The maize GapC4 promoter confers anaerobic reporter gene expression and shows homology to the maize anthocyanin regulatory locus C1

The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) gene family of maize is differentially expressed in response to anaerobic stress. While GapC1 and GapC2 are downregulated, GapC3 and GapC4 are anaerobically induced. We have sequenced and analyzed a 3073 bp promoter fragment of GapC4. The promoter confers anaerobic induction of a reporter gene construct in a transient gene expression system in maize. Deletion analysis of the GapC4 promoter revealed a 270 bp long DNA region required for anaerobic induction. This region contains sequence motifs resembling the cis-acting sequences of the anaerobically induced maize Adh1 and Adh2 genes. Furthermore, the 3073 bp GapC4 promoter fragment displays homology to long terminal repeats of maize retrotransposons and to the 3 region of the maize anthocyanin regulatory locus C1.     

Haplo-diploid gene expression and pollen selection for tolerance to acetochlor in maize

The objectives of this research were to determine if genes controlling the reaction to the herbicide acetochlor in maize (Zea mays L.) are active during both the haploid and the diploid phases of the life cycle and if pollen selection can be utilized for improving sporophytic resistance. Pollen of eight inbred lines, previously characterized through sporophytic analysis for the level of tolerance to acetochlor, showed a differential reaction to the herbicide forin vitro tube length; moreover, such pollen reactions proved to be significantly correlated (r =0.786^*,df=6) with those of the sporophytes producing the pollen. Pollen analysis of two inbred lines (i.e. Mo17, tolerant, and B79, susceptible) and their single cross showed that thein vitro pollen-tube length reaction of the hybrid was intermediate between those of two parents. An experiment on pollen selection was then performed by growing tassels of Mo17xB79 in the presence of the herbicide. Pollen obtained from treated tassels showed a greater tolerance to acetochlor, assessed asin vitro tube length reaction, than pollen obtained from control tassels. Moreover, the backcross [B79 (Mo17xB79)] sporophytic population obtained using pollen from the treated tassels was more tolerant (as indicated by the fresh weight of plants grown in the presence of the herbicide) than was the control backcross population. The two populations did not differ when grown without the herbicide. These findings indicate that genes controlling the reaction to acetochlor in maize have haplodiploid expression; consequently, pollen selection can be applied for improving plant tolerance.     

DNase I hypersensitivity and expression of the Shrunken-1 gene of maize

The local chromatin structure of the Shrunken-1 (Sh) gene of maize was probed by analyzing DNase I hypersensitivity. Sh encodes the gene for sucrose synthetase, a major starch biosynthetic enzyme, which is maximally expressed in the endosperm during seed maturation. In addition to general DNase I sensitivity, specific DNase I hypersensitive sites were identified in endosperm chromatin that mapped near the 5 end of the Sh gene. The pattern of hypersensitive sites and their relative sensitivity were altered in other non-dormant tissues that produce little or no enzyme. However, some changes in chromatin structure appear to be independent of Sh gene expression and may reflect general alterations associated with plant development. The chromatin structure of several sh mutations, induced by Ds controlling element insertions, was also analyzed. Although the insertions perturbed expression of the gene, there were no notable effects on local chromatin structure.     

The maize cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family: organ-specific expression and genetic analysis

Summary The distribution of the cytosolic glyceraldehyde-3-phosphate dehydrogenase gene family (Gpc) in the maize genome was investigated; a genetic variant of glyceraldehyde-3-phosphate dehydrogenase activity is also described. Restriction fragment length polymorphism analysis of an F₂ population shows that the variant is not linked to the three known Gpc genes. However, this trait is linked to one of two genomic DNA fragments that hybridize to a fragment of the Gpc3 coding region, implying the existence of a fourth Gpc gene. Antibodies and cDNA clones were used to investigate the organ-specific expression of the Gpc genes. Results were compared with the expression of the alcohol dehydrogenase 1 (Adh1) gene. RNA and protein levels were examined in seedling roots and shoots, as well as the leaves, developing endosperm and embryo, and the aleurone. In general, it was found that Gpc3 expression behaves in parallel with Adh1 in these organs, and protein levels closely parallel that of RNA for each gene examined. Both Gpc3 and Adh1 show a marked increase in expression during endosperm development, reaching a maximum 15 days after pollination, but no expression is detected in the leaf. Gpc1 expression is similar to that of Gpc2, with an overall decrease in the level of RNA during endosperm development. This expression is discussed in terms of the common sequences found upstream of genes expressed in the developing maize seed.     

uidA gene transfer and expression in maize microspores using the biolistic method

Summary The ability to recover male gametophyte derived plants, which is necessary to get transformed haploid plants, was verified for a hybrid of maize. Using the isolated microspore culture technique, a 9 × 10^–5 plant regeneration frequency was obtained. Maize microspores were bombarded with tungsten particles using a PDS He/1000 apparatus. GUS expression in the microspores was maximum with 1.1 m diameter tungsten microprojectiles for 1100 and 1350 psi helium pressures at a 6 cm distance between the launch point and the target cells. Increasing the amount of DNA coated on the microparticles from 1.66 to 4 g DNA/mg of particles allowed a two-fold and four-fold increase of the GUS-expressing microspore frequency for 1100 and 1350 psi helium pressure bombardment, respectively. Optimal concentration of solidifying agent in the bombardment support culture medium was found to be 1%. Cell density ranging from 25000 microspores/bombardment to 100000 microspores/bombardment did not affect the frequency of GUS-expressing microspores. Using these optimal conditions, the maximum frequency of GUS-expressing microspores was found to be about 9 × 10^–4, while maintaining an embryo formation frequency about 5 × 10^–4.Abbreviations GUS -glucuronidase - PEG polyethylene glycol     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derivedEmu andCaMV 35S in cells of barley,maize and tobacco

The particle gun approach was used for the quantification of promoter efficiency in a test system for transient gene expression. β-Glucuronidase was used as reporter gene for determining promotote strength. The variability inherent in this gene transfer system was considerably reduced by calculating a transformation efficiency factor given by the expression of a cotransferred second reporter gene (firefly luciferase). The calibration of β-glucuronidase activity by the transformation efficiency factor caused a lower statistical variance of the values and allowed reliable results to be obtained with a smaller set of repetitions. The CaMV 35S promoter (as a control) and the monocot-specific promoters for maize polyubiquitin1, rice actin 1 and the maize-derivedEmu were characterized and compared with respect to expression strength, as tested under identical conditions in suspension cell cultures of maize, barley and tobacco. Compared to the 35S promoter, the monocot-specific promoters show up to 15-fold higher expression in maize and barley but give only weak expression in tobacco. No expression was found for the rice actin 1 promoter in tobacco. The level of reporter gene expression is influenced by the osmotic potential in the agar medium. For theEmu promoter, the calibrated β-glucuronidase activities remained mearly constant at low sucrose concentrations. Above 8% sucrose, the calibrated activities increased steadily with increasing osmotic conditions, reaching a three-to four-fold higher level at the highest sucrose concentration (32%) as compared to the standard concentration (4% sucrose) in the medium.     

Gene expression studies on developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene

Previous studies have identified two tissue- and cell-specific, yet functionally redundant, sucrose synthase (SuSy) genes, Sh1 and Sus1, which encode biochemically similar isozymes, SH1 and SUS1 (previously referred to as SS1 and SS2, respectively). Here we report evidence for a third SuSy gene in maize, Sus3, which is more similar to dicot than to monocot SuSys. RNA and/or protein blot analyses on developing kernels and other tissues show evidence of expression of Sus3, although at the lowest steady-state levels of the three SuSy gene products and without a unique pattern of tissue specificity. Immunoblots of sh1sus1-1 embryos that are either lacking or deficient for the embryo-specific SUS1 protein have shown a protein band which we attribute to the Sus3 gene, and may contribute to the residual enzyme activity seen in embryos of the double mutant. We also studied developing seeds of the double mutant sh1sus1-1, which is missing 99.5% of SuSy enzyme activity, for evidence of co-regulation of several genes of sugar metabolism. We found a significant reduction in the steady-state levels of Miniature-1 encoded cell wall invertase2, and Sucrose transporter (Sut) mRNAs in the double mutant, relative to the lineage-related sh1Sus1 and sh1Sus1 kernels. Down-regulation of the Mn1 gene was also reflected in significant reductions in cell wall invertase activity. Co-regulatory changes were not seen in the expression of Sucrose phosphate synthase, UDP-glucose pyrophosphorylase, and ADP-glucose pyrophosphorylase.     

Relationship between gene expression and hybrid vigor in primary root tips of young maize (Zea mays L.) plantlets

Summary To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F₁ primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F₁ hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines.