Enzymatic properties of the 20S proteasome in wheat endosperm and its biochemical characteristics after seed imbibition

The 20S proteasome from wheat ( Triticum aestivum L., Yangmai 158) endosperm was purified to apparent homogeneity by three sequential centrifugations and gradient PAGE (GPAGE). The purified 20S proteasome clearly cleaved peptidyl-arylamide bonds in the model synthetic substrates Z-GGL-AMC and Z-GGR-AMC, which are used to reflect chymotrypsin-like and trypsin-like activity, respectively. For both substrates, the optimum pH was 8.0, but the optimum temperatures for chymotrypsin-like and trypsin-like activity were 55 °C and 37 °C, respectively. Both enzyme activities were clearly inhibited by MG115 and PMSF. Polyubiquitinated proteins remained constant from 0 to 7 days after seed imbibition, but caseinolytic activity and the amount of the 20S proteasome associated with the aleurone layer decreased from 1 to 2 days after imbibition (DAI), then increased from 2 to 4 DAI, and reached a maximum at 4 DAI that was retained until 7 DAI. An increase was seen in the mRNA level of the β5 subunit of the 20S proteasome from 2 DAI, and caseinolytic activity and the amount of the 20S proteasome increased from 3 DAI onwards. In addition, the main storage proteins of the wheat endosperm could not be hydrolyzed by the 20S proteasome. The evidence suggests that the main role of the 20S proteasome may not be to degrade massive proteins of the wheat endosperm after seed imbibition.     

缺磷胁迫下的小麦根系形态特征研究

研究了缺磷条件下不同基因型小麦（Triticum aestivum L.)苗期根系形态学适应特征,以明确环境因子对根系不同组分（根轴和侧根）生长发育调控作用的强度和根系形态与磷营养效率关系。在缺P环境中,小麦根轴数量和侧根长度明显减小,同化物向根部的分配比例增加,根轴长度、侧根数量和根系长度等均有显著提高。供试基因型小麦的根轴数量及其长度的差异在每个供磷水平及不同供磷水平之间均呈显著,说明这两种性状的差异是由基因型和环境因素共同决定的;而侧根特征的差异只在不同供磷水平间显著,表明侧根性状主要受环境因素的控制。对6种基因型小麦的研究表明,根轴数量、根轴长度、根生长角度和根系长度根角之间存在着显著的基因型差异。相关分析表明,小麦的相对产量与缺磷条件下的小麦苗期根系形态指标的交互作用之间具有显著的线性关系。这种关系说明根系形态性状可作为早期有效地筛选磷高效小麦品种的指标。     

Antioxidant defense mechanism under salt stress in wheat seedlings

The present study was carried out to study the effect of salt stress on cell membrane damage, ion content and antioxidant enzymes in wheat (Triticum aestivum) seedlings of two cultivars salt-tolerant KRL-19 and salt-sensitive WH-542. Seedlings (4-d-old) were irrigated with 0, 50 and 100 mM NaCl. Observations were recorded on the 3^rd and 6^th day after salt treatment and 2^nd day after salt removal. The relative water content declined with induction of salt stress, more in WH-542 than in cv. KRL-19. K⁺/Na⁺ ratio in KRL-19 was higher than in WH-542. WH-542 suffered greater damage to cellular membranes due to lipid peroxidation as indicated by higher accumulation of H₂O₂, MDA and greater leakage of electrolytes than KRL-19. The activities of catalase, peroxidase and ascorbate peroxidase and glutathione reductase increased with increase in salt stress in both the cultivars, however, superoxide dismutase activity declined. Upon desalanization, partial recovery in the activities of these enzymes was observed in KRL-19 and very slow recovery in WH-542.     

Na+-K+ transport in roots under salt stress

Salinity causes billion dollar losses in annual crop production. So far, the main avenue in breeding crops for salt tolerance has been to reduce Na⁺ uptake and transport from roots to shoots. Recently we have demonstrated that retention of cytosolic K⁺ could be considered as another key factor in conferring salt tolerance in plants. A subsequent study has shown that Na⁺-induced K⁺ efflux in barley root epidermis occurs primarily via outward rectifying K⁺ channels (KORC). Surprisingly, expression of KORC was similar in salt- tolerant and sensitive genotypes. However, the former were able to better oppose Na⁺-induced depolarization via enhanced activity of plasma membrane H⁺-ATPase (thus minimizing K⁺ leak from the cytosol). In addition to highly K⁺-selective KORC channels, activities of several types of non-selective cation channels were detected at depolarizing potentials. Here we show that the expression of one of them, NORC, was significantly lower in salt-tolerant genotypes. As NORC is capable of mediating K⁺ efflux coupled to Na⁺ influx, we suggest that the restriction of its activity could be beneficial for plants under salt stress.Key words: salinity tolerance, barley, ion flux, K⁺ homeostasis, KOR, non-selective channels, patch-clamp     

Purification and characterization of the 20S proteasome from ostrich skeletal muscle

The proteasome is a high molecular weight, multisubunit and multicatalytic enzyme. Here we report the purification and characterization of ostrich skeletal muscle 20S proteasome. It was purified to homogeneity with Mr 700,000, pI 6.67 and a 'ladder' of 22.2-33.5 kDa bands on SDS-PAGE. The amino acid composition and amino-terminal sequences showed large identities to those of other species. For the three major activities, pH and temperature optima ranged between 8.0-11.0 and 40-70 degrees C, and stabilities between 5-12 and up to 40-60 degrees C. Substrate specificity and inhibitory effects were also studied. Many similarities to other sources were shown, with a few significant differences.     

Naegleria fowleri and Naegleria gruberi 20S proteasome: identification and characterization

The Naegleria are ubiquitous free-living amoebae and are characterized by the presence of three phases in their biological cycle: trophozoite, cyst and flagellate. Of this genus, only Naegleria fowleri has been reported as pathogenic to humans. The proteasome is a multi-catalytic complex and is considered to be the most important structure responsible for the degradation of intracellular proteins. This structure is related to the maintenance of cellular homeostasis and, in pathogenic microorganisms, to the modulation of their virulence. Until now, the proteasome and its function have not been described for the Naegleria genus. In the current study, using bioinformatic analysis, protein sequences homologous to those reported for the subunits of the 20S proteasome in other organisms were found, and virtual modelling was used to determine their three-dimensional structure. The presence of structural and catalytic subunits of the 20S proteasome was detected by Western and dot blot assays. Its localization was observed by immunofluorescence microscopy to be mainly in the cytoplasm, and a leading role of the chymotrypsin-like catalytic activity was determined using fluorogenic peptidase assays and specific proteasome inhibitors. Finally, the role of the 20S proteasome in the proliferation and differentiation of Naegleria genus trophozoites was demonstrated.     

Proteome analysis of roots of wheat seedlings under aluminum stress

The root apex is considered the first sites of aluminum (Al) toxicity and the reduction in root biomass leads to poor uptake of water and nutrients. Aluminum is considered the most limiting factor for plant productivity in acidic soils. Aluminum is a light metal that makes up 7 % of the earth’s scab dissolving ionic forms. The inhibition of root growth is recognized as the primary effect of Al toxicity. Seeds of wheat cv. Keumkang were germinated on petridish for 5 days and then transferred hydroponic apparatus which was treated without or with 100 and 150 μM AlCl₃ for 5 days. The length of roots, shoots and fresh weight of wheat seedlings were decreased under aluminum stress. The concentration of K⁺, Mg²⁺ and Ca²⁺ were decreased, whereas Al³⁺ and P₂O₅ ^? concentration was increased under aluminum stress. Using confocal microscopy, the fluorescence intensity of aluminum increased with morin staining. A proteome analysis was performed to identify proteins, which are responsible to aluminum stress in wheat roots. Proteins were extracted from roots and separated by 2-DE. A total of 47 protein spots were changed under Al stress. Nineteen proteins were significantly increased such as sadenosylmethionine, oxalate oxidase, malate dehydrogenase, cysteine synthase, ascorbate peroxidase and/or, 28 protein spots were significantly decreased such as heat shock protein 70, O-methytransferase 4, enolase, and amylogenin. Our results highlight the importance and identification of stress and defense responsive proteins with morphological and physiological state under Al stress.     

Interaction of Hsp90 with 20S proteasome: thermodynamic and kinetic characterization

The proteasome and heat shock proteins have been found in the centrosome. The evidence of their copurification reported by several studies suggests that they form stable complex. In addition, Hsp90 is involved in the loading of proteasome-generated antigenic peptides to the class I major histocompatibility complex. In this article, we report a detailed thermodynamic and kinetic characterization of the Hsp90-20S proteasome interaction, using a surface plasmon resonance technique. The modulation exerted by protons in solution has been investigated, and the results have been discussed, taking into account structural motifs characterizing the binding interface between the two macromolecules.     

Assembly of the 20S proteasome

The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation.     

Amount and activity changes of 20S proteasome modified by oxidation in salt-treated wheat root tips

To study the response of 20S proteasome in wheat (Triticum aestivum L.) roots to salt stress, the root tips from wheat seedlings treated with 200 mM NaCl for different times were used for studying its carbonyl level, caseinolytic activity, protein abundance and other biochemical characteristics. The contents of carbonylated and ubiquitinated proteins (Ub-P) were also investigated. During this stressed process, both the productive rate of O₂ ⁻ and the content of H₂O₂ gradually increased, with the concomitant increase in carbonyl level of total soluble proteins and 20S proteasome, together with the gradual increase in the activities of the total and 20S proteasome in salt-treated root tips. However, the amounts of 20S proteasome decreased particularly during this process. Moreover, metal-catalyzed oxidation of proteins from control plants in vitro validated that the oxidative modification also could increase the activity of 20S proteasome, but decrease its abundance. In addition, the amounts of Ub-P with molecular weights above 35 kDa remained similar to the control plants, but that below 35 kDa decreased significantly in treated root tips. The changes in the proteasome activity and amount argue in favor of the active involvement of this proteolytic system in salt-stressed plants.     

Proteome analysis of soybean leaves,hypocotyls and roots under salt stress

Background  

Salinity is one of the most widespread agricultural problems in arid and semi-arid regions that makes fields unproductive, and soil salinization is a serious problem in the entire world. To determine the effects of salt stress on soybean seedlings, a proteomic technique was used.     

三角叶滨藜根吸水特点与其抗盐性的关系

采用压力室和冰点渗透压计测定了三角叶滨藜在不同浓度NaCl的根系环境溶液中根木质部的压力势和伤流液的渗透势,并利用原子吸收分光光度计测定了植株和伤流液以及环境溶液中Na 含量。结果表明:随着根环境溶液NaCl浓度的增加,三角叶滨藜植株和木质部伤流液中Na 含量虽呈上升趋势,但根系的过滤系数和体内Na 相对累积量逐渐降低,说明三角叶滨藜根细胞对盐分有很强的过滤作用;木质部伤流液的渗透势随着环境溶液渗透势的降低而降低,但根木质部溶液的水势则逐渐高出根外环境溶液的渗透势;表明三角叶滨藜能够利用较低的木质部负压来抵抗根外溶液的低渗透势而反渗透吸水,并利用根细胞对盐分的过滤作用来避免从环境摄取过量的盐分。     

Influence of silicon on spring wheat seedlings under salt stress

The aim of the study was to examine the effect of silicon on spring wheat subjected to salt stress. The experiment was conducted in hydroponic conditions on 10-day old wheat seedlings. Salt stress was induced by sodium chloride at the concentration of 70 and 100 mM added to nutrient medium. Silicon (H₄SiO₄) at the doses of 1.0 and 1.5 mM significantly increased the shoots and roots weight of wheat seedlings and the content of photosynthetic pigments (chlorophyll a and b, as well as carotenoids) in leaves. It reduced a detrimental effect of salt stress and restricted peroxidation of membrane lipids. We also observed a greater accumulation of nitrates and the decrease in malondialdehyde concentration in plant tissues as a result of silicon addition. Under osmotic stress, silicon did not change the content of sugars in wheat shoots and roots. Silicon did not clearly affect proline content. In general, the obtained results point out that silicon can be used for the alleviation of adverse effect of salinity on plants status.     

Development of the Casparian strip in primary roots of maize under salt stress

The Casparian strip in the endodermis of vascular plant roots appears to play an important role in preventing the influx of salts into the stele through the apoplast under salt stress. The effects of salinity on the development and morphology of the Casparian strip in primary roots of maize (Zea mays L.) were studied. Compared to the controls, the strip matured closer to the root tip with increase in the ambient concentration of NaCl. During growth in 200 mM NaCl, the number and the length of the endodermal cells in the region between the root tip and the lowest position of the endodermal strip decreased, as did the apparent rate of production of cells in single files of endodermal cells (the rate of cell formation being equal to the rate at which cells are lost from the meristem). The estimated time required for an individual cell to complete the formation of the strip after generation of the cell in the presence of 200 mM NaCl was not very different from that required in controls. Thus, salinity did not substantially affect the actual process of formation of the strip in individual cells. The radial width of the Casparian strip, a morphological parameter that should be related to the effectiveness of the strip as a barrier, increased in the presence of 200 mM NaCl. The mean width of the lignified region was 0.92 m in distilled water and 1.33 m in 200 mM NaCl at the lowest position of the strip. The mean width of the strip relative to that of the radial wall at this position was significantly greater after growth in the presence of 200 mM NaCl than in the controls, namely, 20.5% in distilled water and 33.9% in 200 mM NaCl. These observations suggest that the function of the strip is enhanced under salt stress.     

Purification and characterization of a protein inhibitor of the 20S proteasome (macropain).

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.     

Alleviation of exogenous oligochitosan on wheat seedlings growth under salt stress

Hydroponic experiments were carried out to study the role of oligochitosan in enhancing wheat (Triticum aestivum L.) resistance to salt stress. Data were collected on plant biomass, chlorophyll content, photosynthetic rate (P _n), stomatal conductance (g _s), proline content, antioxidant enzyme activities, and malondialdehyde (MDA) content. Under 150 mM salt stress, plant growth was significantly inhibited. Shoot length, root length, and dry weight were sharply reduced by 26%, 31%, and 20%, respectively, of the control. Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were increased by 28%, 13%, and 26%, respectively, of the control and MDA content largely accumulated, which was 1.5-fold of the control. However, 0.0625% oligochitosan pretreatment alleviated the adverse effects of salt stress, which was reflected by increasing root length, shoot length, dry weight, chlorophyll content, P _n, and g _s. Furthermore, it also showed that oligochitosan pretreatment significantly increased antioxidant enzyme (SOD, CAT and POD) activities, and reduced MDA content in leaves. Meanwhile, the accumulation of proline was markedly accelerated. The results indicated that oligochitosan pretreatment ameliorated the adverse effects and partially protected the seedlings from salt stress during the following growth period.