrogenase NADP+(IDH, spot 37) play important roles in carbohydrate metabolism[21]. Of these, GHF belongs to the glycosyl hydrolase family and catalyzes the hydrolysis of polysaccharides to monosaccharides[22]. Fresh-cut significantly increased the expression of GHF during storage,suggesting an enhanced carbohydrate catabolism. FRKs,BIPM and enolase are involved in glycolysis[23], IDH is the key enzyme in the tricarboxylicacidcycle acid (TCA)cycle[24], while TK is the key enzyme of the pentose phosphate pathway[25]. Glycolysis, TCA cycle and pentose phosphate pathways provide energy and inter-conversion building blocks for metabolites synthesis[11]. The downregulated expressions of these proteins after fresh-cut operation implied that the supplyment of energy decreased.
The plant cell walls are crucial for providing shape to many different cell types, forming the interface between adjacent cells, as well as resisting the invasion of pathogens[26]. Three proteins related to cell structure were identified, including profilin (spot 1), actin (spot 18) and actin depolymerisation factor/cofilin-like domains (ADF,spot 27). Actin is a ubiquitous protein involved in the formation of filaments that are a major component of the cytoskeleton[24]. Profilin may link the cytoskeleton with major signaling pathways by interacting with components of the phosphatidylinositol cycle and Ras pathway, which plays a central role in the assembly of branched actin filament networks[27], while ADF acting as a family member of essential eukaryotic actin regulatory proteins could enhance the turnover rate of actin and interact with actin monomers as well as actin filaments[28]. In contrast to actin and profilin,the expression of ADF increased in fresh-cut Z. latifolia during storage, suggesting that fresh-cut accelerated the cell structure disassembly, which was further validated by cell ultrastructural observation using transmission electron microscope (unpublished data).
Stress response, which is mediated via profound changes in gene expression, causes changes in composition of plant transcriptome, proteome and metabolome[29]. Of the eleven stress response and defense proteins, eight were related to disease resistance. Plant basic secretory protein (BSP, spot 2)is believed to be part of the plants defense mechanism against pathogens[30], β-1,3-glucanase precursor (spot 3 and 5), glucan endo-1,3-β-glucosidase (spot 4) and β-1,3-glucanase (spot 28) may provide a degree of protection against microbial invasion through their ability to degrade fungal cell wall polysaccharides[31], universal stress protein family (USP, spot 12) is a small cytoplasmic bacterial protein whose expression is enhanced when the cell is exposed to stress agents[10],thaumatin-like protein isoform 2 (TLP, spot 21) plays a role in host defense and developmental processes[32], while chitinase class I (spot 34) catalyzes the hydrolysis of the β-1,4-N-acetyl-D-glucosamine linkages in chitin polymers of fungal cell walls[33]. The significant up-regulation of these proteins, except chitinase class I, provide a good indication that fresh-cut induced the improvement of anti-pathogen capability compared to the control.
Senescence is a vital aspect of vegetable life cycles, and directly affects vegetable quality and resistance to pathogens.Reactive oxygen species (ROS), as the primary mediators of oxidative damage in plants, are involved in senescence[10].Furthermore, superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR),glutathione S-transferase (GST) and peroxidases (POD) are antioxidant enzymes related to ROS metabolism[34]. In this study, three ROS metabolism related proteins identified were GST (spot 22), class III peroxidases (PODIII, spot 24) and DHAR (spot 33). GST and DHAR are involved in the ascorbate-glutathione cycle, which has been regarded as one of the most important antioxidant pathways for hydrogen peroxide (H2O2) detoxification[34]. PODIII is responsible for the physiological function of indole-3-acetic acid degradation, lignin biosynthesis, wound healing,general stress response and pathogen defense as well as H2O2detoxification[24]. In previous studies, POD activity increased in fresh-cut Z. latifolia during storage at 1 ℃. This result was further validated by POD expression pattern in the present study. Based on these results, we speculated that ROS damage could be implicated in senescence and quality deterioration of fresh-cut Z. latifolia.
The regulation of protein synthesis plays a key role in the control of cell growth, proliferation, and apoptosis[35]. In this study, four proteins involved in protein synthesis, namely glycine-rich RNA-binding protein (GRP, spot 11), putative chaperonin 21 precursor (C21P, spot 13), putative ribosomal protein S12 (RPS12, spot 15) and RNA recognition motif(RRM, spot 31) were identified. GRP is suggested to play a role in response to environmental stresses[36], C21P helps nascent peptide chain precursor reconciliation folded protein peptide chain folds into a protein having a biological function conformation[37], RPS12 located at the subunit interface is proposed to play critical roles in interacting with the tRNA substrates and the large subunit[38], RRM, a highly abundant domain of the proteins in eukaryotes, participates in posttranscriptional gene expression processes, including mRNA and rRNA processing, RNA export, and RNA stability[39]. All of these proteins were significantly down-regulated by freshcut during storage, indicating that fresh-cut accelerated the protein dissimilation in Z. latifolia.
Senescence, caused by pathogens, environmental stresses, or inherent physiological changes, is a collective term for the deteriorative changes in living organisms which lead to death. Two proteins, cysteine proteases (CPs, spot 16)and papain-like CPs (spot 23), were supposed to be related with the senescence. In this report, CPs (spot 16) and papainlike CPs (spot 23) significantly increased in both whole and fresh-cut Z. latifolia during storage. It is likely that the wounding stress caused by fresh-cut enhanced the protein catabolism, which may play a major role in senescence and quality deterioration of Z. latifolia.
Four spots subjected to mass spectrometry were identified as DREPP plasma membrane polypeptide (spot 9),WD40 domain (spot 29), LbH_gamma_CA_like (spot 35)and RicinB_lectin_2 (spot 36). Although they were searched by BLASTP matches of the corresponding protein accessions,their functions were still unclear. The expressions of spot 9, 29, and 36 showed similar expression pattern in both whole and fresh-cut Z. latifolia during storage; nevertheless,LbH_gamma_CA_like (spot 35), which was down-regulated in whole stem, increased in fresh-cut tissue during storage.These results suggested that LbH_gamma_CA_like might play directly/indirectly role in response to wounding stress.
This study demonstrated that when compared to integral stem, the dynamic proteome changes of 35 proteins identified were significantly altered after fresh-cut operation in association with the accelerated senescence and quality deterioration. Based on their functions, the possible mechanisms of fresh-cut operation accelerating senescence and quality deterioration of Z. latifolia might be due to: 1)enhancement of production and transduction of wounding signals; 2) enhancement of the carbohydrate, nucleotide and protein catabolism, but attenuation of the protein assimilation,glycolysis, TCA cycle and pentose phosphate pathways,thus reducing the energy supplement; 3) inducement of the expression of pathogenesis-related proteins and antioxidant enzymes; 4) acceleration of the cell structure disassembly.This study has put forward interpretable data and provided new perspectives on the mechanism of fresh-cut operations accelerating senescence and quality deterioration of Z. latifolia. However, further detailed investigation of these proteins’ specific roles and their functional correlation with wounding response should be performed to better understand the mechanism of fresh-cut operations accelerate senescence and quality deterioration.
References:
[1] FAN X R, REN X R, LIU Y L, et al. Genetic structure of wild rice Zizania latifolia and the implications for its management in the Sanjiang Plain, Northeast China[J]. Biochemical Systematics and Ecology, 2016, 64(1): 81-88. DOI:10.1016/j.bse.2015.11.017.
[2] HAN S F, ZHANG H, ZHAI C K. Protective potentials of wild rice(Zizania latifolia (Griseb) Turcz) against obesity and lipotoxicity induced by a high-fat/cholesterol diet in rats[J]. Food and Chemical Toxicology, 2012, 50(7): 2263-2269. DOI:10.1016/j.fct.2012.04.039.
[3] CHOI J H, SUZUKI T, KAWAGUCHI T, et al. Makomotines A to D from Makomotake, Zizania latifolia infected with Ustilago esculenta[J]. Tetrahedron Letters, 2014, 55(26): 3596-3599.DOI:10.1016/j.tetlet.2014.04.125.
[4] YAN N, WANG X Q, XU X F, et al. Plant growth and photosynthetic performance of Zizania latifolia are altered by endophytic Ustilago esculenta infection[J]. Physiological and Molecular Plant Pathology,2013, 83(6): 75-83. DOI:10.1016/j.pmpp.2013.05.005.
[5] LING J G, LI J, KANG M L, et al. Application of slightly acidic electrolyzed water (SAEW) in preservation of Zizania latifolia stems[J]. Food Science, 2015, 36(22): 250-254. DOI:10.7506/spkx1002-6630-201522047.
[6] LUO Haibo, BAO Y H, JIANG J, et al. Proteome changes of fresh-cut Zizania latifolia during refrigerated (1 ℃) storage[J]. European Food Research and Technology, 2012, 235(6): 1011-1021. DOI:10.1007/s00217-012-1828-2.
[7] ZHOU T, XU S Y, WANG Z, et al. Effects of MAP on quality of minimally processed water bamboo shoot and its mathematical model set-up[J]. Food Science, 2006, 27(3): 235-238. DOI:10.3321/j.issn:1002-6630.2006.03.057.
[8] LAŠTOVIČKOVÁ M, BOBÁLOVÁ J. MS based proteomic approaches for analysis of barley malt[J]. Journal of Cereal Science,2012, 56(3): 519-530. DOI:10.1016/j.jcs.2012.07.013.
[9] LÓPEZ-VIDAL O, CAMEJO D, RIVERA-CABRERA F, et al.Mitochondrial ascorbate-glutathione cycle and proteomic analysis of carbonylated proteins during tomato (Solanum lycopersicum) fruit ripening[J]. Food Chemistry, 2016, 194(3): 1064-1072. DOI:10.1016/j.foodchem.2015.08.055.
[10] LUO H B, JIANG L, BAO Y H, et al. Changes of protein profile in Zizania latifolia during cooling storage[J]. Transactions of the Chinese Society of Agricultural Engineering, 2014, 30(1): 285-292.DOI:10.3969/j.issn.1002-6819.2014.01.036.
[11] HUANG J S, ZHANG Y X, JIANG L, et al. Comparative proteomics analysis of differential proteins in response to 6-benzylaminopurine treatment in Pteridium aquilinum senescence[J]. Postharvest Biology and Technology, 2016, 116(6): 66-74. DOI:10.1016/j.postharvbio.2015.12.030.
[12] MITHOE S C, MENKE F L H. Phosphoproteomics perspective on plant signal transduction and tyrosine phosphorylation[J]. Phytochemistry,2011, 72(10): 997-1006. DOI:10.1016/j.phytochem.2010.12.009.
[13] JIANG L, KANG R Y, ZHANG L, et al. Differential protein profiles of postharvest Gynura bicolor DC leaf treated by 1-methylcyclopropene and ethephon[J]. Food Chemistry, 2015, 176(1): 27-39. DOI:10.1016/j.foodchem.2014.11.081.
[14] HOPFF D, WIENKOOP S, LÜTHJE S. The plasma membrane proteome of maize roots grown underlow and high iron conditions[J].Journal of Proteomics, 2013, 91(10): 605-618. DOI:10.1016/j.jprot.2013.01.006.
[15] SWIGONSKA S, WEIDNER S. Proteomic analysis of response to long-term continuous stress in roots of germinating soybean seeds[J].Journal of Plant Physiology, 2013, 170(5): 470-479. DOI:10.1016/j.jplph.2012.11.020.
[16] ANTAL C E, CALLENDER J A, KORNEV A P, et al. Intramolecular C2 domain-mediated autoinhibition of protein kinase CbII[J]. Cell Reports, 2015, 12(8): 1252-1260. DOI:10.1016/j.celrep.2015.07.039.
[17] HURKMAN W J, VENSEL W H, TANAKA C K, et al. Effect of high temperature on albumin and globulin accumulation in the endosperm proteome of the developing wheat grain[J]. Journal of Cereal Science,2009, 49(1): 12-23. DOI:10.1016/j.jcs.2008.06.014.
[18] KHRAIWESH B, ZHU J K, ZHU J. Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants[J]. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2012, 1819(2):137-148. DOI:10.1016/j.bbagrm.2011.05.001.
[19] JIANG J, JIANG L, ZHANG L, et al. Changes of protein profile in fresh-cut lotus tuber before and after browning[J]. Journal of Agricultural and Food Chemistry, 2012, 60(15): 3955-3965.DOI:10.1021/jf205303y.
[20] OLCZAK M, CIURASZKIEWICZ J, WÓJTOWICZ H, et al.Diphosphonucleotide phosphatase/phosphodiesterase (PPD1)from yellow lupin (Lupinus luteus L.) contains an iron-manganese center[J]. FEBS Letters, 2009, 583(19): 3280-3284. DOI:10.1016/j.febslet.2009.09.024.
[21] BERG J, TYMOCZKO J, STRYER L. Biochemistry[M]. 5th ed. New York: W. H. Freeman and Company, 2002: 93-126.
[22] BAZARGANI M M, SARHADI E, BUSHEHRI A A S, et al. A proteomics view on the role of drought-induced senescence and oxidative stress defense in enhanced stem reserves remobilization in wheat[J]. Journal of Proteomics, 2011, 74(10): 1959-1973.DOI:10.1016/j.jprot.2011.05.015.
[23] TURNER J F, TURNER D H. The regulation of glycolysis and the pentose phosphate pathway[J]. Metabolism & Respiration, 1980, 2:279-316. DOI:10.1016/B978-0-12-675402-5.50013-1.
[24] KONG F J, OYANAGI A, KOMATSU S. Cell wall proteome of wheat roots under flooding stress using gel-based and LC MS/MS-based proteomics approaches[J]. Biochimica et Biophysica Acta, 2010,1804(1): 124-136. DOI:10.1016/j.bbapap.2009.09.023.
[25] YIN X J, SAKATA K, NANJO Y, et al. Analysis of initial changes in the proteins of soybean root tip under flooding stress using gel-free and gel-based proteomic techniques[J]. Journal of Proteomics, 2014,106(6): 1-16. DOI:10.1016/j.jprot.2014.04.004.
[26] FANG X J, GAO H Y, SONG L L, et al. Hypobaric storage maintains postharvest quality and regulates cell wall metabolism in water bamboo shoot[J]. Transactions of the Chinese Society of Agricultural Engineering, 2013, 29(12): 257-263. DOI:10.3969/j.issn.1002-6819.2013.12.032.
[27] DIDRY D, CARLIER M F, PANTALONI D. Synergy between actin depolymerizing factor/cofilin and profilin in increasing actin filament turnover[J]. Journal of Biological Chemistry, 1998, 273(40): 25602-25611. DOI:10.1074/jbc.273.40.25602.
[28] ALLWOOD E G, SMERTENKO A P, HUSSEY P J. Phosphorylation of plant actin-depolymerising factor by calmodulin-like domain protein kinase[J]. FEBS Letters, 2001, 499(1): 97-100. DOI:10.1016/S0014-5793(01)02528-5.
[29] PÉREZ-CLEMENTE R M, VIVES V, ZANDALINAS S I,et al. Biotechnological approaches to study plant responses to stress[J]. BioMed Research International, 2013: 1-10.DOI:10.1155/2013/654120.
[30] DEVNATH S, KATAOKA T, MIURA K, et al. Cgr11 encodes a secretory protein involved in cell adhesion[J]. European Journal of Cell Biology, 2009, 88(9): 521-529. DOI:10.1016/j.ejcb.2009.04.003.
[31] FINNIE C, BAK-JENSEN K S, LAUGESEN S, et al. Differential appearance of isoforms and cultivar variation in protein temporal profiles revealed in the maturing barley grain proteome[J]. Plant Science, 2006, 170(4): 808-821. DOI:10.1016/j.plantsci.2005.11.012.
[32] XU C P, SULLIVAN J H, GARRETT W M, et al. Impact of solar Ultraviolet-B on the proteome in soybean lines differing in flavonoid contents[J]. Phytochemistry, 2008, 69(1): 38-48. DOI:10.1016/j.phytochem.2007.06.010.
[33] WANG L Y, WANG Y S, CHENG H, et al. Cloning of the Aegiceras corniculatum class I chitinase gene (AcCHI I) and the response of AcCHI I mRNA expression to cadmium stress[J]. Ecotoxicology,2015, 24(7/8): 1705-1713. DOI:10.1007/s10646-015-1502-0.
[34] ZHANG Y J, WANG W, YANG H L, et al. Molecular properties and functional divergence of the dehydroascorbate reductase gene family in lower and higher plants[J]. PLoS ONE, 2015, 10(12): e0145038.DOI:10.1371/journal.pone.0145038.
[35] THOMPSON J E, HOPKINS M T, TAYLOR C, et al. Regulation of senescence by eukaryotic translation initiation factor 5A: implications for plant growth and development[J]. Trends in Plant Science, 2004,9(4): 174-179. DOI:10.1016/j.tplants.2004.02.008.
[36] CIUZAN O, HANCOCK J, PAMFIL D, et al. The evolutionarily conserved multifunctional glycine-rich RNA-binding proteins play key roles in development and stress adaptation[J]. Physiologia Plantarum,2015, 153(1): 1-11. DOI:10.1111/ppl.12286.
[37] WEN F P, ZHANG Z H, BAI T, et al. Proteomics reveals the effects of gibberellic acid (GA3) on salt-stressed rice (Oryza sativa L.)shoots[J]. Plant Science, 2010, 178(2): 170-175. DOI:10.1016/j.plantsci.2009.11.006.
[38] APHASIZHEVA I, MASLOV D A, APHASIZHEV R. Kinetoplast DNA-encoded ribosomal protein S12: a possible functional link between mitochondrial RNA editing and translation in Trypanosoma brucei[J]. RNA Biology, 2013, 10(11): 1679-1688. DOI:10.4161/rna.26733.
[39] TOBA G, WHITE K. The third RNA recognition motif of Drosophila ELAV protein has a role in multimerization[J]. Nucleic Acids Research, 2008, 36(4): 1390-1399. DOI:10.1093/nar/gkm1168.
完整和鲜切茭白常温贮藏期间的比较蛋白质组学研究
王韦华1，姜 丽1，陈 伟2，王利斌1，罗海波3,*，郁志芳1,*
（1.南京农业大学食品科学技术学院，江苏 南京 210095；2.浙江万里学院生物与环境学院，浙江 宁波 315100；3.浙江医药高等专科学校食品学院，浙江 宁波 315100）
摘 要：为探讨切分加速茭白衰老和品质劣变的分子生物学机制，应用双向电泳（two-dimensional electrophoresis，2-DE）结合质谱鉴定技术，研究了完整（对照）和鲜切茭白常温（25 ℃）贮藏期间蛋白质组的动态变化。结果显示：2-DE凝胶上可检测到约660 个蛋白点，其中鲜切茭白39 个蛋白表达量达到2.0 倍以上显著差异（P＜0.05），经基质辅助激光解吸电离飞行时间质谱分析成功鉴定出35 个蛋白。按照其功能可分为7 类，即信号转导（8.6%）、代谢（22.9%）、细胞结构（8.6%）、胁迫响应与防御（31.4%）、蛋白质合成（11.4%）、衰老（5.7%）和功能未知蛋白（11.4%）。完整茭白贮藏期间，14 个蛋白表达上调，21 个蛋白表达下调。在21 个表达下调的蛋白中，鲜切诱导了其中6 个蛋白（植物基础分泌蛋白、腺苷激酶、C2结构域蛋白、Ⅲ类过氧化物酶、LbH_gamma_CA_like、小G蛋白Ras）的上调表达，表明这6 个蛋白可能在伤害响应中发挥重要作用。同时，鲜切还显著增强了14 个上调表达蛋白和其余15 个下调表达蛋白的上升或下降趋势。以上差异表达蛋白的功能分析表明，鲜切加速茭白衰老和品质劣变可能与伤害信号产生和转导、自由基损伤加剧、碳水化合物和核酸分解代谢加速、能量代谢平衡失调及细胞结构降解密切相关。
收稿日期：2017-01-13
基金项目：国家自然科学基金青年科学基金项目（31401612）；浙江省自然科学基金面上项目（LY14C200005）；浙江省重中之重学科开放基金项目（KF2012006）
第一作者简介：王韦华（1993—），女，硕士研究生，研究方向为采后生物学。E-mail：weihuawang_0216@126.com
*通信作者简介：罗海波（1979—），男，副教授，博士，研究方向为果蔬采后生物学与处理技术。E-mail：luohaibo_1216@126.com郁志芳（1960—），男，教授，博士，研究方向为果蔬采后生物学与贮藏加工。E-mail：yuzhifang@njau.edu.cn
关键词：茭白；鲜切；衰老；品质劣变；蛋白质组学
DOI:10.7506/spkx1002-6630-201813002
中图分类号：Q816
文献标志码：A
文章编号：1002-6630（2018）13-0007-10
引文格式：WANG Weihua, JIANG Li, CHEN Wei, et al. Comparative proteomics study on whole and fresh-cut Zizania latifolia during storage at room temperature[J]. 食品科学, 2018, 39(13): 7-16.
DOI:10.7506/spkx1002-6630-201813002. http://www.spkx.net.cn
WANG Weihua, JIANG Li, CHEN Wei, et al. Comparative proteomics study on whole and fresh-cut Zizania latifolia during storage at room temperature[J]. Food Science, 2018, 39(13): 7-16. (in English with Chinese abstract)
DOI:10.7506/spkx1002-6630-201813002. http://www.spkx.net.cn