The chloroplast nucleoids of the bundle sheath and mesophyll cells of Zea mays

Dimorphic chloroplasts of Zea mays L. cv. GH5004 from bundle sheath and mesophyll cells contained similar amounts of DNA, while bundle sheath chloroplasts contained twice the number of nucleoids compared to mesophyll chloroplasts. On average bundle sheath nucleoids were half the size of mesophyll nucleoids and contained half as much DNA. Electron microscope autoradiography of the chloroplasts showed that the nucleoid DNA is associated with the thylakoids and in the case of mesophyll chloroplasts preferentially with the grana. These observations suggest that the differences in nucleoid distribution may be due to differences in membrane morphology, with the small nucleoids of agranal bundle sheath chloroplasts being widely dispersed.     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Isolation and culture of mesophyll protoplasts from Rosa hybrida

After 1 h plasmolysis in CPW13M solution, highly viable (>75%) protoplasts were isolated from leaves of axenic shoot cultures of Rosa hybrida L. cv. Abraham Darby using an enzyme mixture containing 1.0% (w/v) Hemicellulase, 0.1% (w/v) Macerozyme, 1.0 (w/v) Cellulase RS, 0.05% (w/v) Pectolyase Y23 and 1.0% (w/v) PVP-10 and from cv. Marie Pavié using an identical mixture but with Cellulase RS and Pectolyase Y23 at 0.7% (w/v) and 0.1% (w/v), respectively. With both cvs., sustained protoplast division was achieved after plating in agarose beads with modified KM8p medium containing 1.0% (w/v) polyvinylpyrrolidone (mol. wt. 10 000; PVP-10), 8.91 μM naphthaleneacetic acid (NAA) and 4.44 μM 6-benzyladenine (BA). Protoplast-derived callus gave rise to roots after transfer to SH medium containing 14 μM 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation and characterization of Azotobacter chroococcum from the roots of Zea mays

Abstract Bacteria showing rapid growth on a nitrogenfree medium and acetylene-reducing activity were isolated from maize roots collected from agricultural soils in Spain. The isolates were Gram-negative motile rods and were identified as Azotobacter chroococcum . Acetylene-reducing activity and microbial counts were determined on root segments from 7- and 30-day-old plants. Rates obtained were in the range of 0.0053–0.848 nmol C₂H₂· g⁻¹· h⁻¹. Root populations were 1.4–6.0 × 10⁴ micro-organisms · g⁻¹. These results showed that there was an association between A. chroococcum strains and roots of maize planted in some Spanish soils.     

Multiple forms of phosphoenolpyruvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba

The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg⁻¹ protein h⁻¹) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg⁻¹ protein h⁻¹, respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the M_r 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of M_r 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements.     

Flavonol content of guard cell and mesophyll cell protoplasts isolated from Vicia faba leaves

Guard cells of the lower epidermis of leaflets of Vicia faba L. cv. Weißkernige Hangdown contain several kaempferol 3,7-O-glycosides. This was demonstrated for the first time by the use of isolated, highly purified guard cell protoplasts for flavonol estimation and quantitation. From a total of ca 12 kaempferol glycosides, three were identified by comparative thin layer chromatography and high performance liquid chromatography as kaempferol 3-O-glucoside 7-O-rhamnoside (major component), 3-O-rhamnogalactoside 7-O-rhamnoside and 3,7-O-bisglucoside (minor components). On average, the total flavonol content was estimated to be 85 fmol protoplast⁻¹. From comparative investigations including alkaline-induced (green) fluorescence characteristics of flavonols and UV-microscopical studies we suggest that kaempferol glycosides are present in guard cells and epidermal cells in similar quantities, and that these compounds are in the vacuole.
By contrast, mesophyll protoplasts have a low flavonol content (one sixth that of guard cells). In spite of the different total flavonol contents, individual components of each cell-type are the same. However, they show differences in their quantitative distribution.     

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K _m (phosphoenolpyruvate) and K _i (malate) were compared. Malate sensitivity was much higher (K _i malate 0.4 mol m^–3) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K _i malate 4.2 mol m^–3). The stimulation of activity by glucose-6-phosphate in the presence of malate (deinhibition) was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.Abbreviations Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

Isolation and sugar uptake characteristics of protoplasts from maize endosperm suspension cultures

The isolation and sugar uptake characteristics of protoplasts from maize ( Zea mays L.) endosperm-derived suspension cultures are described. In contrast with protoplasts from intact developing endosperm, which by virtue of their large size and high starch content are too fragile for sugar uptake experiments, suspension cultures yielded protoplasts capable of withstanding the necessary handling and centrifugations. Intactness of the protoplasts was demonstrated by dye exclusion or accumulation and latency of malate dehydrogenase activity. Uptake of radioactivity from [³H]-inulin did not increase with time, but that from [¹⁴C]-sugars increased over a wide range of external concentrations. Kinetics of fructose, glucose and sucrose uptake were biphasic, and the saturable components of uptake were eliminated by p -chloromercuribenzene sulfonate (PCMBS). Rates of uptake of sucrose and 1'-fluorosucrose were similar, confirming that hydrolysis by cell wall invertase contributes to sucrose uptake by the suspension cultures. The isolation of protoplasts from this tissue source will enable experimental access to plasma membrane sugar carriers which may exist in the intact maize endosperm.     

Purification and characterization of glutathione reductase from corn mesophyll chloroplasts

Differences in the apparent molecular weights of the subunits of glutathione reductase (EC 1.6.4.2) from pea chloroplasts and corn mesophyll chloroplasts have been recently reported. In order to more fully describe the differences between the enzymes from these two sources, glutathione reductase from the mesophyll chloroplasts of corn seedlings ( Zea mays L. cv. G-4507) has been purified 200-fold by affinity chromatography using adenosine 2',5'-disphosphate agarose. The purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)^-1 min^-1. The native enzyme had a relative molecular weight of 190 ± 30 kDa and exhibited polypeptides of 65, 63, 34, and 32 kDa when separated on sodium dodecylsulfate-polyacrylamide gels. Comparisons of the results from electroblotting, native molecular weight and subunit molecular weight analyses suggest that the enzyme exists as a heterotetramer. Optimal enzyme activity was obtained at pH 8 in N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES-NaOH) buffer. The sulfhydryl reagent, n -ethylmaleimide, inhibited enzymatic activity when incubated in the presence of NADPH while no inhibition was detected with oxidized glutathione in the incubation mixture. Reduced glutathione (5 m M ) inactivated the enzyme by 50%. This inactivation followed first order kinetics with a rate constant of 0.0028 s^-1. The enzyme was also inactivated by NADPH. The inactivation reached ca 90% within 30 min and followed first order kinetics with a rate constant of 0.0015 s^-1.     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

Selected cell wall proteins from Zea mays: Assessment of their role in wall hydrolysis

Coleoptile cell wall proteins from Zea mays L. hybrid B 37 × Mo 17 were extracted and fractionated. Three enzymes identified in that extract were examined to determine their role in cell wall hydrolysis with a goal of evaluating the extent to which they participated in autohydrolytic reactions. Two separate proteins were identified as endo- and exo-glucanases. Incubation of these enzymes with heat inactivated cell walls, liberates products derived from the constitutive (1→3), (1→4)-β- d -glucan. The release of sugars from walls resembles that of cell wall autolysis. A third cell wall protein degraded polysaccharides in a more general manner, releasing carbohydrates containing xylose, arabinose, galactose and glucose. Polyclonal antibodies raised against the exoglucanase protein suppressed autolytic reactions of isolated cell wall.     

CO2 gas exchange and phosphoenolpyruvate carboxylase activity in leaves of Zea mays L.

Important gas exchange characteristics of C4 plants depend on the properties of phophoenolpyruvate carboxylase (PEPC), the enzyme catalysing the primary fixation of CO2 during C4 photosynthesis. In this study, the relationship between intracellular resistance for CO2 fixation (r_i) at high photosynthetically active photon flux densities (PPFD) and maximum PEPC activity in vitro (V_pm) was examined in leaves of Zea mays L. The analysis allowed the estimation of the Michaelis constant K_p of the enzyme for CO2 (or the equivalent number for bicarbonate) in vivo. At low PPFD (below 100 mol m-2 s-1) the initial slopes of the curves describing net CO2 uptake rate A as a function of intercellular CO2 concentration c_i increased with increasing PPFD. The increase (i. e. a decrease in r_i) was interpreted as due to a reversible activation of PEPC by light. Including this assumption into a model of C4 photosynthesis enabled us to reproduce A(c_i) response curves measured at low levels of PPFD. Fitting the model to experimental data resulted in values for K_I, the PPFD at which PEPC reaches half of its full activation, of about 200 mol m-2 s-1. Similar results were derived from the dependence of r_i on PPFD. The analysis of the relationships between r_i and V_pm and between r_i and PPFD, as well as fitting of the model to gas exchange data all gave rise to estimates for the resistance for CO2 transfer within mesophyll cells that are comparable with those known from C3 plants.     

Inhibition of phosphoenolpyruvate carboxylase from maize by 2-phosphoglycollate

The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg²⁺ suggestting that it binds only to enzyme-Mg²⁺ complex.     

玉米DEAD-box RNA解旋酶基因的克隆及分析

DEAD-box RNA解旋酶参与RNA转录、前体mRNA剪切、核糖体发生、核质运输、蛋白质翻译、RNA降解等重要的生命活动.根据本室在S-Mo17Rf3Rf3cDNA芯片研究中,检测到花粉发育后期RNA解旋酶上调表达的结果,应用RACE技术从S-Mo17Rf3Rf3花粉中克隆得到该RNA解旋酶基因全长cDNA,命名为ZmRH2并在GenBank注册登记 (DQ327709).序列分析表明：该cDNA全长1 652bp,从第163 bp开始到1 386bp含有一个开放阅读框,编码407个氨基酸.其编码的蛋白质具有DEAD-box RNA解旋酶特有的9个保守模体,与水稻、拟南芥和豌豆中的DEAD-box RNA解旋酶的氨基酸序列存在着很高的同源性.RT-PCR分析表明,该基因在近等基因系S-Mo17Rf3Rf3和S-Mo17rf3rf3的叶、根、和雌穗中的表达没有差异,但在花丝和花粉中有明显差异.     

Exogenous application of abscisic acid or triadimefon affects the recovery of Zea mays seedlings from heat shock

Maize seedlings ( Zea mays L. cv. DK 246) grown for 1–4 days in the presence of abscisic acid (ABA) or triadimefon (a fungicide) demonstrated an enhanced ability to withstand the effects of a 3-h sub-lethal (40°C) or lethal (45°C) heat shock. Both the ABA and triadimefon treatments were applied solely to the roots of seedlings; however, the ability to withstand a heat shock was induced in both the root and the shoot. The level of protection provided by these agents was dependent upon the time that plants were exposed to them; prolonged exposure reduced tolerance to subsequent stress.     

Genome size and numerical polymorphism for the B chromosome in races of maize (Zea mays ssp. mays, Poaceae)

Twenty-one native populations (1120 individuals) of maize from Northern Argentina were studied. These populations, which belong to 13 native races, were cultivated at different altitudes (80-3620 m). Nineteen of the populations analyzed showed B chromosome (Bs) numerical polymorphism. The frequency of individuals with Bs varied from 0 to 94%. The number of Bs per plant varied from 0 to 8 Bs, with the predominant doses being 0, 1, 2, and 3. Those populations with varying number of Bs showed a positive and statistically significant correlation of mean number of Bs with altitude. The DNA content, in plants without Bs (A-DNA)(2n = 20), of 17 populations of the 21 studied was determined. A 36% variation (5.0-6.8 pg) in A-DNA content was found. A significant negative correlation between A-DNA content and altitude of cultivation and between A-DNA content and mean number of Bs was found. This indicates that there is a close interrelationship between the DNA content of A chromosomes and doses of Bs. These results suggest that there is a maximum limit to the mass of nuclear DNA so that Bs are tolerated as long as this maximum limit is not exceeded.     

Temporal accumulation and ultrastructural localization of dehydrins in Zea mays

Immunolocalization using polyclonal antibodies raised against a conserved dehydrin amino acid sequence was used to establish the temporal and spatial patterns of dehydrin accumulation in embryo tissue of Zea mays L. (var. Ohio 43) kernels imbibed in the presence of abscisic acid. The temporal pattern of accumulation indicated an increase in dehydrins over time (particularly between 15 and 30 h) and with maximum levels detected 48 h after the onset of imbibition. Dehydrins were first evident, and also the most concentrated, in the cytosol throughout the accumulation period suggesting that the primary function of dehydrins involves the cytosol and the structures contained therein. Only after an accumulation of dehydrins in the cytosol was there an increase in the abundance of nuclear dehydrins. In addition, dehydrins were also observed in association with the proteinaceous matrix of protein bodies and membranes of protein and lipid bodies; these findings have not been reported previously. The observed localization at a number of sites indicates that the specific biochemical roles of dehydrins are likely to be diverse.     

Direct gene transfer inPetunia hybrida electroporated protoplasts: Evidence for co-transformation with a phosphoenolpyruvate carboxylase cDNA from sorghum leaf

Summary Co-transformation experiments were carried out onPetunia hybrida protoplasts. The method used was electroporation with two plasmids: one confering kanamycin resistance, and the other harbouring a phosphoenolpyruvate carboxylase (PEPC) cDNA fromSorghum vulgare leaves. Southern blot analysis of the selected lines demonstrated a high co-transformation frequency.Abbreviations PEPC phosphoenolpyruvate carboxylase - NPT neomycin phosphotransferase - ATF absolute transformation frequency - PEG polyethylenglycol - BA 6-benzyl-aminopurine - IAA 3-indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effects of low temperature on lateral diffusion in plasma membranes on maize (Zea mays L.) root cortex protoplasts: relevance to chilling sensitivity

The effects of temperature on the lateral diffusion of fluorescent phospholipids, sterols and proteins in the plasma membranes of maize root cortex protoplasts were monitored using fluorescence photobleaching recovery (FPR). Diffusion parameters were measured in two cultivars of maize having different chilling tolerance. Hydrodynamic theory predicts that the diffusion coefficient, D, should increase with increasing temperature. In the more chilling-tolerant cultivar, however, D for all three probes was nearly insensitive to temperature. In the more chilling-sensitive cultivar, D was also insensitive to temperature over the range from 12 to 21°C, but D for the lipid probes tended to be higher and more variable at lower temperatures. The proportion of probe molecules free to diffuse in the membrane was less than 1 for all probes, and increased significantly with increasing temperature for the protein probe. These results, taken together, support the concept that the plasma membrane contains domains having differing diffusional characteristics. Temperature effects on membrane diffusion are moderated by the existence of these domains to limit significant changes. The observed tendency for higher diffusion coefficients at low temperatures in the chilling-sensitive cultivar may correlate to morphological changes observed with protoplasts of that cultivar at low temperatures.