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PubMed2000

Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize.

Dof proteins are transcription factors that appear to be unique to plants. Maize Dof1 has been suggested to be a regulator for C4 photosynthetic phosphoenolpyruvate carboxylase (C4PEPC) gene expression. The present study demonstrates that Dof1 also enhances transcription from the promoters of both cytosolic orthophosphate dikinase (cyPPDK) genes and a non-photosynthetic PEPC gene, which are not present in animals. Expression of Dof1-specific antisense RNA or the DNA-binding domain of Dof1 alone reduced the activities of these promoters in maize leaf protoplasts. Electrophoretic mobility shift assays revealed several Dof1-binding sites in these promoters. The cyppdk1 promoter contained two Dof1-binding sites, one of which was linked to the binding site of a plant bZIP protein. By using deleted or mutated cyppdk1 promoters, both Dof1-binding sites were shown to be functional. Furthermore, Dof1 elevated the activities of the cyppdk and pepc promoters more strongly in greening protoplasts than in etiolated protoplasts, in accordance with the different activities of these promoters in two types of protoplasts. Another Dof protein of maize, Dof2, suppressed the activity of the C4pepc promoter but was able to activate certain other promoters. These results suggest that Dof proteins may play regulatory roles in multiple gene expressions associated with the plant-specific pathway for carbon metabolism in maize. In addition, the primary characteristic of Dof proteins, i.e. different activities in distinct types of cells and opposite actions on promoters in different contexts, suggests the potential of Dof proteins to differentially regulate diverse promoters in a variety of plant tissues. Speculation raised by these results concerning the evolution of the C4pepc gene is also discussed.

Arabidopsis Proteins↗

PubMed2002

A nucleus-encoded maize protein with sigma factor activity accumulates in mitochondria and chloroplasts.

Plants contain nuclear gene families that encode proteins related to the principal sigma factors of eubacteria. As sigma factors function in transcription, the plant proteins have been presumed or demonstrated to associate with the eubacteria-like RNA polymerase of chloroplasts. In maize, five sig cDNA sequences have been reported, and four of the products are present in plastids as predicted. However, in vitro chloroplast import assays and computer algorithms gave ambiguous results with the fifth protein, ZmSig2B. Unlike the other maize sigma factors, ZmSig2B is expressed throughout developing seedling leaves, as well as in roots and etiolated tissues. To determine the subcellular location of ZmSig2B, we have now used immunoblot assays to show that it co-purifies with both mitochondria and plastids. Its NH2-terminal 153 amino acids, translationally fused to green fluorescent protein (GFP), targeted GFP to chloroplasts and mitochondria in bombarded maize leaves. A putative role for ZmSig2B in mitochondrial transcription is supported by its presence in a maize mitochondrial transcription extract. ZmSig2B also exhibits the expected properties of a chloroplast sigma factor: recombinant ZmSig2B binds to a chloroplast promoter and initiates transcription in vitro when combined with Escherichia coli core RNA polymerase. Therefore ZmSig2B is an unusual nucleus-encoded sigma factor that appears to function in both chloroplasts and mitochondria.

Amino Acid Sequence↗

PubMed2003

Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants.

We have compared the site-by-site N-glycosylation status of human lactoferrin (Lf) produced in maize, a monocotyledon, and in tobacco, used as a model dicotyledon. Maize and tobacco plants were stably transformed and recombinant Lf was purified from both seeds and leaves. N-glycopeptides were generated by trypsin digestion of recombinant Lf and purified by reverse-phase HPLC. The N-glycosylation pattern of each site was determined by mass spectrometry. Our results indicated that the N-glycosylation patterns of recombinant Lf produced in maize and tobacco share common structural features. In particular, both N-glycosylation sites of each recombinant Lf are mainly substituted by typical plant paucimannose-type N-glycans, with beta1,2-xylose and alpha1,3-linked fucose at the proximal N-acetylglucosamine. However, tobacco Lf shows a significant amount of processed N-glycans with one or two beta1,2GlcNAc linked to the trimannose core, which are weakly expressed in maize Lf. Finally, no Lewisa epitope was observed on tobacco Lf.

Asparagine↗

PubMedDate not supplied

Unsubstituted hydrolyzed waxy maize as a potential agent for centrifugal leukacytapheresis.

In order to determine whether newer, possibly safer erythrocyte sedimenting agents could be used during centrifugal leukacytapheresis, we tested seven varieties of plant starch. Only waxy maize prepared by partial acid hydrolysis appeared suitable, and it was tested extensively on an analytical device developed especially for testing erythrocyte sedimentation under the conditions of residence time, force, and sedimenting agent concentration used in a cell separator. This species of waxy maize, however, was not quite as effective a sedimenting agent when compared to hydroxyethyl starch (HES 450/0.70) under the test conditions used. Although chromatographic analysis showed the average molecular weights of the unsubstituted waxy maize were considerably higher than those of HES 450/0.70, degradation of waxy maize to smaller molecular weight fragments occurred more rapidly. The rapid degradation of this material may offer significant advantages during centrifugal leukacytapheresis procedures, but additional studies of its storage stability are needed.

Amylases↗

PubMedDate not supplied

Selection of relevant non-target herbivores for monitoring the environmental effects of Bt maize pollen.

Genes of Bacillus thuringiensis var. kurstaki (Berliner) that encode lepidopteran-specific toxins were engineered into maize for protection against the European Corn Borer, Ostrinia nubilalis (Hbn.). Recent data suggest that Lepidoptera may be negatively affected, if maize pollen contains high amounts of Bt toxin and is diposited on host plants near maize fields. Monitoring the environmental effects of commercial Bt maize fields requires effective use of limited financial and logistical resources. The aim of this study was to develop and apply tools for selecting relevant herbivore species for the field monitoring of environmental Bt toxin effects via pollen deposition. We first present a theoretical selection tree based on "risk index of Bt pollen for herbivores" (I(Btp)). Our index consists of five classes from zero (not relevant) to four (highly relevant) derived from data on potential temporal and spatial coincidence of pollen exposure (A), feeding mode (B), susceptibility to lepidopteran-specific Bt toxins (C) and hazard to rare and/or endangered species ("Red List") (D). We then screened the Macrolepidoptera database LEPIDAT to identify relevant species in Germany. Finally, we also applied the index to species found in a local biocoenotic field study (Bonn, Western Rhineland, Germany). Approximately 7% of the German Macrolepidoptera species mainly occur in farmland areas and were selected as being potentially affected by Bt pollen exposure. Of these species, 14% (= 1% of total) were found to be potentially exposed on a regional scale. The combination of I(Btp) and database screening enables us to pre-select species for monitoring purposes.

Animals↗

PubMed2006

Differential regulation of five Pht1 phosphate transporters from maize (Zea mays L.).

Maize is one of the most important crops in the developing world, where adverse soil conditions and low fertilizer input are the two main constraints for stable food supply. Understanding the molecular and biochemical mechanisms involved in nutrient uptake is expected to support the development of future breeding strategies aimed at improving maize productivity on infertile soils. Phosphorus is the least mobile macronutrient in the soils and it is often limiting plant growth. In this work, five genes encoding Pht1 phosphate transporters which contribute to phosphate uptake and allocation in maize were identified. In phosphate-starved plants, transcripts of most of the five transporters were present in roots and leaves. Independent of the phosphate supply, expression of two genes was predominant in pollen or in roots colonized by symbiotic mycorrhizal fungi, respectively. Interestingly, high transcript levels of the mycorrhiza-inducible gene were also detectable in leaves of phosphate-starved plants. Thus, differential expression of Pht1 phosphate transporters in maize suggests involvement of the encoded proteins in diverse processes, including phosphate uptake from soil and transport at the symbiotic interface in mycorrhizas, phosphate (re)translocation in the shoot, and phosphate uptake during pollen tube growth.

Base Sequence↗

PubMed2005

microRNA172 down-regulates glossy15 to promote vegetative phase change in maize.

Shoot development in many higher plant species is characterized by phase change, where meristems and organs transition from one set of identities to another. The transition from a juvenile to adult leaf identity in maize is regulated by the APETALA2-like gene glossy15 (gl15). We demonstrate here that increasing gl15 activity in transgenic maize not only increases the number of leaves expressing juvenile traits, but also delays the onset of reproductive development, indicating that gl15 plays a primary role in the maintenance of the juvenile phase. We also show that the accumulation of a maize microRNA homologous to miR172 increases during shoot development and mediates gl15 mRNA degradation. These data indicate that vegetative phase change in maize is regulated by the opposing actions of gl15 and miR172, with gl15 maintaining the juvenile phase and miR172 promoting the transition to the adult phase by down-regulation of gl15. Our results also suggest that the balance of activities between APETALA2-like genes and miR172 may be a general mechanism for regulating vegetative phase change in higher plants.

Base Sequence↗

PubMed2006

Remarkable variation in maize genome structure inferred from haplotype diversity at the bz locus.

Maize is probably the most diverse of all crop species. Unexpectedly large differences among haplotypes were first revealed in a comparison of the bz genomic regions of two different inbred lines, McC and B73. Retrotransposon clusters, which comprise most of the repetitive DNA in maize, varied markedly in makeup, and location relative to the genes in the region and genic sequences, later shown to be carried by two helitron transposons, also differed between the inbreds. Thus, the allelic bz regions of these Corn Belt inbreds shared only a minority of the total sequence. To investigate further the variation caused by retrotransposons, helitrons, and other insertions, we have analyzed the organization of the bz genomic region in five additional cultivars selected because of their geographic and genetic diversity: the inbreds A188, CML258, and I137TN, and the land races Coroico and NalTel. This vertical comparison has revealed the existence of several new helitrons, new retrotransposons, members of every superfamily of DNA transposons, numerous miniature elements, and novel insertions flanked at either end by TA repeats, which we call TAFTs (TA-flanked transposons). The extent of variation in the region is remarkable. In pairwise comparisons of eight bz haplotypes, the percentage of shared sequences ranges from 25% to 84%. Chimeric haplotypes were identified that combine retrotransposon clusters found in different haplotypes. We propose that recombination in the common gene space greatly amplifies the variability produced by the retrotransposition explosion in the maize ancestry, creating the heterogeneity in genome organization found in modern maize.

Base Sequence↗

PubMed2002

Intraspecific violation of genetic colinearity and its implications in maize.

Although allelic sequences can vary extensively, it is generally assumed that each gene in one individual will have an allelic counterpart in another individual of the same species. We report here that this assumption does not hold true in maize. We have sequenced over 100 kb from the bz genomic region of two different maize lines and have found dramatic differences between them. First, the retrotransposon clusters, which comprise most of the repetitive DNA in maize, differ markedly in make-up and location relative to the genes in the bz region. Second, and more importantly, the genes themselves differ between the two lines, demonstrating that genetic microcolinearity can be violated within the same species. Our finding has bearing on the underlying genetic basis of hybrid vigor in maize, and possibly other organisms, and on the measurement of genetic distances.

Alleles↗

PubMed1978

Maize plastid gene expressed during photoregulated development.

RNAs larger than about 6 S prepared from etioplasts of dark-grown maize seedlings, and from plastids at later stages of light-induced development, were labeled in vitro and hybridized to separated fragments of maize chloroplast DNA digested with endonucleases. The major nonribosomal RNA present in developing plastids, but virtually undetectable in etioplasts, hybridizes to chloroplast DNA Bam fragment 8 and has been mapped on the maize plastid chromosome. Other aliquots of RNA from plastids were translated in a rabbit reticulocyte-derived system. Developing plastids, and mature chloroplasts, but not etioplasts, contain mRNA for an approximately 34,500 dalton polypeptide. The simultaneous appearance, during light-induced maize plastid development, of RNA which hybridizes to Bam 8 and is translated into a 34,500 dalton protein indicates that photoregulated expression of a single gene is being observed.

Journal Article↗

PubMed1986

Transposition of the maize controlling element "Activator" in tobacco.

Transposition of the maize autonomous controlling element Activator (Ac) and a nonautonomous derivative, Dissociation (Ds), was investigated in tobacco cells. Tobacco protoplasts were transformed with Ti-plasmid vectors that contained Ac or Ds flanked by short maize wx gene sequences. The structures of the elements and surrounding wx and T-DNA sequences were investigated in nine Ac and five Ds tobacco transformants by digestion with restriction enzymes, Southern blotting, and hybridization using specific probes. In four of the nine Ac transformed lines, Ac had excised from its original position in the T-DNA and inserted at new sites in the tobacco genome. Ds did not excise from its original T-DNA position in any of the transformants examined. Two Ac fragments and cellular flanking sequences were cloned from a line of tobacco in which Ac had transposed. Fragments, comprised of sequences flanking the newly integrated Ac elements, were used as hybridization probes to normal tobacco DNA and to the tobacco DNA from which they were isolated. The Ac copies were integrated into repetitive tobacco DNA sequences. Two tobacco fragments containing empty Wx donor sites were cloned from the DNA of the same Ac transformant and sequenced. Both sequences are among the types of excision products observed to result from Ac-catalyzed excision events in maize. Our results indicate that the maize controlling element Ac is capable of self-catalyzed transposition in tobacco.

Journal Article↗

PubMed1987

Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis.

Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA.

Base Sequence↗

PubMed1989

Deletion analysis of the maize mitochondrial superoxide dismutase transit peptide.

The maize mitochondrial superoxide dismutase (SOD; EC 1.15.1.1), a nuclear gene product, has been previously shown to be imported into maize mitochondria. The cDNA for maize mitochondrial SOD was subcloned into a vector containing the T7 promoter. Deletions were made in the transit peptide coding region of the cDNA. The undeleted and deleted proteins were synthetically produced by transcription and translation in vitro. Undeleted preSOD-3 is translocated into isolated maize mitochondria with an efficiency of approximately 30%. Mature SOD-3 subunits are recovered from the matrix but not the membranes of subfractionated mitochondria. These subunits are assembled into the tetrameric holoenzyme. The modified SOD-3 precursors are imported into mitochondria at lower efficiencies than undeleted preSOD-3. The relative import efficiency appears to be dependent upon the deletion size. To our knowledge such analysis of a plant mitochondrial precursor protein has not been reported previously.

Amino Acid Sequence↗

PubMed1990

Maize chloroplast RNA polymerase: the 180-, 120-, and 38-kilodalton polypeptides are encoded in chloroplast genes.

Prominent polypeptides with apparent molecular masses of 180, 120, 85, and 38 kDa are found in an extensively purified preparation of maize chloroplast DNA-dependent RNA polymerase that retains the capacity to initiate transcription of the cloned chloroplast gene rbcL correctly and the requirement for a supercoiled DNA template for specific and active transcription. Amino-terminal amino acid sequences of the 180-, 120-, and 38-kDa polypeptides have been determined and found to correspond precisely to the sequences deduced from the 5' ends of the maize chloroplast rpoC2, rpoB, and rpoA genes, respectively. These experiments show that these chloroplast rpo genes encode the prominent polypeptides in the highly enriched maize chloroplast RNA polymerase preparation and support the conclusion that these polypeptides are functional components of the enzyme. The rpoB, rpoC1, and rpoC2 genes have been mapped on the maize chloroplast chromosome.

Amino Acid Sequence↗

PubMed1992

Differential response of maize catalases to abscisic acid: Vp1 transcriptional activator is not required for abscisic acid-regulated Cat1 expression.

In this paper we describe the distinctive responses of the maize catalases to the plant growth regulator abscisic acid (ABA). We analyzed RNA and enzyme accumulation in excised maize embryos and found that each catalase responded differently to exogenously applied ABA. Levels of Cat1 transcript and enzyme activity rapidly increased. In contrast, levels of Cat2 transcript and protein decreased, while Cat3 transcript levels were not affected. In developing kernels of the ABA-deficient/biosynthetic viviparous mutant vp5, lower levels of Cat1 RNA correlated with lower endogenous ABA levels when compared to measured levels in comparably aged wild-type siblings from the same ear. The maize vp1 mutant line is morphologically insensitive to normal endogenous levels of ABA. Analysis of the response of Cat1 to exogenously applied ABA in mutant and wild-type vp1 sibling embryos suggests that, unlike other ABA-responsive genes analyzed to date, the Vp1 gene product is not essential for the ABA-mediated regulation of Cat1. The significance of these responses to ABA in defining the roles of the various CATs in maize is discussed.

Abscisic Acid↗

PubMed1993

Tissue-specific light-regulated expression directed by the promoter of a C4 gene, maize pyruvate,orthophosphate dikinase, in a C3 plant, rice.

Pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) activity is abundant in leaves of C4 plants, while it is difficult to detect in leaves of C3 plants. Recent studies have indicated that C3 plants have a gene encoding PPDK, with a structure similar to that of PPDK in C4 plants. However, low expression makes PPDK detection difficult in C3 plants. This finding suggests that high PPDK expression in C4 plants is due to regulatory mechanisms which are not operative in C3 plants. We have introduced a chimeric gene consisting of the gene encoding beta-glucuronidase (GUS; EC 3.2.1.31) controlled by the PPDK promoter from a C4 plant, maize, into a C3 cereal, rice. The chimeric gene was exclusively expressed in photosynthetic organs, leaf blades and sheaths, and not in roots or stems. Histochemical analysis of GUS activity demonstrated high expression of the chimeric gene in photosynthetic organs, localized in mesophyll cells, and no or very low activity in other cells. GUS expression was also regulated by light in that it was low in etiolated leaves and was enhanced by illumination. These observations indicate that the mechanisms responsible for cell-specific and light-inducible regulation of PPDK observed in C4 plants are also present in C3 plants. We directly tested whether rice has DNA-binding protein(s) which interact with a previously identified cis-acting element of the C4-type gene. Gel retardation assays indicate the presence in rice of a protein which binds this element and is similar to a maize nuclear protein which binds PPDK in maize. Taken together, these results indicate that the regulatory system which controls PPDK expression in maize is not unique to C4 plants.

DNA, Recombinant↗

PubMed1997

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Amino Acid Sequence↗

PubMed1997

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF).

Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3-1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3-2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long alpha-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 A, respectively, with corresponding bond angles of 99.5 degrees and 113 degrees, close to the optimum for a strong hydrogen bond.

Actin Depolymerizing Factors↗