Changes in the expression and the enzymic properties of the 20S proteasome in sugar-starved maize roots. evidence for an in vivo oxidation of the proteasome

The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of alpha 3 and beta 6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations.     

Sensitivity of chickpea and faba bean to root‐zone hypoxia,elevated ethylene,and carbon dioxide

During soil waterlogging, plants experience O₂ deficits, elevated ethylene, and high CO₂ in the root‐zone. The effects on chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.) of ethylene (2 μL L^?1), CO₂ (2–20% v/v) or deoxygenated stagnant solution were evaluated. Ethylene and high CO₂ reduced root growth of both species, but O₂ deficiency had the most damaging effect and especially so for chickpea. Chickpea suffered root tip death when in deoxygenated stagnant solution. High CO₂ inhibited root respiration and reduced growth, whereas sugars accumulated in root tips, of both species. Gas‐filled porosity of the basal portion of the primary root of faba bean (23%, v/v) was greater than for chickpea (10%), and internal O₂ movement was more prominent in faba bean when in an O₂‐free medium. Ethylene treatment increased the porosity of roots. The damaging effects of low O₂, such as death of root tips, resulted in poor recovery of root growth upon reaeration. In conclusion, ethylene and high CO₂ partially inhibited root extension in both species, but low O₂ in deoxygenated stagnant solution had the most damaging effect, even causing death of root tips in chickpea, which was more sensitive to the low O₂ condition than faba bean.     

The activity of the 20S proteasome is maintained in detached wheat leaves during senescence in darkness

In the present paper, we studied the participation of the 20S proteasome, the proteolytic component of the ubiquitin–proteasome pathway, in the remobilization of bulk proteins in senescing wheat leaves. The detached leaves of 15-d-old plants were incubated in darkness for several days, and various proteolytic activities were analysed in soluble extracts prepared at 0, 48 and 96 h after detachment. The endoproteolytic activity, measured at pH 7.5 and 5.4, increased more than 10-fold and the total peptidasic activity increased up to 5-fold after 96 h of incubation in the dark, when expressed as specific activity. In the same period, the leaf-protein content decreased to less than 50% of that present at the initial time. The 20S proteasome chymotrypsin-like activity remained constant when it was expressed as activity per leaf fresh weight and resulted 2-fold higher in terms of specific activity. The western blot analysis showed that the amount of 20S proteasome protein and ubiquitin–protein conjugates also remained constant until 4 d of incubation in darkness. These results indicate that the ubiquitin–proteasome pathway remains functional until the late phases of senescence suggesting that it may participate in the regulatory aspects of the process rather than in the massive protein breakdown.     

Intracellular pH in rice and wheat root tips under hypoxic and anoxic conditions

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with ³¹P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt = 10^−pHcyt) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader''s attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.Key words: anoxia, hypoxia, rice, wheat, cytoplasmic pH regulation     

Simultaneous oxygen production and nitrogenase activity of an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis in a co-culture with wheat

Summary A mutant strain of Anabaena variabilis, strain SA-1 that supported growth of wheat plants in a hydroponic co-culture in nitrogen (N) free medium also produced enough oxygen (O₂) to support root respiration. The steady-state concentration of net O₂ in the co-culture was dependent on incident light intensity. At an incident photosynthetic photoflux (PPF) of 1000 mol photons·m^–2·s^–1, net O₂ evolution by the co-culture in the root zone reached a maximum value of about 220 mol O₂ evolved·h^–1·mg chlorophyll (Chl)^–1. The O₂ concentration in the rhizosphere of the co-culture stayed above the ambient air level. O₂ uptake in the dark by strain SA-1-supplemented wheat roots washed free of cyanobacterium was higher than the root respiration of nitrate-grown plants. Nitrate-grown plants required aeration for maximum growth while the wheat-cyanobacterial co-culture can be cultured without aeration. These results show that O₂ produced by strain SA-1 can be used to supply the O₂ needs for root respiration of wheat. Respiration reduced net O₂ evolution by the mutant SA-1, decreasing the partial pressure of O₂ at the sites of cyanobacterial attachment to the roots. This led to an increase in the specific activity of nitrogenase of the co-culture at the high light intensities used to support wheat growth. This activity of about 30 mol ethylene produced·mg Chl^–1·h^–1 was three-fold higher than the activities obtained with the free-living strain SA-1 assayed at the same light intensity. In the co-culture, ammonia produced by the mutant strain SA-1 was not detectable. The NH _inf4 ^sup+ produced by strain SA-1 was used by the wheat plants and, under these conditions, the total N content of the plants reached as high as 85% of the total N content of nitrate-grown plants. In the co-culture system the metabolism of wheat and the cyanobacterium complemented each other, leading to higher plant growth in N-free medium. Offprint requests to: M. Gunasekaran     

Nickel-induced Inhibition of Wheat Root Growth is Related to H2O2 Production, but not to Lipid Peroxidation

Effects of exogenous nickel (Ni: 10 and 200 μM) on growth, mitotic activity, Ni accumulation, H₂O₂ content and lipid peroxidation as well as the activities of various antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GSH-Px) were investigated in wheat roots. A considerable Ni accumulation in the roots occurred at both the concentrations. Although Ni at 10 μM did not have any significant effect on root growth, it strongly inhibited the root growth at 200 μM. Mitotic activity in the root tips was not significantly affected by exposure of the seedlings to 10 μM Ni; however, it was almost completely inhibited at 200 μM treatment. Ni stress did not result in any significant changes in CAT and APX activities as well as lipid peroxidation. However, H₂O₂ concentration increased up to 82% over the control in the roots of seedlings exposed to 200 μM Ni. There was a significant decline in both SOD (50%) and GSH-Px (20–30%) activities in the roots when the seedlings were treated with 200 μM Ni. The results indicated that a strong inhibition of wheat root growth caused by Ni stress was not due to enhanced lipid peroxidation, but might be related to the accumulation of H₂O₂ in root tissue.     

Molecular cloning and characterization of OsUPS,a U-box containing E3 ligase gene that respond to phosphate starvation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41–79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P_i). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P_i signaling pathway through the ubiquitin-26S proteasome system.     

Hypoxic Induction of Anoxia Tolerance in Root Tips of Zea mays

When root tips of fully aerobic, intact maize (Zea mays L.) seedlings are made anaerobic, viability normally is only 24 hours or less at 25°C. We find that viability can be extended to at least 96 hours if seedlings are given a hypoxic pretreatment for 18 hours by sparging the solution with 4% O₂ in nitrogen (v/v) before anoxia. Fully aerobic root tips (sparged with 40% O₂) had very low alcohol dehydrogenase (ADH) activity (per gram root fresh weight), and the level remained low under anoxia. In hypoxically pretreated roots, however, high levels of ADH activity were induced, and activity rose further during the initial 24 hours of anoxia, and then remained high at about 20 times that of controls in 40% O₂. ADH activity in roots in solution sparged with air (21% O₂) was about three times that in 40% O₂. Improved viability of hypoxically pretreated root tips was associated with maintenance of a high energy metabolism (ATP concentration, total adenylates, and adenylate energy charge). Roots that were not pretreated lost 94% of the total adenylates and ATP at 24 hours of anoxia. The relation between induced ADH activity, energy metabolism, and improved anoxia-tolerance in acclimated maize root tips is discussed.     

Identification and biochemical characterization of 20S proteasome in wheat roots under salt stress

In order to gain a better understanding of proteases state in wheat roots under salt stress, the roots of wheat (Triticum aestivum L., H6756) exposed to 300 mM NaCl were investigated at different days after treatment (DAT). The results showed that the content of soluble proteins decreased continuously, but the activity of total proteases increased gradually, and five root endopeptidase isoenzymes (RE1-5) were detected by natural gradient polyacrylamide gel electrophoresis (GPAGE) with supplement of gelatin as a substrate. Among five REs, RE1 was not only detected in salt-treated roots, but also obviously possessed the higher activity, whereas its protein amount decreased gradually during the salt treatment process. The biochemical characters of RE1 were examined afterwards. The results showed that its molecular mass was about 740 kDa and its optimal temperature was about 40°C. But the optimal pH was 5 to substrate gelatin or 7–8 to substrate casein. Its activity was obviously inhibited by 10 mM PMSF and 20 μM MG115, and RE1 was further confirmed as 20S proteasome by Western blotting. The results in this study suggested that 20S proteasome of wheat roots might be an important protease accumulated in roots in response to salt stress.     

NO和H2O2诱导大豆根尖和边缘细胞耐铝反应的作用

 NO和H₂O₂是参与植物抗非生物胁迫反应的重要信号分子, 为了确定NO和H₂O₂在大豆(Glycine max)根尖和根边缘细胞(root border cells, RBCs)耐铝反应中的作用及其相互关系, 以‘浙春3号’大豆为材料, 研究了铝毒胁迫下大豆根尖内源NO和H₂O₂的变化, 以及外源NO和H₂O₂诱导大豆根尖和RBCs的耐铝反应。结果表明, 50 μmol·L^–1 Al处理48 h显著抑制大豆根的伸长, 提高Al在根尖的积累, 同时显著增加根尖内源NO和H₂O₂含量。施加0.25 mmol·L^–1外源NO供体亚硝基铁氰化钠(Na₂[Fe(CN)₅NO]·2H₂O, sodium nitroprusside, SNP)和0.1 mmol·L^–1H₂O₂, 能有效地缓解Al对大豆根伸长的抑制、根尖Al积累和RBCs 的死亡, 该缓解作用可以被0.05 mmol·L^–1 NO清除剂2-(4- 羧基苯)-4,4,5,5- 四甲基咪唑-1- 氧-3- 氧化物, 钾盐(C₁₄H₁₆N₂O₄·K, carboxy-PTIO, cPTIO)和150 U·mL^–1 H₂O₂清除酶(catalase, CAT)逆转。并且外源NO能够显著促进根尖H₂O₂的积累, 而外源H₂O₂对根尖NO的含量无显著影响。这表明NO和H₂O₂是诱导大豆根尖及RBCs耐铝反应的两种信号分子, NO可能通过调控H₂O₂的形成, 进而诱导大豆根尖及RBCs的耐铝反应。     

Proteins bearing oxidation-induced carbonyl groups are not preferentially ubiquitinated

A substantial part of soluble, oxidized proteins are degraded by the proteasome. However, it is still under debate whether these oxidized proteins are degraded by the 26S proteasome in an ubiquitin-dependent way or in an ubiquitin-independent way by the 20S proteasome. Therefore, we treated cells with H₂O₂ and UV-A irradiation and detected protein carbonyls and ubiquitination by immunoblotting. Separation of ubiquitinated proteins from non-ubiquitinated reveals that most oxidized proteins are not ubiquitinated.     

Boron toxicity is alleviated by hydrogen sulfide in cucumber (Cucumis sativus L.) seedlings

Boron (B) is an essential micronutrient for plants, which when occurs in excess in the growth medium, becomes toxic to plants. Rapid inhibition of root elongation is one of the most distinct symptoms of B toxicity. Hydrogen sulfide (H₂S) is emerging as a potential messenger molecule involved in modulation of physiological processes in plants. In the present study, we investigated the role of H₂S in B toxicity in cucumber (Cucumis sativus) seedlings. Root elongation was significantly inhibited by exposure of cucumber seedlings to solutions containing 5 mM B. The inhibitory effect of B on root elongation was substantially alleviated by treatment with H₂S donor sodium hydrosulfide (NaHS). There was an increase in the activity of pectin methylesterase (PME) and up-regulated expression of genes encoding PME (CsPME) and expansin (CsExp) on exposure to high B concentration. The increase in PME activity and up-regulation of expression of CsPME and CsExp induced by high B concentration were markedly reduced in the presence of H₂S donor. There was a rapid increase in soluble B concentrations in roots on exposure to high concentration B solutions. Treatment with H₂S donor led to a transient reduction in soluble B concentration in roots such that no differences in soluble B concentrations in roots in the absence and presence of NaHS were found after 8 h exposure to the high concentration B solutions. These findings suggest that increases in activities of PME and expansin may underlie the inhibition of root elongation by toxic B, and that H₂S plays an ameliorative role in protection of plants from B toxicity by counteracting B-induced up-regulation of cell wall-associated proteins of PME and expansins.     

Comparative proteomic analysis of Cd-responsive proteins in wheat roots

Cadmium (Cd) is a major environmental toxicant to plant cells due to its potential inhibitory effects on many physiological processes. To gain a comprehensive understanding of plant response to Cd, wheat seedlings were exposed to a range of Cd concentrations (10, 100 and 200 μM) for 1 week and a combination of physiological and proteomic approaches were used to evidence Cd effects and to access the plant response to Cd toxicity. Root and shoot elongation was decreased, whereas the H₂O₂ and malondialdehyde content in wheat seedlings was increased significantly at higher Cd concentration. Protein profiles analyzed by two-dimensional electrophoresis revealed that 46 protein spots showed 1.5-fold change in protein abundance following Cd exposure; 31 protein spots were up-regulated and 15 protein spots were down-regulated; 25 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As expected, most of the up-regulated proteins are involved in heavy metal detoxification and antioxidant processes. Enzyme activity analysis revealed that ascorbate peroxidase and glutathione S-transferase activity was stimulated by Cd treatment. Abundance changes of these proteins, together with their putative functions provide us a new insight that can lead to an integrated understanding of the molecular basis of Cd responses in plants.     

Responses of gas exchange,cellular membrane integrity,and antioxidant enzymes activities of salinity-stressed winter wheat to ozone pollution

A sand-culture experiment was conducted in open-top chambers which were constructed in a greenhouse to investigate the responses of salt-stressed wheat (Triticum aestivum L.) to O₃. Plant seeding of JN17 (a popular winter wheat cultivar) was grown in saltless (−S) and saline (+S, 100 mM NaCl) conditions combined with charcoal-filtered air (CF, < 5 ppb O₃) and elevated O₃ (+O₃, 80 ± 5 ppb, 8 h day⁻¹) for 30 d. O₃ significantly reduced net photosynthetic rate (PN), stomatal conductance, chlorophyll contents and plant biomass in -S treatment, but no considerable differences were noted in those parameters between +O₃+S and CF+S treatments. O₃-induced loss in cellular membrane integrity was significant in -S plants, but not in +S plants evidenced by significant elevations being measured in electrolyte leakage (EL) and malondialdehyde (MDA) content in -S plants, but not in +S plants. Both O₃ and salinity increased proline content and stimulated antioxidant enzymes activities. Soluble protein increased by salinity but decreased by O₃. Abscisic acid (ABA) was significantly elevated by O₃ in -S plants but not in +S plants. The results of this study suggested that the specificity of different agricultural environments should be considered in order to develop reliable prediction models on O₃ damage to wheat plants.     

Capillary electrophoresis for screening of 20S proteasome inhibitors

A method for studying 20S proteasome inhibitors by capillary electrophoresis (CE) has been developed. Proteasome plays a fundamental role in degrading key regulatory proteins. The 20S proteasome can degrade intrinsically disordered proteins in an ATP-independent manner without additional “helper” molecules. The discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy. In this study, the inhibitory effects of MG132 and MG115 on the 20S proteasome were evaluated by CE for the first time. The optimized CE conditions were as follows: fused-silica capillary of 30 cm effective length and 75 μm internal diameter, pressure injection of 0.5 psi for 5 s, 50 mM Hepes buffer (pH 7.6) with 2% dimethyl sulfoxide, constant voltage of 20 kV, and detection wavelength at 340 nm. Also, the new method was used to study the inhibitory effects of 30 novel peptidyl vinyl ester derivatives of MG132. The 50% inhibition concentrations (IC₅₀ values) of MG132 and MG115 were 40.0 and 84.7 nM, respectively. Two new compounds, XP32 and XP35, showed considerable inhibitory effects on the 20S proteasome. When the concentrations of them were fixed at 172 nM, their inhibition rates were 36.2% and 29.1%, respectively. The results showed that the CE method was powerful, sensitive, and fast and required little sample. It could be employed as one of the reliable drug screening methods for 20S proteasome inhibitors.     

Characterisation of plant growth‐promoting rhizobacteria from rhizosphere soil of heat‐stressed and unstressed wheat and their use as bio‐inoculant

• High temperature induces several proteins in plants that enhance tolerance to high temperature shock. The fate of proteins synthesised in microbial cells or secreted into culture media by interacting microbes has not been fully elucidated. The present investigation aimed to characterise plant growth‐promoting rhizobacteria (PGPR) isolated from the rhizosphere of wheat genotypes (differing in tolerance to high temperature stress) and evaluate their performance as bioinoculant for use in wheat.
• Four bacterial strains, viz. Pseudomonas brassicacearum, Bacillus thuringiensis, Bacillus cereus strain W6 and Bacillus subtilis, were isolated from the rhizosphere of heat‐stressed and unstressed wheat genotypes. The wheat genotypes were exposed to high temperature stress at 45 °C for 10 days (3 h daily) at pre‐anthesis phase. Isolates were identified on the basis of morphology and biochemical characteristics, 16S rRNA gene sequencing and whole cell protein profiles. Results were further complemented by size exclusion chromatography (SEC) with fast protein liquid chromatography (FPLC) and SDS PAGE of 80% ammonium sulphate precipitates of the cell‐free supernatants.
• Isolates were positive for catalase, oxidases and antimicrobial activity . P. brassicacearum from the rhizosphere of the heat‐tolerant genotype was more efficient in phosphate solubilisation, bacteriocin production, antifungal and antibacterial activity against Helminthosporium sativum, Fusarium moniliforme and Klebsiella pneumonia, respectively. The inoculated seedlings had significantly higher root and shoot fresh weight, enhanced activity of antioxidant enzymes, proline and protein content. Total profiling of the culture with SDS‐PAGE indicated expression of new protein bands in 95 kDa in P. brassicacearum.
• Temperature‐induced changes in PGPR isolates are similar to those in the host plant. P. brassicacearum may be a good candidate for use in biofertiliser production for plants exposed to high temperature stress.

     

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.     

Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon Methanosarcina thermophila

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α₇ ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr¹ residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys³³ with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys³³ in polarization of the Thr¹ N, which serves to strip a proton from the active-site Thr¹ Oγ nucleophile. Replacement of Asp⁵¹ with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.     

Differential distribution of the cognate and heat-stress-induced isoforms of high Mr cis-trans prolyl peptidyl isomerase (FKBP) in the cytoplasm and nucleoplasm

Wheat root tips express a 73 kDa cognate isoform and a 77 kDa heat-shock-induced isoform of peptidyl prolyl cis-trans isomerase (FK506 binding protein; FKBP) that is part of a chaperone complex with hsp90. The 73 kDa and 77 kDa FKBPs have very similar sequences, differing primarily in the N- and C-terminal 20 amino acids. In order to define the potential functional roles of these proteins, the 73 kDa and 77 kDa FKBPs were localized in root tips using antigen-affinity purified antibodies as a probe. The cognate 73 kDa FKBP is localized in the cytoplasm and appears enriched around the periphery of the early vacuole and vesicles exiting the trans-Golgi. Parallel assays with antibodies directed against tonoplast aquaporin and pyrophosphatase confirmed the association of FKBP with an early vacuole compartment. Sucrose gradient centrifugation analysis of root tip lysates also showed that 73 kDa FKBP is co-fractionated with tonoplast aquaporin and V-ATPase in a light compartment near the top of the gradient. Heat-shock treatment of root tips induces the accumulation of 77 kDa FKBP while the abundance of 73 kDa FKBP remains constant. Quantitative EM immunogold assays of the intracellular distribution of FKBP over an 8 h heat-shock time-course showed that FKBP is initially present in the cytoplasm, but is transported into the nucleus where it accumulates in the nucleoplasm and into specific subnuclear domains. The results of this study show that the intracellular distribution of the high Mr FKBPs in wheat root tips differs at normal and elevated temperatures, indicating different functional roles for the FKBP isoforms.     

Influence of Rhizobial inoculation on yield of wheat (Triticum aestivum L.)

Summary Soil + charcoal (1∶3) carrier based and liquid cultures of Rhizobia were used to inoculate wheat seed cv. HD2329. The plants received 100 kg N in two equal splits and 60 kg P₂O₅ and 40 kg K₂₀ ha⁻¹. Inoculation with rhizobia had little effect on grain yield of wheat. Significant increase in straw yield and N-uptake occurred due to inoculation. A comparison of results of a similar experiment conducted during 1983–84, showed that inoculation with the same strains of rhizobia and application 50 kg N ha⁻¹ as basal dressing, was more effective in increasing yield and N-uptake in wheat cv. HD2329. It appears reasonable to assume occurrence of nitrogen fixation by root nodule bacteria in rhizosphere of wheat.