Development patterns of telomerase activity in barley and maize

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.     

Activation of low and null activity isozymes of maize alcohol dehydrogenase by antibodies

Summary Antisera were raised against several purified, high specific acitivity isozymes of maize alcohol dehydrogenase (ADH1). The various antisera had different effects on the activity of immunoprecipitated ADH. One antiserum completely inactivated maize ADH. This inactivation could be blocked by preicubation of the enzyme with NAD⁺, its cofactor, or with NADP. The different antisera were used to analyze variant froms of ADH1. Isozymes having lowered specific activity were activated to wild-type levels by precipitation of the enzymes with noninactivating antisera. Isozymes having no detectable ADH activity (CRM⁺ nulls) were activated by immunoprecipition with noninactivating antisera when preincubated with NAD⁺ or NADP. All of the CRM⁺ nulls were shown to be unable to bind NAD⁺, a flaw which can account for their lack of activity. The results indicate that a conformational equilibrium between active and inactive forms of maize ADH in solution controls the specific activity of the various isozymes. Both controls the specific activity of the various isozymes. Both NAD⁺ and antibodies raised against high specific activity enzymes can interact with low activity isozymes to shift the balance of the equilibrium toward the active form, thus increasing their specific activity.     

Glutamine synthetase isozymes in germinating barley seeds

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of ammonia assimilation in higher plants. In the present study the subunit composition and localization of GS in germinating barley ( Hordeum vulgare ) seed have been clarified. Analysis of the GS polypeptide composition by immunoblotting revealed two different polypeptides. A and B, with a molecular mass of 42 and 40 kDa, respectively. In the scutellum subunit A was already present in the ungerminated seed and remained unchanged, whereas subunit B appeared on day 2 and increased about 5-fold during germination. Polypeptide B also appeared later during germination in the aleurone layer, roots and weakly in the etiolated shoots. By immunogold microscopy, GS was detected in the scutellum and the aleurone layer of barley seeds during germination. Subcellular localization of GS on ultrathin cryosections showed that a cytosolic isozyme was present in the scutellum. Our study confirms that only a cytosolic GS is expressed in barley seed, and its subunit composition changes during germination.     

Haploid formation in maize, barley, flax, and potato

Summary. The article is reviewing some significant features and issues in the process of haploid formation in two important monocotyledonous crop plants – maize and barley – and in two dicotyledonous plants – flax and potato. Exotic maize lines with higher androgenic response turned up as a good source for this heritable trait and this valuable trait can be incorporated into elite maize lines via crossing. Lots of attempts were devoted to identifying some cytological and/or morphological markers for androgenic response in maize microspore cultures. The “starlike” organization of the cytoplasm inside the induced maize microspores together with the enlarged size of induced microspores can be considered as morphological markers for androgenic response. In barley, microspores with rich cytoplasm that was of granular appearance with the nucleus located near the cell wall and with no visible vacuole had the largest survival rate and many of these cells continued in development and produced embryos. In flax, a dramatic increase of induction rate in anther cultures (up to 25%) was achieved when flax anthers were pretreated for 3 days at 4 °C and afterwards kept for 1 day at 35 °C. Also gynogenesis in flax has been reported already and complete plants were obtained. In potato microspore cultures, formation of two dissimilar cells indicated a strong polarization in the system and as a result of this polarization a prominent suspensor developed that persisted until the torpedo stage of the androgenic embryo. This was the first time the formation of a well developed suspensor was described in connection with androgenesis. Correspondence and reprints: Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, P.O. Box 39A, 950 07 Nitra, Slovak Republic.     

The effect of altered codon usage on luciferase activity in tobacco, maize and wheat

A comparison of the wild-type firefly luciferase reporter gene to a codon-modified gene, available from Promega, demonstrates that in tobacco cell cultures, an increase in G+C content of 1.8%, as a consequence of 36 A/T→G/C synonymous codon alterations and removal of the lysosomal targeting sequence, has no significant effect on expression. In maize Black Mexican Sweet cells and wheat scutellum, increases in activity of 14- to 23-fold and 53- to 59-fold, respectively, are obtained using the codon-modified luciferase with the UBI1 promoter and its leader intron. The observed increase in luc+ expression is most likely a consequence of differences in codon usage reflecting tRNA abundance rather than an increase in the efficiency of intron splicing resulting from the small increase in the G+C content of the coding sequence. This difference in light emission between the wild-type and codon-modified luciferases can be clearly visualised in a low-light imaging camera, making the latter a much more sensitive and useful reporter gene for detecting luciferase activity in vivo. Received: 7 September 1996 / Revision received: 28 November 1996 / Accepted: 6 December 1996     

Evaluation of a catalase inhibitor in the developmental regulation of maize catalase

A protein which has been shown to inhibit catalase in vitro appears to vary inversely with catalase activity in the maize scutellum during early sporophytic development when assayed using a catalase inhibition assay. This result suggested that the inhibitor protein may play a direct role in regulating catalase activity during this time period. Four experimental approaches were used to evaluate this putative regulatory role, including immunological quantitation of individual catalase isozymes during germination using rocket immunoelectrophoresis, perturbation of normal catalase expression with hydrogen peroxide or allylisopropylacetamide (AIA), examination of a mutant line with an altered catalase developmental program, and direct radioimmunoassay of the inhibitor protein during germination. The results of these experiments indicate that the quantitative changes in catalase activity during development are not mainly due to changes in the expression of the catalase inhibitor. Other possible roles of this protein in catalase regulation are discussed.     

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.     

3个桉树无性系过氧化氢酶活性及同工酶比较研究

本文研究杂交桉树(Eucalyptus urophylla×Eucalyptus camaldulensis)LH21无性系、LH22无性系和尾叶桉(Eucalyptus urophylla)U6无性系的过氧化氢酶(CAT)活性及同工酶的差异。结果表明,3个桉树无性系同一器官的CAT活性存在差异,同一个无性系中不同器官的CAT活性差异很显著。采用聚丙烯酰胺不连续凝胶垂直板电泳比较分析CAT同工酶,发现3个无性系的CAT同工酶存在着一定的差异,其中LH21和LH22叶片有相同的谱带,但根的谱带与叶片有差异;而U6各器官的CAT谱带与LH21和LH22有差异。3个无性系的CAT同工酶都在一定程度上具有器官的特异性。     

Genetic control of alcohol dehydrogenase isozymes in maize

By means of horizontal gel electrophoresis and the zymogram technique, genetic variants and the formation of a hybrid molecule of the enzyme alcohol dehydrogenase (ADH) have been found in Zea mays. Each inbred homozygous stock examined showed two types of ADH isozyme patterns: a fast faint zone and a slower deeply staining zone, both anode-migrating at pH 8.5. The variants found differed in that each of the ADH zones varied in its electrophoretic mobility when compared to its counterpart in the other strain. When appropriate genetic crosses were made, the resulting heterozygotes showed the parental ADH zones, and, in addition, a band of intermediate mobility was formed between the deep-staining ADH bands. However, in the fast-moving zone only the parental isozymes were represented in the heterozygote. The formation of the hybrid molecule and the apparent gene dosage effects support the hypothesis that ADH-2 in maize exists as a dimer, whereas ADH-1 may exist as a monomer.This work was supported by the U.S. Atomic Energy Commission, under contract No. AT(11-1)-1338.     

Molecular cloning,characterization and expression analysis of two catalase isozyme genes in barley

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and-3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).     

Genetic control of malate dehydrogenase isozymes in maize

At least six nuclear loci are responsible for the genetic control of malate dehydrogenase (L-malate: NAD oxidoreductase; EC 1.1.1.37; MDH) in coleoptiles of maize. Three independently segregating loci (Mdh1, Mdh2, Mdh3) govern the production of MDH isozymes resistant to inactivation by ascorbic acid and found largely or solely in the mitochondria. A rare recessive allele found at a fourth nuclear locus (mmm) causes increased electrophoretic mobility of the MDH isozymes governed by the Mdh1, Mdh2 and Mdh3 loci.—Two loci (Mdh4, Mdh5) govern MDH isozymes that are selectively inactivated by homogenization in an ascorbic acid solution and that appear to be nonmitochondrial (soluble). Mdh4 and Mdh5 segregate independently of each other and independently of Mdh1, Mdh2 and Mdh3. However, there is close linkage between the migration modifier and Mdh4.——Multiple alleles have been found for all of the Mdh loci except the migration modifier, and electrophoretically "null" or near "null" alleles (as expressed in standardized sections of maize coleoptile) have been found for all loci except Mdh4. Duplicate inheritance commonly occurs for Mdh1 and Mdh2 and also for Mdh4 and Mdh5.——Inter- and intragenic heterodimers are formed between sub-units specified by the three loci governing the mitochondrial MDH isozymes. The same is true of the alleles and nonalleles at the two loci governing the soluble variants. No such heterodimers are formed by interactions between mitochondrial and soluble MDH isozymes.     

Developmental expression of a catalase inhibitor in maize

The expression of an endogenous catalase inhibitor has been studied during development of Zea mays. In the 3-day seedling, the inhibitor is expressed primarily in the scutellum and in the aleurone layer of the endosperm. These tissues also show the highest catalase activity at this stage. Inhibitor expression has also been studied temporally in the scutellum, roots, and shoot over the first 12 days of germination. Inhibitor expression shows an inverse relationship with catalase activity in the scutellum and in the shoot. The relationship is less rigid in the root, due probably to the low levels of inhibitor found in that tissue. The role of the inhibitor in catalase regulation is discussed.     

盐胁迫条件下γ-氨基丁酸对玉米幼苗SOD、POD及CAT活性的影响

逆境下植物体内积累氨基丁酸(GABA)。盐胁迫严重影响玉米种子的萌发,而加入外源GABA可明显提高玉米种子的萌发率。外源GABA能迅速提高SOD、POD、和CAT这三种酶的活性。鉴于超氧化物歧化酶、过氧化氢酶和过氧化物酶是植物抗氧化保护系统中重要的组成部分,推测,盐胁迫条件下,GABA可通过提高保护酶系统活性而缓解盐胁迫对植物的伤害。     

The mitigating role of environmental factors in seedling injury and chill-dependent depression of catalase activity in maize leaves

In pot experiments performed on maize seedlings chilled at 5 °C, leaf injury was diminished by the application of elevated temperature (1 or 5 h at 15 or 20°C, “warm breaks” treatment) in a dose-dependent manner. The lower the injury count, the higher the catalase (CAT) activity. In a separate experiment, the application of 100 % relative humidity also protected the plants from chilling injury and water loss, increased their gas exchange and variable to maximum chlorophyll fluorescence ratio (F_v/F_m), but did not influence CAT activity. Another protective environmental factor, elevated atmospheric CO₂ concentration [700 μmol(CO₂) mol⁻¹(air)] diminished CAT activity inhibition, but only in plants of chilling-resistant cultivar. The positive impact of specific environmental factors accompanying chilling is not obviously related to the suppression of the inhibition of CAT activity, although the enzyme is considered as chilling-sensitive.