Genetic control and racial variation of β-glucosidase isozymes in maize (Zea mays L.)

-Glucosidase (-D-glucoside glucohydrolase, E.C. 3.2.1.21, -Glu) isozyme variants were studied in a large number of inbred lines, crosses, and races of maize (Zea mays L.). The pattern of Mendelian inheritance demonstrated for -GLU variants indicated that they are under nuclear gene control. Twenty-two allelic forms at a single locus were identified in the materials studied by starch gel electrophoresis. Genetic data indicate that -GLU in maize is functionally a dimer. Variation of -GLU isozymes in 51 racial collections of maize from Mexico showed little correlation with morphological or geographical data. In 39 collections from Central America, variation patterns appeared to have some association with altitude.This work was supported in part by NIH Research Grant GM 11546.Paper No. 5040 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Optimisation of DNA transfer and transientβ-glucuronidase expression in electroporated maize (Zea mays L.) microspores

Summary The ability to deliver free DNA into microspores of a highly androgenic hybrid of maize was assessed by electroporation, using a square wave pulse discharge apparatus. The electroporation medium was chosen according to its ability to maintain a high level of regeneration. Nuclease activities were analyzed and were inhibited by the addition of 100 mM KNO₃ and MgSO₄ in the electroporation medium. Seven expression vectors withUid A as the reporter gene under the control of cauliflower mosaic virus 35S, Lat 52-7, Zmg 13, Emu, Ubiq-1, Al, or Actl promoters were tested in relation to the level of ß-glucuronidase expression in maize microspores. The highest level of expression was obtained when theUid A gene was driven by the Actl promoter. Therefore, this vector was further used to define optimal conditions leading to highest levels of ß-glucuronidase expression. The parameters determined in this study could provide an ideal starting point for the obtention of transgenic maize plants from electroporated microspores.Abbreviations DH diplohaploid - PEG polyethylene glycol - GUS ß-glucuronidase - EDTA Ethylene-diaminetetra Acetic acid - CaMV Cauliflower mosaic virus     

Plastid targeting of E. coli β-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells

Summary Dicot and monocot chloroplast targeting peptides (CTPs) were evaluated for their effect on targeting, processing, and expression of two reporter proteins in maize cells. When tested transiently in maize leaf protoplasts, the maize ribulose bisphosphate carboxylase small subunit CTP required the inclusion of the amino terminus of mature small subunit protein to target -glucuronidase (GUS) to the plastid. To remove this amino terminal extension from GUS after import and processing, a repeat of the native processing site was inserted between the native mature protein and the reporter protein. This repeat processing site was used with less efficiency than the native site. Parallel constructs using the Arabidopsis thaliana small subunit and maize granule-bound starch synthase CTPs also localized GUS, but varied in repeat site use and GUS expression levels. Data from the CTP fusions with GUS were generally confirmed with fusions to an allosteric variant of E. coli ADP-glucose pyrophosphorylase. Plastid targeting of this enzyme was required for starch enhancement of transgenic BMS cells.Abbreviations BMS maize Black Mexican Sweet suspension culture cells - CTP chloroplast targeting peptide - glgC16 an allosteric variant of E. coli ADP-glucose pyrophosphorylase - GUS -glucuronidase - LUX luciferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SSU small subunit of ribulose bisphosphate carboxylase     

l-tryptophan-assisted PGPR-mediated induction of drought tolerance in maize (Zea mays L.)

Drought is an important abiotic stress that limits the plant growth and productivity. Present investigation was aimed that plant growth-promoting rhizobacteria (PGPR) isolated from moisture-stressed area impart drought tolerance in plants and tryptophan may improve their efficiency. Pseudomonas sp. (1), Bacillus cereus and Bacillus pumilus (B. pumilus) were isolated from maize rhizosphere grown in irrigated fields, semi-arid region and arid region, respectively. Proteus sp. and Pseudomonas sp. (2) were isolated from rice rhizosphere grown in irrigated fields and raised bed. B. pumilus produced 5× more abscisic acid (ABA) in culture media than Pseudomonas sp. (1) by the addition of l-tryptophan. These inoculants also modulated the phytohormone content of maize leaves in a pot experiment. Higher ABA was produced by the application of B. pumilus and Pseudomonas sp. (2), while indole 3-acetic acid and gibberellic acid were found higher in Pseudomonas sp. (1) and Proteus sp. treated plants. Addition of l-tryptophan increased the concentration of all phytohormones in soil and leaves of maize. Maximum increase in relative water content, osmotic potential, protein content and photosynthetic pigments was recorded in B. pumilus treated maize plants. Under irrigated condition, response of Pseudomonas sp. co-inoculated with B. pumilus from arid field superseded while under drought stress the effect of later predominated. Bacillus pumilus can be used in the formulation of biofertilizer to alleviate drought stress in arid and semi-arid regions.     

Characterisation of the cDNA clones of two β-tubulin genes and their expression in the potato plant (Solanum tuberosum L.)

The cDNA clones of two potato -tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar -tubulin genes, designated TUBST1 and TUBST2. The expression pattern of -tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding -tubulin probes revealed that there are multiple -tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3 non-coding sequences. Phylogenetic analysis of plant -tubulin genes is described.     

In vitro pollination of maize (Zea mays L.) — Proof of double fertilization

Isolated ovules from the maize homozygous recessive brown midrib (bm₃) were in vitro pollinated by pollen carrying the dominant allele — and the purple embryo marker (PEM). The colour characters of the embryos and kernels corresponded to the results of control pollination and confirmed the process of double fertilization. The question whether the method is useful for obtaining haploids is discussed.     

Intracellular immunolocalization of adenosine 5′-diphosphoglucose pyrophosphorylase in developing endosperm cells of maize (Zea mays L.)

We present immuno-electron microscopic evidence to show that ADPglucose pyrophosphorylase (AGP, EC 2.7.7.27) encoded by the Sh2 (shrunken 2) and the Bt2 (brittle 2) genes is localized to amyloplasts in developing endosperm of maize. The three AGP antibodies, including the two maize antisera, each raised against the Sh2encoded large or the Bt2-encoded small subunit and the spinach leaf protein, showed strong immuno-gold signals on developing starch grains in amyloplasts. The maize antibodies, but not the spinach, detected additional cross-reactivity sites to endosperm cell wall. Similar endosperm sections of the sh2-null mutant, lacking the Sh2-encoded subunit, yielded a drastic reduction in immuno signal on both starch grains and cell wall with the Sh2 anti-serum. However, the Bt2 and the spinach antisera showed no detectable difference between the sh2-null and the wild-type genotypes, except that the spinach antisera showed no reactivity to the cell wall in either of the two genotypes. Because the Bt2 epitope was readily detectable on the sh2-null starch grains, we suggest that the Bt2 subunit of this heteromeric enzyme is able to target itself to the organelle. The amyloplastic localization of the AGP protein in our studies is given additional significance by recent molecular data which indicate that full-length cDNA clones of the Sh2 and Bt2 genes show no cleavable transit-peptide signal sequence in their deduced aminoacid sequences. The observed disparity between the molecular and immunocytochemical data described here is discussed in the context of other proteins engaged in intracellular translocation with and without the known signal sequences.     

A tandem of α-tubulin genes preferentially expressed in radicular tissues from Zea mays

The identification of a cDNA (MR19) corresponding to a maize -tubulin and homologous genomic clones (MG19/6 and MG19/14) is described. The cDNA has been isolated by differential screening of a cDNA maize root library. We have found two -tubulin genes in a tandem arrangement in the genomic clones, separated by approximately 1.5 kbp. One of the genes (gene I) contains an identical nucleotide sequence which corresponds to the cDNA clone. The two deduced proteins from DNA sequences are very similar (only two conservative replacements in 451 amino acids) and they share a high homology as compared with the published -tubulin sequences from other systems and in particular with the Arabidopsis thaliana and Chlamydomonas reinhardtii sequences reported. The structure of both genes is also very similar; it includes two introns, of 1.7 kbp and 0.8 kbp respectively, in each gene and only one intron placed at a homologous position in relation to Arabidopsis thaliana genes. By using specific 3 probes it appears that both genes are preferentially expressed in the radicular system of the plant. The -tubulin gene family of Zea mays seems to be represented by at least 3 or 4 members.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Evolution of female sexuality in the maize ear (Zea mays L. subsp.mays—Gramineae)

The discovery of staminodes within the female inflorescences, or “ears,” of some Mexican maize races, and of feminized male inflorescences in annual Mexican teosinte, provides additional support for the theory that the ears of maize evolved from the male primary lateral branch tassels of teosinte by sexual transmutation, and that teosinte is the wild ancestor of maize.     

Measurement of N2 fixation in maize (Zea mays L.)—ricebean (Vigna umbellata [Thunb.] Ohwi and Ohashi) intercrops

The yield of N in maize (Zea mays L.) and ricebean (Vigna umbellata [Thumb.] Ohwi and Ohashi) were compared on a Tropoqualf soil in North Thailand in 1984 and 1985. Both species were grown in field plots in monoculture or as intercrops at a constant planting density equivalent to 8 maize or 16 ricebean plants per m². The contribution of symbiotic N₂ fixation to ricebean growth was estimated from measurements of the natural abundance of¹⁵N (δ¹⁵N) in shoot nitrogen and from analysis of ureides in xylem sap vacuumextracted from detached stems. The natural abundance of¹⁵N in the intercropped ricebean was found to be considerably less than that in monoculture in both growing seasons. Using maize and a weed (Ageratum conyzoides L.) as non-fixing¹⁵N reference plants the proportions (P ¹⁵N) of ricebean shoot N derived from N₂ fixation ranged from 0.27 to 0.36 in monoculture ricebean up to 0.86 when grown in a 75% maize: 25% ricebean intercrop. When glasshouse-derived calibration curves were used to calculate plant proportional N₂ fixation (Pur) from the relative ureide contents of field collected xylem exudates, the contribution of N₂ fixation to ricebean N yields throughout the 1985 growing season were greater in intercrop than in monocrop even at the lowest maize:legume ratio (25∶75). Seasonal patterns of sap ureide abundance indicated that N₂ fixation was greatest at the time of ricebean podset. The averagePur andP ¹⁵N in ricebean during the first 90 days of growth showed identical rankings of monocrop and intercrop treatments in terms of N₂ fixation, although the two sets ofP values were different. Nonetheless, seasonal estimates of N₂ fixation during the entire 147 days of legume growth determined from ureide analyses indicated that equivalent amounts of N could be fixed by ricebean in a 75∶25 intercrop and in monoculture despite the former being planted at one-quarter the density.