Differential induction of apoplastic peroxidase and chitinase activities in susceptible and resistant wheat cultivars by Russian wheat aphid infestation

The intercellular peroxidase and chitinase activities of three wheat cultivars [Triticum aestivum L. cvs `Tugela DN', `Molopo DN' (Gariep) and `Betta DN'] containing the Dn-1 gene for resistance to the Russian wheat aphid (RWA) Diuraphis noxia (Mordvilko) and the corresponding near-isogenic susceptible cultivars (`Tugela', `Molopo' and `Betta') were studied under conditions of infestation and non-infestation. The aim was to gain information on the mechanism of resistance. The resistance response was induced by RWA infestation. Infestation rapidly induced the activities of both enzymes selectively in resistant wheat to levels of magnitudes higher than those in susceptible wheat. The genetic background in which the Dn-1 resistance gene is bred played a role and the level of activity corresponded to the level of resistance. Immunologic studies confirmed that the induction of enzyme activities was due to the induction of higher protein levels. These results indicate that peroxidase and chitinase may have a role in insect resistance. Received: 20 June 1997 / Revision received: 9 April 1998 / Accepted: 5 June 1998     

Characterizing plasma membrane H+-ATPase in two varieties of coffee leaf (Coffea arabica L.) and its interaction with an elicitor fraction from the orange rust fungus (H. vastatrix Berk and Br.) race II.

Early intercellular signaling in Coffea arabica L.-Hemileia vastatrix host-pathogen interaction was studied, using inside-out plasma membrane from two varieties of coffee leaf and a fungal fraction to determine the plant's biochemical responses. Microsomal pellets (100,000 x g) from the susceptible (Caturra) and resistant (Colombia) coffee leaf varieties were purified by partitioning in two-polymer DEX (6.3% w/w) and PEG (6.3% w/w) system aqueous phase. Fungal material was obtained from orange rust Hemileia vastatrix Berk and Br. race II urediospore germ tubes. Plasma membrane vesicles were preferentially localized to PEG phase, as indicated by its enzyme marker distribution. Both H(+)-ATPase activities displayed similar kinetic and biochemical characteristics, comparable to those described for P-type ATPases. Several enzymes may play pivotal roles in plants regarding early interaction with fungal elicitors. Studies of fungal fractions' effects on H(+)-ATPase and both varieties' proton pumping activities were thus carried out. Concentration as low as 0.1 Gluc eq. ml(-1) fungal fraction induced specific inhibition of H(+)-ATPase and the resistant variety's proton pumping activities. The present work describes characterizing the H(+)-ATPase plasma membrane from two Coffea arabica L. varieties (Caturra and Colombia) for the first time and the race specific inhibitory effect of a crude fungal fraction on both H(+)-ATPase and the resistant variety's proton pumping activities.     

Responses of Ribulose-1,5-Bisphosphate Carboxylase,Cytochrome f,and Sucrose Synthesis Enzymes in Rice Leaves to Leaf Nitrogen and Their Relationships to Photosynthesis

The photosynthetic gas-exchange rates and various biochemical components of photosynthesis, including ribulose-1,5-bisphosphate carboxylase (Rubisco) content, cytochrome (Cyt) f content, and the activities of two sucrose synthesis enzymes, were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically in different nitrogen concentrations. The light-saturated rate of photosynthesis at an intercellular CO2 pressure of 20 Pa (CO2-limited photosynthesis) was linearly dependent on leaf nitrogen content, but curvilinearly correlated with Rubisco content. This difference was due to a greater than proportional increase in Rubisco content relative to leaf nitrogen content and the presence of a CO2 transfer resistance between the intercellular air spaces and the carboxylation sites. CO2-limited photosynthesis was proportional to Cyt f content, one of the key components of electron transport, but was not proportional to the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, the two regulatory enzymes of sucrose synthesis. Light-saturated photosynthesis above an intercellular CO2 pressure of 60 Pa (CO2-saturated photosynthesis) was curvilinearly dependent on leaf nitrogen content. This CO2-saturated photosynthesis was proportional to Cyt f content in the low- and normal-nitrogen leaves, and correlated better with the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase in the high-nitrogen leaves. The increase in the activities of these two enzymes with increasing leaf nitrogen was not as great as the increase in Cyt f content. Thus, as leaf nitrogen increased, the limitation caused by the activities of sucrose synthesis enzymes came into play, which resulted in the curvilinear relationship. However, this limitation by sucrose synthesis enzymes did not affect photosynthesis under normal ambient air.     

Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress

Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens.     

Lipids as bioeffectors in the immune system

Lipids, in addition to serving as fuel stores and structural components of cell membranes, act as effectors and second messengers in a variety of biological processes including those associated with the immune system. These lipid mediators and regulators differ in structural composition and exert a diverse array of effects on cellular functional activities including those linked to homeostasis, immune responsiveness, and inflammation. They function as intercellular mediators and at the intracellular level act as critical conduits of external stimuli in signal transduction cascades. Lipid derived messengers and their receptors also may interact with other signaling molecules. Exogenous compounds such as cannabinoids share functionally relevant receptor binding domains with those for endogenous lipid signaling ligands and have the potential to alter transductional cascades linked to immune functional activities.     

Intercellular 27 kD protein is a chitinase induced by water stress or Pseudoperonospora cubensis in cucumber leaves]

Cucumber seedlings were drought-stressed or inoculated with Pseudoperonospora cubensis. After 3 or 6 d the intercellular fluids of treated cucumber leaves were extracted and analyzed. Protein contents increased after pathogen inoculation and a 27-kD protein was found in intercellular fluids (Figs.1, 7). Both 27 kD proteins were purified from the intercellular fluids of cucumber leaves after drought stress or pathogen inoculation by SDS-PAGE and electro-elution protocol respectively (Fig.2, 3). Purified proteins from drought-stressed and P. cubensis infected seedlings were analyzed by MALDI-TOF MS and their peptide mass fingerprinting (PMF) results were obtained (Figs.4, 5). The PMF results were compared with protein database using the software Profound. The results show that the 27 kD proteins from seedlings after drought stress and after P. cubensis infection were the same protein, i.e. an acidic chitinase (Tables 1, 2; Fig.6). The activities of chitinase in the intercellular fluids of cucumber leaves after pathogen inoculation and in those drought stress were also analyzed. Results showed that both treatments induced the increase in chitinase activity (Fig.8), which indicated that chitinase may be involved in the protection of cucumber plant against both pathogen attack and water stress.     

Redox state of ascorbic acid in the apoplast of stems of Kalanchoë daigremontiana

The aqueous phase of cell walls in stems of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie (apoplast) contained ascorbic acid (AA) and dehydroascorbic acid (DHA). Ratios of AA/(AA + DHA) were 0.31 ± 0.12 (SD, n = 4), whereas those of whole stems (tissues plus apoplast) were >0.9. The amounts of (AA + DHA) in the stems were 1970 ± 190 (SD, n = 4) nmol g⁻¹ fresh weight and those in the apoplast were 14 ± 2 (SD, n = 4) nmol g⁻¹ fresh weight of stems. Ratios of AA/(AA + DHA) differed in different tissues of the stems. The ratios of AA/(AA + DHA) of apoplast plus symplast were in the following order: pith ⋍ epidermis plus cortex > vascular bundle system, and those of apoplast were: pith > epidermis plus cortex > vascular bundle system. Ratios of AA/(AA + DHA) in the apoplast of the different tissues decreased to about 1/3 of the original values after wounding, while the amounts of (AA + DHA) remained largely unaffected. In contrast, soluble apoplastic peroxidase activities increased 30- to 70-fold on wounding. Hydrogen peroxide infiltrated into stems caused a rapid oxidation of AA. Coniferyl alcohol was oxidized by peroxidase in intercellular washing fluid and by cell wall-bound peroxidase. The oxidation of coniferyl alcohol by peroxidase in intercellular washing fluid was completely inhibited as long as AA was present in reaction mixtures. The oxidation of the coniferyl alcohol by cell wall-bound peroxidase was partially inihibited by AA and the degree of inhibition was dependent upon the concentration of AA. The possible functions of AA in the apoplast are discussed in relation to the control of peroxidase-dependent oxidation of phenolics.     

A freeze-fracture study of two types of collagen-phagocytosing cell in the post-partum rat endometrium

In the post-partum rat endometrium, ultrastructural distinction could be made between stromal cells (fibroblast-like cells) and macrophages, especially by the freeze-fracture technic. The stromal cells were characterized by a well-developed rough-surfaced endoplasmic reticulum (RER) and intercellular junctions, while the macrophages had many vacuoles and vesicles, but no intercellular contact with each other. The freeze-fracture image showed that the stromal cells had many low linear elevations and gap junctions on the cleaved plane of the cell membranes, while the macrophages had no linear elevations or intercellular junctions. The cell membranes of the stromal cells had more intramembranous particles (IMP) (P-face 697 +/- 63/micrometers 2, E-face 303 +/- 52/micrometers 2) than those of the macrophages (P-face 467 +/- 50/micrometers 2, E-face 217 +/- 35/micrometers 2). It was confirmed that these two types of cell phagocytosed collagen fibrils.     

Intercellular chitinase and peroxidase activities associated with resistance conferred by geneLr35to leaf rust of wheat

The effect of leaf rust (Puccinia triticina) infection on intercellular chitinase (EC 3.2.1.14) and peroxidase (EC 1.11.1.7) activities was studied in resistant [RL 6082 (Thatcher/Lr35)] and susceptible (Thatcher) near isogenic wheat (Triticum aestivum L.) lines at seedling, stem elongation and flag leaf stages of plant growth. The levels of activity of these enzymes were low during the seedling and stem elongation stages. Resistant plants at the flag leaf stage, during which the Lr35 resistance gene was maximally expressed, exhibited high constitutive levels of chitinase and peroxidase activities, in contrast to the lower constitutive levels of susceptible plants. The results suggest that chitinase and peroxidase, constitutively present in the intercellular spaces of Thatcher/Lr35 wheat leaves, may play a role in Lr35 mediated resistance to leaf rust.     

黄萎病菌粗毒素接种对感病和抗病茄子品种的一些酶类活性和光合特性的影响

以黄萎病菌粗毒素接种不同茄子品种的结果表明:接种后抗病品种比感病品种的过氧化物酶(POD)、超氧化物歧化酶(SOD)活性高,而多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性则相对稳定;前者叶的净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)和细胞间CO2浓度(Ci)的变化幅度比后者小,但72 h后二者的叶片Pn、Gs、Tr都明显呈下降趋势.     

Photosynthesis, carbohydrate levels and chlorophyll fluorescence-estimated intercellular CO2 in water-stressed Casuarina equisetifolia Forst. & Forst.

The effect of long-term water stress on photosynthetic carbon metabolism in Casuarina equisetifolia Forst. & Forst. was analysed by measuring CO₂ assimilation, stomatal conductance, the quantum yield of photosystem II ( Φ _PSII), enzyme activities, and the levels of photosynthetic intermediates and carbohydrates. CO₂ assimilation decreased under water stress while the intercellular CO₂ concentration ( C _i) as estimated by gas exchange measurements remained high. However, the estimates of C _i from measurements of Φ _PSII suggest that the decrease in photosynthesis can be explained in terms of stomatal closure. Water stress decreased total stromal fructose-1,6-bisphosphatase activity and did not alter the activities and activation states of ribulose bisphosphate carboxylase oxygenase and NADP-dependent malate dehydrogenase (NADP-MDH). The concentration of photosynthetic metabolites, glucose, fructose and sucrose decreased, whereas starch concentrations increased under drought conditions.     

Intercellular communication of transformed and non-transformed rat liver epithelial cells : Modulation by TPA

Gap-junctional intercellular communication of transformed and non-transformed rat liver epithelial cell lines was compared using a dye transfer method in the presence and absence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Whereas non-transformed cells (IAR 20, non-tumorigenic in newborn rats and in nude mice) showed very high communication capacity throughout a culture period of 3 weeks, transformed cells (IAR 6-1, tumorigenic in newborn rats and in nude mice) were less able to communicate. Similar correlation between intercellular communication and expression of transformed phenotypes were also found in newly cloned epithelial cell lines, IAR 27 E and IAR 27F. When TPA was added to culture medium at 100 ng/ml, intercellular communication in all lines tested was reduced within 60 min. However, communication recovered completely from the effect within 10 h after addition of TPA. Further addition of TPA to the cultures every 24 h for 3 weeks had no effect on intercellular communication (measured 30 min after each TPA addition), suggesting that a single application of TPA made these cells refractory to further doses. A known stimulator of gap-junctional communication, db-cAMP, also increased dye transfer in IAR 20 and IAR 6-1 cells. TPA added to db-cAMP-treated cultures of IAR 20 and IAR 6-1 cells inhibited intercellular communication, suggesting that cAMP is not an antagonist of the effect of TPA on intercellular communication in these cell lines. These results are in sharp contrast to those obtained with the fibroblast cell line BALB/c 3T3, in which db-cAMP antagonized TPA effect [1] and inhibition by TPA of intercellular communication was transient only when administered during their growth phase, and was stable and continuous when TPA was applied at confluence [2], and suggest that TPA may not be an effective tumour promoter in rat liver.     

Occurrence of two endoproteinases in wheat leaf intercellular washing fluid

The presence of endoproteinases in the intercellular washing fluid of the first wheat ( Triticum aestivum ) leaf 13 days after sowing was investigated. Two activities were detected after separation of the intercellular fluid proteins by SDS-PAGE using slab gels containing gelatin as substrate. Their sizes were 70 and 100 kDa. Both enzymes hydrolyzed gelatin and casein. They also seem to be involved in the degradation of a 40 kDa protein component of the intercellular fluid. These results indicate that both enzymes are endoproteinases. They were also obtained from leaves previously sumitted to intercellular fluid extraction. However, the 100 kDa enzyme yield was low, indicating that it is mainly located in the intercellular washing fluid. Assays performed with either specific substrates or inhibitors indicate that both enzymes are serine proteinases.     

NaCl胁迫对西瓜嫁接苗叶片抗氧化酶活性及光合特性的影响

以耐盐性较强的葫芦品种‘超丰抗生王’为砧木,耐盐性较弱的西瓜品种‘秀丽’为接穗,采用营养液水培法,研究了NaCl胁迫对西瓜自根苗和嫁接苗保护酶活性、膜质过氧化及光合特性的影响。结果表明,NaCl胁迫下,嫁接苗和自根苗生物量显著下降,但嫁接苗下降幅度小于自根苗;NaCl胁迫抑制了西瓜自根苗和嫁接苗的气体交换参数,但是嫁接苗的净光合速率(Pn)、气孔导度(Gs)、胞间CO2浓度(Ci)及叶绿素含量显著高于自根苗;NaCl胁迫下西瓜嫁接苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均显著高于自根苗,丙二醛含量较自根苗低。上述结果表明,NaCl胁迫下嫁接苗通过维持较高的抗氧化酶活性来提高清除活性氧(ROS)的能力,从而降低氧化损伤,并保持较高光合速率,从而增强西瓜幼苗对盐胁迫的耐性。     

Peroxidase isoenzymes in the defense response of Capsicum annuum to Phytophthora capsici

Quantitative and qualitative changes in isoperoxidase patterns from stems of three cultivars of pepper ( Capsicum annuum L.). one susceptible, one intermediate and one resistant, were found upon inoculation with Phytophthora capsici using a decapitation method. The peroxidase activity was determined in the intercellular fluid as well as in the cytosolic fraction of the necrotic, healthy and intermediate zones of stems of the three cultivars, 6 days after inoculation. In the intercellular fluid, peroxidase activity of the susceptible cv. Yolo Wonder increased somewhat from 4.7 (healthy zone) to 12.9 (intermediate zone) μmol mg⁻¹ protein min⁻¹, whereas in the intermediate cv. Americano, the peroxidase activity decreased from 123 (healthy zone) to 78 (intermediate zone) μmol mg⁻¹ protein min⁻¹. The most dramatic increase (5.7 to 662 μmol mg⁻¹ protein min⁻¹) in intercellular peroxidase activity was found in the resistant cv. Smith-5. This, in conjunction with the appearance of an additional acidic isoperoxidase (pI 4.4) specific for the cv. Smith-5, could be the reason for the resistance of this cultivar against the fungus attack. The release of peroxidase into the intercellular space as a defense reaction was confirmed by histochemical analysis, showing that peroxidase activity occurred in the intercellular spaces of those stems of the resistant cultivar that had not yet been invaded by the fungus, but was detected neither in the other cultivars nor in the intercellular spaces of such stems of the intermediate and susceptible cultivars that contained growing mycelium of P. capsici. The lack of staining in the intercellular spaces of the susceptible cultivars could be attributed to their low content in peroxidase.     

脱落酸对UV-C胁迫下小麦幼苗光合特性及抗氧化酶活性的影响

研究了UV-C辐射下短期和长期脱落酸（ABA）处理对小麦幼苗CO₂同化作用、羧化效率、光合CO₂响应以及抗氧化酶活性等的影响.结果表明,在无UV-C辐射情况下,短期和长期ABA处理能提高光合速率,比对照增加14.69%和20.46%,降低气孔导度,比对照降低14.74%和17.31%,但对胞间CO₂浓度和羧化效率影响不大.当受到UV-C辐射时,光合速率、羧化效率、气孔导度和胞间CO₂浓度逐渐降低.长期ABA处理变化最小,其次为ABA短期处理,对照降低最大.ABA处理能够提高小麦光合对CO₂的响应,UV-C辐射抑制光合对CO₂的响应.ABA处理能够提高小麦抗氧化酶（CAT、SOD、POD）活性而降低MDA含量.在UV-C辐射下,CAT活性先升高随后降低,在辐射处理1 h时活性达最大值,ABA处理的SOD和POD活性先升高后降低,且ABA长期处理比短期处理增加明显,对照则逐渐降低.ABA处理可能通过提高小麦CO₂同化作用和抗氧化酶活性增强对UV-C胁迫的抗性,且ABA长期处理比短期处理效果更明显.     

Intercellular location of glycoprotein in soybean nodules: effect of altered rhizosphere oxygen concentration

Abstract. A glycoprotein which occludes intercellular spaces in the inner cortex of legume nodules may be involved in controlling oxygen diffusion into rhizobial-infected cells. Here we investigated this possibility by localizing the glycoprotein using monoclonal antibodies and immunogold labelling in nodulated roots of soybean cv. Clarke inoculated with Bradyrhizobium japonicum strain RCR3442 exposed to atmospheres with either 10, 21 (control) or 40% oxygen for 28d. Infected cells showed evidence of premature senescence when grown in above or below ambient pO₂ particularly at 10% oxygen, although cortical cells appeared to be little altered by oxygen treatment. In the inner cortical cells, more glycoprotein was seen to be occluding intercellular spaces of those nodules subjected to 40% oxygen and less in those nodules exposed to 10% oxygen, when compared to controls. This observation, made at the light microscope level (using silver enhancement) was confirmed under the TEM using immunogold labelling. Therefore, it is suggested that intercellular space glycoprotein is one of the structural components of the diffusion resistance in the cortex of legume nodules.     

Balance between anionic and cationic extracellular peroxidase activities in Sedum album leaves after ozone exposure. Analysis by high-performance liquid chromatography

Peroxidase activity was assayed with different electron donors (guaiacol, ascorbate, syringaldazine) in the intercellular fluid of Sedum album L. leaves after ozone exposure. Anionic and cationic peroxidases were separated and purified by high performance ion-exchange and gel permeation chromatography. Both isoperoxidases were tested as regards their molecular weight and apparent kinetic constants with different substrates. Ascorbate peroxidase activity was rapidly stimulated after ozone exposure, whereas syringaldazine peroxidase activity reached its maximum 24 h later. Increases in ascorbate and syringaldazine peroxidase activities occurred simultaneously with increases in cationic and anionic peroxidase activities, respectively. Apparent K_m values indicate a high affinity of cationic peroxidases for ascorbate and of anionic peroxidases for syringaldazine. The metabolic role of this balance between cationic and anionic peroxidases after ozone exposure is discussed.     

HeLa细胞中缝隙连接蛋白Cx26/Cx32的表达及其功能调控

目的 在HeLa宫颈癌细胞中研究不同浓度的多西环素对缝隙连接蛋白Cx26/Cx32表达及由其形成的缝隙连接通讯功能的影响.方法 采用Western印迹检测HeLa细胞中Cx26/Cx32的蛋白表达;荧光示踪实验用于检测HeLa细胞中由Cx26/Cx32形成的缝隙连接通讯功能.结果 Western印迹结果显示多西环素在0.01～1 μg/ml的范围内,随着剂量的增加,Cx26/Cx32蛋白表达水平增加;荧光示踪实验结果显示HeLa细胞之间的荧光传递随着多西环素增加也相应增强.结论 采用加入不同浓度多西环素的方法,可制备缝隙连接通讯功能强弱不同的细胞模型.