Udo Wienand

The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.

The structure of the wild‐type c1 locus of Zea mays was determined by sequence analysis of one genomic and two cDNA clones. The coding region is composed of three exons (150 bp, 129 bp and one, at least 720 bp) and two small introns (88 bp and 145 bp). Transcription of the mRNAs corresponding to the two cDNA clones cLC6 (1.1 kb) and cLC28 (2.1 kb) starts from the same promoter. Both cDNAs are identical except that cLC28 extends further at its 3′ end. A putative protein, 273 amino acids in length was deduced from the sequence of both transcripts. It contains two domains, one basic and the other acidic and might function as a transcriptional activator. The basic domain of this c1‐encoded protein shows 40% sequence homology to the protein products of animal myb proto‐oncogenes.

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

SummaryThe c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.

Molecular cloning of the c locus of Zea mays: a locus regulating the anthocyanin pathway

The c locus of Zea mays, involved in the regulation of anthocyanin biosynthesis, has been cloned by transposon tagging. A clone (#18En) containing a full size En1 element was initially isolated from the En element‐induced mutable allele c‐m668655. Sequences of clone #18En flanking the En1 element were used to clone other c mutants, whose structure was predicted genetically. Clone #23En (isolated from c‐m668613) contained a full size En1 element, clone #3Ds (isolated from c‐m2) a Ds element and clone #5 (isolated from c+) had no element on the cloned fragment. From these data we conclude that the clones obtained contain at least part of the c locus. Preliminary data on transcript analysis using a 1‐kb DNA fragment from wild‐type clone #5 showed that at least three transcripts are encoded by that part of the locus, indicating that c is a complex locus.

The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated; evidence for translational control of Whp expression by the anthocyanin intensifying gene in.

Two chalcone synthase genes in maize have been cloned and molecularly characterized to be the C2 and the Whp (white pollen) locus. The two genes have highly homologous exon sequences but differ considerably in sequences 5′ upstream and 3′ downstream of the coding region, as well as in their introns. Northern and Western experiments of chalcone synthase expression in various tissues and in different genotypes indicated that C2 and Whp are differently regulated. The expression of Whp in maize aleurone is dependent on the presence of the recessive allele of the gene intensifier (in). The regulatory effect of in on Whp expression is not detectable at the transcriptional level, but seems to take place during translation.

Chalcone synthase genes in plants: A tool to study evolutionary relationships

SummaryChalcone synthase (CHS) is the key enzyme of the anthocyanin biosynthesis pathway in plants. cDNAs specific for CHS have been isolated and sequenced for the following species:Hordeum vulgare (1477 bp),Magnolia liliiflora (1359 bp),Petunia hybrida (1335 bp),Ranunculus acer (1334 bp and 1358 bp), andZea mays (1461 bp). Comparison of the coding regions of these CHS cDNA sequences including the sequences ofAntirrhinum majus (Sommer and Saedler 1986) andPetroselinum hortense (Reimold et al. 1983) reveals a similarity higher than 66% at the nucleotide and higher than 80% at the amino acid level. The CHS transcript is G/C rich in monocotyledons (65.7%–69.3%), but not in dicotyledons (45.5%–53.9%). The monocotyledonous plants show a strong codon bias preferring codons with a G or C in the third position. A phylogenetic tree was constructed on the basis of nucleotide sequence comparison; it is evident that the branching order ofRanunculus acer andPetroselinum hortense is changed as compared to the morphological order. Splitting of the CHS coding region into two parts represented by the common position of one intron seems to indicate that the first exon ofPetroselinum hortense evolved in a way different from the same exon of all other species.

The maize (Zea mays L.) roothairless3 gene encodes a putative GPI-anchored, monocot-specific, COBRA-like protein that significantly affects grain yield

Summary The rth3 (roothairless 3) mutant is specifically affected in root hair elongation. We report here the cloning of the rth3 gene via a PCR-based strategy (amplification of insertion mutagenized sites) and demonstrate that it encodes a COBRA-like protein that displays all the structural features of a glycosylphosphatidylinositol anchor. Genes of the COBRA family are involved in various types of cell expansion and cell wall biosynthesis. The rth3 gene belongs to a monocot-specific clade of the COBRA gene family comprising two maize and two rice genes. While the rice (Oryza sativa) gene OsBC1L1 appears to be orthologous to rth3 based on sequence similarity (86% identity at the protein level) and maize/rice synteny, the maize (Zea mays L.) rth3-like gene does not appear to be a functional homolog of rth3 based on their distinct expression profiles. Massively parallel signature sequencing analysis detected rth3 expression in all analyzed tissues, but at relatively low levels, with the most abundant expression in primary roots where the root hair phenotype is manifested. In situ hybridization experiments confine rth3 expression to root hair-forming epidermal cells and lateral root primordia. Remarkably, in replicated field trials involving near-isogenic lines, the rth3 mutant conferred significant losses in grain yield.

A general method to identify plant structural genes among genomic DNA clones using transposable element induced mutations

SummarySeveral genomic clones from Petroselinum hortense, Zea mays and Antirrhinum majus all homologous to cloned Petroselinum chalcone synthase cDNA were isolated using the λgt WES cloning system.Clones containing the chalcone synthase structural gene were identified by hybridization to cDNA from Petroselinum hortense, genomic wildtype, mutant and revertant DNA.Among the 5 different clones from Petroselinum hortense, PH3 is the most likely candidate to contain at least a portion of the chalcone synthase gene.None of the 4 Zea mays clones appeared to contain part of the chalcone synthase gene.Among the 2 different clones from Antirrhinum majus, AM3 contains the portion of the chalcone synthase structural gene which is altered in the mutant nivea recurrens (nivrec). This mutant is considered to be due to the integration of a transposable element. In revertants of nivrec to niv+ the wildtype locus is restored molecularly.

Zein specific restriction enzyme fragments of maize DNA

Zein is the major storage protein of maize endosperm and is synthesized in great amounts at defined times of endosperm development [ 11. It consists primarily of two size classes of Mr -19 000 and 2 1 000 with considerable charge heterogeneity [2]. The two major protein classes are coded for by separate nonhomologous mRNA [3,4]. CDNA clones constructed from zein mRNAs have revealed 3 classes of mRNAs which can be distinguished on the basis of hybridisation experiments [ 51. However, 1.5 non-crosshybridizing mRNA were suggested [6] as a result of reassociating zein mRNA with cDNA. At the DNA level, by nucleic acid reassociation studies, the presence of 120 zein genes was proposed [63. To allow a more direct analysis of zein specific sequences in the maize genome, an investigation of zein specific restriction enzyme fragments was undertaken by applying the Southern technique [7]. The results obtained can be taken as an indication for the presence of a multigene system.

Electrophoretic elution of nucleic acids from gels adapted for subsequent biological tests: Application for analysis of mRNAs from maize endosperm

Gel electrophoresis has become a powerful tool for the analytical investigation of nucleic acid mixtures and for their preparative fractionation. However, the recovery of biologically active nucleic acids after electrophoresis has posed serious problems. The method of crushing the gel followed by elution often results in varying yields and partial loss of biological activity [ 1,2]. The method of electrophoretic elution of nucleic acids overcomes these problems. But, as applied so far, this method requires elaborate equip ment and skilled handling and can lead to dilute solutions [3-51. In all cases, the processing of several samples at the same time is rather time consuming. These disadvantages are overcome by the electrophoretic elution method described here. The procedure allows, without additional equipment, simultaneous elution of up to 20 samples/elution gel and has shown to yield, directly and with good recovery, biologically active mRNA, plasmid DNA and double-stranded DNA fragments. The performance of the method is demonstrated with mRNAs from maize endosperm and with DNA restriction fragments from a hybrid Col-plasmid.

An Essential Pentatricopeptide Repeat Protein Facilitates 5′ Maturation and Translation Initiation of rps3 mRNA in Maize Mitochondria

This work finds that a maize PPR protein (MPPR6) localized to the mitochondria is directly involved in 5′ maturation and translation initiation of rps3 mRNA. This dual role supports a general principle of action for PPR proteins in RNA processing and translation. Pentatricopeptide repeat (PPR) proteins are members of one of the largest nucleus-encoded protein families in plants. Here, we describe the previously uncharacterized maize (Zea mays) PPR gene, MPPR6, which was isolated from a Mutator-induced collection of maize kernel mutants by a cDNA-based forward genetic approach. Identification of a second mutant allele and cosegregation analysis confirmed correlation with the mutant phenotype. Histological investigations revealed that the mutation coincides with abnormities in the transfer cell layer, retardation of embryo development, and a considerable reduction of starch level. The function of MPPR6 is conserved across a wide phylogenetic distance as revealed by heterologous complementation of the Arabidopsis thaliana mutant in the orthologous APPR6 gene. MPPR6 appeared to be exclusively present in mitochondria. RNA coimmunoprecipitation and in vitro binding studies revealed a specific physical interaction of MPPR6 with the 5′ untranslated region of ribosomal protein S3 (rps3) mRNA. Mapping of transcript termini showed specifically extended rps3 5′ ends in the mppr6 mutant. Considerable reduction of mitochondrial translation was observed, indicating loss of RPS3 function. This is consistent with the appearance of truncated RPS3 protein lacking the N terminus in mppr6. Our results suggest that MPPR6 is directly involved in 5′ maturation and translation initiation of rps3 mRNA.