Cell wall-associated malate dehydrogenase activity from maize roots

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1 M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn²⁺ and Cu²⁺ in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn²⁺ and Cu²⁺ in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.     

Occurrence of aflatoxin in three maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) hybrids over 5 years in Northern Mississippi

Aflatoxins are produced as secondary metabolites under conducive climatic conditions by Aspergillus flavus. The incidence of aflatoxin varies with environmental conditions, genotype, and location. An expanded understanding of the interaction of the plant, fungus, and weather conditions is needed to further elucidate the field infection process of maize by A. flavus and subsequent aflatoxin contamination. One of the problems in evaluating maize hybrids for resistance to kernel infection and aflatoxin contamination is identifying a time period and environmental conditions that are most advantageous. Three maize genotypes (Pioneer Brand 3223, Mo18W × Mp313E, and Mp313E × Mp420) were evaluated from 1998 to 2002 in response to A. flavus inoculation and aflatoxin contamination and weather conditions favorable for aflatoxin contamination were identified. The highest aflatoxin levels were observed in 1998 and 2000 (1186 and 901 ng g⁻¹; P < 0.0001); while the lowest levels were detected in 1999 (39 ng g⁻¹). Pioneer 3223 had significantly higher levels (1198 ng g⁻¹) than Mp313E × Mp420 (205 ng g⁻¹), and Mo18W ×Mp313E (161 ng g⁻¹; P < 0.0001). The hybrids had six weather-related variables in common that were positively correlated with aflatoxin accumulation. Four of these occurred during 65–85 days after planting and were temperature-related. These results suggest that regardless of the hybrid’s maturity or physiological development, the time from 65 to 85 days after planting may be indicative of a period of stress which leads to greater aflatoxin accumulation at harvest. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts

Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.     

Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an α zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.     

Ethanol production from maize silage as lignocellulosic biomass in anaerobically digested and wet-oxidized manure

In this communication, pretreatment of the anaerobically digested (AD) manure and the application of the pretreated AD manure as liquid medium for the simultaneous saccharification and fermentation (SSF) were described. Furthermore, fermentation of pretreated maize silage and wheat straw was investigated using 2 l bioreactors. Wet oxidation performed for 20 min at 121 °C was found as the most suitable pretreatment conditions for AD manure. High ammonia concentration and significant amount of macro- and micro-nutrients in the AD manure had a positive influence on the ethanol fermentation. No extra nitrogen source was needed in the fermentation broth. It was shown that the AD manure could successfully substitute process water in SSF of pretreated lignocellulosic fibres. Theoretical ethanol yields of 82% were achieved, giving 30.8 kg ethanol per 100 kg dry mass of maize silage.     

Light affects competition for inorganic and organic nitrogen between maize and rhizosphere microorganisms

Effects of light on the short term competition for organic and inorganic nitrogen between maize and rhizosphere microorganisms were investigated using a mixture of amino acid, ammonium and nitrate under controlled conditions. The amount and forms of N added in the three treatments was identical, but only one of the three N forms was labeled with ¹⁵N. Glycine was additionally labeled with ¹⁴C to prove its uptake by maize and incorporation into microbial biomass in an intact form. Maize out-competed microorganisms for during the whole experiment under low and high light intensity. Microbial uptake of ¹⁵N and ¹⁴C was not directly influenced by the light intensity, but was indirectly related to the impact the light intensity had on the plant. More was recovered in microbial biomass than in plants in the initial 4 h under the two light intensities, although more ¹⁵N-glycine was incorporated into microbial biomass than in plants in the initial 4 h under low light intensity. Light had a significant effect on uptake by maize, but no significant effects on the uptake of or ¹⁵N-glycine. High light intensity significantly increased plant uptake of and glycine ¹⁴C. Based on ¹⁴C to ¹⁵N recovery ratios of plants, intact glycine contributed at least 13% to glycine-derived nitrogen 4 h after tracer additions, but it contributed only 0.5% to total nitrogen uptake. These findings suggest that light intensity alters the competitive relationship between maize roots and rhizosphere microorganisms and that C4 cereals such as maize are able to access small amounts of intact glycine. We conclude that roots were stronger competitor than microorganisms for inorganic N, but microorganisms out competed plants during a short period for organic N, which was mineralized into inorganic N within a few hours of application to the soil and was thereafter available for root uptake.     

Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars

Effects of cadmium (Cd(2+)) on photosynthetic and antioxidant activities of maize (Zea mays L.) cultivars (3223 and 32D99) were investigated. Fourteen-day-old cultivar seedlings were exposed to different Cd concentrations [0, 0.3, 0.6 and 0.9mM Cd(NO(3))(2).4H(2)O] for 8 days. The results of chlorophyll fluorescence indicated that different levels of Cd affected photochemical efficiency in 3223 much more than that in 32D99. In parallel, the level of Cd at 0.9mM caused oxidative damage but did not indicate cessation of PSII activity of the cultivars; plant death was not observed at highly toxic Cd levels. Additionally, the increase in Cd concentration caused loss of chlorophylls and carotenoid and membrane damage in both cultivars, but greater membrane damage was observed in 3223 than in 32D99. Depending on Cd accumulation, a significant reduction in dry biomass was observed in both cultivars at all Cd concentrations. The accumulation of Cd was higher in roots than in leaves for both cultivars. Nevertheless, cultivar 3223 transferred more Cd from roots to leaves than did 32D99. On the other hand, our results suggest that there were similar responses in SOD, APX and GR activities with increasing Cd concentrations for both cultivars. However, POD activity significantly increased at highly toxic Cd levels in 32D99. This result may be regarded as an indication of better tolerance of the Z. mays L. cultivar 32D99 to Cd contamination.     

A member of the maize isopentenyl transferase gene family, <Emphasis Type="Italic">Zea mays isopentenyl transferase 2</Emphasis> (<Emphasis Type="Italic">ZmIPT2</Emphasis>), encodes a cytokinin biosynthetic enzyme expressed during kernel development

Cytokinins (CKs) are plant hormones that regulate a large number of processes associated with plant growth and development such as induction of stomata opening, delayed senescence, suppression of auxin-induced apical dominance, signaling of nitrogen availability, differentiation of plastids and control of sink strength. In maize, CKs are thought to play an important role in establishing seed size and increasing seed set under normal and unfavorable environmental conditions therefore influencing yield. In recent years, the discovery of isopentenyl transferase (IPT) genes in plants has shed light on the CK biosynthesis pathway in plants. In an effort to increase our understanding of the role played by CKs in maize development and sink-strength, we identified several putative IPT genes using a bioinformatics approach. We focused our attention on one gene in particular, ZmIPT2, because of its strong expression in developing kernels. The expression of the gene and its product overlays the change in CK levels in developing kernels suggesting a major role in CK biosynthesis for kernel development. We demonstrate that at 8–10 days after pollination (DAP) the endosperm and especially the basal transfer cell layer (BETL) is a major site of ZmIPT2 expression, and that this expression persists in the BETL and the developing embryo into later kernel development stages. We show that ectopic expression of ZmIPT2 in calli and in planta created phenotypes consistent with CK overproduction. We also show that ZmIPT2 preferentially uses ADP and ATP over AMP as the substrates for dimethylallyl diphosphate (DMAPP) IPT activity. The expression pattern of ZmIPT2 in the BETL, endosperm and embryo during kernel development will be discussed with an emphasis on the suggested role of CKs in determining sink-strength and grain production in crop plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteomic changes in maize roots after short-term adjustment to saline growth conditions

It is of fundamental importance to understand adaptation processes leading to salt resistance. The initial effects on maize roots in the first hour after the adjustment to saline conditions were monitored to elucidate initial responses. The subsequent proteome change was monitored using a 2‐D proteomic approach. We found several new salt‐inducible proteins, whose expression has not been previously reported to be modulated by salt. A set of phosphoproteins in maize was detected but only ten proteins were phosphorylated and six proteins were dephosphorylated after the application of 25 mM NaCl for 1 h. Some of the phosphorylated maize proteins such as fructokinase, UDP‐glucosyl transferase BX9, and 2‐Cys‐peroxyredoxine were enhanced, whereas an isocitrate‐dehydrogenase, calmodulin, maturase, and a 40‐S‐ribosomal protein were dephosphorylated after adjustment to saline conditions. The initial reaction of the proteome and phosphoproteome of maize after adjustment to saline conditions reveals members of sugar signalling and cell signalling pathways such as calmodulin, and gave hint to a transduction chain which is involved in NaCl‐induced signalling. An alteration of 14‐3‐3 proteins as detected may change plasma membrane ATPase activity and cell wall growth regulators such as xyloglucane endotransglycosylase were also found to be changed immediately after the adjustment to salt stress.