Cooperative action of SLR1 and SLR2 is required for lateral root-specific cell elongation in maize.

Lateral roots play an important role in water and nutrient uptake largely by increasing the root surface area. In an effort to characterize lateral root development in maize (Zea mays), we have isolated from Mutator (Mu) transposon stocks and characterized two nonallelic monogenic recessive mutants: slr1 and slr2 (short lateral roots1 and 2), which display short lateral roots as a result of impaired root cell elongation. The defects in both mutants act specifically during early postembryonic root development, affecting only the lateral roots emerging from the embryonic primary and seminal roots but not from the postembryonic nodal roots. These mutations have no major influence on the aboveground performance of the affected plants. The double mutant slr1; slr2 displays a strikingly different phenotype than the single mutants. The defect in slr1; slr2 does not only influence lateral root specific cell elongation, but also leads to disarranged cellular patterns in the primary and seminal roots. However, the phase-specific nature of the single mutants is retained in the double mutant, indicating that the two loci cooperate in the wild type to maintain the lateral root specificity during a short time of early root development.

Structure of genes encoding chromosomal HMG1 proteins from maize.

The high mobility group (HMG) proteins of the HMG1 family are architectural proteins in chromatin that are considered to facilitate the formation of complex nucleoprotein structures in various biological processes such as transcription and recombination. Plants express a variety of these non-sequence-specific DNA-bending proteins. The sequences encoding the maize HMGa and HMGc1 proteins were isolated from a genomic DNA library. Determination of the nucleotide sequences of these genes revealed that the coding region of both genes has a similar genomic structure, comprising seven exons and six introns. The positioning of the introns is conserved between the two genes, whereas the number of introns and their positions are entirely different in the related animal genes. In the 5' flanking region of the hmgc1 gene, a copia-like retrotransposon was identified. In addition to the genes encoding HMGa and HMGc1, several genomic fragments (retropseudo gene, fragments of the genes) were isolated and characterised.

Plant chromosomal HMGI/Y proteins and histone H1 exhibit a protein domain of common origin.

The chromosomal high-mobility-group (HMG) proteins of the HMGI/Y family interact with A/T-rich stretches in duplex DNA, and are considered assistant factors in transcriptional regulation. A cDNA encoding an HMGI/Y protein of 190 amino acid residues was isolated from maize and characterized. Like other plant HMGI/Y proteins, the maize HMGI/Y protein contains four copies of the AT-hook DNA-binding motif and an amino-terminal 'histone H1-like region' with a similarity to the globular domain of H1. The maize hmgi/y gene that was isolated from a genomic DNA library contains a single intron that is localized in the region of sequence similarity to histone H1. Interestingly, the genes encoding plant H1 contain an intron at exactly the same relative position, indicating an evolutionary relationship of the plant genes encoding HMGI/Y and H1 proteins.

Root-specific expression of a Zea mays gene encoding a novel glycine-rich protein, zmGRP3.

The isolation and characterization of a cDNA clone from Zea mays coding for a novel glycine-rich protein (GRP) is described. The corresponding 1.4 kb mRNA accumulates exclusively in roots (primary, lateral seminal and crown roots) of young maize seedlings, following developmentally specific patterns. In agreement with previously described GRPs from other plant species the derived protein sequence exhibits a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Genomic Southern analysis indicates that the zmGRP3 gene is present in the maize genome as one or two copies or at a low copy number.

Early post-embryonic root formation is specifically affected in the maize mutant lrt1.

The isolation and detailed characterisation of the maize mutant lrt1 , which is completely deficient in the initiation of lateral roots at the primary and seminal lateral roots and of the crown roots at the coleoptilar node is described. The monogenic and recessive mutant was isolated from a segregating EMS mutagenised population, maps to the short arm of chromosome 2, and acts independently of the nodal root deficient rtcs locus. Histological analysis revealed that the mutation acts at a very early stage of root initiation, as indicated by the absence of primordia formation in the affected roots. At later stages of plant development lateral and crown root initiations recover leading to fertile plants. If grown in the dark, the mutant does not form an elongated mesocotyl, although the photomorphogenic response appears to be normal in the mutant. Furthermore, the wild-type cannot be rescued from mutants by the application of auxin to germinating kernels. The gene impaired in lrt1 seems to be of great importance for the general mechanism of early post-embryonic root initiation, both from root and nodal tissues, since lateral and crown root initiation are both affected to the same extent and in the same transient time pattern.

Genomic structure of the maize TATA-box binding protein 1 (TBP-1): conserved exon/intron structure in eukaryotic TBP genes.

The gene system of the TATA-box binding protein (TBP) is well suited for the study of the evolutionary conservation of essential components of eukaryotic transcription initiation. In this context we have isolated and sequenced the maize TBP gene for a comparison with TBP genes from other organisms. In particular, a molecular phylogenetic analysis of the exon/intron structure of these genes including the archaeal TBP homolog (Thermococcus celer) was performed, revealing that the intron insertion probably occurred after the early appearance of the characteristic tandem repeat within the highly conserved C-terminal domain of all known TBPs, but before separation of the eukaryotic progenitor into the different kingdoms.

Characterization of a maize ribosomal P2 protein cDNA and phylogenetic analysis of the P1/P2 family of ribosomal proteins.

The nucleotide sequence of a full-length ribosomal P2 protein cDNA from maize was determined and used for a sequence comparison with the P2 and P1 proteins from other organisms. The integration of these data into a phylogenetic tree shows that the P proteins separated into the subspecies P1 and P2 before the eukaryotic kingdoms including plants developed from their ancestor.

Coordinated Transcriptional Regulation of Storage Product Genes in the Maize Endosperm.

We have demonstrated that expression of genes involved in starch and storage protein synthesis of the maize (Zea mays L.) endosperm are coordinated. Genetic lesions altering synthetic events in one biosynthetic pathway affect expression of genes in both pathways. Initial studies focused on shrunken2 (sh2) and brittle2 (bt2) mutants because these genes encode subunits of the same enzyme, ADP-glucose pyrophosphorylase. Analysis of various sh2- and bt2- mutant alleles showed that the most severe mutations also conditioned the largest increase in transcripts. The analysis was extended by monitoring the transcripts of the genes, shrunken1 (sh1, structural gene for Suc synthase), sh2, bt2, waxy1 (wx1, structural gene for starch synthase), and those of the large and small zeins in isogenic maize lines at 14, 22, and 30 d postpollination. Endosperms were wild type for all of these genes or contained sh1-, sh2-, bt1-, bt2-, opaque2 (o2-), or amylose-extender1 (ae1-) dull1 (du1-) wx1- mutations. Transcripts increased continually throughout kernel development in the mutants relative to the standard W64A used. Variation in the amount of Suc entering the developing seed also altered transcript amounts. The results indicate that starch and protein biosynthetic genes act in a concerted manner, and both are sensitive to mutationally induced differences.

The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility.

The abundant maize high-mobility group protein HMGa belongs to the chromosomal, non-histone proteins and consists of a basic region containing the HMG-box DNA-binding domain and a highly acidic carboxy-terminal tail. The full-length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA-bending in a ligase mediated circularization assay with short DNA fragments. It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA-binding. They bind furthermore preferentially to A/T-rich linear DNA or bent DNA structures such as four-way junctions and DNA minicircles. The DNA-binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites.

Stability of the maize chromosomal high-mobility-group proteins, HMGa and HMGb, in vivo.

Chromosomal non-histone high-mobility-group (HMG) proteins represent essential components of eukaryotic chromatin and have also been isolated from a variety of plants. In maize, studies on structure and function of the two larger of the four major HMG proteins have recently been performed and are now extended by analysis of their in vivo stability using pulse-chase experiments in a cell suspension culture. The half-life of the analyzed HMGa and HMGb proteins was found to be 65 h or more than 78 h, respectively.

Comparative analysis of chromosomal HMG proteins from monocotyledons and dicotyledons.

Chromosomal high-mobility-group (HMG) proteins occur ubiquitously in eukaryotes and their common structural and biochemical features indicate a critical role. In this context, we compared structural and functional aspects of HMG proteins from the monocotyledonous plant maize and the dicotyledonous plant Vicia faba. Besides biochemical similarities and immunological differences found between these proteins, the isolation and characterization of a cDNA encoding the V. faba homologue of the maize HMGa protein revealed great similarities between these two proteins, including the HMG-box DNA-binding motif and an acidic domain. Therefore, like the maize HMGa protein, the V. faba HMG protein belongs to the vertebrate HMG1 family, which consists of HMG proteins and transcription factors of various eukaryotes.

Phylogenetic relationships of HMG box DNA-binding domains.

HMG boxes were initially identified as DNA-binding domains of the human RNA polymerase I (pol I) transcription factor hUBF and the animal high-mobility-group (HMG) protein family HMG1. Since then, numerous sequences of HMG-box-containing HMG proteins and other DNA-binding proteins from several species have become available. By sequence comparisons of a selected range of HMG boxes from these proteins and the construction of phylogenetic trees we show that the HMG box is highly conserved between DNA-binding proteins of organisms from all three eukaryotic kingdoms and that HMG boxes are linked by distinct evolutionary relationships. In addition, most HMG boxes display comparable hydropathy profiles and amino acid arrangements, which could serve as nuclear targeting sequences.

Stimulatory effect of the maize HMGa protein on reporter gene expression in maize protoplasts.

The high mobility group (HMG) proteins represent a class of chromosomal non-histone proteins with an assumed influence on transcription. In this context, the effect of the maize HMGa protein on reporter gene expression was examined. Transient co-transformation experiments in maize protoplasts with plasmid constructs directing the synthesis of the maize HMGa protein and with a luciferase reporter plasmid demonstrated a stimulatory effect of the HMGa protein on the reporter gene expression. Additional experiments with HMGa deletion constructs indicated that the HMG-Box DNA-binding motif is important for the observed effect, while the acidic carboxy-terminal domain of the HMGa protein appears to be dispensable.

Repeat units from a maize rDNA external spacer region exhibit DNA curvature and interact with high-mobility-group proteins.

The 3-kb external spacer from a maize (Zea mays L. cv. A619) nuclear rRNA gene unit which contains nine highly homologous 200-bp repeat elements was found to include a region with DNA-curvature properties. The centre of curvature was localized within repeats 5 and 6 using a circular permutation assay. A 60-bp-long subfragment of this region was found to interact with nuclear proteins, including high-mobility-group (HMG) proteins, and with the maize HMGa protein synthesized in Escherichia coli from a recombinant plasmid. The potential influence of the binding of the HMG proteins on the conformation of this subfragment was studied with a permutation assay based on a bending vector.

Two different cDNAs encoding TFIID proteins of maize.

Two different complementary DNAs (cDNAs) encoding maize TFIID proteins were isolated from a maize leaf cDNA. Both cDNA sequences reveal two types of TFIID, each encoding an open reading frame of 200 amino acids. The two cDNAs are 76% identical at the DNA level and their putative amino acid sequences differ at only three amino acids. Like TATA box binding proteins from other organisms they show a bipartite structure containing a specific N-terminal region and a highly conserved C-terminal domain expected to be necessary and sufficient for the essential TFIID functions in transcriptional initiation.

Functional analysis of the -300 region of maize zein genes.

A 43 bp fragment containing the -300 region upstream control element common to the endosperm expressed zein genes of Zea mays L. has been analyzed by in vivo and in vitro techniques. Transient transformation studies with protoplasts from a maize endosperm culture indicate that the element positively affects CaMV 35S promoter-driven gene expression, and that this effect is both orientation- and position-dependent. Band-shift and Southwestern blotting experiments demonstrate that the element is specifically bound by different sets of DNA-binding proteins from seedling and endosperm nuclei. A 2 bp substitution within the most conserved region of the element both reduces the stimulatory effect on transcription and alters the binding of nuclear proteins from both tissues.

Isolation and characterization of maize cDNAs encoding a high mobility group protein displaying a HMG-box.

cDNAs encoding a nonhistone chromosomal high mobility group (HMG) protein corresponding to the animal HMG1 family were isolated from a maize cDNA library using an immunoscreening approach. The cDNAs revealed an open reading frame of 471 base pairs together with 413 base pairs of flanking region, in agreement with the size of mRNA detected by Northern analysis of maize endosperm RNA. Like its animal counterparts the 17146 Da maize HMG protein contains a basic aminoterminus and an acidic carboxyterminus. The HMG-box region of this plant HMG protein shows striking sequence similarity to members of the vertebrate HMG1 family. Based on Southern blot hybridization analysis of genomic DNA, the isolated cDNA appears to be derived from a single or low copy gene.

Isolation and characterization of high-mobility-group proteins from maize.

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5' flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.

Characterization of a maize endosperm culture expressing zein genes and its use in transient transformation assays.

An endosperm derived tissue culture of maize (Zea mays L.) variety A636 has been characterised for its ability to synthesize zein protein and respond to a zein gene regulatory element. Western analysis with zein specific antibodies revealed the distinct presence of zein proteins of the 15, 19 and 21 kDa classes in this tissue, in contrast to an embryo-derived Black Mexican Sweet variety tissue culture which exhibited no zein proteins. Transient transformation studies with a cauliflower mosaic virus 35S promoter and luciferase reporter gene construct demonstrate that protoplasts from this tissue culture, but not from the embryo-derived culture, respond positively to the potential enhancer which is located approximately 300 base pairs upstream of the coding region in most zein genes of maize, thus establishing the usefulness of this culture for studies of tissue specific gene regulation.