Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [214-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.

Use of the pyrophosphatase activity as a reliable tonoplast marker in maize roots.

Membranes of maize (Zea mays L., cv LG 11) roots were fractionated by sucrose (in presence or absence of Mg2+) or dextran density gradient centrifugations and the locations of organelles were determined using marker enzymes. Latent UDPase was used as a Golgi marker, catalase for the peroxysomes, cytochrome c oxidase for the mitochondria, UDP-Gal-galactosyltransferase for the amyloplast membranes and NADH-cytochrome c reductase for the ER. Two markers were selected for the plasmalemma, the vanadate-sensitive ATPase and UDP-Glc-sterolglucosyltransferase. The distributions of the PPase and vacuolar ATPase were found to be similar after density gradient centrifugation. The PPase and vacuolar ATPase activities were clearly separated from almost all the other markers tested, however, a partial association of both activities with the ER cannot be completely ruled out. The PPase of maize roots is more active and easier to measure than the vacuolar ATPase and is therefore an excellent candidate for use as a tonoplast marker.

A Ca2+/H+ antiport system driven by the tonoplast pyrophosphate-dependent proton pump from maize roots.

Calcium uptake by tonoplast enriched membrane vesicles from maize (Zea mays L. cv. LG 11) primary roots was studied. A pH gradient, measured by the fluorescence quenching of quinacrine, was generated across sealed vesicles driven by the pyrophosphate-dependent proton pump. The fluorescence quenching was strongly inhibited by Ca2+; moreover, when increasing Ca2+ concentrations were added to vesicles at steady-state, a concomitant decrease in the proton gradient was observed. Ca2+ uptake using Ca-45(2+) was linear from 10 min when oxalate (10 mM) was present, while Ca2+ uptake was completely inhibited with proton ionophores (FCCP and monensin), indicating a Ca2+/H+ antiport. Membranes were further fractionated using a linear sucrose density gradient (10-45%) and were identified with marker enzymes. Ca2+ uptake co-migrated with the tonoplast pyrophosphate-dependent proton pumping, pyrophosphatase and ATPase activities: the Ca2+/H+ antiport is consequently located at the tonoplast.

Tonoplast localization of a calmodulin-stimulated Ca2+-pump from maize roots.

The subcellular localization of a calmodulin-stimulated calcium (Ca2+)-ATPase activity from maize roots (Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca2+ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure Ca-45(2+) transport in sealed vesicles. Most of the ATP-dependent Ca2+ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H+/Ca2+ antiport) and the plasma membrane. When the Ca2+ transport was measured in the presence of exogenous calmodulin (1 muM), a 3-5-fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, ruling out a direct effect of calmodulin on the H+/Ca2+ antiport. In conclusion, four different Ca2+ transporters are present in young maize root cells. A Ca2+/H+ antiport system is present on the tonoplast, whereas, the plasma membrane and the ER possess each a calmodulinin-sensitive Ca2+-ATPase. Finally, a calmodulin-stimulated Ca2+-ATPase is associated with the tonoplast.

Characterization of the tonoplast Ca2+/H+ antiport system from maize roots.

The tonoplast calcium Ca2+/H+ antiport system of maize (Zea mays L. cv LG 11) roots was characterized using the ''pH jump'' technique in order to avoid interference from the tonoplast proton and Ca2+ pumps. Ca2+ uptake was recorded in the presence of different inhibitors and divalent ions. Chemical modification of amino acid residues of the antiport was used to elucidate the amino acid residues participating in the Ca2+ transport activity. The Ca2+/H+ antiport activity was found to be strongly inhibited by ruthenium red and verapamil, whereas diethylstilbestrol was less effective. Vanadate, erythrosin B, cyclopiazonic acid, bafilomycin, thapsigargin, N,N'-dicyclohexylcarbodiimide (DCCD) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) were without effect. Lanthanum and divalent ions were strongly inhibitory (Cd2+ > Mn2+ > Sr2+ > Ba2+). While reagents modifying sulfhydryl groups (N-ethylmaleimide and 5,5'-dithio-bis(2-nitrobenzoate)) did not affect the antiport activity, modification of trytophan residues (N-bromosuccinimide) was strongly inhibitory. We conclude that ruthenium red, verapamil, lanthanum and divalent cations directly inhibit Ca2+ uptake independent of the function of the proton and Ca2+ pumps. Moreover, the results of chemically modified amino acid residues suggest that sulfhydryl groups are not involved in Ca2+ transport, while tryptophan residues seem important for this translocation.

Quantification of abscisic Acid in a single maize root.

Quantitative analyses of abscisic acid in the elongating zone of a single maize root (Zea mays L. cv LG 11) were performed by gas chromatography-mass spectrometry using negative chemical ion ionization. Data showed that the more abscisic acid, the slower the growth, but a large dispersion of individual values was observed. We assume that abscisic acid is perhaps not correlated only to the growth rate.

Localization in sucrose gradients of the pyrophosphate-dependent proton transport of maize root membranes.

A maize (Zea mays L. cv LG 11) root homogenate was prepared and centrifuged to sediment the mitochondria. The pellet (6 KP) and the supernatant (6 KS) were collected and fractionated on linear sucrose density gradients. Marker enzymes were used to study the distribution of the different cell membranes in the gradients. The distribution of the ATP- and pyrophosphate-dependent proton pumping activities was similar after 3 hours of centrifugation of the 6 KS or the 6 KP fraction. The pumps were clearly separated from the mitochondrial marker cytochrome c oxidase and the plasmalemma marker UDP-glucose-sterolglucosyl-transferase. The pyrophosphate-dependent proton pump might be associated with the tonoplast, as the ATP-dependent pump, despite the lack of a specific marker for this membrane. However, under all the conditions tested, the two pumps overlapped the Golgi markers latent UDPase and glucan synthase I and the ER marker NADH-cytochrome c reductase. It is therefore not possible to exclude the presence of proton pumping activities on the Golgi or the ER of maize root cells. The two pumps (but especially the pyrophosphate-dependent one) were more active (or more abundant) in the tip than in the basal part of maize roots, indicating that these activities might be important in growth processes.

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast.

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.

Survey of ear flies (Diptera, Ulidiidae) in maize (Zea mays L.) and a new record of Euxesta mazorca Steyskal in Brazil

Survey of ear flies (Diptera, Ulidiidae) in maize (Zea mays L.) and a new record of Euxesta mazorca Steyskalin Brazil. Species of Euxesta (Diptera, Ulidiidae), known as silk flies or ear flies, are becoming increasingly important as maize insect pests in South America, although very little is known about them in Brazil. The larvae of some species of this genus initially damage female reproductive tissues, and then the developing kernels on the ear. As a result of feeding, fermentation and associated odors cause complete loss of the grain because it is no longer fit for human or livestock consumption. The main objective of this work was to evaluate the incidence of Euxesta spp. in Brazilian maize fields and to determine the most prevalent species using two different hydrolyzed protein foods attractants, BioAnastrepha® (hydrolyzed maize protein) and Torula, placed inside McPhail traps. The two species identified were E. eluta Loew and E. mazorca Steyskal, the latter being a new record from Brazil. Between the two species, E. eluta was the more abundant in maize fields. Both attractants were efficient in capturing the two species. However, BioAnastrepha® captured significantly more insects than Torula.

Larval development of Spodoptera eridania (Cramer) fed on leaves of Bt maize expressing Cry1F and Cry1F + Cry1A.105 + Cry2Ab2 proteins and its non-Bt isoline

This study aimed to evaluate, in controlled laboratory conditions (temperature of 25±2 °C, relative humidity of 60±10%, and 14/10 h L/D photoperiod), the larval development of Spodoptera eridania (Cramer, 1784) (Lepidoptera, Noctuidae) fed with leaves of Bt maize expressing Cry1F and Cry1F + Cry1A.105 + Cry2Ab2 insecticide proteins and its non-Btisoline. Maize leaves triggered 100% of mortality on S. eridania larvae independently of being Bt or non-Bt plants. However, it was observed that in overall Bt maize (expressing a single or pyramided protein) slightly affects the larval development of S. eridania, even under reduced leaf consumption. Therefore, these results showed that Cry1F and Cry1F + Cry1A.105 + Cry2Ab2 can affect the larval development of S. eridania, although it is not a target pest of this plant; however, more research is needed to better understand this evidence. Finally, this study confirms that non-Bt maize leaves are unsuitable food source to S. eridania larvae, suggesting that they are not a potential pest in maize fields.

Does Bt maize cultivation affect the non-target insect community in the agro ecosystem?

ABSTRACT The cultivation of genetically modified crops in Brazil has led to the need to assess the impacts of this technology on non-target species. Under field conditions, the potential effect on insect biodiversity was evaluated by comparing a homogeneous corn field with conventional and transgenic maize, expressing different Bt proteins in seven counties of Minas Gerais, Brazil. The richness pattern of non-target insect species, secondary pests and natural enemies were observed. The results do not support the hypothesis that Bt protein affects insect biodiversity. The richness and diversity data of insects studied were dependent on the location and other factors, such as the use of insecticides, which may be a major factor where they are used.

Growth and metabolic activity of the extramatrical mycelium of endomycorrhizal maize plants

The objective of this experiment was to quantify the extramatrical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus etunicatum (Becker & Gerdemann) grown on maize (Zea mays L. var. Piranão) provided with various levels of phosphate fertilizer and harvested at 30, 60 and 90 days after planting (DAP). Total extramatrical mycelium (TEM) was extracted from soil using a modified membrane filtration method, followed by quantification using a grid intersection technique. Active extramatrical mycelium (AEM) proportion was determined using an enzymatic method which measured dehydrogenase activity by following iodonitrotetrazolium reduction. At low levels of added P, there was relatively less TEM than at high levels of added P, but the AEM proportion at low soil P availability was significantly greater than at high soil P.

Nitrogen application timing and soil inorganic nitrogen dynamics under no-till oat/maize sequential cropping

The timing of N application to maize is a key factor to be considered in no-till oat/maize sequential cropping. This study aimed to evaluate the influence of pre-planting, planting and sidedress N application on oat residue decomposition, on soil N immobilisation and remineralisation and on N uptake by maize plants in no-till oat/maize sequential cropping. Undisturbed soil cores of 10 and 20 cm diameter were collected from the 0-15 cm layer of a no-till Red Latossol, when the oat cover crop was in the milk-grain stage. Two greenhouse experiments were conducted simultaneously. Experiment A, established in the 10 cm diameter cores and without plant cultivation, was used to asses N dynamics in soil and oat residues. Experiment B, established in the 20 cm diameter cores and with maize cultivation, was used to assess plant growth and N uptake. An amount of 6.0 Mg ha-1 dry matter of oat residues was spread on the surface of the cores. A rate of 90 kg N ha-1 applied as ammonium sulphate in both experiments was split in pre-planting, planting and sidedress applications as follows: (a) 00-00-00 (control), (b) 90-00-00 (pre-planting application, 20 days before planting), (c) 00-90-00 (planting application), (d) 00-30-60 (split in a planting and a sidedress application 31 days after emergence), (e) 00-00-00* (control, without oat residue) and (f) 90-00-00* (pre-planting application, without oat residue). The N concentration and N content in oat residues were not affected during decomposition by N fertilisation. Most of the fertiliser NH4+-N was converted into NO3--N within 20 days after application. A significant decrease in NO3--N contents in the 0-4 cm layer was observed in all treatments between 40 and 60 days after the oat residue placement on the soil surface, suggesting the occurrence of N immobilisation in this period. Considering that most of the inorganic N was converted into NO3- and that no immobilisation of the pre planting fertiliser N occurred at the time of its application, it was possible to conclude that pre-planting applied N was prone to losses by leaching. On the other hand, with split N applications, maize plants showed N deficiency symptoms before sidedress application. Two indications for fertiliser-N management in no-till oat/maize sequential cropping could be suggested: (a) in case of split application, the sidedress should be earlier than 30 days after emergence, and (b) if integral application is preferred to save field operations, this should be done at planting.

Maize response to nitrogen fertilization timing in two tillage systems in a soil with high organic matter content

No-tillage systems, associated to black oat as preceding cover crop, have been increasingly adopted. This has motivated anticipated maize nitrogen fertilization, transferring it from the side-dress system at the stage when plants have five to six expanded leaves to when the preceding cover crop is eliminated or to maize sowing. This study was conducted to evaluate the effects of soil tillage system and timing of N fertilization on maize grain yield and agronomic efficiency of N applied to a soil with high organic matter content. A three-year field experiment was conducted in Lages, state of Santa Catarina, from 1999 onwards. Treatments were set up in a split plot arrangement. Two soil tillage systems were tested in the main plots: conventional tillage (CT) and no-tillage (NT). Six N management systems were assessed in the split-plots: S1 - control, without N application; S2 - all N (100 kg ha-1) applied at oat desiccation; S3 - all N applied at maize sowing; S4 - all N side-dressed when maize had five expanded leaves (V5 growth stage); S5 - 1/3 of N rate applied at maize sowing and 2/3 at V5; S6 - 2/3 of nitrogen rate applied at maize sowing and 1/3 at V5. Maize response to the time and form of splitting N was not affected by the soil tillage system. Grain yield ranged from 6.0 to 11.8 t ha-1. The anticipation of N application (S2 and S3) decreased grain yield in two of three years. In the rainiest early spring season (2000/2001) of the experiment, S4 promoted an yield advantage of 2.2 t ha-1 over S2 and S3. Application of total N rate before or at sowing decreased the number of kernels produced per ear in 2000/2001 and 2001/2002 and the number of ears produced per area in 2001/2002, resulting in reduced grain yield. The agronomic efficiency of applied N (kg grain increase/kg of N applied) ranged from 13.9 to 38.8 and was always higher in the S4 than in the S2 and S3 N systems. Short-term N immobilization did not reduce grain yield when no N was applied before or at maize sowing in a soil with high organic matter content, regardless of the soil tillage system.