Influence of phosphorus and formononetin on isozyme expression in the Zea mays‐Glomus intraradices symbiosis

Maize (Zea mays L. cv. Great Lakes 586) plants were either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith, or grown in the presence of the isoflavone formononetin or were provided with both G. intraradices and formononetin. All plants were grown in soil containing one of five levels of inorganic P (between 8 and 110 µg g^?1 soil). By 3 weeks there were significant differences in a number of enzyme activities and in the pattern of isoenzymes in roots colonized by the VAM fungus or treated with formononetin. One NAD-malate dehydrogenase (MDH) isozyme was expressed only in mycorrhizal roots, whether treated or not with formononetin. Despite differences in the soil P level, the expression of this isozyme was not observed in non-mycorrhizal roots, indicating specific expression in the mycorrhizae. We suggest that MDH isozyme could serve as a specific, early indicator of the Zea-Glomus symbiosis. Differences in the esterase (EST) isozyme pattern were not detectable between VAM and non-VAM roots, suggesting that this enzyme system is not a good parameter for the evaluation of mycorrhizal colonization. As available P in the soil increased, total EST activity appeared to increase as well. Interestingly, total peroxidase (POX) activity increased along with P suggesting that as plant P nutrition improved, both cell wall ramification and the quantity of defense peroxidases increased as well. Total POX activity from mycorrhizal roots was inversely correlated with root colonization, indicating that there was suppression of POX activity by the host under low soil P. Most interestingly, formononetin further decreased POX activity regardless of the level of P or mycorrhizal status. This may suggest one mechanism by which formononetin enhances root VAM colonization. The presence of this isoflavone suppressed POX activity in mycorrhizal roots allowing a rapid penetration and spread of the fungus in the root cortex. The interplay between host root, soil P levels, secondary metabolites and endogenous host enzyme activities and a particular VAM fungus has a profound effect on the efficiency, duration and functioning of an endomycorrhizal symbiosis.     

Arbuscular mycorrhizae and nitrogen assimilation in maize after drought and recovery

In a greenhouse experiment, the effect of arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck & Smith) colonization on N assimilation in maize (Zea mays L.) was examined after well-watered, drought and recovery periods. Seeds of selection cycles C0 (drought-sensitive) and C8 (drought-resistant) of the tropical maize cultivar Tuxpeño sequía were used for this study. Maize plants were exposed or not to drought stress for 3 weeks (45-65 days after sowing, DAS) followed by 3 weeks of recovery (66-86 DAS) at the preflowering stage. Root and shoot samples harvested at the end of the drought or well-watered and recovery periods were determined for key enzymes involved in N assimilation (NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase), protein and amino acid concentrations, and total N contents. Drought stress significantly (P ≤ 0.01 or P ≤ 0.001) decreased all the enzyme activities except NiR in the roots and shoots of both cultivars. After 3 weeks of drought, the AM roots of both cultivars had higher activities of NR (C0, 45%; C8, 26%), GS (C0, 76%; C8, 33%) and GOGAT (C0, 41%; C8, 53%) than non-AM roots and were comparable to well-watered plants. These enzyme activities were also enhanced in drought-stressed AM shoots of C0 and C8. Total amino acid concentrations in AM plants of C0 were 4.6 and 1.6 times higher in roots and shoots, respectively, compared to non-AM plants. The predominant amino acids detected were Ala, Arg, Asn, Asp, Gln and Glu which constituted approximately 56 and 75% of the total pool in roots and shoots, respectively. Soluble proteins and total N contents were also higher in AM plants than non-AM plants under drought conditions. The enhancement of N-assimilating enzymes and nitrogenous compounds in maize may indicate a transfer of NO₃^? through the extraradical mycelium or increased N assimilation due to the AM symbiosis. Our overall results suggest that AM association plays an important role in enhancing N assimilation or N nutritional status which enables the host plant to withstand drought conditions and recover after stress is relieved.     

Thiols of Cu-treated maize plants inoculated with the arbuscular-mycorrhizal fungus Glomus intraradices

Mycorrhizal colonization of roots, fresh weight, content of cysteine, γ-glutamylcysteine (γEC). glutathione (GSH), thiol groups in Cu-binding peptides (CuBP), and the uptake of Cu were measured in roots and shoots of maize ( Zea mays L., cv. Honeycomb F-1) grown in quartz sand, with Cu at 0, 4.5, 9, 15 and 30 μg g⁻¹ added with or without inoculum of the arbuscular-mycorrhizal fungus (AMF) Glomus intraradices . In control plants (no Cu added) AMF significantly reduced shoot growth, but did not affect root growth. At an external Cu supply of 9 μg (g quartz sand)⁻¹ or higher, both mycorrhizal colonization and growth of roots and shoots of mycorrhizal and non-mycorrhizal plants were significantly reduced.
With up to 9 μg Cu g⁻¹, mycorrhizal colonization increased the content of cysteine, γEC and GSH in the roots. However, the amount of thiols in CuBPs was not increased by mycorrhizal colonization in Cu-treated plants and no differences in Cu uptake were detected between non-mycorrhizal and mycorrhizal plants. A CuBP-complex with a relative molecular mass of 7300 and a SH:Cu ratio of 1.77:1 was separated on a Sephadex G-50 column from both non-inoculated and inoculated roots of Cu-treated plants. HPLC chromatography of the CuBPs of both non-inoculated and inoculated roots resulted in a similar peak pattern, indicating that no additional CuBPs were formed by the fungus. In conclusion, our results do not support the idea that AMF protects maize from Cu-toxicity.     

European and African maize cultivars differ in their physiological and molecular responses to mycorrhizal infection

Physiological and molecular responses to phosphorus (P) supply and mycorrhizal infection by Glomus intraradices were compared in European (River) and African (H511) maize (Zea mays) cultivars to examine the extent to which these responses differed between plants developed for use in high- and low-nutrient-input agricultural systems. Biomass, photosynthetic rates, nutrient and carbohydrate contents, mycorrhizal colonization and nutrient-responsive phosphate transporter gene expression were measured in nonmycorrhizal and mycorrhizal plants grown at different inorganic phosphorus (P(i)) supply rates. Nonmycorrhizal River plants grew poorly at low P(i) but were highly responsive to mycorrhizal infection; there were large increases in biomass, tissue P content and the rate of photosynthesis and a decline in the expression of phosphate transporter genes. Nonmycorrhizal H511 plants grew better than River plants at low P(i), and had a higher root : shoot ratio. However, the responses of H511 plants to higher P(i) supplies and mycorrhizal infection were much more limited than those of River plants. The adaptations that allowed nonmycorrhizal H511 plants to perform well in low-P soils limited their ability to respond to higher nutrient supply rates and mycorrhizal infection. The European variety had not lost the ability to respond to mycorrhizas and may have traits useful for low-nutrient agriculture where mycorrhizal symbioses are established.     

Regulation of glutamate dehydrogenase activity by amino acids in maize seedlings

Root or secondary leaf segments from maize ( Zea mays L. cv. Ganga safed-2) seedlings were incubated with 9-amino acids and two amides separately, each at 5 m M for 24 h, to study their effects on glutamate dehydrogenase (GDH) activity. Most of the compounds tested inhibited the specific activity of NADH-GDH and increased that of NAD⁺-GDH in the roots in the presence as well as in the absence of ammonium. In the leaves, such effects were recorded only with a few amino acids. Total soluble protein in the root and leaf tissues increased with the supply of most of the amino compounds. The effect of glutamate on enzyme activity and protein was concentration dependent in both tissues. When the enzyme extracts from root or leaf tissues were incubated with some of the amino acids, NADH-GDH declined while NAD⁺-GDH increased in most cases. The inhibition of NADH-GDH increased with increasing concentration of cysteine from 1 to 5 m M . The experiments demonstrate that most of the amino acids regulated GDH activity, possibly through some physicochemical modulation of the enzyme molecule.     

Effect of salicylic acid on nitrate reductase activity in maize seedlings

The effect of different concentrations of salicylic acid on total Kjeldahl nitrogen and nitrate reductase activity in the maize ( Zea mays L.) seedling was studied. The total nitrogen of the maize embryonic axis (root + shoot) from seedlings raised with 10 m M Ca(NO₃)₂ for 5 days was substantially higher than that from the control when 0.01 m M salicylic acid was supplied. As supply of high (1 m M ) concentrations of salicylic acid decreased the accumulation of organic nitrogen. The in vivo activity of nitrate reductase in the roots increased at low concentrations of salicylic acid, while high concentrations were inhibitory. The stimulative concentration of the acid protected in vivo loss of nitrate reductase activity under non-inducing conditions, whereas it had no effect on in vitro loss of enzyme. It is suggested that salicylic acid increases in vivo enzyme activity indirectly, to some extent by protecting the natural inactivation of the enzyme.     

Community analysis of arbuscular mycorrhizal fungi and bacteria in the maize mycorrhizosphere in a long-term fertilization trial

In this study, we investigated the impact of organic and mineral fertilizers on the community composition of arbuscular mycorrhizal (AM) fungi and bacteria in the mycorrhizosphere of maize in a field experiment established in 1956, in south-east Sweden. Roots and root-associated soil aggregates were sampled four times during the growing season in 2005, in control plots and in plots amended with calcium nitrate, ammonium sulphate, green manure, farmyard manure or sewage sludge. Fungi in roots were identified by cloning and sequencing, and bacteria in soil aggregates were analysed by terminal-restriction fragment length polymorphism, cloning and sequencing. The community composition of AM fungi and bacteria was significantly influenced by the different fertilizers. Changes in microbial community composition were mainly correlated with changes in pH induced by the fertilization regime. However, other factors, including phosphate and soil carbon content, also contributed significantly to these changes. Changes in bacterial community composition and a reduction in bacterial taxon richness throughout the growing season were also manifest. The results of this study highlight the importance and significant effects of the long-term application of different fertilizers on edaphic factors and specific groups of fungi and bacteria playing a key role in arable soils.     

Influence of genotype and texture on zein content in endosperm of maize grains

The effect of genotypes and texture on the content of proteins in maize grains was examined by assessing absolute amounts of six protein fractions in the whole endosperms of four wild‐type lines with high protein content and four quality protein maize (QPM) varieties and for hand‐dissected hard and soft endosperm regions from eight other lines. As previously reported for six wild‐type lines and their opaque‐2(o2) versions, zeins were predominant for all genetic backgrounds and all types of endosperms. From these data and others the amounts of zeins and true proteins (crude proteins free of non‐protein nitrogen) in developing and mature endosperms of wild‐type lines were correlated. The data points for zeins from hard endosperms lay between the regression line and the upper limit of confidence area. Those for zeins from soft endosperms were located at the lower part of confidence area and on a level with the points corresponding to the most immature endosperms. Furthermore, some data points for zeins from o2 and QPM samples lay near the lower limit while the others were outside the confidence area. This suggested an initial zein accumulation dependent on the genotype at a low relative rate, followed by an accumulation at higher rate. The conditions used for isolating and quantitating zeins are discussed.     

The effect of abscisic acid on cell growth, cell division and DNA synthesis in the maize root meristem

Cis -abscisic acid (ABA), when applied to maize ( Zea mays L. cv. LG 11) roots, decreases the rates of cell growth and cell division in the meristem. It also decreases the rate at which nuclei become labelled with [³H]-thymidine and enter mitosis. Removing the root cap accelerates the entry of nuclei into the DNA synthetic phase of the mitotic cycle and enhances the rate of cell proliferation in the quescent centre. ABA diminishes these effects, but does not suppress them. Thus, ABA cannot wholely substitute for the presence of a cap. One of the primary effects of applied ABA is to retard cell enlargement which may in turn affect the rate of cell division; natural endogenous ABA may act similarly. ABA might in this way assist in maintaining the quiescent centre in intact roots, but cannot be the sole agent involved.     

Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in maize tissues

The activity of the enzyme 3-hydroxy-3-methlglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) is highly expressed in 4-day-old etiolated seedlings of normal (cv. DeKalb XL72AA), dwarf ( d ₅) and albino ( lw ₃) maize ( Zea mays L.). HMGR activity of maize seedlings appeared to be exclusively associated with the microsomal rather than the plastidic fraction of maize cells. Maize tissues with high meristematic activity such as germinating seeds, leaf bases, root tips and the site of origin of lateral roots contained high levels of microsomal HMGR activity. The activity of HMGR extracted from leaf tips of normal, dwarf and albino maize seedlings is regulated by light. Microsomal HMGR activity from leaf tips of 4-day-old maize seedlings was inhibited significantly following exposure to strong light (600 μmol m⁻² s⁻¹) for more than 10 h. By comparison, microsomal HMGR activity from leaf bases and root tips of maize was not inhibited by exposure to strong light. These results suggest that the microsomal HMGR which is highly expressed in maize may be related to sterol biosynthesis and membrane biogenesis rather than plastidic-associated isoprenoid synthesis and that light may regulate HMGR activity indirectly by increasing cell differentiation.     

Expression and low pH increase the activity of an apoplastic acid phosphatase in growing tissues from Zea mays

Auxin‐induced secretion of an acid phosphatase (EC 3.1.3.2) leads to the hypothesis that this enzyme may be involved in plant cell elongation growth (W. Pfeiffer. 1996. Physiol. Plant. 98: 773–779). Elongation growth can be characterized by the effects of pH, phosphate and citrate, and the correlation with a particular region of the root: the elongation region. Therefore, it was investigated whether these parameters may reveal further correlations between acid phosphatase and elongation growth. The following results were obtained. (1) An extracellular acid phosphatase with high substrate affinity was characterized (Michaelis‐Menten constant, 0.03 m M for 4‐methylumbelliferyl phosphate; pH optimum, 3.0). The pH dependence of the enzyme was similar to that of elongation growth of coleoptile segments after pretreatment with phosphate (U. Kutschera and P. Schopfer. 1985. Planta 163: 483–493). (2) Phosphate inhibited both the acid phosphatase and coleoptile growth. Phosphate was a competitive inhibitor of the acid phosphatase (inhibitor constant, 2.5 m M ). (3) Citrate inhibited coleoptile growth and the acid phosphatase in a similar way (inhibitor constant, 21 mM). (4) The elongation region of maize roots contained more apoplastic acid phosphatase than adjacent regions (170%). The pH dependence of the enzyme suggests that the low pH reported for the elongation region would result in an additional increase of the enzymatic activity (pH optimum at 3.0).     

Heat stress effects on sink activity of developing maize kernels grown in vitro

The objective of this study was to determine the effect of short-term (4 days) and long-term (8 days) heat stress (35°C) on sink activity of maize (Zea mays L.) kernels. Beginning at 3 days after pollination (DAP) kernels were grown in vitro at 25°C and 24 h later were transferred to 35°C for either 4 or 8 days. Each treatment had a control that was maintained continously at 25°C. Two experiments were designed to examine the uptake and distribution of ¹⁴C among hexoses, sucrose and starch in the pedicel placento-chalazal (pedicel/p-c). endosperm, and pericarp tissues of kernels exposed to heat stress for 4 or 8 days. Kernels cultured in vitro were placed in ¹⁴C-sucrose medium either during the period of heat stress (experiment 1; 5 to 13 DAP) or immediately following heat-stress treatments (experiment 2; 10 to 22 DAP). In both experiments no significant effect of heat stress was observed on the total radioactivity accumulated in the kernels until about 17 DAP, after which heat-stressed kernels accumulated less ¹⁴C than the control. During the linear fill period, the endosperm of kernels exposed to heat stress accumulated more radioactivity associated with hexoses and sucrose and less radioactivity incorporated into starch, as compared to the control. Kernels heat stressed for 4 days showed a partial recovery in starch synthesis by 21 DAP, but to levels of only 65% of that of the control. Kernels heat stressed for 8 days did not recover. When ¹⁴C-sucrose was supplied during the heat stress period (5–13 DAP). kernels from all treatments accumulated more hexoses that sucrose in the pedicel/p-c. However, during the period following heat stress (10–22 DAP), pedicel/p-c accumulated sucrose, but only in kernels exposed to long-term heat stress. Soluble invertase activity was inhibited by both short-term and long-term heat stress, whereas the activity of insoluble invertase was affected only by long-term heat stress. These results support the hypothesis that the disruption of kernel growth and more particularly endosperm starch biosynthesis, in response to heat stress, is mainly associated with changes in carbon utilization and partitioning between the different nonstructural carbohydrates within the endosperm rather than with a limitation in carbon supply to the kernel. Therefore, the effect on sink activity does not seem to be attributable to a thermal disruption of kernel uptake of sugars, but rather it is a consequence of heat perturbation of other physiological processes such as endosperm sugar metabolism and starch biosynthesis.     

Glomus intraradices causes differential changes in amino acid and starch concentrations of in vitro strawberry subjected to water stress

The effect of colonization of tissue-cultured strawberry ( Fragaria × ananassa Duch. cv. Kent) plantlets in vitro by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices on plantlet response to poly(ethylene glycol) (PEG)-8000-induced water stress was investigated. The plantlets were inoculated axenically and co-cultured with the AMF for 4 wk, then transferred to 15% PEG-8000 solutions for 4, 8 and 12 h. Relative water content, water potential, osmotic potential, leaf conductance for water vapour diffusion and photosynthetic efficiency as estimated by chlorophyll a fluorescence were all affected by the PEG treatment and its duration but not by the presence of the intraradical phase of the AMF. However, distinct differences in PEG-induced changes in amino acid content were observed between nonmycorrhizal and mycorrhizal plantlets. In the latter, the treatment with PEG caused a substantial decrease in asparagine levels in leaves that was accompanied by a marked increase in asparagine concentration in roots. The opposite was observed in nonmycorrhizal plantlets. Furthermore, concentrations of aspartic acid, serine, threonine, amino- N -butyric acid, alanine and starch increased in roots of mycorrhizal and decreased in nonmycorrhizal plantlets. Our results suggest the presence of a mobile pool of asparagine that can be translocated from leaves to roots or vice versa in response to PEG-induced water stress, depending on the mycorrhizal status of the plantlets. These opposite patterns suggest different strategies of mycorrhizal and nonmycorrhizal plantlets to water stress, which seem to involve different adjustments in nitrogen and carbon metabolism.     

Effect of arbuscular mycorrhizal fungal inoculation on heavy metal accumulation of maize grown in a naturally contaminated soil

A pot culture experiment was carried out to study heavy metal (HM) phytoaccumulation from soil contaminated with Cu, Zn, Pb, and Cd by maize (Zea mays L.) inoculated with arbuscular mycorrhizal (AM) fungi (AMF). Two AM fungal inocula--MI containing only one AM fungal strain (Glomus caledonium 90036) and MII consisting of Gigaspora margarita ZJ37, Gigaspora decipens ZJ38, Scutellospora gilmori ZJ39, Acaulospora spp., and Glomus spp.--were applied to the soil under unsterilized conditions. The control received no mycorrhizal inoculation. The maize plants were harvested after 10 wk of growth. MI-treated plants had higher mycorrhizal colonization than MII-treated plants. Both MI and MII increased P concentrations in roots, but not in shoots. Neither MI nor MII had significant effects on shoot or root dry weight (DW). Compared with the control, shoot Cu, Zn, Pb, and Cd concentrations were decreased by MI but increased by MII. Cu, Zn, Pb, and Cd uptake into shoots and roots all increased in MII-treated plants, while in MI-treated plants Cu, Zn, and Pb uptake into shoots and Cd uptake into roots decreased but Cu, Zn, and Pb uptake into roots and Cd into shoots increased. MII was more effective than MI in promoting HM extraction efficiencies. The results indicate that MII can benefit HMphytoextraction and, therefore, show potential in the phytoremediation of HM-contaminated soils.     

Inhibition of the activity of aminoacyl-tRNA synthetases in pollen grains of maize

Extracts from pollen grains of maize ( Zea mays L.) show a low activity of aminoacyltRNA synthetases (EC 6. 1. 1). They also contain a specific factor inhibiting the activity of these enzymes. The molecular mass of this factor, which may be a short peptide, is about 3000 Da as determined by column chromatography on Sephadex G-25 Fine. The Michaelis constant (K_m), determined for the amino acid in the presence of this factor, suggests its allosteric influence on the affinity of the enzyme. Short-term incubation of the factor with pronase R resulted in conversion of the inhibiting action into a stimulating one. Kinetics of aminoacylation reactions confirm inhibitory and stimulative influences of the effector on the enzyme activity. High performance liquid chromatography shows that inhibition of the activity of aminoacyl-tRNA synthetases is affected by a group of compounds of similar molecular masses.