Analysis of protein changes during grape berry ripening by 2-DE and MALDI-TOF

Grape berry, a nonclimacteric fruit, during ripening turns from green, hard and acidic to coloured, soft and sweet. Many studies have focused on dynamic changes of mRNA levels, metabolites, sugars or individual proteins, but this is the first report of a proteomic approach applied to the screening of the most prominent variations that take place during berry ripening. Vitis vinifera cv. 'Nebbiolo Lampia' berries were collected at 10-day intervals, starting 1 month after flowering to complete ripe stage; total protein extracts from deseeded berries were separated by 2-DE. A total of 730 spots were detected in the 2-DE gels. 118 protein spots, differentially expressed during berry development, were subjected to MALDI-TOF analysis. Ninety-three of them were identified, corresponding to 101 proteins. The majority of proteins were linked to metabolism, energy and protein synthesis and fate. In comparison to published surveys of major berry proteins, fewer proteins related to stress response and more proteins related to cell structure were differentially expressed. Our data confirm a general decrease of glycolysis during ripening, and an increase of PR proteins in the range of 20-35 kDa. They furthermore suggest that oxidative stress decreases during ripening while extensive cytoskeleton rearrangement takes place in this period.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

Proteomic analysis of differentially accumulated proteins during ripening and in response to 1-MCP in papaya fruit

Papaya (Carica papaya L.) is a climacteric fruit susceptible to postharvest losses due to the ethylene-induced ripening. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has been used worldwide as a safe postharvest non-toxic agent, but the physiological and biochemical modifications induced by 1-MCP are not well understood. Using the 2-DE analysis, we report the changes in the protein profiles after 6 and 18 days of postharvest and the effect of the effect of 1-MCP treatment on fruits. Twenty seven protein spots showing differences in abundance during ripening were successfully identified by nano-LC-ESI/MS/MS. Some spots corresponded to the cell wall degrading enzymes related to fruit ripening; others were involved in oxidative damage protection, protein folding, and cell growth and survival that were induced by 1-MCP. This is the first proteomic report analyzing the effect of 1-MCP in papaya ripening. The present data will help to shed light on papaya fruit ripening process.     

Regulation of the expression of lipoxygenase genes in <Emphasis Type="Italic">Prunus persica</Emphasis> fruit ripening

Three genes of the lipoxygenase (LOX) family in peach (Prunus persica var. compressa cv. Ruipan 4) were cloned, and their expression patterns during fruit ripening were analyzed using real-time quantitative PCR. All of the three peach LOX genes had been expressed during fruit ripening; however, their expression patterns were significantly different. During the normal ripening of peach fruits, the expression levels of PpLox1, PpLox2 and PpLox3 increased in varying degrees accompanying upsurge of ethylene evolution. After treated by methyl jasmonic acid (MeJA), the peak of ethylene releasing occurred in advance, and the declining rate of fruit hardness was accelerated, the expression level of the three peach LOX genes in fruits markedly enhanced at the early stage of storage, but significantly decreased at the late storage stage. So, it could be suggested that all three LOXs relate to fruit ripening; however, their functions might be different. PpLox1 expression increase along with the upsurge of ethylene evolution in both control and MeJA-treated peach fruits suggested that PpLox1 probably played a major role in the peach fruit ripening. Expression peak of PpLox2 appeared at the 1 DAH (days after harvest) in both control and MeJA-treated peach fruits, while obvious changes in ethylene evolution and fruit hardness was not observed, which suggested that the rise of PpLox2 expression can be induced by certain stimulation related to ripening, such as harvesting stress and MeJA treatment. The expression of PpLox3 kept a lower level in the natural ripening fruits, whereas raced up at the early stage of storage in the fruits treated with MeJA, which indicated that PpLox3 was expressed inductively and had minor roles during the normal ripening of peach fruits, but when encountered with external stimulation, its expression level would rapidly enhance and accelerate the ripening of peach fruit.     

Cloning of 9-cis-epoxycarotenoid dioxygenase (NCED) gene and the role of ABA on fruit ripening

In order to understand more details about the role of abscisic acid (ABA) in fruit ripening and senescence, six 740 bp cDNAs (LeNCED1, LeNCED2, PpNCED1, VVNCED1, DKNCED1 and CMNCED1) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) as a key enzyme in ABA biosynthesis, were cloned from fruits of tomato, peach, grape, persimmon and melon using an RT-PCR approach. A Blast homology search revealed a similarity of amino acid 85.76% between the NCEDs. A relationship between ABA and ethylene during ripening was also investigated. At the mature green stage, exogenous ABA treatment increased ABA content in flesh, and promoting ethylene synthesis and fruit ripening, while treatment with nordihydroguaiaretic acid (NDGA), inhibited them, delayed fruit ripening and softening. However, ABA inhibited the ethylene synthesis obviously while NDGA promoted them when treated the immature fruit with these chemicals. At the breaker, NDGA treatment cannot block ABA accumulation and ethylene synthesis. Based on the results obtained in this study, it was concluded that ABA plays different role in ethylene synthesis system in different stages of tomato fruit ripening.Key words: tomato, NCED gene, ABA, ethylene, fruit ripening, peach, grape, persimmon, melon     

A DIGE-based quantitative proteomic analysis of grape berry flesh development and ripening reveals key events in sugar and organic acid metabolism

Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components, such as sugars, acids, flavours, anthocyanins, tannins, etc., are accumulated during the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance the understanding of the berry development and ripening processes. Here, the developmental analysis of V. vinifera cv. Muscat Hamburg berries is reported at protein level, from fruit set to full ripening. A top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry led to identification and quantification of 156 and 61 differentially expressed proteins in green and ripening phases, respectively. Two key points in development, with respect to changes in protein level, were detected: end of green development and beginning of ripening. The profiles of carbohydrate metabolism enzymes were consistent with a net conversion of sucrose to malate during green development. Pyrophosphate-dependent phosphofructokinase is likely to play a key role to allow an unrestricted carbon flow. The well-known change of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch in abundance between sucrose synthase and soluble acid invertase. The role of the identified proteins is discussed in relation to their biological function, grape berry development, and to quality traits. Another DIGE experiment comparing fully ripe berries from two vintages showed very few spots changing, thus indicating that protein changes detected throughout development are specific.     

Postharvest temperature influences volatile lactone production via regulation of acyl-CoA oxidases in peach fruit

The biosynthesis of volatile compounds in plants is affected by environmental conditions. Lactones are considered to be peach‐like aroma volatiles; however, no enzymes or genes associated with their biosynthesis have been characterized. White‐fleshed (cv. Hujingmilu) and yellow‐fleshed (cv. Jinxiu) melting peach (Prunus persica L. Batsch) fruit were used as materials in two successive seasons and responses measured to four different temperature treatments. Five major lactones accumulated during postharvest peach fruit ripening at 20 °C. Peach fruit at 5 °C, which induces chilling injury (CI), had the lowest lactone content during subsequent shelf life after removal, while 0 °C and a low‐temperature conditioning (LTC) treatment alleviated development of CI and maintained significantly higher lactone contents. Expression of PpACX1 and activity of acyl‐CoA oxidase (ACX) with C16‐CoA tended to increase during postharvest ripening both at 20 °C and during shelf life after removal from cold storage when no CI was developed. There was a positive correlation between ACX and lactones in peach fruit postharvest. Changes in lactone production in response to temperatures are suggested to be a consequence of altered expression of PpACX1 and long‐chain ACX activity.     

桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

Reversible Inhibition of Tomato Fruit Gene Expression at High Temperature (Effects on Tomato Fruit Ripening)

The reversible inhibition of three ripening-related processes by high-temperature treatment (38[deg]C) was examined in tomato (Lycopersicon esculentum L. cv Daniella) fruit. Ethylene production, color development, and softening were inhibited during heating and recovered afterward, whether recovery took place at 20[deg]C or fruit were first held at chilling temperature (2[deg]C) after heating and then placed at 20[deg]C. Ethylene production and color development proceeded normally in heated fruit after 14 d of chilling, whereas the unheated fruit had delayed ethylene production and uneven color development. Levels of mRNA for 1-aminocyclopropane-1-carboxylic acid oxidase, phytoene synthase, and polygalacturonase decreased dramatically during the heat treatment but recovered afterward, whereas the mRNA for HSP17 increased during the high-temperature treatment and then decreased when fruit were removed from heat. As monitored by western blots, the HSP17 protein disappeared from fruit tissue after 3 d at 20[deg]C but remained when fruit were held at 2[deg]C. The persistence of heat-shock proteins at low temperature may be relevant to the protection against chilling injury provided by the heat treatment. Protein levels of 1-aminocyclopropane-1-carboxylic acid oxidase and polygalacturonase also did not closely follow the changes in their respective mRNAs. This implied both differences in relative stability and turnover rates of mRNA compared to protein and nontranslation of the message that accumulated in low temperature. The results suggest that high temperature inhibits ripening by inhibiting the accumulation of ripening-related mRNAs. Ripening processes that depend on continuous protein synthesis including ethylene production, lycopene accumulation, and cell-wall dissolution are thereby diminished.     

Deciphering the metabolic pathways influencing heat and cold responses during post‐harvest physiology of peach fruit

Peaches are highly perishable and deteriorate quickly at ambient temperature. Cold storage is commonly used to prevent fruit decay; however, it affects fruit quality causing physiological disorders collectively termed ‘chilling injury’ (CI). To prevent or ameliorate CI, heat treatment is often applied prior to cold storage. In the present work, metabolic profiling was performed to determine the metabolic dynamics associated with the induction of acquired CI tolerance in response to heat shock. ‘Dixiland’ peach fruits exposed to 39 °C, cold stored, or after a combined treatment of heat and cold, were compared with fruits ripening at 20 °C. Dramatic changes in the levels of compatible solutes such as galactinol and raffinose were observed, while amino acid precursors of the phenylpropanoid pathway were also modified due to the stress treatments, as was the polyamine putrescine. The observed responses towards temperature stress in peaches are composed of both common and specific response mechanisms to heat and cold, but also of more general adaptive responses that confer strategic advantages in adverse conditions such as biotic stresses. The identification of such key metabolites, which prime the fruit to cope with different stress situations, will likely greatly accelerate the design and the improvement of plant breeding programs.     

Assessment of Prunus persica fruit softening using a proteomics approach

Fruit ripening in Prunus persica involves a number of physiological changes, being one of the most significant the mesocarp softening in melting varieties. In order to get a better understanding of the molecular processes involved in this phenomenon, the protein accumulation patterns in firm and soft fruit of three peach and two nectarine melting flesh varieties were assessed using 2D gel analysis. A General Linear Model (GLM) two-way analysis of variance determined that 164 of the 621 protein spots analyzed displayed a differential accumulation associated with the softening process. Among them, only 14 proteins changed their accumulation in all the varieties assessed, including proteins mostly involved in carbohydrates and cell wall metabolism as well as fruit senescence. The analysis among varieties showed that 195 and 189 spots changed within the firm and soft fruit conditions, respectively. Despite the changes in relative abundance in the spot proteins, the proteome is conserved among varieties and during the transition from firm to soft fruit. Only two spots proteins exhibited a qualitative change in all the conditions assessed. These results are in agreement with the notion that Prunus persica commercial varieties have a narrow genetic background.     

Comparative proteomic analysis reveals differentially expressed proteins in Caenorhabditis elegans pgl-1 mutants grown at 20°C and 25°C

PGL-1 is an RNA-binding protein component of germ granules and essential for fertility in Caenorhabditis elegans. To clarify the molecular function of PGL-1, we performed comparative proteomic analysis using 2-D DIGE and LC-MS/MS. Five groups of synchronized adult hermaphrodites were analyzed: (1) wild-type N2 grown at 20°C, (2) pgl-1(bn101) mutants grown at 20°C, (3) pgl-1(bn101) mutants grown at 20°C then upshifted to 25°C after the L1 stage, (4) pgl-1(ct131) mutants grown at 20°C, and (5) pgl-1(ct131) mutants grown at 20°C then upshifted to 25°C after the L1 stage. The five groups were divided into two experimental sets for 2-D DIGE: set A included N2 and pgl-1(bn101) mutants, and set B included N2 and pgl-1(ct131) mutants. Dunnett's test indicated 90 and 100 specific spots, respectively, with significantly different expression levels from the rest of the experimental set (q≤0.1). Among them, 69 and 58 spots, respectively, were analyzed by LC-MS/MS. Finally, we identified 19 proteins from 24 specific spots common to both the experimental sets. RNAi analysis indicated that decreased eef-1G expression is strongly associated with the temperature-sensitive sterile phenotype of pgl-1. Our results suggest that PGL-1 is closely involved in translational processes during C. elegans germline development.