Thermal inactivation of pectin methylesterase,polygalacturonase, and peroxidase in tomato juice

Thermal inactivation kinetics have been determined for pectin methylesterase (PME), polygalacturonase (PG), and peroxidase (POD) in tomato juice. Two parameters, the inactivation rate constant (k) at a reference temperature and the activation energy for inactivation (E(a)), were determined for each enzyme. For PME and PG, the k and E(a) values reported here do not agree with those in several previously published reports. These differences can be explained either by the differences in pH values used for inactivation determinations or by inadequacies in the heating methods used in some previous studies. POD showed simple first-order inactivation kinetics and was less thermally stable than either PME or PG. When different cultivars of tomatoes were evaluated, there was no difference in the thermal inactivation kinetics of these enzymes.     

番茄果实中成熟衰老相关small RNAs的最新研究进展

番茄(Lycopersicon esculentum)是研究果实成熟衰老的模式材料,其成熟是一个多基因调控的高度协调的遗传调控过程,伴随着色泽、质地、风味、香气和其他代谢等许多显著的生理生化变化.近年来,small RNAs作为一种新型的转录后调控因子受到越来越多的关注,几乎参与了动植物中所有的生理和生化过程.在植物中参与生长发育、果实成熟、生物和非生物胁迫反应等过程.本文系统地综述了番茄果实中small RNAs的分离鉴定现状,对番茄果实中与乙烯信号途径、颜色、风味、软化等代谢途径相关的small RNAs进行了系统总结,并展望了未来small RNAs在果实成熟过程中的相关研究方向.     

分离自陕西泾阳番茄的番茄黄化曲叶病毒(TYLCD)的分子特征

从陕西泾阳地区的番茄(Lycopersicon esculentum)上采集到73份表现矮化、黄化和曲叶症状的番茄黄化曲叶病(Tomato yellow leaf curl disease,TYLCD)样品,利用双生病毒简并引物PA和PB及番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的通用引物TYT-F和TYT-R进行PCR扩增,共有70个样品检测呈阳性;利用双生病毒DNA-B及卫星DNA的通用引物,未扩增到相应目标条带;随机选取样品SX8,利用滚环扩增PCR(RCA-PCR)、克隆及测序等技术获得病毒DNA-A的全基因组序列,该DNA分子全长含2781bp(GenBank登录号:JN412854),与来自山东番茄的TYLCV分离物SD2(缩写为TYLCV-[SD2])(GenBank登录号:GU199587)的核苷酸序列相似性最高,为99.9%;系统进化分析发现,该病毒分离物与我国已报道的TYLCV各分离物亲缘关系较近。这些结果表明,陕西番茄黄化曲叶病是由TYLCV引起,该病毒可能来源于我国山东番茄上的TYLCV。     

根围促生细菌(PGPR)蜡质芽胞杆菌AR156对番茄的诱导耐旱性研究

根围促生细菌(PGPR)蜡质芽胞杆菌(Bacillus cereus)AR156是具有很高应用前景的生防菌剂,为了研究其对番茄(Lycopersicon esculentum)的诱导耐旱机理,本研究通过检测干旱胁迫条件下叶片相对含水量、丙二醛(MDA)含量、相对电导率、叶绿素含量和根系还原能力,抗氧化酶活性以及胞质抗坏血酸还原酶基因(cAPX)、单脱氢抗坏血酸还原酶基因(MDHAR)和核酮糖-1,5-二磷酸羧化酶(Rubisco)小亚基基因(rbcS)的表达情况,从表型、生理水平和分子水平上进行阐释.研究发现,干旱胁迫条件下AR156培养上清诱导后,番茄叶片相对含水量为82％,与对照组相比提高了44％;MDA含量为2.2x10-3μmol/g FW,降低了21.4％,相对电导率为56.0％,降低了28.2％;叶绿素a、叶绿素b和总叶绿素含量分别增加了16％、20％和20.2％;根系还原强度为0.336 mg/g/h,是对照组的两倍;另外,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性也显著提高;同时,分子水平的检测发现rbcS在干旱处理过程中—直保持较高水平,cA PX和MDHAR的表达在干旱处理后期也显著高于对照组;并且干旱胁迫处理20 d后重新浇水24 h,番茄存活率达到90.91％,比对照组高1.6倍.结果表明,AR156培养上清可以通过保护细胞膜的完整性、维持较高水平的光合效率和提高抗氧化酶活性来提高番茄的系统耐旱性,初步阐释了PGPR AR156的诱导耐旱机理,为AR156作为优良生物类耐旱诱导因子提供了理论依据.     

低温对番茄种子萌发的影响

在人工气候箱中模拟了3个低温处理(24h最低~最高温度分别设定为7~12℃、5~10℃和3~8℃),分析了播种后不同天数低温对番茄种子(品种:红贞女)萌发的影响。结果表明:与对照(18~25℃)相比,日均温低于10.0℃(相当于低温胁迫处理7~12℃)时,番茄种子萌发开始受影响,温度越低影响越大。播种后越早经受低温番茄种子萌发受影响越大,播种后3d内经受低温胁迫,番茄种子发芽势、发芽指数和萌发率均有较大的下降,播种6d后低温胁迫对其萌发指标的影响较小。低温导致了番茄种子α-淀粉酶活力下降,相对离子渗漏率增加。低温影响了番茄种子萌发贮存物质的降解与转化,增加了细胞膜的透性,导致外渗物增多,从而降低了种子的发芽势、发芽指数和萌发率。因此,露地育苗时应避免在萌发的早期受低温影响,以提高番茄的出苗和壮苗率。     

Purification and thermal and high-pressure inactivation of pectinmethylesterase isoenzymes from tomatoes (Lycopersicon esculentum): a novel pressure labile isoenzyme

Tomato pectinmethylesterase (PME) was successfully purified by a two-step method consisting of affinity chromatography followed by cation exchange chromatography. According to this procedure, four different isoenzymes were identified representing molar masses around 34.5-35.0 kDa. Thermal and high-pressure inactivation kinetics of the two major isoenzymes of tomato PME were studied. A striking difference between their process stability was found. The thermostable isoenzyme was completely inactivated after 5.0 min at 70 degrees C, whereas for the thermolabile isoenzyme, temperatures at around 60 degrees C were sufficient for complete inactivation. The thermostable isoenzyme was also found to be pressure stable since no inactivation was observed after 5.0 min of treatment at 800 MPa and 20 or 40 degrees C. The thermolabile isoenzyme appeared to be pressure labile since it could be completely inactivated after 5.0 min of treatment at 700 MPa and 20 degrees C or 650 MPa and 40 degrees C. Inactivation kinetics at pH 6.0 could be accurately described by a first-order model.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Partial purification,characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.)

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.     

类番茄茄基因渗入栽培番茄的研究

用栽培番茄作母本 ,类番茄茄作父本杂交 ,结实率为 40 %。通过离体胚培养获得杂种 1代 ,用栽培番茄作母本与之回交 ,得到回交 1代种子 ,结实率达 1 5 6% ,获得种子发育成苗 2 3株。但该回交 1代植株自交不结实 ,与栽培番茄 2次回交表现不亲和。用类番茄茄与栽培番茄的杂交 1代作母本 ,潘那利作父本杂交 ,结实率为 1 1 % ,得到复合杂种植株 5株 ,用该复合杂种作母本 ,潘那利作父本进行回交 ,结实率达 1 5 6% ,得到回交后代 2 5株 ,该代植株自交结实 ,并与栽培番茄杂交亲和。     

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.     

外源硫化氢对盐胁迫下加工番茄幼苗光合参数及叶绿素荧光特性的影响

为探究外源硫化氢(H_2S)对盐胁迫下加工番茄幼苗光合作用和光合荧光参数的影响,以耐盐性不同的2个加工番茄KT-7(耐盐性强)和KT-32(耐盐性弱)为材料,采用分光光度法、CIRAS-3型光合仪和Imaging-PAM调制荧光成像系统分别测定光合色素含量、光合参数和叶绿素荧光参数。结果表明,外源H_2S提高了盐胁迫下加工番茄幼苗的生长及叶绿素a、叶绿素b、胡萝卜素含量、净光合速率(P_n)、气孔导度(G_s)、叶片蒸腾速率(Tr)、PSⅡ最大光化学效率(F_v/F_m)和PSⅡ实际光化学效率(ΦPSⅡ);降低了胞间CO_2浓度(Ci)、PSⅡ调节性能量耗散的量子产额[Y(NPQ)]和非光化学猝灭系数(NPQ);而PSⅡ非调节性能量耗散的量子产额[Y(NO)]稳定在较低水平。此外,外源H_2S对KT-32的缓解效应强于KT-7。综上所述,外源H_2S通过提高加工番茄幼苗叶片的光合色素量和光合电子传递效率,有效地缓解盐胁迫对加工番茄叶片PSⅡ的伤害。本研究结果为探究外源H_2S增强加工番茄耐盐性研究提供了科学依据。     

Purification and glycosylation analysis of an acidic pectin methylesterase in jelly fig (Ficus awkeotsang) achenes

An acidic pectin methylesterase (PME) is responsible for the gelation of water extract from jelly fig (Ficus awkeotasang) achenes. A new, fast and efficient, method has been developed to purify this acidic PME. The method includes preparing jelly curd by traditional hand washing, extracting proteins from the curd, and separating PME by anion-exchanger. The purified PME exists as a monomer of 38 kDa determined by gel filtration, and exerts enzymatic activity over a broad pH range, particularly in acidic environments where most known PME enzymes from various species are inactivated. Chemical staining and enzymatic cleavage suggest that the jelly fig PME is an N-linked glycoprotein. Fluorophore-assisted carbohydrate electrophoresis reveals that the polysaccharide of this glycoprotein putatively consists of 22 hexoses including 16 mannose, 4 N-acetylglucosamine, and 2 galactose residues.     

Occurrence and distribution of organochlorine pesticides (OCPs) in tomato (Lycopersicon esculentum) crops from organic production

Organochlorine pesticides (OCPs) were quantified by GC-ECD in tomato (Lycopersicon esculentum) during a vegetation period. Plants were harvested at 15, 60, and 151 days after seed germination. Leaves, stem, roots, and fruit (peel and flesh) were analyzed separately. The results showed that tomato plants were able to accumulate OCPs from soils, and a trend to reach the equilibrium among tissues at mature stages was also observed. Endosulfans comprised the main OCP group, probably due to its spray during summer months in the surrounding areas. Banned pesticides such as DDTs, heptachlor, and dieldrin were found. OCPs levels in the fruit were below the maximum residues limits (MRL) considered by the Codex Alimentarius. DDE/DDT and alpha-/gamma-HCH ratios of <1 would indicate recent inputs of DDT and lindane in the environment. The occurrence of OCPs in the study farm, where agrochemicals have never been used, is a result of atmospheric deposition of those pesticides.     

Partial purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectin methylesterase (PME) is present in acerola fruit and was partially purified by gel filtration on Sephadex G-100. The results of gel filtration showed different PME isoforms. The total PME (precipitated by 70% salt saturation) and one of these isoforms (fraction from Sephadex G-100 elution) that showed a molecular mass of 15.5 +/- 1.0 kDa were studied. The optimum pH values of both forms were 9.0. The total and the partially purified PME showed that PME specific activity increases with temperature. The total acerola PME retained 13.5% of its specific activity after 90 min of incubation at 98 degrees C. The partially purified acerola (PME isoform) showed 125.5% of its specific activity after 90 min of incubation at 98 degrees C. The K(m) values of the total PME and the partially purified PME isoform were 0.081 and 0.12 mg/mL, respectively. The V(max) values of the total PME and the partially purified PME were 2.92 and 6.21 micromol/min/mL/mg of protein, respectively.     

Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

Nutritional value of cherry tomatoes (Lycopersicon esculentum Cv. Naomi F1) harvested at different ripening stages

The average content of some classes of antioxidants is generally higher in cherry tomatoes than in normal-sized berries. The aim of this work was to assess the nutritional value of cherry tomato (cv. Naomi F1) by investigating the compositional pattern of berries harvested at different ripening stages and evaluating, in particular, all of the main antioxidants (carotenoids, ascorbic acid, phenolic compounds, and alpha-tocopherol) and the antioxidant activity of the water-soluble and water-insoluble fractions. Results confirmed the relatively high level of carotenoids in cherry tomato but showed that not all biologically active compounds necessarily increase in tomatoes picked at later stages of ripeness. Cherry tomatoes harvested at full ripeness exhibited the highest level of carotenoids and antioxidant activity in the water-insoluble fraction. On the other hand, no significant differences in ascorbic acid content were observed at different ripening stages, whereas the main phenolics content and the antioxidant activity of water-soluble fraction showed slight, but significant, decreases at later stages of ripeness.     

Detection and quantitation of resveratrol in tomato fruit (Lycopersicon esculentum Mill.)

Resveratrol is a stilbene phytoalexin well-known for its presence in grape, wine, and peanut. As a result of its antioxidant and chemopreventative properties, it has gained much attention as a functional food ingredient. A gas chromatography-mass spectrometry method for the detection of resveratrol, its 3-glucopyranoside piceid, and the cis isomers of both compounds has been developed and used to quantitate the levels of these compounds in the skin of commercially available tomato fruit (Lycopersicon esculentum Mill.). The resveratrol concentration remains relatively stable during fruit maturation, reaching a maximum concentration in the skin of 18.4 +/- 1.6 microg/g dry weight at 4 weeks postbreaker. No stilbenes were detected in the flesh of tomato fruit.     

Metabolism of fenitrothion and conjugation of 3-methyl-4-nitrophenol in tomato plant (Lycopersicon esculentum)

The metabolism of (14)C-labeled fenitrothion (Sumithion, [O,O-dimethyl-O-(3-methyl-4-nitrophenyl)phosphorothioate]) in tomato plant (Lycopersicon esculentum Mill., cv. Ponderosa) grown in the greenhouse equipped with quartz glass was conducted to investigate the effect of sunlight on the behavior of fenitrothion and to elucidate the detailed structure of conjugated metabolites. Tomato plants (BBCH 85) were topically treated with (14)C-labeled fenitrothion twice with a 2 week interval between applications. At 15 days after the second application, more than half of the recovered (14)C was detected as unaltered fenitrothion, glucose, and cellobiose esters of 3-methyl-4-nitrophenol (NMC) in extracts from tomato fruit. The photoinduced formation of the S-methyl isomer of fenitrothion via thiono-thiolo rearrangement was detected only in the surface rinse but at trace amounts. In the whole tomato fruit, fenitrothion, the S isomer, NMC-beta-glucoside, and NMC cellobioside were detected at 34.16, 1.28, 7.47, and 15.07% of the recovered (14)C, respectively. Trace amounts of the oxon analogue of fenitrothion were detected only on tomato leaves. The chemical structure of the cellobiose conjugate of NMC, 1-O-beta-d-glucopyranosyl-(1-->4)-beta-d-glucopyranosyl-3-methyl-4-nitrophenol, was determined by spectroscopic analyses (liquid chromatography-mass spectrometry, NMR), using the metabolite obtained from leaves and stems of tomato plant hydroponically grown with (14)C-labeled NMC.     

Growth,Ion Uptake,and Yield Responses of Three Indigenous Small-Sized Greek Tomato (Lycopersicon esculentum L.) Cultivars and Four Hybrids of Cherry Tomato under NaCl Salinity Stress

The effect of three sodium chloride treatments (0, 75, and 150 mM) on the growth, yield, and ion uptake of three small-sized Greek tomato cultivars (Santorini Authentic, Santorini Kaisia, and Chios) and four cherry tomato hybrids (Cherelino, Scintilla, Delicassi, and Zucchero) was studied by using a completely randomized block design with seven replicates. The results indicated that Santorini Authentic under 75 mM sodium chloride (NaCl) had reduced its total biomass production (total plant + fruits fresh weight) by only 22% while the relevant percentage decreases of some hybrids were found to be duplicated. Regarding yield characteristics, Zucchero under 75 mM NaCl, followed by Santorini Kaisia and Chios, showed the lowest reduction of the marketable total fruit fresh weight compared to Cherelino and Delicassi. The greater tolerance of Santorini Authentic might be due to the greater sodium and chlorine retention by the roots in combination with the nonsignificant decrease of the leaf potassium.