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Differential antioxidant responses to norflurazon-induced oxidative stress in maize.

This study examined the contribution of catalase (CAT) and superoxide dismutase (SOD) in the overall antioxidant response to norflurazon (NF)-induced oxidative stress in leaves, mesocotyls and scutella of maize (Zea mays). Maize catalase null mutants were used to provide insights into the role(s) of these isozymes. A substantial increase in Cat1 and Cat2 transcript levels occurred in NF-treated leaves in all maize lines examined. However, these two transcripts did not show a particular pattern of change in NF-treated scutella from 5-day postimbibition (dpi) and 18-day postpollination (dpp) maize. The NF-induced increase in Cat1 appeared to be dependent on excessive light energy caused by a lack of photoprotectant carotenoids. especially in leaves. In NF-treated leaves, the chloroplastic Cu/Zn-SOD-1 isozyme responded strongly compared to the cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD-3 isozymes, suggesting the critical role of SOD-1 as a major component in chloroplastic antioxidant defenses. All SOD isozymes in the NF-treated scutella of various maize lines were consistent in their response to NF. The most significant increase was observed with Sod1 in NF-treated leaves; however, no significant Sod1 changes were observed in similarly treated scutella at 5 dpi and 18 dpp. These results suggest that the response of the Cat and Sod genes to NF is likely developmental and tissue-specific.

Antioxidants↗

Duplicate FLORICAULA/LEAFY homologs zfl1 and zfl2 control inflorescence architecture and flower patterning in maize.

The homologous transcription factors FLORICAULA of Antirrhinum and LEAFY of Arabidopsis share conserved roles in flower meristem identity and floral patterning. While roles for FLORICAULA/LEAFY homologs in flower development have been demonstrated in numerous dicots, little is known about the function of these meristem identity genes in the more distantly related flowering plants, the monocots. We used reverse genetics to investigate the role of two duplicate FLORICAULA/LEAFY homologs in maize (Zea mays L. ssp. mays) - a monocot species with dramatically different flower and inflorescence morphology from that of dicot species. Transposon insertions into the maize genes, zfl1 and zfl2, led to a disruption of floral organ identity and patterning, as well as to defects in inflorescence architecture and in the vegetative to reproductive phase transition. Our results demonstrate that these genes share conserved roles with their dicot counterparts in flower and inflorescence patterning. The phenotype of zfl1; zfl2 double mutants suggests that these maize FLORICAULA/LEAFY homologs act as upstream regulators of the ABC floral organ identity genes, and this along with previously published work, indicates that the transcriptional network regulating flower development is at least partially conserved between monocots and dicots. Our data also suggest that the zfl genes may play a novel role in controlling quantitative aspects of inflorescence phyllotaxy in maize, consistent with their candidacy for quantitative trait loci that control differences in inflorescence structure between maize and its progenitor, teosinte.

Amino Acid Sequence↗

A biotechnological process involving filamentous fungi to produce natural crystalline vanillin from maize bran.

A new process involving the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus has been designed for the release of ferulic acid by enzymic degradation of a cheap and natural agricultural byproduct (autoclaved maize bran) and its biotransformation into vanillic acid and/or vanillin with a limited number of steps. On the one hand, the potentialities of A. niger I-1472 to produce high levels of polysaccharide-degrading enzymes including feruloyl esterases and to transform ferulic acid into vanillic acid were successfully combined for the release of free ferulic acid from autoclaved maize bran. Then vanillic acid was recovered and efficiently transformed into vanillin by P. cinnabarinus MUCL39533, since 767 mg/L of biotechnologic vanillin could be produced in the presence of cellobiose and XAD-2 resin. On the other hand, 3-d-old high-density cultures of P. cinnabarinus MUCL39533 could be fed with the autoclaved fraction of maize bran as a ferulic acid source and A. niger I-1472 culture filtrate as an extracellular enzyme source. Under these conditions, P. cinnabarinus MUCL39533 was shown to directly biotransform free ferulic acid released from the autoclaved maize bran by A. niger I-1472 enzymes into 584 mg/L of vanillin. These processes, involving physical enzymic, and fungal treatments, permitted us to produce crystallin vanillin from autoclaved maize bran without any purification step.

Aspergillus niger↗

Clonal mosaic analysis of EMPTY PERICARP2 reveals nonredundant functions of the duplicated HEAT SHOCK FACTOR BINDING PROTEINs during maize shoot development.

The paralogous maize proteins EMPTY PERICARP2 (EMP2) and HEAT SHOCK FACTOR BINDING PROTEIN2 (HSBP2) each contain a single recognizable motif: the coiled-coil domain. EMP2 and HSBP2 accumulate differentially during maize development and heat stress. Previous analyses revealed that EMP2 is required for regulation of heat shock protein (hsp) gene expression and also for embryo morphogenesis. Developmentally abnormal emp2 mutant embryos are aborted during early embryogenesis. To analyze EMP2 function during postembryonic stages, plants mosaic for sectors of emp2 mutant tissue were constructed. Clonal sectors of emp2 mutant tissue revealed multiple defects during maize vegetative shoot development, but these sector phenotypes are not correlated with aberrant hsp gene regulation. Furthermore, equivalent phenotypes are observed in emp2 sectored plants grown under heat stress and nonstress conditions. Thus, the function of EMP2 during regulation of the heat stress response can be separated from its role in plant development. The discovery of emp2 mutant phenotypes in postembryonic shoots reveals that the duplicate genes emp2 and hsbp2 encode nonredundant functions throughout maize development. Distinct developmental phenotypes correlated with the developmental timing, position, and tissue layer of emp2 mutant sectors, suggesting that EMP2 has evolved diverse developmental functions in the maize shoot.

Base Sequence↗

Effect of teosinte cytoplasmic genomes on maize phenotype.

Determining the contribution of organelle genes to plant phenotype is hampered by several factors, including the paucity of variation in the plastid and mitochondrial genomes. To circumvent this problem, evolutionary divergence between maize (Zea mays ssp. mays) and the teosintes, its closest relatives, was utilized as a source of cytoplasmic genetic variation. Maize lines in which the maize organelle genomes were replaced through serial backcrossing by those representing the entire genus, yielding alloplasmic sublines, or cytolines were created. To avoid the confounding effects of segregating nuclear alleles, an inbred maize line was utilized. Cytolines with Z. mays teosinte cytoplasms were generally indistinguishable from maize. However, cytolines with cytoplasm from the more distantly related Z. luxurians, Z. diploperennis, or Z. perennis exhibited a plethora of differences in growth, development, morphology, and function. Significant differences were observed for 56 of the 58 characters studied. Each cytoline was significantly different from the inbred line for most characters. For a given character, variation was often greater among cytolines having cytoplasms from the same species than among those from different species. The characters differed largely independently of each other. These results suggest that the cytoplasm contributes significantly to a large proportion of plant traits and that many of the organelle genes are phenotypically important.

Crosses, Genetic↗

Phylogenetic analysis of 5'-noncoding regions from the ABA-responsive rab16/17 gene family of sorghum, maize and rice provides insight into the composition, organization and function of cis-regulatory modules.

Phylogenetic analysis of sequences from gene families and homologous genes from species of varying divergence can be used to identify conserved noncoding regulatory elements. In this study, phylogenetic analysis of 5'-noncoding sequences was optimized using rab17, a well-characterized ABA-responsive gene from maize, and five additional rab16/17 homologs from sorghum and rice. Conserved 5'-noncoding sequences among the maize, sorghum, and rice rab16/17 homologs were identified with the aid of the software program FootPrinter and by screening for known transcription-factor-binding sites. Searches for 7 of 8 (7/8)bp sequence matches within aligned 5'-noncoding segments of the rab genes identified many of the cis-elements previously characterized by biochemical analysis in maize rab17 plus several additional putative regulatory elements. Differences in the composition of conserved noncoding sequences among rab16/17 genes were related to variation in rab gene mRNA levels in different tissues and to response to ABA treatment using qRT-PCR. Absence of a GRA-like element in the promoter of sorghum dhn2 relative to maize rab17 was correlated with an approximately 85-fold reduction of dhn2 RNA in sorghum shoots. Overall, we conclude that phylogenetic analysis of gene families among rice, sorghum, and maize will help identify regulatory sequences in the noncoding regions of genes and contribute to our understanding of grass gene regulatory networks.

5' Untranslated Regions↗

An analysis of genetic diversity across the maize genome using microsatellites.

How domestication bottlenecks and artificial selection shaped the amount and distribution of genetic variation in the genomes of modern crops is poorly understood. We analyzed diversity at 462 simple sequence repeats (SSRs) or microsatellites spread throughout the maize genome and compared the diversity observed at these SSRs in maize to that observed in its wild progenitor, teosinte. The results reveal a modest genome-wide deficit of diversity in maize relative to teosinte. The relative deficit of diversity is less for SSRs with dinucleotide repeat motifs than for SSRs with repeat motifs of more than two nucleotides, suggesting that the former with their higher mutation rate have partially recovered from the domestication bottleneck. We analyzed the relationship between SSR diversity and proximity to QTL for domestication traits and observed no relationship between these factors. However, we did observe a weak, although significant, spatial correlation for diversity statistics among SSRs within 2 cM of one another, suggesting that SSR diversity is weakly patterned across the genome. Twenty-four of 462 SSRs (5%) show some evidence of positive selection in maize under multiple tests. Overall, the pattern of genetic diversity at maize SSRs can be explained largely by a bottleneck effect with a smaller effect from selection.

DNA, Plant↗

Genetic architecture of flowering time in maize as inferred from quantitative trait loci meta-analysis and synteny conservation with the rice genome.

Genetic architecture of flowering time in maize was addressed by synthesizing a total of 313 quantitative trait loci (QTL) available for this trait. These were analyzed first with an overview statistic that highlighted regions of key importance and then with a meta-analysis method that yielded a synthetic genetic model with 62 consensus QTL. Six of these displayed a major effect. Meta-analysis led in this case to a twofold increase in the precision in QTL position estimation, when compared to the most precise initial QTL position within the corresponding region. The 62 consensus QTL were compared first to the positions of the few flowering-time candidate genes that have been mapped in maize. We then projected rice candidate genes onto the maize genome using a synteny conservation approach based on comparative mapping between the maize genetic map and japonica rice physical map. This yielded 19 associations between maize QTL and genes involved in flowering time in rice and in Arabidopsis. Results suggest that the combination of meta-analysis within a species of interest and synteny-based projections from a related model plant can be an efficient strategy for identifying new candidate genes for trait variation.

Chromosome Mapping↗

Structure and evolution of the r/b chromosomal regions in rice, maize and sorghum.

The r1 and b1 genes of maize, each derived from the chromosomes of two progenitors that hybridized >4.8 million years ago (MYA), have been a rich source for studying transposition, recombination, genomic imprinting, and paramutation. To provide a phylogenetic context to the genetic studies, we sequenced orthologous regions from maize and sorghum (>600 kb) surrounding these genes and compared them with the rice genome. This comparison showed that the homologous regions underwent complete or partial gene deletions, selective retention of orthologous genes, and insertion of nonorthologous genes. Phylogenetic analyses of the r/b genes revealed that the ancestral gene was amplified independently in different grass lineages, that rice experienced an intragenomic gene movement and parallel duplication, that the maize r1 and b1 genes are descendants of two divergent progenitors, and that the two paralogous r genes of sorghum are almost as old as the sorghum lineage. Such sequence mobility also extends to linked genes. The cisZOG genes are characterized by gene amplification in an ancestral grass, parallel duplications and deletions in different grass lineages, and movement to a nonorthologous position in maize. In addition to gene mobility, both maize and rice regions experienced recent transposition (<3 MYA).

Chromosome Mapping↗

Monitoring and modeling of field infestation and damage by the maize ear borer Mussidia nigrivenella (Lepidoptera: Pyralidae) in Benin, West Africa.

In many countries in West Africa, the pyralid ear borer Mussidia nigrivenella Ragonot occasionally causes severe damage to pre- and postharvest maize. Between 1992 and 1995, the distribution of and damage caused by M. nigrivenella were studied in Benin using survey data and an on-station field experiment. The borer was distributed across the whole country, and at maturity an average 25% of the ears sampled in maize fields were infested. Damage levels varied with agro-ecological zones and were highest in the Guinea Savannas. However, borer-related yield losses were comparatively low. Three applications of cypermethrin over the growing season did not provide sufficient control in the on-station field experiment. A model was developed to estimate maize losses caused by M. nigrivenella, using the percentage of infested ears, which explained 93% of the variance. Extrapolation of field data indicated a 25% yield loss once a 100% infestation of maize ears was reached. For surveys in maize fields the model is a valid tool for a rapid assessment of crop losses caused by M. nigrivenella.

Animals↗

A component of maize pollen that stimulates larval mosquitoes (Diptera: Culicidae) to feed and increases toxicity of microbial larvicides.

To explain how larval Anopheles arabiensis Patton feed effectively in the turbid water in which they frequently develop, we determined whether an extractable component of maize, Zea mays L., pollen enhances feeding by these mosquitoes. Maturing maize produces a copious amount of wind-borne pollen that is nutritious enough and produced over a sufficient period to support the development of at least one generation of anopheline mosquitoes. Larval An. arabiensis readily ingest the contents of maize pollen or the intact pollen grains themselves. An aqueous extract of maize pollen markedly accelerates the rate at which larval An. arabiensis ingest inert particles and strongly enhances the effectiveness of Bti against larval An. arabiensis. We conclude that the ability of larval anopheline mosquitoes to feed on maize pollen in turbid water is enhanced by the release from these pollen grains of a water-soluble phagostimulatory component (or components), which may be used to increase ingestion of microbial entomotoxins.

Animals↗

Claviceps cyperi, a new cause of severe ergotism in dairy cattle consuming maize silage and teff hay contaminated with ergotised Cyperus esculentus (nut sedge) on the Highveld of South Africa.

During December/January 1996/97 typical summer syndrome (hyperthermia and a 30% drop in milk yield) occurred in succession in two Holstein dairy herds (n=240 and n=150 milking cows, respectively) on the South African Highveld. These farms are situated in the midst of the prime maize and dairy farming areas of South Africa where this condition had never been diagnosed before. The individual components of the concentrate on both farms were negative for ergot alkaloids. Endophytic fungi and/or ergot infestation of teff and other grasses fed to the cows were then suspected of being involved, but neither endophytes nor ergot alkaloids could be implicated from these sources. By measuring the serum prolactin levels of groups of sheep (n=5) fed the first farm's total mixed ration (TMR) or its three individual fibre components for a period of 11 days, the source of the ergot alkaloids was identified. A statistically significant decrease in the level of this hormone occurred only in the group on maize silage (which constituted 28% on dry matter base of the TMR). The involvement of the maize silage was further chemically confirmed by the high levels of total ergot alkaloids, predominantly ergocryptine, found by LC-MS in the silage as well as in the TMR (115-975 ppb and 65-300 ppb, respectively). The ergot alkaloid content (mainly ergocryptine) of the maize silage on the second affected farm was 875 ppb. Withdrawal of contaminated silage resulted in gradual recovery of stock on both farms. Nut sedge (Cyperus esculentus and Cyperus rotundus of the family Cyperaceae) has a world-wide distribution and is a common weed in annual crops, and can be parasitized by Claviceps cyperi. Careful examination of the maize silage from both farms revealed that it was heavily contaminated with nut sedge and that it contained minute sclerotia, identified as those of Claviceps cyperi, originating from the latter. Nut sedge was abundant on both farms and it is believed that late seasonal rain had resulted in mature, heavily ergotised nut sedge being cut with the silage. Claviceps cyperi sclerotia, collected on the affected fields in the following autumn contained 3600-4000 ppm ergocryptine. That the dominant alkaloid produced by this particular fungus was indeed ergocryptine, was confirmed by negative ion chemical ionization MS/MS. In one further outbreak in another Holstein herd, teff hay contaminated with ergotised nut sedge and containing 1200 ppb alkaloids, was incriminated as the cause of the condition. This is the first report of bovine ergotism not associated with the Poaceae infected with Claviceps purpureum or endophytes but with the family Cyperaceae and this particular fungal phytopathogen.

Animal Feed↗

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

Agrobacterium tumefaciens↗

Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures.

In maize leaves growth at low temperatures causes decreases in maximum catalytic activities of photosynthetic enzymes and reduced amounts of proteins, rather than effects on regulation or co-ordination of the photosynthetic processes. To test the hypothesis that differential localization of antioxidants between the different types of photosynthetic cell in maize leaves is a major determinant of the extreme sensitivity of maize leaves to chilling damage, oxidative damage to proteins, induced by incubation of maize leaves with paraquat, has been measured and compared with the effects incurred by growth at low temperatures. While the increase in protein carbonyl groups caused by paraquat treatment was much greater than that caused by low temperature growth conditions, most carbonyl groups were detected on bundle sheath proteins in both stress conditions. With one or two exceptions proteins located in the mesophyll tissues were free of protein carbonyl groups in both situations. Paraquat treatment caused a complete loss of the psaA gene products, modified the photosystem II reaction centre polypeptide, D1, and increased the number of peptides arising from breakdown of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco). In contrast, growth at 15 degrees C increased the abundance (but not number) of Rubisco breakdown products and decreased that of the psaB gene product while the psaA gene product and PEP carboxylase were largely unaffected. Since bundle sheath proteins are more susceptible to oxidative damage than those located in the mesophyll cells, strategies for achieving a more balanced system of antioxidant defence may be effective in improving chilling tolerance in maize.

Adaptation, Physiological↗

Expression of maize eukaryotic initiation factor (eIF) iso4E is regulated at the translational level.

Mechanisms for regulation of gene expression at the translational level have been reported at specific developmental stages in eukaryotes. Control of eukaryotic initiation factor (eIF) 4E availability by insulin/growth factors constitutes a main point of translational regulation. The aim of the present research was to understand the regulatory mechanism(s) behind the differential expression of two main 4E factors present in maize embryonic axes during germination. De novo synthesis of eIFiso4E initiates earlier and is faster than that of eIF4E in maize axes. Insulin addition to maize axes stimulated de novo synthesis of the eIFiso4E protein, but not that of eIF4E. Specific recruitment of the eIFiso4E transcript into polysomes was observed in these axes after insulin stimulation. Inhibitors of the insulin signal-transduction pathway, wortmannin and rapamycin, reversed the insulin effect. In vitro translation of maize poly(A)(+) RNAs by S6 ribosomal protein (rp)-phosphorylated ribosomes demonstrated a strong increase in eIFiso4E synthesis, as compared with its translation by S6 rp-non-phosphorylated ribosomes. Other mRNAs from the poly(A)(+) RNA set, including the eIF4E mRNA, did not show differential translation with regard to the S6-phosphorylated status of the ribosomes. The overall results indicate that eIFiso4E, but not eIF4E, cell content is regulated by de novo synthesis in maize axes during germination, most probably by specific mRNA recruitment into polysomes via a signal-transduction pathway involving S6 rp phosphorylation.

Eukaryotic Initiation Factor-4E↗

Natural enemies of lepidopterous borers on maize and elephant grass in the forest zone of Cameroon.

The importance, geographical and temporal distributions of parasitoids of lepidopterous borers on maize and elephant grass, Pennisetum purpureum, were assessed during surveys in farmers' fields in six villages and two on-station trials in the forest zone of Cameroon between 1995 and 1996. The borer species encountered were Busseola fusca (Fuller), Sesamia calamistis Hampson, Eldana saccharina Walker on both host plants, and Mussidia nigrivenella Ragonot on maize only. Busseola fusca was the predominant host accounting for 44-57% and 96% on maize and elephant grass, respectively, followed by E. saccharina on maize with 27-39%. Fifteen hymenopterous, two dipterous and one fungal species were found on these stem and cob-borers. Among those were six pupal, six larval, four egg, one larval-pupal parasitoid and four hyperparasitoids. The scelionid parasitoids Telenomus busseolae Gahan and T. isis Polaszek were found on B. fusca eggs in all locations. During the first season, mean egg parasitism was low and ranged between 3.1% and 27% versus 54-87% during the second season. Species belonging to the Tetrastichus atriclavus Waterston complex were recovered from all four borer species. The majority and most common larval and pupal parasitoid species belonged to the ingress-and-sting guild. Larval and pupal parasitism were very erratic and on more than 50% of the sampling occasions no parasitoids were recovered. Parasitoid diversity was higher on elephant grass than maize.

Animals↗

Molecular and physiological approaches to maize improvement for drought tolerance.

Average maize yields have increased steadily over the years in the USA and yet the variations in harvestable yield have also markedly increased. Much of the increase in yield variability can be attributed to (1) varying environmental stress conditions; (2) improved nitrogen inputs and better weed control; and (3) continuing sensitivity of different maize lines to the variation in input supply, especially rainfall. Drought stress alone can account for a significant percentage of average yield losses. Yet despite variable environments, new commercially available maize hybrids continue to be produced each year with ever-increasing harvestable yield. Since many factors contribute to high plant performance under water deficits, efforts are being made to elucidate the nature of water-stress tolerance in an attempt to improve maize hybrids further. Such factors include better partitioning of biomass to the developing ear resulting in faster spikelet growth and improved reproductive success. An emphasis on faster spikelet growth rate may result in a reduction in the number of spikelets formed on the ear that facilitates overall seed set by reducing water and carbon constraints per spikelet. To understand the molecular mechanisms for drought tolerance in improved maize lines better, a variety of genomic tools are being used. Newer molecular markers and comprehensive gene expression profiling methods provide opportunities to direct the continued breeding of genotypes that provide stable grain yield under widely varied environmental conditions.

Acclimatization↗

Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules.

Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops.

DNA, Plant↗